"Metabolomic studies in smokers, non-smokers and quitters"
Michael Göttel1,2, Daniel Müller1,2, Reinhard Nießner1,
Nikola Pluym2, Gerhard Scherer2, Max Scherer2
1Technische Universität München
2ABF, Analytisch-Biologisches Forschungslabor GmbH, München
5th Munich Biomarker Conference
Munich
1st - 2nd December, 2015
Theoretical section
Introduction to metabolomics
Review of metabolomics study: S vs NS
Objectives
Experimental section
Study design of smoking cessation study
Compliance
Fingerprinting
Summary & outlook
Overview
Cascade of ‘omics‘
The metabolome represents the
entity of all ‘small’ molecules
(< 1500 Dalton) and is most
predictive of the phenotype of an
organism.
Metabolomics is the study of
the entire set of small molecules
in a biological sample.
Identification
Quantification
Validation
Metabolomic strategies
Metabolomic fingerprinting:
Untargeted screening and
identification of as many
metabolites in a sample as
possible
Ideally identification of the
related metabolic pathways.
Metabolomic profiling:
Identification and quantification
of a selective number of
predefined metabolites, which
are normally related to a
specific metabolic pathway.
The M
eta
bolo
me
Ta
rge
ted
Holis
tic
Smoking related health issues
Tobacco smoke contains ~5000 chemicals1
> 250 are toxic
> 70 are carcinogenic
Main health issues:
Cancer
Cardiovascular diseases
Respiratory diseases
1) Rodgman et al., Int J Environ Res Public Health, 2011.
5
Recap: Main finding from metabolomics study S vs NS
A diet controlled clinical study with 25 smokers and 25 non-
smokers was conducted.
A GC-TOF-MS metabolomics fingerprinting method was
successfully established.
Plasma and urine:
Mainly smoking-related alterations in fatty acid, amino acid and
energy metabolism.
Saliva:
Mainly smoking-related alterations in tyramine, purine, lipid and
energy metabolism.
Fatty acid profile of S and NS
Mueller et al. Diss. Apr. 2014. In cooperation with Dr. C. Degen: Institute of Nutrition, Dept. of Nutritional
Physiology, Friedrich Schiller University, Jena, Germany.
0
10
20
30
40
FA
12
:0
FA
14
:0
FA
14
:1 n
-5
FA
15
:0
FA
16
:0
FA
16
:1 n
-7*
FA
17
:0*
FA
17
:1 c
/t*
FA
18
:0**
FA
18
:1 n
-9**
*
FA
18:1
n-7
*
FA
18
:2 n
-6
γ-F
A 1
8:3
n-6
α-F
A 1
8:3
n-3
FA
20
:0
FA
20
:1 n
-9**
FA
20
:2 n
-6
FA
20
:3 n
-6
FA
20
:4 n
-6
FA
20
:4 n
-3
FA
20
:5 n
-3
FA
22
:0
FA
22
:4 n
-6
FA
22
:5 n
-6
FA
22
:5 n
-3
FA
22
:6 n
-3**
FA
24
:0
FA
24
:1 n
-9*
[%]
NS S
N=
25
N=
25
↑Elevated in S; ↓ Decreased in S. *p < 0.05, **p < 0.01, *** p < 0.001
Normalized to total measured FA content in plasma
7
Nicotine leads to a transcription factor activated increase of the enzyme
stearoyl Co-enzyme desaturase 1 (SCD1), which catalyzes the
desaturation of saturated fatty acids to mono unsaturated fatty acids.
Müller et al. JCB, Mar 2014.
Müller et al. Diss. Apr. 2014.
Hypothesis for altered FA metabolism
8
FA 17:1 ▲
FA 18:0 ▼
FA 16:1 ▲ FA 18:1▲
LXRSREBP -1c
LXR
PPAR
Nicotine
+
Nicotine
Transcription Factor
Enzyme
SCD1
▲ Elevated levels found in smokers in this study
▼Decreased levels found in smokers in this study
FA 17:0 ▼FA 16:0
+
LXR: liver X receptorSREBP-1c:Sterol regulatory element binding protein-1c
Current objectives
Clinical study: smoking cessation
Untargeted metabolomic fingerprinting by GC-TOF-MS
In plasma, saliva and urine
Identify altered biochemical pathways and biomarkers for smokingcessation
Metabolomic profiling
Study design: overview
Subject requirements:
60 healthy volunteers
Males: age 20-50 years
BMI: 18-29 kg/m2
Smokers: >15 cigarettes/d during the last year
Strong intention to stop smoking
Study design:
3 months
After day 1, subjects have to quit smoking
4 x 24 h stationary visits
Controlled diet
Several ambulant visits for compliance check
Study design: timeline
1st
stationaryvisit
TP0
5 ambulant
visits
2nd
stationaryvisit
TP1
3 ambulant
visits
3rd
stationaryvisit
TP2
6 ambulant
visits
4th
stationaryvisit
TP3
1 week 1 month 3 months
1st
day
Plasma, saliva and urine
Saliva and urine
Compliance
Biomarkers for nicotine and tobacco product consumption:
Carbon monoxide in exhaled breath (COex)
Exclusion criterion COex > 5 ppm
Cotinine in saliva
Exclusion criterion Salivary cotinine > 15 ng/mL
Urinary cotinine for confirmation
Exclusion criterion Urinary cotinine > 50 ng/mL
Compliance: final status
Included subjects: 60
Drop outs: 21
Compliant subjects 39
Dropout reasons: Number
Protocol violation 4
Cotinine measurement 12
Withdrawn study agreement 3
Missed visit 2
13
MS Analysis
- GC-TOF-MS (Almsco)
- GC: Dimethyl-polysiloxane
(30m x 250µm i.d.)
Biological
Sample
Urine,
Plasma,
Saliva
Sample
clean-up
- IS-spiking (d4-ttMA)
- Urea digestion (only Urine)
- Protein precipitation
- Methoximation, Silylation
Fingerprinting: Analytical workflow for the untargeted biomarker identification by GC-TOF-MS
Data
processing
- Baseline correction ProtoTOF
(Almsco)
- Mass detection, peak alignment
(Mzmine)
- Normalization to IS
Statistical
analysis
&
target hit
identification
Statistics: PLS-DA
Mann-Whitney-U test,
fold change
Identification:
Deconvolution,
Databases (NIST, Golm)
Reference compounds
Mueller et al. JPR, Dec 2013.
Mueller et al. JCB, Mar 2014.
Plasma fingerprinting: quality control
Quality control strategy during 10 d measurement batches:
20 blank runs
5 reagent blanks Result quality control samples:
14 quality control matrix samples
Spiked with : L-Alanine
trans-trans Muconic acid (ttMA),
D-Glucose
Adenosine
Internal standard d4-ttMA
Analyte
Plasma
CV [%]
L-Alanine 11.5
ttMA 13.5
D-Glucose 15.7
Adenosine 19.5
15
rel.
are
a
QC Sample No
QC plot: L-Ala
Results of QC samples Target Target +/-15 %
Compound Possible classification Significance Fold change(TP0/TP1)
Retention time[min]
Identification
1 Morpholine TSC * 1.22 ▲ 5.717 Library
2 Hexanoic acid TSC * 1.08 ▲ 6.950 Library3 3-Hydroxybutyric acid TSC ** 1.61 ▲ 8.382 Library4 2-Oxoisoleucine Isoleucine metabolism * 1.50 ▲ 8.784 Library5 Lysine Proteinogenic amino acid * 1.81 ▲ 15.440 Library6 Glycerol 3-phosphate Glycolysis * 0.66 ▼ 16.059 Library7 1H-Indole-3-propionic acid Tryptophan metabolism * 0.93 ▼ 18.829 Library8 Uric acid Purine metabolism * 0.68 ▼ 19.285 Library9 Kynurenine Tryptophan metabolism ** 0.71 ▼ 20.003 Library10 5-Hydroxytryptophan Tryptophan metabolism * 1.14 ▲ 22.012 Library
*p < 0.05 , **p < 0.01 Mann-Whitney-U Test
Plasma fingerprinting: Potential targets after 1 week
16TSC: Tobacco smoke component Libraries used: Nist and GOLM
Compound Possible classification Significance Fold change(TP0/TP2)
Retention time[min]
Identification
1 2-Oxo-3-methylbutanoic acid Precursor to Leu; Val * 1.62 ▲ 7.462 Library
2 Phenylpropanoic acid Phenylalanine metab. * 1.75 ▲ 6.950 Library3 Dihydrouracil Uracil metabolism * 4.00 ▲ 12.526 Library4 Cysteine Proteinogenic amino acid * 0.56 ▼ 13.639 Library5 Pyrophosphate Energy metabolism * 1.58 ▲ 14.812 Library6 Glycerol 3-phosphate Glycolysis * 0.71 ▼ 16.059 Library7 Uric acid Purine metabolism * 0.79 ▼ 19.285 Library8 FA 17:0 FA metabolism * 1.23 ▲ 19.567 Library9 Kynurenine Tryptophan metabolism ** 0.60 ▼ 20.003 Library
10 FA18:1 FA metabolism * 1.65 ▲ 20.172 Library11 Cystine Cysteine metabolism * 0.64 ▼ 21.055 Library12 FA 20:4 FA metabolism * 0.74 ▼ 21.401 Library13 Pseudouridine Nucleoside interaction * 0.69 ▼ 21.511 Library14 Octadecenamide Signaling; e. storage * 1.29 ▲ 21.723 Library15 FA 20:1 FA metabolism * 2.91 ▲ 21.841 Library16 Lathosterol Cholesterol metabolism ** 0.39 ▼ 27.805 Library
*p < 0.05 , **p < 0.01 Mann-Whitney-U Test
Plasma fingerprinting: Potential targets after 1 month
17FA: Fatty acid Libraries used: Nist and GOLM
Compound Possible classification Significance Fold change(TP0/TP3)
Retention time[min]
Identification
1 Urea Amino acid metabolism * 0.62 ▼ 8.865 Library
2 Aminomalonic acid Protein interaction * 0.58 ▼ 12.571 Library3 Aspartic acid Proteinogenic amino acid ** 1.54 ▲ 13.236 Library4 alpha-Ketoglutaric acid Citric acid cycle * 0.65 ▼ 13.665 Library5 2,3,4-Trihydroxybutanoic acid Metabolite of ? ** 2.80 ▲ 13.910 Library6 Glutamic acid Proteinogenic amino acid ** 1.51 ▲ 14.390 Library7 Glycerol 3-phosphate Energy metabolism * 0.45 ▼ 16.059 Library8 FA 14:0 FA metabolism * 1.61 ▲ 16.731 Library9 Histidine Proteinogenic amino acid * 0.59 ▼ 17.425 Library10 FA 16:1 FA metabolism * 2.98 ▲ 18.390 Library11 FA 16:0 FA metabolism *** 1.58 ▲ 18.656 Library12 Uric acid Purine metabolism * 0.67 ▼ 19.285 Library13 Kynurenine Tryptophan metabolism *** 0.64 ▼ 20.003 Library14 FA 18:2 FA metabolism ** 1.55 ▲ 20.132 Library15 FA 18:1 FA metabolism * 1.39 ▲ 20.209 Library16 Tryptophan Proteinogenic amino acid * 0.41 ▼ 20.240 Library17 Adenosine monophosphate Nucleotide * 1.36 ▲ 26.736 Library18 Cholesterol Cell membrane, signaling * 0.57 ▼ 27.336 Library
*p < 0.05 , **p < 0.01, ***p < 0.001 Mann-Whitney-U Test
Plasma fingerprinting: Potential targets after 3 months
18FA: Fatty acid Libraries used: Nist and GOLM
Summary: plasma fingerprinting
Smoker vs after 1 week of cessation
Amino acid metabolism
Tryptophan metabolism
Smoker vs after 1 month of cessation
Amino acid metabolism
Fatty acid metabolism
Unsaturated FAs decreased
FA 20:4
Smoker vs after 3 months of cessation
Amino acid metabolism
Fatty acid metabolism
Unsaturated FAs decreased
Summary & Outlook
Clinical smoking cessation study
Compliance measurements
Fingerprinting measurement of plasma, saliva and urine
Dataprocessing and target hit identification: plasma
Fatty acid profiling method developed
Dataprocessing and statistical evaluation: saliva and urine
Complete fatty acid profilng: method validation and measurement
Further profiling methods (e.g. amino acids)
Acknowledgement
TUM
Prof. Dr. Reinhard Nießner
ABF
Prof. Dr. Gerhard Scherer
Dr. Max Scherer
Dr. Nikola Pluym
ABF Team
Sponsor: Imperial Tobacco Group
Thank you for your attention.
Visit www.abf-lab.com
24 h-urine sample from each subject will be made up of aliquots of the 3 fractions
EDTA-plasma: cooled vacutainer, immediately centrifuged 4°C, frozen with dry ice
Saliva: modified unstimulated spitting method by Navazesh, ANN NY ACAD SCI, 1993.
Study design: stationary visits
8 am
1st day
start
9 am
breakfast
A
10 am
saliva
COex
12 am
lunch
B
2 pm
saliva
(COex)
C
6 pm
saliva
(COex)
7 pm
dinner
D
10 pm
saliva
(COex)
E
8 am
saliva
(COex)
plasma
Fraction 1:
urine pool
Fraction 2: spot urine
Fraction 1: urine pool
Fraction 3 spot urine
Smoking ad libitum
(8am-11pm; 1st study day)
No smoking allowed
(11pm; 1st day – 8am 90thday)