ANALYTICAL TECHNIQUES TO IDENTIFY SLOW AND FAST ACETYLATORS OF NAT-2 ENZYME USING ISONIAZID

Sara Sattar 1 , * Dr. Zain-ul-Abadin 2 , Muzamil Aftab 3 and Dr. Anees Qureshi 4 . 1. M.Phil (Pharmacology), Pharm-D, Punjab Healthcare Commission. Lahore. 2. MBBS, RHC, Narang Mandi.Shiekhupura. 3. Pharm-D, College of Pharmacy, Lahore Medical & Dental College. Lahore. 4. MBBS, M.Sc (Medical Administration), Punjab Healthcare Commission. Lahore. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In Pakistan, every ethnic group of the South Asian region is present (Vree T. B., 1980;Zaid R. B., 2007). Pakistani population consists of 31.8% of fast acetylators, proved from a study conducted on healthy volunteers and tuberculosis patients, following an oral dose of INH and examining their plasma with drug-free background to avoid any interference in the results. This study concluded that the rate of INH inactivation does not affect the drug response, if INH is taken twice or thrice daily. And the ratio of fast and slow acetylators was similar in healthy individuals and tuberculosis patients(Mohammad Saleem, 1989). Furthermore,60% of Pakistani female population is fast and 40% is slow acetylators while in males, fast and slow acetylators are 62% and 38%, respectively (Akhtar N., 2011). Within Punjab˗Pakistan, 42.7% were reported to be fast and 57.3% were slow acetylators, after examining the plasma and urine sample of healthy and tuberculosis patients spectrophotometrically, following a dose of INH. This study indicated that tuberculosis disease has no influence on the acetylation capacity, which is genetically controlled (Abdul Sattar Sohrani, 1992).

Methods Used to Determine NAT-2 Phenotypes:-Ion˗Exclusion Column˗Chromatography:-
Initially, different procedures were developed for determining the phenotypic acetylation of NAT-2such as ion˗exclusion column˗chromatographic procedure had been employed to separate INH and its metabolites (Peters et al., 1965). But it was laborious and was not sensitive to minute concentration of those compounds that are found in serum/plasma of patients taking INH.

Colorimetric & Fluorimetric Methods:-
It was developed to determine INH in serum or urine by incorporating extraction procedures before and after the reaction with p-dimethylaminobenzaldehyde(Maher J. R., 1957). And another similar fluorimetric method was also developed (H., 1960). Whereas, Scott & Wright developed a more sensitive fluorimetric method for detection of INH in serum that cannot be used for urine samples (Scott E. M. & Wright, 1967). First, the reactions were done after solvent extraction instead of on a protein-free filtrate of serum. Secondly, salicylaldehyde was used as its own 'internal' pH indicator to facilitate the control of the pH. Thirdly, (NH4)2SO4 was added to avoid having to centrifuge the butan-1-ol extract.Venkataraman had developed a colorimetric method for the determination of acetylisoniazid in urine but this method cannot be used for serum samples (Venkataraman P., 1968). Results of both methods are compared in the following table-I: Isonicotinic acid can also be determined in urine by reaction with cyanogens chloride and barbituric acid, and measurement of the extinction of the respective polymethine dyes at 600 and 620nm (L., 1959a; Nielsch, 1959b). But this method is not sensitive enough for serum detection of the compound.

Figure-1:-Chromatograms obtained from plasma spiked with INH and AcINH
One of the most reliable,liquid chromatography-mass spectrometry (LC˗MS) method was used for determination of INH in human plasma, using single-ion monitoring technique with the help of mass-spectrometer (X. Chen, 2005). All of these analytical methods use one of the following procedures:

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Different studies were also conducted on INH, using Gas Chromatography (GC) (A. Calo, 1976;Zbinden, 1975). Then, J. A. Timbrell developed a very precise GC method for quantification of INH and its hydrazine metabolites, by using human urine samples (J. A. Timbrell, 1977).
Another reliable and a very sensitive GC˗MS method was used for the quantification of INH and acetylhydrazine, in the serum(Georg Karlaganis, 1987), by using capillary column (Grob, 1983).This study indicated that INH prevents the acetylation and detoxification of acetylhydrazine. And the acetylation of INH as well as acetylhydrazine is controlled by genetic polymorphism as both of them have similar half˗life. Thus, INH competes with acetylhydrazine for its acetylation and therefore, reduces the elimination rate of acetylhydrazine.
Similarly, a study involving quantitative determination of INH and its metabolites in human urine samples was conducted, by using GC and GC˗MS techniques, which quantified minute quantities of various metabolites of INH in healthy volunteers as well as patients on INH treatment (A. Noda, 1978), which is not possible by using other techniques as mentioned earlier. The urine samples were collected at 0˗2, 2˗4, 4˗6 and 6˗8 hours, after oral INH dose (Table III). INH, acetyl INH and diacetylhydrazine were detected from GC technique, whereas the analysis of hydrazine and monoacetylhydrazine involved mass fragmentography, by using GC˗MS technique. Mutagenesis test of INH metabolites was also performed using Salmonella typhimurium, as given in Ames' method (B. N. Ames, 1975). The result showed significant mutagenesis produced by hydrazine compound. This study produced highly accurate results (K. Matsuyama, 1977). It determined the slow and rapid inactivators of INH as well as found the production of free hydrazine mutagen which excreted slowly in urine, after oral dose of INH. But studies say that the more precise way of determining the acetylation phenotype is to measure the metabolite ratio to drug fractions in plasma as compared to calculating the urinary metabolite ratios (DW, 2002; G. A. Ellard, 1973). So, in another study, hydrazine concentration in plasma of healthy males were calculated, following an oral dose of INH, using sensitive stable isotope dilution GC˗MS method. Hydrazine was detected in plasma, formed by either hydrolysis of INH or hydrolysis of acetylhydrazine. In slow acetylators, hydrazine was in higher concentration as compared to fast acetylators(I. A. Blair, 1985).
A similar study was conducted for the determination of hydrazine metabolites, acetyl INH, acetylhydrazine and diacetylhydrazine in human plasma using GC˗MS (Fig. 2). But in this way,acetylhydrazine and diacetylhydrazine cannot be measured, irrespective of their appearance in solvent front. This method can also be used for the extraction of metabolites in urine, by doing acid hydrolysis of their hydrazones in urine(Bernhard H. Lauterburg, 1981).