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Mood Insomnia Fatigue
A New Model of Diagnosisand Treatment
Decker Weiss NMD FASA
Naturopathic Cardiologist
Scottsdale Heart Institute
Senior Fellow Artis Research
PTSD and Radicalization Researcher Jordan Syria Iraq Gaza Israel
Financial Disclosure Nothing to Disclose
Mood
Depression in
America
1 in 10
The diagnosis is INCREASING 20 per year
80 of depressed persons do not seek treatment
Depression varies by state Oklahoma Mississippi Louisiana Alabama W Virginia are amongst the highest
Healthline InfographicPosted as of 29Feb2016 httpwwwhealthlinecomhealthdepressionstatistics-infographic
Depression and Women
ldquoCompared to men women are twice as
likely to develop depressionrdquo
Psychology Today The 7 Reasons Why Depression is More Common in Women Compared to men women are twice as likely to develop depression
Published on May 17 2012 by Neel Burton MD in Hide and Seek
httpwwwpsychologytodaycombloghide-and-seek201205the-7-reasons-why-depression-is-more-common-in-women as reported 30 Sept 2014
Depression in Teens
The Old Model
Educated link to a diagnoses educated
guess to a treatment than repeat
Common Interventions for Depression
Reuptake inhibitors Serotonin
Sertraline
Citalopram
Escitalopram
Reuptake inhibitors
Norepinephrine Dopamine
Bupropion
Venlafaxine
Cyclics
Trazodone
Mirtazapine
Atypical
Ariprazole
Olanzapine
Quetiapine
7
Psychiatry Failure in the Press
The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental
Disorders (DSM) shows that US psychiatry has no intention of changing course
Edward Shorter- University of Toronto- WSJ
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mood
Depression in
America
1 in 10
The diagnosis is INCREASING 20 per year
80 of depressed persons do not seek treatment
Depression varies by state Oklahoma Mississippi Louisiana Alabama W Virginia are amongst the highest
Healthline InfographicPosted as of 29Feb2016 httpwwwhealthlinecomhealthdepressionstatistics-infographic
Depression and Women
ldquoCompared to men women are twice as
likely to develop depressionrdquo
Psychology Today The 7 Reasons Why Depression is More Common in Women Compared to men women are twice as likely to develop depression
Published on May 17 2012 by Neel Burton MD in Hide and Seek
httpwwwpsychologytodaycombloghide-and-seek201205the-7-reasons-why-depression-is-more-common-in-women as reported 30 Sept 2014
Depression in Teens
The Old Model
Educated link to a diagnoses educated
guess to a treatment than repeat
Common Interventions for Depression
Reuptake inhibitors Serotonin
Sertraline
Citalopram
Escitalopram
Reuptake inhibitors
Norepinephrine Dopamine
Bupropion
Venlafaxine
Cyclics
Trazodone
Mirtazapine
Atypical
Ariprazole
Olanzapine
Quetiapine
7
Psychiatry Failure in the Press
The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental
Disorders (DSM) shows that US psychiatry has no intention of changing course
Edward Shorter- University of Toronto- WSJ
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Depression in
America
1 in 10
The diagnosis is INCREASING 20 per year
80 of depressed persons do not seek treatment
Depression varies by state Oklahoma Mississippi Louisiana Alabama W Virginia are amongst the highest
Healthline InfographicPosted as of 29Feb2016 httpwwwhealthlinecomhealthdepressionstatistics-infographic
Depression and Women
ldquoCompared to men women are twice as
likely to develop depressionrdquo
Psychology Today The 7 Reasons Why Depression is More Common in Women Compared to men women are twice as likely to develop depression
Published on May 17 2012 by Neel Burton MD in Hide and Seek
httpwwwpsychologytodaycombloghide-and-seek201205the-7-reasons-why-depression-is-more-common-in-women as reported 30 Sept 2014
Depression in Teens
The Old Model
Educated link to a diagnoses educated
guess to a treatment than repeat
Common Interventions for Depression
Reuptake inhibitors Serotonin
Sertraline
Citalopram
Escitalopram
Reuptake inhibitors
Norepinephrine Dopamine
Bupropion
Venlafaxine
Cyclics
Trazodone
Mirtazapine
Atypical
Ariprazole
Olanzapine
Quetiapine
7
Psychiatry Failure in the Press
The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental
Disorders (DSM) shows that US psychiatry has no intention of changing course
Edward Shorter- University of Toronto- WSJ
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Depression and Women
ldquoCompared to men women are twice as
likely to develop depressionrdquo
Psychology Today The 7 Reasons Why Depression is More Common in Women Compared to men women are twice as likely to develop depression
Published on May 17 2012 by Neel Burton MD in Hide and Seek
httpwwwpsychologytodaycombloghide-and-seek201205the-7-reasons-why-depression-is-more-common-in-women as reported 30 Sept 2014
Depression in Teens
The Old Model
Educated link to a diagnoses educated
guess to a treatment than repeat
Common Interventions for Depression
Reuptake inhibitors Serotonin
Sertraline
Citalopram
Escitalopram
Reuptake inhibitors
Norepinephrine Dopamine
Bupropion
Venlafaxine
Cyclics
Trazodone
Mirtazapine
Atypical
Ariprazole
Olanzapine
Quetiapine
7
Psychiatry Failure in the Press
The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental
Disorders (DSM) shows that US psychiatry has no intention of changing course
Edward Shorter- University of Toronto- WSJ
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Depression in Teens
The Old Model
Educated link to a diagnoses educated
guess to a treatment than repeat
Common Interventions for Depression
Reuptake inhibitors Serotonin
Sertraline
Citalopram
Escitalopram
Reuptake inhibitors
Norepinephrine Dopamine
Bupropion
Venlafaxine
Cyclics
Trazodone
Mirtazapine
Atypical
Ariprazole
Olanzapine
Quetiapine
7
Psychiatry Failure in the Press
The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental
Disorders (DSM) shows that US psychiatry has no intention of changing course
Edward Shorter- University of Toronto- WSJ
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
The Old Model
Educated link to a diagnoses educated
guess to a treatment than repeat
Common Interventions for Depression
Reuptake inhibitors Serotonin
Sertraline
Citalopram
Escitalopram
Reuptake inhibitors
Norepinephrine Dopamine
Bupropion
Venlafaxine
Cyclics
Trazodone
Mirtazapine
Atypical
Ariprazole
Olanzapine
Quetiapine
7
Psychiatry Failure in the Press
The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental
Disorders (DSM) shows that US psychiatry has no intention of changing course
Edward Shorter- University of Toronto- WSJ
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Common Interventions for Depression
Reuptake inhibitors Serotonin
Sertraline
Citalopram
Escitalopram
Reuptake inhibitors
Norepinephrine Dopamine
Bupropion
Venlafaxine
Cyclics
Trazodone
Mirtazapine
Atypical
Ariprazole
Olanzapine
Quetiapine
7
Psychiatry Failure in the Press
The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental
Disorders (DSM) shows that US psychiatry has no intention of changing course
Edward Shorter- University of Toronto- WSJ
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Psychiatry Failure in the Press
The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental
Disorders (DSM) shows that US psychiatry has no intention of changing course
Edward Shorter- University of Toronto- WSJ
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Waiting on a
Breakthrough
ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities
Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry
The New Yorker Posted as of 22Feb 2016
SEPTEMBER 3 2013The Psychiatric Drug Crisis
BY GARY GREENBERG
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Fatigue
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Fatigue Fatigue is a common complaint with a
prevalence in population-based surveys in Britain
and the United States of between 60 and 75
percent
A cross-sectional survey of United States workers
found the two-week period prevalence of fatigue
to be 38 percent with an estimated annual cost
to employers exceeding $136 billion in lost
productive work time
In addition an estimated 21 to 33 percent of
patients seeking attention in primary care settings
report significant fatigue resulting in
approximately seven million office visits per year in
the United States
Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Fatigue
Mayo
Treatment includes
antidepressants
sleeping pills
Pace yourself
Graded Exercise
Psychological counseling
httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Insomnia
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender
As reported Sept 1 2017
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most
are white female educated or 50 or older according to the first
government study of its kind
The CDC estimates that ldquo50-70 million US adults have sleep or
wakefulness disorderrdquo These numbers are striking because they
essentially mean that for every adult with a sleep disorder a doctor is
writing a prescription for sleeping pills
Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to
60 million in the US About 38 million of those prescriptions were for drugs
containing zolpidem (including the brand name Ambien) according
to IMS Health
The United States insomnia pharmacological treatment market is
projected to reach $424 billion by 2021 from $338 billion in 2016
httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mood Fatigue Insomnia Theory
Mood Insomnia and Fatigue Issues are
inflammatory issues which are measurable
and reversible even with a poor genomic
outlook
-Decker Weiss
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
The Three Stooges of Mood Fatigue
and Insomnia
Inflammation causing Neurotransmitter Dysfunction
Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses
Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
DepressionInsomniaFatigue
GlutamatergicExcess
Many Causes Similar Result
NoradrenergicDeficiency
SerotonergicDeficiency
Belmaker R H and Agam G Major depressive
disorder (1-3-2008) NEnglJMed 358(1) 55-
68
GutBrainDysfunction
Mitochondrial Dysfunction
18
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Neurotransmitters
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
PVN
C1
LC
Pituitary
DR
Thoracic
Lumbar
Cortisol
DHEAEpinephrine
Norepinephrine
CRF
ACTH
Traditional Stress Response
Hypothalamus is the master controller of the HPA axis (endocrine model)
Traditionally the start of the HPA axis
Pituitary gland has two parts that bridge the brain and body
Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF
Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone
Two distinct parts of the adrenal gland produce hormones and neurotransmitters
Adrenal Cortex = cortisol and DHEA
Adrenal Medulla = norepinephrine and epinephrine
Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709
Are you
considering
the adrenal
medulla
20
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
PVN
C1
LC
Hypothalamus
Pituitary
DR
Cortex
Medulla
Thoracic
Lumbar
CRF
NE
Cortisol
Glutamate
Epinephrine
ACh
Glutamate activates Thoracic pathway to
Adrenals
Sympathetic Chain
Serotonin
21
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
The ldquoBackward RuleMatch Diagnosis to Labs
Neurotransmitter Subject 1 Subject 2 Subject 3
Serotonin
GABA
Glycine
Glutamate
Histamine
PEA
Dopamine
Norepinephrine
Epinephrine
Cortisol
22
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Biomarkers for Early Stage 2
3
23
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Clinical Presentation19 year old Israeli Soldier during OPE
OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Biomarkers for Mid Stage
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Clinical Presentation36 Year old Israeli Nurse during OPE
Wired but TiredDepressionInsomnia
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Biomarkers for End Stage
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Clinical Presentation
ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors
Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Genomics
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of
the brain responsible for personality inhibition of behaviors abstract thinking
and emotion1
The COMT val158Met polymorphism has been shown to be associated with phobic
anxiety 2 neuroticism 3 and panic disorders 4
Results indicate a relationship of COMT polymorphism rs4680 with immune
dysregulation in CFS providing a potential link for the association between stress and
infection susceptibility in CFS(chronic fatigue syndrome)
1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype
on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J
Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8
27
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
17 yo radicalized female with anger
antisocial and rage issues(similar not exact due to security)
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Methylene-tetrahydrofolate reductase
(MTHFR SNPrsquos)
Creates methylated folate
the active folate in the
body
Mutations have been
associated with vascular
disease dementia cancer
and autism
Methylation is the genetics
elevated homocysteine is
the genomics 29
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
End Point of Poor Methylation and
Oxidation
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
Genetic Expression on Mitochondrial
Function
(ROS Degradation)
bull Superoxide dismutase 1
(SOD1)
bull Superoxide dismutase 2
(SOD2)
bull Superoxide dismutase 3
(SOD3)
bull Glutathione peroxidase 1
(GPx1)
bull Catalase (CAT)
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Superoxide Dismutase 1
SOD1 (CuZnSOD)(Encoded Chrom 21)
Found in cytoplasmic and mitochondrial
intermembrane space(inserted)
Converts superoxide radicals (O2-) to hydrogen
peroxide (H2O2)
Needs copper and zinc as cofactors
Apoptosis Oxidative Stress ALS Myocardial
Infarction Macular Degeneration a marker for
heavy Metal toxicity
O2- H2O2
SOD1SOD1
Cu Zn
1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2
2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160
3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Superoxide Dismutase 2
SOD2 (MnSOD) (Chrom 6)
Found in mitochondria
Converts superoxide radicals (O2-)
to hydrogen peroxide (H2O2)
Needs manganese as a cofactor
Heart Disease Myocardial
Infarction Tumor metastasis
neurodegenerative diseases
O2- H2O2SOD2SOD2
Mn
Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Superoxide Dismutase 3SOD3
Found extracellularly
Converts superoxide radicals (O2-) to
hydrogen peroxide (H2O2)
Needs copper and zinc as cofactors
Chronic skin disorders Diabetic
Neuropathy Folate Metabolism Ischemic
Heart Disease
O2- H2O2SOD3SOD3
Cu Zn
Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Glutathione Peroxidase 1
GPx1(Chrom 3)
Found in the cytoplasm of nearly all mammalian tissues
Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)
Uses glutathione an antioxidant as a cofactor
Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression
H2O2H2O O2GPx1GPx1
Glutathione
Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and
Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Catalase
CAT(Chrom 11)
Found in the cytoplasm of cells
Needs iron and selenium as cofactors
Reduce hydrogen peroxide (H2O2) to water (H2O)
High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second
Hypertension Type 2 Diabetes Acatalasemia
httpsghrnlmnihgovgeneCATconditions
H2O2 H2O O2CATCAT
Fe Se
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Function Assessment
Mitochondrial Superoxide (O2-)
Plasma Peroxide Assay (PPA)
Mitochondrial Membrane
Potential (MMP) Assay with
Oxidative Challenge
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Superoxide (O2-)
Superoxide is considered to be a major
factor in oxidant toxicity and
mitochondrial MnSOD enzymes
constitute an essential defense against
superoxide
High levels indicate the proanti-oxidant
system is out of balance and oxidative
stress is present
Mitochondria are the major source of
superoxide
Superoxide is the origin of reactive
oxygen (ROS)and nitrogen species (RNS)
and as such causes various redox
related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo
123Hsiu-Chuan Yen
4Ikuo Nakanishi
5Ken-ichiro Matsumoto
5Masato Tamura
6Yumiko Nagano
6Hirofumi Matsui
6Oleg Gusev
789Richard Cornette
9Takashi
Okuda9
Yukiko Minamiyama10
Hiroshi Ichikawa11
Shigeaki Suenaga1
Misato Oki2
Tsuyoshi Sato1
Toshihiko Ozawa12
Daret K St Clair3
and Hideyuki J Majima12dagger
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Retrospective Study Sample Distribution
To
tal S
ub
jects
(n
=86)
Lo
w (
n=10)
No
rmal (n
=27)
Ele
vate
d (
n=19)
Hig
h (
n=30)
0
1
2
3
4
5
Mit
oS
OA
R (
RF
U)
3 5
2 2
2 7
1 2
M ito S O A R
R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n
MitoSOARtrade (RFU)
lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects
N 10 27 19 3086
12 31 22 35
57 of the tested population has
superoxide levels in the
elevatedhigh range leading to high
risk for certain clinical conditions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Functional Genetics
CT
CC
0 5
1 0
1 5
2 0
2 5
Mit
oS
OA
R (
RF
U)
S O D 2 H e tro z y g o u s g e n o ty p e (C T )
S O D 2 H o m o z y g o u s g e n o ty p e (C C )
P lt 0 0 5
M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e
(S O D 2 ) P o ly m o rp h is m
Individuals who are homozygous
recessive(CC) for SOD2 may
have impaired SOD2 function
resulting in higher levels of the
free radical superoxide and
require antioxidant support
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Superoxide Levels
Assessment of Redox status
Inflammation
Viral or Bacterial Infection
Monitoring treatment of a
disease or disorder
Borrelia infection
Treatment of Inflammation
Functional Effect
Improvement verifies positive clinical effect
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Plasma Peroxide Assay
Low Levels may indicate oxidative stress and
possible need for antioxidants
High levels may indicate immune challenges
Can correlate with uric acid
Potential hypertension risk
Related to lipid peroxides
Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13
Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Plasma Peroxidase
To
tal S
ub
jects
(n
=86)
Lo
w (
n=30)
No
rmal (n
=24)
Ele
vate
d (
n=11)
Hig
h (
n=16)
0
2 0
4 0
6 0
8 0
Pla
sm
a P
erix
ida
se
Ac
tiv
ity
(m
Um
l)
2 0
1 4
3 0
3 7
M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s
R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e
S a m p le D is tr ib u tio n
Plasma Peroxidase Activity (mUmL)
lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects
N 30 24 11 1681
37 30 14 20
37 of the population studied had low
peroxidase activity indicating an
impaired ability to defend against free
radicals
34 of the studies population had
elevatedhigh activity potentially
indicating the presence of oxidative
stress
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Assessment of Baseline Potential
Starting point for defense of excessive
ROS
Effect of Environmental Insults
Assessment of Capacity to defend against
excessive ROS
Assessment and monitoring treatment of a
disease or disorder
Viral or Bacterial infection
Cardiovascular disease
Mitochondrial Membrane Potential
Oxidative Challenge
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Membrane
PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential
JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential
Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)
Fibroblast cells loaded with JC-1
A Healthy unchallenged mitochondria
B Membrane potential collapse after
challenge with ROS (Hydrogen
peroxide)httpwwwmedunipgitimagelabmitochhtml
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative
Stress
N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )
7 5
8 0
8 5
9 0
9 5
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
()
bull Assessed baseline potential between non-smokers and current smokers
bull Significantly different of p lt 0005
Low baseline mitochondrial membrane
potential results in a decreased capacity
to defend against excessive ROS and
may lead to Oxidative Stress
Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives
2002
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Membrane Potential Biological Variability
S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5
5 0
6 0
7 0
8 0
9 0
1 0 0
B io lo g ic a l V a r ia b ility
I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
ntia
l (
)Subject Day 1 Day 2 Day 3 Mean SD CV
1 91 91 91 91 0 0
2 88 91 88 89 2 2
3 90 93 90 91 1 2
4 93 93 92 93 1 1
5 90 91 92 91 1 2
There is very little variability
between health subjects
indicating a good marker
for clinical utility
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Membrane Potential
Reference Interval
Copyright 1991-2012 Data Innovations LLC
Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710
EP Evaluator 1000517
Baseline
Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)
Central 95 Interval(N = 48)
Lower UpperValue 90 CI Value 90 CI Ratio
Confidence
Parametric 8139 7984 to 8294 9627 9472 to 9783 021
Alternatives
Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016
Transformed Parametric -- -- -- -- --
Confidence Limits for Nonparametric Index method computed by exact formula
True Gaussian SDI
3210-1-2-3
Ba
selin
e (
)
95
90
85
Nonparametric 90 CI Parametric
(Original Data)
Probability Plot
No transformation found
that significantly improves
fit to Gaussian distribution
(Transformed Data)
Probability Plot
Reference Interval Estimation Combined
Selection Criteria
Bounds None
Filter None
Statistics
Mean 88831
SD 3797
Median 89250
Range 8090 to 9640
N 48 of 48
Distinct Values 42
Zeroes 0
Central 95 Index 12 to 478
Analyst BNP
Expt Date 02 Feb 2016
Baseline ()
110100908070
25
20
15
10
5
0
Histogram
Normalizing Transformation
Exponent --
Constant --
Accepted by
DateSignature
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Membrane Potential
Novel Biomarker of CVD-Derived Oxidative
Stressbull Males and Females age
45-65
bull All diagnosed with Cardiovascular disease
bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics
NO antioxidants NO Tanning or Excessive Sun
bull Non-Smokers and NO Excessive use of Alcohol
bull Fatigue was a major complaint
7 5
8 0
8 5
9 0
9 5
B a se l in e
Mit
oc
ho
nd
ria
l M
em
br
an
e P
ote
nti
al
() H e a lth y (5 5 )
C a rd ia c (1 3 )
Maack and Bohm 2011 Madamanchi et al 2005
Patients with CVD
have low
membrane
potential The
integrity of their
mitochondria has
been
compromised by
oxidative stress
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Mitochondrial Membrane
Potential Assessment and
Treatment Monitoringbull The influence of HIV infection
and antiretroviral therapy on the mitochondrial membrane potential
bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002
Schmid Antivir Ther 2007
Mitochondrial membrane potential
can be improved and can be
used to monitor treatment
effectiveness
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
BrainGut or GutBrain
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Signs and Symptoms of Food
Intolerance
Colitis
Gastrointestinal issues
Irritable Bowel Syndrome
Bloating
Ear infections
Arthritis
Chronic inflammation
Chronic pain
Autism
Fatigue
Frequent illness
Eczema
Sinusitis
Dermatitis
Weight gain
Anxiety
Headaches
Hypersensitivity
Hyperactivity
Attention difficulties
Sleep difficulties
Depression
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Itrsquos all About the Gut
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Pro-Inflammatory Cytokines
Several pro-inflammatory cytokines directly increase intestinal permeability
IL-6
TNF-alpha
IL-17
Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311
HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Clinical Pearl
ldquoIgG food sensitivity symptoms are typically
delayed up to 72 hours after ingesting the
offending foodrdquo
ldquoA need to know for reintroduction symptomsrdquo
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Case Analysis
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Case Study 56 year old male
Primary complaints
Severe insomnia
Irritability
Mild anxiety
Joint pain
Gas and Bloating
Multivitamin
Fish omegas
58
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Individual Lab Results
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
SOD1
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
SOD 1 (another)
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
SOD 2
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
SOD 3
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
GPX-1
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
CAT
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
bull Strategybull Modified paleo diet
with allergens removed
bull 90 days to six months
bull Alkalinization
bull Consider SIBOFODMAP
reset
bull Consider detox
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
PLAN First 90 days
Waking N-A-C 600 mgs am and noon to support Glutathione synthesis
Ubiquinol 100 mgs am and noon to support mitochondria
Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa
for free radical scavenging (Get inflammation under control)
BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo
fat soluble antioxidant
Concentrated Theanine 2 at bedtime (push down Glutamate )
Taurine 1000 mgs at bedtime
AM exercise
Implement exercise plan
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
90 Day Follow Up
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Wrap Up
Within 3 weeks sleep arrived
Joint pain almost resolved
Occasional anxiety (resolves with
glutamate reduction lozenge)
Needed testosterone to increase
stamina and energy
Dependent on antioxidants
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Case Study
FatigueDepression
49 year old Female
Menopause
Depression
Fatigue
Stopped SSRI due to
ineffectiveness
Bio-identical hormones
improved Vaginal dryness
but nothing else
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Clinical Pearl
Sex hormones can cover
up neurotransmitter
depletion symptoms
such as mood issues
and hot flashes
Menopause hormone
reduction exposes the
issue
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Clinical Pearl
Mitochondrial
dysfunction directly
decreases sex hormone
and neurotransmitter
production
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
90 Day Protocol Modified SIBOFODMAP diet
Catecholamine support am and noon (NT report)
Ubiquinol am and noon (SOD 2 support)
Stop hormones except a testestriol suppository
5-HTP lozenge as needed to cool off
SerotoninGABAMelatonin support at bedtime
Concentrated Theanine at bedtime
3 week liver detox with colon hydrotherapy X 6
IV antixidants X 6
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
90 DAY Follow UP
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
ldquoThe Fog is Liftedrdquo
Patient states that the
ldquofog liftedrdquo in two weeks
Started exercise on her
own
Sex drive slowly returning
True story new hair-style
and new boyfriend
Questions
Questions