Steven R. Mason

Fluorimetric detection of serum corticosterone using high-performance liquid chromatography

A simple, sensitive, specific and reproducible method for the determination of corticosterone concentrations in rat serum using high-performance liquid chromatography (HPLC) with fluorimetric detection is described. Corticosterone is detectable down to 0.1 ng injected onto the HPLC column. Cortisol is used as an internal standard. Ethyl acetate was used for both initial serum corticosteroid extraction and the subsequent fluorophore extraction after sulfuric acid hydrolysis; thus sulfuric acid does not enter the HPLC system. The resultant fluorophores for both corticosterone and cortisol are stable for at least two weeks at ambient temperature not requiring storage at -20 degrees C. The procedure is highly suitable for use with HPLC systems utilising automatic sample injectors. The method is specific for corticosterone; dexamethasone, cortisone and gonadal steroids are not detectable and do not interfere in this assay.

Evidence for induction of hepatic microsomal cytochrome P-450 by cimetidine: Binding and kinetic studies

The interaction of cimetidine with liver microsomes has been examined by spectral and equilibrium partition studies. First, difference spectroscopy has been used to evaluate the proportion of cytochrome P-450 in rat liver microsomes that exhibits an affinity for cimetidine in the pharmacologically relevant, low micromolar range of drug concentration. The value of 0.45 so obtained has confirmed that a substantial proportion of rat liver cytochrome P-450 has a high binding affinity for this drug. Second, a study of the binding of cimetidine to human liver microsomes by difference spectroscopy and partition equilibrium has detected a similar interaction, thus providing direct support for the postulate that the clinically observed impairment of oxidative drug metabolism may be due in part to inhibition of cytochrome P-450 monooxygenase by cimetidine. Hepatic microsomes from cimetidine-pretreated rats have been shown to exhibit elevated cytochrome P-450 specific content but a decreased proportion of sites with high affinity for the drug; this finding has been shown not to be the consequence of cimetidine-mediated, time-dependent, irreversible monooxygenase inhibition. Although cimetidine pretreatment caused enhanced specific activity of 7-ethoxyresorufin O-dealkylation, the specific activities for O-dealkylation of 7-ethoxycoumarin and 4-nitroanisole were decreased, as were those for the N-dealkylation of morphine, ethylmorphine, aminopyrine, and dimethylnitrosamine. Since cimetidine pretreatment was shown to cause no change in the Michaelis constants for oxidation of morphine or 7-ethoxyresorufin, it is argued that these results provide strong presumptive evidence for changes in the relative abundance of isoenzymes catalyzing these various oxidations. Thus, a dual role of cimetidine, acting both as inhibitor and inducer of the cytochrome P-450 system, is proposed to account for the impaired oxidative metabolism of some drugs that occurs during coadministration with this H2-receptor antagonist.

Proteomic comparison of Hypnale hypnale (Hump-Nosed Pit-Viper) and Calloselasma rhodostoma (Malayan Pit-Viper) venoms

UNLABELLED Treatment of Hypnale hypnale bites with commercial antivenoms, even those raised against its sister taxon Calloselasma rhodostoma, has never been clinically successful. As these two genera have been separated for 20million years, we tested to see whether significant variations in venom had accumulated during this long period of evolutionary divergence, and thus could be responsible for the failure of antivenom. Proteomic analyses of C. rhodostoma and H. hypnale venom were performed using 1D and 2D PAGE as well as 2D-DIGE. C. rhodostoma venom was diverse containing large amounts of Disintegrin, Kallikrein, l-amino acid oxidase, Lectin, phospholipase A2 (acidic, basic and neutral) and Snake Venom Metalloprotease. In contrast, while H. hypnale also contained a wide range of toxin types, the venom was overwhelmingly dominated by two molecular weight forms of basic PLA2. 2D-DIGE (2-D Fluorescence Difference Gel Electrophoresis analysis) showed that even when a particular toxin class was shared between the two venoms, there were significant molecular weights or isoelectric point differences. This proteomic difference explains the past treatment failures with C. rhodostoma antivenom and highlights the need for a H. hypnale specific antivenom. BIOLOGICAL SIGNIFICANCE These results have direct implications for the treatment of envenomed patients in Sri Lanka. The unusual venom profile of Hypnale hypnale underscores the biodiscovery potential of novel snake venoms.

Ethanol and leucine oxidation--II. Leucine oxidation by rat tissue in vitro.

The effect of ethanol upon leucine oxidation by rat tissues in vitro is reported. The activities of branched chain amino acid aminotransferase and 2-oxo acid dehydrogenase were decreased by chronic administration of ethanol (20% v/v solution as drinking water for 35 d) in muscle and kidney but were increased, although not significantly, in liver. Acute administration of ethanol (8 g kg-1 body-weight 0.73) did not affect enzyme activities. Tissue NAD+:NADH ratios, calculated from lactate:pyruvate ratios, were significantly decreased in the liver and kidney of rats receiving ethanol acutely. These data are consistent with the view that ethanol decreases leucine oxidation by decreasing availability of NAD+ when given acutely and by decreasing enzyme activity when administered chronically.

Effects of exercise and antioxidant supplementation on endothelial gene expression

BACKGROUND The molecular mechanisms of exercise training induced cardiovascular protection are poorly understood. There is growing evidence that reactive oxygen species may be involved in a number of these adaptations and that antioxidants may be used to investigate this effect. OBJECTIVE To determine the effects of exercise training and/or antioxidant supplementation on myocardial endothelium and vascular endothelium gene expression. METHODS Male Wistar rats were divided into four groups: i) control; ii) exercise trained (90 min of treadmill running 4d per week, 14 weeks); iii) antioxidant-supplemented (α-tocopherol 1000 IU kg(-1) diet and α-lipoic acid 1.6 g kg(-1) diet, mixed with rat chow) and iv) exercise trained and antioxidant-supplemented. RESULTS cDNA microarray analysis showed diverse expression changes in both left ventricular and coronary artery endothelial cells. In particular, RT-PCR analysis showed that a gene involved in cardiovascular disease progression, Ras homolog gene family member A, was down-regulated by exercise, and up-regulated by antioxidant supplementation in left ventricular endothelial cells. Furthermore, an important gene involved in inflammation, IL-6, was down-regulated by all treatments. CONCLUSIONS Exercise training and/or antioxidant supplementation affects cardiac endothelial cell gene expression, and their effects on genes such as ras homolog gene family member A and IL-6 provides insight into the molecular mechanisms of their influences on cardiovascular diseases.

Effects of ethinylestradiol and testosterone implants on hepatic microsomal cytochrome P450 monooxygenases of birth gonadectomized male and female Dark Agouti rats

Monooxygenases in the cytochrome P450 IIIA subfamily are induced by a number of their xenobiotic substrates and by testosterone, an endobiotic substrate of importance in their regulation. 17 alpha-Ethinylestradiol (EE) is also metabolized by these enzymes and in this study Dark Agouti rats were used to examine the effects of subcutaneous implantation of controlled release silastic capsules containing EE to determine if this steroid also induces these enzymes. Data were compared with results obtained from equivalent groups of animals implanted with capsules containing testosterone propionate (TP). Liver microsomes prepared from male and female rats were used to identify intrinsic gender differences in the monooxygenases studied and gender differences in the responses to the implanted steroids were also determined. Effects due to imprinting of growth hormone secretion patterns were controlled by using male and female birth gonadectomized animals. Results obtained from groups with blank implants showed there were no effects due to the silastic implant material itself on the monooxygenases studied. The specific activities of erythromycin N-demethylation in liver microsomes of both EE and TP implanted male and female birth gonadectomized animals were enhanced relative to corresponding blank implanted controls consistent with both steroids having an effect to induce activity attributable to cytochrome P450 IIIA isoforms. Immunoinhibition studies using microsomes from EE treated female rats with erythromycin as substrate provided further evidence for this steroid having this induction effect. The specific activity of ethylmorphine N-demethylation was however not increased in microsomes prepared from the EE implanted female animals and was decreased in the corresponding male preparations. These findings distinguished the response to this steroid from that to TP and suggested induction by this estrogen of an isoform(s) having a more limited range of substrates than has characteristically been found in this subfamily. EE treatment also caused an increase in diazepam C3 hydroxylase consistent with an effect to induce P450 IIIA activity but this was found only in microsomes from birth gonadectomized female animals. This was in contrast to the effect of TP treatment which produced increases in this monooxygenase in both male and female animals. Another gender specific effect of EE was a striking decrease in morphine N-demethylase activity seen only in birth gonadectomized male rats. This again contrasted with the effect of TP which caused a marked increase in this activity in liver microsomes of both male and female birth gonadectomized animals consistent with the proposal that testosterone is important in the regulation of this activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluorimetric detection of microsomal lauric acid hydroxylations using high-performance liquid chromatography after selective solvent partitioning and esterification with 1-pyrenyldiazomethane

Abstract A novel and simple method for determining microsomal lauric acid hydroxylase activity is presented. Lauric acid and hydroxy-metabolites are separated using differential acid/base solubilities coupled to solvent partitioning. After esterification with 1-pyrenyldiazomethane, metabolites are quantitated using isocratic high-performance iiquid chromatography with fluorimetric detection. Column washing and equilibration between samples is not required. The method was verified by measuring the induction, in rats, of microsomal lauric acid hydroxylase activity by clofibrate. The method has clear advantages over published radiochemical procedures for measuring the formation of hydroxylated metabolites of lauric acid.

Protein turnover in subcellular fractions of brain from the ethanol-fed rat.

The effect of ethanol consumption (27% of energy intake) for 21 days, on protein synthesis and degradation in subcellular fractions of rat brain was investigated using the Na2(14)CO3 labelling procedure. Ethanol administration elicited an increase in the rate of protein synthesis in all fractions which was accompanied by an increase of similar magnitude in the rate of breakdown. These results imply that whilst ethanol increases the rate of protein turnover it is without effect on the rate of protein accretion.

A radiochemical method for determination of ethanol oxidation

A radiochemical method for the measurement of ethanol oxidation by tissue preparations is described. Ethanol oxidation is determined from the production of [14C]acetaldehyde, quantified as the semicarbazone derivative, from [1-14C]ethanol. The assay is quantitative, reproducible and highly correlated with the NADH-enzymic-spectrophotometric procedure.

Mechanism-based inhibition of rat liver microsomal diazepam C3-hydroxylase by mifepristone associated with loss of spectrally detectable cytochrome P450

Since initial studies with the steroids norethindrone and ethynylestradiol, reported by White and Muller-Eberhard in 1977 (Biochem. J. 166, 57-64), there has been continuing interest in xenobiotics that bear terminal or sub-terminal acetylenic groups which can cause catalysis-dependent inhibition of CYP monooxygenases associated either with loss of prosthetic group heme or protein adduct formation. Mifepristone is a synthetic steroid bearing a propyne substitution on carbon 17 and this suggested to us that it may act as a mechanism-based inhibitor of the CYP isoforms responsible for its metabolism. In human and rat liver, CYP3A isoforms have been implicated in mifepristone clearance and mifepristone administration to rats has also been shown to induce CYP3A enzymes and the associated diazepam C3-hydroxylase activity (Cheesman, Mason and Reilly, J. Steroid Biochem. Mol. Biol., 58, 1996, 447-454). With microsomes prepared from the livers of untreated female rats and others in which diazepam C3-hydroxylase has been induced, we show here that mifepristone can cause catalysis-dependent inhibition of this monooxygenase. In addition, incubation of microsomes with mifepristone in the presence, but not in the absence, of NADPH caused loss of spectrally detectable cytochrome P450. These results suggest that heme adduct formation may result from mifepristone metabolism by CYP3A monooxygenases which undergo self-catalysed irreversible inactivation with this drug as substrate. Since mifepristone administration in vivo is able also to cause induction of the synthesis of hepatic CYP3A apoprotein, mifepristone may have the potential in human medicine for complex interactions with other co-administered drugs which are also substrates for CYP3A monooxygenases.