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Biosynthesis of steroid hormones in human endocrine tissue and in the rat testis Ford, Henry Crawford

Abstract

This thesis reports the results of studies on steroid metabolism in a subject with an adrenocortical carcinoma and hypoglycemia, in the testes obtained from a subject with virilizing male pseudohermaphroditism and in the testes of the normal rat. Incubations of cell-free homogenates of an adrenocortical carcinoma from a 51 year old female with severe hypoglycemia were performed using ³H-pregnenolone and ¹⁴C-progesterone as substrates. Transformation of ³H-pregnenolone to progesterone, dehydroepiandrosterone and androstenedione was observed; no metabolites of ¹⁴C-progesterone were detected. The excretion rate in urine of 3α ,17,21-trihydroxy-5β -pregnan-20-one, a metabolite of cortexolone, was elevated which suggests that a defect in 11β -hydroxylase activity was present. The excretion rates in urine of total 17-ketosteroids, 17-hydroxycorticoids and 17-ketogenic steroids were elevated; the excretion rates of testosterone, dehydroepiandrosterone, pregnandiol, pregnanetriol and free cortisol were not elevated. The etiology of the hypoglycemia that may accompany some adrenocortical tumors remains unknown. It was not possible to relate the results of the investigations of steroid metabolism reported herein to the hypoglycemia that was present. Steroid biosynthesis in vitro was investigated in testes obtained during puberty from a 14 year old subject with virilizing male pseudohermaphroditism. Cell-free homogenates of gonadal tissue efficiently metabolized ³H-pregnenolone, ¹⁴C-progesterone and ¹⁴C-androstenedione to testosterone; formation of estrone and estradiol-17β was not detected. 16α-Htdroxyprogesterone was formed from both ³H-pregnenolone and ¹⁴C-progesterone. The results are similar to those of others who have investigated the steroidogenic capacity of gonadal tissue in patients with male pseudohermaphroditism and feminization at puberty. A defect in the formation of progesterone from pregnenolone has been suggested to explain the results of a previous study in which the gonadal tissue obtained from a patient with virilizing male pseudohermaphroditism was incubated with ³H-pregnenolone as substrate. In the investigations reported herein, transformations of ³H-pregnenolone to testosterone and androstenedione occurred both via 17-hydroxypregnenolone and dehydroepiandrosterone and via progesterone and 17-hydroxyprogesterone. The failure of patients with virilizing male pseudohermaphroditism to masculinize during embryonic development contrasts with the virilization that occurs during puberty. A biochemical abnormality may exert a transient effect during embryonic development. Alternatively, the sensitivity to androgenic hormones may be subnormal in certain tissues and normal in other tissues of patients with virilizing male pseudohermaphroditism. The biosynthesis of testosterone from progesterone and pregnenolone was investigated in the rat testis. Time studies were performed using cell-free homogenates and ³H-progesterone and ¹⁴C-17-hydroxyprogesterone in combination as substrates. It was demonstrated that the side-chain cleavage of 17-hydroxyprogesterone is the rate-limiting reaction in the biosynthesis of testosterone from progesterone and the evidence suggested that 17-hydroxyprogesterone was present as a bound intermediate (at least in part). The progesterone 17-hydroxylase and the 17-hydroxyprogesterone side- chain cleavage enzyme of the rat testis can be solubilized by treatment of lyophilized microsomes with Triton N-101. Both enzymes displayed maximal activity at pH 6.8 and at 37°. Progesterone rather than pregnenolone is the preferred substrate for the 17α-hydroxylase. Either NADH or NADPH can serve as the reductant for active 17-hydroxylation of progesterone and for side-chain cleavage of 17-hydroxyprogesterone. The soluble fraction contains NADPH dehydrogenase, non-heme iron protein and cytochrome P-450. The presence of these compounds in association with the 17α-hydroxylase and the side-chain cleavage enzyme activities suggests that these reactions are catalyzed by elaborate enzymatic systems analogous to those required for 11β-hydroxylation and cholesterol side-chain cleavage in adrenal mitochondria

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