Aniline Hydroxylation Research Articles

Changes in certain kinetic properties of hepatic microsomal aniline hydroxylase and ethylmorphine demethylase associated with postnatal development and maturation in male rats.

1. Changes in certain kinetic properties (V(max.) and apparent K(m)) of hepatic microsomal mixed-function oxidases have been studied as a function of postnatal development and maturation in male rats. 2. Microsomal cytochrome P-450 content changed only slightly between 1 and 12 weeks of age. 3. Aniline hydroxylase activity (V(max.)) increased abruptly between 1 and 2 weeks of age to greater than adult activities and then returned to a plateau value between 4(1/2) and 12 weeks of age. Ethylmorphine demethylase activity remained low and relatively constant between 1 and 3 weeks of age and then increased markedly ( approximately 100%) between 3 and 4(1/2) weeks. 4. The apparent Michaelis constant (K(m)) for aniline hydroxylation increased almost linearly with time between 1 and 6 weeks of age and tended to reach a plateau value thereafter. The apparent K(m) for ethylmorphine demethylation increased between 1 and 3 weeks of age and then decreased abruptly to a constant value between 6 and 12 weeks. 5. The data indicate that developmental changes in the activity of these microsomal oxidases do not correlate temporally with each other or with changes in microsomal cytochrome P-450 content. 6. The most dramatic changes in enzyme activity were associated with early development (1-3 weeks) and weaning (3-4 weeks). 7. Changes in weight of seminal vesicle, a criterion of sexual maturation in male rats, were most prominent between 6 and 8 weeks of age and thus appeared to be separated in time from the prominent changes in enzyme activity.

Microsomal hydroxylation of decane

1. 1. Aliphatic hydrocarbons (hexane, octane and decane) were oxidized in the microsomes from the livers of mouse, rat, rabbit, beef, pigeon and chick embryo. 2. 2. The oxidation of decane by mouse liver microsomes required NADPH and O 2. Decanol, decanoic acid, and decamethyleneglycol were identified as the major decane oxidation products, suggesting that the decane oxidation is initiated by the hydroxylation of decane to decanol. 3. 3. The Michaelis constant for the decane hydroxylation was 0.5 mM. The pH optimum was approx. 8.1. The reaction was strongly inhibited by CO, SKF 525-A, or tert.-butyl isocyanide. The decane hydroxylation system was considerably unstable, in comparison with the decanoate ω-oxidation system. These results suggest that the two hydroxylation systems are not identical. 4. 4. As with drug hydroxylation, the activity of decane hydroxylation was increased markedly by treatment of rats with phenobarbital. However, unlike aniline hydroxylation, the decane hydroxylation was highly active in the microsomes from kidney and lung as well as in those from liver.

Relationship between the interconversion of cytochrome P-450 and P-420 and its activities in hydroxylations and demethylations by P-450 oxidase systems

The relationship between the interconversion of microsomal cytochrome P-450 and P-420 and the hydroxylation and demethylation activities of the P-450 oxidase system was studied. The hydroxylation and demethylation activities could only be partly restored by complete reconversion of P-420 to P-450 by addition of polyols by dilution or by washing. The P-420 produced by detergents, monohydric alcohols or anilines did not retain hydroxylation and demethylation activities. Cytochrome P-450 is not rate limiting in the overall reaction for hydroxylation and demethylation of compounds such as anilines, nitroanisoles and aminopyrine. The substrate specificities of the hydroxylations and demethylations by the P-450 oxidase systems of different tissues differ. The rate of hydroxylation of anilines at the p-position was influenced by not only the hydrophobic character, but also by electronic and steric effects of the substrate. The magnitude of the spectral changes of the microsomes induced by the substrate corresponded to the ESR signal height at g m = 2.25 of microsomal Fe x rather than to the absorption peak of the CO complex of P-450 at 450 mμ. The hydroxylation and demethylation activities were observed with either NADPH or NADH. Evidence is presented that microsomal NADPH-cytochrome c reductase is reduced by NADH and the K m for the reductase with NADH is greater than the K m for NADPH.

The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells.

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3-5), per mug. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1.7) or per unit of NADPH-cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH-cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.

Acetone enhancement of microsomal aniline para-hydroxylase activity

Acetone in concentrations of 0.045–1.8 m markedly enhanced the para-hydroxylation of aniline by hepatic microsomal fractions derived from rats, mice, rabbits and dogs. Similarly acetone enhanced the hydroxylation of acetanilide and N-butylaniline but not the N-demethylation of ethylmorphine, N-methyl or N,N-dimethylaniline nor the O-demethylation of p-nitroanisole. The degree of enhancement increased with increasing pH. In the presence of acetone, both the Michaelis constant and maximal velocity for the hydroxylation of aniline increased. SKF 525-A and piperonyl butoxide were more potent inhibitors of aniline hydroxylation in the presence of acetone. Finally, evidence is presented that acetone produces its enhancement of aniline hydroxylation by a mechanism different from that of ethyl isocyanide.

The oxidation of drugs by fishes

1. 1. Fish liver microsomal systems have been found to catalyze the hydroxylation of aniline and acetanilide, the N-demethylation of aminopyrine and the O-dealkylation of phenacetin. 2. 2. These systems are similar to the corresponding mammalian enzymes and they may be considered to be mixed function oxidase since they require NADPH and oxygen. An absolute requirement for oxygen, however, was difficult to demonstrate for the hepatic phenacetin cleavage system from fish. 3. 3. Microsomal drug metabolizing systems from fish have temperature optima which are considerably lower than those of corresponding mammalian systems.

On the use of nicotinamide in assays for microsomal mixed-function oxidase activity

Nicotinamide, an additive to almost all media used to study oxidative metabolism of substrates of hepatic microsomal mixed-function oxidase, is shown to inhibit the metabolism of known substrates of this system. Inhibition of aminopyrine demethylation appears to be competitive, while inhibition of aniline hydroxylation appears to be a mixture of competitive and noncompetitive inhibition. It is shown that an accurate assay of substrate metabolism can be obtained in the absence of nicotinamide if the assay time is kept short, NADPH concentration is maintained at a high level, and the concentration of microsomes in the medium is kept low.

Biochemical Studies on the Drug Metabolism (V)

The effects of food preservatives on the metabolism of drugs were investigated using female rats. Among five preservatives dehydroacetic acid (DHA) showed some stimulating effect on the metabolism of drugs. The pretreatment of female rats with 200mg/kg of DHA daily for 7 or 10 days shortened the duration of hypnosis by 100mg/kg of hexobarbital. The liver preparation from DHA-treated rat had higher activity of N-demethylation of aminopyrine, hydroxylation of hexobarbital or cyclobarbital, aromatic hydroxylation of aniline, and hydrolysis of parathion than control. DHA increased glycogen content but did not change the amount of crude fat in liver. Electronmicroscopical picture of liver from DHA-treated rat showed a marked proliferation of smooth-surfaced endoplasmic reticulum in the hepatic cell. These observations were discussed in connection with the inductive effect of some drugs on the metabolism of drugs.

Species differences in the hydroxylation of aniline and N-ethylaniline by liver microsomes.

The velocity of the N- and p-hydroxylation of aniline and N-ethylaniline by NADPH-dependent hydroxylases in guinea pig liver microsomes was found to be as high as in rabbit liver microsomes. Microsomes prepared from cat livers were less active.

THE EFFECT OF CARBON MONOXIDE ON THE HYDROXYLATION OF ANILINE AND N-ETHYLANILINE BY MICROSOMAL ENZYMES.

The N-hydroxylation of aniline and N-ethylaniline by NADPH-dependent enzymes in rabbit liver microsomes was not inhibited by carbon monoxide.

FURTHER FACTORS AFFECTING THE HYDROXYLATION OF ANILINE AND SOME OF ITS DERIVATIVES BY LIVER MICROSOMES.

FURTHER FACTORS AFFECTING THE HYDROXYLATION OF ANILINE AND SOME OF ITS DERIVATIVES BY LIVER MICROSOMES.

Effect of ascorbic acid in aniline poisoning.

ASCORBIC acid has been utilized extensively in large doses in the therapy of industrial methaemoglobinaemia, although Halsted1 commented that at developments greater than 60 per cent methaemoglobinaemia, it is ineffective because of its lack of ability to bring on rapidly a reversion mechanism. But the rapidity of reversion is only the first problem. The second problem to be settled is whether, in chemical cyanosis, the effect of ascorbic acid may not be restricted only to bring about the reduction. It is known that ascorbic acid promotes the rate of hydroxylation of aniline to p-aminophenol2, which is a potent agent in the production of Heinz bodies not only in vivo but also in vitro3, and ascorbic acid is able to produce Heinz bodies in vitro without any toxic agents4. We have found that in rats poisoned with aniline the formation of Heinz bodies is promoted by treatment with ascorbic acid.