The Department of Medicine, University of Miami School of Medicine, The Veterans Administration Hospital, Miami, Florida and The Departments of Investigative and Experimental Medicine, McGill University, Montreal, Canada

METABOLISM OF [4-14C] BY HUMAN ADRENAL GLANDS IN VITRO AND ITS INHIBITION BY METYRAPONE

By Andres Carballeira, Su Chiau Cheng and Lawrence M. Fishman

ABSTRACT

The in vitro conversion of [4-14C] cholesterol1) to hormones was studied in 1 normal, 1 adenomatous and 2 hyperplastic, surgically re- sected, human adrenals. The degree of overall conversion per gram of tissue was similar (4.4\p=n-\5.6%) in NADPH-supplemented homogenates from normal or moderately hyperactive adrenals; a 2-fold increase was found in a markedly hyperfunctioning gland. Only labelled cortisol,

* Address reprint requests to A. Carballeira, M. D., Ph. D., Veterans Administration Hospital, 1201 N. W. 16th Street, Miami, Florida 33125. ** Present address: Department of Biochemistry, University of Southern California School of Medicine, Los Angeles, California. ') In the present report, the following trivial names and abbreviations have been used: metyrapone, 2-methyl-l,2-bis(3-pyridyI)-l-propanone; aminoglutethimide, 2- (p-aminophenyl)-2-ethylglutarimide; o,p'-DDD, 2,2-bis(2-chlorophenyl-4-chloro- phcnyl)l,l-dichloroethane; cholesterol, 5-choIesten-3/?-ol; pregnenolone, 3/?-hydroxy- 5-pregnen-20-one; 17-hydroxypregnenolone, 3/-?,17-dihydroxy-5-pregnen-20-one; pro¬ gesterone, 4-pregnene-3,20-dione; 11-deoxycorticosterone, 21-hydroxy-4-pregnene- 3,20-dione; corticosterone. ll/5,21-dihydroxy-4-pregnene-3,20-dione; aldosterone, ll/?,21-dihydroxy-18-oxo-4-pregnene-3,20-dione; 11-deoxycortisol, 17,21-dihydroxy- 4-pregnene-3,20-dione; cortisol, ll/?,17,21-trihydroxy-4-pregnene-3,20-dione; de- hydroepiandrosterone, 3/?-hydroxy-5-androsten-l 7-one; androstenedione, 4-androst- ene-3,17-dione. ACTH is the abbreviation for adrenocorticotrophic hormone.

Downloaded from Bioscientifica.com at 09/24/2021 02:01:04AM via free access corticosterone, 11-deoxycortisol, 11-deoxycorticosterone and androstene- dione were isolated from incubations with [4-14C] cholesterol, but cortisol accounted for slightly more than 50 % of overall substrate conversion in the normal adrenal, while 11-deoxycortisol and androstenedione pre- dominated in the abnormal glands. "Apparent 11\g=b\-hydroxylaseactivity" was considerably lower in the abnormal glands than in normal tissue when assessed using [4-14C] cholesterol as substrate, but this deficit was not observed in comparable incubations with 14C-labelled pregnenolone, progesterone or 11-deoxycorticosterone. [4-14C] Cholesterol was a more efficient precursor of androstenedione than either [4-14C] pregnenolone or [4-14C] progesterone. In parallel studies, metyrapone (1.0 mM) depressed the formation of both 11-oxy and 11-deoxymetabolies from [4-14C] cholesterol, overall inhibi- tion ranging from 66 to 86%. The conversion of [4-14C] 11-deoxy- corticosterone to corticosterone was inhibited by 93 % under identical conditions. Metyrapone did not, however, impair the overall transforma- tion of either [4-14C]pregnenolone or [4-14C]progesterone, since the in- hibition of cortisol and corticosterone biosynthesis was associated with an increment in 11-deoxycortisol (with [4-14C] pregnenolone) or 11-deoxy- corticosterone (with [4-14C]progesterone). From these studies it appears likely that the additional site of metyrapone inhibition of steroid bio- synthesis suggested by others on the basis of clinical observations involves the rate-limiting, ACTH-regulated conversion of cholesterol to preg- nenolone.

The utilization of labelled cholesterol for steroid biosynthesis by adrenal in vitro preparations is well documented in numerous vertebrates (Hechter 1958; Halkerston et al. 1961; Constantopoulos 8c Tchen 1961; Shimizu et al. 1961; Carballeira 8c Venning 1964; Karaboyas 8c Koritz 1965; Sandor et al. 1965; Carballeira 8c Durnhofer 1968; Mehdi 8c Carballeira 191la,l912). In the human adrenal cortex, such reports are limited to isolated studies, mostly involving virilizing neoplasms: Homogenates from two adrenal adenomas have been shown to transform [4-14C] cholesterol into dehydroepiandrosterone (Guai et al. 1962) or into 17-hydroxyprogesterone and other minor metabolites (Neville et al. 1969). More recently, the incubations of two adrenal carcinomas (Hochberg et al. 1971; Burstein et al. 1971) with [26-14C] cholesterol have been reported to result in the formation of labelled C(¡ side chain fragments. This paucity of data has prompted us to explore the conversion of [4-14C]- cholesterol into and androgens by NADPH-supplemented homo¬ genates from four human adrenal glands obtained from patients with and without overt hypercorticism. Homogenates from one hyperfunctioning gland were also separately incubated with HC-labelled pregnenolone, progesterone and 11-deoxycorticosterone. These studies represent the only data thus far reported relating to the conversion of cholesterol to corticosteroids and andro¬ gens in hyperplastic, ACTH-dependent human adrenals.

Downloaded from Bioscientifica.com at 09/24/2021 02:01:04AM via free access The results of parallel studies conducted in the presence of metyrapone are also reported. Previous work (Cheng 8c Carballeira 1969) has suggested that, in addition to its well recognized action as an inhibitor of mitochondrial, NADPH-mediated 11/3-hydroxylase (Gaunt et al. 1968; Neher 8c Kahnt 1962; Domínguez 8c Samuels 1963; Kraulis 8c Birmingham 1965; Williamson 8c O'Donnell 1967), metyrapone may also act as an inhibitor of the mitochondrial, NADPH-mediated cholesterol oxidase system.

MATERIALS AND METHODS

Reagents. The following [4-14C] labelled substrates were employed: cholesterol, pregnenolone,- progesterone and 11-deoxycorticosterone (specific activities 30-50 mCi/ mmole). Stable and 3H-labelled were used as carriers or reference compounds. Radioactive compounds were purified in paper (PC) and thin-layer (TLC) Chromato¬ graphie systems and unlabelled carriers (Ikapharm), used for final identification, were recrystallized 3 times from pentane:ethanol (Axelrod et al. 1965). Fumarate nicotin- amide, nicotinamide adenine dinucleotide phosphate (NADP) and glucose 6-phosphate (G6P) were purchased from Sigma Chemical Co. Metyrapone was generously supplied by the Ciba Pharmaceutical Co. Solvents were purified and redistilled in accordance with procedures recommended for minimal decomposition of radioactive materials (Frankel Sc Nalbandov 1966). Adrenal glands. AD-l - normal adrenals (12 g), obtained from a 40-year old - woman with carcinoma of the breast and multiple skeletal métastases. The patient showed no clinical manifestations of adrenal dysfunction and hormonal assays were normal. Pathologic examination showed no evidence of métastases in either adrenal. AD-Il left adrenal (10 g), with 3 adenomata 0.6-0.8 cm in diameter, excised from - a 40-year old woman suffering from and marked weakness but lacking clinical features of hypercortisolism or androgen excess. Urinary aldosterone was nor¬ mal (10-12 Mg/24 h) on an unrestricted sodium intake but was not modified by re¬ stricting (200 mg/day) or loading with (12 g/day) dietary sodium. Baseline values of urinary 17-hydroxycorticosteroids (17-OHCS), 17-ketogenic steroids (17-KGS) and 17- ketosteroids (17-KS) were normal and were suppressed normally by low doses (0.5 mg every 6 h for 3 days) of dexamethasone. AD-lll moderately hyperplastic adrenals (17 g), removed from a 49-year old woman with classic- stigmata of Cushing's syndrome (truncal obesity, moon face, marked hirsutism, hypertension, diabetes, osteoporosis and oligomenorrhoea). Baseline urinary steroids were: 17-OHCS 14.6 mg, 17-KGS 32.2 mg, 17-KS 11.6 mg and free cortisol 675//g/24 h. Plasma cortisol (8 a.m.) was 31.0 µgl 100 ml. These values were only slightly decreased by low doses of dexamethasone, but following 2.0 mg every 6 h for 3 days, were as follows: urinary 17-OHCS 3.3 mg, 17-KGS 8.7 mg, 17-KS 6.2 mg, free cortisol 135.0 /(g/24 h; plasma cortisol (8 a.m.) 14 /(g/100 ml. AD-IV markedly hyperplastic adrenals (28 g), removed from a 55-year

- old woman with bronchogenic carcinoma complicated by progressive cutaneous pig¬ mentation and recent onset of hypertension and diabetes. Pathologic examination showed no evidence of métastases in the resected adrenals. Baseline urinary steroids were: 17-OHCS 52.7 mg, 17-KGS 71.3 mg, 17-KS 27.2 and free cortisol 9900 µg/24 h. Plasma cortisol (8 a. m.) was 213 Mg/100 ml. These values were unaffected by either low or high doses of dexamethasone.

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Downloaded from Bioscientifica.com at 09/24/2021 02:01:04AM via free access The clinical data and adrenal tissue were made available through the courtesy of Drs. S. Solomon, E. E. McGarry and M. M. Hoffman of the Royal Victoria Hospital, Montreal (AD-I, HI and IV) and Dr. A. J. Blair, Jr. of the Wayne County General Hospital, Detroit (AD-II). In each case, adrenal resection was carried out at least 10 days after completion of clinical assessment of adrenal function. Fresh tissues from AD-I, III and IV were processed and incubated within 90 min of their surgical re¬ moval. AD-II was frozen immediately after operation and shipped to the laboratory, where the specimen was thawed and processed after 2 days in transit. Experimental procedures. Duplicate observations were carried out in the absence

- (control) and in the presence of metyrapone. [4-14C] Cholesterol was used as substrate with AD-I, II, III and IV; with AD-IV, separate incubations were also carried out with 14C-labelled pregnenolone, progesterone and 11-deoxycorticosterone. Adrenal homogenates were prepared in the cold with Krebs-Ringer phosphate buffered solu¬ tions containing fumarate and nicotinamide (0.04 M each) at pH 7.4. Each incubation vessel contained: 2.0 fid of substrate dissolved in 0.2 ml propylene glycol; NADP (0.6 mM); G6P (1.7 mM); an aliquot of adrenal homogenate representing 1 g of tissue; where indicated, metyrapone (1.0 mM); and additional buffer to adjust the volume to 10.0 ml. Aerobic incubations (95% 02-5% CO¿) were carried out at 37°C for 2 h, fol¬ lowing which acetone was added to stop enzymatic reactions and precipitate protein (Carballeira Se Venning 1964). In order to account for procedural losses during sub¬ sequent extraction and initial resolution of the various metabolites found, measured amounts of 3H-labelled pregnenolone and progesterone were added to the incubation flasks. Previous studies (Mehdi 8c Carballeira 19715) have demonstrated that the re¬ coveries of these labelled compounds closely parallel those of various other tritiated steroids when subjected to the present methods of extraction and Chromatographie resolution. Acetone was removed by vacuum distillation and the aqueous residue ex¬ tracted exhaustively with ethyl acetate and chloroform; the pooled organic phases were washed several times with aqueous solutions (Mehdi Se Carballeira 197 lö). Radio- assays of the remaining aqueous media and the washings showed less than 0.5 °/o of the total radioactivity recovered. The dried acetate-chloroform extract was partitioned between n-hexane and 85 °/o aqueous methanol (Carballeira Se Durnhofer 1968). The «-hexane extract contained only unconverted radiocholesterol when chromato¬ in graphed system PC-12); the identity of this material was further established by the demonstration of constant 3H/14C ratios following the addition of [7-3H]cholesterol and successive chromatography in TLC-1, 2 and 3. The methanol extract was analyzed

2) The following abbreviations were used for paper Chromatographie solvent systems: PC-1, ligroin/propylene glycol; PC-2, toluene/ethylene glycol; PC-3, toluene:ethyl acetate/methanol:water (20:10/50:50); PC-4, isooctane/i-butanol:water (100/50:90); PC-5, benzene/formamide; PC-6, benzene/methanohwater (100/50:50); PC-7, iso- octane:toluene/methanol:water (50:50/105:45); and PC-8. methylcyclohexane:toluene/ methanobwater (100:25/80:20). The following abbreviations were used for thin- layer Chromatographie solvent systems: TLC-1, cyclohexane:ethyl acetate:methanoI (45:45:10); TLC-2, benzene:ethyl acetate (90:10); TLC-3, petroleum ethendiethyl- ether:acetic acid (60:40:2); TLC-4, acetone:benzene (50:50); TLC-5, cyclohexane:ethyl acetate:ethanol (45:45:10); TLC-6, chloroform-ethanol (90:10); and TLC-7, acetone: benzene (20:80). Details and references for these Chromatographie systems have been given elsewhere (Carballeira Se Venning 1964; Mehdi Se Carballeira 19715).

Downloaded from Bioscientifica.com at 09/24/2021 02:01:04AM via free access using a recent modification (Mehdi Se Carballeira 19715) of a steroid separating PC scheme previously described in detail (Carballeira Se Venning 1964). Each of the isolated l4C-labelled metabolites (cortisol, corticosterone, 11-deoxycortisol, 11-deoxy¬ corticosterone and androstenedione) was mixed with its corresponding 3H-labelled carrier and subjected to repetitive chromatography in suitable PC and TLC systems (Carballeira 8- Venning 1964; Mehdi 8c Carballeira 19715) until 3H/14C ratios were constant. The identity and purity of each metabolite were further established by crystallizations and derivative formation (Table 1), as previously reported (Mehdi & Carballeira 19715). Radioactive materials were detected by autoradiography and radio- chromatogram scanning (Carballeira Se Durnltofer 1968). Duplicate radioassays were carried out in a liquid scintillation spectrometer (Packard model 3372) with the fol¬ lowing efficiencies: ,4C = 87 °/o, 3H = 34 °/o (single label) and »C = 55 «Vo, 3H = 22 °/o (double label). Substrate incorporation into metabolites has been expressed as corrected DPM/g tissue/2 h incubation. Procedural losses amounted to 28 ± 4.6 °/o (mean ± sd). Of the radioactivity incubated in each experiment, from 78 to 91 *Vo could be accounted for by the sum of unreacted substrate and reaction products isolated. "Apparent 11/?- hydroxylase activity" has been expressed as [ll-oxymetabolites/(ll-oxymetabolites + 11-deoxymetabolites)] 100 (Nicolis Se Gabrilove 1969).

RESULTS AND DISCUSSION

In the control studies (incubations without metyrapone), the degree of overall conversion of [4-14] cholesterol per gram of adrenal tissue was very similar in homogenates from human adrenals without (AD-I, AD-II) or with moderate (AD-III) in vivo hyperactivity (Table 2); in homogenates from the markedly hyperfunctioning gland (AD-IV), the degree of overall substrate transforma¬ tion was increased by only two-fold (Table 3). This demonstration of the comparable capacities of normal and hyperplastic adrenal tissue to convert [4-14C] cholesterol into steroid hormones under standardized in vitro conditions is consistent with the present concept that the adrenal hyperactivity in Cushing's disease and in the ectopie ACTH syndrome is not a reflection of intrinsic adrenal dysfunction but rather of abnormal extra-adrenal stimulation (Liddle 1967). The pattern of metabolites elaborated from [4-14C] cholesterol was uniform in all control homogenates: only labelled cortisol, corticosterone, 11-deoxy¬ cortisol, 11-deoxycorticosterone and androstenedione were identified in each instance (Tables 2 and 3). In contrast to 5-15 min incubations of rat adrenal mitochondria (Carballeira et al. 1974), pregnenolone was not detected as a conversion product of [4-14C] cholesterol in the present studies. The complete array of microsomal enzymes (i. e., A5, 3/5-hydroxysteroid dehydrogenase-iso- merase, 17a- and 21-hydroxylases and C-17, C-20-lyase) present in human adrenal homogenates and the longer period of incubation (2 h) used here probably account for the absence of this readily metabolized intermediate. Such considerations might also account for the absence of other expected

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Downloaded from Bioscientifica.com at 09/24/2021 02:01:04AM via free access metabolites (progesterone or 17-hydroxyprogesterone) not only in the studies with labelled cholesterol but also in those with labelled pregnenolone and progesterone. The formation of individual metabolites from [4-14C] cholesterol followed two distinct trends; cortisol accounted for 50 °/o of overall [4- 14C]cholesterol conversion in the normal AD-I, while in AD-II, 111 and TV the predominant metabolites formed were 11-deoxycortisol and androstenedione (Tables 2 and 3). These differences resulted in values for "apparent llß-hydroxylase activity" in the normal AD-I that were considerably higher than in AD-II, 111 and IV. When hyperplastic AD-IV was incubated with 14C-labelled preg¬ nenolone, progesterone or 11-deoxycorticosterone, however, the values for "apparent ] l/j-hydroxylase activity" were again similar to those calculated for the normal AD-I incubated in this study with [4-I4C] cholesterol and to those calculated from an analogous incubation of a hyperplastic adrenal with labelled pregnenolone and progesterone reported by Villee (1964). This discrepancy in 11/J-hydroxylation between the studies with labelled cholesterol and those with labelled pregnane-precursors was not further explored. It is possible, however, that in pathologic adrenal tissue both the cholesterol cleavage and the 11/5- hydroxylating systems compete for the available supply of the same or closely related species of mitochondrial cytochrome P-450 (Harding et al. 1968), which may become a limiting factor under certain conditions. The data relating to the formation of androstenedione merit special com¬ ment. Although this androgen is present in normal adrenal tissue and venous effluent (Baulieu et al. 1967), it was unexpected to find it as the second major product elaborated from cholesterol in the normal AD-I (Table 2), inasmuch as adrenal homogenates from other cortisol-producing species have not been shown to transform cholesterol into C19 end-products (Carballeira 8c Venning 1964). Of further interest is the observation that in AD-IV the formation of androstenedione was three times greater from [4-14C] cholesterol than from either [4-,4C]pregnenolone or [4-14C]progesterone, the more proximal precursors (Table 3). This occurred despite the fact that the overall degree of transforma¬ tion of these labelled pregnane-precursors far exceeded that of [4-14C] chole¬ sterol, in keeping with their respective metabolic positions in the biosynthetic scheme of steroidogenesis (Hechter 1958; Karaboyas 8c Koritz 1965). A possible explanation for these findings is an alternate C27 -* C19 pathway, bypassing C21 intermediates (Jungmann 1968). Caution is indicated in this interpretation, since such a direct route of androgen biosynthesis has not been confirmed by other investigators (Hochberg et al. 1971; Burstein et al. 1971). Free dehydroepiandrosterone was not detected in these studies, an observa¬ tion that is consistent with its absence or presence in only minute traces in normal or pathologic adrenal tissue or adrenal venous blood (Baulieu et al. 1967). While dehydroepiandrosterone sulphate is present in adrenal tissue and

Downloaded from Bioscientifica.com at 09/24/2021 02:01:04AM via free access effluent (Baulieu et al. 1967), its possible formation in the present incubations was not definitively examined in view of the negligible radioactivity found in the aqueous phases. It is thus evident that the in vitro metabolism of [4-14C]- cholesterol occurred predominantly through the A3, 3-ketone pathway of bio¬ synthesis in all four adrenals studied. The deficiency in A5, 3/?-hydroxysteroid dehydrogenase-isomerase associated with some virilizing forms of adrenal hyperfunction (Guai el al. 1962; Neville et al. 1969) was not encountered in the hyperplastic adrenals obtained here from patients who exhibited clinical (AD-III) or biochemical (AD-IV) evidence of excessive androgen production, a finding further corroborated in the incubation of AD-IV with [4-14C]preg¬ nenolone (Table 3). As would be expected (Domínguez 8c Samuels 1963; Kraulis 8c Birmingham 1965; Williamson 8c O'Donnell 1967), metyrapone resulted in a marked in¬ hibition of 11/3-hydroxylation in the studies with all substrates (Tables 2 and 3). This inhibition, however, did not impair the total yield of radioactive meta¬ bolites resulting from [4-14C]pregnenolone or [4-14C]progesterone (Table 3). With these substrates, the metyrapone-induced interference with the formation of cortisol and corticosterone was counterbalanced by the accumulation in the in vitro system of an 11-deoxy intermediate. Compensation for the metyrapone blockage of 11-oxymetabolites was achieved, however, through different means with the two precursors: with [4-14C]pregnenolone, it occurred mainly as a result of an increase in 11-deoxycortisol; with [4-14C]progesterone, ex¬ clusively by an increment in 11-deoxycorticosterone. These findings suggest that, in the studies with [4-14C]pregnenolone as substrate, 11-deoxycortisol was preferentially synthesized via 17-hydroxypregnenolone rather than via pro¬ gesterone, providing additional evidence for the concept that progesterone is not an obligatory intermediate in the biogenesis of corticosteroids in the human adrenal cortex (Weliky 8c Engel 1963). In contrast, metyrapone depressed the overall transformation of [4-14C]- cholesterol by the 4 human adrenals, impairing the elaboration of all meta¬ bolites, whether oxygenated at C-ll or not (Tables 2 or 3). Since the overall conversion of [4-14C]pregnenolone proceded without impairment under iden¬ tical conditions (Table 3), it appears likely that metyrapone blocked the meta¬ bolism of labelled cholesterol by preventing scission of its side chain. This assumption has been confirmed in experiments with rat adrenal mitochondria reported in the accompanying paper (Carballeira et al. 1974). A comparison of the data obtained with [4-14C] cholesterol and [4-14C] 11-de¬ oxycorticosterone (Table 3) indicates that 1.0 mM metyrapone results in quan¬ titatively similar inhibition of the side chain cleavage (83%) and the 11/3- hydroxylating (93 °/o) activities, although a dissociation between these in¬ hibitory effects can be demonstrated under other conditions (Carballeira et al. 1974). This dual in vitro inhibition, however, is not unique to metyrapone:

Downloaded from Bioscientifica.com at 09/24/2021 02:01:04AM via free access ascorbic acid (Hayano et al. 1956; Shimizu 1970) and , '-DDD (Hart et al. 1971) also affect adversely both enzymatic complexes. Moreover, amino- glutethimide, a highly selective inhibitor of adrenal cholesterol utilization in vitro (Cohen 1968; Kowal 1969) and in vivo (Dexter et al. 1967; Fishman et al. 1967), also impedes the 11/3-hydroxylation of 11-deoxycorticosterone under the in vitro conditions reported by Kowal (1969). The inhibition of both cholesterol cleavage and 11/j-hydroxylation shown here for metyrapone in vitro is not generally evident when this agent is ad¬ ministered to normal subjects or to patients with Cushing's disease in the course of tests of pituitary-adrenal function (Liddle et al. 1959; Liddle 1967). In patients with unimpaired pituitary-adrenal reserve, the inhibition by metyra¬ pone of 11/3-hydroxylation appears to be the only effect seen, as manifested by the replacement of a significant fraction of circulating cortisol by 11-deoxy¬ cortisol. In fact, the major increases in 11-deoxycortisol regularly found under such circumstances would appear to be inconsistent with an inhibitory effect of metyrapone on the conversion of cholesterol to corticosteroids. These clinical observations are nevertheles not in conflict with the in vitro inhibitory effect of metyrapone on both 11/3-hydroxylation and cholesterol oxidation reported here. In the clinical situations cited, increased levels of circulating ACTH, induced by the inhibitory effect of metyrapone on cortisol formation (Liddle 1967), accelerate the conversion of cholesterol to pregneno¬ lone and thereby tend to overcome the inhibition by metyrapone of this rate- limiting reaction; since the rate of 11/3-hydroxylation is unaffected by acute changes in ACTH, the inhibition of this reaction is apparent as an accumula¬ tion of the 11-deoxy steroid precursor. The hypothesis that inhibitory effects on the cholesterol -> pregnenolone reaction can, for the most part, be com¬ pensated for by increases in circulating ACTH is indirectly supported by studies with aminoglutethimide. Fishman et al. (1967) have demonstrated that, in normal subjects given aminoglutethimide, cortisol secretion is maintained at or near control values, as a consequence of augmented endogenous ACTH release; this phenomenon is not observed in patients with autonomous adrenal tumours given this inhibitor. Further support for this formulation is provided by the observation that the administration of metyrapone to patients unable to respond with increments in ACTH release usually results in interference with total secretion. Such findings led Liddle et al. (1959) and Hudson 8c Evans (1962) to suggest soon after the introduction of metyrapone that this agent might exert an inhibitory effect at an earlier step in cortisol synthesis, in addition to its interference with 11/3-hydroxylation. The data presented here exclude the possibility that metyrapone might inter¬ fere in the human adrenal with hydroxylation at C-17 or C-21. It is also apparent that A'3, 3/3-hydroxysteroid dehydrogenase-isomerase activity proceeds

Downloaded from Bioscientifica.com at 09/24/2021 02:01:04AM via free access without impairment in the presence of this agent. The present studies do suggest, however, that the additional site of metyrapone inhibition first sus¬ pected by Liddle et al. (1959) on the basis of their clinical observations in¬ volves the rate-limiting, ACTH-regulated conversion of cholesterol to preg¬ nenolone.

ACKNOWLEDGMENTS

The authors wish to acknowledge to Professor J. S. L. Browne his encouragement of this investigation. We are grateful to Dr. Kenneth D. Savard for his valuable criticism of the manuscript. These studies were supported by the U. S. Veterans Administration and by grants from the Banting Research Foundation, the Medical Research Council of Canada and the U. S. Public Health Service-National Institutes of Health (5 R01 AM-12570).

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Received on October 24th, 1973.

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