Genotype and Phenotype of Caffeine Metabolism: A Biochemistry

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Genotype and Phenotype of Caffeine Metabolism: A Biochemistry Laboratory Experiment Julie T. Millard,* Tenzin Passang, Jiayu Ye, Gabriel M. Kline, Tina M. Beachy, Victoria L. Hepburn, and Edmund J. Klinkerch Department of Chemistry, Colby College, Waterville, Maine 04901, United States

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S Supporting Information *

ABSTRACT: An experiment for the upper-division undergraduate biochemistry laboratory is described in which students investigate the influence of genetic variations of cytochrome P450 1A2 on drug metabolism, using caffeine as a model compound. Saliva samples from human subjects are characterized for a single-nucleotide polymorphism in the CYP1A2 gene (genotyping) and for the rate of caffeine clearance (phenotyping). This experiment has clinical significance in the area of personalized medicine. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Collaborative/Cooperative Learning, Drugs/Pharmaceuticals, Gas Chromatography, Metabolism, Nucleic Acids/DNA/RNA

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optimize caffeine dosing,9 although other scenarios involving drug metabolism or performance enhancement could be developed according to instructor interest. Several other undergraduate laboratory experiments have been published to genotype medically relevant loci, including variants involved in type 2 diabetes,10 susceptibility to cancer,11,12 blood type,13 lactose intolerance,14 and color vision.15 However, the experiment reported herein combines several advantageous elements, including its use of noninvasive DNA samples, rather than blood, its determination of an easily measurable phenotype during the same exercise, and its focus on a topic of great relevance to students, with the added bonus that they can determine their own genotypes. Caffeine metabolism phenotyping via HPLC analysis of urine samples has also been reported, although that experiment did not include genotyping.16 In this experiment, caffeine metabolism genotyping was performed for CYP1A2*1F, a site containing a C/A singlenucleotide polymorphism (SNP [rs762551]) that has been reported to affect the amount of enzyme.17,5 In addition to being associated with higher CYP1A2 activity, the AA

INTRODUCTION Caffeine is the most widely used psychoactive drug in the world, with the majority of the world’s population consuming it on a daily basis (for a review of caffeine pharmacology, see Benowitz1). In addition to its activity as a central nervous system stimulant, caffeine is also used clinically to treat premature neonatal apnea2 and to enhance the analgesic effect of pain relievers such as acetaminophen.3 Despite its widespread use, individuals respond very differently to caffeine, with some people experiencing anxiety after a single cup of coffee and others consuming several cups even in the evening without any sleep disturbances. Both environmental and genetic factors are believed to modulate individual responses to caffeine.4,5 One factor influencing caffeine’s biological effects is its lifetime in the body, mediated principally by the liver enzyme cytochrome P450 1A2 (CYP1A2).6 The activity of CYP1A2, which also metabolizes many other substrates, can vary between individuals by more than 10-fold.7 Both genetics and personal habits, such as smoking, contribute to this variation.8 In this biochemistry laboratory experiment, students characterize CYP1A2 genotype and phenotype of test subjects to examine the role of this enzyme in caffeine metabolism. The motivation for the work is development of a point-of-care test in the neonatal intensive care unit (NICU) in order to © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: May 2, 2018 Revised: July 5, 2018

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DOI: 10.1021/acs.jchemed.8b00318 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. Student-generated results for CYP1A2*1F genotyping of subjects 1, 2, and 3 via PCR-RFLP and sequencing. The SNP of interest is a C/ A within the sequence GGGCCC, which is the ApaI cleavage site, or GGGCAC. (A) PCR products were incubated with ApaI and analyzed on a 1.2% agarose gel. The undigested product is represented by one 919 bp band (subjectt 3; AA genotype), with digestion resulting in 206 bp and 713 bp fragments (subject 1; CC genotype). Subject 2 has all three band sizes, suggesting that he is a heterozygote (AC genotype). (B) The reverse strand was sequenced, so position 179 is a G or a T. Subject 2 is a heterozygote with both G and T at this position, confirming the PCR-RFLP results.

participants confidential and using a blind numbering system for student samples. The first laboratory period takes about 3 h to perform the ELISA test and to set up PCR reactions. Each pair of students analyzes samples from one test subject, testing saliva for caffeine content and cheek swab samples for DNA. Willing colleagues, and the course instructors, were screened ahead of time to identify test subjects of different genotypes, although the CC genotype is rare, occurring in only 10% of individuals.17 Replicate analyses for each test subject were performed across each lab section in case of failure. Students also genotyped their own DNA samples on a voluntary basis, which was one of the reported highlights of the experiment. For phenotyping, competitive ELISA was used, in which caffeine in the saliva competes with a caffeine−enzyme conjugate for binding to an immobilized antibody. Saliva caffeine concentrations were determined from a standard curve prepared by each pair of students. The second laboratory period, which also takes about 3 h, involves digesting PCR products with a restriction enzyme and analyzing the fragments via agarose gel electrophoresis. The A allele is not digested, whereas the C allele gives two fragments. Students also prepared PCR products for commercial sequencing to verify genotype assignments. The third laboratory period was used for GC−MS analysis of the saliva samples for comparison with ELISA data, although this confirmatory test is optional. A standard curve for caffeine was run by the instructor before lab. Length of time depends on the availability of an autosampler, with sample preparation via solid-phase extraction taking less than 1 h. (In the absence of an autosampler, groups of students can work together to ensure that data for critical time points are acquired for all subjects, with each sample taking about 20 min to run.) Students then wrote a formal journal-style report

genotype has also been linked to a lower risk of myocardial infarction18 and a higher enhancement of athletic performance upon caffeine ingestion.19 Genotyping of DNA from cheek swab samples was achieved via PCR-RFLP,20 with sequencing used to confirm assignments. Phenotyping was performed on time-course saliva samples taken from test subjects after caffeine ingestion. The use of saliva in lieu of blood samples has been validated as a safe, noninvasive, and effective method for monitoring caffeine clearance.21−23 We used two methods to determine caffeine levels in each subject over time: a simple commercially available enzyme-linked immunosorbent assay (ELISA) and gas chromatography−mass spectrometry (GC−MS). Students compared the two methods for ease of use and reliability in order to evaluate the better method for monitoring caffeine levels in human subjects. They then assessed the relationship between CYP1A2 genotype and the relative rate of caffeine metabolism in human subjects in order to make a recommendation for the optimization of treatment regimens in the NICU.

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METHODS AND MATERIALS This modular experiment was designed for an upper-division undergraduate biochemistry course and requires up to three laboratory periods to complete. Necessary equipment includes a thermal cycler, agarose gel boxes, power supplies, plate reader, and a gas chromatograph−mass spectrometer. Detailed procedures for students and notes for instructors are provided in the Supporting Information. The use of human subjects requires prior approval of an Institutional Review Board (IRB), and instructors should consult with their own IRBs for appropriate procedures, such as keeping the identities of B




Figure 2. Salivary caffeine concentrations as a function of time for an individual with the AA genotype as determined by two different methods. Eight time-course samples (0, 30, 60, 120, 180, 240, 300, and 360 min) were analyzed by competitive ELISA (−▲−) and GC−MS (−■−). Halflives determined by the two methods were 3.7 and 5.3 h, respectively. Data for an individual with the CC genotype as determined by competitive ELISA (−●−) are shown for comparison (half-life, 6.5 h).

half-life). Tmax occurred at about 60 min for all subjects, consistent with previous studies of absorption rates.25 The two methods of caffeine determination gave somewhat different values of half-life. Two significant sources of error for the ELISA are cross-reactivity of the antibody with caffeine metabolites and the dilutions required because of the increased sensitivity (∼0.5−12 μg/L) relative to that of GC−MS (∼0.5− 50 mg/L). Peak physiological caffeine concentrations after a 200 mg dose are typically about 3 mg/L.25

in which they were expected to put their work into the context of the NICU by assessing the strengths and weaknesses of each test for optimizing caffeine-dosing protocols.

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HAZARDS Although standard precautions do not apply to saliva,24 students should wear gloves and protective eyewear throughout. Isopropanol, methanol, and acetonitrile are flammable solvents that are hazardous if ingested, inhaled, or absorbed through the skin. They should be handled in a fume hood. Guanidine hydrochloride is very hazardous in the case of skin contact, eye contact, or ingestion. DNA samples should be discarded after completion of the experiment for privacy of subjects.

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DISCUSSION This experiment provides an excellent introduction to personalized medicine and pharmacogenomics. Drug metabolism is an important topic that is often covered only briefly, if at all, in undergraduate biochemistry courses (e.g., during discussion of oxidative phosphorylation26). CYP1A2 plays an important role in the metabolism of several drugs, and variations in its activity can lead to difficulties in achieving optimum therapeutic levels.5,7,8 The effect of a SNP previously shown to influence induction of CYP1A217 was examined, with enzyme activity assessed by monitoring saliva caffeine levels via ELISA and GC. HPLC is most often used to quantitate caffeine in biological samples and could be used in this experiment if desired,16 although there is a precedent for plasma caffeine determination via GC in the literature.27−29 The rate of caffeine clearance has previously been shown to correlate to liver CYP1A2 content.30 Alternatively, phenotyping can be achieved through monitoring the ratio of caffeine to its principal metabolite, paraxanthine,16,21,30 both of which can be quantitated via GC or HPLC at the same time. This experiment was performed in successive years in our biochemistry course. Overall, student feedback was very positive, with particular appreciation of the real-world relevance of the work. Favorite aspects included comparing different techniques for analysis and having the opportunity to obtain their own genotypes. Negative comments mostly focused on the disagreement between the ELISA and chromatographic data for phenotyping, providing an oppor-

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RESULTS In this experiment, students used the relatively simple method of PCR-RFLP to determine CYP1A2*1F genotype. Because heterozygotes can be difficult to distinguish from incomplete restriction digestion, a commercial sequencing service was used to verify results. (The price of DNA sequencing is a few dollars per sample, and the turn-around time is about 1 day, making it very feasible in the teaching laboratory setting. Alternatively, sequencing could be done in-house or simply omitted from the exercise.) In each laboratory section, there were one or two amplification failures, likely due to some students being poor “shedders” of cheek cells or not swabbing vigorously enough. However, because our test subjects were typed ahead of time, ensuring that their DNA samples were robust, all groups successfully determined the genotype of their subject through both methods (Figure 1). Phenotyping was performed by monitoring the disappearance of caffeine from human saliva over time after a single 200 mg dose of caffeine (8 time points; 0−6 h). Students plotted caffeine concentration versus time (Figure 2) and used these plots to determine key pharmacokinetic parameters, including Cmax (maximal concentration), Tmax (the time to maximal concentration), ke (the elimination rate constant), and t1/2 (the C




(3) Renner, B.; Clarke, G.; Grattan, T.; Beisel, A.; Mueller, C.; Werner, U.; Kobal, G.; Brune, K. Caffeine Accelerates Absorption and Enhances the Analgesic Effect of Acetaminophen. J. Clin. Pharmacol. 2007, 47, 715−726. (4) Yang, A.; Palmer, A. A.; de Wit, H. Genetics of Caffeine Consumption and Responses to Caffeine. Psychopharmacology 2010, 211, 245−257. (5) Zhou, S.-F.; Yang, L.-P.; Zhou, Z.-W.; Liu, Y.-H.; Chan, E. Insights into the Substrate Specificity, Inhibitors, Regulation, and Polymorphisms and the Clinical Impact of Human Cytochrome P450 1A2. AAPS J. 2009, 11, 481−494. (6) Perera, V.; Gross, A. S.; McLachlan, A. J. Measurement of CYP1A2 Activity: A Focus on Caffeine as a Probe. Curr. Drug Metab. 2012, 13, 667−678. (7) Faber, M. S.; Jetter, A.; Fuhr, U. Assessment of CYP1A2 Activity in Clinical Practice: Why, How, and When? Basic Clin. Pharmacol. Toxicol. 2005, 97, 125−134. (8) Gunes, A.; Dahl, M.-L. Variations in CYP1A2 Activity and its Clinical Implications: Influence of Environmental Factors and Genetic Polymorphisms. Pharmacogenomics 2008, 9, 625−637. (9) Aranda, J. V.; Beharry, K.; Valencia, G. B.; Natarajan, G.; Davis, J. Caffeine Impact on Neonatal Morbidities. J. Matern.-Fetal Neonat. Med. 2010, 23 (S3), 20−23. (10) Legorreta-Herrera, M.; Mosqueda-Romo, N. A.; HernandezClemente, F.; Soto-Cruz, I. Detection of an ABCA1 Variant Associated with Type 2 Diabetes Mellitus Susceptibility for Biochemistry and Genetic Laboratory Courses. Biochem. Mol. Biol. Educ. 2013, 41, 409−418. (11) Soto-Cruz, I.; Legorreta-Herrera, M. Analysis of a p53 Mutation Associated with Cancer Susceptibility for Biochemistry and Genetic Laboratory Courses. Biochem. Mol. Biol. Educ. 2009, 37, 236−242. (12) Zhang, X.; Shao, M.; Gao, L.; Zhao, Y.; Sun, Z.; Zhou, L.; Yan, Y.; Shao, Q.; Xu, W.; Qian, H. A Comprehensive Experiment for Molecular Biology: Determination of Single Nucleotide Polymorphism in Human REV3 Gene using PCR-RFLP. Biochem. Mol. Biol. Educ. 2017, 45, 299−304. (13) Martin, M. P.; Detzel, S. M. A Laboratory Exercise to Determine Human ABO Blood Type by Noninvasive Methods. Biochem. Mol. Biol. Educ. 2008, 36, 139−146. (14) Schultheis, P. J.; Bowling, B. V. Analysis of a SNP Linked to Lactase Persistence. Biochem. Mol. Biol. Educ. 2011, 39, 133−140. (15) Wilson, B.; Grant, K. B.; Lubin, I. M. Allele-Specific Polymerase Chain Reaction-Based Genotyping of a Normal Variation in Human Color Vision. J. Chem. Educ. 2003, 80, 1289−1291. (16) Furge, L. L.; Fletke, K. J. HPLC Determination of Caffeine and Paraxanthine in Urine. Biochem. Mol. Biol. Educ. 2007, 35, 138−144. (17) Sachse, C.; Brockmöller, J.; Bauer, S.; Roots, I. Functional Significance of a C→A Polymorphism in Intron 1 of the Cytochrome P450 CYP1A2 Gene Tested with Caffeine. Br. J. Clin. Pharmacol. 1999, 47, 445−449. (18) Cornelis, M. C.; El-Sohemy, A.; Campos, H. Genetic Polymorphism of CYP1A2 Increases the Risk of Myocardial Infarction. J. Med. Genet. 2004, 41, 758−762. (19) Womack, C. J.; Saunders, M. J.; Bechtel, M. K.; Bolton, D. J.; Martin, M.; Luden, N. D.; Dunham, W.; Hancock, M. The Influence of CYP1A2 Polymorphism on the Ergogenic Effects of Caffeine. J. Int. Soc. Sports Nutr. 2012, 9, 7−12. (20) Ota, M.; Fukushima, H.; Kulski, J. K.; Inoko, H. Single Nucleotide Polymorphism Detection by Polymerase Chain ReactionRestriction Fragment Length Polymorphism. Nat. Protoc. 2007, 2, 2857−2864. (21) Fuhr, U.; Rost, K. L. Simple and Reliable CYP1A2 Phenotyping by the Paraxanthine/Caffeine Ratio in Plasma and in Saliva. Pharmacogenetics 1994, 4, 109−116. (22) Dobson, N. R.; Liu, X.; Rhein, L. M.; Darnall, R. A.; Corwin, M. J.; McEntire, B. L.; Ward, R. M.; James, L. P.; Sherwin, C. M. T.; Heeren, T. C.; Hunt, C. E. Salivary Caffeine Concentrations are Comparable to Plasma Concentrations in Preterm Infants Receiving

tunity for students to consider reasons why two different techniques for measuring the same parameter may differ. Anticipated learning outcomes included understanding both specific techniques (i.e., RFLP and competitive ELISA) and general applications of drug metabolism (e.g., personalized medicine), with pre- and postlab surveys supporting that learning goals were achieved (Table 1). Furthermore, the high Table 1. Comparison of Survey Results before and after This Experiment Average Scores,a N = 25 Statements for Student Response: I Understand

Prelab

Postlab

The RFLP technique and its uses. The competitive ELISA technique and its uses. What “personalized medicine” is. Why drugs affect individuals differently.

2.0 2.5 3.5 3.5

4.3b 4.4b 4.3b 4.4b

a

Scale ranges from 1 (“strongly disagree”) to 5 (“strongly agree”). Significant increase (p < 0.05) as determined through a paired, onetailed t-test.

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quality of the final papers reinforced our belief that student engagement with the theme of this experiment improved motivation and learning. Students successfully used the primary literature to set the context of the work, recognized that different analytical techniques can be used to achieve the same outcome, and made reasonable recommendations for point-of-care testing based on their data.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00318. Instructor notes (PDF, DOCX) Student handout (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Julie T. Millard: 0000-0001-6825-0207 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank BC368 students in 2017 and 2018 for testing this experiment, especially Danielle and Erika Smith, and Megan Watts, Cathy Bevier, Whitney King, Allison Moloney, Das Thamattoor, and Eric Thomas for helpful contributions. Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant P20GM103423.

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REFERENCES

(1) Benowitz, N. L. Clinical Pharmacology of Caffeine. Annu. Rev. Med. 1990, 41, 277−288. (2) Natarajan, G.; Lulic-Botica, M.; Aranda, J. V. Clinical Pharmacology of Caffeine in the Newborn. NeoReviews 2007, 8, e214−e221. D




Extended Caffeine Therapy. Br. J. Clin. Pharmacol. 2016, 82, 754− 761. (23) Zylber-Katz, E.; Granit, L.; Levy, M. Relationship between Caffeine Concentrations in Plasma and Saliva. Clin. Pharmacol. Ther. 1984, 36, 133−137. (24) CDC Perspectives in Disease Prevention and Health Promotion Update: Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and Other Bloodborne Pathogens in Health-Care Settings. MMWR 1988, 37, 377−388. (25) Kamimori, G. H.; Karyekar, C. S.; Otterstetter, R.; Cox, D. S.; Balkin, T. J.; Belenky, G. L.; Eddington, N. D. The Rate of Absorption and Relative Bioavailability of Caffeine Administered in Chewing Gum versus Capsules to Normal Healthy Volunteers. Int. J. Pharm. 2002, 234, 159−167. (26) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 7th ed.; MacMillan Learning: New York, 2017; pp 744−475. (27) Grab, F. L.; Reinstein, J. A. Determination of caffeine in plasma by gas chromatography. J. Pharm. Sci. 1968, 57, 1703−1706. (28) Newton, R.; Broughton, L. J.; Lind, M. J.; Morrison, P. J.; Rogers, H. J.; Bradbrook, I. D. Plasma and Salivary Pharmacokinetics of Caffeine in Man. Eur. J. Clin. Pharmacol. 1981, 21, 45−52. (29) Carregaro, A. B.; Mataqueiro, M. I.; Soares, O. A. B.; QueirozNeto, A. Study of Caffeine in Urine and Saliva of Horses Subjected to Urinary Acidification. J. Appl. Toxicol. 2004, 24, 513−518. (30) Fuhr, U.; Rost, K. L.; Engelhardt, R.; Sachs, M.; Liermann, D.; Belloc, C.; Beaune, P.; Janezic, S.; Grant, D.; Meyer, U. A.; Staib, A. H. 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.

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Genotype and Phenotype of Caffeine Metabolism: A Biochemistry

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