Yoshiro Miura

A new analytical method for stereoisomers of methamphetamine and amphetamine and its application to forensic toxicology

We have developed a rapid and sensitive high-performance liquid chromatographic method for the determination of low concentrations of the stereoisomers of methamphetamine (MAMP) and the demethylated metabolite, amphetamine (AMP). The acetyl derivatives of the stereoisomers (d and l-form) of MAMP and AMP smuggled by gangsters or extracted from biological specimens (hair) of drug addicts were clearly separated on two stereoisomer-analytical columns (Chiralcel OB and OJ, Daicel Ind. Co., Japan) connected in series at 50 degrees C. The mobile phase was a mixture of n-hexane and isopropanol (9:1, v/v) and the UV detector was set at 220 nm. The practical limit of sensitivity for the analysis was 62.5 ng of the stereoisomers of MAMP and AMP. Our analytical method for MAMP and AMP in human hair is very useful for the diagnosis of abuse of the drug. The determination of the d/l ratio of MAMP and AMP (the fingerprint of MAMP and AMP) is applicable to the investigation of smuggling routes of these drugs.

The biological significance of ω-oxidation of fatty acids

The author focuses on the biological significance of ω-oxidation of fatty acids. Early studies revealed that there is a subsidiary pathway for β-oxidation of fatty acids when β-oxidation is blocked. Many studies demonstrated that the ω-oxidation serves to provide succinyl-CoA for the citric acid cycle and for gluconeogenesis under conditions of starvation and diabetes. Acylglucosylceramides which are composed of linoleic acid, long chain ω-hydroxy fatty acids, eicosasphingenine (or trihydroxyeicosasphingenine) and glucose, are responsible for normal epidermal permeability function in the skin. It is observed that ω- and (ω-1)-oxidation of fatty acids are related to energy metabolism in some laboratory animals such as musk shrews and Mongolian gerbils. Studies confirmed that ω- and (ω-1)-oxidation of fatty acids play crucial roles in the production of insect pheromones of honeybees and in the formation of biopolyesters of higher plants. In addition, the biological significance of ω-oxidation of prostaglandins and leukotrienes is described.

Effect of inhibitors on ω-and (ω-1)-hydroxylation of lauric acid by frog liver microsomes

To investigate the involvement of different cytochrome P-450 monooxygenases in fatty acid hydroxylation in frog liver microsomes, the effect of various inhibitors of cytochrome P-450 monooxygenases on the ω- and (ω-1)-hydroxylation of laurate was examined. The ω/ω-1-hydroxylation ratios were changed significantly by various levels of carbon monooxide (CO) inhibition; the formation of ω-hydroxylaurate was more sharply inhibited by various levels of CO than was the formation of (ω-1)-hydroxylaurate. On the contrary, metyrapone inhibited only the formation of (ω-1)-hydroxylaurate and stimulated the formation of ω-hydroxylaurate. 7,8-Benzoflavone as well as CO was more inhibitory to the ω-hydroxylation of laurate. At low concentrations of KCN (0.2 and 0.1 mM), the (ω-1)-hydroxylase activity was stimulated, but both the ω- and (ω-1)-hydroxylase activities were inhibited at the higher concentrations (5–10 mM). The effect of drugs and hydroxylaurate isomers on the ω- and (ω-1)-hydroxylation was also examined. Aminopyrine showed a stimulative effect on ω-hydroxylase activity and no effect on the (ω-1)-hydroxylase activity, whilep-nitroanisole inhibited the (ω-1)-hydroxylase activity and showed almost no effect on the ω-hydroxylase activity. 12-Hydroxylaurate inhibited both the ω- and (ω-1)-hydroxylase activities, but the ω-hydroxylase activity was inhibited to a much greater extent. 11-Hydroxylaurate had no effect on either hydroxylation. These findings strongly support the hypothesis that different cytochrome P-450 species are involved in the hepatic microsomal hydroxylation of laurate at ω- and (ω-1)-positions in the frog.

Effect of detergents on the microsomal cytochrome P-450-dependent monooxygenases from frog and rat liver

Abstract 1. 1. A frog liver microsomal hydroxylase system was more susceptible to inhibition by five detergents (sodium cholate, sodium deoxycholate, Triton X-100, Emulgen 913 and Tween 20) in the assay for laurate hydroxylation than the rat liver microsomal hydroxylase system. This suggests that liver microsomal cytochrome P-450-dependent monooxygenases from different sources may not have the same response to detergents. 2. 2. Emulgen 913 was the most effective detergent for the solubilization of cytochrome P-450 from frog and rat liver microsomes among the five detergents. 3. 3. The average level of cytochrome P-450 as determined in the frog ( 0.401 ± 0.023 nmol/mg protein ) is lower than that in the rat (0.60 or 1.19 ± 0.10 ), rabbit ( 1.45 ± 0.35 ), swine ( 0.59 ± 0.16 ), and approximately the same as in the human ( 0.39 ± 0.14 ) and the cat ( 0.380 ± 0.085 ).

Oxidation of Fatty Acids by Kidney Microsomes of Musk Shrew (Suncus murinus)

Substrate specificity and other properties of a fatty acid monooxygenase system in kidney microsomes of the Japanese house musk shrew (Suncus murinus) were examined. The suncus kidney microsomes catalyzed the hydroxylation of various saturated and unsaturated fatty acids to the omega- and (omega-1)-hydroxy derivatives. Laurate was most effectively hydroxylated among saturated and unsaturated fatty acids. The specific activity (53.79 +/- 5.59 [mean +/- SD, n = 6] nmol/nmol cytochrome P450/min) of laurate in suncus kidney microsomes was very high compared with that in liver and kidney microsomes of other species. C18 unsaturated fatty acids were converted to epoxides by a cytochrome P450-dependent fatty acid monooxygenase system in suncus kidney microsomes, in addition to omega- and (omega-1)-hydroxylation products. The monooxygenase system metabolized arachidonic acid only to omega- and (omega-1)-hydroxylation products, not to epoxidation products.

Detergent Induced Changes in Serum Lipid Composition in Rats

The influence ofin vivo administration of detergents on serum lipid composition was studied in rats. Male Wistar rats received 50 mg Emulgen 913 (polyoxyethylene nonylphenylether, a nonionic detergent) or SDS (sodium dodecylsulfate, an anionic detergent) per kg of body weight intraperitoneally for 3 consecutive days. Emulgen 913 and SDS administration increased the level of cholesterol esters and phospholipids, respectively. But Emulgen 913 administration reduced the level of triglycerides in the Serum, and SDS administration reduced also the levels of triglycerides and cholesterol esters. In spite of the changes in serum lipid composition, the administration of these detergents did not affect the amount of total lipids in rat serum. The proportion of palmitic, oleic, and docosahexaenoic acids in phospholipids was decreased by the administration of Emulgen 913 while the level of arachidonic acid was raised. However, the level SDS administration had no effect on the fatty acid composition of the serum phospholipids. On the other hand, both Emulgen 913 and SDS administration showed an effect on the fatty acid composition of triglycerides. It is postulated that liver damage due to administration of detergents is responsible for the changes in serum lipid and fatty acid composition in detergent-treated rats.

ω- and (ω-1)-hydroxylation of 1-dodecanol by frog liver microsomes

Frog liver microsomes catalyzed the hydroxylation of 1-dodecanol into the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation rate for 1-dodecanol was much lower than that for lauric acid. Both NADPH and O2 were required for hydroxylation activity. NADH had no effect on the hydroxylation. The hydroxylating system was inhibited 49% by CO at a CO∶O2 ratio of 4.0. The formation of ω-hydroxydodecanol was more sharply inhibited by CO than was the formation of (ω-1)-hydroxydodecanol, implying that more than one cytochrome P-450 was involved in the hydroxylation of 1-dodecanol and that CO has a higher affinity for the P-450 catalyzing the ω-hydroxylation. The formation of laurate during the incubation of 1-dodecanol with frog liver microsomes suggests that a fatty alcohol oxidation system is also present in the microsomes. NAD+ was the most effective cofactor for the oxidation of 1-dodecanol and NADP+ had a little effect. Pyrazole (an inhibitor of alcohol dehydrogenase) had a slight inhibitory effect on the oxidation and sodium azide (an inhibitor of catalase) had no effect.

Comparative aspects of microsomal cytochrome P-450-dependent monooxygenase activities in musk shrew (Suncus murinus), mongolian gerbil (Meriones unguiculatus), harvest mouse (Micromys minutus) and rat

1. The cytochrome P-450 content (0.75 +/- 0.13 nmol/mg microsomal protein) in musk shrew (suncus, Suncus murinus) liver microsomes was lower than that (1.30 +/- 0.26) in rat liver microsomes, but it is approximately the same level as in the Mongolian gerbil (Meriones unguiculatus, 1.18 +/- 0.14), harvest mouse (Micromys minutus, 1.11 +/- 0.02) and rat. 2. The hydroxylation activity (based on cytochrome P-450) of medium-chain fatty acids (otanoic, decanoic, lauric and tridecanoic acids) is much higher in suncus, Mongolian gerbil and harvest mouse than in rat, with the exception of the activity of decanoic and tridecanoic acids in Mongolian gerbil. 3. This suggests that cytochrome P-450 species catalyzing the hydroxylation of medium-chain fatty acids are present in these laboratory animals in higher concentrations. 4. The aminopyrine N-demethylation activity based on microsomal protein or cytochrome P-450 in suncus is significantly lower than that in rat, but the N-demethylation activity in Mongolian gerbil and harvest mouse is approximately 1.7-2.0-fold greater than that in rat.

Hydroxylation of fatty acids and alcohols by hepatic microsomal cytochrome P-450 system from the Mongolian gerbil

The liver microsomes of the Mongolian gerbilMeriones unguiculatus catalyzed the hydroxylation of various saturated fatty acids (C8−C18), alcohols (C12 and C16) and hydrocarbon (C12) to the corresponding ω- and (ω-1)-hydroxy derivatives. Lauric acid was hydroxylated most effectively among saturated fatty acids and the order of activity as hydroxylation substrates was C12>C14>C13>C16>C10>C18>C8. The specific activity of laurate hydroxylation (5.99 nmol/mg microsomal protein/min) in gerbil liver microsomes was higher than that observed in other species. 1-Dodecanol was also hydroxylated very effectively (4.58 nmol/mg microsomal protein/min) by gerbil liver microsomes, but in general the hydroxylation rates for fatty alcohols were much lower than those for the corresponding acids. It was found from both inhibitor and cofactor studies that the enzyme catalyzing the hydroxylation of fatty acids and alcohols in the liver microsomes of the Mongolian gerbil was a typical cytochrome P-450-linked monooxygenase, and at least two different cytochrome P-450 species were involved in the hydroxylation.

Seasonal change and sex difference in drug-metabolizing activity in frog liver microsomes.

The drug-metabolizing activities (aminopyrine N-demethylase and p-nitroanisole O-demethylase activities) have been measured at monthly intervals throughout the year in liver microsomes of male and female Japanese bullfrogs, Rana catesbeiana. The aminopyrine N-demethylase activity based on cytochrome P-450 of both sexes was significantly higher in May-July (spring-summer) than in August-October (summer-autumn). On the contrary, the p-nitroanisole O-demethylase activity based on cytochrome P-450 of males was significantly higher in July-October (summer-autumn) than in May-June (spring). However, there was no seasonal changes in the O-demethylase activity in females. There were significant differences between males and females in the N-demethylase activity in August-October and in the O-demethylase activity in May-June (or July-October).