ABSTRACT
The bovine uterus, like other estrogen-responsive organs of man and rat, synthesizes an unusual nonpolar metabolite of estradiol (Schatz, F., and Hochberg, R. B. (1981) Endocrinology 109, 697-703). This compound, the lipoidal derivative of estradiol (LE2), was synthesized from estradiol by bovine endometrial tissue in vitro, and it was purified by extensive chromatography on two adsorption columns, a reversed phase partition celite column and three different high pressure liquid chromatography columns. LE2 was separated into nine fractions which were analyzed by direct probe mass spectroscopy and by gas chromatography mass spectroscopy after cleavage of the lipoidal moieties. In this manner, 10 fatty acid esters of estradiol, exclusively esterified at C-17 of the steroid nucleus, were identified. The unsaturated fatty acid esters of estradiol comprise more than 85% of the total LE2 and estradiol 17 beta-arachidonate is the most abundant component. The cholesterol esters and phospholipids of the bovine endometrium were also purified and it was found that the distribution of fatty acids in these lipids is far different from that of LE2.
Subject(s)
Estradiol/analogs & derivatives , Fatty Acids/metabolism , Uterus/metabolism , Animals , Cattle , Chromatography, High Pressure Liquid , Estradiol/metabolism , Fatty Acids/analysis , Female , Gas Chromatography-Mass Spectrometry , Mass SpectrometryABSTRACT
The incubation of pregnenolone or dehydroisoandrosterone with bovine or rat adrenal homogenates leads to the formation of nonpolar metabolites of these steroids. The enzymatically prepared compounds have properties that are similar to the endogenous lipoidal derivatives of pregnenolone found in bovine adrenals (Hochberg, R. B., Bandy, L., Ponticorvo, L., and Lieberman, S. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 941-945) in that they are much less polar than the parent steroid and yield the parent steroid as a product of treatment with alkali. The lipoidal derivatives of both dehydroisoandrosterone and pregnenolone proved to be chromatographically heterogeneous. 17 alpha-Hydroxypregnenolone, 17 alpha-hydroxyprogesterone, progesterone, and testosterone were not converted into lipoidal derivatives when incubated with the bovine adrenal homogenate.
Subject(s)
Adrenal Glands/metabolism , Dehydroepiandrosterone/analogs & derivatives , Pregnenolone/analogs & derivatives , Animals , Cattle , Dehydroepiandrosterone/biosynthesis , Kinetics , Microsomes/metabolism , Mitochondria/metabolism , Pregnenolone/biosynthesis , Pregnenolone/metabolism , Rats , Structure-Activity Relationship , Subcellular Fractions/metabolismABSTRACT
The lipoidal derivatives of [3H]pregnenolone, prepared biosynthetically, were converted, by incubation with a mitochondrial-microsomal fraction from adrenal cortical tissue, into lipoidal derivatives of 17-hydroxy-pregnenolone and of dehydroisoandrosterone, thus proving that these pregnenolone derivatives can serve as substrates for 17-hydroxylase and for the lyase enzyme that converts a C21-17-hydroxy-20-ketol into a C19-17-ketosteroid. Three synthetically prepared esters of pregnenolone, the oleate, the linoleate, and the arachidonate, were also hydroxylated at C-17 by a similar adrenal preparation. With the synthetic substrates, however, the corresponding esters of dehydroisoandrosterone were not formed.
Subject(s)
Adrenal Glands/metabolism , Microsomes/metabolism , Mitochondria/metabolism , Pregnenolone/analogs & derivatives , Steroid 17-alpha-Hydroxylase/metabolism , Steroid Hydroxylases/metabolism , Animals , Cattle , Crystallization , Pregnenolone/metabolismABSTRACT
Using mass spectrometric, radioisotopic, chromatographic and chemical techniques, five fatty acid esters of 3 beta-hydroxy-5-pregnen-20-one (pregnenolone) have been identified as components of the lipoidal derivatives biosynthesized in vitro with bovine adrenal mitochondria. The five compounds are: pregnenolone arachidonate, pregnenolone linoleate, pregnenolone oleate, pregnenolone palmitate, and pregnenolone stearate. The distribution of the fatty acids among these five esters is different from the previously reported (Cmelik, S.H.W., and Ley, H. (1977) Comp. Biochem. Physiol. 56B, 267-270) fatty acid composition of these organelles.