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The in Vitro Metabolism of Desmosterol with Adrenal and Liver Preparations

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The in Vitro Metabolism of Desmosterol with Adrenal and Liver Preparations THE IN VITRO METABOLISM OF DESMOSTEROL WITH ADRENAL AND LIVER PREPARATIONS DeWitt S. Goodman, … , Joel Avigan, Hildegard Wilson J Clin Invest. 1962;41(12):2135-2141. https://doi.org/10.1172/JCI104671. Research Article Find the latest version: https://jci.me/104671/pdf Journal of Clinical Investigation Vol. 41, No. 12, 1962 THE IN VITRO METABOLISM OF DESMOSTEROL WITH ADRENAL AND) LIVER PREPARATIONS * By DEWITT S. GOODMAN,t JOEL AVIGAN, AND HILDEGARD WILSON (From the Section on Metabolism, National Heart Institute, and National Cancer Institulte, Endocrinology Branch, N.I.H., Bethesda, Md.) (Submitted for publication May 24, 1962; accepted August 9, 1962) IN a recent communication (2), it was demon- acid-Celite (2: 1) as described by Avigan and co-work- strated that in human subjects receiving triparanol ers (3). Chromatography of both samples was contin- therapy, 24-dehydrocholesterol (desmosterol) ued until the cholesterol zones had moved almost the entire length of the column. Only the cholesterol zone could serve as a direct precursor of several physio- was visible in the sample from the normal rats; the sam- logically important compounds normally derived ple from the triparanol-fed rats showed a desmosterol from cholesterol. The results indicated the direct zone fairly well separated from the cholesterol zone. conversion of desmosterol to both bile acids and The contents of both columns Adere extruded, the ceni- to steroid hormones in these patients, and sug- tral portion of the cholesterol zone collected from the sample from the normal rats and the center of the des- gested that desmosterol was similar to cholesterol mosterol zone from the sample from triparanol-fed rats. in its ability to serve as a precursor for these com- The latter was rechromatographed on a smaller column poulnds. to obtain desmnosterol of high purity. The labeled cho- The present study compares the metabolism of lesterol and desmosterol esters were then hydrolyzed, desmosterol and cholesterol in in vitro adrenal and the free sterols extracted, and analyzed for mass and for radioactivity. The yields of the two sterols were: a) liver preparations. The results support the con- cholesterol, 22 mg, with specific radioactivity 74,500 dpm clusion of the previous in vivo studies. per mg; I)) desmosterol, 7 mg with specific radioactivity 57,000 dpm per mg. METHODS A second specimen of C14-desmosterol wvas prepared later by injection of 40 Ac of 2-C'4-mevalonate into one Preparation of labcled sinbstratcs. Both C14-desmosterol triparanol-fed rat. Desmosterol was purified from the and C'4-cholesterol were biosynthetically prepared from nonsaponifiable fraction of the liver by thin-layer chro- 2-C14-mevalonic acid. The C'4-mevalonic acid was pur- matography of the free sterols and of the sterol acetate chased from the Volk Radiochemical Corp. as its di- esters (4). The yield of desmosterol was 6.5 mg, with benzylethylenediamine salt, and was converted to a solu- specific radioactivity 192,000 dpm per mg. This prepara- tion of the potassium salt by alkalinization with KOH, tion was demonstrated to contain pure desmosterol by extraction of the diamine with light petroleum ether, and gas-liquid chromatography. In the experiments reported neutralization. Twenty microcuries of C14-mevalonate below, this second desmosterol preparation was used were injected intravenously into each of two normal rats only in the second experiment with adrenal homogenates. and into two rats that had been given a diet containing Both cholesterol and desmosterol synthesized from 1 per cent triparanol for 2 months. The normal rats 2-C'4-mevalonate contain five labeled carbon atoms, three were used as the source of C14-cholesterol, and the tri- in the sterol nucleus-carbon atoms 1, 7, and 15-and two paranol-fed rats of C'4-desmosterol. After 2.5 hours the in the side-chain-carbon atoms 22 and 27 (5, 6). Since livers were excised and extracted in a small blender with both labeled sterols were synthesized from the same pre- 25 vol (vol/wt) of acetone: ethanol (1: 1, vol: vol). cursor and purified by the same methods, it was felt that The total lipid extract so obtained was saponified with 2 the two substrates were absolutely comparable. per cent KOH in 50 per cent ethanol for one hour under Experiments zenith liver mitochondria. The oxidation nitrogen, and the nonsaponifiable fraction was then ex- of the terminal carbon atoms at the side-chain of cho- tracted with light petroleum ether. Further purification lesterol and desmosterol by mouse liver mitochondria of the sterols was effected by chromatography of their was studied by the method of Horning, Fredrickson, and p-phenylazobenzoyl esters on 40-cm columns of silicic Anfinsen (7). In these experiments, the substrate sterol was added to the incubation mixtures as a serum-albumin * Presented in part at the First International Pharma- stabilized suspension containing 0.9 Amole of sterol per cology Meeting, Stockholm, Sweden, September, 1961 ml of 1 per cent bovine serum albumin. Mitochondria (1). were obtained from livers of adult male mice by homog- t Present address: Department of Medicine, Columbia enization of the livers with a solution of 0.25 M sucrose University College of Physicians and Surgeons, New and 0.01 M nicotinamide in a Potter-Elvehjem homog- York, N. Y. enizer, dilution of the homogenate to 75 ml per 10 g liver, 2135 2136 DEWITT S. GOODMAN, JOEL AVIGAN, AND HILDEGARD WILSON and differential centrifugation to settle the mitochondria about 40 per cent desmosterol and 60 per cent cholesterol. as sediment at 10,000 X G. The mitochondria were The remainder of the homogenate, 4.5 ml, was divided washed once by centrifugation and suspended in a vol- into two equal parts in each of two 25-ml Erlenmeyer ume equal to the original weight of liver. flasks, and to each flasks were added: 5 Emoles TPNH, Incubations were carried out in 25-ml Erlenmeyer 5 /moles TPN, and 20 /umoles glucose-6-phosphate. The flasks sealed with rubber serological caps. From each final volume was 3 ml. The C14-sterol substrate was then cap a small glass well was suspended. Each flask con- added to each flask in 100 uL acetone solution. In the tained, in a volume of 6 ml: 300 /moles Tris-HCl buf- first experiment, 1.65 Amoles C14-cholesterol was added fer at pH 8.4, 100 /Amoles nicotinamide, 4 Amoles DPN, to one flask, and the same amount of C14-desmosterol to 2 /Amoles TPNH, 20 jumoles glutathione, 10 Amoles the second flask. In the second experiment 1 Amole of 5-adenylic acid, 1 ml supernatant fraction of boiled liver each substrate was employed, and the second preparation homogenate-see (7)-, 3 ml washed mitochondrial sus- of C1'-desmosterol was used. The flasks were flushed pension, and 0.9 Emole substrate. After preparation of with oxygen, stoppered, and incubated in a shaking incu- the incubation mixtures, 0.3 ml 10 per cent KOH was bator at 37° C for 2 hours. The incubations were ter- added to each hanging well, and the flasks were sealed minated by the addition of an equal volume of ethanol, and and gently shaken in a metabolic incubator for 3.5 hours the lipids and steroids extracted twice with 10 ml CH2CI2 at 370 C. Subsequently, 0.3 ml 6N H2SO4 was injected and once more with 20 ml acetone: ethanol (1: 1). through the cap into the incubation medium and the Unlabeled carrier steroids were added to each total flasks were shaken for 1 hour at room temperature. extract, and the solvent evaporated under nitrogen. The The caps and wells were then removed and the KOH carrier steroids included approximately 100 lsg each of: quantitatively removed and diluted to 2.5 ml with water. progesterone, pregnenolone, corticosterone (compound B), In order to measure the C1402 trapped in the KOH, 2.0 11-dehydrocorticosterone (compound A), and cortisone.1 ml of the diluted KOH was added to 10 ml of the scintil- The steroids were partially separated from the sub- lation mixture described by Bray (8) and the resultant strate sterols, and from other less polar lipids, by chro- solution assayed in a Packard liquid scintillation spec- matography on a small column packed with 2 g silica gel trometer with an efficiency of about 40 per cent. Cor- (Davison Chemical Co., 100-200 mesh), with a slight rections for quenching were made by again counting the modification of the method of Goldzieher, Baker, and samples after the addition of internal standard. All other, Riha (9). Preliminary standardization showed that radioassays reported employed 0.5 per cent diphenyl- most of the cholesterol was eluted with 15 ml of 5 per oxazole in toluene as scintillation solvent, with a count- cent ethyl ether in benzene. Progesterone, the least ing efficiency of 50 to 55 per cent. The two counting sys- polar steroid expected, remained entirely on the column. tems were compared by assay of the same amount of C1' All steroids, including cortisol, could then be eluted with absolute standard in each. 50 ml ethyl acetate. For chromatography of the samples At the end of the experiments calculations were made, from the incubations, most of the sterols were eluted with from the recovery of C1402, of the percentage conversion 5 ml hexane and 15 ml 5 per cent ethyl ether in benzene. of the terminal carbons of the sterol side-chain to CO2. The steroids were then eluted with 50 ml ethyl acetate. As mentioned above, the terminal carbons of the side-chain Further purification of the steroids was effected by of each sterol contained one-fifth the total radioactivity paper chromatography.
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  • Download Product Insert (PDF)
    PRODUCT INFORMATION 24-dehydro Cholesterol Item No. 14943 CAS Registry No.: 313-04-2 Formal Name: (3β)-cholesta-5,24-dien-3-ol H Synonyms: Desmosterol, NSC 226126 MF: C H O 27 44 H FW: 384.6 Purity: ≥98% H H Supplied as: A crystalline solid Storage: -20°C HO Stability: ≥2 years Information represents the product specifications. Batch specific analytical results are provided on each certificate of analysis. Laboratory Procedures 24-dehydro Cholesterol is supplied as a crystalline solid. A stock solution may be made by dissolving the 24-dehydro cholesterol in the solvent of choice. 24-dehydro Cholesterol is soluble in organic solvents such as ethanol and dimethyl formamide, which should be purged with an inert gas. The solubility of 24-dehydro cholesterol in these solvents is approximately 0.25 and 50 mg/ml, respectively. 24-dehydro Cholesterol is sparingly soluble in aqueous solutions. To enhance aqueous solubility, dilute the organic solvent solution into aqueous buffers or isotonic saline. If performing biological experiments, ensure the residual amount of organic solvent is insignificant, since organic solvents may have physiological effects at low concentrations. We do not recommend storing the aqueous solution for more than one day. Description 24-dehydro Cholesterol is an immediate precursor to cholesterol in the Bloch pathway of cholesterol biosynthesis. Structurally, it varies from cholesterol only by a single double bond at carbon 24 and has been used as cholesterol substitute in cellular membrane studies.1 During brain development, 24-dehydro cholesterol transiently accumulates, composing up to 30% of total brain sterol, where it is poised to rapidly enrich membrane sterols.2 However, defects in cholesterol synthesis can lead to a condition called, desmosterolosis, which results in an accumulation of excess 24-dehydro cholesterol.3 24-dehydro Cholesterol has been reported to activate liver X receptor-target genes in both the liver of cholesterol-free mice and in cholesterol-starved macrophage foam cells in atherosclerotic lesions.4-5 References 1.
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