|Year : 2020 | Volume
| Issue : 1 | Page : 18-23
Plasma chlorzoxazone as a probe for cytochrome 2E1 activity among Hausa/Fulani in northwest Nigeria: Determination of acetaminophen metabolic phenotypes
Muhammad Tukur Umar1, Shaibu O Bello1, Aminu Chika1, Yakubu Abdulmumini2
1 Department of Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, College of Health Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria
2 Department of Medicine, Faculty of Clinical Sciences, College of Health Sciences, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria
|Date of Submission||09-Dec-2019|
|Date of Decision||08-Jan-2020|
|Date of Acceptance||20-Jan-2020|
|Date of Web Publication||23-Oct-2020|
Dr. Muhammad Tukur Umar
Department of Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, College of Health Sciences, Usmanu Danfodiyo University, PMB, Sokoto.
Source of Support: None, Conflict of Interest: None
Background: Acetaminophen is the most common drug consumed globally and toxicity of minute percentage translates to millions of people being affected. Even at recommended dose, acetaminophen can cause liver injury due to cytochrome 2E1 gene polymorphism. Interethnic variability in drug response is a well-recognized phenomenon and Hausa/Fulani provides suitable justification as the most populous ethnic group in Nigeria. Aim: The aim of this study was to determine metabolic phenotype among Hausa/Fulani of northwest Nigeria using plasma chlorzoxazone as a probe for cytochrome 2E1. Materials and Methods: This was an exploratory study involving 20 participants of both sexes selected by criterion sampling. A chlorzoxazone tablet was administered after an overnight fast with distilled water. Three hours post-dose, blood sample was collected for the assay of the parent drug and its metabolite 6-hydroxychlorzoxazone in plasma using reversed phase high-performance liquid chromatography. Metabolic ratio of chlorzoxazone and 6-hydroxychlorzoxazone was determined, probit plot was constructed, trend line added, and resulting polynomial equation was resolved to obtain the anti-mode. Values greater or equal to the anti-mode were considered poor metabolizers, whereas values less than the anti-mode were considered as extensive metabolizers. Statistical Test Used: Frequency histograms and scatter charts using Statistical Package for Social Sciences (IBM* version 25, Armonk, NY, IBM Corp. 2017) were used to analyze the data and results were expressed as proportions with 95% confidence interval. Results: The log metabolic ratio ranged from –0.87 to 2.8 and the value of anti-mode was found to be –1.2. All the participants were found to be in poor metabolizer’s classification. Conclusion: Hausa/Fulani of northwest Nigeria are less susceptible to the toxic effects of N-acetyl-p-benzoquinone imine, a hepatotoxic metabolite of cytochrome 2E1 metabolism of acetaminophen.
Keywords: Acetaminophen, chlorzoxazone, Hausa/Fulani, phenotype
|How to cite this article:|
Umar MT, Bello SO, Chika A, Abdulmumini Y. Plasma chlorzoxazone as a probe for cytochrome 2E1 activity among Hausa/Fulani in northwest Nigeria: Determination of acetaminophen metabolic phenotypes. J Health Res Rev 2020;7:18-23
|How to cite this URL:|
Umar MT, Bello SO, Chika A, Abdulmumini Y. Plasma chlorzoxazone as a probe for cytochrome 2E1 activity among Hausa/Fulani in northwest Nigeria: Determination of acetaminophen metabolic phenotypes. J Health Res Rev [serial online] 2020 [cited 2023 Dec 1];7:18-23. Available from: https://www.jhrr.org/text.asp?2020/7/1/18/298878
| Introduction|| |
In the last few years, it has become clear that acetaminophen is the most common cause of drug-related liver damage, even when taken at recommended doses. It accounts for 51% of acute liver failure. Acetaminophen use during the first year of life is strongly associated with the development of asthma, rhinoconjunctivitis, and eczema later in life. In another vein, it is implicated in adverse drug reactions such as Stevens–Johnson syndrome and toxic epidermal necrolysis. Acetaminophen is reported as the most commonly used antipyretic and analgesic globally and is consumed by millions of people worldwide.
The biotransformation of acetaminophen occurs in the liver, although kidneys and intestines play some appreciable roles. Two main enzymes, Uridyldiphospho-glucuronosyltransferases and sulfotransferases, respectively, are involved in the inactivation of the drug to its metabolites, Acetyl p-amino-phenol-gluc and APAP-sulfate (acetyl p-aminophenol-gluc and acetyl p-aminophenol sulfate, which constitute 52%–57% and 30%–44% of urinary metabolites, respectively). Through these pathways, approximately 90%–95% of the drug is metabolized and only 5%–10% acetaminophen is metabolically activated by cytochrome P450 2E1 to form a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPBQI), which is the main culprit of hepatotoxicity., This metabolite has a wide clinical implication as is associated with the pathogenesis of urinary, nasopharyngeal, colonic, and gastric malignancies.
Chlorzoxazone is recognized as a probe for cytochrome P450 2E1. Its hydroxylation to its metabolite, 6-hydroxychlorzoxazone, is a recognized measure of the in vivo cytochrome P450 2E1 activity. Apart from its known acceptable safety profile, it has the advantage of taking only one blood or urine sample after 2–4 h.,,
The vulnerability of an individual to drug toxicity, to a large extent, depends on the single-nucleotide polymorphisms (SNPs) of genes that encode proteins concerned in absorption, distribution, metabolism, and excretion of drugs., The existence of these SNPs in drug metabolizing enzymes most specifically cytochromes P450 results in a variation of these enzyme activities classified phenotypically as poor metabolizers, intermediate metabolizers, extensive metabolizers, and ultrarapid metabolizers.,
These variations occur in different ethnic populations and give rise to adverse drug reactions, which are a major cause of death globally, ranked the fifth leading cause of death in the USA, and responsible for 7% of hospital admissions. Because phenotype reflects the real individual enzyme activity, it is assumed as a critical tool in predicting treatment failure or toxicity of clinically used drugs.
In Nigeria, there is a paucity of data on acetaminophen toxicity. My Pikin (my child), and also syrup after tainted acetaminophen used in the pediatrics age group for teething problems that claimed 39 lives in 2008 readily comes to mind. This study therefore aimed at determining metabolic phenotypes among the Hausa/Fulani ethnic group in Northwest Nigeria and to the best of our knowledge is the first attempt to determine these phenotypes in the study area.
| Materials and Methods|| |
A presented study was performed at laboratories of Departments of Pharmacology and Therapeutics and Chemical Pathology, College of Health Sciences, Usmanu Danfodiyo University, Sokoto. The study protocol, investigators, study site, informed consent form, and recruiting materials were reviewed and approved by the Ethics Committee of Sokoto State Ministry of Health Nigeria (SMH/1580/V.IV).
A total of 20 participants (10 men and 10 women) were enrolled in the study. The study was exploratory and participants fasted overnight for 11 h, and at 8 AM on the day of the sample taking tablet 250 mg chlorzoxazone was administered orally with 100mL of distilled water. Three hours after dosing, 5mL of whole blood was collected via cubital venipuncture in a heparinized vacuum tube. Thereafter participants were observed for 8 h post-dose for any untoward effects and were discharged uneventfully.
Acetonitrile, 6-hydroxychlorzoxazone (100%), chlorzoxazone (98% pure), acetofenitidin, and β-glucuronidase from Escherichia coli Type VII-A, tetrahydrofuran, and methanol were all high-performance liquid chromatography (HPLC) grade purchased from Sigma-Aldrich (St. Louis, MO).
Population, sampling and exclusion criteria
Only volunteers who expressed written consents, declared Hausa/Fulani descent, and confirmed by one of the investigators (Muhammad Tukur Umar) were included in the study. They were made up of people from suburb of Sokoto in Sokoto state, northwest Nigeria and were selected by the criterion sampling. Participants who consumed alcohol, smoke tobacco, or with suspicion of hypersensitivity to chlorzoxazone (2E1 probe) were excluded from the study. Pregnant women, breastfeeding mothers, and those younger than 18 years were also excluded from the study. They were asked to refrain from any prescription or herbal medicines within 2 weeks before the study.
Blood sample preparation
The blood samples were centrifuged at 3000 r/min for 10 min and plasma was harvested into a plain vacuum tube and stored at –4° before chromatography. Assay of chlorzoxazone and 6-hydroxychlorzoxazone in plasma was carried out using Stiff et al.’s method. The stock, standard solutions, and preparation of individual concentrations for calibration curve are shown in [Table 1].
The calibration curves were generated using drug-free plasma spiked with known concentrations of chlorzoxazone and remained constantly linear (R2 > 0.98) over the concentration range. To 1mL of the plasma, 50 µL of 1 mg/mL of chlorzoxazone was added along with 100 µL each of phenacetin (internal standard) and 6-hydroxychlorzoxazone. Acetonitrile 1mL was also liquated. The same was repeated for solutions v, iv, iii, ii, and i at 50 µL, 100 µL, 150 µL, 200 µL, and 250 µL of chlorzoxazone, respectively.
Drug concentrations determination
HPLC equipped with a Ultraviolet detector was developed for simultaneous estimation of chlorzoxazone and its metabolite 6-hydroxychlorzoxazone in plasma sample (diluted 1:500) treated with β-glucuronidase followed by solid-phase extraction. Simple protein precipitation technique using zinc sulfate (35%) was used in extraction from plasma and acetofenitidin was used as internal standard. Separation of components was achieved with reverse phase HPLC column 18 (10 µm, 3.9 mm × 300 mm) with gradient mobile phase A comprising potassium phosphate buffer 0.01 M, pH 3.0:methanol:tetrahydrofuran (68.5:31:0.5 v/v/v), and mobile phase B comprising methanol:tetrahydrofuran (93.25:6.75 v/v). Detection of components was on the wavelength of 270 nm.
Metabolic ratio (MR) was calculated for each participant from the concentrations of chlorzoxazone and 6-hydroxychlorzoxazone, and the logarithmic values were determined. Frequency histogram was constructed using number of participants and log MR. Probit values were obtained from Z-table and were plotted on the Y-axis against log MR on the X-axis (scatter chart). A trend line was added to the probit plot and polynomial equation was obtained. Anti-mode was determined as the intercept of the X-axis from log MR of plasma.
Participants with anti-mode values greater than or equal to intercept on log MR were regarded as poor metabolizers, whereas those with anti-mode values less than to intercept on log MR were considered extensive metabolizers. The outcome reported as proportions with 95% confidence intervals. Polymorphism was determined graphically as the deviation of the probit values from the line of fit on the graph.
| Results|| |
The age ranges and median age are shown in [Table 2]. Nonparametric statistics were used simply to identify trends, although they may be considered inappropriate for nonrandom data. The distribution of individual participant metabolic phenotype status is shown in [Table 3]. All the participants were poor metabolizers with the anti-mode of –1.2 [Figure 1] and [Figure 2].
|Figure 1: Plasma frequency histogram of Hausa/Fulani phenotype study. MR = metabolic ratio|
Click here to view
|Figure 2: Plasma probit versus log MR plot of Hausa/Fulani phenotype study. Scatter (XY) chart showing trend line with a best linear fit to the data and polynomial equation. At x-intercept where y = 0, the equation becomes –0.052x2+0.347x + 0.505 = 0. The values of x were –1.2 and 7.9 and therefore –1.2 was taken as the anti-mode. Graphically all the probits fit into the trend line|
Click here to view
| Discussion|| |
Chlorzoxazone so far is the only drug recognized as a probe drug for cytochrome 2E1, to determine phenotypes of individual subjects. Previous works on cytochrome 2E1 have shown between-subject variability in enzyme’s activities and consistent ethnic variations in the gene expression. The variability of cytochrome P450 2E1 between individuals is significant and correlated with its enzymatic activity. High expression and the activity of the enzyme have been linked to nonalcoholic fatty liver disease and variously implicated to increased susceptibility to the gastric, nasopharyngeal, colorectal, urinary bladder, and esophageal malignancies among a host of others.,
Due to its unique role in the metabolic activation of procarcinogens and chemical carcinogenesis, most of the studies on this enzyme were on its relationship with malignancies.
The presence of deviations from the line of fitness observed in the probit plot was suggestive of polymorphisms among the participants [Figure 2]. This finding was consistent with that obtained by Kim et al., The correlations of the plasma plots showed an excellent relationship between the variables of probits and log MR as only 0.7% variations were unexplainable from polynomial expressions studied as shown in [Figure 2].
Cytochrome P450 2E1 enzyme activity among the participants was categorized phenotypically into poor and extensive metabolism. All the participants were poor metabolizers based on the anti-mode derived from plasma probit versus log MR plots [Table 3]. This finding sharply differed remarkably with 9.38%, 2.08%, and 3.91% reported among the Shanghai, Xi’an, and Chinese populations’, respectively. Interethnic variability in the function of the drug metabolizing enzymes is a well-documented phenomenon.
This observation may be protective as only approximately 5%–10% of acetaminophen undergoes metabolism to produce the toxic metabolic product NAPBQI. The major pathways of acetaminophen metabolism are glucuronidation and sulfation. Poor metabolism, therefore, ensures non-production of NAPBQI as compared to rapid and ultrarapid metabolism.
| Conclusion|| |
All the participants studied were poor metabolizers of acetaminophen to NAPBQI, Its harmful by-product through metabolism with cytochrome 2E1 enzyme. This reduces the chances of developing toxicities arising from this metabolite in Hausa/Fulani ethnic group of northwest Nigeria.
| Limitation of the Study|| |
Use of convenience sampling technique and sample size limit the generalization of the findings. Further studies involving much large sample size and random sampling technique are desirable.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Hynan LS, et al
.; Acute Liver Failure Study Group. Acetaminophen-induced acute liver failure: Results of a United States Multicenter, Prospective Study. Hepatology 2005;42:1364-72.
Becker KG, Schultz ST Similarities in features of autism and asthma and a possible link to acetaminophen use. Med Hypotheses 2010;74:7-11.
Pena M, Pérez S, Zazo M, Alcalá P, Cuello J, Zapater P, et al
Concerning a case of toxic epidermal necrolysis secondary to acetaminophen in a child. Curr Drug Saf 2015;10:1-1.
Graham GG, Davies MJ, Day RO, Mohamudally A, Scott KF The modern pharmacology of paracetamol: Therapeutic actions, mechanism of action, metabolism, toxicity and recent pharmacological findings. Inflammopharmacology 2013;21:201-32.
Allegaert K, Peeters MY, Beleyn B, Smits A, Kulo A, van Calsteren K, et al
. Paracetamol pharmacokinetics and metabolism in young women. BMC Anesthesiol 2015;15:163.
Caparrotta TM, Antoine DJ, Dear JW Are some people at increased risk of paracetamol-induced liver injury? A critical review of the literature. Eur J Clin Pharmacol 2018;74:147-60.
Bessems JG, Vermeulen NP Paracetamol (acetaminophen)-induced toxicity: Molecular and biochemical mechanisms, analogues and protective approaches. Crit Rev Toxicol 2001;31:55-138.
Lu T, Liang WZ, Han LJ, Kuo CC, Shieh P, Chou CT, et al
Action of chlorzoxazone on Ca2+ movement and viability in human oral cancer cells. Chinese J Physiol 2019;62:123-30.
Zhu B, Ouyang D, Chen X, Huang S, Tan Z, He N, et al
Assessment of cytochrome P450 activity by a five-drug cocktail approach CPT. Clin Pharmacol Ther.2001;70:455-61.
Blakey GE, Lockton JA, Perrett J, Norwood P, Russell M, Aherne Z, et al
. Pharmacokinetic and pharmacodynamic assessment of a five-probe metabolic cocktail for CYPs 1a2, 3a4, 2c9, 2d6 and 2e1. Br J Clin Pharmacol 2004;57:162-9.
Afsar A, Bruckmouller H, Werk AN, Muhammad KN, Ahmad HR, Ingolf C Implication of genetic variation of common drug metaboliszing enzymes and ABC transporters and Pakistani population. Sci Rep 2019;9:7323.
Sissung TM, Troutman SM, Campbell TJ, Pressler HM, Sung H, Bates SE, et al
. Transporter pharmacogenetics: Transporter polymorphisms affect normal physiology, diseases, and pharmacotherapy. Discov Med 2012;13:19-34.
Ma Q, Lu AY Pharmacogenetics, pharmacogenomics, and individualized medicine. Pharmacol Rev 2011;63:437-59.
Johansson I, Ingelman-Sundberg M Genetic polymorphism and toxicology-with emphasis on cytochrome p450. Toxicol Sci 2011;120:1-13.
Eichelbaum M, Ingelman-Sundberg M, Evans WE Pharmacogenomics and individualized drug therapy. Annu Rev Med 2006;57:119-37.
Keller GA, Gago MLF, Diez RA, Di Girolamo G In vivo
phenotyping methods: Cytochrome P450 probes with emphasis on the cocktail approach. Curr Pharm Des 2017;23:2035-49.
Akinyandenu O Counterfeit drugs in Nigeria: A threat to public health. Afr J Pharm Pharmacol 2013;7:2571-6.
Stiff DD, Frye RF, Branch RA Sensitive high-performance liquid chromatographic determination of chlorzoxazone and 6-hydroxychlorzoxazone in plasma. J Chromatogr 1993;613:127-31.
Witt L, Suzuki Y, Hohmann N, Mikus G, Haefeli WE, Burhenne J Ultrasensitive quantification of the CYP2E1 probe chlorzoxazone and its main metabolite 6-hydroxychlorzoxazone in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry after chlorzoxazone microdosing. J Chromatogr B Analyt Technol Biomed Life Sci 2016;1027:207-13.
Bolt HM, Roos PH, Thier R The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals: Consequences for occupational and environmental medicine. Int Arch Occup Environ Health 2003;76:174-85.
Ohtsuki S, Schaefer O, Kawakami H, Inoue T, Liehner S, Saito A, et al
. Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: Comparison with MRNA levels and activities. Drug Metab Dispos 2012;40:83-92.
Tang K, Li X, Xing Q, Li W, Feng G, He L, et al
. Genetic polymorphism analysis of cytochrome P4502E1 (CYP2E1) in Chinese Han populations from four different geographic areas of mainland china. Genomics 2010;95:224-9.
Yao K, Qin H, Gong L, Zhang R, Li L CYP2E1 polymorphisms and nasopharyngeal carcinoma risk: A meta-analysis. Eur Arch Otorhinolaryngol 2017;274:253-9.
Zanger UM, Schwab M Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 2013;138:103-41.
Varshney E, Saha N, Tandon M, Shrivastava V, Ali S Prevalence of poor and rapid metabolizers of drugs metabolized by CYP2B6 in north Indian population residing in Indian national capital territory. Springerplus 2012;1:34.
Kim YJ, Lim KH, Jo EJ, Lee SY, Lee SE, et al
. Cross reactivity to acetaminophen according to the type of Non steroidal anti inflammatory drugs hypersensitivity. Allergy, Asthma & Immunology Research. 2014;6:156.
Li W, Guoxia R, Jingjie L, Linhao Z, Fanglin N, Mengdan Y, et al
. Genetic polymorphism analysis of cytochrome 2E1 in a Chinese Tibetan population. Medicine 2017;96:e8855.
Wu Z, Zhang X, Shen L, Xiong Y, Wu X, Huo R, et al
. A systematically combined genotype and functional combination analysis of CYP2E1, CYP2D6, CYP2C9, CYP2C19 in different geographic areas of mainland china-a basis for personalized therapy. PLoS One 2013;8:e71934.
Johnsrud EK, Koukouritaki SB, Divakaran K, Brunengraber LL, Hines RN, McCarver DG Human hepatic CYP2E1 expression during development. J Pharmacol Exp Ther 2003;307:402-7.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]