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Biomedical subjects

S Shefer

Publications and source records attributed to S Shefer.

At least 181 records · Page 10Linked to original sources
PubMed1978

Gas-liquid chromatography-mass spectrometry of trimethylsilyl ethers of bile alcohols.

This report describes the gas-liquid chromatography-mass spectrometry (GLC-MS) of the trimethylsilyl ethers of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol with mono- or dihydroxy substitution in the side chain. Compounds with 24- and 25-unsaturation in the side chain were also studied. The gas-liquid chromatographic separation of the different bile alcohols was carried out using 3% QF-1 and 1% HI-EFF 8BP as column packings. Both columns were useful in that the retention times of the trimethyl-silyl ethers of the various 5 beta-cholestanetetrols varied linearly with the position of the side-chain trimethylsiloxyl substituent. The major fragmentations in the GLC-MS of all bile alcohols tested were due to the trimethylsiloxyl side-chain substituent(s). A trimethylsiloxyl group at C-22 was the most effective in promoting side-chain fragmentation, followed, in order of decreasing effectiveness, by substituents at carbons 25, 23, 24, and 26. The side-chain fragments generally gave rise to one or two intense mass peaks and a series of weaker peaks, 90 mass units apart, owing to the loss of successive molecules of trimethylsilanol. Most 5 beta-cholestane-triols, -tetrols, and -pentols can be unequivocally identified by their strong side-chain fragmentation peaks, except for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol, the trimethylsilyl ether of which exhibited very low intensity side-chain fragments.

Cholestanols↗
PubMed1977

Cholic acid biosynthesis: conversion of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol into 5beta-cholestane-3alpha,7alpha, 12alpha,24beta,25-pentol by human and rat liver microsomes.

This paper describes the conversion of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol into 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol by liver microsomes. A sensitive radioactive assay for measuring the formation of 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol was developed. Optimal assay conditions for human and rat microsomal systems were established. A higher 24beta-hydroxylation activity was detected in rat than in human liver under the conditions employed. The hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol by the rat liver microsomal fraction fortified with NADPH was stimulated about two-fold by administration of phenobarbital. Phenobarbital treatment also stimulated hydroxylations at C-23, C-24alpha, and C-26. Carbon monoxide markedly inhibited all side-chain hydroxylations. In contrast, side-chain hydroxylase activities were not affected in animals deprived of food for 48 hr. These results are consistent with a previously postulated cholic acid biosynthetic pathway involving 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol as a key intermediate in man and in the rat.

Animals↗
PubMed1976

A 25-hydroxylation pathway of cholic acid biosynthesis in man and rat.

This paper describes a pathway of cholic acid synthesis, in man and in the rat, which involves 25-hydroxylated intermediates and is catalyzed by microsomal and soluble enzymes. The subcellular localization, stereospecificity, and other properties of the enzymes involved were studied with liver fractions of normolipidemic subjects, cerebrotendinous xanthomatosis patients, and rats. 5beta-Cholestane-3alpha,7alpha,12alpha,25-tetrol was converted to 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol by the microsomal fraction in the presence of NADPH and O2. 5beta-Cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol, 5beta-cholestane-3alpha,7alpha,12alpha,-23xi,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,25,26-pentol were also formed. In the presence of NAD, 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol, but not the other 5beta-cholestanepentols formed, was converted to cholic acid by soluble enzymes in good yield. These experiments demonstrate the existence of a pathway for side-chain degradation in cholic acid synthesis which does not involve hydroxylation at C-26 or the participation of mitochondria.

Animals↗
PubMed1976

Synthesis of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 245, 25-pentol.

This paper describes syntheses of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol which give higher yields than previously published methods. In addition, 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol was synthesized by a different procedure, namely via performic acid oxidation of the correspinding unsaturated triol, which gave a lower yield but avoided the formation of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol, which normally tends to contaminate the final product. Structures were confirmed by gas-liquid chromatography, infrared-, proton magnetic resonance- and mass spectrometry, 5beta-Cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol were required for in vivo and in vitro studies of the (hypothetical) 25-hydroxylation pathway of cholic acid biosynthesis.

Methods↗
PubMed1976

C26-Analogs of naturally occurring C27 bile alcohols.

C26 Bile of the 24-nor-5beta-cholestane series were prepared starting from methyl cholate. A Grignard reaction of methyl magnesium iodide with methyl cholate yielded 24-nor-5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol which was dehydrated TO FORM a mixture of 24-nor-5beta-cholest-23-ene-3alpha, 7alpha, 12 alpha-triol and the corresponding delta25 compound. Oxidation of the former with OsO4 yielded 24-nor-5beta-cholestane-3alpha, 7alpha, 12alpha, 23epsilon, 25pentol, while catalytic hydrogenation of a mixture of the delta23 and delta25 triols resulted in the formation of 24-nor-5beta-cholestane-3-alpha-, 7alpha-12alpha-triol. The structures of these new compounds were confirmed by infrared and nuclear megnetic resonance spectrometry and by gas-liquid chromatography-mass spectrometry.

Chromatography, Gas↗
PubMed1975

Sterol balance studies in the rat. Effects of dietary cholesterol and beta-sitosterol on sterol balance and rate-limiting enzymes of sterol metabolism.

Sterol balance measurements using isotopic and chromatographic techniques were carried out in rats fed diets containing beta-sitosterol (0.8%) and cholesterol (1.2%). The activities of the rate-limiting enzymes of cholesterol synthesis (beta-hydroxy-beta-methylglutaryl-CoA reductase, EC 1.1.1.34) and bile acid synthesis (cholesterol 7 alpha-hydroxylase) were determined in the same animals. Cholesterol feeding increased cholesterol absorption from 1.2 to 70 mg/day. The increased absorption was compensated for by inhibition of hepatic cholesterol synthesis, enhanced conversion of cholesterol to bile acids (from 13.7 to 27.3 mg/day) and a slight increase in the excretion of endogenous neutral steroids (from 7.7 to 11.2 mg/day). Despite the adaptation there was accumulation of cholesterol in the liver (from 2.2 to 9.2 mg/g). Beta-Sitosterol feeding inhibited cholesterol absorption (calculated absorption was zero). In these rats there was enhanced cholesterol synthesis (from 20.0 to 28.8 mg/day, but no change in the rates of bile acid formation. Measurements of the activities of the rate-limiting enzymes showed fair correlation with cholesterol-bile acid balance. In cholesterol fed animals, beta-hydroxy-beta-methylglutaryl-CoA reductase was inhibited 80% and cholesterol 7 alpha-hydroxylase was enhanced 61%. In beta-sitosterol-fed animals, the reductase was increased 2-fold and cholesterol 7 alpha-hydroxylase was not significantly different from controls.

Animals↗
PubMed1975

Bile alcohol metabolism in man. Conversion of 5beta-cholestane-3alpha, 7alpha,12alpha, 25-tetrol to cholic acid.

To study the role of C25-HYDROXY BILE ALCOHOLS AS PRECURSORS OF CHOlic acid, [G-3-H]5beta-cholestane-3alpha,7alpha12alpha,25-tetrol was administered intravenously to two subjects with cerebrotendinous xanthomatosis (CTX) and two normal individuals. One day after pulse labeling, radioactivity was present in the cholic acid isolated from the bile and feces of the subjects with CTX and the bile of the normal individuals. In the two normal subjects, the sp act decay curves of [G-3-H]-cholic acid were exponential, and no traces of [G-3-H]-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol were detected. In contrast, appreciable quantities of labeled 5beta-cholestane-3alpha,-7aopha,12alpha,25-tetrol were present in the bile and feces of the CTX subjects. The sp act vs. time curves of fecal [G-3-H]5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and [G-3-H]-cholic acid showed a precursor-product relationship. Although these results suggest that 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol may be a precursor of cholic acid in man, the possibility that C26-hydroxy intermediates represent the normal pathway can not be excluded.

Alcohols↗
PubMed1975

Isotope derivative assay of microsomal cholesterol 7 alpha-hydroxylase.

A rapid method was developed to measure cholesterol 7 alpha-hydroxylase activity of hepatic microsomes by the direct determination of the mass of 7 alpha-hydroxycholesterol formed. The method is based on the quantitative acetylation of the incubation mixture with [-3H]acetic anhydride and the separation of the biosynthetic 7 alpha-hydroxycholesterol as its diacetate by thin-layer chromatography on alumina. Amounts of 7 alpha-hydroxycholesterol as low as 0.1 nmole could be measured. A comparison of the proposed isotope derivative method with the previously used isotope incorporation method showed that the latter underestimated the enzyme activity by about 20 percent.

Acetylation↗
PubMed1975

Identification of pentahydroxy bile alcohols in cerebrotendinous xanthomatosis: characterization of 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 23xi, 25-pentol.

This paper describes studies dealing with the nature of the C27 pentahydroxy bile alcohols present in the bile and feces of two patients with cerebrotendinous xanthomatosis (CTX). The presence of a bile alcohol having the structure 5beta-cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol was confirmed by separation of the two 24-hydroxy epimers of 5beta-cholestane-3alpha,7alpha,12alpha,24,25-pentol and characterization of the dpimers by gas-liquid chromatography and infrared and mass spectrometry. Tentative assignment of the 24alpha and 24beta configuration was made on the basis of molecular rotation differences. A second major bile alcohol excreted by the CTX subjects was 5beta-cholestane-3alpha,7alpha,12alpha,23xi,25-pentol. Its structure was determined by infrared spectrometry, proton magnetic resonance spectrometry, and mass spectrometry because a reference compound was not available.

Bile↗
PubMed1975

Hepatic cholesterol metabolism in patients with gallstones.

Relative rates of cholesterol and bile acid synthesis were estimated in patients with cholesterol gallstones and biliary obstruction by determining the hepatic activities of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase, the respective rate-determining enzymes for cholesterol and bile acid synthesis. As compared with eight control studies, 3-hydroxy-3-methylglutaryl-CoA reductase activity was 27% higher in 12 gallstone subjects, but 75% lower in 5 subjects with biliary obstruction. Cholesterol 7 alpha-hydroxylase activity was reduced in the gallstone (47% lower) and biliary obstruction (78% lower) subjects. Liver cholesterol concentrations were 56% higher in the gallstone and 53% higher in the biliary obstruction subjects than the control group. These findings suggest that the pathogenesis of gallstones is related to both increased cholesterol synthesis and decreased bile acid formation, whereas cholesterol accumulates in biliary obstruction because of defective removal since cholesterol production is low.

Adult↗
PubMed1974

Determination of hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity in man.

Procedures were developed for the determination of the activity of the microsomal enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase, EC 1.1.1.34) in human liver. The enzyme assay could be carried out with as little as 20 mg of fresh liver tissue, thus making the method applicable to specimens obtained by percutaneous liver biopsy. Experiments were carried out to determine optimal assay conditions and to establish the identity and radiopurity of the reaction product formed from 3-(14)C-labeled 3-hydroxy-3-methylglutaryl CoA. The specific activity of the enzyme was measured in a number of patients with different disorders of lipid metabolism.

Alcohol Oxidoreductases↗
PubMed1973

Regulatory effects of dietary sterols and bile acids on rat intestinal HMG CoA reductase.

The specific activity (concentration) of microsomal HMG CoA reductase of intestinal crypt cells was studied in rats fed sterols and bile acids, either singly or in combination. It was found that the basal activity of the reductase was not suppressed by the administration of relatively large amounts of bile acid (taurocholate or taurochenodeoxycholate). Bile acids reduced the specific activity of the reductase only in rats in which the activity of the enzyme had first been enhanced by biliary diversion or by sitosterol feeding. In addition, bile acid feeding abolished the diurnal elevation of reductase activity that normally occurs between midnight and 2 a.m. In no case did bile acids reduce enzyme activity below basal levels. A pronounced (60%) reduction of intestinal HMG CoA reductase activity was observed in rats fed cholesterol and bile acid in combination. This reduction in activity could not be ascribed to an increase in intestinal bile acid flux but was associated with an increase in sterol concentration within the intestinal crypt cells. These results indicate that dietary sterols and bile acids both play a role in the regulation of intestinal HMG CoA reductase.

Alcohol Oxidoreductases↗
PubMed1973

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat.

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.

Alcohol Oxidoreductases↗
PubMed1972

HMG CoA reductase of intestinal mucosa and liver of the rat.

Methods were developed for the determination of HMG CoA (3-hydroxy-3-methylglutaryl CoA) reductase activity in subcellular fractions of intestinal mucosa and liver of Wistar strain rats. In the liver, reductase activity was located exclusively in the microsomal fraction. In the intestinal mucosa, activity was found in both mitochondrial and microsomal fractions of crypt cells but not of villi. The microsomal HMG CoA reductases of liver and intestinal mucosa had similar kinetic characteristics and pH optima. However, the activity of the hepatic enzyme differed with age and sex of the experimental animals while that of the intestinal crypt cells did not. Cholestyramine treatment enhanced the activity of the microsomal HMG CoA reductase in both liver and intestinal mucosa. Reductase activity of the intestinal crypt cells was elevated in both jejunum and ileum. The greatest stimulation, both relatively and absolutely, was observed in the distal half of the jejunum.

Aging↗
PubMed1972

Stimulation of cholesterol 7 -hydroxylase by phenobarbital in two strains of rats.

The effect of phenobarbital administration on the in vitro activity of cholesterol 7alpha-hydroxylase was investigated in two strains of rats. In rats of the Wistar strain the daily injection of phenobarbital (100 mg/kg per day ip for 5 days) produced a 33% increase in hepatic microsomal protein and a sixfold stimulation of specific activity of the enzyme. In rats of the Charles River colony (Sprague-Dawley derived) identical treatment with phenobarbital resulted in a 45% increase in hepatic microsomal protein and no change in the specific activity of cholesterol 7alpha-hydroxylase. Plasma phenobarbital concentrations were three to four times greater in the Wistar rats, suggesting that these strains differ also in their capacity to metabolize phenobarbital.

Animals↗