THE IN VITRO 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 ers (3). Chromatography of both samples was contin- therapy, 24-dehydrocholesterol (desmosterol) ued until the 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 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-. 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 : 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 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. The steroid samples were ap- in the sterol molecule. plied to separate strips of Whatman no. 1 paper, 46 cm in Experiments with adrenal homogenates. The in vitro length.- Polar steroids were separated first with the conversion of C14-cholesterol and C14-desmosterol to Bush Bi system-toluene: ligroin: methanol: water, 5: 5: steroid hormones by homogenates of adrenal glands from 7: 3-(10). These chromatograms contained compound triparanol-fed rats was studied in two experiments. B and compound A, fairly well separated from each other, Adrenal glands were obtained from 8 male Sprague-Daw- and 11,-hydroxy-4-androstenedione near the end of the ley rats. The rats had been fed 1 per cent triparanol for strip. The less polar steroids were all found in the efflu- 2 to 3 weeks, and weighed about 200 g each at the time ents, or runoffs, from these chromatograms. of study. After decapitation of the animals, the adrenal A second set of paper chromatographies was then car- glands were excised, rinsed in ice-cold saline, blotted dry, ried out, in which the effluents from the first chromato- and weighed. The combined weight of the 16 glands was grams, containing the less polar steroids, were reapplied 348 mg in the first experiment, and 388 mg in the sec- to other paper strips and chromatographed with the ond. The glands were homogenized in a small Potter- Bush Al system-ligroin: methanol: water, 10: 9: 1- Elvehjem homogenizer with 5 ml of a solution of 0.05 M (10). Progesterone was found in the distant third of potassium phosphate buffer at pH 7.4, 0.015 M nicotina- these paper strips, and was separated from pregnenolone mide, 0.05 M KCl, and 0.005 M MgCl2. One-half ml of by about 7 cm. The effluents from these papers con- the homogenate was extracted with acetone-ethanol (1: 1) tained any contaminating cholesterol or desmosterol, and and saponified; the nonsaponifiable fraction was then ex- tracted with light petroleum ether and analyzed by gas- 'A4-pregnene-3,20-dione; 3fi-hydroxy-A'-pregnen-20-one; liquid chromatography. In each experiment this analy- A&4-pregnene-11,8,21-diol-3,20-dione; A'-pregnen-21-ol-3,-11,- sis showed the endogenous adrenal sterols to consist of 20-trione; and 17a,21-dihydroxy-A'-pregnene-3,11,20-trione. IN VITRO METABOLISM OF DESMOSTEROL 2137 were combined with the sterol-containing fractions from and C'4-desmosterol as substrates, mitochondria the silica gel columns. -were-prepared -from mice fed 1 per cent triparanol Each paper chromatogram was scanned for radio- -for'12 Since such mitochondria are activity with an Actigraph II paper scanner days. incapa- strip (Nu- ble' clear-Chicago Corp.). Test portions 1 or 2 mm wide of reducing desmosterol to cholesterol (12), were cut off and tested for reducing areas and for 3,s- this ensured that the added C14-desmosterol would hydroxy-A' steroids with the Allen reagent (11). All not be converted to C14-cholesterol during the in- carrier steroids could be visualized under ultraviolet cubation. Any radioactivity appearing as C1402 light. in the incubations with C14-desmosterol would Areas of radioactivity in the same positions as carrier steroids were cut out and eluted by descending capillary therefore-have had to come directly from desmos- flow of ethanol. Adjacent regions of the papers, which terol. The effectiveness of the drug therapy was were devoid of radioactivity, were also eluted as control checked by assay of the serum sterols of one mouse areas, to determine the background level of radioac- after 9 days of drug feeding. Desmosterol com- tivity on the papers. All or part of each eluted sample prised 35 per cent of the total serum sterols, indi- was then evaporated and radioassayed in the scintillation spectrometer. cating the presence of an effective block of des- The pregnenolone formed from C14-desmosterol in the mosterol reductase. first experiment was further characterized. One-tenth of The results of this study are shown in Table I. the eluate from the pregnenolone area was used for ra- Incubations with each labeled sterol were carried dioassay, and the remainder was reduced, to form 5-preg- out in duplicate, together with a control incubation nene-3#,20,6-diol, with 2 mg potassium borohydride in 4 ml 80 per cent ethanol. After standing for 6 hours at lacking mitochondria. The results indicated that room temperature, the mixture was neutralized with both sterols were effective substrates for oxida- acetic acid, diluted with 2 ml water, and the products tion by the mitochondria. Similar yields of C402 extracted with CH2C12. Chromatography on paper re- were obtained with both sterols, with the yield sulted in a band giving a yellow color with the Allen from cholesterol being slightly greater than that reagent and located directly opposite a reference stand- from ard of 5-pregnene-3p,2O0B-diol. The strip scanner showed desmosterol. Even this slight apparent dif- radioactivity over the same area. This region was eluted ference may not have been real, however, since the and radioassayed, together with the eluate from a control liver mitochondria- in each' incubation contained area. almost 1 ,umole of endogenous sterol, 60 to 70 per The corticosterone formed from both C1'-desmosterol cent of which'was desmosterol. The specific ra- and C14-cholesterol in the second experiment was also characterized further. From each chromatogram of dioactivity -of the added C14-desmosterol was there- polar steroids, a relatively wide area, which included fore probably more reduced than that of C"4-cho- all the corticosterone carrier, was eluted. One-tenth lesterol by dilution with endogenous sterol. It is of the eluate was radioassayed and the remainder acetyl- clear that the two sterols were comparable in their ated with acetic anhydride and pyridine. Chromatog- to serve as raphy of the product ability substrates in this system. in the Bush B1 system for 2.5 hours At the end of the showed one band absorbing ultraviolet light directly op- experiment, the incubation posite the compound B acetate reference standard. The mixtures were saponified and the nonsaponifiable strip scanner showed definite peaks of radioactivity in compounds extracted and assayed for cholesterol the same areas in both chromatograms. Areas of weak radioactivity also appeared opposite compound A acetate. TABLE I The regions corresponding to the acetates of compounds Oxidation of the terminal side-chain of cholesterol and B and A were separately eluted, together with adjacent desmosterol by liver mitochondria from control areas, and were radioassayed. triparanol-fed mice

Yield of RESULTS terminal C14 C'4-sterol added C1402 collected as C1402 Sterol side-chain oxidation by liver mitochon- dpm mlAmole % dria. Preliminary experiments with liver mito- Cholesterol 228 38 4.2 chondria from normal mice showed a consistent Cholesterol 247 42 4.6 yield of 4.5 to 6 per cent of the terminal carbons Desmosterol 147 31 3.4 of C14-cholesterol as C1402, when the experiments Desmosterol 163 34.5 3.8 were conducted as described. Control: no enzyme For the definitive comparison of C14-cholesterol + cholesterol 4 <1 <0.1 2138 DrEWITT S. GOODMAN, JOEL AVIGAN, AND HILDEGARD WILSON cild desmosterol by differential colorimetry (3). the steroid radioactivity found in compounds B Evidence that C14-desmosterol had not been con- and A. No radioactivity was detected with the verted to C14-cholesterol was obtained by chro- strip scanner in locations corresponding to other matography of the sterols, from the incubations steroids. Very small amounts of radioactivity in with C14-desmosterol, as their p-phenylazobenzoyl such locations would not have been detected be- esters. No radioactivity was found in the cho- cause of the low sensitivity (efficiency about 6 per lesterol zones from these chromatographies. cent) of the strip scanner. As described above, Steroid hormone biosynthesis by adrenal ho- control elutions were made with portions of each mogenates. The first experiment with adrenal paper strip that were on each side of the locations homogenates showed a definite conversion of both of the compounds listed in Table II. The radio- C14-sterols into C14-steroid hormones. The total activity eluted from the areas corresponding to the yield of steroid hormones was in the range of 4 to compounds was much greater than that from the 6 per cent of each labeled sterol. In each incuba- control areas, and was corrected by subtraction of tion, radioactivity was detected in compound B, the radioactivity in control areas of identical compound A, pregnenolone, and progesterone. length. The greatest amounts of radioactivity were found Further evidence that the observed radioactivity in the chromatographic areas corresponding to actually resided in the steroid hormones was ob- compound B, although considerable radioactivity tained with pregnenolone and with compound B. was found in the areas of both pregnenolone and After reduction of the eluate from the pregneno- progesterone. More radioactivity was found in lone area (in experiment 1) and rechromatog- the latter two steroids with desmosterol as sub- raphy, the peak of radioactivity coincided with strate than with cholesterol. the location of 5-pregnene-3,8,20,/-diol. Almost Because of the relatively low specific radioac- 70 per cent of the total radioactivity used in the tivity of the first preparation of C14-desmosterol, reduction was recovered in this peak. Similarly, and because of some technical difficulties, exact rechromatography of 90 per cent of the eluates quantitative data could not be obtained from the from the areas corresponding to compound B (in first experiment. The second experiment was experiment 2) after acetylation resulted in the re- hence conducted to obtain quantitative data on covery of 60 to 70 per cent of the radioactivity in the yield of each steroid hormone from each C14- areas corresponding to compound B-acetate and to sterol. The results, summarized in Table II, A-acetate (mostly in B-acetate). This is ap- showed conversion of both C14-cholesterol and of proximately the expected recovery for the series C'4-desmosterol into each of the four steroids of manipulations. For tabulation of the radio- listed. In contrast to the first experiment, only activity recovered in each compound (Table II), very small amounts of radioactivity were detected the C14 found in the A-acetate zone was added to in pregnenolone and progesterone, with almost all the radioactivity found in the area of compound A

TABLE II Biosynthesis of steroid hormones from cholesterol and desmosterol by adrenal gland homogenates from triparanol-fed rats

Radioactivity C'4-sterol added Steroid product Observed Corrected * Yield dpn dprn % rn)Anole Cholesterol Compound B 384 640 2.1 22 Cholesterol Compound A 228 379 1.3 13 Cholesterol Pregnenolone 19 32 0.12 1.2 Cholesterol Progesterone 23 38 0.13 1.3 Desmosterol Compound B 972 1620 2.2 22 Dismosterol Compound A 527 876 1.2 12 Desmosterol Pregnenolone 162 271 0.36 3.7 Desmosterol Progesterone 62 102 0.14 1.4 * Corrected for loss of 2 of 5 labeled carbons during side-chain removal (see text). IN VITRO METABOLISM OF DESMOSTEROL 2139 in the first chromatography. The individual yields employed in order to prevent the conversion of C14- listed for compounds B and A may therefore be desmosterol to C"4-cholesterol during the incuba- somewhat inaccurate, although the sum of the tions. In each experiment, analysis of the sterols yields of compounds B plus A is accurate in each present at the end of the incubations indicated that case. conversion of desmosterol to cholesterol had not In Table II the first column of figures lists the occurred. A quantitative comparison of the effec- actual dpm observed (minus appropriate controls) tiveness of cholesterol and desmosterol as sub- in the areas corresponding to each steroid. Since strates was obtained by conducting simultaneous two of the five labeled carbon atoms of the pre- incubations with labeled sterol substrates that cursor sterol are lost during the removal of the were prepared in the same manner. terminal five carbons of the side-chain en route Despite studies by several groups of workers to steroid hormones, the observed dpm have been (7, 13-17), the details of the in vitro oxidation, corrected for this loss in the second column. by liver mitochondria, of the terminal carbons of The data in Table II indicate that C14-choles- the cholesterol side-chain to CO2 are only partly terol and C14-desmosterol were remarkably simi- understood. This fact is reflected in the large lar in their abilities to act as substrates for the number of cofactors employed in the incubation biosynthesis of steroid hormones. The yields of mixtures. It is, however, generally accepted that the different steroids from the two sterols were this oxidation is related to the reactions involved almost identical. in the biosynthesis of bile acids. Evidence exists Direct evidence was again obtained that the that bile acid biosynthesis involves the oxidative added C14-desmosterol was not converted to C14- removal of the terminal three carbons of the sterol cholesterol during the incubation. The neutral side-chain as propionylcoenzyme A (15). There lipids and sterols from each incubation, obtained is also evidence that the in vitro oxidation of the in the first elution from the small silica gel col- terminal side-chain of cholesterol by liver mito- umns, were saponified, the nonsal)onifiable com- chondria involves removal of carbons 25, 26, and pounds extracted, and one-third of each nonsaponi- 27 as a three-carbon unit (14). The three-carbon fiable fraction added to a 1-mg mixture of non- unit is then presumably completely oxidized to radioactive cholesterol and desmosterol. These Co2. mixtures were then acetylated, and cholesterol In the present experiments, very similar yields acetate and desmosterol acetate separated by thin- of C140, were obtained with C14-cholesterol and layer chromatography (4). Ninety-eight per cent with C14-desmosterol. If the production of this of the radioactivity in the nonsaponifiable fraction C1402 followed the removal of carbons 25 to 27 from the incubation with C"4-cholesterol was found as an intact three-carbon unit, these results would in the cholesterol acetate zone. All of the radioac- suggest that the presence of a double bond between tivity in the nonsaponifiables from the incubation carbons 24 and 25 does not significantly affect the with C14-desmosterol was found in the desmosterol reactions involved in the splitting of the sterol acetate zone. These results, therefore, indicated side-chain at this site. It is, of course, also pos- that the radioactive steroids found in each incuba- sible that the effects of the presence of the 24,25 tion must have been directly derived from the sub- double bond are obscured because other reactions strate sterol added. in the total process may be rate-limiting. The experiments with adrenal gland homoge- DISCUSSION nates demonstrated the formation of 35 to 40 m/Anioles of labeled steroid hormone on incuba- In our previous study, indirect evidence was tion with approximately 1 Mmole of each labeled presented for the conversion of desmosterol to sterol precursor. Compound B, the major steroid bile acids and to steroid hormones in triparanol- normally produced by rat adrenal glands, was the treated men (2). The present study provides con- main kMbeled product in each case. Recent stud- firmatory direct evidence for this conclusion by ies on the biosynthesis of adrenal steroid hormones in vitro experiments with tissues from triparanol- (18-22) have revealed that cholesterol is prob- treated rats and mice. Drug-treated animals were ably first hydroxylated to form 20,22-dihydroxy- 2140 DEWITT S. GOODMAN, JOEL AVIGAN, AND HILDEGARD WILSON cholesterol, followed by cleavage of the side-chain pounds may be synthesized directly from sterol to yield pregnenolone and isocaproic aldehyde. precursors of cholesterol. The enzymes involved are located in the adrenal mitochondria and require molecular oxygen and SUM MARY reduced triphosphopyridine nucleotide. It is gen- The metabolism of C"4-labeled 24-dehydrocho- erally accepted that the hydroxylation of the sterol lesterol (desmosterol) and cholesterol was com- side-chain is the rate-limiting reaction in the pro- pared with in vitro liver and adrenal preparations duction of steroid hormones. The finding that from triparanol-fed mice and rats. Both sterol identical yields of steroids were obtained from substrates were biosynthetically prepared from C14-cholesterol and C14-desmosterol therefore sug- 2-C'4-mevalonic acid. The reactions studied in- gests that the enzymes involved in the side-chain cluded the oxidation of the terminal carbons of hydroxylation, and possibly also the side-chain the sterol side-chain to CO2 by mouse liver mito- cleavage enzyme, are unaffected by the presence of chondria and the conversion of sterol to steroid a double bond in the side-chain. hormones by rat adrenal gland -homogenates. Several reports have indicated that adrenal func- Similar yields of C02, and of steroid hormones, tion seems to be reduced during triparanol ad- were obtained with each of the two sterol sub- ministration (23, 24). The present demonstra- strates, demonstrating that desmosterol and cho- tion that desmosterol is as good a steroid precur- lesterol were quantitatively comparable as sub- sor as cholesterol in the triparanol-treated rat adds strates in these in vitro enzyme systems. These support to our previous conclusion, that the de- results confirm our previous in vivo study (2), cline in adrenal activity during triparanol therapy which suggested that desmosterol was similar to is not due to the specific blocking of the conversion cholesterol in its ability to serve as a precursor for of desmosterol to cholesterol. The reason for the bile acids and for steroid hormones in patients on reduced adrenal function is hence not clear. In triparanol therapy. preliminary analyses we have, however, observed that the sterol content of the adrenals of triparanol- REFERENCES and that the rela- treated rats is less than normal, 1. Goodman, DeW. S., Avigan, J., and Wilson, H. N. tive content of free sterol in these adrenals is The metabolism of desmosterol in human subjects much greater than normal. Thus, analyses of two during triparanol therapy. Biochem. Pharmacol. sets of pooled adrenals from triparanol-fed rats 1961, 8, 87. showed a total sterol content of 5 to 10 mg sterol 2. Goodman, DeW. S., Avigan, J., and Wilson, H. of wet The metabolism of desmosterol in human sub- (cholesterol plus desmosterol) per gram jects during triparanol administration. J. clin. weight tissue; the normal sterol concentration is Invest. 1962, 41, 962. close to 30 mg per gram wet weight (25). A re- 3. Avigan, J., Steinberg, D., Vroman, H. E., Thompson, duced sterol concentration in adrenal glands from M. J., and Mosettig, E. Studies of cholesterol triparanol-treated rats has also been reported by biosynthesis. I. The identification of desmosterol Most was the in serum and tissues of animals and man treated Paoletti (26). striking, however, with MER-29. J. biol. Chem. 1960, 235, 3123. finding that 50 per cent or more of the adrenal 4. Avigan, J., Goodman, DeW. S., and Steinberg, D. sterol in the triparanol-treated rats was present as Thin layer chromatography of sterols and steroids. free sterol, with the same ratio of desmosterol to J. Lipid Res. In press. cholesterol in both free and. total sterol. Normal 5. Cornforth, J. W., Cornforth, R. N., Popjak, G., and most of their Gore, I. Y. Studies on the biosynthesis of cho- rat adrenals contain (90 per cent) lesterol. 5. Biosynthesis of from DL-3- sterol as esterified cholesterol (25). It is possible hydroxy-3-methyl- [2-14C] pentano-5-lactone. Bio- that this great relative increase in free sterol in the chem. J. 1958, 69, 146. drug-fed animals is in some way related to the 6. Isler, O., Ruiegg, R., Wuirsch, J., Gey, K. F., and reduced adrenal function in these animals. Pletscher, A. Zur biosynthese des cholesterins that desmosterol is an ef- aus #,8-dihydroxy-,8-methylvaleriansiure. Helv. 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