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Title: Sterol metabolism in extra-hepatic tissues
Author: Raggatt, Peter Rufus
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1967
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It is currently believed that cholesterol is the principal precursor of steroid hormones in endocrine organs and that it is converted to hormones via pregnenolone. The cholesterol molecule is first hydroxylated at C-20, and then at C-22 to yield 20α,22ζ-dihydroxy-cholesterol and this is then cleaved between C-20 and C-22 to yield pregnenolone and isocaproic acid. The enzyme system catalysing this series of reactions (cholesterol oxidase, E.C. has been investigated in some detail using tissue preparations of bovine adrenal cortex and human term placenta. Cholesterol oxidase in both the adrenal cortex and the placenta is associated with the mitochondria but a method was devised for bringing it into solution by means of sonication. Mitochondria, soluble preparations of mitochondria and soluble, steroid-free extracts of acetone-dried preparations have been used as the source of the enzyme in these experiments. A reliable method of assaying cholesterol oxidase by measuring the [5-14C]-isocaproic acid produced from [26-14C]-cholesterol was devised and thin-layer chromatographic methods were developed for the separation and identification of the steroid products. An apparatus was devised for the quantitative recovery of the steroids from the thin-layer chromatograms, and this was also utilised in a method of extraction, separation and assay of the endogenous cholesterol, cholesteryl esters and pregnenolone in adrenal cortex subcellular fractions. The method was capable of detecting 7 μg of cholesterol, 16 μg of cholesteryl ester and 2 μg of pregnenolone, using pure samples of the steroids. In the course of this investigation the following compounds were prepared:- sodium cholesteryl-3β-sulphate, pyridinium cholesteryl-3β- sulphate, sodium [4-14C]-cholesteryl-3β-sulphate, sodium [26-14C]- cholesteryl-3β-sulphate, sodium pregnenolone-3β-sulphate, sodium 25-oxo- 27-nor-cholesteryl-3β-sulphate, [4-14C]-cholesteryl-3β-acetate, [26-14C]- cholesteryl-3β-acetate, [26-14C]-cholest-4-ene-3-one, 27-nor-cholest-4- ene-3,25-dione, dihydrogen cholesteryl-3β-phosphate, diphenyl cholesteryl- 3β-phosphate, cholesteryl-3β-chloride, pregnenolone-3β-palmitate, 20α-hydroxy- cholesterol, 20α-hydroxy-cholesteryl-3β-acetate and pregnenolone- 3β-tetrapyranyl ether. Evidence was obtained that NADPH is required for cholesterol oxidation in adrenal cortex mitochondria and in preparations derived from the mitochondria and that in mitochondria, this may arise by the action of transhydrogenase (E.C. on the NADH formed from Krebs-cycle intermediates. However, adrenal cortex mitochondria as prepared in this work were shown to contain NADP-linked malic enzyme (E.C. and NADP-linked glucose-6-phosphate dehydrogenase (E.C.; in most tissues these enzymes are found mainly in the cytoplasm but if in the adrenal cortex they are associated with the mitochondria, as the results suggest, then they also could give rise to mitochondrial NADPH. The glucose-6-phosphate dehydrogenase of adrenal cortex (but not of yeast) was shown to be inhibited by pregnenolone but not by cholesterol. However, by comparison of the magnitude of this inhibition and the measured pregnenolone content of the adrenal cortex preparations, it was concluded that pregnenolone was unlikely to be a physiological regulator of cholesterol oxidase through its effect on glucose-6-phosphate dehydrogenase and the NADPH supply. Cholesterol was dispersed in aqueous incubation media using N,N-dimethyl formamide and the cholesterol dispersed in this way was found to be absorbed by the mitochondria. This absorption was partly reversible and the amount absorbed increased with the concentration of the added cholesterol. Absorbed cholesterol appeared to be less readily converted into steroid hormones than free cholesterol. The principal steroid product of cholesterol oxidation by adrenal cortex mitochondria, and by extracts made from mitochondria, was pregnenolone, but some progesterone was also formed, especially in experiments with whole mitochondria. The pregnenolone formed was partly retained in the mitochondria and partly released into the extra-particulate fluid; it was not taken up by the microsomes although the enzymes which convert pregnenolone into progesterone are known to be microsomal. The C6-product of cholesterol oxidation by adrenocortical preparations was isocaproic acid. This isocaproic acid was oxidised to carbon dioxide by the mitochondria under the conditions used but to such a slight extent that it did not affect the use of the production of isocaproic acid as a measure of cholesterol oxidase activity. The substrate specificity of cholesterol oxidase was investigated: cholesteryl-3β-sulphate , cholest-4-ene-3-one , cholesteryl-3β-acetate and cholesteryl-3β-linolenate were compared with cholesterol and were found to be less readily oxidised. Evidence was obtained that cholesteryl fatty acyl esters are not oxidised directly to pregnenolone esters but are first hydrolysed to free cholesterol by an esterase and subsequently oxidised to pregnenolone. It is suggested that cholesteryl fatty acyl esters form a reserve of cholesterol which can be metabolised, after hydrolysis, to steroid hormones. Cholesteryl-3β-sulphate was oxidised directly to pregnenolone-3β- sulphate under the experimental conditions employed and no free pregnenolone or other steroids were formed. It is suggested that oxidation of cholesteryl-3β-sulphate forms part of an alternative pathway of steroid hormone biosynthesis in adrenal cortex, but no evidence was obtained that sulphurylation or phosphorylation were obligatory steps in cholesterol oxidation. Cholesteryl-3β-sulphate and free cholesterol were compared as substrates for cholesterol oxidase. Kinetic studies in a variety of tissue preparations indicated that free cholesterol was the preferred substrate with a Km of 1-4 μM whereas the Km for cholesteryl-3β- sulphate was about 500 μM. Cholesterol sulphatase was detected in adrenal cortex and was found to be microsomal. It was inhibited by inorganic phosphate and therefore phosphate was used in experiments designed to measure cholesteryl- 3β-sulphate oxidation. he inhibitor specificity of cholesterol oxidase was investigated. Cholesteryl-3β-esters (phosphate, sulphate, acetate, oleate and linolenate) inhibited cholesterol oxidation competitively but it is suggested that inhibition by fatty acyl esters was due to production of free cholesterol by esterase activity. The products of cholesterol oxidation, pregnenolone and 20α-hydroxy-cholesterol inhibited cholesterol oxidation non-competitively. The Ki for pregnenolone was 80 μM and for 20α-hydroxy cholesterol 10 μM. Evidence was obtained that feed-back inhibition by pregnenolone may be a physiological mechanism for the control of cholesterol oxidation and steroid hormone formation and that this effect is exerted directly on the enzyme cholesterol oxidase. A number of other steroids inhibited cholesterol oxidation and among these was 25-oxo-27-nor-cholesterol, a synthetic steroid which was more potent than pregnenolone (Ki 16 μM, non-competitive). These results suggest that a 3β-hydroxyl group as well as an oxygen function in the side chain are important structural characteristics of an inhibitor of cholesterol oxidase. Cholesterol oxidase was also inhibited by Su 4885 (2-methyl,1,2-di-(pyrid-3-yl)-propan-l-one), a synthetic hydroxylation inhibitor. When cholesterol oxidation was inhibited by pregnenolone, 20αhydroxy- cholesterol, 25-oxo-27-nor-cholesterol or Su 4885, no trace of accumulated intermediates was detected. This supports the theory that 20α-hydroxylation is the first and rate-limiting step of cholesterol oxidation. Certain steroid carboxylic acids (3β-hydroxy-chol-5-enoic acid, 3α-hydroxy-chol-5-enoic acid and 3β-hydroxy-22,23-bisnor-chol-5-enoic acid) stimulated cholesterol oxidation but 3β-hydroxy-androst-5-ene- 17α-carboxylic acid and 3β-acetoxy-22,23-bisnor-chol-5-enoic acid did not. The oxidation of cholesteryl-3β-sulphate by adrenal cortex mitochondria was inhibited by pregnenolone, 20α-hydroxy-cholesterol and 25-oxo-27-nor-cholesterol but not by pregnenolone-3β-sulphate or 25-oxo- 27-nor-cholesteryl-3β-sulphate. This similarity to the inhibitor specificity of cholesterol oxidation suggests that there is only one enzyme system oxidising both cholesterol and cholesteryl-3β-sulphate. This theory is supported by the similarity between the maximum rates of oxidation of cholesterol and cholesteryl-3β-sulphate by soluble, steroid-free cholesterol oxidase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available