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biovis’D I A G N O S T I K
Nitrosative stress and/or mitochondrial co-factor deficiency
www.biovis.de
Expert information 1 /2012 Mitochondrial diseases and nitrosative stress
Mitochondrial DiseasesDiagnostics of acquired mitochondrial diseases and nitrosative stress
In the scope of mitochondrial diagnostics more and more parameters are offe-
red. Often they are expensive and elaborate procedures. But are those parame-
ters really better than those already available or do they simply provoke needless
expenses. biovis has revised its diagnostic spectrum in the range of mitochon-
drial medicine: sensible new parameters were included in our spectrum and old
pre-analytically very elaborate parameters were discontinued. This brochure is
the attempt to provide a guideline for mitochondria diagnostics. What is sensib-
le and what is not? Also pre-analytic snares are shown to avoid mistakes during
specimen collection, which may cause apparently pathological results.
Traditionally mitochondrial diseases are regarded as congenital diseases and
normally diagnoses are confirmed by muscle biopsy. There is a whole variety of
such inborn mitochondrial diseases. They can practically concern every area of
the mitochondrion. For pyruvate dehydrogenase alone there are several diseases
of which most are passed on dominantly x-chromosomal. In addition there are
also disorders of citrate cycle, the respiratory chain or fat burning (e.g. carnitin
transporter deficiency).
Acquired forms are more frequent than genetic mitochondrial diseases. For pa-
tients suffering from acquired mitochondrial diseases the analyses do not yield
characteristic findings. The clinical patterns may vary considerably. Patients of-
ten complain about lack of energy, but also complaints similar to those of CFS,
MCS or fibromyalgia can be observed.
Acquired mitochondrial diseases are triggered by nitrosative stress, which may
damage the structure and genome of mitochondria, and lack of mitochondrial
co-factors like for example co-enzyme Q10, riboflavine or niacin.
Mitochondrial Disease
In the scope of mitochondrial diagnostics more and more parameters are offe-
red. Often they are expensive and elaborate procedures. But are those parame-
ters really better than those already available or do they simply provoke needless
expenses. biovis has revised its diagnostic spectrum in the range of mitochon-
drial medicine: sensible new parameters were included in our spectrum and old
pre-analytically very elaborate parameters were discontinued. This brochure is
the attempt to provide a guideline for mitochondria diagnostics. What is sensib-
le and what is not? Also pre-analytic snares are shown to avoid mistakes during
specimen collection, which may cause apparently pathological results.
Traditionally mitochondrial diseases are regarded as congenital diseases and
normally diagnoses are confirmed by muscle biopsy. There is a whole variety of
such inborn mitochondrial diseases. They can practically concern every area of
the mitochondrion. For pyruvate dehydrogenase alone there are several diseases
of which most are passed on dominantly x-chromosomal. In addition there are
also disorders of citrate cycle, the respiratory chain or fat burning (e.g. carnitin
transporter deficiency).
Acquired forms are more frequent than genetic mitochondrial diseases. For pa-
tients suffering from acquired mitochondrial diseases the analyses do not yield
characteristic findings. The clinical patterns may vary considerably. Patients of-
ten complain about lack of energy, but also complaints similar to those of CFS,
MCS or fibromyalgia can be observed.
Acquired mitochondrial diseases are triggered by nitrosative stress, which may
damage the structure and genome of mitochondria, and lack of mitochondrial
co-factors like for example co-enzyme Q10, riboflavine or niacin.
Mitochondrial Disease
4 5
A whole variety of diseases or complaint patterns were associated with nitrosative
stress in the past. Inflammatory diseases promote the activation of inducible NO-
synthase (iNOS) by releasing cytokines. As a result more NO is developed – leading
to nitrosative stress.
A list of possible mitochondrial and chronic inflammatory diseases:
• Rheumatoidarthritis
• SeronegativeSpondyloarthropathy(e.g. Bekhterev’s disease, reactive arthritis)
• Connectivetissuediseases (e.g. systemic lupus, Sjögren’s syndrome, sclerodermia)
• Polymyalgiarheumatica
• Fibromyalgia
• Chronifiedinfections(e.g. borreliosis, chlamydia, hepatitis)
• Chronicfatigue
• Multiplesclerosis
• Tumourdiseases
• Metabolicsyndrome,diabetes,coronaryheartdiseaseand
allarteriosclerosisdiseases
• Secondarydepressionandanxiety
• Psoriasis
• Allergies,neurodermatitis,asthma
• Variousliverdiseases
• Migraine,chronicheadaches
• Chronicinflammatorygastro-intestinaldiseasesandirritablecolon
Diagnostics – Summary
Diagnostic approaches in case of mitochondrial diseases or nitrosative stress may
be manifold. They can be analyses of NO-development, preconditions of peroxi-
nitrite synthesis or impact on mitochondria. Also important therapeutic factors
have to be considered.
DeterminationofIncreasedNODevelopment • Citrulline in urine
• Amino acids in plasma (arginine and citrulline)
• NO – breath gas test
biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss
Nitrosative stress is generated if increased amounts of nitrogen monoxide (NO)
are developed. In its form as endothelial NO (eNOS) it leads to vascular vasodila-
tation. NO is developed in nerve cells (nNOS) in case of bacterial or viral infections
(iNOS) or in mitochondria (mNOS). NO is a colourless gas of radical nature because
of an unpaired electron. This is the cause of significant biological effects. In case
of oxidative stress it is converted to peroxynitrite after reactions with superoxide
anions. Peroxinitrite is a toxic substance which may damage mitochondria. It is the
main damaging agent in the cascade of nitrosative stress, mitochondrial disease,
immune dysfunction (chronic, often subclinical inflammation) and in many cases
also pains.
Frequent causes of increased NO synthesis or nitrosative stress are listed below.
Causes of nitrosative stress: [Pall et al. 2007, Kuklinski 2006, 2007]
• Pains
• ChronicInflammation
• chronicStress
• EnvironmentalToxins:solvents, pesticides, heavy metals
• Drugs: long-term nitrate, antihypertensive agents (e.g. dihydralazine),
cholesterol synthesis inhibitors, anti-diabetics (mainly metformin),
mitochondria damaging antibiotic agents (e.g. gentamicin, cotrimoxazole)
and others
• Cervical spine traumata
Nitrosative Stress
Complex I
H+
NADH + H+ NAD+
Succinate Fumarate
Pi + ADP ATP + H2O
1/2Os + H+ H2O
Complex II Complex III
H+
Complex IV
H+
ATP-Synthase
Biochemical consequences of increased NO syntheses
Inhibition of the FeS-containing enzymes in the mitochondrial respira-tory chain in complexes I and II reduced ATP development
H+
e- e-
e-
4 5
A whole variety of diseases or complaint patterns were associated with nitrosative
stress in the past. Inflammatory diseases promote the activation of inducible NO-
synthase (iNOS) by releasing cytokines. As a result more NO is developed – leading
to nitrosative stress.
A list of possible mitochondrial and chronic inflammatory diseases:
• Rheumatoidarthritis
• SeronegativeSpondyloarthropathy(e.g. Bekhterev’s disease, reactive arthritis)
• Connectivetissuediseases (e.g. systemic lupus, Sjögren’s syndrome, sclerodermia)
• Polymyalgiarheumatica
• Fibromyalgia
• Chronifiedinfections(e.g. borreliosis, chlamydia, hepatitis)
• Chronicfatigue
• Multiplesclerosis
• Tumourdiseases
• Metabolicsyndrome,diabetes,coronaryheartdiseaseand
allarteriosclerosisdiseases
• Secondarydepressionandanxiety
• Psoriasis
• Allergies,neurodermatitis,asthma
• Variousliverdiseases
• Migraine,chronicheadaches
• Chronicinflammatorygastro-intestinaldiseasesandirritablecolon
Diagnostics – Summary
Diagnostic approaches in case of mitochondrial diseases or nitrosative stress may
be manifold. They can be analyses of NO-development, preconditions of peroxi-
nitrite synthesis or impact on mitochondria. Also important therapeutic factors
have to be considered.
DeterminationofIncreasedNODevelopment • Citrulline in urine
• Amino acids in plasma (arginine and citrulline)
• NO – breath gas test
biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss
Nitrosative stress is generated if increased amounts of nitrogen monoxide (NO)
are developed. In its form as endothelial NO (eNOS) it leads to vascular vasodila-
tation. NO is developed in nerve cells (nNOS) in case of bacterial or viral infections
(iNOS) or in mitochondria (mNOS). NO is a colourless gas of radical nature because
of an unpaired electron. This is the cause of significant biological effects. In case
of oxidative stress it is converted to peroxynitrite after reactions with superoxide
anions. Peroxinitrite is a toxic substance which may damage mitochondria. It is the
main damaging agent in the cascade of nitrosative stress, mitochondrial disease,
immune dysfunction (chronic, often subclinical inflammation) and in many cases
also pains.
Frequent causes of increased NO synthesis or nitrosative stress are listed below.
Causes of nitrosative stress: [Pall et al. 2007, Kuklinski 2006, 2007]
• Pains
• ChronicInflammation
• chronicStress
• EnvironmentalToxins:solvents, pesticides, heavy metals
• Drugs: long-term nitrate, antihypertensive agents (e.g. dihydralazine),
cholesterol synthesis inhibitors, anti-diabetics (mainly metformin),
mitochondria damaging antibiotic agents (e.g. gentamicin, cotrimoxazole)
and others
• Cervical spine traumata
Nitrosative Stress
Complex I
H+
NADH + H+ NAD+
Succinate Fumarate
Pi + ADP ATP + H2O
1/2Os + H+ H2O
Complex II Complex III
H+
Complex IV
H+
ATP-Synthase
Biochemical consequences of increased NO syntheses
Inhibition of the FeS-containing enzymes in the mitochondrial respira-tory chain in complexes I and II reduced ATP development
H+
e- e-
e-
6 7
Special Diagnostics of Nitrosative Stress and Mitochondrial Diseases
Citrulline
Material: 1st morning urine
Standard: < 4 mg/g creatinine
Citrulline values above the standard indicate increased NO-accrual.
NO may lead to peroxynitrite development by reaction with super-
oxide anions. This might damage the mitochondria.
As citrulline development varies considerably (stress-dependent),
negative results do not rule out NO-stress or mitochondrial diseases.
The amount of citrulline developed depends on many omitting factors,
among others on the amount of available arginine.
Determinationofoxidativeandnitrosativestress • Lipid peroxidation, anti-oxidative capacity, glutathione
• Nitrotyrosine, nitrophenyl acetic acid
Determinationofmitochondrialdisorders • LDH isoenzyme
• Mitochondrial membrane potential
• Lactate Stress Test
• (Lactate / Pyruvate Ratio)
• M2PK (CAVE: is also a tumour marker)
DeterminationofInflammationandTH-Shift
• Humoral activity marker: CRP, neopterin, sIL2R, ECP
• Cytokines in serum, TNF, IL-1, IL-6 possible also IL-8 and IL-12
• Stimulated cytokine statuses
MicronutrientDiagnostics • Vitamin (mainly B12, B2, B3, folic acid, biotin , C, 25-OH-D3))
• Whole blood mineral analysis (mainly K, Mg, Zn, Se, amino acid status)
DeterminationofConsequences • Differential blood count, gGT, GPT, creatinine, urea, serum electrolytes,
blood lipids, blood sugar etc.
• L-tryptophan (alternatively serotonin), possibly complete aminogram
DeterminationofBlood-Brain-BarrierDisorders •Protein S100
biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss
Endogenic NO is developed by
the enzyme NOS (NO-Synthase)
from L-arginine.
Arginine + oxygen
NO + citrulline3-Nitrotyrosine
O2 Thiols
S-GSNOS-Nitrosothiol
ONOOPeroxinitrite
Superoxide anions
NO2Nitrite
NO
pH reduced
HN
CH2
CH2
CH2
H2N
OH
C
NH
CHC O
NH2
O
CH2
CH2
CH2
H2N
OH
C
NH
CHC O
NH2
NADPHNOS
l-Arginin
L-CitrullinNADP+ NADPH
H2O
NO-synthesis
FADFMN H4B
L-NMMAL-NAME
Pimagedine (aminoguanidine)
O2
6 7
Special Diagnostics of Nitrosative Stress and Mitochondrial Diseases
Citrulline
Material: 1st morning urine
Standard: < 4 mg/g creatinine
Citrulline values above the standard indicate increased NO-accrual.
NO may lead to peroxynitrite development by reaction with super-
oxide anions. This might damage the mitochondria.
As citrulline development varies considerably (stress-dependent),
negative results do not rule out NO-stress or mitochondrial diseases.
The amount of citrulline developed depends on many omitting factors,
among others on the amount of available arginine.
Determinationofoxidativeandnitrosativestress • Lipid peroxidation, anti-oxidative capacity, glutathione
• Nitrotyrosine, nitrophenyl acetic acid
Determinationofmitochondrialdisorders • LDH isoenzyme
• Mitochondrial membrane potential
• Lactate Stress Test
• (Lactate / Pyruvate Ratio)
• M2PK (CAVE: is also a tumour marker)
DeterminationofInflammationandTH-Shift
• Humoral activity marker: CRP, neopterin, sIL2R, ECP
• Cytokines in serum, TNF, IL-1, IL-6 possible also IL-8 and IL-12
• Stimulated cytokine statuses
MicronutrientDiagnostics • Vitamin (mainly B12, B2, B3, folic acid, biotin , C, 25-OH-D3))
• Whole blood mineral analysis (mainly K, Mg, Zn, Se, amino acid status)
DeterminationofConsequences • Differential blood count, gGT, GPT, creatinine, urea, serum electrolytes,
blood lipids, blood sugar etc.
• L-tryptophan (alternatively serotonin), possibly complete aminogram
DeterminationofBlood-Brain-BarrierDisorders •Protein S100
biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss
Endogenic NO is developed by
the enzyme NOS (NO-Synthase)
from L-arginine.
Arginine + oxygen
NO + citrulline3-Nitrotyrosine
O2 Thiols
S-GSNOS-Nitrosothiol
ONOOPeroxinitrite
Superoxide anions
NO2Nitrite
NO
pH reduced
HN
CH2
CH2
CH2
H2N
OH
C
NH
CHC O
NH2
O
CH2
CH2
CH2
H2N
OH
C
NH
CHC O
NH2
NADPHNOS
l-Arginin
L-CitrullinNADP+ NADPH
H2O
NO-synthesis
FADFMN H4B
L-NMMAL-NAME
Pimagedine (aminoguanidine)
O2
8
Compared to the LDH isoenzymes the analysis of the mitochondral
activity achieves results which can be quantified better – not only for
patient examinations, they can also be used also for in vivo or in vitro
studies (e.g. after giving mitotropic compounds like coenzyme Q10).
Attention: Measuring the mitochondrial activity has proved its
value over the last years. As no granulocytes are measured
there might be “paradox” results for tumour patients in rare cases –
when tumour patients with damaged specific immune defence and
prevailingly unspecific defence show apparently good values, although
apart from granulocytes the she situation looks different. In this case
ATP measurements are to be preferred.
LDH-Isoenzymes
Material: Serum, not frozen! (freezing destroys LDH-5)
Standard: LDH4: < 9,4 %
LDH5: < 10 %
LDH isoenzymes are reasonably priced markers for the evaluation of
mitochondrial functions. LDH-4 (H3M) and before all LDH-5 (4M) increase
if mitochondria are destroyed. Relative shares of LDH-4 or LDH-5
higher 10% indicate mitochondrial diseases (especially in case of normal
total LDH).
Attention: The total LDH should be within or only slightly above the standard range.
Severely increased total LDH may lead to misinterpretations, if liver
enzymes (gGT, GPT) and (heart) muscle enzymes (e.g. CK, troponin) as
well as haemolysis values (e.g. haptoglobin) are not available at the
same time, as LDH-5 increases are also found in case of liver damages.
There is also a relative shift to LDH1/2 in case of are myocardial damages
or haemolysis.
ATP-Measurement
Material: 1 CPDA (by express shipment because of living cell analysis)
Standard: > 500 pmol/ 106 cells
ATP-measurements are to be evaluated similar to those of the mito-
chondrial activity as ATP is developed in complex V of the respiratory
chain. Easily available leukocytes of blood are used for measuring
Nitrotyrosine,NitrophenylAceticAcid
Nitrotyrosine:
Material: EDTA blood (requires EXPRESS SHIPMENT)
Standard: <3.2 nmol/l
Peroxinitrite is toxic and may damage mitochondria. It cannot be
analysed directly. Peroxynitrite leads to nitrosation of aromatic amino
acids. For example 3-nitrotyrosine is developed from tyrosine, which
can be determined in blood. High nitrotyrosine levels indicate increased
peroxinitrite development and thus mitochondrial damage.
Increased nitrotyrosine values confirm the presence of nitrosative
stress (high specificity)
Attention: Just like in case of citrulline inconspicuous nitrotyrosine values do
not exclude mitochondrial damage by nitrosative stress. Reason for
inconspicuous measuring values may for example be tyrosine deficiency
Nitrophenylessigsäure:
Material: 1st morning urine
Standard: < 3,0 µg/g creatinine
3-nitrophenyl acetic acid is a metabolic product of 3-nitrotyrosine.
Positive nitrophenyl acetic acid values reliably confirm nitrosative
stress! Therefore measuring nitrophenyl acetic acid in urine is ana
logue to the nitrotyrosine determination in EDTA blood, but it is less
sensitive.
MitochondrialActivity
Material: EDTA blood (requires EXPRESS SHIPMENT)
Standard: > 90 % active mitochondria
Optimal: > 95 % active mitochondria
The enzymes of the respiratory chain transport protons from the cell
to the intermembrane space. This leads to development of electro-
chemical membrane potential, which can be determined with
fluorescence. Intact mitochondria show significant protone gradients,
inactive mitochondria do not. Flow cytometry procedures can determi
ne the membrane potential. Active and inactive mitochondria can
thus be distinguished. The mitochondrial activity is determined by mar
king granulocytes with a fluorescence colorant, which changes its
fluorescence depending on the electrical potential.
biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss
8 9
100
100
100 100 100 100
100
100
100
Green fluorescence (FL1)
Red
fluor
esce
nce
(FL2
) Staurosporine
Membrane potential can bemeasured by flow cytometry.
Cells with intact membrane potential
Cells with intact membra-ne potential
Increase of green florescence caused by mitochondrial function disorders
100
8
Compared to the LDH isoenzymes the analysis of the mitochondral
activity achieves results which can be quantified better – not only for
patient examinations, they can also be used also for in vivo or in vitro
studies (e.g. after giving mitotropic compounds like coenzyme Q10).
Attention: Measuring the mitochondrial activity has proved its
value over the last years. As no granulocytes are measured
there might be “paradox” results for tumour patients in rare cases –
when tumour patients with damaged specific immune defence and
prevailingly unspecific defence show apparently good values, although
apart from granulocytes the she situation looks different. In this case
ATP measurements are to be preferred.
LDH-Isoenzymes
Material: Serum, not frozen! (freezing destroys LDH-5)
Standard: LDH4: < 9,4 %
LDH5: < 10 %
LDH isoenzymes are reasonably priced markers for the evaluation of
mitochondrial functions. LDH-4 (H3M) and before all LDH-5 (4M) increase
if mitochondria are destroyed. Relative shares of LDH-4 or LDH-5
higher 10% indicate mitochondrial diseases (especially in case of normal
total LDH).
Attention: The total LDH should be within or only slightly above the standard range.
Severely increased total LDH may lead to misinterpretations, if liver
enzymes (gGT, GPT) and (heart) muscle enzymes (e.g. CK, troponin) as
well as haemolysis values (e.g. haptoglobin) are not available at the
same time, as LDH-5 increases are also found in case of liver damages.
There is also a relative shift to LDH1/2 in case of are myocardial damages
or haemolysis.
ATP-Measurement
Material: 1 CPDA (by express shipment because of living cell analysis)
Standard: > 500 pmol/ 106 cells
ATP-measurements are to be evaluated similar to those of the mito-
chondrial activity as ATP is developed in complex V of the respiratory
chain. Easily available leukocytes of blood are used for measuring
Nitrotyrosine,NitrophenylAceticAcid
Nitrotyrosine:
Material: EDTA blood (requires EXPRESS SHIPMENT)
Standard: <3.2 nmol/l
Peroxinitrite is toxic and may damage mitochondria. It cannot be
analysed directly. Peroxynitrite leads to nitrosation of aromatic amino
acids. For example 3-nitrotyrosine is developed from tyrosine, which
can be determined in blood. High nitrotyrosine levels indicate increased
peroxinitrite development and thus mitochondrial damage.
Increased nitrotyrosine values confirm the presence of nitrosative
stress (high specificity)
Attention: Just like in case of citrulline inconspicuous nitrotyrosine values do
not exclude mitochondrial damage by nitrosative stress. Reason for
inconspicuous measuring values may for example be tyrosine deficiency
Nitrophenylessigsäure:
Material: 1st morning urine
Standard: < 3,0 µg/g creatinine
3-nitrophenyl acetic acid is a metabolic product of 3-nitrotyrosine.
Positive nitrophenyl acetic acid values reliably confirm nitrosative
stress! Therefore measuring nitrophenyl acetic acid in urine is ana
logue to the nitrotyrosine determination in EDTA blood, but it is less
sensitive.
MitochondrialActivity
Material: EDTA blood (requires EXPRESS SHIPMENT)
Standard: > 90 % active mitochondria
Optimal: > 95 % active mitochondria
The enzymes of the respiratory chain transport protons from the cell
to the intermembrane space. This leads to development of electro-
chemical membrane potential, which can be determined with
fluorescence. Intact mitochondria show significant protone gradients,
inactive mitochondria do not. Flow cytometry procedures can determi
ne the membrane potential. Active and inactive mitochondria can
thus be distinguished. The mitochondrial activity is determined by mar
king granulocytes with a fluorescence colorant, which changes its
fluorescence depending on the electrical potential.
biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss
8 9
100
100
100 100 100 100
100
100
100
Green fluorescence (FL1)
Red
fluor
esce
nce
(FL2
) Staurosporine
Membrane potential can bemeasured by flow cytometry.
Cells with intact membrane potential
Cells with intact membra-ne potential
Increase of green florescence caused by mitochondrial function disorders
100
cellular ATP. Apparently paradoxical results in case of tumour patients –
as can be observed in mitochondrial activity measurements – do not
occur when measuring cellular ATP.
Basal ATP (initial measurement) is measured first. Then sodium azide
is added to the cells. This reversibly inhibits the synthesis chain in
complex IV. Due to the inhibition the ATP concentration considerably
decreases (Stress ATP). If the inhibitor is now removed again, the elec-
tron transport chain starts to regenerate and ATP development in-
creases (Recovery-ATP). While patients frequently show normal basal
ATP values, the mitochondrial regeneration capacity is significantly
limited in many cases. The recovery-ATP should be at least 25 %
above that of the stress-ATP value. Increases of 50 % and more can
often be observed in healthy people. If increases of 25% are not reached,
one can safely assume disordered mitochondrial regeneration.
ATP-measurementsprovideinformationaboutmitochondrialfunction,
capacityandperformance.
Lactate / Pyruvate Ratio
Material: 1-2 NaF (ship via express)
Attention: As food consumption and physical exercise lead to significant increase
of pyruvate values, the sample has to be taken on an empty
stomach and in resting condition. Stasis should not be longer than
1 minute.
Standard: Ratio < 20:1
Thepyruvatedeterminationisextremelysusceptibletofailure(food,
exercise,bloodstasisinveins)andalsoproblematicwheretransportis
concerned.Duetotheinstabilityofthesamplematerialthismitochon-
drialfunctiontestisonly recommended with restrictions.
ProteinS100
Material: 2 x serum
• blood withdrawal in resting condition
• 10 minutes climbing stairs or head circling
• Renewed blood withdrawal
• Centrifuge whole blood and freeze serum, ship in frozen condition
per express
Standard: > 0.13 (malign melanoma) / > 0.07 µg/l (nitrosative stress)
Each value higher than 0.07 µh/l indicates blood-brain barrier
disorders.
In case of moderate complaints protein S 100 will normally only increase
after strain (climbing stairs, circling head).
Causes of non-specific increases of protein S100 – values
• slight increases: liver cirrhosis, renal insufficiency
• increases up to 2.0 µg/l: severe bacterial infections.
In case of physiologically dark skin colour: Protein S 100 test cannot
be evaluated!!!
• Significant increase (more than 2.0 µg/l): vascular damage,
heart attack, cerebral ischaemia
Attention: Haemolysis leads to false positive results.
OrganicAcidsinUrine
Material: Urine
Profile: Organic Acids of the Citric Acid Cycle
One of the major consequences of nitrosative stress is the destruction
of iron containing enzymes. One of these enzymes is aconitase, which
catalyses the conversion of citric acid (citrate) to isocitric acid (isocitrate).
If the aconitase is destroyed by peroxynitrite this will lead to
citric acid stasis and an interruption of the cancer cycle. Increased
citric acid and simultaneously low isocitric acid may therefore be an
additional indication of mitochondrial diseases.
biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss
10 11
Patient 1
(ATP
)
435pmol
NW: 500 - 1100 Pmol
980pmol
658pmol
Patient 3Patient 2
(ATP
)
1050pmol
40,4 %
100 %
84,5 %
424pmol
887pmol
+ 109 %
Initial Measurement Stress Measurement Regeneration Measurement
Initial Measurement Stress Measurement Regeneration Measurement
(ATP
)950
pmol
35,9 %
100 %
40,5
341pmol
385pmol
+ 12,8 %
Mitochondria – ATP – Initial Measurement
Mitochondria – Stress Test - satisfactory
Mitochondria – Stress Test - deficient
cellular ATP. Apparently paradoxical results in case of tumour patients –
as can be observed in mitochondrial activity measurements – do not
occur when measuring cellular ATP.
Basal ATP (initial measurement) is measured first. Then sodium azide
is added to the cells. This reversibly inhibits the synthesis chain in
complex IV. Due to the inhibition the ATP concentration considerably
decreases (Stress ATP). If the inhibitor is now removed again, the elec-
tron transport chain starts to regenerate and ATP development in-
creases (Recovery-ATP). While patients frequently show normal basal
ATP values, the mitochondrial regeneration capacity is significantly
limited in many cases. The recovery-ATP should be at least 25 %
above that of the stress-ATP value. Increases of 50 % and more can
often be observed in healthy people. If increases of 25% are not reached,
one can safely assume disordered mitochondrial regeneration.
ATP-measurementsprovideinformationaboutmitochondrialfunction,
capacityandperformance.
Lactate / Pyruvate Ratio
Material: 1-2 NaF (ship via express)
Attention: As food consumption and physical exercise lead to significant increase
of pyruvate values, the sample has to be taken on an empty
stomach and in resting condition. Stasis should not be longer than
1 minute.
Standard: Ratio < 20:1
Thepyruvatedeterminationisextremelysusceptibletofailure(food,
exercise,bloodstasisinveins)andalsoproblematicwheretransportis
concerned.Duetotheinstabilityofthesamplematerialthismitochon-
drialfunctiontestisonly recommended with restrictions.
ProteinS100
Material: 2 x serum
• blood withdrawal in resting condition
• 10 minutes climbing stairs or head circling
• Renewed blood withdrawal
• Centrifuge whole blood and freeze serum, ship in frozen condition
per express
Standard: > 0.13 (malign melanoma) / > 0.07 µg/l (nitrosative stress)
Each value higher than 0.07 µh/l indicates blood-brain barrier
disorders.
In case of moderate complaints protein S 100 will normally only increase
after strain (climbing stairs, circling head).
Causes of non-specific increases of protein S100 – values
• slight increases: liver cirrhosis, renal insufficiency
• increases up to 2.0 µg/l: severe bacterial infections.
In case of physiologically dark skin colour: Protein S 100 test cannot
be evaluated!!!
• Significant increase (more than 2.0 µg/l): vascular damage,
heart attack, cerebral ischaemia
Attention: Haemolysis leads to false positive results.
OrganicAcidsinUrine
Material: Urine
Profile: Organic Acids of the Citric Acid Cycle
One of the major consequences of nitrosative stress is the destruction
of iron containing enzymes. One of these enzymes is aconitase, which
catalyses the conversion of citric acid (citrate) to isocitric acid (isocitrate).
If the aconitase is destroyed by peroxynitrite this will lead to
citric acid stasis and an interruption of the cancer cycle. Increased
citric acid and simultaneously low isocitric acid may therefore be an
additional indication of mitochondrial diseases.
biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss biovis Exper t Information 2 /2011 Mi to chondr ial Dis eas e s and Ni tro s ati ve Stre ss
10 11
Patient 1
(ATP
)
435pmol
NW: 500 - 1100 Pmol
980pmol
658pmol
Patient 3Patient 2
(ATP
)
1050pmol
40,4 %
100 %
84,5 %
424pmol
887pmol
+ 109 %
Initial Measurement Stress Measurement Regeneration Measurement
Initial Measurement Stress Measurement Regeneration Measurement
(ATP
)
950pmol
35,9 %
100 %
40,5
341pmol
385pmol
+ 12,8 %
Mitochondria – ATP – Initial Measurement
Mitochondria – Stress Test - satisfactory
Mitochondria – Stress Test - deficient
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