Isoniazid (INH) is a main agent in the treatment of tuberculosis in combination with other drugs or alone as a prophylactic agent. The major pathway for INH metabolism is acetylation by a hepatic enzyme N-acetylotransferase, discovered over 40 years ago following differences observed in tuberculosis patients to isoniazid toxicity and bioavailability. The biochemical basis for this observation relates to substrate specificity and molecular genetics of two distinct N-acetyltranferase isoenzymes NAT1 an NAT2. The genetically polymorphic NAT2 is responsible for INH metabolism and individuals can be classified as rapid and slow acetylators. In our last study in 237 Tb patients treated by standard course of chemotherapy with dose of INH 300 mg/day we observed that 1. majority of the patients (70 %) shown fast type of INH acetylation and 2. after the same doses the three parameters of INH bioavalability in a serum of fast and slow acetylators had significant differences: INH concentrations, pharmacokinetic and biovailability factors. The objective of the present study was to apply the genotyping of the fast and slow acetylators for personalized therapeutic dose. Materials and methods: blood samples were taken from the patients and volunteers before ( time 0 ) and 1,3,6,24 hrs after drug administration. Plasma concentrations of INH were determined with biological method in the authors modification.This method warrants high accuracy and secured repeatable results. The lowest measurable concentration was 0,25 mcg/ml. For marking INH concentrations in human serum was applied as a pattern strain Mycobacterium aurum REB . Two indicators of acetylation rate I₃ and C₆ have been used to determine an acetylation type. Genomic DNA was isolated from the blood samples. DNA extracted by Gustinicich method and amplified by PCR by Spurr with two primers: P1: 5'-GCTGGGTCTGGAAGCTCCTC-3' and P2: 5'-TTGGGTGATACATACACAAGGG-3'. After initial amplification, the PCR product was cut separately with 3 different restriction enzymes: Kpn1, Tag1, and BamH1. A loos of a Kpn1 restriction site denotes NAT2*5 allele, a Tag1 restriction site denotes NAT2*6 allele, and a BamH1 restriction site denotes NAT2*7 allele. We separated the products on non-denaturing poliacrylamide gels and then stained them with ethidium bromide and visualized them on an UV transilluminator. The presence of any 2 mutant alleles defines the slow-acetylator genotype, whereas rapid acetylators have 1 or 2 wild-type NAT2*4 alleles. In a third part of patients with fast acetylation phenotype INH concentration in serum did not reach a value of 1 mcg/ml after 3h from therapeutic dose administration. In a third part of patients with slow acetylation phenotype INH concentration in serum exceeded a value of 2 mcg/ml after 6h from therapeutic dose administration. Four different NAT 2 alleles were detected in the study population. For all treated patients concentrations of isoniazid observed in rapid acetylators were considerably lower the those found in slow acetylators. On the basis of our results we suggest the using of NAT 2 genotyping for discrimination of the fast and slow acetylators in monitoring of tuberculosis therapy.

Praca finansowana z grantu KBN nr. 2 PO5B 136 27

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