Biomedical subjects
Publications and source records attributed to T Inaba.
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A genetic polymorphism characterized by deficient drug oxidation exists for the hydroxylation of mephenytoin. This deficiency was first recognized in a family study that suggested an autosomal recessive pattern of inheritance. To confirm the observation, we investigated 28 relatives of five poor metabolizers. Subjects ingested 50 mg of mephenytoin, and the 24-hr urine was analyzed for hydroxymephenytoin. The pedigree data shown here provide strong evidence that deficient mephenytoin hydroxylation is an autosomal recessive trait.
The genetically controlled mephenytoin p(4')-hydroxylation capacity was determined in 118 Caucasians and 70 Orientals. After an oral dose of 50 or 100 mg of racemic mephenytoin, the amount of p(4')-hydroxymephenytoin in 24 h urine was measured by gas chromatography. Bimodal distribution was found with 9/70 (13%) Orientals and 5/118 (4%) Caucasians demonstrating deficient p(4')-hydroxylation. The statistically significant difference between Orientals and Caucasians (P less than 0.05) was accounted for by the high incidence of poor metabolizers among the Japanese subjects, 7/31 (23%). The frequency among Chinese subjects, 2/39 (5%), was similar to the frequency among Caucasians.
The regulatory role of cAMP-dependent protein kinase in steroidogenesis was examined in luteal cell mitochondria prepared from heavily luteinized prepubertal rat ovaries. The cAMP-dependent protein kinase, localized in luteal mitochondria, comprised 5.5% of the total cellular protein kinase activity (cAMP-dependent). Intact mitochondria supported by a suitable electron-donor substrate and inhibited by isoxazole converted cholesterol to a single steroid product, pregnenolone. Neither (Bu)2 cAMP nor a crude preparation of cytosolic protein kinase stimulated pregnenolone production from cholesterol when added to intact luteal cell mitochondria; however, mitochondria treated with 10 mM CaCl2 became responsive to both (Bu)2 cAMP and protein kinase by showing increased pregnenolone production. Likewise, the addition of cytosol protein kinase to incubations of cholesterol and crude cholesterol sidechain cleavage enzyme (cytochrome P-450cscc) isolated from luteal mitochondria, also stimulated pregnenolone production. Cholesterol-poor mitochondria, depleted of endogenous sterol by prolonged preincubation, when subsequently incubated with Ca+2 plus (Bu)2 cAMP and protein kinase showed significantly increased pregnenolone production. Conversely, mitochondria with greatly increased intramitochondrial cholesterol after preincubation with 200 microM cholesterol and a cytochrome P-450cscc inhibitor (aminoglutethimide) synthesized pregnenolone in significantly higher amounts than either normal or cholesterol-poor mitochondria after removal of the aminoglutethimide block. However, addition of (Bu)2cAMP or protein kinase to Ca+2-treated cholesterol-rich mitochondria failed to additionally stimulate pregnenolone synthesis. We conclude from these observations that the mitochondrial membrane normally excludes protein kinase and (Bu)2cAMP from any stimulatory action on cholesterol side-chain cleavage. Disruption of the mitochondrial membrane by high Ca+2 concentrations eliminates this barrier and permits (Bu)2cAMP and protein kinase stimulation of the CSCC enzyme system. The mechanism of stimulation is not clear. It could involve direct action on the CSCC enzyme. Alternatively, an increase in either intramitochondrial transport or binding of cholesterol substrate to the CSCC enzyme could be facilitated by protein kinase action. Direct stimulation of the enzyme by protein kinase seems less likely, since increased enzyme activity was not observed in the presence of high concentrations of intramitochondrial cholesterol substrate.
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The rate of oxidation of sparteine by the 9000 x g supernatant fraction of a human liver was measured in the presence of various drugs which exert cardiovascular effects. Hexamethonium, ouabain, caffeine and isoproterenol had no effect on this rate, while alprenolol, metoprolol, oxprenolol, propranolol, timolol, pindolol, lidocaine, mexiletine, 17-n-pentyl-sparteine, tolazoline, quinine, quinidine, cinchonine and cinchonidine inhibited the in vitro reaction competitively. Stereoselective inhibition was observed between quinine (Ki = 15 microM) and quinidine (Ki = 0.06 microM). Genetic evidence suggests that the primary metabolism of sparteine depends on a single species of cytochrome P450. In vitro competitive inhibition of sparteine oxidation by a drug indicates that this drug is capable of occupying the same enzymatic site as sparteine. This may mean that the competing drug is also metabolized at that site and thereby subject to the same genetic variation as sparteine's oxidation; absence of inhibition excludes this possibility.
A mobile unit for mass screening of prostatic disease using transrectal ultrasonotomography was developed in December 1980. In the following years we screened 1,396 males over 55 years of age. We have detected 463 cases [33.2%] of benign prostatic hypertrophy [BPH] and eight cases [0.6%] of prostatic cancer. The detection rate of BPH and malignancy of the prostate was shown to be very high. Thus, the unit promises to be of value in preventative medicine projects for prostatic disease in Japan.
The frequency of genetically deficient hydroxylation of mephenytoin (M-defect) was studied in 83 healthy Caucasians living in Toronto. The M-defect was compared with the widely studied genetic polymorphism of sparteine/debrisoquine oxidations (S-defect). After ingestion of mephenytoin and sparteine, urine samples (0 to 24 hr) were analyzed for p(4')-hydroxymephenytoin and urine samples over 0 to 12 hr were analyzed for sparteine and 2-and 5-dehydrosparteine by gas chromatographic methods. Nirvanol, the N-demethylation product of mephenytoin, was determined by a newly developed gas chromatographic/mass spectrometric method. Frequency distributions of both p-hydroxymephenytoin and dehydrosparteine excreted in urine were discontinuous (bimodal), while nirvanol and sparteine data were normally distributed. Two poor metabolizers of mephenytoin excreted 2% to 3% of the dose as p-hydroxymephenytoin and excreted normal amounts of nirvanol, but they were extensive metabolizers of sparteine. Six poor metabolizers of sparteine were found to be extensive metabolizers of mephenytoin (34% to 42% excreted in urine as p-hydroxyme-phenytoin). Thus the M-defect occurs among Canadian Caucasians with a frequency of 2% (0.0% to 7.5% with a confidence limit of 99%) and is independent of the S-defect.
Recent population data suggest independence of the genetic polymorphisms in mephenytoin and sparteine/debrisoquine oxidation. We used human liver preparations to test whether mephenytoin competes with sparteine for binding to the genetically variable cytochrome P-450, which mediates metabolism of both sparteine and debrisoquine. Mephenytoin failed to inhibit in vitro sparteine oxidation. This provides biochemical evidence that the polymorphism of sparteine/debrisoquine metabolism is not related to that of mephenytoin.
A radiometric method to assay debrisoquine 4-hydroxylase activity in human liver microsomes was established. Following incubation with 14C-labelled debrisoquine, unreacted debrisoquine was extracted with chloroform; 4-hydroxydebrisoquine was derivatized with hexafluoroacetylacetone, extracted, and subjected to high performance thin-layer chromatography (HP-TLC) followed by liquid scintillation counting. The Km values for debrisoquine 4-hydroxylase were 70 and 120 microM and the Vmax values were 8 and 24 pmol per milligram microsomal protein per minute. By application of this assay, it was possible to show that 4-hydroxydebrisoquine formation was competitively inhibited by sparteine. Antipyrine up to a concentration of 4 mM had no effect.
The present study compares the debrisoquine monooxygenase and the sparteine monooxygenase activities of human liver microsomes. In the presence of 14 competitive inhibitors, apparent inhibition constants (Ki) as determined by these two activities ranged over four orders of magnitude with a correlation coefficient 0.99. These in vitro results represent the strongest evidence to date that the debrisoquine monooxygenase and the sparteine monooxygenase are identical and involve a single isozyme of cytochrome P-450.
The disposition and metabolism of iodochlorhydroxyquin (clioquinol), an amebicidal drug with neurotoxic properties, were studied in dogs and rats with 14C-labelled drug. Pharmacokinetic studies in the dog demonstrated that the compound was well absorbed; the bioavailability was 36% of the dose of 1 mg/kg. The serum half-life was 1.3-1.8 h. In both the dog and the rat, biliary excretion was a major route of elimination. The dog excreted 27% of an intravenously administered dose (1 mg/kg) in the bile within 2 h; the rat excreted 39% of the dose (5 mg/kg i.v.) in less than 3 h. Elimination via the renal route was also substantial in both species. Urinary and biliary metabolites were separated by TLC (thin layer chromatography) and identified as sulfate and glucuronide conjugates in both species. No evidence for any other metabolites was found. A significant difference was observed between the dog and the rat in the extent of conjugation; the percentage radioactivity in the urine accounted for by the unchanged compound was six to twenty times greater for the dog than for the rat. The species differences in the disposition and metabolism of the compound might explain its greater toxicity in the dog than in the rat.
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Automatic calculation of control charts for precision and accuracy of radioimmunoassay was reported by Faure, et al. Duplicate control samples independently measured was assumed to have a bivariate normal distribution. In this case they assumed that the correlation coefficient between each value of the pairs of control samples is zero. Our experience using this method revealed that a considerable number of assayed samples distributed outside the calculated control limits in case of "accuracy control". It was considered that this happened because in radioimmunoassay the between-assays precision is usually larger than the within-an-assay precision and there is a significant correlation between values of duplicates. We also found equal probability density did not make a true circle but a long circle. Therefore in the present paper we proposed for control charts of radioimmunoassay an equal probability long circle calculated by bivariate analysis of Mahalanobis' generalized distance. It was found that a Mahalanobis' long circle could explain the density distribution of radioimmunoassay with a reasonable percent of samples outside the calculated control limits. What happened here can be interpreted by a large between-assays variability shown by some commercial kits. This automatic calculation method could be applied not only for quality control but also for evaluation and comparison of radioimmunoassay system or commercial kits. Control survey could also be analyzed by such a method.