"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)