Metabolic pathways for caffeine(in mice - human overlaps underlined)

Caffeine is broken down in humans by several enzymes from the Cytochrome P450 (CYP) superclass of enzymes. These CYP enzymes are important in activating or eliminating many medications. The evaluation of caffeine metabolites in a patient has been proposed as a means of estimating the activity of some CYP enzymes, contributing to genetics-based personalized medicine.

The frequency and distribution of polymorphisms in inbred strains of mice often mirrors the variety in genotypes found in human populations.

This project hopes to determine whether four inbred strains have enough inter-strain differences in both their metabolite phenotypes and expression of mouse CYP enzymes to support additional investigations.

1. Introduction

Caffeine metabolism and Cytochrome P450 enzyme mRNA expression levels of genetically diverse inbred mouse strains

Neal Addicott - CSU East Bay, Michael Malfatti - Lawrence Livermore National Laboratory, Gabriela G. Loots - Lawrence Livermore National Laboratory

2. Background

4. Results

5. Discussion

References

Lawrence Livermore National Laboratory: Nicole Collette, Deepa Muguresh,Vicky WalsworthCal State East Bay: David Stronck, Kathy HahnThis material is based upon work supported by the S.D. Bechtel, Jr. Foundation and by the National Science Foundation under Grant No. 0952013 and Grant No. 0934931. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the S.D. Bechtel, Jr. Foundation or the National Science Foundation.

Acknowledgements

3. Methods

Overall process of project

Cytochrome P450 Enzymes

Image from: http://en.wikipedia.org/wiki/File:CytP450Oxidase-1OG2.png

.0 C57BL BALB CBA/J

0.06

0

0.01

0.02

0.03

0.04

0.05

Mouse Strain

The

obro

min

e Pe

ak A

rea

/ C

affei

ne P

eak

Are

a

.0 C57BL BALB CBA/J DBA/2J

0.06

0

0.01

0.02

0.03

0.04

0.05

Mouse Strain

The

ophy

lline

Pea

k A

rea

/ C

affei

ne P

eak

Are

a

.0 C57BL BALB CBA/J DBA/2J

0.06

0

0.01

0.02

0.03

0.04

0.05

Mouse Strain

Para

xant

hine

Pea

k A

rea

/ C

affei

ne P

eak

Are

a

Paraxanthine/Caffeine Theophylline/Caffeine

Metabolites 30 minutes after dose(n=6 per strain)

C57BL/6J BALB/cJ CBA/J

para

xant

hine

pea

k ar

ea /

caffe

ine

peak

are

a

DBA/2J C57BL/6J BALB/cJ CBA/J

theo

phyl

line

peak

are

a /c

affe

ine

peak

are

a

DBA/2J

Mouse Strain

*Theobromine/Caffeine

C57 BALB CBA

theo

brom

ine

peak

are

a /c

affe

ine

peak

are

a

DBA

(error bars represent a 90% confidence interval using Student's t = 2.015)

• The DBA/2J strain may be a slow metabolizer by a significant amount. Other time points besides 30 minutes after dose may be able to confirm.

• qPCR data is still pending as of 8/4/2011.

Caffeine (1,3,7 - trimethylxanthine)

N

NN

N

O

O

CH3

CH3

CH3

N

NN

N

O

O

CH3

CH3

1

23

4

56

7

8

9

Paraxanthine(1,7 - dimethylxanthine)

17

Cyp1a2(3-N-demethylization)

3

N

NN

N

O

O

CH3

1

3

7H

Cyp1a2(1-N-demethylization)

Theobromine(3,7 - dimethylxanthine)

N

NN

N

O

O

CH3

CH3

CH3

1

3

7

8 OH

1,3,7 - trimethyluricacid

Cyp3a4(8-hydrolyzation)

AFMU

AAMU

? Nat1Nat2Nat3

spontaneous

1-methylxanthine

Cyp1a2

1-methyluricacid

Xdh

1,7-dimethyluricacid

Cyp1a2XdhCyp2a12Cyp2a4Cyp2a5

7-methylxanthine

7-methyluricacid

Xdh

3,7-dimethyluricacid

Xdh

3,6,8-trimethylallantoinCH3

N

NN

N

O

O

CH3

H

CH3

Theophylline(1,3 - dimethylxanthine)

1

3

7

(7-N-demethylization)

H

100 1 2 3 4 5 6 7 8 9

100

0

25

50

75

X Axis

%

Elimination Routes for Top 200 Prescribed DrugsPrimary

Elimination Route

Hepatic (liver)

PrincipalHepatic Pathway

P450

P450 Involved in Metabolism

CYP1A2CYP3A4/5(figure adapted from Zanger et. al. 2008)

Several polymorphic CYP proteins outside of this study's scope have already been characterized in Caucasian populations with enough detail to warrant clinical dosing decisions based on CYP genotype1. The current understanding of CYP1A2 and CYP3A4/5 genetics still needs more development before being applied to personalized medicine. Caffeine metabolism (specifically Paraxanthine/Caffeine ratios) serves as a clinically recognized indicator of CYP1A2 activity2.

70%75%

9%

37%

Inject mouse with caffeine

Wait 30 minutes

Collect Blood

Analyze plasma for caffeine/metabolites with HPLC

Collect Liver and Brain tissue

Check for mRNA expression with rtPCR

Quantify mRNA expression with qPCR

Check for statistical correlation between gene expression and metabolism

(Plasma)

Images from:http://ohioline.osu.edu/vme-fact/0023.htmlhttp://www.flickr.com/photos/_intellinuts_/212714053/http://www.flickr.com/photos/fiddledydee/3267271237/

Overall process of project

400 5 10 15 20 25 30 35

100

0

20

40

60

80

Time from injection (minutes)

% B

uffer

A

Solvent B (Organic)non-polar

Solvent A (Aqueous)polar

C-8-hydroxylation CYP1A2 > CYP2B6 > CYP2E1 > CYP2D6 > CYP2C18 > CYP3A4

CYP1A2 > CYP2D6 > CYP2C9 > CYP2E1 > CYP2A6 > CYP3A57-N-demethylation CYP1A2 > CYP3A4 > CYP2C9 > CYP2C8 > CYP2D6 > CYP2E13-N-demethylation CYP1A2 > CYP2D6 > CYP2C8 > CYP2C19 > CYP3A51-N-demethylationPreference of human CYP Enzymes for caffeine catalysisPathway

(Source: Kot and Daniel. 2008)

Relative CYP strengths metabolizing caffeine in human liver microsomes

CYP1A2 is the primary enzyme responsible for caffeine metabolism in mice and humans - eliminating 87% of caffeine at normal concentrations in mice3. CYP2A6, CYP2E1, and CYP3A4 have been shown to play a role in caffeine metabolism in humans, and in wild-type mice the orthologous genes are expressed in similar patterns4 but may not have the exact same roles in caffeine metabolism. Rat caffeine metabolism has significantly different characteristics.

Cyp2e1

Cyp2a5

Cyp3a11

Orthologous CYP in mouse

Cyp1a2CYP1A2

9%

CYP

CYP2E16-17%CYP2A6

18-40%

% of CYP in human liver

CYP3A4

15-18%

(Source: Hrycay and Bandiera 2009

3. Buters J, Tang B, Pineau T, Gelboin H, Kimura S, and Gonzalez F. Role of CYP1A2 in caffeine pharmacokinetics and metabolism: studies using mice deficient in CYP1A2. Pharmacogenetics 1996; 6: 291-296.

5. Kot M, and Daniel W. The relative contribution of human cytochrome P450 isoforms to the four caffeine oxidation pathways:An in vitro compareative study with cDNA-expressed P450s including CYP2C isoforms . Biochemical Pharmacology 2008; 76: 543-551.

2. Fuhr U, Rost K, Engelhardt R, Sachs M, Liermann D, Belloc C, Beaune P, Janezic S, Grant D, Meyer U, and Staib A. Evaluation of caffeine as a test drug for CYP1A2, NAT2, and CYP2E1 phenotyping in man by in vivo versus in vitro correlations. Pharmacogenetics 1996; 6, 159-176.

4. Hrycay EG, and Bandiera SM. Expression, function and regulation of mouse Cytochrome P450 Enzymes: Comparison with Human Cytochrome P450 Enzymes. Current Drug Metabolism 2009; 10: 1151-1183.

1. Zanger U, Turpeinen M, Klein K, and Schwab M. Functional pharmacogenetics/genomics of human cytochromes P450 involved in drug biotransformation. Analytical and Bioanalytical Chemistry 2008; 392: 1093-1108.

2110 11 12 13 14 15 16 17 18 19 20

1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Time (minutes)

Abso

rban

ce U

nit

(AU

) at

27

7nm

Paraxanthine Theophylline

Representation of a typical Chromatogram Result

Caffeine

High Performance Liquid Chromatography (HPLC)• Blood sample was centrifuged to isolate plasma• Proteins were precipitated using 100% Methanol (200 !l of MeOH + 100 !l plasma)• Protein-free precipitate was evaporated and reconstituted in 50!l 10% Methanol• 40!l of reconstituted sample was injected into HPLC device. Non-polar compounds are attracted to the analytical column and take longer to reach the detector. More polar compounds (metabolites) are forced off the column by increasing concentrations of the organic buffer.* Solvent A: 99.99% HPLC grade H20 and 0.01% Acetic Acid (pH 4) * Solvent B: 90% Methanol, 10% Acetonitrile and 0.01% Acetic Acid (pH4)

Reverse Transcriptase Polymerase Chain Reaction (rtPCR) andQuantitative Real Time Polymerase Chain Reaction (qPCR)• Tissue Samples were preserved after collection in 3ml of RNALater® and stored at -4°C• ~30mg of tissue was homogenized in TRIzol and purified to total mRNA using QIAGEN RNeasy columns and reagents. Yield ranged from 1.2-4.0 !g mRNA/!l water.• Total cDNA for each sample was made using Invitrogen's SuperScript™ First-Strand Synthesis System. • cDNA was amplified using a standard PCR protocol and the primers listed above.• qPCR was conducted with the same cDNA primers were ordered from Applied Biosystems for use with a TaqMan® qPCR device and reagents.

% B

uffe

r A

Time from sample injection (min) Time from sample injection (min)

Abs

orba

nce

Uni

ts (A

U) a

t 277

nm

Sample gel electrophoresis of rtPCR products

Protocol for HPLC Samples

This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344 LLNL-POST-491655

3455' AACAGAAAGCCATAGCGCAGGG 3'5' GCTTCGAATGATGCTGGGAAGCT 3'Cyp2a5

269

495

366

223

Product Length (bp)

5'TAGCCCGTATCTGGTGCTGAAGGA3'

5' AAACCTCCGCACGTCCTTCCAT 3'

5'TGTGACAGCAAGGAGAGGCGTTT3'

Reverse Primer (-)

5' TCCTCTGCACGTTAGGCCATGT 3'Cyp1a2

5'AAGACCGGGAGAGAACAGCACAAC3'

Gene

NAT2

5' ACCGTTGCCTTGCTTGTCTGGA 3'Cyp2e1

5'ACAAACAAGCAGGGATGGACCTGG3'

Forward Primer (+)

Cyp3a11

5' AGAGGTTGGCCACTTCGAACCA 3'

Cyp1a2 - 495bp

C57

BL/6

JBA

LB/c

J

CBA

/JD

BA/2

J

C57

BL/6

JBA

LB/c

J

CBA

/JD

BA/2

J

C57

BL/6

JBA

LB/c

J

CBA

/JD

BA/2

J

C57

BL/6

JBA

LB/c

J

CBA

/JD

BA/2

J

C57

BL/6

JBA

LB/c

J

CBA

/JD

BA/2

J

Cyp2e1 - 366bpNAT2 -223bp

Cyp3a11- 269bp Cyp2a5- 345bp

• While the theobromine levels appear reliable within each strain, the amounts detected are close to the limits of the device's detection range and should not be weighted too heavily in the consideration of the data.

Adapted from: http://www.genome.jp/kegg-bin/show_pathway?

• The BALB/cJ strain metabolized caffeine into theophylline by a significantly larger amount than other strains. BALB/cJ's paraxanthine level was not significantly greater than C57BL/6J or CBA/J, but significant differences may arise through additional sampling or at other time points.

• rtPCR (not a quantitative method) did not reveal any obvious inter-strain differences in gene expression (no strains are null for a gene).* See "sample gel electrophoresis" figure.

*While retention times were similar, not all sample peaks were recognized as theobromine by the HPLC software.

This work represents the first steps of a larger investigation, and the BALB/cJ and DBA/2J strains have emerged as candidate model organisms for human CYP polymorphisms detectable by caffeine metabolite levels. In the next stage, eleven additional mouse strains will be examined for mRNA expression levels.

While paraxanthine was expected to be the primary metabolite, after thirty minutes theophylline was detected at similar levels. Data will need to be collected two hours after injection for a closer comparison to published paraxanthine/caffeine ratios. A 1,3,7-Trimethyluric acid standard will be prepared and checked against existing chromatogram data. A small peak near theobromine is a likely possibility.

Identified candidate mouse strains will have their mRNA sequences determined to more carefully characterize polymorphisms that could influence catalytic activity.