MEASUREMENT OF CYP1A2 PHENOTYPE USING FEMALE VOLUNTEER PLASMA: A FOCUS ON CAFFEINE AND PARAXANTHINE AS A PROBE
Saima Rehman, Shagufta Kamal, Shumaila Kiran, Ismat Bibi, Iqbal Hussain
Department of Applied and Biochemistry, Government College University, Faisalabad-38000, Pakistan . Department of Chemistry, Islamia University, Bahawalpur 63100, Pakistan. Department of Botany, Government College University, Faisalabad-38000, Pakistan
Keywords: Cytochrome P-450, caffeine, metabolism, cancer, volunteers
Caffeine neither causing didn't decrease the risk of cancer, yet it used just to note the activity of cytochrome P-4501A2 (CYP1A2) by converting into its metabolite i.e., paraxanthine. The purpose of the present study was to determine the caffeine and its metabolite phenotypes and their relation to cancer risk in healthy female volunteers of local population in Pakistan. The average value of metabolic ratio [(1,7-dimethylxanthine (17X) and Caffeine 1,3,7-trimethylxanthine (137X)] was found to be 1.182995 ± 0.21137. BMI (used to categorize into different groups, i.e., overweight, underweight etc.) of all volunteers were found to be 19.93Kg/m2. Retention time was 15 and 37 min for 1,7-dimethylxanthine (17DMX) and 1,3,7 trimethylxanthine (137TMX), respectively. The linearity of calibration curve of 137TMX and 17DMX were covered 0–12 µg/mL (R2 = 0.994). A significant positive correlation was observed between metabolic ratio and cancer risk factors. We could concluded that all the volunteers are fast metabolizers having a greater risk of cancerous diseases.
of CYP1A2 using plasma: a focus on caffeine and paraxanthine as a probe
Caffeine (137TMX), one of the most commonly ingested
compounds throughout the world, is an alkaloid unsurprisingly found in fruit of
plants, leaves or seeds such as coffee (Coffea sp.), tea (Camellia sinensis),
cocoa (Theobroma cacao), kola (Cola acuminate), Yerba mate (Ilex
paraguariensis), and Guarana (Paullinia cupana). On
the other hand, a variety of common beverages and certain medications, contain
caffeine[2-3]. Caffeine acts as an inhibitor of phosphodiesterase,
?-aminobutyric acid (GABA) receptors and decreases anxiety and fatigue [4-5].
Caffeine elevates catecholamine levels, basal
metabolic rate (BMR), vigilance, fortitude and neuromuscular
coordination, improve mood, cognitive performance and also promotes lipolysis .
Complex metabolic pathway of caffeine involves the formation of three principal
metabolite i.e theobromine (37DMX:
3,7-dimethylxanthine), theophylline (13DMX:
1,3-dimethylxanthine) and paraxanthine (17DMX) and mainly restricted to the
liver which are primarily formed by CYP
1A2. About 80% of the metabolic pathway for caffeine
comprises its conversion to 17DMX metabolite. Due to lipophilic properties of
caffeine and low plasma protein binding, caffeine easily penetrates all
biological membranes, including placental and blood-brain barrier 
with a half-life of about 4 to 6 h and a total clearance of approximately 2
mL/min/kg. Due to its safe, inexpensive, rapid, complete
absorption and short half-life, caffeine is considered an ideal probe drug for
estimation of in vivo activity of CYP1A2.
Drug specific phenotyping of CYP1A2
alongside caffeine have been analyzed by many pharmaceuticals.
Efforts to associate the genotype with CYP1A2
metabolic phenotypes have been enduring. However, prediction of the metabolic
phenotype based on genotype with CYP1A2
has been inveterate to be challenging and is an area of research that creates
continued hullabaloo [11-12].
Phenotyping using plasma and saliva samples to measure
the paraxanthine to caffeine (17DMX/137TMX) ratio correlates fit with many
measures of CYP1A2
activity. Various urinary metabolic ratios for caffeine phenotyping have been
proposed, but shortcomings have been demonstrated for all the proposed urinary
metabolic ratios . Nutrition is an ecological factor that
greatly affects the activity of P4501A2. In addition to previously mentioned
char-grilled meats, Crucifereae vegetables (including broccoli and cauliflower)
also have an effect on the metabolic activity of CYP1A2. Apiaceae
vegetables (carrot, celery, fennel, parsley and parsnip) exhibited an
inhibitory influence, whereas; Allium vegetables (garlic, onions, scallions,
leeks, and chives) have also shown effects on CYP1A2 activity when compared with a
control diet lacking vegetables . Apart from these environmental
factors, age and gender have also been affected the metabolic activity of CYP1A2 with
children having higher activity than adults, and men have higher activity than
women, although this latter observation is disputed.
CYP Isozymes reduce the half life and duration of exposure
to xenobiotics and prevent accumulation of the parent compounds. At the same
time, excessive CYP
activity without adequate concomitant conjugating activity may also be a risk
factor for cancer. Geonatics has a geographical influence on
genetics and manifested by dissimilar biochemical, physiological and
pharmacological parameters when local populations are compared with foreign
counterparts. The differences have been shown to affect the fate
of drug, therapeutics standards arid dose regimens on the basis of indigenous
investigations. The current study focused on to investigate the
activity of CYP1A2 by using the drug i-e Caffeine with the help of HPLC technique, determination of risk factors of
cancer related to CYP 1A2 and comparison of the activity of CYP1A2 in the local
population and their foreign counterparts.
MATERIALS AND METHODS
General Experimental Details
The study was designed to also elaborate the
biotransformation of caffeine, which act as a probe drug to study the activity
of CYP1A2 in eleven unrelated healthy female volunteers of age between 17-24
years. All the volunteers were advised to refrain from any form of caffeine
intake, including tea, coffee and chocolate, etc. for at least 24 h prior to
the study. After completing the washout period, volunteers received a single
cup of coffee (Nescafe) which contains 400 mg of coffee. Six hours after
caffeine intake, 5mL of venous blood sample was drawn into EDTA (ethylenediaminetetraacetic acid)
containing centrifuge tube. Then blood sample was centrifuged at 3000 rpm for
10 min and plasma separated. The plasma was collected in glass tubes with the
help of a dropper. Glass tubes containing plasma were stored at -20°C
until analyzed. Volunteer having any type of abnormalities in their history was
not included in this study. The demographic data (Age, body weight, height,
blood pressure, body temperature and BMI) of eleven healthy female volunteers
are given in Table 1.
Chemicals All the chemicals, i.e., paraxanthine (17DMX), (D5385,
Sigma, USA), caffeine (137TMX) (Merck,
Darmstadt, Germany) and acetic acid (Sigma Aldrich, Buchs, Switzerland) of
analytical grade were used for sample preparation
Preparation of standard solutions
A stock solution of 10 mg of caffeine and paraxanthine in
the eluent was used to prepare the standard solutions of caffeine and
paraxanthine. All the standard solutions were prepared by diluting this stock
A simple extraction and quantification procedure were
developed that used reverse phase chromatography (RP-HPLC) as described by.
The extraction procedure was as follows: the plasma proteins were precipitated
by addition of 300µ Lacetonitrile. The mixture was placed in a vortex blender
for 10 seconds, followed by centrifugation at 3500g for 10 minutes. The supernatant was evaporated to dryness by
using nitrogen evaporation (speed vac plus, savant instruments, Inc, Holbrook,
NY) and reconstituted in a 100 µL mobile phase before 20 µL was injected onto a
Table 1. Values of Age, Body
weight, Height, Blood Pressure, Body Temperature and BMI of eleven healthy
temperature; SD: standard deviation; BMI:
body mass index
The method of analysis was performed using the following
HPLC (Hitachi) system, flow control valve (FCV-10AL), Degasser unit (DGU-12A),
System controller (SCL-10A), Spectral detector UV (L-2400) and pump (L-2130).
The chromatographic data were collected by using CSW-32 software. Separation
was achieved at ambient temperature with a Shim Pack CLC-ODS (150 *4.6 mm, 5 µm
pore size) column and analytes were detected at 273 nm. Flow Rate was 1.0
mL/min and run time was 40 min.
Mobile phase was prepared by using 15% methanol in 25
mmol/L sodium acetate buffer (pH: 4.0). The flow rate was 1.0 mL/min and
detection wavelength was 273 nm. Mobile phase was filtered in a vacuum
filtration assembly having a cellulose acetate filter, diameter was 47 mm, pore
size 0.45 µm, then sonicated (Elma-Sonic
E-60) for 15 to 20 min.
Peak areas were compared for quantification of caffeine
and paraxanthine using acetic acid as an internal standard
of Caffeine metabolites
Regression equation was used to determine concentration
of caffeine metabolites,
X= (Y-b)/a, where X= drug
concentration; Y=Peak area
RESULTS AND DISCUSSION
In the present study, paraxanthine was selected as a
metabolite of caffeine and its values were used to calculate the activity of
CYP 1A2 in female volunteers. Representative chromatogram of standard
concentrations of 17X and caffeine is given in Fig.1-2. Figure 3 reveals that the level of 17X is high after 6 h of
caffeine administration. Retention time for each analyte is given in Table 2. Representative chromatograms indicated that all the
peaks 17X and 137X were not disturbed from each other and solvent peak
indicating good performance of each standard determination in the same sample.
The calibration curves of 137X and 17X were linear over the concentration range
0-12µg/mL and R2 was 0.993 and 0.994 respectively, as shown in Fig. 4-5, which is very close to Fig.
1. The concentration of caffeine exhibited various ranges
starting from 4.670 to 6.384 with mean 5.570639 ± 0.518101055507µg/mL.
Concentration of paraxanthine in the serum of healthy female volunteers ranges
from 3.117296 to 6.386429 with a mean of 4.8357 ± 1.055507µg/mL.
Retention Time in minutes for each analyte
Table 3. Metabolic ratio for
P-450 1A2 activity
detected; SD : standard deviation; 137TMX: 1,3,7
trimethylxanthine; 17DMX: 1,7-dimethylxanthine; MR (metabolic ratio) = paraxanthine/Caffeine
Representative chromatogram for the standard value of 17X (paraxanthine)
Representative chromatogram for the standard value of 137 X (Caffeine)
Representative chromatogram after six hour of caffeine administration
4. Calibration curve for caffeine in female volunteer serum
Calibration curve for dimethylxanthine in female volunteer serum
Results depicted in Table
3 to show the different values of caffeine and 17DMX.
These values could be compared with the values reported earlier. The metabolic
ratio ?0.5 was considered as fast metabolizer and metabolic
ratio ?0.5 was considered as slow metabolizers .
As all the values depicted in table 3 is ?0.5 since they all were referred as fast metabolizers.
Our results are in consonance with the findings of . These
scientists reported that caffeine consumers showed a high metabolic ratio because
caffeine has positive relationships with ALT (alanine transaminase) and AST (aspartate transaminase).
Several methods of determining an individual's CYP1A2 activity by using
caffeine as a probe drug has been developed with various metabolic ratios such
as 17DMX×17DMU/137TMX; (17DMU: 1,7-dimethyluric acid).We
selected paraxanthine/caffeine (17DMX/137TMX) as a metabolic ratio due to
largest amounts (84%) of paraxanthine produced as a result of caffeine
biotransformation. There are different risk factors of cancer; one of these is
Isozyme of CYP1A2. The MR (metabolic
rate) and CYP1A2
activity are directly proportional to each other. The high value of CYP leads
to higher chances of cancer to occur.
For sample with paraxanthine concentrations below detection
level, no MR value was calculated. Volunteer 1 with lower BMI does not show any
metabolic ratio, thus cytochrome P-450 1A2 is lowest and chances of cancer will
be less as compared to all other volunteers. Volunteer 3 with BMI values 18.9
show highest metabolic ratio 1.498 thus the activity of CYP 1A2 is high and
this Isozyme serve as a risk factor of cancer. The average value of metabolic
ratio with ± S.D. 1.182995±0.21137 which is high as compared to the values
reported earlier (MR= 0.5). It is reported that Chinese women showed the
highest values of CYP1A2 when determined by the caffeine metabolic ratio
indicating a higher risk of lung cancer . The rapid cytochrome
CYP 1A2 phenotype increases the risk of developing colorectal cancer in humans [25-26].
In conclusion, this simplified method can be used to
determine CYP1A2 activity and cancer risk factors in the population exposed the
CYP1A2 interference agents.
Le Marchand L, Hankin JH, Wilkens LR, Pierce LM,
Franke A, Kolonel LN, Donlon T. Combined effects of well-done red meat,
smoking, and rapid N-acetyltransferase 2 and CYP1A2 phenotypes in increasing
colorectal cancer risk. Cancer Epidem. Biomark. Prevent. (2001);10(12):
N, Mashev N. Antioxidant activity and polyphenols content of some herbal
teas of lamiaceae family from Greece and Bulgaria. Oxid. Communications,
- Mandel HG. Update on caffeine consumption,
disposition and action. Food. Chem.
Toxicol. (2002); 40(9): 1231-1234
A, Palmer AA, de Wit H. Genetics of caffeine consumption and responses to
caffeine. Psychopharmacol. (2010);211(3): 245-257
- Chou T. Wake up and smell the coffee. Caffeine,
coffee, and the medical consequences. West. J. Medicine, (1992); 157(5):
- Fredholm BB.
Notes on the history of caffeine use. In: Methylxanthines. Springer
Berlin Heidelberg. (2011); pp: 1-9
- Ferré S. An update on the mechanisms of the
psychostimulant effects of caffeine. J. Neurochem. (2008);105(4):
M, Daniel WA. Caffeine as a marker substrate for testing cytochrome P450
activity in human and rat. Pharmacol.
Reports (2008); 60 (789):
- George SE,
Ramalakshmi K, Mohan Rao LJ. A
perception on health benefits of coffee. Crit. Rev. Food Sci. Nutr. (2008)
W, Tang BK. The use of caffeine for enzyme assays: a critical appraisal. Clin. Pharm. Therap. (1993); 53(5): 503-514.
KL, Hon YY, Penzak SR, Lam YW, Spratlin V, Jann MW. Correlation of
cytochrome P450 (CYP)1A2 activity using caffeine phenotyping and olanzapine
disposition in healthy volunteers. Neuropsychopharmacol.
K, Saruwatari J, Nakada N, Nakagawa M, Fukuda K, Tanaka F, Nakagawa K.
Phenotype-genotype analysis of CYP1A2 in Japanese patients receiving oral
theophylline therapy. Eur. J.
Clin. Pharmacol. (2006);62(1):
- Sharma MC. Application of RP-HPLC and extractive
spectrophometric methods for the determination of lumefantrine in
pharmaceutical dosage form. Oxid. Communications. (2013); 36: 379
NMK. Caffeine metabolic ratios for the in
vivo evaluation of CYP1A2, N-acetyltransferase 2, xanthine oxidase and
CYP2A6 enzymatic activities. Curr. drug metabol. (2009);10(4): 329-338
- Walters DG, Young PJ, Agus C, Knize MG, Boobis AR,
Gooderham NJ, Lake BG. Cruciferous vegetable consumption alters the
metabolism of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo
[4,5-b] pyridine (PhIP) in humans. Carcinogen. (2004); 25(9): 1659-1669
- Lampe JW, King IB, Li S, Grate MT, Barale KV, Chen
C, Potter JD. Brassica vegetables increase and apiaceous vegetables
decrease cytochrome P4501A2 activity in humans: changes in caffeine
metabolite ratios in response to controlled vegetable diets. Carcinogen.
(2000); 21(6): 1157-1162.
- Krul C, Hageman G. Analysis of urinary caffeine
metabolites to assess biotransformation enzyme activities by
reversed-phase high-performance liquid chromatography. J. Chromatogr. B
Biomed. Sci. Appl. (1998);709(1): 27-34.
- Ioannides C Ed. Cytochromes P450: metabolic and
toxicological aspects. CRC Press. (1996).
- Durak A, Gawlik UG. Bioacccessibility and
interaction between natural antioxidants as main factors determining
biological activity of plant derived food. Oxid. Commun. (2014); 37: 492
- Nawaz M, Iqbal T, Nawa R.
Genetical consideration in disposition kinetic evaluation of
chemotherapeutic agents. Vol. 2 Cong. Europ. Assoc. Vet. Pharmacol.
Therap. Budapest (1988);2: 206-260
- Heydorn K, Anglov
T, Byrialsen K, Carstensen JK,Christensen F, Christensen S, Winther, H. Uncertainty budget for final assay of a
pharmaceutical product based on RP-HPLC. Accred Quality Assur. (2003); 8(5): 225
- Begas , Kouvaras E, Tsakalof A, Papakosta S,
Asprodini EK. In vivo evaluation of CYP1A2, CYP2A6, NAT-2 and xanthine oxidase activities in a Greek population sample by the
RP-HPLC monitoring of caffeine metabolic ratios.
Biomed. Chromat. (2007);21(2): 190–200
Y, Chaudhary VK, Bhatnagar R, Misra UK. Mixed function oxidases in
response to quality and quantity of dietary protein. Biochem. Inter. (1988); 17(1): 1-8
- Butler MA, Lang NP, Young JF,
Caporaso NE, Vineis P, Hayes RB, Kadlubar FF. Determination of CYP1A2 and
NAT2 phenotypes in human populations by analysis of caffeine urinary metabolites. Pharmacogenet. Genom.
(1992); 2 (3): 116–127
A, Zhao B, Lee EJ, Poh WT, The M, Eng P, Lee HP. Cytochrome P4501A2
(CYP1A2) activity and lung cancer risk: a preliminary study among Chinese
women in Singapore. Carcinogen. (2001); 22(4): 673-677
- Lang NP, Butler MA, Massengill J, Lawson M, Stotts
RC, Hauer-Jense M, Kadlubar FF. Rapid metabolic phenotypes for
acetyltransferase and cytochrome P4501A2 and putative exposure to
food-borne heterocyclic amines increase the risk for colorectal cancer or
polyps. Cancer Epidem. Biomark. Prevent. (1994);3(8): 675-682
© 2016 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license.
You are free to:
Share — copy and redistribute the material in any medium or format
Adapt — remix, transform, and build upon the material for any purpose, even commercially.
The licensor cannot revoke these freedoms as long as you follow the license terms.
Under the following terms:
Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made.
You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use.
No additional restrictions
You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits