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Human N-acetyltransferase 2 (NAT2) catalyzes the N-acetylation of numerous aromatic amine drugs such as sulfamethazine (SMZ) and hydrazine drugs such as isoniazid (INH). NAT2 also catalyzes the N-acetylation of aromatic amine carcinogens such as 2-aminofluorene and the O- and N,O-acetylation of aromatic amine and heterocyclic amine metabolites. Genetic polymorphism in NAT2 modifies drug efficacy and toxicity as well as cancer risk. Acetyltransferase catalytic activities and heat stability associated with six novel NAT2 haplotypes (NAT2*6C, NAT2*14C, NAT2*14D, NAT2*14E, NAT2*17, and NAT2*18) were compared with that of the reference NAT2*4 haplotype following recombinant expression in Escherichia coli. N-acetyltransferase activities towards SMZ and INH were significantly (p < 0.0001) lower when catalyzed by the novel recombinant human NAT2 allozymes compared to NAT2 4. SMZ and INH N-acetyltransferase activities catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.001) than catalyzed by NAT2 6C and NAT2 14E. N-Acetylation catalyzed by recombinant human NAT2 17 was over several hundred-fold lower than by recombinant NAT2 4 precluding measurement of its kinetic or heat inactivation constants. Similar results were observed for the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and the intramolecular N,O-acetylation of N-hydroxy-N-acetyl-2-aminofluorene. The apparent V max of the novel recombinant NAT2 allozymes NAT2 6C, NAT2 14C, NAT2 14D, and NAT2 14E towards AF, 4-aminobiphenyl (ABP), and 3,2'-dimethyl-4-aminobiphenyl (DMABP) were each significantly (p < 0.001) lower while their apparent K m values did not differ significantly (p > 0.05) from recombinant NAT2 4. The apparent V max catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.05) than the apparent V max catalyzed by NAT2 6C and NAT2 14E towards AF, ABP, and DMABP. Heat inactivation rate constants for recombinant human NAT2 14C, 14D, 14E, and 18 were significantly (p < 0.05) higher than NAT2 4. These results provide further evidence of genetic heterogeneity within the NAT2 slow acetylator phenotype.

Collections of sputum from 105 patients with newly diagnosed pulmonary tuberculosis were made before and at 1 and 2 d after the start of chemotherapy with isoniazid (INH) alone given to groups of patients in doses of 600 mg, 300 mg, 150 mg, 75 mg, 37.5 mg, 18.75 mg, and 9 mg daily, as well as from an untreated group. Counts of colony forming units (cfu) of Mycobacterium tuberculosis in the collections were set up on plates of selective 7H10 medium. The early bactericidal activity (EBA) of INH was defined as the decrease in log10 cfu/ml sputum/day during the first 2 d of treatment. A smooth curve relating EBA to log dose was obtained, with 600 mg INH yielding the highest mean EBA of 0.539, and 18.75 mg INH yielding the lowest EBA (0.111) that could be distinguished from the bactericidal activity of the untreated group. The ratio of the usual dose of 300 mg INH to the lowest dose, of 18.75 mg, that produced a detectable EBA, termed the therapeutic margin, was therefore 16, in contrast to the lower therapeutic margin of 4 for rifampin. The EBA was related to the INH acetylator genotype of patients treated with 600 mg or 9 mg INH.

Isoniazid, a highly effective drug for the chemoprophylaxis and treatment of tuberculosis, is associated with severe hepatotoxicity in 1-2% of individuals. In a rabbit model of isoniazid-induced hepatotoxicity, we have measured hepatic necrosis (quantitated by elevation of plasma argininosuccinic acid lyase (ASAL) activity), hepatic steatosis (quantitated by elevation of hepatic triglyceride content), and elevation in plasma triglyceride concentration in 15 rabbits. Eight of 15 rabbits were male, and 14 of 15 were rapid acetylators of sulfamethazine. Administration of isoniazid to rabbits resulted in a 27-fold increase in plasma ASAL activities, a 7.5-fold increase in hepatic triglyceride content, and a 13-fold increase in plasma triglyceride levels. This study demonstrated no effect of gender on these three pathological changes that occur in this model of isoniazid-induced hepatotoxicity in rabbits.

Suspensions of isolated liver cells were prepared from rat livers perfused with Ca++-free buffer and 0.05% collagenase. Primary cell suspensions (containing both parenchymal and nonparenchymal liver cells) metabolized sulfadimidine, sulfanilamide, p-aminobenzoic acid, and isoniazid approximately at first order kinetics for at least 4 hr. Suspensions of parenchymal cells had the same metabolic capacity, although the metabolism of isoniazid proceeded at a somewhat reduced rate compared to the primary cell suspensions. Suspensions of nonparenchymal cells did not metabolize sulfadimidine, sulfanilamide, or p-aminobenzoic acid during 4 hr, although such suspensions acted upon isoniazid to some degree. It was concluded that parenchymal rat liver cells may metabolize (acetylate) all four drugs tested, whereas nonparenchymal cells metabolize only isoniazid to any considerable extent.

Sulphadimidine acetylation was determined in 110 cases in samples obtained from urine and blood. A trimodal distribution was observed by both the methods. The correlation co-efficient "r" for the two methods was 0.46.

The most important factor determining the speed with which isoniazid is eliminated from the body is the rate of its acetylation in the liver. There are large differences between individuals in the rates at which isoniazid is acetylated. Over 98% of subjects can be clearly characterized as being either rapid of slow acetylators. Among the many satisfactory procedures for determining the acetylator phenotype of subjects, the simple sulphamethazine method is probably the most convenient. The proportions of rapid acetylators among different populations vary from about 40% among those of European and South Indian descent to over 85% among Japanese and Eskimos. The isoniazid acetylator phenotype of tuberculosis patients treated with isoniazid-containing regimens is without prognostic significance when treatment is given daily and only of doubtful importance when weak twice-weekly regimens are employed. However if treatment is given on a once-weekly basis, the response of rapid acetylators is generally much less satisfactory than that of slow acetylators. Since isoniazid is eliminated from the body predominantly by metabolism its clearance is not greatly diminished in the event of renal failure. As a consequence there are no grounds for reducing the dosage of isoniazid given to patients with impaired renal function. The incidence of the commonly encountered asymptomatic increases in serum transaminases associated with isoniazid treatment is similar in rapid and slow acetylators. Clinically important hepatic toxicity manifested by jaundice only occurs in 1-2% of patients treated with isoniazid-containing regimens. Despite earlier suggestions to the contrary, it is no more common in rapid than among slow acetylators. Since all patients can be treated effectively and safely with standard isoniazid dosages, determining patients' isoniazid serum/plasma levels or acetylator phenotype is hardly ever worthwhile. By contrast, since poor patient compliance is a major cause of therapeutic failure, monitoring the regularity with which patients self-administer their prescribed isoniazid treatment using simple urine-test methods can be of considerable value.

N-acetyltransferase (NAT) have been demonstrated to be localized in sinusoïdal cells and not in hepatocytes. Acetylation activities increased after pretreatment with Zymosan (40 mg/kg, i.v., 24 h before phenotyping) as well in vivo and in isolated perfused liver and in cytosolic fractions prepared from several rats organs. These data favor the localization of NAT-activity in reticulo-endothelial system. Increased protein levels in liver, gut, spleen and lungs cytosolic fractions (100 000 g, 120 mn) 48 h after Zymosan administration, suggest and inducing effect of this drug on NAT activity.

Great intraindividual variation of N-acetyltransferase activity was found in human beings. Between nutritional factors and human or rat liver N-acetyltransferase activity a correlation has been observed. The proportion of rapid acetylators in human population correlates to fish protein supply (p less than 0.01). Laboratory rats of different inbred and outbred strains displayed altered enzyme activities when they were fed with different "standard diets". Furthermore, N-acetyltransferase is inhibited by a lot of naturally occurring compounds. In rats N-acetyltransferase activity values are polymorphically distributed; acetylation activity seems to be controlled by a mechanism different from man.

The pharmacokinetics of sulfamethazine (SMZ) was studied in 44 healthy volunteers. After an oral dose of 10 mg/kg we found the usual distribution of rapid and slow acetylators. The differences in the t1/2s between the phenotypes were not due to variations in renal clearance. Repeat studies at this dose showed very little intraindividual variation in t1/2s. After a 40 mg/kg dose there was an increase in the t1/2 of SMZ in every subject. The implications of these results on methods of phenotyping are discussed.

Human liver N-acetyltransferase (NAT) activity undergoes rapid inactivation after death. Thus, in vitro determination of acetylator phenotype in liver autopsies may be unreliable. In the present investigation, the relative stabilities of monomorphic and polymorphic NAT activity were compared in situ in human and rabbit liver of rapid and slow acetylator phenotype. In both phenotypes, the monomorphic NAT activity was considerably less stable than the polymorphic, enabling it to be utilized as an enzyme marker to assure reliable in vitro phenotype classification.

The influence of concomitant administration of isoniazid (INH) on the acetylation of sulphadimidine has been studied in 6 healthy volunteers, previously identified as having the fast acetylator phenotype. INH was administered in a slow release form (500 mg tablet) 1 hour before the sulphadimidine. Acetylation of sulphadimidine was measured in plasma 6 h after its intake and in urine collected between 5 and 6 hours. INH significantly decreased the acetylated fraction of sulphadimidine in plasma from 69.0 to 54.0 and in urine from 85.9 to 81.2%. This was reflected in a significantly higher plasma concentration of unconjugated sulphadimidine and reduced urinary excretion of acetylated sulphadimidine. It is concluded that concomitant administration of INH inhibits acetylation of sulphadimidine. Fast acetylators at the border line of discrimination, may be misclassified if phenotyped with sulphadimidine during concomitant administration of INH.

Drug acetylation polymorphism is of clinical importance in slow and rapid acetylators. There are therapeutic and toxic consequences of interindividual drug acetylation of, e.g. isoniazid, procainamide and various sulfonamides. For comparing pharmacokinetic and pharmacogenetic data it is necessary to establish a useful model in experimental animals: 1. We determined the half-lives of isoniazid and sulfadimidine in man and rats using a simple loading test and we compared these data with those of in vitro acetylation. In man the half-lives of both substances correlate very well and correspond to acetylation values in vitro. 2. There is no sufficiently good correlation of sulfadimidine concentration in blood and saliva. Furthermore we found no strict correlation between drug concentration in venous and capillary blood. This should be should be considered in pharmacogenetic research. 3. The sulfadimidine half-lives in male Sprague-Dawley and Wistar rat are similar. Both half-lives and acetylation rates in vivo are normally distributed. Sulfadimidine half-lives and the rate of acetylation in vitro do not correlate. Compared with human values the investigated rats are rapid acetylators. Young rats reach their total acetylation capacity in vivo 65 days post partum. 4. Many organs of the rat acetylate sulfadimidine (kidney, lung, intestinal mucosa) with the exception of the spleen. 5. Excess substrate inhibits acetylation of sulfadimidine in vitro. Results of further inhibition studies suggest two or more N-acetyltransferases. 6. Isoniazid inhibits the sulfadimidine acetylation in rat liver homogenates and vice versa. To sum up all data, Sprague-Dawley and Wistar rats seem to be a useful model for human sulfadimidine metabolism pharmacokinetic and, in part, pharmacogenetic studies. Furthermore drug analysis in human saliva can be helpful in pharmacogenetic and pharmacokinetic research in the future.

The effects of rifampicin and isoniazid on liver function have been studied in 63 patients with pulmonary tuberculosis; 29% showed abnormalities of serum aspartate aminotransferase (SGOT) and a similar percentage abnormalities of serum bilirubin. These usually occurred during the first 12 weeks of therapy. The average duration of the abnormalities was 14(1/2) days, irrespective of whether treatment was interrupted or not.The relationship between raised SGOT and acetylator phenotype in a small number of patients suggests that those with raised SGOT are usually slow acetylator phenotypes. It seems that hepatic reactions in patients with previously normal liver function are usually mild and non-specific. However, patients who continue with rifampicin should be kept under close biochemical observation.

Investigations in the rabbit have indicated the existence of more than one N-acetyltransferase (EC 2.3.1.5). At least two enzymes, possibly isoenzymes, were partially characterized. The enzymes differed in their tissue distribution, substrate specificity, stability and pH characteristics. One of the enzymes was primarily associated with liver and gut and catalysed the acetylation of a wide range of drugs and foreign compounds, e.g. isoniazid, p-aminobenzoic acid, sulphamethazine and sulphadiazine. The activity of this enzyme corresponded to the well-characterized polymorphic trait of isoniazid acetylation, and determined whether individuals were classified as either ;rapid' or ;slow' acetylators. Another enzyme activity found in extrahepatic tissues readily catalysed the acetylation of p-aminobenzoic acid but was much less active towards isoniazid and sulphamethazine. The activity of this enzyme remained relatively constant from individual to individual. Studies in vitro and in vivo with both ;rapid' and ;slow' acetylator rabbits revealed that, for certain substrates, extrahepatic N-acetyltransferase contributes significantly to the total acetylating capacity of the individual. The possible significance and applicability of these findings to drug metabolism and acetylation polymorphism in man is discussed.