Eur J Clin Pharmacol (2015) 71:687–690 DOI 10.1007/s00228-015-1852-9
SPECIAL ARTICLE
Distribution of xanthine oxidase activity in a Nigerian population Ayorinde Adehin 1 & Oluseye Oladotun Bolaji 1
Received: 8 February 2015 / Accepted: 24 April 2015 / Published online: 5 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Background Xanthine oxidase (XO) is one of the two interconvertible forms of xanthine oxidoreductase and wellstudied for its role in purine catabolism and that of other purine analogues, drugs especially. Our study investigated the incidence of polymorphism in phenotypes along with the influence of gender and age on enzyme activity in a Nigerian population. Methods Caffeine (110 mg) was administered to each of 129 healthy, unrelated subjects who were nonsmokers. Urine voided within 7 h after dosing was collected for a highperformance liquid chromatographic analysis of metabolites, and the urinary molar ratio of metabolites was used as marker for enzyme activity. Statistical analysis of data was carried out to identify the prevalent phenotypes and also assessed the influence of age and sex on enzyme activity. Result A sevenfold variation in XO activity with a population mean (±SD) molar ratio of 0.43±0.15 and median (interquartile range) of 0.42 (0.16) was observed. Distinctly higher enzyme activity was also recorded in 8 % of the study population, and there was no correlation (P>0.05) between enzyme activity and the studied covariates. Conclusions Our study confirmed the existence of polymorphism in xanthine oxidase activity in Nigerians and also the incidence of individuals with distinctly higher XO activity in the population.
* Ayorinde Adehin [email protected]; [email protected] 1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria
Keywords Xanthine oxidase . Phenotypes . Nigerian population
Xanthine oxidase (XO) is one of the two interconvertible forms of xanthine oxidoreductase (XOR) widely distributed throughout various organs including the liver, gut, lung, kidney, heart, brain, and plasma [1]. Its role in purine catabolism and that of other purine analogues has been extensively studied, and well documented schemes are those describing the formation of uric acid from xanthine and hypoxanthine. In addition, certain other physiological functions such as the in vivo inhibition of bacterial growth as well as some pathophysiological properties have been attributed to XO [2]. A few drugs metabolized by XO are acyclovir, mercaptopurine, azathioprine, methotrexate, doxorubicin, daunomycin, and mitomycin C [3]. It has also been identified as a significant contributor to the level of superoxides in human blood vessels, and elevated XO activity has been reported in patients with chronic heart failure [4]. Considering its roles in biological systems, polymorphisms in XO activity as a consequence carry a potential for lack of therapeutic benefit, adverse drug reactions, toxicity due to elevated levels of harmful superoxides, and impaired metabolism of relevant endogenous substrates which has been associated with some disease states. As a result, XO has over time become the target of certain pharmaceuticals such as the case is with the treatment of hyperuricemia and gout [1]. The study of XO phenotypes in some populations have reported marked interindividual and interethnic differences in activity just as gender-related differences in the level of hepatic XO activity in humans have also been observed [5]. Thus, the aim of the present study was to investigate the variability of XO activity in the Nigerian population and as
688
Eur J Clin Pharmacol (2015) 71:687–690
well report on the influence of gender and age on activity in the same population. The study was approved by the ethics committee of the Obafemi Awolowo University Teaching Hospital, Ile-Ife (Nigeria). One hundred and twenty-nine healthy, unrelated subjects who were nonsmokers consisting of 85 males and 44 females with a median age (range) of 22 years (20) were recruited for the study after written consents had been provided. The subjects were required to stay off caffeinated items for 3 days prior to the commencement of the study, and spot urine samples were collected to verify adherence. Coffee (equivalent of 110 mg caffeine) was administered and urine collected for 7 h post administration. Extraction and analysis of metabolites [1methylxanthine (1X) and 1-methyluric acid (1U)] in urine was carried out as earlier described by Djordjevic et al. [3] with some modifications. Briefly, caffeine, IX, IU, and the internal standard (phenacetin) were extracted with dichloromethane/isopropanol (90:10 v/v) and analyzed using an Agilent series 1100 high-performance liquid chromatographic system (Agilent technologies, Palm Alto, USA). Sample injection was through a Rheodyne 7725i injector valve (Cotati, CA, USA) fitted with a 20 μl loop, and separation of analytes was effected on a C8 Zorbax Eclipse XDB column (4 μm, 150×4.6 mm i.d.; Agilent technologies, Palm Alto, USA) at 26 °C. The eluent consisted of water, methanol, and acetonitrile in varying proportions in a step gradient mode as described in Table 1. The effluent was monitored at 274 nm, and the chromatograms were recorded with a HP ChemStation software. Standard calibration curves for the analytes were constructed at concentrations between 10 and 240 μM. The urinary molar ratio of metabolites, 1U/(1X+1U), was used as marker for enzyme activity. Statistical analysis of the population data which included a Rosin-Rammler-SperlingWeibull distribution (RRSW) analysis [6], KolmogorovSmirnov test for normality, Spearman rank-order correlation test, and a chi-square test for association was carried out using Minitab 17 software (LEAD Technologies, Inc.). A P value less than 0.05 was considered as statistically significant.
Table 1 HPLC flow program for the separation of caffeine and its metabolite
The metabolites of interest and the internal standard were well separated without interference from caffeine and endogenous materials in the urine. Plotted calibration curves for the metabolites were linear in range of 10 to 240 μM with a limit of detection of 6.23 μM for 1U and 3.73 μM for 1X, while the back calculated concentrations of the calibration standards were less than ± 7 % for both 1U and 1X, respectively. Assay recovery at 10, 40, and 150 μM was 63–73 % for 1U and 65–75 % for 1X. Coefficient of variation for both metabolites was less than 7 and 14 % for the intra- and inter-day precision studies, respectively. A sevenfold variation in enzyme activity with a mean (±SD) urinary molar ratio of 0.43±0.15 and median (interquartile range) of 0.42 (0.16) was observed in the population. Incidence of phenotypes in the population via RRSW analysis (Fig. 1a), however, returned a breakpoint at 0.69 suggesting the presence of 92 % extensive metabolizers and a unique 8 % with markedly higher XO activity compared with others in the population. A frequency distribution histogram (Fig. 1b) also further displays this stratification of phenotypes in the study population. Normality of distribution (P>0.150) was observed, and association between age and XO activity was not significant (P=0.754). Similarly, no correlation was observed between gender and XO activity (P=1.000). Several populations have been investigated for variability in XO activity using caffeine as a probe and either 1U/1X or 1U/(1U+1X) urinary molar ratio as marker for activity. Unimodally distributed phenotypes have been reported in Danish, Greeks, and Serbs [3], while some other studies have reported bimodal distribution of activity that suggest the presence of poor metabolizers ranging from 4 % in Spaniards [7] to about 11 % in Ethiopians [8]. Table 2 gives a summary data of XO phenotypes in some studied populations. The present study in Nigerians, however, reports an interesting distribution of phenotypes in the population. This observed distribution in the population suggests the presence of a distinct metabolizer phenotype constituting 8 % of the study population. We find this a noteworthy observation as the mean urinary molar ratio for 92 % of the population was comparable with some reported
Time (min)
Water (%)
Methanol (%)
Acetonitrile (%)
Flow rate (ml/min)
0.00 2.20 4.00 4.50 6.00 8.20 11.00
85.0 80.0 80.0 70.0 70.0 85.0 85.0
15.0 20.0 20.0 30.0 0.0 15.0 15.0
0.0 0.0 0.0 0.0 30.0 0.0 0.0
0.600 0.900 1.300 1.300 1.300 1.000 0.600
Eur J Clin Pharmacol (2015) 71:687–690
689
Fig. 1 Plots of XO activity data. a A Weibull plot of the observed phenotypes for xanthine oxidase activity in 129 healthy Nigerians; b A frequency distribution histogram of the population data showing the breakpoint at 0.69
(a) 9 8
0.69
Frequency
7 6 5 4 3 2 1 0 0.2
0.4
0.6
0.8
1.0
1U/(1U+1X)
(b)
extensive metabolizers’ values [3, 9] in literature with similar activity marker scale. Reports on functional variations in the XO gene is rare, and some non-genetic factors (gender, diet and cigarette smoking), though with conflicting reports, have been considered as possible determinants of activity [3, 5, 7]. Our findings rule out the influence of gender and smoking in addition to the null influence of age on activity. The Nigerian
population, however, has not been previously studied for the prevalent genotypes of XO, and we therefore suspect that likely genetic contributors (within or maybe outside the XO gene) yet to be determined in the population or dietary components may be responsible for the observed higher level of XO activity. Moreover, a previous genetic study by Kudo et al. [5] had established the identity of some alleles (Ile703Val and
690
Eur J Clin Pharmacol (2015) 71:687–690
Table 2 Summary data of xanthine oxidase activity in some populations
Conflict of interest The authors declare that they have no competing interests and source of funding.
Xanthine oxidase phenotypes (%) Population Poor metabolizers Extensive metabolizers References
References Nigerians
–
Greeks Serbs Danish Spaniards Ethiopians Japanese Swedes Koreans
Unimodal Unimodal Unimodal 4 11 11 6.4 9.5
a
92a
Present study
96 91 91 93.6 90.5
[3] [3] [3] [7] [8] [9] [10] [10]
1.
2.
3.
4.
Remaining 8 % showed distinctly higher activity
His1221Arg) of the XO gene responsible for about twofold higher XO activity than was observed in the wild type. XO has been reported to show higher affinity for thiopurines (especially 6-mercaptopurine) than they do for xanthines and play a significant role in their first pass metabolism [5]. Hence, the administration of standard doses of such drugs in the Nigerian population may be relatively devoid of adverse drug reactions as majority will be extensive metabolizers. However, the identified group of metabolizers with higher XO activity, though few, may require dose adjustment for optimum therapeutic benefit. This group of metabolizers may also be prone to generating elevated levels of harmful superoxides that have previously been correlated with chronic heart failure and adult respiratory distress syndrome (ARDS) [4, 11]. In conclusion, this study, the first in Nigerians, reports the existence of a high-activity XO phenotype in the population. Also, age and sex had no observed influence on enzyme activity in the population.
5.
6.
7.
8.
9.
10.
11.
Pacher P, Nivorozhkin A, Szabo C (2006) Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol Rev 58(1):87–114 Berry CE, Hare JM (2004) Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol 555(Pt 3):589–606 Djordjevic N, Carrillo A, Gervasini G, Jankovic S, Aklillu E (2010) In vivo evaluation of CYP2A6 and xanthine oxidase enzyme activities in the Serbian population. Eur J Clin Pharmacol 66:571–578 Landmesser U, Spiekermann S, Dikalov S et al (2002) Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: role of xanthine-oxidase and extracellular superoxide dismutase. Circulation 106(24):3073–3078 Kudo M, Moteki T, Sasaki T, Konno Y, Ujiie S, Onose A, Mizugaki M, Ishikawa M, Hiratsuka M (2008) Functional characterization of human xanthine oxidase allelic variants. Pharmacogenet Genomics 18(3):243–251 Jack DB (1983) Statistical analysis of polymorphic drug metabolism data using the Rosin Rammler Sperling Weibull distribution. Eur J Clin Pharmacol 25:443–448 Carrillo JA, Benitez J (1994) Caffeine metabolism in a healthy Spanish population: N-acetylator phenotype and oxidation pathways. Clin Pharmacol Ther 55:293–304 Aklillu E, Carrillo JA, Makonnen E, Bertilsson L, IngelmanSundberg M (2003) Xanthine oxidase activity is influenced by environmental factors in Ethiopians. Eur J Clin Pharmacol 59: 533–536 Saruwatari J, Nakagawa K, Shindo J, Tajiri T, Fujieda M, Yamazaki H, Kamataki T, Ishizaki T (2002) A population phenotyping study of three drug-metabolizing enzymes in Kyushu, Japan, with use of the caffeine test. Clin Pharmacol Ther 72:200–208 Djordjevic N, Carrillo JA, Roh H, Karlsson S, Ueda N, Bertilsson L, Aklillu E (2012) Comparison of Nacetyltransferase-2 enzyme genotype-phenotype and xanthine oxidase enzyme activity between Swedes and Koreans. J Clin Pharmacol 52:1527–1534 Grum CM, Ragsdale RA, Ketai LH, Simon RH (1987) Plasma xanthine oxidase activity in patients with adult respiratory distress syndrome. J Crit Care 2(1):22–26
SPECIAL ARTICLE
Distribution of xanthine oxidase activity in a Nigerian population Ayorinde Adehin 1 & Oluseye Oladotun Bolaji 1
Received: 8 February 2015 / Accepted: 24 April 2015 / Published online: 5 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Background Xanthine oxidase (XO) is one of the two interconvertible forms of xanthine oxidoreductase and wellstudied for its role in purine catabolism and that of other purine analogues, drugs especially. Our study investigated the incidence of polymorphism in phenotypes along with the influence of gender and age on enzyme activity in a Nigerian population. Methods Caffeine (110 mg) was administered to each of 129 healthy, unrelated subjects who were nonsmokers. Urine voided within 7 h after dosing was collected for a highperformance liquid chromatographic analysis of metabolites, and the urinary molar ratio of metabolites was used as marker for enzyme activity. Statistical analysis of data was carried out to identify the prevalent phenotypes and also assessed the influence of age and sex on enzyme activity. Result A sevenfold variation in XO activity with a population mean (±SD) molar ratio of 0.43±0.15 and median (interquartile range) of 0.42 (0.16) was observed. Distinctly higher enzyme activity was also recorded in 8 % of the study population, and there was no correlation (P>0.05) between enzyme activity and the studied covariates. Conclusions Our study confirmed the existence of polymorphism in xanthine oxidase activity in Nigerians and also the incidence of individuals with distinctly higher XO activity in the population.
* Ayorinde Adehin [email protected]; [email protected] 1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria
Keywords Xanthine oxidase . Phenotypes . Nigerian population
Xanthine oxidase (XO) is one of the two interconvertible forms of xanthine oxidoreductase (XOR) widely distributed throughout various organs including the liver, gut, lung, kidney, heart, brain, and plasma [1]. Its role in purine catabolism and that of other purine analogues has been extensively studied, and well documented schemes are those describing the formation of uric acid from xanthine and hypoxanthine. In addition, certain other physiological functions such as the in vivo inhibition of bacterial growth as well as some pathophysiological properties have been attributed to XO [2]. A few drugs metabolized by XO are acyclovir, mercaptopurine, azathioprine, methotrexate, doxorubicin, daunomycin, and mitomycin C [3]. It has also been identified as a significant contributor to the level of superoxides in human blood vessels, and elevated XO activity has been reported in patients with chronic heart failure [4]. Considering its roles in biological systems, polymorphisms in XO activity as a consequence carry a potential for lack of therapeutic benefit, adverse drug reactions, toxicity due to elevated levels of harmful superoxides, and impaired metabolism of relevant endogenous substrates which has been associated with some disease states. As a result, XO has over time become the target of certain pharmaceuticals such as the case is with the treatment of hyperuricemia and gout [1]. The study of XO phenotypes in some populations have reported marked interindividual and interethnic differences in activity just as gender-related differences in the level of hepatic XO activity in humans have also been observed [5]. Thus, the aim of the present study was to investigate the variability of XO activity in the Nigerian population and as
688
Eur J Clin Pharmacol (2015) 71:687–690
well report on the influence of gender and age on activity in the same population. The study was approved by the ethics committee of the Obafemi Awolowo University Teaching Hospital, Ile-Ife (Nigeria). One hundred and twenty-nine healthy, unrelated subjects who were nonsmokers consisting of 85 males and 44 females with a median age (range) of 22 years (20) were recruited for the study after written consents had been provided. The subjects were required to stay off caffeinated items for 3 days prior to the commencement of the study, and spot urine samples were collected to verify adherence. Coffee (equivalent of 110 mg caffeine) was administered and urine collected for 7 h post administration. Extraction and analysis of metabolites [1methylxanthine (1X) and 1-methyluric acid (1U)] in urine was carried out as earlier described by Djordjevic et al. [3] with some modifications. Briefly, caffeine, IX, IU, and the internal standard (phenacetin) were extracted with dichloromethane/isopropanol (90:10 v/v) and analyzed using an Agilent series 1100 high-performance liquid chromatographic system (Agilent technologies, Palm Alto, USA). Sample injection was through a Rheodyne 7725i injector valve (Cotati, CA, USA) fitted with a 20 μl loop, and separation of analytes was effected on a C8 Zorbax Eclipse XDB column (4 μm, 150×4.6 mm i.d.; Agilent technologies, Palm Alto, USA) at 26 °C. The eluent consisted of water, methanol, and acetonitrile in varying proportions in a step gradient mode as described in Table 1. The effluent was monitored at 274 nm, and the chromatograms were recorded with a HP ChemStation software. Standard calibration curves for the analytes were constructed at concentrations between 10 and 240 μM. The urinary molar ratio of metabolites, 1U/(1X+1U), was used as marker for enzyme activity. Statistical analysis of the population data which included a Rosin-Rammler-SperlingWeibull distribution (RRSW) analysis [6], KolmogorovSmirnov test for normality, Spearman rank-order correlation test, and a chi-square test for association was carried out using Minitab 17 software (LEAD Technologies, Inc.). A P value less than 0.05 was considered as statistically significant.
Table 1 HPLC flow program for the separation of caffeine and its metabolite
The metabolites of interest and the internal standard were well separated without interference from caffeine and endogenous materials in the urine. Plotted calibration curves for the metabolites were linear in range of 10 to 240 μM with a limit of detection of 6.23 μM for 1U and 3.73 μM for 1X, while the back calculated concentrations of the calibration standards were less than ± 7 % for both 1U and 1X, respectively. Assay recovery at 10, 40, and 150 μM was 63–73 % for 1U and 65–75 % for 1X. Coefficient of variation for both metabolites was less than 7 and 14 % for the intra- and inter-day precision studies, respectively. A sevenfold variation in enzyme activity with a mean (±SD) urinary molar ratio of 0.43±0.15 and median (interquartile range) of 0.42 (0.16) was observed in the population. Incidence of phenotypes in the population via RRSW analysis (Fig. 1a), however, returned a breakpoint at 0.69 suggesting the presence of 92 % extensive metabolizers and a unique 8 % with markedly higher XO activity compared with others in the population. A frequency distribution histogram (Fig. 1b) also further displays this stratification of phenotypes in the study population. Normality of distribution (P>0.150) was observed, and association between age and XO activity was not significant (P=0.754). Similarly, no correlation was observed between gender and XO activity (P=1.000). Several populations have been investigated for variability in XO activity using caffeine as a probe and either 1U/1X or 1U/(1U+1X) urinary molar ratio as marker for activity. Unimodally distributed phenotypes have been reported in Danish, Greeks, and Serbs [3], while some other studies have reported bimodal distribution of activity that suggest the presence of poor metabolizers ranging from 4 % in Spaniards [7] to about 11 % in Ethiopians [8]. Table 2 gives a summary data of XO phenotypes in some studied populations. The present study in Nigerians, however, reports an interesting distribution of phenotypes in the population. This observed distribution in the population suggests the presence of a distinct metabolizer phenotype constituting 8 % of the study population. We find this a noteworthy observation as the mean urinary molar ratio for 92 % of the population was comparable with some reported
Time (min)
Water (%)
Methanol (%)
Acetonitrile (%)
Flow rate (ml/min)
0.00 2.20 4.00 4.50 6.00 8.20 11.00
85.0 80.0 80.0 70.0 70.0 85.0 85.0
15.0 20.0 20.0 30.0 0.0 15.0 15.0
0.0 0.0 0.0 0.0 30.0 0.0 0.0
0.600 0.900 1.300 1.300 1.300 1.000 0.600
Eur J Clin Pharmacol (2015) 71:687–690
689
Fig. 1 Plots of XO activity data. a A Weibull plot of the observed phenotypes for xanthine oxidase activity in 129 healthy Nigerians; b A frequency distribution histogram of the population data showing the breakpoint at 0.69
(a) 9 8
0.69
Frequency
7 6 5 4 3 2 1 0 0.2
0.4
0.6
0.8
1.0
1U/(1U+1X)
(b)
extensive metabolizers’ values [3, 9] in literature with similar activity marker scale. Reports on functional variations in the XO gene is rare, and some non-genetic factors (gender, diet and cigarette smoking), though with conflicting reports, have been considered as possible determinants of activity [3, 5, 7]. Our findings rule out the influence of gender and smoking in addition to the null influence of age on activity. The Nigerian
population, however, has not been previously studied for the prevalent genotypes of XO, and we therefore suspect that likely genetic contributors (within or maybe outside the XO gene) yet to be determined in the population or dietary components may be responsible for the observed higher level of XO activity. Moreover, a previous genetic study by Kudo et al. [5] had established the identity of some alleles (Ile703Val and
690
Eur J Clin Pharmacol (2015) 71:687–690
Table 2 Summary data of xanthine oxidase activity in some populations
Conflict of interest The authors declare that they have no competing interests and source of funding.
Xanthine oxidase phenotypes (%) Population Poor metabolizers Extensive metabolizers References
References Nigerians
–
Greeks Serbs Danish Spaniards Ethiopians Japanese Swedes Koreans
Unimodal Unimodal Unimodal 4 11 11 6.4 9.5
a
92a
Present study
96 91 91 93.6 90.5
[3] [3] [3] [7] [8] [9] [10] [10]
1.
2.
3.
4.
Remaining 8 % showed distinctly higher activity
His1221Arg) of the XO gene responsible for about twofold higher XO activity than was observed in the wild type. XO has been reported to show higher affinity for thiopurines (especially 6-mercaptopurine) than they do for xanthines and play a significant role in their first pass metabolism [5]. Hence, the administration of standard doses of such drugs in the Nigerian population may be relatively devoid of adverse drug reactions as majority will be extensive metabolizers. However, the identified group of metabolizers with higher XO activity, though few, may require dose adjustment for optimum therapeutic benefit. This group of metabolizers may also be prone to generating elevated levels of harmful superoxides that have previously been correlated with chronic heart failure and adult respiratory distress syndrome (ARDS) [4, 11]. In conclusion, this study, the first in Nigerians, reports the existence of a high-activity XO phenotype in the population. Also, age and sex had no observed influence on enzyme activity in the population.
5.
6.
7.
8.
9.
10.
11.
Pacher P, Nivorozhkin A, Szabo C (2006) Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol Rev 58(1):87–114 Berry CE, Hare JM (2004) Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol 555(Pt 3):589–606 Djordjevic N, Carrillo A, Gervasini G, Jankovic S, Aklillu E (2010) In vivo evaluation of CYP2A6 and xanthine oxidase enzyme activities in the Serbian population. Eur J Clin Pharmacol 66:571–578 Landmesser U, Spiekermann S, Dikalov S et al (2002) Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: role of xanthine-oxidase and extracellular superoxide dismutase. Circulation 106(24):3073–3078 Kudo M, Moteki T, Sasaki T, Konno Y, Ujiie S, Onose A, Mizugaki M, Ishikawa M, Hiratsuka M (2008) Functional characterization of human xanthine oxidase allelic variants. Pharmacogenet Genomics 18(3):243–251 Jack DB (1983) Statistical analysis of polymorphic drug metabolism data using the Rosin Rammler Sperling Weibull distribution. Eur J Clin Pharmacol 25:443–448 Carrillo JA, Benitez J (1994) Caffeine metabolism in a healthy Spanish population: N-acetylator phenotype and oxidation pathways. Clin Pharmacol Ther 55:293–304 Aklillu E, Carrillo JA, Makonnen E, Bertilsson L, IngelmanSundberg M (2003) Xanthine oxidase activity is influenced by environmental factors in Ethiopians. Eur J Clin Pharmacol 59: 533–536 Saruwatari J, Nakagawa K, Shindo J, Tajiri T, Fujieda M, Yamazaki H, Kamataki T, Ishizaki T (2002) A population phenotyping study of three drug-metabolizing enzymes in Kyushu, Japan, with use of the caffeine test. Clin Pharmacol Ther 72:200–208 Djordjevic N, Carrillo JA, Roh H, Karlsson S, Ueda N, Bertilsson L, Aklillu E (2012) Comparison of Nacetyltransferase-2 enzyme genotype-phenotype and xanthine oxidase enzyme activity between Swedes and Koreans. J Clin Pharmacol 52:1527–1534 Grum CM, Ragsdale RA, Ketai LH, Simon RH (1987) Plasma xanthine oxidase activity in patients with adult respiratory distress syndrome. J Crit Care 2(1):22–26
