THE METABOLISM OF DESMOSTEROL IN HUMAN SUBJECTS DURING TRIPARANOL ADMINISTRATION DeWitt S. Goodman, … , Joel Avigan, Hildegard Wilson J Clin Invest. 1962;41(5):962-971. https://doi.org/10.1172/JCI104575. Research Article Find the latest version: https://jci.me/104575/pdf Journal of Clinical Investigation Vol. 41, No. 5, 1962 THE METABOLISM OF DESMOSTEROL IN HUMAN SUBJECTS DURING TRIPARANOL ADMINISTRATION * BY DEWITT S. GOODMAN, JOEL AVIGAN AND HILDEGARD WILSON (From the Section on Metabolism, National Heart Institute, and the National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.) (Submitted for publication October 25, 1961; accepted January 25, 1962) Recent studies with triparanol (1-[p-,3-diethyl- Patient G.B. was a 55 year old man with known arterio- aminoethoxyphenyl ]-1- (p-tolyl) -2- (p-chloro- sclerotic heart disease and mild hypercholesterolemia; phenyl)ethanol) have demonstrated that this com- since 1957 he had maintained a satisfactory and stable cardiac status. At the time of the present study he had pound inhibits cholesterol biosynthesis by blocking been taking 250 mg triparanol daily for 4 weeks, and had the reduction of 24-dehydrocholesterol (desmos- a total serum sterol level in the high normal range. terol) to cholesterol (2-4). Administration of Patient F.A. was a 40 year old man with a 4- to 5-year triparanol to laboratory animals and to man re- history of gout and essential hyperlipemia. At the time sults in of of this study he had been on an isocaloric low purine diet the accumulation desmosterol in the for several weeks, and both the gout and hyperlipemia plasma and tissues, usually with some concom- were in remission. He received 250 mg triparanol daily itant lowering of the plasma total sterol concen- for 2 weeks prior to the start of this study. tration (2, 5). In studies with human subjects Design of study. Each study was begun in the fasting it was observed that after about 2 weeks the des- patient by the rapid injection of 20 /Ac of 2-C14-mevalonic mosterol concentration tended to plateau at an acid, in isotonic saline, into an antecubital vein. Patient F.A. also received, in the same injection, 20 jsc of 7-a H'- average level of 27 per cent of the total circulating cholesterol, previously incorporated into his own serum sterols (5). The ratios of desmosterol to cho- lipoproteins by the method of Avigan (6). Blood sam- lesterol in animal tissues (2) differed somewhat ples were collected 3 hours later from the opposite ante- from the ratios in plasma. In addition, injection of cubital vein, and at varying intervals thereafter. We ob- the cholesterol precursor 2-C14-mevalonic acid dur- tained bile in the early mornings by duodenal intubation, employing intravenous cholecystokinin when necessary, ing triparanol administration resulted in the rapid to ensure an adequate sample, and also collected 24-hour appearance of labeled desmosterol in blood, while urine samples, using small amounts of toluene as pre- no significant radioactivity appeared in cholesterol servative. in the first few days (5). Serum sterols. Sera were extracted with 25 vol of The object of the present experiments was to alcohol: acetone, 1: 1 (vol/vol), and the free sterols were precipitated from these extracts as their digitonide com- study the metabolism of desmosterol in patients plexes. After collection of the digitonide precipitates, the receiving triparanol. Since cholesterol serves as supernatants were saponified with ethanolic KOH, neu- the biosynthetic precursor for several physiologi- tralized with acetic acid, and the hydrolyzed ester sterols cally important compounds, including bile acids were precipitated as digitonides. The sterol digitonides and steroid hormones, the question arose whether thus derived from the free and ester sterol fractions desmosterol could also function as a precursor for were then washed, cleaved with pyridine, and the sterols extracted, washed, and dried as described by Steinberg, these compounds without first being transformed Avigan and Feigelson (5). The sterol fractions so ob- into cholesterol. The studies reported herein dem- tained, consisting of mixtures of desmosterol and choles- onstrate the direct conversion of desmosterol to terol, will subsequently be referred to as unfractionated both bile acids and to steroid hormones, and also free or ester sterols. Small portions of the unfractionated information sterols were used for the measurement of cholesterol and provide about the over-all metabolism desmosterol in the sample, by the differential colori- of this sterol in the triparanol-treated man. metric method of Avigan and co-workers (2). Other portions of the sterols were directly assayed for radio- EXPERIMENTAL activity, and the specific radioactivities were determined. Clinical material. Both of the patients studied were The remaining unfractionated sterols of each sample hospitalized on a metabolic ward of the Clinical Center. were separated into cholesterol and desmosterol by chro- matography of their p-phenylazobenzoyl esters on col- * Presented in part at the First International Pharma- umns of silicic acid: Celite, 2: 1 (2). The purified cho- cology Meeting, Stockholm, Sweden, August, 1961 (1). lesterol and desmosterol fractions were then individually 962 DESMOSTEROL METABOLISM DURING TRIPARANOL 963 assayed for radioactivity and were also colorimetrically oids were obtained from the 24-hour urine samples by analyzed for cholesterol and desmosterol. Cholesterol incubation of the urine with p-glucuronidase, followed by was usually obtained free of desmosterol, but the des- acidification, continuous ether extraction for 72 hours, mosterol samples were generally contaminated with small and appropriate washings of the ether extract to re- amounts of cholesterol. As discussed elsewhere, how- move acids and phenolic compounds (11, 12). In the ever (5), by knowing the analytical values for the puri- first study (Patient G.B.), the neutral extract was then fied sterols in the samples counted, it was possible to directly oxidized with chromic acid (CrO3) solution by calculate with precision the individual specific radioac- a slightly modified procedure of Wotiz, Lemon and tivities of cholesterol and desmosterol, despite the con- Marcus (13). The CrO3 oxidation converts most corti- tamination of the desmosterol samples with small amounts sol metabolites to etiocholanetrione; the other metabo- of cholesterol. An internal check on the accuracy of lites giving significant amounts of this product are the these determinations was provided by the specific radio- C1903 steroids (e.g., 1 1-hydroxyetiocholanolone). This activity measurements of the unfractionated sterols. The procedure, consequently, provided enough of a single calculated and measured values for specific radioactivity compound for accurate determination of specific radio- of the unfractionated sterols agreed closely in all cases. activity. The excess CrO3 was reduced with 0.2 per Bile. Bile samples were extracted with 25 vol of cent NaHSO3 and the steroids extracted with methylene ethanol. Each ethanol extract was evaporated nearly to chloride. This extract was washed with 0.1 N NaOH dryness and saponified with NaOH under pressure. The and with water, and then prepared for paper chromatog- unsaponifiable fraction was then extracted with ethyl raphy. ether, followed by acidification of the sample and extrac- In the second study (Patient F.A.) the entire extract tion of the acidic fraction with ethyl ether. Cholic acid of each urine was first subjected to partition column was purified from the acidic fraction of the bile of Pa- chromatography as described elsewhere (11), with a tient G.B. by reversed-phase partition chromatography, column of 15 g aluminum silicate and 50 per cent with methanol-water as moving phase and isooctanol- ethanol as stationary phase. The elution sequence was chloroform as stationary phase, as described by Norman simplied so that only two fractions were collected: 1) (7) and Sjovall (8). A mixture of chenodeoxy- and 200 ml 15 per cent chloroform in hexane; and 2) 260 deoxycholic acids was purified from the acidic fractions ml 30 per cent chloroform in hexane, followed by 120 ml of the bile samples of Patient F.A. by partition chroma- 50 per cent chloroform in hexane. The first fraction tography as described by Mosbach, Zomzely and Ken- contained all C,,O2 and C,903 (and any C2,04) steroids; dall (9), with acetic acid on Celite as stationary phase the second fraction contained all C2105 and C210A metabo- and isopropyl ether-petroleum ether mixtures as mobile lites present (i.e., all the cortisol metabolites and little phase. The amount of purified bile acid obtained was de- or nothing else). Paper chromatograms run on aliquots termined by weighing the material after crystallization of both fractions showed that in all cases the column had and drying; verification of these values was secured by effected the desired separation. These spot tests also spectrophotometric analyses of aliquots of the samples by proved that the patient was excreting the normal ma- the method of Mosbach, Kalinsky, Halpern and Kendall jor steroid metabolites in his urine. Thus the cortisol (10). The specific radioactivity of the bile acids was metabolite fraction included both THF and THE; the obtained by radioassay of a measured aliquot of the C390, steroids present included 11-ketoetiocholanolone, 11- purified sample. hydroxyetiocholanolone (in lesser amounts), and 11-hy- In some samples sterols were precipitated from the droxyandrosterone; the C1902 steroids were represented unsaponifiable fraction of bile as their digitonide com- by a band corresponding to etiocholanolone and andros- plexes, analyzed colorimetrically, and counted to deter- terone (which would not separate in the system used). mine specific radioactivity. Each column fraction from the urine samples of Pa- Urinary steroids.' Ether extracts of the neutral ster- tient F.A. was then subjected to chromic acid oxidation, 1 as described above, followed by paper chromatography of Trivial names of the steroids used in this paper fol- The oxidized steroid fractions, ex- THF: the oxidized steroids. low. Etiocholanetrione: etiocholane-3,11,17-trione.