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Multiple N-Acetyltransferases and Drug Metabolism TISSUE DISTRIBUTION, CHARACTERIZATION and SIGNIFICANCE of MAMMALIAN N-ACETYLTRANSFERASE by D

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Multiple N-Acetyltransferases and Drug Metabolism TISSUE DISTRIBUTION, CHARACTERIZATION and SIGNIFICANCE of MAMMALIAN N-ACETYLTRANSFERASE by D Biochem. J. (1973) 132, 519-526 519 Printed in Great Britain Multiple N-Acetyltransferases and Drug Metabolism TISSUE DISTRIBUTION, CHARACTERIZATION AND SIGNIFICANCE OF MAMMALIAN N-ACETYLTRANSFERASE By D. J. HEARSE* and W. W. WEBER Department ofPharmacology, New York University Medical Center, 550 First Avenue, New York, N. Y. 10016, U.S.A. (Received 6 October 1972) Investigations in the rabbit have indicated the existence of more than one N-acetyl- transferase (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 ofp-aminobenzoic acid but was much less active towards isoniazid and sulpha- methazine. 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 re- vealed that, for certain substrates, extrahepatic N-acetyltransferase contributes signifi- cantly to the total acetylating capacity of the individual. The possible significance and applicability ofthese findings to drugmetabolism and acetylation polymorphism in man is discussed. Liver N-acetyltransferase catalyses the acetylation purified by the same procedure and their pH charac- of a number of commonly used drugs and foreign teristics, heat stabilities, kinetic properties, substrate compounds such as isoniazid, sulphamethazine, specificities and reaction mechanisms are indis- sulphadiazine, p-aminobenzoic acid, diamino- tinguishable. Thus Jenne (1965) has proposed that diphenylsulphone and hydralazine in man and certain the same enzyme is present in the liver of both other mammalian species (Weber, 1971b). The act- phenotypes and that the slow acetylator has less of it. ivity of this enzyme varies widely between individ- The following observations are difficult to account uals. Geneticstudies with man (Evans et al., 1960) and for by this concept. rabbit (Frymoyer & Jacox, 1963a) have shown that (1) In man and rabbit some drugs are acetylated individuals are either 'rapid' or 'slow' acetylators. polymorphically whereas others are acetylated mono- Slow acetylators have low N-acetyltransferase activity morphically. In man, polymorphically acetylated and are homozygous for an autosomal recessive gene. drugs are isoniazid, sulphapyridine (Schroder & The identification of the acetylator phenotype of an Evans, 1972), sulphamethazine, hydralazine, diamino- individual as either rapid or slow has been clearly diphenylsulphone and probably also phenelzine (see demonstrated in population studies in which the Weber, 1971b). Monomorphically acetylated drugs ability of individuals to acetylate isoniazid (Evans include p-aminobenzoic acid, p-aminosalicylic acid et al., 1960), sulphamethazine (Evans & White, and sulphanilamide (see Weber, 1971b). Certain other 1964) and sulphadiazine (Frymoyer & Jacox, 1963a) drugs such as sulphamethoxypyridazine (White & has been measured and clear bimodal frequency- Evans, 1968) appear to follow the isoniazid-acetyla- distribution histograms have been obtained. This tion pattern to some extent. phenomenon has become known as the isoniazid- (2) Slow acetylators are especially susceptible to acetylation polymorphism. dose-related toxicity from polymorphically acetylated Previous studies (Jenne, 1965; Weber, 1971b) of drugs owing to their low acetylating capacity (Hughes the biochemical basis of the acetylation poly- et al., 1954; Devadatta et al., 1960; Evans et al., 1965; morphism have not shown any difference in the Perry et al., 1967). Susceptibility to adverse effects characteristics of N-acetyltransferases prepared from among slow acetylators, however, is subject to con- rapid- and slow-acetylator livers. The enzymes can be siderable variability. This is evident from the obser- * Present address: Department of Biochemistry, Im- vation that the incidence of toxicity among this perial College, London SW7 2AY, U.K. group is appreciably lower than their frequency in Vol. 132 520 D. J. HEARSE AND W. W. WEBER populations of patients exposed to these drugs Reagents (Harris, 1961). Isoniazid, sulphamethazine, sulphadiazine, p- (3) The difference in liver N-acetyltransferase aminobenzoic acid and acetyl-CoA were obtained activity in rapid and slow acetylators is much greater from Nutritional Biochemicals Corp., Cleveland, than the difference in their acetylating capacity in vivo. Ohio, U.S.A. N-1-(Naphthyl)ethylenediamine di- In the rabbit (Frymoyer & Jacox, 1963a,b), the ratio hydrochloride and routine laboratory chemicals were ofthe average drug-acetylation rates measured in vivo obtained from Fisher Scientific Co., Springfield, N.J., in rapid- versus slow-acetylator animals is small (2: 1) U.S.A. DEAE-cellulose was obtained from Nutri- compared with the ratio of drug-acetylating-enzyme tional Biochemicals Corp. activity measured in vitro (80:1) in liver samples from the two acetylator phenotypes. Much less infor- Determination ofacetylator phenotype mation from humans is available for comparison, but The half-life ofsulphadiazine in the blood ofrabbits from what is available a similar disparity appears to was determined by a modification of the method exist (Evans et al., 1960; Jenne, 1965). It should be described by Frymoyer & Jacox (1963a). Sulpha- recognized in connexion with these differences that diazine (20mg/kg body wt.) was administered intra- the values determined in vitro only take into account venously via a marginal vein in one ear and blood the metabolic contribution of the liver whereas the samples (0.l00ml) were obtained at approx. 20, 30,40, values in vivo reflect the contribution of extrahepatic 50, 60 and 70min after injection. Experimental as well as hepatic drug-metabolizing systems. In animals in which the half-life for sulphadiazine was addition, the value in vivo is further affected by greater than 70-80min were classified as slow renal mechanisms of elimination of drug from the acetylators and those in which the half-life was body. shorter than 35min were classified as rapid acetyl- These observations indicate that individual vari- ators. It has been shown (Frymoyer & Jacox, 1963a) ability in the rate and pattern of drug acetylation that slow acetylators are characterized by a half-life depends on factors additional to the isoniazid-acetyl- for sulphadiazine of greater than 60-70min. ator status. The drug-acetylating enzymes of liver clearly make an important contribution to the total Enzyme purification acetylating capacity ofsome individuals. Investigating the importance ofrenal factors, Jenne etal. (1961) sug- Animals were killed (by injection of 20ml of air gested that they are likely to be small compared with into a marginal ear vein) and tissues were removed, the acetylation of the drug. As such, renal factors homogenized (25%, w/v) in 0.1M-sodium pyro- would probably not account for the disparity between phosphate buffer, pH6.8 at 4°C, and centrifuged at methods for assessing the acetylating capacity of an lOOOOOg for 60min; the supernatant fraction was individual in vivo and in vitro. Several investigators retained for analysis. Additional purification of the (Evans & White, 1964; Motulsky, 1964; Jenne et al., enzyme was required for some studies and was carried 1961) have obtained evidence for the existence of out as described by Weber (1971a). For the prepara- drug-acetylating enzymes in extrahepatic tissues and tion of enzyme from the gut, the duodenum was they have suggested that they may be important in removed, washed, incised longitudinally and the explaining this disparity, but a comprehensive study mucosal tissue was scraped away with a sharp scalpel of these enzymes and their possible significance in blade. The muscular gut-wall tissue was discarded the acetylation of drugs was not carried out. and the mucosa preparation was homogenized as The studies reported here were designed to deter- described above. mine: (a) whether more than one N-acetylating en- zyme contributes to the total drug-acetylating capa- Determination ofacetylating activity in vitro city of the rabbit; (b) the relative importance of In these studies two substrates, sulphamethazine hepatic and extrahepatic N-acetylating enzymes to and p-aminobenzoic acid, were used. In contrast with the total drug-acetylating capacity oftissues obtained the determinations in vivo and to obtain maximal from rapid and slow isoniazid-acetylator rabbits; assay sensitivity sulphamethazine was used in (c) the distribution and some of the characteristics of preference to sulphadiazine, additional studies partially purified N-acetylating activity in the tissues (D. J. Hearse & W. W. Weber, unpublished work) ofrapid and slow isoniazid-acetylator rabbits. having demonstrated that there was a close correla- tion between the results obtained with sulphadiazine Experimental and sulphamethazine. Purified samples (1000OOg-supernatant fraction) Animals were suitably diluted and assayed for sulphameth- New Zealand White rabbits (5
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