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VIBEKE JENSEN, PAMELA CARSON and N. DESHPANDE

It is now generally accepted that dehydroepiandrosterone (DHA) is one of the androgens secreted by the human adrenal gland and that it arises from the side-chain cleavage of 17α-hydroxypregnenolone (Soloman, Carter & Lieberman, 1960; Gaul, Lemus, Kline, Gut & Dorfman, 1962; Deshpande, Jensen, Carson, Bulbrook & Doouss, 1970; Jones, Groom & Griffiths, 1970). Although the biosynthetic pathways by which the hormone is synthesized have been established, the precise requirements for the side-chain cleavage of 17α-hydroxypregnenolone and the factors affecting the reaction are as yet unknown. For this reason examination of the kinetics of the enzyme involved in the side-chain cleavage of 17α-hydroxypregnenolone (17-desomolase) was undertaken.

Human adrenal glands, obtained at operation from patients with advanced breast cancer, were fractionated in a Beckman Ultra Centrifuge (Model L2–65B) according to the procedure of Allfrey, Littan & Mursky (1964). Tritiated 17α-hydroxypregnenolone (sp.act. 19·9 Ci/mm) used as substrate and [14C]DHA (sp.act. 57·1

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N. DESHPANDE, PAMELA CARSON and SHEILA HARLEY

SUMMARY

An in-situ continuous infusion technique was used to study the biogenesis of androgens and cortisol in the guinea-pig adrenal gland after the infusion of 3H- and 14C-labelled precursors of these compounds. Pregnenolone was converted to 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone and cortisol. The results of infusion of a combined dose of [3H]17α-hydroxypregnenolone and [14C]progesterone indicated that the major pathway to cortisol is via 17α-hydroxypregnenolone. No conclusive proof could be obtained regarding synthesis of dehydroepiandrosterone or androst-4-ene-3,17-dione. The only androgen detected in the guinea-pig adrenal venous blood was 11β-hydroxyandrost-4-ene-3,17-dione. The major and minor biosynthetic pathways involved in the synthesis of these hormones are discussed.

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VIBEKE JENSEN, PAMELA CARSON and N. DESHPANDE

SUMMARY

The control of dehydroepiandrosterone (DHA) synthesis in the human adrenal gland was studied by investigating the biosynthesis of its immediate precursor, 17-hydroxypregnenolone (3β,17α-dihydroxy-pregn-5-en-20-one) and the side-chain cleavage. Both conversions take place in the microsomal fraction and there is an obligatory requirement for the co-factor, reduced nicotinamide adenine dinucleotide phosphate (NADPH). Under optimal conditions, 17-hydroxylation is controlled only by the availability of the substrate and the co-factor. By using the same kinetic approach it was observed that the synthesis of DHA is controlled by the availability of substrate and co-factor. However, the reaction was inhibited by the oxidized form (NADP+) and when the ratio of NADP+:NADPH reached 1, the maximum velocity was halved.

Two major metabolites of 17-hydroxypregnenolone, namely DHA and 17-hydroxyprogesterone, are non-competitive inhibitors of the reaction. The inhibition constants are of the magnitude of the tissue concentration of these steroids in the adrenal gland which suggests that non-competitive inhibition takes place in vivo.

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N. DESHPANDE, VIBEKE JENSEN, PAMELA CARSON, R. D. BULBROOK and T. W. DOOUSS

SUMMARY

A variety of 14C and 3H-labelled steroids have been perfused through the human adrenal gland in situ and their metabolic products isolated from adrenal venous blood.

Progesterone, dehydroepiandrosterone and cortisol were isolated after infusion of [3H]pregnenolone; 17α-hydroxyprogesterone, dehydroepiandrosterone and cortisol after infusion of [3H]17α-hydroxypregnenolone and [14C]progesterone; androstenedione and cortisol after infusion of [3H]17α-hydroxyprogesterone and [14C]dehydroepiandrosterone; and 11β-hydroxyandrostenedione after infusion of [3H]androstenedione and [14C]cortisol.

From a consideration of the incorporation of radioactivity into the metabolic products, the3H: 14C ratios and the tissue pool sizes it was concluded that the major biosynthetic pathway to cortisol in the human adrenal glands was: pregnenolone→ 17α-hydroxypregnenolone → cortisol. Progesterone was not an important intermediary.

Androstenedione was mainly formed by way of 17α-hydroxypregnenolone → 17α-hydroxyprogesterone → androstenedione. 11β-Hydroxyandrostenedione was formed mainly from cortisol and only a minor amount came from the hydroxylation of androstenedione.

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N. DESHPANDE, VIBEKE JENSEN, PAMELA CARSON, R. D. BULBROOK and A. A. LEWIS

SUMMARY

The production rate of cortisol based on both the plasma clearance of the hormone (plasma production rate) and the urinary metabolite method (urinary production rate) was simultaneously measured in early or advanced breast cancer patients and in controls. Higher production of the hormone was observed by both these methods in patients with advanced breast cancer.

There was a significant correlation between the plasma production rate of cortisol and its urinary production based on the specific activities of three urinary metabolites, namely, cortisol, tetrahydrocortisol (THF) and tetrahydrocortisone (THE). However, the values obtained for the urinary production rate differed considerably in about one-third of the patients due to differences in the specific activities of THE and THF. It is postulated that, in some cases, there may be a second precursor of urinary THE and THF which contributes significant amounts to the excretion of these metabolites.

No correlation was found between the cortisol production rate and urinary total 17-hydroxycorticosteroids (17-OHCS).