THE OF DESMOSTEROL IN HUMAN SUBJECTS DURING 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 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- 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: , 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 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 (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 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 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. the + fractions of F.A., were chromato- THE: 3a,- cept C,,03 C1903 3a,llp,17a,21-tetrahydroxypregnane-20-one. on paper with a Bush "A2" system (ligroin: 17a,21-trihydroxypregnane-1 1,20-dione. I1-Ketoetiocho- graphed methanol: water, 100: 70: 30) for 6 hours (14). Test lanolone: 3a-hydroxyetiocholane-11,17-dione. 11- Hy- strips were analyzed by the Zimmermann reaction for 17- droxyetiocholanolone: 3a,11,8-dihydroxyetiocholane-17-one. . The oxidized extracts showed a strong 11-Hydroxyandrosterone: 3a,11,B-dihydroxyandrostane-17- band corresponding to etiocholanetrione, and a weaker - one. Etiocholanolone: 3a - hydroxyetiocholane 17-one . to adrenosterone. The etiocholane- Androsterone: 3a-hydroxyandrostane-1 7-one. Adrenoster- band corresponding trione bands were cut out and eluted with ethanol by de- one: A' - androstene - 3,11,17 - trione. : andro- scending capillary flow. An aliquot was assayed by the stane-3a,17a-diol, dihydroandrosterone. Cortisol: 1 lp,17a,- 21-trihydroxy-4-pregnene-3,20-dione. Corticosterone:118,- micro-Zimmermann reaction (15), and the remainder 21 -dihydroxy-4 -pregnene-3,20-dione. Etiocholanedione: was used for radioassay. etiocholane-3,17-dione. : -3,17- The oxidized C190, + C,,03 fractions of F.A. were dione. chromatographed on paper with the Bush "Al" system 964 DEWITT S. GOODMAN, JOEL AVIGAN AND HILDEGARD WILSON

TABLE I Vitrum Pharmaceutical Co. of Stockholm. The isotope- Specific radioactivity of serum sterols and of sterol metabolites containing solutions for intravenous injection were pre- after C14-mevalonate, during treatment with triparanol, pared by the Pharmacy Department of the Clinical Cen- in Patient G.B. ter of the National Institutes of Health. Correction of results. Cholesterol molecules synthe- Specific Sample radio sized from 2-C"-mevalonate contain five labeled carbon Compound analyzed obtained at: activity atoms: carbon atoms 1, 7, and 15 in the sterol nucleus and carbon atoms 22 and 27 in the side chain (16, 17). Des- cpm/,Jmole Serum free desmosterol 3 hrs 452 mosterol synthesized from 2-C"-mevalonate should have Serum free cholesterol 3 hrs 0 the same pattern of labeling. On conversion of this kind Serum unfract. free sterols 3 hrs 154 of labeled cholesterol, or desmosterol, to bile acids, the one labeled carbon atom in the terminal isopropyl group Serum free desmosterol 3 days 99.5 Serum free cholesterol 3 days 5.1 (carbon atom 27) is lost. Similarly, two of the five la- Serum unfract. free sterols 3 days 33.6 beled atoms (nos. 22 and 27) are lost during the removal of the terminal six carbons of the side chain, en route to Red cells, unfract. free steroid hormones (18). The results that follow have 3 36.4 sterols days been corrected for the above losses by multiplying the Cholic acid from bile 3 days 30.1 measured bile acid derivs. from urine 3-4 37.7 C1' specific radioactivities by 5/4, and Cortisol days the corresponding steroid C1' specific activities by 5/3.

(ligroin: methanol: water, 100: 90:10). These chromato- RESULTS grams showed bands corresponding to etiocholanetrione (from C,,O, metabolites) and also strong, less polar For Patient G.B. the specific activities of the se- bands containing mixtures of androstanedione (oxidation rum sterols were determined after 3 hours and after product of androsterone and androstanediol) and etiocho- 3 days. Table I demonstrates that at 3 hours there lanedione (oxidation product of etiocholanolone and etio- was considerable radioactivity in serum free des- cholanediol); both "diones" (to be referred to as "an- mosterol (452 cpm per but no measurable drogen derivatives") are derived from urinary C,,O, ster- pmole) oids and do not separate in the chromatographic system radioactivity in cholesterol. After 3 days the used. These bands were eluted, assayed against an an- specific activity of desmosterol had decreased to drostanedione reference standard (the chromogenic value 99.5 cpm per Atmole and a little radioactivity (5.1 of etiocholanedione is very close to that of androstane- cpm per umole) was detectable in cholesterol. The dione), and radioassayed. specific activity of the unfractionated sterols at both Radioassay was performed with a Packard liquid scintil- lation spectrometer. Sterols and oxidized steroids were 3 hours and 3 days reflects the fact that in this pa- dissolved in 15 ml 0.5 per cent diphenyloxazole (DPO) tient about one-third of the serum sterols was des- in toluene for counting; bile acids were first dissolved in mosterol and two-thirds cholesterol. 1 ml p-dioxane, followed by the addition of 15 ml DPO The ratio of desmosterol to cholesterol in the in toluene. All samples from F.A., together with ab- solute C1' and H' standards, were simultaneously counted unfractionated red cell sterols obtained after 3 days for C" and H' by using appropriate settings for the dis- was very similar to that in plasma. This fact, criminators of the two channels. The quenching of each plus the closeness of the specific radioactivities of isotope was individually corrected by counting the samples the unfractionated free sterols of serum and red again after the addition of a H' internal standard, and cells (see Table I), suggests that desmosterol and then after addition of a C" internal standard. Counting efficiencies for the double-isotope procedure were 35 per cholesterol of serum and red cells were in isotopic cent for C" and 18 per cent for H'; when C" was equilibrium. counted alone, the efficiency was 52 per cent. After 3 days, bile and urine were collected, from Materials. DL-2-C"-mevalonic acid (specific activity, which cholic acid and the cortisol derivatives (plus 2.7 mc per mmole) was obtained from the Volk Radio- chemical Corp. 7-a-H'-cholesterol (specific activity, 5.4 C190, derivatives, as etiocholanetrione: vide supra) mc per mmole) was a product of the New England Nu- were respectively isolated. Both cholic acid and clear Corp. Pure desmosterol, for colorimetric stand- the cortisol derivatives had specific radioactivity ard, was isolated from livers of rats fed triparanol, as much greater than that of serum cholesterol (see previously described (3). Cholic and deoxycholic acids, Table I), demonstrating that a significant part of for use as analytic and chromatographic standards, were obtained from Nutritional Biochemicals Corp. The refer- these materials must have been synthesized di- ence steroids were obtained from USP reference stand- rectly from desmosterol. ards. Cholecystokinin (Cecekin) was obtained from the The second study, with Patient F.A. as subject, DESMOSTEROL METABOLISM DURING TRIPARANOL 965

was more elaborate in that samples were collected 700 at 1- or 2-day intervals for a week. In addition, 600 as already mentioned, H3-labeled cholesterol was 500 injected simultaneously with the C14-mevalonate. 400 *-0 BILE ACIDS (MIXTURE w CHENODEOXY-AND DEOXY- For the sake of clarity, however, the C"A data from J 0 300 CHOLIC ACIDS) this study will initially be presented alone. -- - REFERENCE CURVE: Figure 1 shows the C14 specific radioactivity of 200 _ SERUM FREEDESMOSTEROL the serum free and esterified desmosterol and un- CL fractionated sterols. The C14 specific radioactivity of all cholesterol samples was below 10, and hence i- 100 is not shown in this semilogarithmic plot. The C"A 70 specific radioactivity of cholesterol ranged from 60 6.2 at 3 hours to 7.7 cpm per umole at 7 days for 50 free cholesterol, and from 2.3 to 7.3 cpm per umole 40 n for esterified cholesterol. These low values con- w a- 30 stituted, however, a significant portion of the total co) (SE RUM CHOLESTEROL: ALL<10) radioactivity by 7 days. The ratio of desmosterol I 1 2 3 4 5 6 to cholesterol was remarkably constant during the DAYS study period; desmosterol comprised 32 to 34 FIG. 2. C' SPECIFIC RADIOACTIVITY OF THE BILE ACIDS per cent of the total serum sterols in all six OF PATIENT F.A. DURING TRIPARANOL ADMINISTRATION, samples. AFTER INJECTION OF 2-C -MEVALONATE. Figure 2 shows the corrected C14 specific activity of the bile acids from Patient F.A., compared with terol. Because of a laboratory accident the cholic the specific activity curve of serum free desmos- acid samples could not be obtained, and mixtures of chenodeoxy- and deoxycholic acids were puri- fied and analyzed instead. It is evident that, 600 _ throughout the period of study, the bile acid C14 500 specific radioactivity was much greater than that of 400 serum cholesterol. This indicates that at least part 300 0 0 FREE of these acids must have been made directly from A~~~t A ESTER 0 desmosterol. 200 Figure 3 shows similar data for the cortisol a:~~~' derivatives, analyzed as etiocholanetrione, and for the androgen derivatives, analyzed as "androstane- 100 U DESMOSTEROL dione," isolated from the urine samples of Pa- tient F.A. The C14 specific radioactivity of both 70 N O60- classes of steroids was much higher than that of 50'AA cholesterol throughout the study, demonstrating 40 that part of these steroids must have been made directly from desmosterol. a. 30 ( OuNFRAcTN TED Let us now consider the double-isotope data obtained from Patient F.A. Figure 4 again shows the C14 specific radiactivity curves for se- rum free and esterified desmosterol, and also

. v shows the H3 specific radioactivity curves for free 1 2 3 4 5 6 7 and esterified cholesterol isolated from the same DAYS serum samples. The disappearance rates of cho- FIG. 1. C' SPECIFIC RADIOACTIVITY OF FREE AND ESTERI- lesterol and desmosterol are fairly similar, desmos- FIED STEROLS, DURING TRIPARANOL ADMINISTRATION, IN a little more than THE SERUM OF PATIENT F.A. AFTER INJECTION OF 2-C - terol disappearance being rapid MEVALONATE. that of cholesterol. 966 DEWITT S. GOODMAN, JOEL AVIGAN AND HILDEGARD WILSON

71t00_loo terol in the entire metabolically active pool (i.e., 6 the plasma-red cell-liver pool) was similar to 00 _ that in plasma. This assumption is reasonable, 400 -x *-* CORT/SOL DERIV'S since this ratio was very similar in red cells and w -J 300O _\ANDROGEN^ DER/V'S plasma, and since even a moderate difference in 0 --- REFERENCE CURVE: the liver should have only a small effect on the SERUM FREE 2100 DESMOSTEROL sterol ratio of the entire pool. By then using the a.* 0 three sets of measurements-1) the rate of disap- It pearance of C14 in desmosterol, 2) the rate of dis- Id00 appearance of H3 in cholesterol, and 3) the rate 0- of appearance of C14 in cholesterol-a calculation 70 was made of the fraction of the disappearing OC- U 60 _- E 50 -... desmosterol that reappeared as C14-cholesterol.2 U 40- The calculation showed that in this patient 21 ± 4 z per cent (SE of mean) of the desmosterol which a.w 30 V, disappeared from the metabolically active pool re- (SERUM CHOLESTEROL: ALL<10) 20 1 2 3 4 5 6 TABLE II DAYS Specific radioactivity ratios of H3 to C'4 (cpm/,umole), after C14-mevalonate and H3-cholesterol during triparanol FIG. 3. C' SPECIFIC RADIOACTIVITY OF THE URINARY therapy in Patient F.A. STEROID DERIVATIVES OF PATIENT F.A. DURING TRAPARANOL ADMINISTRATION, AFTER INJECTION OF 2-C'-MEVALONATE. Serum unfrac- Urinary steroids tionated sterols Sample Cortisol Androgen From these data information can be derived collected at: Free Ester derivs. derivs. about the total metabolism of desmosterol in this 3 hrs 2.64 2.98 patient. For this purpose it is necessary to as- 1 day 1.74 1.75 0.79 1.09 2 days 2.27 1.74 0.67 0.78 sume that the mass ratio of cholesterol to desmos- 3 days 2.12 2.12 5 days 2.41 1.71 0.97 0.73 7 days 2.40 2.07 0.91 0.87 700 600 500 DESMOSTEROL (C'4) appeared therein as cholesterol. More than three- fourths of desmosterol disappearance therefore 400 \ O-O FREE A ---- ESTER occurred via other metabolic pathways.3 J 300 - CHOLESTEROL (H3) 0 The use of H3-cholesterol together with C14- 0*---- FREE mevalonate also provided additional information 200 A-A ESTER about the relationship of the serum sterols to the 2 The difference between the disappearance rates of C' rate of z~~~~~~~~~~z and H' in serum cholesterol represented the syn- thesis of C"-cholesterol from C"4-desmosterol. The dis- appearance rate of C"4-desmosterol (in cpm per mg), mul- 70- mass 60- tiplied by the desmosterol: cholesterol ratio, gave

50- the maximum possible increment in C"4-cholesterol spe- cific radioactivity, should cholesterol be the only end iC. 40 5 product of desmosterol metabolism. Comparison of the RUw 30Q -.ATETFADRN RPRNO DIITA observed and maximum possible C"-cholesterol incre- 0. ments then yielded the fraction of disappearing desmosterol 201 that reappeared as cholesterol in the metabolically active 2 3 4 5 6 pool. DAYS SThis is the maximum possible value for desmosterol FIG. 4. SPECIFIC RADIOACTIVITY OF FREE AND ESTERIFIED disappearance via noncholesterol pathways, since some of DESMOSTEROL (C") AND CHOLESTEROL (H') IN THE SE- the disappearing desmosterol might have been converted RUM OF PATIENT F.A. DURING TRIPARANOL ADMINISTRA- to cholesterol in tissue pools that are not in equilibrium TION, AFTER INJECTION OF 2-C'-MEVALONATE AND 7-H'- with the serum cholesterol. Such conversion probably CHOLESTEROL. In cpm of either C" or H' per usmole sterol. occurs only to a minor extent. DESMOSTEROL METABOLISM DURING TRIPARANOL 967 urinary steroid metabolites in Patient F.A. Table tent does the specific radioactivity of the serum II presents the ratios of H3 to C'4, in counts per sterols reflect that of the precursor sterols in the minute per micromole for each isotope, observed organs synthesizing the metabolites under study? in the unfractionated serum sterols and in the uri- For the case of bile acid synthesis the serum free nary steroid derivatives. Both the cortisol and cholesterol has been shown to closely reflect the the androgen derivatives contained significant liver precursor sterol pool. Thus it is known that amounts of both HM and C14 throughout the study. the liver free cholesterol pool is in rapid isotopic This indicates that both the serum cholesterol and equilibrium with serum free cholesterol, and that desmosterol pools served as precursors for the two the bile acids derived from the former are in rapid classes of steroids. The ratio of H3 to C14 was isotopic equilibrium with both (23). Further- decidedly lower in the steroid derivatives than in more, in the studies with Patient F.A. it was found the unfractionated serum sterols. As expected, that the specific radioactivities of both desmosterol only minute amounts of H3 were found in the bile and cholesterol isolated from bile were fairly close acids from F.A. This agrees with the finding of to the corresponding specific radioactivities of the Bergstrom and Lindstedt and their associates that serum sterols. In these samples desmosterol rep- 7-a-hydroxylation is an integral part of bile acid resented an average of 41 per cent of the total biosynthesis and that the formation of deoxycholic biliary sterols. It is thus certain that most of the acid is due to subsequent bacterial action on cholic radioactivity observed in the bile acids was di- acid in the intestinal lumen (19, 20). The min- rectly derived from desmosterol. It is also pos- ute amounts of H3 (less than 15 per cent of the sible that during the early hours of the studies la- C14 cpm per ,umole) observed in the bile acid sam- beled bile acids were formed from sterol precur- ples might reflect some slight isotopic nonhomo- sors of desmosterol; no evidence is available on geneity in the labeling of the 7-a-H3-cholesterol this subject. It is exceedingly unlikely, however, used. that any significant part of the radioactivity of the bile acids was derived from cholesterol, since this DISCUSSION would require a precursor cholesterol pool of The present study has demonstrated the direct much higher specific radioactivity than the en- conversion of desmosterol into bile acids and into tire liver cholesterol pool, contrary to the studies steroid hormones during triparanol therapy. This quoted above. This would, moreover, require the conclusion can be drawn with a high degree of existence of a special fraction of liver desmosterol confidence from the finding that the C14 specific reductase which was much less inhibited by tri- radioactivity of these two classes of metabolites, paranol than was the main body of this enzyme. after C'4-mevalonate injection, was considerably Studies with animals and with liver homogenates higher than that of the serum cholesterol. do not support this possibility (2, 3). Two possible criticisms may be raised here. Comparable detailed information about the First, was the radioactivity observed in the bile adrenal (and testicular) sterol pools is not avail- acids and urinary steroids solely derived from able for man. Animal studies have indicated that sterol precursors? Under normal conditions the guinea pig adrenal gland cholesterol is never in natural D-isomer of mevalonic acid is rapidly and isotopic equilibrium with plasma cholesterol (24), efficiently converted into cholesterol (21), and the probably because of considerable endogenous latter serves as the normal precursor of the me- adrenal sterol biosynthesis, coupled with slow tabolites considered here. The products of the equilibration. Urinary cortisol was, however, in other quantitatively important pathway of mevalo- isotopic equilibrium with adrenal cholesterol (24). nate metabolism, prenoic acids, do not give rise In contrast, adrenal gland and plasma cholesterol to the steroidal ring structure (22). There is no did reach isotopic equilibrium in the rat (25). In basis for an assumption that treatment with tri- the present studies, therefore, the plasma sterol paranol should introduce new metabolic pathways specific radioactivities cannot be assumed to reflect for mevalonic acid, particularly since in these pa- closely those of the precursor adrenal sterol pools. tients labeled mevalonate was rapidly converted The data in Table II do, in fact, suggest that the into a sterol (desmosterol). Second, to what ex- plasma and adrenal precursor sterol pools might 968 DEWITT S. GOODMAN, JOEL AVIGAN AND HILDEGARD WILSON have been different during the period of study with that most of the C14 steroids were synthesized di- Patient F.A. This derives from the persistently rectly from desmosterol. large difference in the H3 to C14 ratio of the uri- An approximate comparison of the abilities of nary steroids and that of the plasma unfractionated desmosterol and cholesterol to serve as precursors sterols. One explanation for this difference is the for bile acids can be obtained from the specific possibility that there was significant endogenous radioactivity data. In the case of Patient G.B. adrenal sterol biosynthesis from C14-mevalonate the specific radioactivity of cholic acid was close during the early hours of the study. If there were to that of the serum (and red cell) unfractionated also a preference for endogenously synthesized free sterols. This suggests that desmosterol and sterol for the synthesis of steroid hormones, or cholesterol were fairly similar in their ability to very poor mixing of the endogenous and plasma serve as precursors. In the case of Patient F.A., sterol pools, or both, the observed lower H3 to C14 the C14 specific radioactivity of the bile acids was, ratio in the urinary steroids would result. There in fact, greater than that of the serum unfraction- are, of course, other possible explanations. Thus, ated sterols throughout most of the study. It is if desmosterol were a better steroid precursor than thus clear that desmosterol is at least comparable is cholesterol, the lower ratio would result. In with cholesterol in its ability to serve as a precursor addition, a variety of other factors might be in- of bile acids. volved, such as different ratios of desmosterol to This conclusion is also consonant with the data cholesterol in the different pools, variable rates of on the over-all metabolism of desmosterol in Pa- equilibration between the sterol pools, or a pos- tient F.A., indicating that approximately three- sible loss of H3 occurring during the conversion of fourths of desmosterol disappeared by routes other 7-H3-cholesterol to steroids because of labilization than conversion to cholesterol. It is almost cer- of the hydrogens at carbon 7 during the double tain that these routes were the same ones normally bond shift from 5,6 to 4,5. involved in cholesterol metabolism, with conver- Despite the absence of detailed information sion of sterol to bile acids and excretion of sterol about these factors, the data presented here pro- in the feces being the quantitatively important vide strong evidence that desmosterol (perhaps processes. together with its precursor sterols) can effectively Animal studies support this conclusion. Blohm, serve as a direct precursor for steroid hormone Kariga and Mackenzie (26) found that, after the synthesis. This derives from the fact that the injection of C14-mevalonate into triparanol-treated urinary steroid C14 specific radioactivity was very rats, a considerable part of the radioactivity ad- much higher than that of plasma cholesterol ministered appeared in the feces as bile acids. throughout the study. In order for C14 to appear Furthermore, in vitro studies from this laboratory in the steroids via cholesterol it would be neces- (to be published in detail elsewhere) have directly sary for the adrenal precursor cholesterol pool to demonstrated that desmosterol and cholesterol are have had a much higher C14 specific radioactivity comparable substrates for oxidation by liver mito- than the corresponding plasma pool had, particu- chondria from triparanol-treated mice. larly early in the study. This in turn would have Because of the uncertainty about the relations required a lack of inhibition of adrenal desmosterol of the various pools involved in steroid biosyn- reductase by triparanol, in order to permit the thesis, only a less satisfactory estimate can be made early formation of C14-cholesterol from mevalo- of the comparative abilities of the two sterols to nate. Animal studies (unpublished) have shown serve as precursors of steroid hormones. The that desmosterol accumulation in adrenal glands data do suggest, however, that the two sterols are is comparable with its accumulation in other tis- probably comparable, since in both patients the sues, indicating an inhibition of adrenal des- steroid specific activities were similar to those of mosterol reductase comparable with that found the bile acids and the unfractionated plasma sterols. elsewhere. It is hence highly unlikely that the At any rate, it is reasonably clear that desmosterol C14-labeled steroids were synthesized via choles- can serve as an effective precursor for steroid terol. Since desmosterol is the major labeled biosynthesis. adrenal sterol after C'4-mevalonate, it is probable Since desmosterol is an effective precursor for DESMOSTEROL METABOLISM DURING TRIPARANOL 969

these sterol metabolites, it is apparent that the en- body. On the other hand, these results add new zymes involved in the splitting of the cholesterol information on the great similarity that exists be- side chain in the biosynthesis of bile acids and tween desmosterol and cholesterol. Avigan and steroids do not require a great deal of structural Steinberg and their associates have already com- specificity in the sterol side chain. In addition, mented upon the similarities of these two sterols these results raise the interesting possibility that in their physical and chemical properties, and have even under normal conditions some bile acid and noted the widespread distribution of desmosterol steroid biosynthesis takes place from sterol pre- in many different tissues, and the comparable rates cursors other than cholesterol. In other words it of esterification of the sterols in vivo (2, 5). It is possible, during the bioysnthetic sequence from is now apparent that the two sterols also enter to cholesterol, that some of the inter- other biosynthetic reaction sequences in a similar mediate sterols could be used directly for the syn- fashion. It is hence likely that desmosterol will thesis of bile acids or steroids. Whether this nor- resemble cholesterol in its relationship to the de- mally occurs to any extent is, of course, not velopment of atheromatous lesions or clinical known. atherosclerosis. Recent observations in this lab- Several recent reports indicate that adrenal oratory (Avigan, unpublished) have in fact indi- function seems to be reduced during triparanol cated that desmosterol can be recovered from both administration. Melby, St. Cyr and Dale found normal and atheromatous aortae of animals being that the production of cortisol and aldosterone was treated with triparanol. It is therefore probable significantly reduced in normal subjects at a dos- that, as previously suggested (5), the best short- age of 1,000 mg per day (27). Hollander, Cho- term indicator of the potential value of triparanol banian and Wilkins noted a slight but statistically therapy resides in its effect on the total sterol significant decrease in urinary 17-hydroxycorti- level, rather than on the cholesterol level per se. costeroid excretion during treatment of patients An average depression of total circulating sterols with 250 mg triparanol daily; 17- ex- of 15 per cent has been reported (5). In addi- cretion was, however, normal (28). Reduced tion, the long-term biological effects of this therapy adrenal vein corticosterone has also been ob- still require further consideration. served during the administration of relatively much larger doses of triparanol to rats (29). In SUMMARY view of the present finding that desmosterol is a The conversion of 24-dehydrocholesterol (des- precursor of a quali- suitable for steroid hormones hormones normal composition, it is quite possible mosterol) to bile acids and to steroid tatively has been studied that this decline in adrenal activity during tri- during triparanol administration in two male subjects. In each study, DL-2-C14- paranol therapy is not due to the blocking of the mevalonic acid was injected intravenously and the conversion of desmosterol to cholesterol per se, C14 specific radioactivity of serum desmosterol but to some other factor. This factor might well and cholesterol was determined after 3 hours and be relatively nonspecific, possibly related to a va- of toxic reactions noted in this laboratory at 1- to 3-day intervals thereafter. Radioactivity riety with (unpublished) during administration of relatively rapidly appeared in desmosterol peak specific obtained within a few hours; cholesterol large doses of triparanol to animals. These effects activity much lower include reduced weight gain and listlessness and C14 specific activity was very during first few of each study. Bile samples induction of abortion in rats. the days duodenal intubation and the bile Somewhat opposing therapeutic implications were collected by acids extracted and purified by column chroma- can be drawn from the results of these studies. tography. The urinary metabolites of cortisol On the one hand, the fact that desmosterol can were extracted from 24-hour urine samples and serve as a satisfactory precursor for bile acids one or two and steroid hormones suggests that the partial re- purified by chromatography in systems. placement of cholesterol by desmosterol during tri- In one study, derivatives of the urinary C,,O0 paranol administration is not likely in itself to steroids were separately isolated and purified. In disrupt seriously the over-all physiology of the both patients the C'4 specific radioactivity of the 970 DEWITT S. GOODMAN, JOEL AVIGAN AND HILDEGARD WILSON bile acids and steroids was much higher than that synthesis and on blood sterol levels in man. J. of cholesterol throughout the study, demonstrat- clin. Invest. 1961, 40, 884. 6. Avigan, J. A method for incorporating cholesterol ing that a significant part of these compounds and other lipides into serum lipoproteins in vitro. must have been. made directly from desmosterol. J. biol. Chem. 1959, 234, 787. In one study 7-a-H3-cholesterol, previously in- 7. Norman, A. Separation of conjugated bile acids by corporated into the patient's serum lipoproteins, partition chromatography. Bile acids and steroids was injected together with the C14-mevalonate, re- 6. Acta chem. scand. 1953, 7, 1413. 8. Sjovall, J. On the separation of bile acids by par- sulting in the labeling of cholesterol and desmos- tition chromatography; bile acids and steroids. terol with different isotopes. Comparison of the Acta physiol. scand. 1953, 29, 232. serum specific activity curves for the two sterols 9. Mosbach, E. H., Zomzely, C., and Kendall, F. E. (C14-desmosterol and H3-cholesterol) showed Separation of bile acids by column-partition chro- that their disappearance rates were fairly similar, matography. Arch. Biochem. 1954, 48, 95. 10. Mosbach, E. H., Kalinsky, H. J., Halpern, E., and desmosterol disappearance being a little more rapid Kendall, F. E. Determination of deoxycholic and than that of cholesterol. Calculation of the over- cholic acids in bile. Arch. Biochem. 1954, 51, 402. all metabolism of desmosterol from the double- 11. Wilson, H., Borris, J. J., and Garrison, M. M. isotope data indicated that in this patient only Chromatographic procedure for the determination 21 ± 4 per cent of the desmosterol that disap- of urinary corticosteroids and C,9 steroids. J. clin. from Endocr. 1958, 18, 643. peared the metabolically active pool reap- 12. Wilson, H., Lipsett, M. B., and Butler, L. C. Steroid peared therein as cholesterol. Approximately excretion in hypophysectomized women, and the three-fourths of desmosterol disappearance, there- initial effects of corticotropin: A study in urinary fore, occurred via noncholesterol pathways. steroid patterns. J. clin. Endocr. 1960, 20, 534. The specific activity data and other considera- 13. Wotiz, H. H., Lemon, H. M., and Marcus, P. De- to cho- termination of urinary 11-oxygenated 17-ketogenic tions suggest that desmosterol is similar steroids. J. clin. Endocr. 1957, 17, 116. lesterol in its ability to serve as a direct precursor 14. Bush, I. E. Methods of paper chromatography of for several sterol metabolites-namely, bile acids steroids applicable to the study of steroids in mam- and steroid hormones. malian blood and tissues. Biochem. J. 1952, 50, 370. ACNOWLEDGM ENT 15. Wilson, H. Chromogenic values of various keto- steroids in a micro modification of the Zimmer- The authors are greatly indebted to Dr. Daniel Stein- mann reaction: Comparison with the macro pro- berg for advice in the design and conduct of these stud- cedure. Arch. Biochem. 1954, 52, 217. ies, and to Mr. Hugh Vroman and Mr. David W. Ryan 16. Cornforth, J. W., Cornforth, R. H., Popjak, G., and for expert technical assistance. Gore, I. Y. Studies on the biosynthesis of cho- lesterol. 5. Biosynthesis of from DL-3- REFERENCES hydroxy-3-methyl- (2-14C) pentano-5-lactone. Bio- 146. 1. Goodman, D. S., Avigan, J., and Wilson, H. N. The chem. J. 1958, 69, metabolism of desmosterol in human subjects dur- 17. Isler, O., Ruegg, R., Wuirsch, J., Gey, K. F., and ing triparanol administration. Biochem. Pharma- Pletscher, A. Zur Biosynthese des Cholesterins aus col. 1961, 8, 87. p,8- Dihydroxy-,8-Methyl -Valeriansaure. Helv. 2. Avigan, J., Steinberg, D., Vroman, H. E., Thompson, chim. Acta 1957, 40, 2369. M. J., and Mosettig, E. Studies of cholesterol bio- 18. Constantopoulos, G., and Tchen, T. T. Cleavage of synthesis. I. The identification of desmosterol in cholesterol sidechain by adrenal cortex. I. Cofac- serum and tissues of animals and man treated with tor requirement and product of cleavage. J. biol. MER-29. J. biol. Chem. 1960, 235, 3123. Chem. 1961, 236, 65. 3. Steinberg, D., and Avigan, J. Studies of cholesterol 19. Bergstrom, S., Lindstedt, S., Samuelson, B., Corey, biosynthesis. II. The role of desmosterol in the E. J., and Gregoriou, G. A. The stereochemistry biosynthesis of cholesterol. J. biol. Chem. 1960, of 7-a-hydroxylation in the biosynthesis of cholic 235, 3127. acid from cholesterol. J. Amer. chem. Soc. 1958, 4. Frantz, I. D., Mobberley, M. L., and Schroepfer, 80, 2337. G. J., Jr. Effects of MER-29 on the intermediary 20. Lindstedt, S., and Sjovall, J. The formation of de- metabolism of cholesterol. Progr. cardiovasc. oxycholic acid from cholic acid in the rabbit. Acta Dis. 1960, 2, 511. chem. scand. 1957, 11, 421. 5. Steinberg, D., Avigan, J., and Feigelson, E. B. Ef- 21. Popjak, G., and Cornforth, J. W. The biosynthesis fects of triparanol (MER-29) on cholesterol bio- of cholesterol. Advanc. Enzymol. 1960, 22, 281. DESMOSTEROL METABOLISM DURING TRIPARANOL 971 22. Christophe, J., and Popjak, G. Studies on the bio- 26. Blohm, T. R., Kariya, T., and Mackenzie, R. D. Ex- synthesis of cholesterol. XIV. The origin of pre- cretory products of the sterol biosynthetic path- noic acids from allyl pyrophosphates in liver en- way in the MER-29 treated rat. Excretion of zyme systems. J. Lip. Res. 1961, 2, 244. MER-29. Progr. cardiovasc. Dis. 1960, 2, 519. 23. Rosenfeld, R. S., and Hellman, L. The relation of 27. Melby, J. C., St. Cyr, M., and Dale, S. L. Reduction plasma and biliary cholesterol to bile acid syn- of adrenal-steroid production by an inhibitor of thesis in man. J. clin. Invest. 1959, 38, 1334. cholesterol biosynthesis. New Engl. J. Med. 1961, 24. Werbin, H., and Chaikoff, I. L. Utilization of 264, 583. adrenal gland cholesterol for synthesis of cortisol 28. Hollander, W., Chobanian, A. V., and Wilkins, by the intact normal and the ACTH-treated guinea R. W. The effects of triparanol (MER-29) in pig. Arch. Biochem. 1961, 93, 476. subjects with and without coronary artery dis- 25. Morris, M. D., and Chaikoff, I. L. The origin of ease. J. Amer. med. Ass. 1960, 174, 5. cholesterol in liver, small intestine, adrenal gland, 29. Holloszy, J., and Eisenstein, A. Effect of MER/29 and testis in the rat: Dietary versus endogenous (triparanol) on corticosterone secretion by rat contributions. J. biol. Chem. 1959, 234, 1095. adrenals. Fed. Proc. 1961, 20, 176.

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