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Biomedical subjects

G F Gibbons

Publications and source records attributed to G F Gibbons.

At least 109 records · Page 6Linked to original sources
PubMed1980

Metabolism of hydroxy sterols by rat liver.

5 alpha-[16-3H]lanost-8-ene-3 beta,15 alpha-diol and 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol were both extensively metabolised by rat liver enzymes in vitro. Quantitatively, the most important product in both cases was a more polar compound, tentatively identified as a 5 alpha-lanost-8-enetriol. In addition, 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol gave rise to the corresponding 3 beta,15beta-diol diester, whilst with 5 alpha-[16-3H]lanost-8-ene-3 beta,15 alpha-diol only the 3 beta-hydroxyl group was esterified. The enzymes involved may normally be responsible for metabolising spontaneously produced non-enzymic oxidation products of dietary or cellular cholesterol. High concentrations of 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol stimulated ester formation. With both substrates, carbon monoxide inhibited formation of the polar sterol metabolite but stimulated ester formation. Under all conditions, cholesterol was a relatively minor metabolic product of either of the 5 alpha-lanost-8-ene-3 beta,15-diols.

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PubMed1979

Effects of various oxygenated sterols on cellular sterol biosynthesis in Chinese hamster lung cells resistant to 25-hydroxycholesterol.

The effects of a wide variety of oxygenated sterols upon sterol biosynthesis and hydroxymethylglutaryl-CoA reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) activity in a wild-type clone and in a 25-hydroxycholesterol-resistant clone of Chinese hamster lung (Dede) cells are described. Derivatives of cholesterol which were oxygenated in the 6, 7 or 15 positions of the sterol nucleus or in the 20, 22, 24 or 25 positions of the sterol side chain were shown to be potent inhibitors of sterol synthesis and reductase activity in the wild-type cells but none of these substitutions had any effect on the 25-hydroxycholesterol-resistant A2 clone. A 32-hydroxylated derivative of lanosterol also suppressed sterol synthesis and reductase activity in wild-type cells but had no significant effect upon the A2 line. It was also appraent that a complete sterol side chain was necessary for inhibitory activity. Studies of a wide range of inhibitory sterols indicated that there was a close correlation between their effects upon sterol synthesis and reductase activity and that their inhibitory action was specific for sterol biosynthesis since little effect was observed upon fatty acid or CO2 synthesis. Previous studies had shown that the uptake of 25-hydroxycholesterol by the resistant A2 line was unimpaired and the present results indicate that metabolism of this oxygenated sterol is also unaltered. These results, in conjunction with previous studies, suggest that the resistant A2 line is defective in feedback regulation of cholesterol synthesis and that all of the oxygenated sterols tested suppress the biosynthetic pathway through at least one common step.

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PubMed1979

Utilization of endogenous and exogenous sources of substrate for cholesterol biosynthesis by isolated hepatocytes.

The rates of cholesterol biosynthesis in isolated rat hepatocytes were determined by using a method based on measurement of the rate of formation of desmosterol (cholesta-5,24-dien-3beta-ol), which accumulates during inhibition of cholesterogenesis by the drug triparanol. Incubation of cells from normal or 24h-starved animals in a medium containing albumin, glucose, amino acids and acetate as the only organic constituents led to an accelerating rate of sterol formation during the earlier stages of a 6h incubation period. The contribution of exogenously added acetate (initial concentration 3.34mm) to sterol synthesis in both types of cells reached an early maximum and then continually declined. Exogenously added pyruvate and lactate were more efficient sources of sterol carbon than was acetate. Exogenous glucose even at relatively high concentrations (11.1mm) was incapable of providing more than 6% of the total sterol carbon. Although the proportion of total sterol carbon supplied from exogenous acetate increased with increasing concentrations of the extracellular substrate, the rates of total sterol synthesis in both types of cell remained unchanged. Similar observations were made when lactate or pyruvate was the cholesterogenic precursor in normal cells. These studies suggest that, although exogenous substrates were capable of expanding an intracellular pool of cholesterol precursor, the normal supply of intermediary metabolites was not rate-limiting for cholesterogenesis.

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PubMed1977

Measurement of the absolute rates of cholesterol biosynthesis in isolated rat liver cells.

Triparanol [2-(4-chlorophenyl)-1-(4-diethylaminoethoxyphenyl)-1-p-tolylethanol] at a concentration of 2 micronm has no effect on the overall conversion of [2=14C]acetate into C27 sterols by isolated liver cells. In the presence of triparanol, however, the formation of radioactive cholesterol is inhibited by 85-90% and the balance of radioactivity appears in the C27 sterol desmosterol (cholesta-5,24-dien-3beta-ol). The very small weights of desmosterol which accumulate under these conditions were, as a routine, quantitatively converted into the heptafluorobutyrate 3-enol ester of cholesta-4,24-dien-3-one. This derivative has a high electron-capturing capability, a property that enables extremely small quantities (less than 0.25pmol) of the material to be accurately measured by gas chromatography with electron-capture detection. Measurements of the mass and specific radioactivity of the newly biosynthesized desmosterol formed in the presence of triparanol provides an accurate assessment of the amount of cholesterol that would be synthesized by the liver cells in the absence of the drug.

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PubMed1976

Lanosterol 14alpha-demethylase. Similarity of the enzyme system from yeast and rat liver.

The chemical synthesis of 24,25-dihydro[32-14C]lanosterol is described. The incubation of this material with a cell-free system from Saccharomyces cerevisiae or with a microsomal preparation from rat liver resulted in both cases in the release of [14C]formic acid. This result suggests that in the biosynthesis of ergosterol in yeast, as well as in that of cholesterol in higher animals, the 14alpha-methyl group of lanosterol is removed as formic acid. In both systems, the measurement of the rate of release of [14C]formic acid from 24,25-dihydro[32-14C]lanosterol provides a simple and direct assay of lanosterol 14alpha-demethylase. Carbon monoxide inhibited both yeast and liver 14alpha-demethylase.

Animals↗
PubMed1975

Effect of trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride and carbon monoxide on hepatic cholesterol biosynthesis from 4,4,-dimethyl sterols in vitro.

The drug trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) almost completely inhibited the conversion of [2-14C] mevalonic acid, dihydro[14C]lanosterol, 4,4-dimethyl-5alpha-[2-3H2]cholesta-8,14-dien-3beta-ol and 4,4-dimethyl-5alpha-[2-3H2]cholest-8(14)-en-3beta-ol to 5alpha-cholest-7-en-3beta-ol and cholesterol by cell-free systems of rat liver. With the first three precursors, the inhibition was accompanied by an accumulation of radioactive 5alpha-cholesta-8,14-dien-3beta-ol, but this material could not be detected during inhibition of cholesterol biosynthesis from 4,4-dimethyl-5alpha-[2-3H2] cholest-8(14)-en-3beta-ol. Regardless of the nature of the precursor, trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride did not result in the accumulation of any delta5,7 sterols. Non-radioactive 5alpha-cholest-8(14)-en-3beta-ol inhibited the conversion of dihydro[14C]lanosterol to 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol. Carbon monoxide resulted in a decrease in the rate of conversion of dihydro[14C]lanosterol to 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol but had no effect on the rate of conversion of 4,4-dimethyl-5alpha-[2-3H2]cholesta-8,14-dien-3beta-ol to 5alpha-cholest-7-en-3beta-ol and cholesterol suggesting that cytochrome P-450 is involved neither in the oxidative removal of the 4-methyl groups nor in the oxidative introduction of the delta5 bond during cholesterol biosynthesis. In addition, the process of cholesterol and 5alpha-cholest-7-en-3beta-ol biosynthesis from 4,4-dimethyl-5alpha-[2-3H2]cholest-8(14)-en-3beta-ol was inhibited by carbon monoxide at a stage after the formation of 5alpha-cholest-8(14)-en-3beta-ol.

Animals↗
PubMed1974

The metabolic sequence by which some 4,4-dimethyl sterols are converted into cholesterol.

Cholest-8(14)-enol is the major radioactive component of the 4-di-demethyl sterol fraction biosynthesized from 4,4-dimethyl[2-(3)H(2)]cholest-8(14)-enol by rat liver microsomal fractions, and therefore the first steps in the biosynthesis of cholesterol from the latter compound probably involve removal of the 4-methyl groups. 4,4-Dimethylcholesta-8,14-dienol therefore is not an intermediate in this process, although its presence in the incubation medium at a concentration of 0.146mm almost completely inhibits the demethylation of 4,4-dimethyl[2-(3)H(2)]cholest-8(14)-enol. Nor is cholesta-8,14-dienol an intermediate in the conversion of cholest-8(14)-enol into cholest-7-enol and cholesterol. With 4,4-dimethyl[2-(3)H(2)]cholesta-8,14-dienol as the cholesterol precursor, 4,4-dimethylcholest-8(9)-enol becomes heavily labelled and there is very little radioactivity associated with cholesta-8,14-dienol. In this case, the most heavily labelled 4-di-demethyl sterols are cholest-7-enol and cholesterol with the former predominating. There is little or no radio-activity associated with cholest-8(14)-enol. A similar labelling pattern amongst the 4-di-demethyl sterols was observed with dihydro[(14)C]lanosterol as the precursor. The first step therefore in the synthesis of cholesterol from the 4,4-dimethyl[2-(3)H(2)]dienol is reduction of the Delta(14(15)) bond and not removal of the 4alpha-methyl group. Depending on the nature of the precursor, addition of the soluble fraction of the cell to the microsomal fraction resulted in a two- to four-fold stimulation of 4-di-demethyl sterol biosynthesis from the 4,4-dimethyl sterols studied. Under these conditions, 4,4-dimethylcholesta-8,14-dienol is the most efficient precursor of cholesterol and cholest-7-enol, and dihydrolanosterol is better than 4,4-dimethylcholest-8(14)-enol.

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PubMed1973

The effect of carbon monoxide on the nature of the accumulated 4,4-dimethyl sterol precursors of cholesterol during its biosynthesis from (2-14C)mevalonic acid in vitro.

Cholesterol biosynthesis was studied in rat liver subcellular fractions incubated with dl-[2-(14)C]mevalonic acid under gas phases consisting of either N(2)+O(2) (90:10) or CO+O(2) (90:10). CO inhibits cholesterol biosynthesis from [2-(14)C]mevalonic acid and results in a large accumulation of radioactive 4,4-dimethyl sterols. Separation of the components of the 4,4-dimethyl sterol fraction showed that lanosterol and dihydrolanosterol are the major components that accumulate during cholesterol biosynthesis in an atmosphere containing CO, whereas 14-demethyl-lanosterol and 14-demethyldihydrolanosterol are the major components of the much less intensely radioactive 4,4-dimethyl sterol fraction isolated from incubations with N(2)+O(2) as the gas phase. The identities of lanosterol, dihydrolanosterol and 14-demethyldihydrolanosterol were confirmed by both radiochemical and physicochemical methods, including g.l.c. and combined g.l.c.-mass spectrometry. CO therefore results in a qualitative as well as a quantitative difference in the 4,4-dimethyl sterol fraction which arises during cholesterol biosynthesis from mevalonic acid. The specific radioactivity of the [(14)C]lanosterol biosynthesized in the presence of CO was lower than that of its companion, [(14)C]dihydrolanosterol. The relative amounts of 4,4-dimethyl-Delta(24)-sterols and 4,4-dimethyl-24,25-dihydrosterols present in each type of incubation suggest that enzymic reduction of the sterol side chain occurs predominantly at a stage after that of lanosterol.

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