Plasma chlorzoxazone as a probe for cytochrome 2E1 activity among Hausa/Fulani in northwest Nigeria: Determination of acetaminophen metabolic phenotypes

Muhammad Tukur Umar; Shaibu O Bello; Aminu Chika; Yakubu Abdulmumini

doi:10.4103/jhrr.JHRR_59_19

ORIGINAL ARTICLE

Year : 2020 | Volume : 7 | 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

Correspondence Address:
Dr. Muhammad Tukur Umar
Department of Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, College of Health Sciences, Usmanu Danfodiyo University, PMB, Sokoto.
Nigeria

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jhrr.JHRR_59_19

Abstract

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.^[1] It accounts for 51% of acute liver failure.^[1] Acetaminophen use during the first year of life is strongly associated with the development of asthma, rhinoconjunctivitis, and eczema later in life.^[2] In another vein, it is implicated in adverse drug reactions such as Stevens–Johnson syndrome and toxic epidermal necrolysis.^[3] Acetaminophen is reported as the most commonly used antipyretic and analgesic globally^[4] 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).^[5] 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.^[6],[7] 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.^[8],[9],[10]

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.^[11],[12] 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.^[13],[14]

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.^[15] 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.^[16]

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.^[17] 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

Study design

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.^[18] The stock, standard solutions, and preparation of individual concentrations for calibration curve are shown in [Table 1].

Table 1: Preparation of individual concentrations for calibration curve

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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.

Statistical analysis

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

Demographic profile

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].

Table 2: Demographic characteristics of participants (n = 20)

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Table 3: Hausa/Fulani plasma phenotype parameters (n = 20)

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Figure 1: Plasma frequency histogram of Hausa/Fulani phenotype study. MR = metabolic ratio

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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

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Discussion

Chlorzoxazone so far is the only drug recognized as a probe drug for cytochrome 2E1, to determine phenotypes of individual subjects.^[19] Previous works on cytochrome 2E1 have shown between-subject variability in enzyme’s activities and consistent ethnic variations in the gene expression.^[20] The variability of cytochrome P450 2E1 between individuals is significant and correlated with its enzymatic activity.^[21] 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.^[22],[23]

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.^[24]

The presence of deviations from the line of fitness observed in the probit plot was suggestive of polymorphisms among the participants^[25] [Figure 2]. This finding was consistent with that obtained by Kim et al.^[26],[27] 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.^[28] Interethnic variability in the function of the drug metabolizing enzymes is a well-documented phenomenon.^[29]

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

Nil.

Conflicts of interest

There are no conflicts of interest.

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