Laboratory Experiment

Cite This: J. Chem. Educ. 2018, 95, 1856−1860 pubs.acs.org/jchemeduc

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
*
S Supporting Information
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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

■ 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
© 2018 American Chemical Society and
Division of Chemical Education, Inc.
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 single-
nucleotide 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
Received: May 2, 2018
Revised: July 5, 2018
Published: August 9, 2018
1856 DOI: 10.1021/acs.jchemed.8b00318
J. Chem. Educ. 2018, 95, 1856−1860
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.

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.
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

■
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
1857
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
DOI: 10.1021/acs.jchemed.8b00318
J. Chem. Educ. 2018, 95, 1856−1860
Journal of Chemical Education
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 (−■−). Half-
lives 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).
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.
■ HAZARDS
Although standard precautions do not apply to saliva,24
students should wear gloves and protective eyewear through-
out. 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.
■
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 disappear-
ance 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
1858
Laboratory Experiment
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
■ DISCUSSION
This experiment provides an excellent introduction to
personalized medicine and pharmacogenomics. Drug metabo-
lism 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, phenotyp-
ing 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-
DOI: 10.1021/acs.jchemed.8b00318
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Journal of Chemical Education
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
The RFLP technique and its uses. 2.0
The competitive ELISA technique and its uses. 2.5
What “personalized medicine” is. 3.5
Why drugs affect individuals differently. 3.5
a Polymorphisms. Pharmacogenomics 2008, 9, 625−637.
Scale ranges from 1 (“strongly disagree”) to 5 (“strongly agree”).
b
Significant increase (p < 0.05) as determined through a paired, one-
tailed t-test.
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.
■
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)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: jtmillar@colby.edu.
ORCID
Julie T. Millard: 0000-0001-6825-0207
Notes
The authors declare no competing financial interest.
■
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.
■
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.
Laboratory Experiment
(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.
Postlab (7) Faber, M. S.; Jetter, A.; Fuhr, U. Assessment of CYP1A2 Activity
4.3b in Clinical Practice: Why, How, and When? Basic Clin. Pharmacol.
4.4b Toxicol. 2005, 97, 125−134.
4.3b (8) Gunes, A.; Dahl, M.-L. Variations in CYP1A2 Activity and its
4.4b
Clinical Implications: Influence of Environmental Factors and Genetic
(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.; Hernandez-
Clemente, 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 Poly-
morphism 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 Reaction-
Restriction 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
1859 DOI: 10.1021/acs.jchemed.8b00318
J. Chem. Educ. 2018, 95, 1856−1860
Journal of Chemical Education Laboratory Experiment

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 Trans-
mission 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.; Queiroz-
Neto, 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.

1860 DOI: 10.1021/acs.jchemed.8b00318

J. Chem. Educ. 2018, 95, 1856−1860