Natasa Djordjevic

Induction of CYP1A2 by heavy coffee consumption is associated with the CYP1A2 −163C>A polymorphism

ObjectivesTo investigate the association of CYP1A2 genetic polymorphisms with the inducing effect of heavy coffee consumption on CYP1A2 activity in Serbian and Swedish populations, and to determine the frequency of the CYP1A2 genetic polymorphisms in Serbs.MethodsUsing PCR-RFLP and the tag-array minisequencing method, 126 Serbian healthy volunteers were genotyped for −3860G>A, −2467delT, −739T>G, −729C>T, −163C>A, 2159G>A, and 4795G>A. For 64 nonsmoking participants, the data on CYP1A2 activity (plasma paraxanthine/caffeine ratio) and coffee consumption habit were available from our previous study. The data on CYP1A2 genotype, enzyme activity, and coffee consumption from 114 Swedish healthy nonsmoking subjects were included in the analyses.ResultsIn Serbs, CYP1A2 polymorphisms −3860G>A, −2467delT, −739T>G, −729C>T, −163C>A, and 2159G>A were found at the frequencies of 0.4, 5.0, 3.4, 0.7, 61.1, and 56.0%, respectively, while 4795G>A was not detected. Significant association of heavy coffee consumption with high CYP1A2 enzyme activity was observed only in carriers of −163 A/A. Increasing effect of −163C>A on CYP1A2 inducibility was found in both Serbian (P = 0.022) and Swedish (P = 0.016) nonsmoking heavy coffee consumers. There was no significant difference in CYP1A2 enzyme activity among genotypes in non–heavy coffee consumers. The results indicate that 22 and 14% of the phenotypic variability among Serbian and Swedish heavy coffee consumers, respectively, might be explained by −163C>A polymorphism.ConclusionsCYP1A2 polymorphism −163C>A has an important increasing effect on CYP1A2 inducibility by heavy coffee consumption and may possibly be a contributing factor for interindividual variations in CYP1A2 enzyme activity.

N‐acetyltransferase‐2 (NAT2) Gene Polymorphisms and Enzyme Activity in Serbs: Unprecedented High Prevalence of Rapid Acetylators in a White Population

The aim of this study was to investigate N‐acetyltransferase 2 (NAT2) genetic polymorphism and enzyme activity in Serbs, and to examine the influence of NAT2 genotype, sex, and smoking on the phenotype. Genotyping for 190C>T, 282C>T, 341T>C, 403C>G, 411T>A, 481C>T, 590G>A, 803A>G, and 857G>A in the NAT2 gene, was performed in 140 healthy Serbs. NAT2 activity was determined as AFMU/(AFMU + 1X + 1U) urinary ratio in 100 subjects using caffeine as a probe. The most frequent NAT2 haplotypes were NAT2*5B (38.2%), NAT2*6A (26.0%), and NAT2*4 (24.4%). The log‐transformed NAT2 activity indices exhibited trimodal distribution with 9%, 36%, and 55% of slow, intermediate, and rapid acetylators, respectively. Significant NAT2 genotype‐phenotype correlation was observed (P < .0001). The frequency of NAT1*10 and NAT1*11 were 27.5% and 6.9%, respectively. There was no significant influence of sex or cigarette smoking on NAT2 enzyme activity. Eight subjects displayed rapid NAT2 acetylators phenotype despite being homozygous for NAT2 slow alleles, and NAT1 fast acetylators genotype (NAT1*10 and NAT1*11) had no implication. In contrast to other white populations described hitherto, rapid acetylator is the predominant NAT2 phenotype in Serbs. NAT2 genotype, but not sex and cigarette smoking, influence enzyme activity. NAT1 fast acetylators genotypes do not contribute for NAT2 genotype‐phenotype discordance.

High CYP2A6 Enzyme Activity as Measured by a Caffeine Test and Unique Distribution of CYP2A6 Variant Alleles in Ethiopian Population

CYP2A6 metabolizes clinically relevant drugs, including antiretroviral and antimalarial drugs of major public health importance for the African populations. CYP2A6 genotype-phenotype relationship in African populations, and implications of geographic differences on enzyme activity, remain to be investigated. We evaluated the influence of CYP2A6 genotype, geographical differences, gender, and cigarette smoking on enzyme activity, using caffeine as a probe in 100 healthy unrelated Ethiopians living in Ethiopia, and 72 living in Sweden. CYP2A6 phenotype was estimated by urinary 1,7-dimethyluric acid (17U)/1,7-dimethylxanthine or paraxanthine (17X) ratio. The frequencies of CYP2A6*1B, *1D, *2, *4, *9, and *1x2 in Ethiopians were 31.3, 29.4, 0.6, 0.6, 2.8, and 0.3%, respectively. The overall mean±SD for log 17U/17X was 0.12±0.24 and coefficient of variation 199%. No significant difference in the mean log 17U/17X ratio between Ethiopians living in Sweden versus Ethiopia was observed. Analysis of variance revealed CYP2A6 genotype (p=0.04, F=2.01) but not geographical differences, sex, or cigarette smoking as predictors of CYP2A6 activity. Importantly, the median (interquartile range) of 17U/17X ratio in Ethiopians 1.35 (0.99 to 1.84) was 3- and 11-fold higher than the previously reported value in Swedes 0.52 (0.27 to 1.00) and Koreans 0.13 (0.0 to 0.35), respectively (Djordjevic et al., 2013). Taken together, we report here the relevance of CYP2A6 genotype for enzyme activity in this Ethiopian sample, as well as high CYP2A6 activity and unique distribution of the CYP2A6 variant alleles in Ethiopians as compared other populations described hitherto. Because Omics biomarker research is rapidly accelerating in Africa, CYP2A6 pharmacogenetics and clinical pharmacology observations reported herein for the Ethiopian populations have clinical and biological importance to plan for future rational therapeutics efforts in the African continent as well as therapeutics as a global science.

Comparison of N‐Acetyltransferase‐2 Enzyme Genotype‐Phenotype and Xanthine Oxidase Enzyme Activity Between Swedes and Koreans

The aim of this study was to compare xanthine oxidase (XO) and N‐acetyltransferase‐2 (NAT2) genotype and phenotype between Swedes (n = 113) and Koreans (n = 150), as well as to investigate the effect of sex, smoking, age, and oral contraceptive (OC) use on enzyme activities, using caffeine as a probe. XO and NAT2 activities were estimated by 1U/(1U+1X) and AFMU/(AFMU+1X+1U) urinary ratios, respectively. Participants were genotyped for 191G>A, 341T>C, 590G>A, and 857G>A NAT2 polymorphisms. There was no significant difference in XO activity between Swedes and Koreans. In Swedes, higher XO activity was observed in women (P < .003). There were significant differences in NAT2 genotype and phenotype between Swedes and Koreans. Koreans display significantly higher frequency of NAT2 fast acetylator genotype (89%), whereas the slow acetylator genotype is predominant (62%) in Swedes (P < .0001). Significantly higher NAT2 activity was observed in Koreans compared to Swedes (P < .0001). Having the same NAT2 fast acetylator genotype, Koreans display higher enzyme activity than Swedes (P < .004). OC use significantly increased NAT2 activity in Swedish women. In conclusion, Koreans display higher NAT2 activity than Swedes regardless of NAT2 genotype. Ethnicity, OC use, and genotype determine NAT2 activity, whereas sex is the only determinant of XO activity.

Caffeine consumption patterns and determinants among adolescents in Serbia.

Abstract Background: The number of adolescents who use caffeine is constantly increasing. As juvenile age is vulnerable, it is reasonable to expect that they will differently perceive reason and react to caffeine use than adults, and be more prone to unwanted physiological and psychological consequences of its consumption. Aim: Analysis of the scope and pattern of caffeine consumption among adolescents in Serbia. Study design: The cross-sectional survey was implemented in the study population of 191 Serbian adolescents during 2010. Results: The median daily intake of caffeine was 95.6 mg. The major source of caffeine was brewed coffee, and the most common reasons for caffeine intake were leisure, peer influence, or habit. Only 57.6% of the subjects were aware that caffeine is present in consumed beverages. Sex affected the pattern, but not the overall level, of caffeine consumption. No association between caffeine consumption and smoking status, frequency of caffeine use in the family, or negative personal experience with caffeine effects was observed. Conclusion: Our investigation provides first and rather detailed insight into caffeine-containing beverage consumption scope and pattern among Serbian adolescents. For accurate estimation and analysis of caffeine intake in this population, randomized studies with prospective longitudinal design, caffeine content measurement, and more subjects involved are warranted.

CYP3A5 Polymorphism In Serbian Paediatric Epileptic Patients On Carbamazepine Treatment

Abstract Carbamazepine exhibits significant inter-individual variability in its efficacy and safety, which leads to unpredictable therapy outcomes for the majority of patients. Although its complex biotransformation depends on CYP3A5 activity, evidence of association between carbamazepine treatment outcomes and CYP3A5 functional variations remains inconclusive. The aim of the present study was to investigate the distribution of two of the functionally important CYP3A5 variants *2 and *3 as well as their effects on carbamazepine dose requirements, plasma concentrations and clearance in a Serbian population. The study involved 40 paediatric epileptic patients on steady-state carbamazepine treatment. Genotyping was conducted using the PCR-RFLP method, and carbamazepine plasma concentrations were determined using the HPLC method. CYP3A5*2 and *3 polymorphisms were found at frequencies of 0.0% and 97.5%, respectively, which corresponds well to previously published data for Caucasians. No differences in CYP3A5*3 allele frequencies were detected among epileptic patients in comparison to healthy volunteers within similar ethnic populations (p>0.08), indicating that CYP3A5 polymorphism does not represent a risk factor for epilepsy development. There was an observed tendency towards lower dosage requirements (mean±SD: 15.06±4.45 mg/kg vs. 18.74±5.55 mg/kg; p=0.26), higher plasma concentrations (mean±SD: 0.45±0.13 mg/kg vs. 0.38±0.03 mg/kg; p=0.47) and lower clearance (mean±SD: 0.14±0.05 mg/kg vs. 0.15±0.01 mg/kg; p=0.79) of carbamazepine in homozygous carriers of CYP3A5*3/*3 compared to heterozygous CYP3A5*1A/*3 Serbians. Because these genotype groups did not differ significantly in terms of their carbamazepine pharmacokinetics parameters, the proposed effects of CYP3A5*3 on carbamazepine metabolism could not be confirmed.

CFTR IVS8 Poly-T Variation Affects Severity of Acute Pancreatitis in Women

BackgroundCystic fibrosis transmembrane conductance regulator (CFTR) is important for normal pancreatic function. Its coding gene is polymorphic, and the variations have been associated with the increased risk for acute pancreatitis. However, their impact on the disease severity is still unknown. Therefore, the aim of our study was to determine the functional importance of common cystic fibrosis transmembrane conductance regulator variations IVS8-poly T, R117H, and M470V for the severity of acute pancreatitis.MethodThe study involved 98 acute pancreatitis patients. The severity of the disease was determined based on the Atlanta Classification system. IVS8-poly T, R117H, and M470V genotyping was performed using PCR-RFLP method.ResultsIVS8-5T, IVS8-7T, IVS8-9T, and M470V alleles were found at the frequencies of 5.7, 75.5, 18.9, and 55.7%, respectively, while R117H was not observed. Among women, the severe form of the disease was more frequent in carriers of at least one IVS8 9T allele (RR for 9T/9T + 9T/non-9T vs. non-9T/non-9T: 2.115; 95% CI: 1.241–3.605). This association was not detected in men and was not affected by M470V. In addition, co-morbidities increased the severity of acute pancreatitis (p = 0.022).ConclusionOur study reveals that IVS8 poly-T variation affects severity of acute pancreatitis in women and that existent co-morbidities worsen the clinical course of the disease.

Clinical and laboratory parameters associated with death in acute pancreatitis

Introduction. Acute pancreatitis is an inflammatory condition having the significant mortality rate in the case of severe forms of disease. The aim of this study was to investigate putative factors of increased mortality with contradictory prior evidence, and to reveal factors that were insufficiently explored previously. Methods. A prospective cohort study with nested case/control design included all adult patients treated for acute pancreatitis in Clinical Center of Kragujevac, Serbia, during the 3-year period (from October 2011 to December 2014). The cases (n=19) were patients who died, while the controls (n=113) were patients who survived. The associations between putative risk factors and the study outcomes were tested by univariate and multivariate logistic regressions, and expressed as crude and adjusted odds ratios (OR) with corresponding 95% confidence intervals (CI). Results. Significant association with the lethal outcome in acute pancreatitis was found for advanced age (adjusted OR 1.12, 95%CI 1.02-1.23), presence of significant comorbidities (adjusted OR 10.62, 95%CI 1.01-111.39), higher interleukin-8 (IL-8) value on third day from onset of symptoms (adjusted OR 1.05, 95%CI 1.02-1.08), use of tramadol and/or morphine (adjusted OR 47.34, 95%CI 3.21-699.08), BISAP score ≥ 3 in the first 24 hours (adjusted OR 48.11, 95%CI 3.14-736.29), and prophylactic use of antibiotics (adjusted OR 0.07, 95%CI 0.01-0.85). Conclusion. Advanced age, significant comorbidities, use of tramadol and/or morphine and more severe disease as assessed by BISAP score can increase the risk for death in acute pancreatitis, while prophylactic use of antibiotics may have a protective role. [Projekat Ministarstva nauke Republike Srbije, br. 175007]

Lack Of PRSS1 And SPINK1 Polymorphisms In Serbian Acute Pancreatitis Patients

Abstract Acute pancreatitis represents an acute nonbacterial inflammation of the pancreas caused by a premature and ectopic activation of pancreatic digestive enzymes. Two of the most important genes in pancreatic autodigestion, PRSS1 and SPINK1, were implicated in the earliest discoveries of the genetic background of pancreatitis. However, the distribution of their variations displays interethnic variability, which could significantly affect the magnitude of their proposed effects on this disease worldwide. The aim of the present study was to investigate the distribution of the most important functional variations of PRSS1 (86A>T and 365G>A) and SPINK1 (101A>G), and their influence on the clinical course of acute pancreatitis in Serbian patients. The study enrolled 81 subjects, the severity of disease course was determined using the Atlanta Classification system, and the genotyping was conducted using a PCR-RFLP method. PRSS1 86A>T and 365G>A SNPs were not observed in the study population, while SPINK1 101A>G was present with the frequency of 0.62% (95% CI: 0.00, 3.83%). Due to extremely low frequencies or absences of examined variations, the proposed effect of these SNPs on the severity of acute pancreatitis could not be confirmed. The results do not support routine genotyping of either PRSS1 or SPINK1 in Serbs.