Wendell W. Weber

Dose-dependent changes in sulfamethazine kinetics in rapid and slow isoniazid acetylators.

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 t½s between the phenotypes were not due to variations in renal clearance. Repeat studies at this dose showed very little intraindividual variation in t½s. After a 40 mg/kg dose there was an increase in the t½ of SMZ in every subject. The implications of these results on methods of phenotyping are discussed.

PURIFICATION AND PROPERTIES OF N‐ACETYLTRANSFERASE FROM MAMMALIAN LIVER

Acetylation is a major route for the metabolism of aromatic amines in many different species (Williams, 1959). Early in vitro studies of acetylation of sulfanilamide by rabbit liver slices were reported by Klein and Harris (1938). Acetylation of sulfanilamide by rabbit and pigeon liver homogenates was reported by Lipmann who used the acetylating enzyme system as an analytical tool in the investigations which led to the identification of coenzyme A (Lipmann, 1948). This system was also used by Lynen (1952) in his work on the isolation and identification of acetyl coenzyme A. Early studies of the properties of the acetylating enzyme itself indicated that the enzyme from pigeon liver catalyzes the acetylation of isoniazid ( INH) (Johnson, 1954) as well as sulfanilamide. Jenne and Boyer (1962) reported on the kinetic characteristics of the acetylation of INH by a pigeon liver extract. In their studies, the acetyl coenzyme A generating system, which had been used in earlier studies, was replaced by purified acetyl coenzyme A. They suggested that an acetyl-enzyme was formed in the acetylation of INH and they proposed a mechanism for this reaction. The properties of the acetylating enzyme system from human liver were reported by Evans (1962; Evans & White, 1964) from studies in which INH and various other substrates were utilized in investigations of the human acetylation polymorphism. He showed that there is a correlation between the rate of acetylation of 1NH i11 v i v o and the level of activity of the liver acetylating enzyme system. Frymoyer and Jacox (1963; 1963a) reported that a polymorphism in the rate of acetylation of INH exists in the rabbit. They found a correlation between the rate of acetylation in vivo and the level of activity in liver homogenates which was similar to the correlation found in man. Studies of partially purified human N-acetyltransferase prepared from liver of rapid and of slow inactivators of INH were reported by Jenne (1965). The purified enzyme preparations from the liver of slow inactivators of INH had less N-acetyltransferase activity than those from the liver of rapid inactivators. He found that the enzyme obtained from slow inactivators had kinetic and temperature stability characteristics similar to the enzyme obtained from rapid inactivators. He suggested that the difference between slow and rapid inactivator phenotypes “may be due to differences in amount of an identical enzyme molecule.” This report summarizes our studies of partially purified N-acetyl-transferase from 4 mammalian species-rabbit, human, monkey and rat-and from the slow INH-inactivator rabbit (Weber & Cohen, 1967, 1967a; Cohen & Steinberg, 1967; Weber et al., 1967).

Changes in the physicochemical characteristics of rabbit liver N-acetyltransferase during post-natal development

Abstract This study was undertaken to evaluate the characteristics of liver N -acetyltransferase, a drug conjugating enzyme, at different stages of development. This enzyme catalyzes the acetylation of isonicotinic acid hydrazide, various sulfonamides and other aromatic amines. Partially purified enzyme was prepared for these studies from the livers of 6–12-day-old, 3-week-old, and adult rabbits. One-week-old rabbits have less than 10% of the activity per g of liver found in mature animals. The relative activity increased to the adult level at about 4 weeks of age. Two distinguishing characteristics were noted in studies of enzyme from the youngest animals. These were: limiting K m for isonicotinic acid hydrazide, a kinetic parameter; and the activation energy for inactivation by heat, a physical characteristic. Enzyme from 3-week-old animals had the same limiting K m as that from adult animals. It is postulated that in addition to the different forms of N -acetyltransferase that exist in different tissues 1 there are different forms of the hepatic drug conjugating N -acetyltransferase that are specific for various developmental stages.

Isotope exchange studies on rabbit liver N-acetyltransferase

Abstract N -Acetyltransferase from mammalian liver catalyzes the acetylation of isonicotinic acid hydrazide, various sulfonamides and other aromatic amines. Initial velocity and product inhibition studies reported previously indicate that this reaction proceeds according to a simple ping pong BiBi mechanism. It follows that enzymatic acetylation of a drug should consist of two consecutive reactions, the first of which leads to formation of an acetyl-enzyme intermediate and the second to acetylation of the drug with regeneration of the enzyme. In the present study, the existence of these two reactions has been demonstrated by isotope exchange techniques and an acetyl- N -acetyltransferase intermediate capable of transferring acetyl groups to isonicotinic acid hydrazide has been isolated.

An improved micro spectrofluorometric assay for determining isoniazid in serum

Abstract A highly sensitive micro modification of a fluorometric procedure for measuring isoniazid levels in serum is described. This modification permits the use of as little as 25 μl of serum and simplifies the measurement of isoniazid in infants, primates, and small laboratory animals.