Detection in extracts of bovine brain of lipophilic complexes of sulfate esters of cholesterol and beta-sitosterol.

Evidence indicating that there exist in bovine brains hitherto-unrecognized lipophilic conjugates of sterol sulfates is presented. These conjugates are soluble in nonpolar solvents and, when heated in methanol containing pyridine, yield polar sterol conjugates. These polar substances have the chromatographic mobility of sterol sulfates and are cleaved to free sterols when subjected to a solvolytic process known to be specific for sulfate esters. The brain sterols that have been identified in this way are cholesterol and beta-sitosterol.

Identification of 24-hydroxycholesterol in bovine adrenals in both free and esterified forms and in bovine brains as its sulfate ester.

24-Hydroxycholesterol was found to be present in bovine adrenals in a concentration of 2-3 micrograms/kg and in larger amounts (10-12 micrograms/kg) as a saponifiable ester. No solvolyzable sulfate ester was detected. In bovine brains, however, the sulfate ester of this sterol was found in a concentration of 84 micrograms/kg. A procedure for the synthesis of [7-3H] 24S-hydroxycholesterol is described.

The state of neutral lipids in normal and cataractous human lenses.

A simple and rapid high pressure liquid chromatographic method (HPLC) was developed to analyse the neutral lipid fraction from normal and cataractous human lenses. No differences were found between old normal and cataractous lenses suggesting that membrane deterioration in cataracts is not due to oxidation of these components. Several new lens components were isolated and identified including cholesterol metabolites, and vitamin E (alpha-tocopherol).

Formation of 20 beta-dihydrosteroids by anaerobic bacteria.

Cortisol was metabolized to a variety of products, among them small amounts of cortol by fecal flora of humans and rats. A microorganism, Bifidobacterium adolescentis, isolated from both sources, synthesized a 20-hydroxysteroid dehydrogenase which reduced cortisol to 20 beta-dihydrocortisol. The metabolite was reduced to cortol by Clostridium paraputrificum. The 20-hydroxysteroid dehydrogenase showed a wide substrate specificity; it was independent of the 4-ene and the configuration at C3, C-11, C-17 and C-21. Cortol was resistant to any further alteration by human fecal flora, i.e. it is a metabolic end product. As expected, B. adolescentis effectively prevented 21-dehydroxylation of cortisol by Eubacterium lentum.

Estradiol fatty acid esters. The isolation and identification of the lipoidal derivative of estradiol synthesized in the bovine uterus.

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.

Identification of fatty acid esters of pregnenolone and allopregnanolone from bovine corpora lutea.

The lipoidal derivatives of steroids present in bovine corpora lutea have been identified and quantified. Nonpolar steroid derivatives were extracted from the tissue with a mixture of chloroform and methanol. The extract was subjected to column and high performance liquid chromatography in order to remove phospholipids, cholesterol, triglycerides, cholesteryl esters, and unesterified steroids. A portion of the purified nonpolar material was subjected to methanolysis. By gas-liquid chromatographic analysis, the products were shown to consist of the steroids 3 beta-hydroxy-5 alpha-pregnan-20-one (80%) and pregnenolone (20%), and the methyl esters of palmitic (34%), stearic (27%), oleic (22%), linoleic (7%), arachidonic (7%), palmitoleic (3%), and eicosatrienoic (1%) acids. Mass spectrometric analysis of the intact lipoidal derivatives and of their methoximes confirmed the presence of seven esters of allopregnanolone and pregnenolone: allopregnanolone palmitate, pregnenolone palmitate, allopregnanolone stearate, allopregnanolone oleate, allopregnanolone linoleate, allopregnanolone eicosatrienoate, and pregnenolone arachidonate. The mass spectral data suggested that four other esters were also present: pregnenolone stearate, pregnenolone oleate, pregnenolone linoleate, and allopregnanolone arachidonate.

Characterization of the lipoidal derivatives of pregnenolone prepared by incubation of the steroid with adrenal mitochondria.

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.

Bacterial metabolism of corticoids with particular reference to the 21-dehydroxylation.

Clostridium paraputrificum, an obligate anaerobe recovered from human feces, reduces the alpha,beta-unsaturated carbonyl of deoxycorticosterone, corticosterone, cortisone, and cortisol. The same steroids are 21-dehydroxylated by Culture 116, recently isolated from human feces, and by a closely related organism, Eubacterium lentum. The 21-dehydroxylase has no effect on hydroxyl groups at carbon atoms 11 and 17.

Detection in bovine adrenal cortex of a lipoidal substance that yields pregnenolone upon treatment with alkali.

Bovine adrenal cortical tissue contains a lipoidal derivative of pregnenolone (3beta-hydroxy-pregn-5-en-20-one) from which the free steroid can be liberated by treatment with alkali. Evidence for the presence of such an entity comes from examination of a nonpolar extract of tissue from which pregnenolone and its sulfate had been removed by chromatography. Treatment of the nonpolar fraction with alkali followed by exhaustive chromatographic analysis led to the detection of pregnenolone. The steroid was identified by both gas chromatography/mass spectrometry and double isotope procedures. Quantitative analysis indicated that the three forms of pregnenolone are present in bovine adrenal cortical tissue in the following amounts (mug/kg): lipoidal derivative, 290; free steroid, 435; and sulfate, 65. Because the only known metabolic function of pregnenolone is to serve as a precursor of the steroid hormones, these findings have far-reaching implications for steroid hormone biochemistry.

Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol.

Incubation of (20R)-20-phenyl-5-pregnene-3beta,20-diol, an aromatic analog of (23S)-20-hydroxycholesterol, with an adrenal mitochondrial preparation leads to the formation of four compounds: pregnenolone, phenol, a C8 ketone, acetophenone, and a nonpolar C19 compound. This latter compound has now been identified by reverse isotope dilution analysis and by gas chromatography/mass spectrometry as 17-methyl-18-norandrosta-5,13(17)-dien-3beta-ol. From these results it is evident that enzymatic fission of the C-17,20 bond of this synthetic derivative occurs. On the other hand, when (20S)-20-hydroxy[21-14C]cholesterol was used as substrate, the analogous cleavage did not take place. Thus, substitution of an aromatic group on C-20 facilitates side chain cleavage between that carbon atom and the nucleus whereas neither of the naturally occuring precursors, cholesterol or its 20-hydroxylated counterpart, are metabolized to a C8 fragment.