Acetylation polymorphism of caffeine in a Japanese population The frequency distribution of N-acetylation of caffeine was determined in 140 unrelated healthy Japanese subjects by measuring the amount of two main metabolites of caffeine, 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methyhanthine (IX), in urine after an oral dose of caffeine. N-Acetylation capacity for caffeine appeared to be polymorphic: 15 subjects (10.7%) were phenotyped as slow acetylators, whereas 125 subjects (89.3%) were phenotyped as rapid ones. The urinary molar excretion ratio of AFMU (AFMU/1X) in 2 hours-urine samples ranged from 0.03 (slow acetylators) to 2.66 (rapid acetylators). The frequency of slow acetylators in this study was similar to that reported previously for the isoniazid and dapsone polymorphism in Japanese populations. (CLIN PHARKAcoL THER 1992;52:274-6.)
Masayuki Hashiguchi, PhD, and Akio Ebihara, MD Oita and Tochigi, Japan Polymorphisms of drug acetylation and oxidation have been well recognized in humans in the metabolism of various clinically useful drugs such as isoniazid, procainamide, certain sulfonamides, tricyclic antidepressants, and p-blockers." These polymorphisms are known to be under monogenic control." It has been reported that the frequency of the poor metabolizer phenotype of debrisoquin is between 0% (Japanese population5) and 10% (Swedish population6), whereas the poor metabolizer phenotype of mephenytoin is between 0% (Cuna Amerindians') and 23% (Japanese population8'9) and the poor metabolizer phenotype of isoniazid ranges from 0% to 80%.4 There are pronounced interethnic differences in the frequency of the poor metabolizer phenotype. The frequency of defective drug acetylation capacity appears to be larger than that of defective drug oxidation capacity. As the therapeutic effects and the risk of some drug-related toxic responses are closely associated with individual genetic drug-metabolizing capacity, the knowledge of pharmacogenetics in different population is very important in the assessment of optimum therapeutic response and the incidence of adverse drug reaction. From the Department of Clinical Pharmacology and Therapeutics, Oita Medical University, Oita, and the Department of Clinical Pharmacology and Therapeutics, Jichi Medical School, Tochigi. Received for publication Jan. 28, 1992; accepted May 21, 1992. Reprint requests: Masayuki Hashiguchi, PhD, Department of Clinical Pharmacology and Therapeutics, Oita Medical University, 1-1, Idaigaoka, Hasama-machi, Oita-gun, Oita 879-55, Japan. 13/1/39833
274
Recently, caffeine metabolism has been developed as a new, safe, noninvasive agent to determine acetylator phenotype.") We have reported a simplified acetylator phenotyping method using 2-hour urine samples collected from 2 to 4 hours after ingestion of caffeine." The acetylator phenotype in Japanese populations has been previously reported with use of only isoniazidu or dapsone.13 The purpose of this study was to determine the frequency of acetylator phenotype in a Japanese population with use of caffeine by the previously reported 2-hour urine method.11
MATERIAL AND METHODS Subjects. We studied 140 unrelated healthy Japanese men (age, 24.3 ± 3.1 years [mean ± SD]; age range, 22 to 41 years; body weight, 65.1 ± 7.6 kg; weight range, 52 to 86 kg; height, 171.2 ± 4.9 cm, height range, 156 to 185 cm). Written informed consent was obtained from each subject after the purpose, procedures, and risks of the study were explained. Protocol. The subjects abstained from tobacco, xanthine derivativecontaining foods and beverages such as coffee, tea, chocolate, cola, bananas, and vanilla ice cream for 24 hours before and during the study. They each took 150 mg caffeine dissolved in 150 ml instant decaffeinated coffee (Nescafe). Two hours after ingestion of the coffee, each subject emptied his bladder and collected urine for the following 2 hours. Analytic procedures. The volume of each urine sample was recorded, and aliquots of urine were frozen immediately at 20° C until analyzed. All sam-
VOLUME 52 NUMBER 3
Acetylation of caffeine metabolites by Japanese subjects
275
8
'
.1.
2
0
0.5
1.0
2.0
1.5
2.5
3.0
AFMU/1X Fig. 1. Probit plot and frequency distribution histogram of urinary molar excretion ratio of AFMU/1X in 140 Japanese subjects.
pies were analyzed within 3 days. Urinary concentration of 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (1X) were measured by HPLC by use of the method described previ-
type for each subject was carried out by frequency distribution histogram and probit plot with use of the urinary molar excretion ratios of AFMU to IX (AFMU/1X). Statistical significance was assessed by use of the )(2 test, with or without Yates' correction. Statistical significance was defined as p < 0.05.
pattern and the histogram indicated an AFMU/1X ratio of 0.3 discriminated between slow acetylators and rapid acetylators. The probit plot also separated two different acetylator phenotypes. The subjects with a low urinary molar excretion ratio of AFMU/1X (15 of 140 subjects) were classified as the slow acetylator phenotypes, with a AFMU/1X ratio between 0.03 and 0.17 (10.7%; 95% confidence interval, 5.6% to 15.8%). The other subjects (125 subjects, 89.3%) had a urinary molar excretion ratio of AFMU/1X in the range 0.40 to 2.66 and were classified as the rapid acetylator phenotypes.
RESULTS The frequency distribution histogram and the probit plot of urinary molar excretion ratio of AFMU to IX (AFMU/1X) in 140 unrelated healthy Japanese subjects are shown in Fig. 1. In our subjects the urinary molar excretion ratio of AFMU/1X ranged from 0.03 to 2.66 and the observed distribution displayed a ninetyfold interindividual range. The distribution showed an apparently bimodal
DISCUSSION To our knowledge, pharmacogenetic studies of N-acetylation in Japanese populations have been carried out with isoniazid 12 and dapsone" as test drugs. Recently, caffeine has been noted as a safe and ubiquitous test drug for determination of acetylator phenotype.I° However, no data are available regarding the N-acetylation polymorphism of caffeine in a Japanese population. Therefore, we investigated the acetylation
ously." Data analysis. The assessment of acetylator pheno-
CL1N PHARMACOL THER SEPTEMBER 1992
276 Hashiguchi and Ebihara Table I. Frequency of slow acetylators in a Japanese population with use of various test drugs Slow acetylators
Reference
Test drug
No. of study subjects
No.
Sunahara et al.1 2 Horai et al." Present study
Isoniazid Dapsone Caffeine
1808 182 140
207 12 15
Frequency (%) 11.5*
6.6* 10.7
95% Confidence interval
10.0-12.9 3.8-11.2
5.6-15.8
*No statistically significant differences compared with the present study by use of caffeine.
polymorphism in a Japanese population using the 2-hour urine method" as described previously. The present data indicate that the distribution of acetylator phenotype with use of caffeine as a test drug is polymorphic in a Japanese population. Both the frequency distribution histogram and the probit plot showed an apparent bimodal distribution, and 15 of 140 subjects (10.7%) were phenotyped as slow acetylators. The frequency of acetylator phenotype with use of isoniazidI2 and dapsone" as test drugs in a Japanese population are shown in Table I. The frequency of slow acetylator phenotype with use of caffeine was consistent with that observed with isoniazid or dapsone. We are grateful to Yoshitomi Pharmaceutical Co., Ltd., Osaka, Japan, for providing 5-acetylamino-6-formylamino3-methyluracil (AFMU).
References I. Drayer DE, Reidenberg MM. Clinical consequences of polymorphic acetylation of basic drugs. CLAN PHARMACOL THER 1977;22:251-8. Weber WW, Hein DW. N-Acetylation pharmacogenetics. Pharmac Rev 1985;37:25-79. Eichelbaum M. Defective oxidation of drugs: pharmacokinetic and therapeutic Implications. Clin Pharmacokinet 1982;7:1-22. Clark DWJ. Genetically determined variability in acetylation and oxidation. Therapeutic implications. Drugs 1985;29:342-75.
Nakamura K, Goto F, Ray WA, et al. Interethnic differences in genetic polymorphism of debrisoquine and mephenytoin hydroxylation between Japanese and Caucasian populations. CLIN PHARMACOL THER 1985;38: 402-8. Steiner E, Iselius L, Alvan G, Lindstein J, Sjoqvist F. A family study of genetic and environmental factors determining polymorphic hydroxylation of debrisoquin. CLIN PHARMACOL THER 1985;38:394-401. Inaba T, Jorge LF, Arias TD. Mephenytoin hydroxylation in the Cuna Amerindians of Panama. Br J Clin Pharmacol 1985;19:483-7. Jurima M, Inaba T, Kadar D, Kalow W. Genetic polymorphism of mephenytoin p(4')-hydroxylation: difference between Orientals and Caucasians. Br J Clin Pharmacol 1985;19:483-7. Horai Y, Nakano M, Ishizaki T, et al. Metoprolol and mephenytoin oxidation polymorphism in Far Eastern Oriental subjects: Japanese versus mainland Chinese. CLIN PHARMACOL THER 1989;46:198-207. Grant DM, Tang BK, Kalow W. A simple test for acetylator phenotype using caffeine. Br J Clin Pharmacol 1984;17:459-64. Hashiguchi M, Tsutsumi K, Nakashima H, Ebihara A. Assessment of a simple method for acetylator phenotyping with caffeine. Jpn J Clin Pharmacol Ther 1990;21: 425-31. Sunahara S, Urano M, Ogawa M. Genetical and geographic studies on isoniazid inactivation. Science 1961;134:1530-1. Horai Y, Ishizaki T. N-Acetylation polymorphism of dapsone in a Japanese population. Br J Clin Pharmacol 1988;25:487-94.
Masayuki Hashiguchi, PhD, and Akio Ebihara, MD Oita and Tochigi, Japan Polymorphisms of drug acetylation and oxidation have been well recognized in humans in the metabolism of various clinically useful drugs such as isoniazid, procainamide, certain sulfonamides, tricyclic antidepressants, and p-blockers." These polymorphisms are known to be under monogenic control." It has been reported that the frequency of the poor metabolizer phenotype of debrisoquin is between 0% (Japanese population5) and 10% (Swedish population6), whereas the poor metabolizer phenotype of mephenytoin is between 0% (Cuna Amerindians') and 23% (Japanese population8'9) and the poor metabolizer phenotype of isoniazid ranges from 0% to 80%.4 There are pronounced interethnic differences in the frequency of the poor metabolizer phenotype. The frequency of defective drug acetylation capacity appears to be larger than that of defective drug oxidation capacity. As the therapeutic effects and the risk of some drug-related toxic responses are closely associated with individual genetic drug-metabolizing capacity, the knowledge of pharmacogenetics in different population is very important in the assessment of optimum therapeutic response and the incidence of adverse drug reaction. From the Department of Clinical Pharmacology and Therapeutics, Oita Medical University, Oita, and the Department of Clinical Pharmacology and Therapeutics, Jichi Medical School, Tochigi. Received for publication Jan. 28, 1992; accepted May 21, 1992. Reprint requests: Masayuki Hashiguchi, PhD, Department of Clinical Pharmacology and Therapeutics, Oita Medical University, 1-1, Idaigaoka, Hasama-machi, Oita-gun, Oita 879-55, Japan. 13/1/39833
274
Recently, caffeine metabolism has been developed as a new, safe, noninvasive agent to determine acetylator phenotype.") We have reported a simplified acetylator phenotyping method using 2-hour urine samples collected from 2 to 4 hours after ingestion of caffeine." The acetylator phenotype in Japanese populations has been previously reported with use of only isoniazidu or dapsone.13 The purpose of this study was to determine the frequency of acetylator phenotype in a Japanese population with use of caffeine by the previously reported 2-hour urine method.11
MATERIAL AND METHODS Subjects. We studied 140 unrelated healthy Japanese men (age, 24.3 ± 3.1 years [mean ± SD]; age range, 22 to 41 years; body weight, 65.1 ± 7.6 kg; weight range, 52 to 86 kg; height, 171.2 ± 4.9 cm, height range, 156 to 185 cm). Written informed consent was obtained from each subject after the purpose, procedures, and risks of the study were explained. Protocol. The subjects abstained from tobacco, xanthine derivativecontaining foods and beverages such as coffee, tea, chocolate, cola, bananas, and vanilla ice cream for 24 hours before and during the study. They each took 150 mg caffeine dissolved in 150 ml instant decaffeinated coffee (Nescafe). Two hours after ingestion of the coffee, each subject emptied his bladder and collected urine for the following 2 hours. Analytic procedures. The volume of each urine sample was recorded, and aliquots of urine were frozen immediately at 20° C until analyzed. All sam-
VOLUME 52 NUMBER 3
Acetylation of caffeine metabolites by Japanese subjects
275
8
'
.1.
2
0
0.5
1.0
2.0
1.5
2.5
3.0
AFMU/1X Fig. 1. Probit plot and frequency distribution histogram of urinary molar excretion ratio of AFMU/1X in 140 Japanese subjects.
pies were analyzed within 3 days. Urinary concentration of 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (1X) were measured by HPLC by use of the method described previ-
type for each subject was carried out by frequency distribution histogram and probit plot with use of the urinary molar excretion ratios of AFMU to IX (AFMU/1X). Statistical significance was assessed by use of the )(2 test, with or without Yates' correction. Statistical significance was defined as p < 0.05.
pattern and the histogram indicated an AFMU/1X ratio of 0.3 discriminated between slow acetylators and rapid acetylators. The probit plot also separated two different acetylator phenotypes. The subjects with a low urinary molar excretion ratio of AFMU/1X (15 of 140 subjects) were classified as the slow acetylator phenotypes, with a AFMU/1X ratio between 0.03 and 0.17 (10.7%; 95% confidence interval, 5.6% to 15.8%). The other subjects (125 subjects, 89.3%) had a urinary molar excretion ratio of AFMU/1X in the range 0.40 to 2.66 and were classified as the rapid acetylator phenotypes.
RESULTS The frequency distribution histogram and the probit plot of urinary molar excretion ratio of AFMU to IX (AFMU/1X) in 140 unrelated healthy Japanese subjects are shown in Fig. 1. In our subjects the urinary molar excretion ratio of AFMU/1X ranged from 0.03 to 2.66 and the observed distribution displayed a ninetyfold interindividual range. The distribution showed an apparently bimodal
DISCUSSION To our knowledge, pharmacogenetic studies of N-acetylation in Japanese populations have been carried out with isoniazid 12 and dapsone" as test drugs. Recently, caffeine has been noted as a safe and ubiquitous test drug for determination of acetylator phenotype.I° However, no data are available regarding the N-acetylation polymorphism of caffeine in a Japanese population. Therefore, we investigated the acetylation
ously." Data analysis. The assessment of acetylator pheno-
CL1N PHARMACOL THER SEPTEMBER 1992
276 Hashiguchi and Ebihara Table I. Frequency of slow acetylators in a Japanese population with use of various test drugs Slow acetylators
Reference
Test drug
No. of study subjects
No.
Sunahara et al.1 2 Horai et al." Present study
Isoniazid Dapsone Caffeine
1808 182 140
207 12 15
Frequency (%) 11.5*
6.6* 10.7
95% Confidence interval
10.0-12.9 3.8-11.2
5.6-15.8
*No statistically significant differences compared with the present study by use of caffeine.
polymorphism in a Japanese population using the 2-hour urine method" as described previously. The present data indicate that the distribution of acetylator phenotype with use of caffeine as a test drug is polymorphic in a Japanese population. Both the frequency distribution histogram and the probit plot showed an apparent bimodal distribution, and 15 of 140 subjects (10.7%) were phenotyped as slow acetylators. The frequency of acetylator phenotype with use of isoniazidI2 and dapsone" as test drugs in a Japanese population are shown in Table I. The frequency of slow acetylator phenotype with use of caffeine was consistent with that observed with isoniazid or dapsone. We are grateful to Yoshitomi Pharmaceutical Co., Ltd., Osaka, Japan, for providing 5-acetylamino-6-formylamino3-methyluracil (AFMU).
References I. Drayer DE, Reidenberg MM. Clinical consequences of polymorphic acetylation of basic drugs. CLAN PHARMACOL THER 1977;22:251-8. Weber WW, Hein DW. N-Acetylation pharmacogenetics. Pharmac Rev 1985;37:25-79. Eichelbaum M. Defective oxidation of drugs: pharmacokinetic and therapeutic Implications. Clin Pharmacokinet 1982;7:1-22. Clark DWJ. Genetically determined variability in acetylation and oxidation. Therapeutic implications. Drugs 1985;29:342-75.
Nakamura K, Goto F, Ray WA, et al. Interethnic differences in genetic polymorphism of debrisoquine and mephenytoin hydroxylation between Japanese and Caucasian populations. CLIN PHARMACOL THER 1985;38: 402-8. Steiner E, Iselius L, Alvan G, Lindstein J, Sjoqvist F. A family study of genetic and environmental factors determining polymorphic hydroxylation of debrisoquin. CLIN PHARMACOL THER 1985;38:394-401. Inaba T, Jorge LF, Arias TD. Mephenytoin hydroxylation in the Cuna Amerindians of Panama. Br J Clin Pharmacol 1985;19:483-7. Jurima M, Inaba T, Kadar D, Kalow W. Genetic polymorphism of mephenytoin p(4')-hydroxylation: difference between Orientals and Caucasians. Br J Clin Pharmacol 1985;19:483-7. Horai Y, Nakano M, Ishizaki T, et al. Metoprolol and mephenytoin oxidation polymorphism in Far Eastern Oriental subjects: Japanese versus mainland Chinese. CLIN PHARMACOL THER 1989;46:198-207. Grant DM, Tang BK, Kalow W. A simple test for acetylator phenotype using caffeine. Br J Clin Pharmacol 1984;17:459-64. Hashiguchi M, Tsutsumi K, Nakashima H, Ebihara A. Assessment of a simple method for acetylator phenotyping with caffeine. Jpn J Clin Pharmacol Ther 1990;21: 425-31. Sunahara S, Urano M, Ogawa M. Genetical and geographic studies on isoniazid inactivation. Science 1961;134:1530-1. Horai Y, Ishizaki T. N-Acetylation polymorphism of dapsone in a Japanese population. Br J Clin Pharmacol 1988;25:487-94.
