Further characterization of human microsomal 3alpha-hydroxysteroid dehydrogenase.

This manuscript reports further characterization of the recently discovered human short-chain alcohol dehydrogenase, proposed to oxidize 3alpha-androstanediol to dihydrotestosterone in testis and prostate (M. G. Biswas and D. W. Russell, 1997, J. Biol. Chem. 272, 15959-15966). Enzyme expressed using the Baculovirus System localized in the microsomal fraction and catalyzed oxidation and reduction of the functional groups on steroids at carbons 3 and 17. Autoradiography assays revealed that the enzyme was most efficient as a 3alpha-hydroxysteroid oxidoreductase. High affinity of the enzyme for NADH (Km of 0.18 microM), lack of stereospecificity in the reductive direction, and poor efficiency for 3beta- versus 3alpha-hydroxyl oxidation could account for the observed transient accumulation of 3beta-stereoisomers in the oxidative reaction. Consistent with the 65% sequence identity with RoDH dehydrogenases, the enzyme oxidized all-trans-retinol with the Km value of 3.2 microM and Vmax value of 1.2 nmol/min per milligram microsomes. 13-cis-Retinol and all-trans-retinol bound to the cellular retinol-binding protein were not substrates. Neurosteroid allopregnanolone was a better substrate than all-trans-retinol with the Km and Vmax values of 0.24 microM and 14.7 nmol/min per milligram microsomes. Northern blot analysis revealed that the corresponding mRNA was present in adult human brain (caudate nucleus, amygdala, hippocampus, substantia nigra, thalamus) and spinal cord in addition to other tissues. The message was also detected in fetal lung, liver, and brain. Antibodies against the enzyme recognized a protein of approximately 35 kDa in the particulate fraction of human tissues. This study presents new information about enzymatic properties, substrate specificity, and tissue distribution of this enzyme, and provides a better insight into its possible physiological function(s).

Binding and hydrolysis of meperidine by human liver carboxylesterase hCE-1.

Human liver carboxylesterases catalyze the hydrolysis of apolar drug or xenobiotic esters into more soluble acid and alcohol products for elimination. Two carboxylesterases, hCE-1 and hCE-2, have been purified and characterized with respect to their role in cocaine and heroin hydrolysis. The binding of meperidine (Demerol) and propoxyphene (Darvon) was examined in a competitive binding, spectrophotometric assay. The hCE-1 and hCE-2 bound both drugs, with Ki values in the 0.4- to 1.3-mM range. Meperidine was hydrolyzed to meperidinic acid and ethanol by hCE-1 but not hCE-2. The Km of hCE-1 for meperidine was 1.9 mM and the kcat (catalytic rate constant) was 0.67 min-1. Hydrolysis of meperidine by hCE-1 was consistent with its specificity for hydrolysis of esters containing simple aliphatic alcohol substituents. Hence, hCE-1 in human liver microsomes may play an important role in meperidine elimination. Propoxyphene was not hydrolyzed by hCE-1 or hCE-2. This observation is consistent with the absence of a major hydrolytic pathway for propoxyphene metabolism in humans.

The quantitative determination of R- and S-salsolinol in the striatum and adrenal gland of rats selectively bred for disparate alcohol drinking.

To explore the hypothesis that endogenous 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) might be involved in the etiology of alcoholism, its concentration was determined in the striatum and adrenal gland of rats bred selectively for disparate alcohol drinking. The alcohol-naive alcohol-preferring (P) and the high-alcohol-drinking (HAD) lines of rats demonstrated significantly lower striatal and adrenal salsolinol content when compared with the alcohol-non-preferring (NP) and the low-alcohol-drinking (LAD) lines. In the P-line of rats, 4 weeks of free-choice alcohol drinking had no significant effect on striatal salsolinol levels, although adrenal levels of salsolinol were significantly higher. The salsolinol assayed in the striatum of all lines of rats occurred as a racemic mixture of enantiomers that was unchanged following 4 weeks of alcohol exposure. Unlike striatal tissue, the adrenals of alcohol naive P-rats contained significantly more S- than R-salsolinol (ratio S/R = 83/17) and alcohol consumption resulted in the formation of a nearly racemic mixture of enantiomers. These results suggest a role for genetic factors in the formation of endogenous salsolinol and its potential regulation by short-term alcohol intake.

Simultaneous measurement of ethanol and ethyl-d5 alcohol by stable isotope gas chromatography-mass spectrometry.

Efforts to define gender- and ethnic-dependent differences in ethanol first-pass metabolism by gastric mucosa and liver have been limited by a lack of analytical tools that distinguish ethanol concurrently administered by oral and intravenous routes. A stable isotope gas chromatography-mass spectrometry method for simultaneous measurement of ethanol and ethyl-d5 alcohol in serum was developed to meet this need. The assay was linear from 1 to 30 mmol/L. The limit of quantification was 1 mmol/L. Analytical imprecision (CV) was <10%. Analytical recovery was >90%. Specificity was based on retention time and reproducibility of ion ratios. The assay was free from interference by other volatile alcohols. Simultaneous oral administration of ethanol and ethyl-d5 alcohol produced nearly identical pharmacokinetic profiles. Simultaneous oral ingestion of ethanol and intravenous infusion of ethyl-d5 alcohol, adjusted for gastric emptying time, revealed decreased bioavailability of ethanol by the oral route. The method described is sufficient to study the first-pass metabolism of ethanol.

Simultaneous determination of primaquine and carboxyprimaquine in plasma using high-performance liquid chromatography with electrochemical detection.

A selective and sensitive high-performance liquid chromatographic method with electrochemical detection is described for the simultaneous quantitation of primaquine and carboxyprimaquine, its primary metabolite, in plasma. After addition of internal standard, plasma was deproteinized by addition of acetonitrile. Nitrogen-dried supernatants, resuspended in mobile phase were analyzed on a C8 reversed-phase column. Limits of detection for primaquine and carboxyprimaquine were 2 and 5 ng/ml with quantitation limits of 5 and 20 ng/ml, respectively. None of 47 tested antimicrobial agents interfered. In contrast to previously reported methods, the assay sensitivity and specificity are sufficient to permit quantitation of primaquine in plasma for pharmacokinetics following low dose (30 mg, base) oral administration of primaquine, typically used in the treatment of malaria and Pneumocystis carinii pneumonia.

Effects of ethanol on cocaine metabolism: formation of cocaethylene and norcocaethylene.

The coabuse of cocaine and ethanol occurs with high frequency and increases the risk of cocaine-related morbidity and mortality. The mechanisms mediating the toxic interactions of cocaine and ethanol are not clearly defined. This study examined the effects of acute ethanol administration on the metabolism of cocaine in the male Wistar rat. Intraperitoneal administration of 2 g/kg ethanol 30 min prior to administration of 25 mg/kg cocaine resulted in the formation of two ethylated derivatives of cocaine, benzoylecgonine ethyl ester (cocaethylene) and benzoylnorecgonine ethyl ester (norcocaethylene) in liver, brain, and serum. Fifteen minutes after cocaine administration, the tissue levels of cocaethylene were 22, 10, and 9% of the cocaine recovered from liver, serum, and brain, respectively. Ethanol pretreatment increased cocaine concentrations in liver and benzoylnorecgonine concentrations in liver and serum. The increased morbidity and hepatotoxicity seen with acute combined administration of cocaine and ethanol may be due to the formation of the toxic ethylated and N-demethylated metabolites of cocaine. Ethanol pretreatment decreased benzoylecgonine concentrations in serum and liver. The most important consequence of ethanol-induced inhibition of the normally rapid hydrolysis of cocaine to benzoylecgonine may be a decrease in benzoylecgonine-mediated vasoconstriction.