PHARMACOKINETICS AND DISPOSITION

Is there any difference between acetylator phenotypesin tuberculosis patients and healthy subjects?

Hossein Khalili & Simin Dashti-Khavidaki &Mohsen Amini & Reza Mahjub &

Mahboobeh Hajiabdolbaghi

Received: 2 July 2009 /Accepted: 3 October 2009# Springer-Verlag 2009

AbstractBackground and purpose Many studies have been done todetermine the distribution of acetylator phenotypes amongpopulations of different geographic origin. The goal of thisstudy was to investigate the acetylator phenotypes of theIranian population and compare them between tubercularpatients and healthy subjects.Methods The study population consisted of two groups; thefirst group included 100 newly diagnosed tubercularpatients and the second group consisted of 100 healthysubjects. Acetylator phenotype was determined from themetabolic ratio of acetyl-isoniazid to isoniazid in theplasma samples. Metabolic ratio was used to classifysubjects as slow (≤0.70) or fast acetylators (>0.70).Results In the tubercular patients, the frequencies of slow andfast acetylator phenotypes were 62 and 38%, respectively. Ofthe healthy individuals, 45% were found to be slowacetylators and the remaining 55% were fast acetylators.

Conclusion It seems that tubercular patients metabolizeisoniazid more slowly than healthy individuals.

Keywords Acetylation . Phenotype . Tuberculosis .

Isoniazid

Introduction

Isoniazid (INH) is a first-line antituberculosis drug that hasvaluable effects in prophylaxis and treatment of tuberculosis(TB) [1]. After oral administration, INH is absorbed rapidlyand relatively completely from the gastrointestinal tract.However, it can be influenced by first pass metabolism in theliver, which may alter the plasma concentrations of the drug[2]. The main route of INH metabolism is through the liverbut a small amount of the intact drug is also eliminated fromthe kidneys [3]. The metabolism of INH occurs throughacetylation by N-acetyltransferase (NAT), resulting inacetyl-INH [4].

In fast acetylators, serum concentrations of INH are lowerthan in slow acetylators and the desired efficacy may not beachieved [5, 6]. The presence of a correlation betweenacetylator phenotypes and incidence of hepatotoxicity iscontroversial [4, 7, 8]. In slow acetylators, peripheralneuropathy due to INH may be more prevalent [9].

Different proportions of rapid and slow acetylatorphenotypes have been reported based on ethnic or geo-graphic population origin. Most populations in Europe andNorth America have 40–70% slow acetylators, whereaspopulations along the Pacific Asian littoral (e.g., Japanese,Chinese, Korean, and Thai) have only 10–30% slowacetylators [5, 10–12].

There is no case-control study on acetylator phenotypesin an Iranian population. The goal of this study was to

H. Khalili : S. Dashti-Khavidaki : R. MahjubDepartment of Pharmacotherapy, School of Pharmacy,Tehran University of Medical Sciences,Tehran, Iran

M. AminiDepartment of Medical Chemistry, School of Pharmacy,Tehran University of Medical Sciences,Tehran, Iran

M. HajiabdolbaghiDepartment of Infectious Disease, Faculty of Medicine,Tehran University of Medical Sciences,Tehran, Iran

H. Khalili (*)School of Pharmacy, Tehran University of Medical Sciences,P.O. Box: 14155/6451, Tehran Postal Code: 1417614411, Irane-mail: khalilih@tums.ac.ir

Eur J Clin PharmacolDOI 10.1007/s00228-009-0745-1

investigate the acetylator phenotypes of the Iranian popu-lation and compare those of tubercular patients with healthysubjects.

Materials and methods

The study population consisted of two groups; the firstgroup (case) included 100 newly diagnosed tubercularpatients over 18 years old who were referred to theinfectious disease ward of Imam Hospital (affiliated withTehran University of Medical Sciences, Tehran, Iran) fromMay 2007 to December 2008. The infectious disease wardof Imam Hospital is a referral center for management oftuberculosis in Tehran. Tuberculosis was diagnosed if apatient (1) had a positive culture for Mycobacteriumtuberculosis, (2) was culture negative but PCR positivewith clinical or radiological features and response totreatment consistent with TB, or (3) had histologicalfindings and response to treatment consistent with TB [13].

The second study group (control) consisted of 100healthy subjects defined as individuals over 18 years oldwithout any medical problems, chronic diseases, or acuteinfections, with negative history of alcohol ingestion orsmoking, and negative tuberculin skin test. The controlgroup was age- and sex-matched relatives of patients whoaccompanied or visited them in the hospital.

Patients with human immunodeficiency virus (HIV)infection, hepatic insufficiency (ALT or AST > 2× upperlimit normal or clinical symptoms of liver disease such asjaundice and ascites) and renal insufficiency (creatinineclearance < 50 ml/min based on Cockcroft-Gault equation),history of smoking (based on individual expression), orchronic alcohol consumption (anyone that may have ahistory of regular alcohol drinking of at least 30 g daily for6 months before hospital admission) were excluded fromthe study. There is no evidence that acute alcohol intake hasany impact on the conversion of INH to acetyl-INH [14].The purpose of the study was explained to all participants,and the research was approved by the ethics committee ofthe hospital. The healthy subjects were provided withinformation about INH and its probable adverse effects.Informed consent was obtained from all participants prior toenrollment in the study.

Standard treatment of tuberculosis in Iran is based onWHO recommendations—2 months of INH, rifampin,pyrazinamide, and ethambutol and then 4 months of INHand rifampin. At the beginning of tuberculosis treatmentand after an overnight (8 h) fasting, each subject received5 mg/kg of INH (generic product of Kharazmi Company,Tehran, Iran) orally under observation, and then 5 ml ofvenous blood sample was taken exactly 3 h after. Thesamples were collected into EDTA tubes and immediately

centrifuged. The plasma samples were then separated andkept frozen at −70°C until analysis. After sampling, otherantituberculosis drugs were started for tubercular patients.

Purified INH was prepared from Sigma Chemical.Acetyl-INH and iproniazid (internal standard) weresynthesized [15–17]. The structure and purity wereascertained by nuclear magnetic resonance (NMR) andmelting point determination.

A gradient HPLC method was used that involved a C18stainless steel column (tracer excel 5 μm, 15×0.46 ODSA)(Technokroma, Barcelona, Spain). TheHPLC system suppliedfrom Knauer (Berlin, Germany) consisted of a Knauer onlinedegasser, a solvent organizer (k-1500), a pump (Maxi-starK-1001), and a detector (K-2600) that was set at 266 nm.

Mobile phase included solvents A, B, and C, whichconsisted of phosphate buffer (0.01 M, pH=7.9), wateradjusted to a pH of 3 with 17% phosphoric acid solution,and methanol (HPLC-grade), respectively. This mobilephase was programmed to deliver with a flow rate of1.5 ml/min as follows: The mobile phase was 100% ofsolvent A for 5 min. At this time, solvent Awas stopped for30 s, and the mobile phase was shifted to 100% of solventB. Then immediately after 5 min, solvent C was increasedlinearly from 0 to 10% through the 20th minute (thecomposition of the mobile phase at the 20th minute was90% solvent B and 10% solvent C). After the 20th minute,solvent C was increased linearly from 10% to 30% throughthe 25th minute, and this composition was held until theend of the analysis.

For sample preparation, the method described byHutchings et al. was used [18]. Iproniazid solution (25 μl;50 µg/ml) and 250 µl of 0.5 M phosphate buffer (pH=7.4)solution that was saturated with sodium chloride were addedto 1 ml of plasma. The extraction was carried out with 5 ml ofa mixture of chloroform and 1-butanol (70:30). Aftercentrifugation at a rate of 2,000 rpm for 4 min, the aqueousphase was discarded, and 500 µl of a solution containingphosphoric acid (0.01 M) was added to the organic phase,mixed for 10 min, and re-extracted. The samples werecentrifuged at a rate of 1,300 rpm for 3 min, and 50 µl ofthe supernatant aqueous phase was injected to HPLC.

A sample plasma chromatogram is shown in Fig. 1. Thepeak at the 11th minute relates to an unknown impurity inthe plasma and does not interfere with the analysis, so wedecided to continue analysis despite the presence of thispeak. Peaks at 5.08, 6.02, and 25.02 min were related toacetyl-INH, INH, and iproniazid (IS). All of these peakshave suitable resolution, tailing, and capacity factors, andthere was no evidence of interference from the peak at the11th minute.

Lower and upper limits of quantification were calculatedto be 0.33 µg/ml (0.5 µg/ml) and 20 µg/m (16 µg/ml),respectively, for INH (acetyl-INH). The interday and intraday

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assays for standard calibration of INH and acetyl-INH arepresented in Table 1.

Based on equations carried out using Excel, it has beendemonstrated that (1) all calibration curves have an R valuegreater than 0.99, and (2) all calibration curves have anintercept equal to 0 with 95 and 99% probabilities.

ICH guidelines for validation of an analytical methodinclude determining specificity, accuracy, precision (repeat-ability, intermediate precision, and reproducibility), limitsof detection, and qualification linearity and range. Weperformed all of these tests except reproducibility (variationamong different labs) due to some limitations.

Acetylator phenotype was determined from the metabolicratio (MR) of acetyl-INH to INH concentration in the plasmasamples. Based on the distribution of the MRs of healthy andtubercular patients, an antimode of 0.7 was selected, and

subjects with MR≤0.7 and MR>0.7 were considered slow orfast acetylators, respectively.

For comparing other variables between two groups, weused SPSS software (version 13.5) and independent samplet-test, chi-square, and Fisher-exact tests. Linear regressionwas used for qualifying the effect of cofactors andcovariates such as age, sex, comorbidity, and comedication.A P value less than 0.05 was considered as significant.

Results

The tubercular patients’ (male: n=56, female: n=44) agerange was 18–63 years with mean age ± SD equal to 43.96±18.57 years. The age of healthy volunteers (male: n=58,female: n=42) ranged from 18 to 58 years with mean age ±

Table 1 Accuracy and precision in spiked plasma for isoniazid and acetyl-isoniazid

Parameters Concentrationadded (µg/ml)

Isoniazid Acetyl-isoniazid

Concentration found(mean ± SD; µg/ml)

CV (%) Accuracy (%) Concentration found(mean ± SD; µg/ml)

CV (%) Accuracy (%)

Intraday (n=3) 1 0.96±0.06 6.34 96.3 0.92±0.02 2.17 92.0

5 4.96±0.22 4.53 99.3 4.64±0.12 2.58 92.8

10 10.29±0.2 1.99 102.9 10.36±0.23 2.26 103.6

15 14.61±0.39 2.66 97.4 15.64±0.26 1.67 104.3

20 20.15±0.36 1.82 100.8 19.34±0.42 2.17 96.7

Interday (n=3) 1 1.02±0.09 9.31 102.0 0.87±0.03 3.44 87.0

5 5.23±0.39 7.45 104.7 4.52±0.46 10.17 90.4

10 10.25±0.42 4.18 102.5 9.57±0.67 7 95.7

15 13.58±0.68 4.9 92.3 15.08±0.50 3.31 100.5

20 20.69±0.66 3.18 103.4 20.23±0.74 3.65 101.2

Fig. 1 Chromatogram of HPLCof a plasma sample belonging toone patient. INH, acetyl-INH,and MR calculated for thispatient, considered a fast acety-lator, were 3.76, 6.39, and1.69 µg/ml. Peaks at 5.08, 6.20,and 25.02 min were related toacetyl-INH, INH, and internalstandard (iproniazid),respectively

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SD equal to 41.05±11.73. There were not significantdifferences in sex (P=0.21) or age (P=0.13) between thetwo groups. Based on individual reports, the ethnicity of thestudy population was as follows: 61% Fars, 35% Azeri, 3%Mazandarani and Gilaki, and 1% Kurds. Since the controlgroup individuals were the relatives of case group patients,there were not ethnic differences between the two groups.Patients had not received any medication for tuberculosismanagement before admission, and if they were given anymedications, i.e., antibiotics, bronchodilators, antitussives,analgesics (acetaminophen, ibuprofen, naproxen), multivita-mins, and herbal medicines, all were stopped beforetuberculosis work-up. Comorbidities and comedications ofthe tubercular patients are shown in Table 2.

The mean INH and acetyl-INH serum concentrations,MRs, and comparisons between groups are shown inTable 3. The MR distribution pattern is shown in Fig. 2.The acetylator phenotype pattern of distribution was found tobe bimodal in healthy and tubercular groups. The data showthat the frequencies of slow and fast acetylator phenotypeswere 62 and 38%, respectively, among tubercular patients. Inhealthy subjects, 45% were found to be slow and 55% fastacetylators. In regression analysis there were no significantrelations between acetylator phenotype and patient age, sex,comorbidity, or comedication.

Discussion

Many studies have been performed to determine thedistribution of acetylator phenotype in populations ofdifferent geographic origins. Some drugs such as caffeine,dapsone, sulfamethazine, procainamide, and INH have beenused for assessment of acetylation capacity in individuals.Since INH is one of the drugs in the treatment regimen oftuberculosis and because maintaining caffeine abstinence isa problem in individuals entered into studies, INH wasselected for the study of polymorphism.

Some researchers have used INH half-life in plasma fordetermination of the activity of NAT, which requires repeatedblood sampling [19, 20]. In the current study, we used MR asa determining factor for the estimation of enzyme activity,which requires only one blood sample. Studies have shownthat a good correlation exists between determination ofacetylator phenotype by MR and INH half-life in plasma [19].

Distribution of acetylator phenotype can differ widelyamong different populations. Several studies have describedthe distribution of acetylator phenotypes among populationsof different geographic locations. In Asia, the distributionof slow acetylators is 13.1% among Japanese pulmonarytubercular patients [21], 14.6% among Indian pulmonarytubercular and nontubercular chest-disease subjects [22],94.9% among Saudi Arabian healthy subjects [23], and67.5% among Jordanian healthy individuals [24].

In the present study, the prevalence of slow acetylatorphenotypes was 62 and 45% in the tubercular patients andthe healthy subjects, respectively. In another study, slowacetylators were more common in tuberculosis patients thanhealthy subjects [25]. Concentration of urine-intact INH inJapanese tuberculosis patients was less than in the healthypopulation following INH ingestion. This difference wasdescribed in terms of the effects of concomitant drugs andINH gastrointestinal absorption differences but may berelated to different rates of INH metabolism in the studypopulations [26].

Our results are different from some of the previousstudies that have been done in the Iranian population. In thestudy of Hassanzadeh et al., 56% of healthy Iranian andAfghan populations were slow acetylators based on thepercentage of acetyl-INH excreted in urine sample [27]. Inthat study, urine acetyl-INH concentrations were deter-mined by spectrophotometer, which is not comparable withblood samples and the precise HPLC technique used in thepresent study.

In the study of Bakayev et al., it was revealed that 32.9,48.9, and 18.2% of the healthy general population ofTehran, Iran, were slow, intermediate, and fast acetylators,respectively [28]. The results of that study were reportedbased on genotyping evaluation and are not comparablewith the results of present study. Genotyping and phenotypingresults can be discordant [29]. Based on detection ofacetylated sulphadimidine in urine, 14.6% of Indian tuber-culosis patients were slow acetylators [22], but in agenotyping study, 55% of Indian population individualswere slow acetylators, and determined genotypes werecorrelated with INH serum concentration [30].

Based on PCR genotyping in Iranian healthy individuals,the percentage of slow, intermediate, and fast acetylatorswas reported to be 49.3, 41.47, and 9.17%, respectively, inthe Torkaman et al. study [31].

Parameter Tubercular patients (n)

Comorbidity Hypertension (2), diabetes (1), rheumatoid arthritis (1), thyroid dysfunction (1),osteoporosis (2), skin disorders (1), osteoarthritis (1), gastritis or gastric ulcer (3)

Comedication Atenolol (1), captopril (2), atorvastatin (1), hydrochlorothiazide (2), glibenclamide (1),prednisolone (3), naproxen (4), levothyroxine (1), calcium-D (3), hydroxyzine (2),ranitidine (3), antacids (2)

Table 2 Comorbidities andcomedications in the tubercularpatients

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0

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0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4[Acetyl-INH/INH] (MR)

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0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4[Acetyl-INH/INH](MR)

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Healthy Sybjects

Fig. 2 Histograms of MRdistribution in healthy subjectsand tubercular patients. MRMetabolic ratio, INH isoniazid

Table 3 Acetylator phenotypes in patients and healthy subjects

Parameter Acetylator status Patients Healthy P value

INH serum concentration (μg/ml) Slow 9.76±1.42 (7.58–11.34) 7.05±0.78 (6.44–8.77) <0.04

Fast 4.97±0.77 (4.42–5.80) 3.67±0.81 (2.25–4.56) <0.02

P value <0.001 <0.001

Acetyl-INH serum concentration (μg/ml) Slow 3.53±1.14 (2.87–5.46) 3.01±0.70 (2.32–4.010 0.04

Fast 8.03±0.84 (7.35–8.53) 6.63±0.89 (5.25–7.15) 0.01

P value <0.001 <0.001

MR Slow 0.51±0.17 (0.35–0.70) 0.34±0.05 (0.29–0.40) 0.03

Fast 1.56±0.28 (1.34–1.80) 1.83±0.21 (1.59–1.97) 0.04

P value <0.001 <0.001

Patients Slow 62 (62%) 45 (45%) <0.001

Fast 38 (38%) 55 (55%) <0.001

P value <0.001 0.01

Values are mean ± SD (95% CI) or n (%). Independent sample t-test was used for comparison of the means betweens groups

MR Metabolic ratio, INH isoniazid

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The present study was a case-control one, and plasmasample analysis was done with a precise HPLC method.Some of the previous studies did not have a controlgroup, and in another study, individual acetylatorphenotypes were determined based on urine sample testsor spectrophotometery, which are not as precise as theHPLC method.

The results of the current study have shown that thedistribution of INH elimination is bimodal among Iraniantubercular patients and healthy subjects. The time of plasmasampling is critical in interpreting INH elimination patterns.Some studies have shown that the time of sampling is animportant factor for determination of whether distributionof INH elimination is bimodal or trimodal. It has beenreported that the distribution of INH elimination is trimodalin 3-h post-dose sampling, but it is bimodal in 2-h and6-h post-dose samples [5].

We emphasize that the blood sample collection was doneexactly 3 h after INH oral consumption in all individuals.The difference in the pattern of INH metabolism pheno-types in the tuberculosis patients and the healthy subjectsmay be related to tuberculosis disease. An effect of diseasestatus on acetylator phenotype determination has beenreported in another study [29–32]. That study suggestedthat changes in metabolic capacity may be caused byinfection-mediated cytokine release. Tuberculosis is achronic inflammatory disease, and it has been reportedthat levels of some cytokines including gamma interferonand tumor necrosis factor α were elevated in patientswith tuberculosis [32]. These cytokines may causereduction of acetyltransferase activity and differentpatterns of acetylator phenotypes among patients andhealthy individuals [33].

Our study has some limitations as well. The sample sizeof the study was small. Results of the study would beimproved if the findings of phenotyping could be comparedto those of genotyping. If it were possible to compare theresults of the phenotype study with a patient’s genotypeparameters, it would be more reliable. The principal roleof baseline disease on drug metabolism, especially intuberculosis patients, needs to be evaluated in designedcontrolled studies with sufficient sample size.

Acknowledgement This study was supported by PharmaceuticalSciences Research Center of Tehran University of Medical Sciencesand there is no conflict of interest.

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