Spironolactone Inhibition of Cortisol Production by Guinea Pig Adrenocortical Cells
Summary Prior investigations with adrenal subcellular fractions demonstrated that the diuretic, spironolactone (SL), was converted to a reactive metabolite by adrenal microsomes, resulting in the degradation of microsomal cytochrome(s) P-450. Studies were done to evaluate the effects of SL and 7a-thio-SL, a putative intermediate in the activation pathway, on Cortisol production by intact guinea pig adrenocortical cells. Preincubation of adrenal cells with SL or 7a-thio-SL caused time-dependent and concentration-dependent decreases in subsequent ACTH-stimulated Cortisol production. 7a-Thio-SL was a far more potent inhibitor than SL. In the absence of a preincubation period, neither SL nor 7a-thio-SL affected Cortisol production. The results indicate that the effects of SL on adrenal microsomal cytochrome(s) P-450 compromise steroid synthesis by intact adrenal cells and lend support to the hypothesis that metabolism of the drug is required for the inhibition of steroidogenesis. Key words Spironolactone — Adrenal Cortex — Cortisol — Steroidogenesis — Cytochrome P-450
Introduction Spironolactone (SL) is a potassium sparing diuretic used widely in the treatment of congestive heart failure, cirrhosis and other edematous states (Saunders and Alberti 1978). In recent years, SL has also been commonly employed to treat hirsutism (Givens 1985). The side effects of SL include destruction of adrenal and testicular cytochromes P-450 (Greiner, Kramer, Jarrell and Colby 1976; Menard, Guenthner, Kon and Gillette 1979), the terminal oxidases for several steroidogenic enzymes (Hall 1985). The actions of SL on cytochromes P-450 are apparently mediated by a reactive metabolite produced by adrenal and testicular microsomal enzymes. Recent studies indicate that the deacetylation of SL to 7a-thioSL is an obligatory first step in the activation pathway, but the further metabolism of 7a-thio-SL is required for P-450 degradation (Sherry, O'Donnell, Flowers, LaCagnin and Colby 1986). Horm. metab. Res. 22(1990) 573-575 ©Georg Thieme Verlag Stuttgart-New York
Although the decline in adrenal cytochrome(s) P-450 caused by SL decreases the maximal activities of several steroidogenic enzymes (Greiner et al. 1976; Menard et al. 1979), little is known about the extent to which these changes impact on adrenal steroid synthesis and/or secretion. The rate-limiting step for adrenal steroidogenesis is cholesterol sidechain cleavage, a mitochondrial enzyme (Hall 1985), but it is microsomal cytochromes P-450 that are most dramatically affected by SL (Greiner et al. 1976; Menard et al. 1979). Changes in plasma corticosteroid levels as a result of spironolactone treatment have been noted by several investigators (Abshagen, Sporl, Schoneshofer and Oelkers 1977; Serafini and Lobo 1985; Tuck, Sowers, Fittingoff, Fisher, Berg, Asp and Mayes 1981). However, studies concerning the actions of SL in vivo on adrenal steroid secretion are complicated by the potential for extraadrenal effects which can secondarily influence plasma steroid levels. Prior in vitro investigations have been done with adrenal subcellular fractions which are useful for mechanistic studies but which are unphysiological preparations and their use precludes the possibility of evaluating changes in overall steroid synthesis from normal endogenous precursors. Therefore, the following studies were done to evaluate the direct effects of SL and 7a-thio-SL on Cortisol production by intact guinea pig adrenocortical cells. Methods Adult, male English Short Hair guinea pigs weighing approximately 800-1000 g were obtained from Camm Research Institute, Wayne, NJ and used in all experiments. Animals were housed under standardized conditions of light (0600—1800) and temperature (22 °C) and received food and water ad lib. All animals were killed by decapitation between 0800—0900. Adrenal glands were quickly removed, trimmed free of fat, and placed in cold Minimal Essential Medium (MEM). Adrenals were then bisected longitudinally and the tan outer zone, consisting of zona glomerulosa and zona fasciculata, was gently dissected from the dark inner zone, consisting of zona reticularis, as described previously (Martin and Black 1982; Eacho and Colby 1983). Isolated adrenal cells were prepared from the outer zone preparations by collagenase dispersion (Nishikawa and Strott 1984). We previously demonstrated that the outer zone is the major source of Cortisol in the guinea pig adrenal cortex (Eacho and Colby 1983). The final cell pellet was resuspended in MEM containing 2% bovine serum albumin and 25 mM Hepes buffer (pH 7.4). Cell numbers were determined with a hemocytometer and cell viability by trypan blue exclusion (Eacho and Colby 1983). Viability was usually about 85—90 %. For time-course and concentration-dependence studies on the effects of SL or 7a-thio-SL, the isolated adrenal cells were Received: 9 Nov. 1989
Accepted: 21 March 1990 after revision
Downloaded by: National University of Singapore. Copyrighted material.
Kelly A. Rourke and H. D. Colby Department of Pharmacology and Toxicology, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania, U. S. A.
Horm. metab. Res. 22 (1990)
Kelly A. RourkeandH.
O •
40
Time (min)
20
Spironolactone 7a-Thio-SL
40
60
80
100
Drug Concentration (/iM)
Fig. 1 Time-course for the preincubation effects of spironolactone (SL) on Cortisol production by isolated adrenal cells in response to ACTH (10 ng). All flasks were preincubated with SL or ethanol vehicle and then incubated with ACTH as described in the Methods. Cortisol was measured by radioimmunoassay. Values are means + the SE of 4 experiments.
Fig. 2 Effects of preincubating isolated adrenal cells with varying concentrations of spironolactone (SL) or 7a-thio-SL for 60 minutes on subsequent Cortisol production in response to ACTH (10 ng). Control flasks received ethanol vehicle only. Values are expressed as mean % of control ±SE of 4 experiments. Control values (100%) were 7.2±0.4 ng cortisol/105 cells/90 min.
resuspended to a final concentration of 1 x 10 cells/ml. SL or 7athio-SL (in 5 or 10 JJ,1 of ethanol) was preincubated with aliquots of the cell suspension in a rotary shaker bath at 37 °C under air for 60 minutes unless otherwise indicated. Control flasks received equal volumes of vehicle only. After the preincubation period, cells were pelleted by centrifugation for 5 minutes at 100 xg. Pellets were washed twice with MEM-BSA medium and aliquots (1 x 105 cells) were then incubated with and without 10 ng adrenocorticotropic hormone (ACTH 1—24 fragment, Sigma) for 90 minutes at 37 °C under air in a total volume of 2 ml. The results of preliminary experiments indicated that 10 ng ACTH caused a maximal Cortisol secretory response irrespective of the preincubation conditions. After incubation, steroids were extracted from the medium with 5 ml methylene dichloride and reconstituted in 50 u.1 ethanol after evaporation of the methylene dichloride.
These observations confirm and extend earlier studies on SL actions in broken adrenal cell preparations {Greiner et al. 1976; Menard et al. 1979). The use of adrenal subcellular fractions has been necessary for a detailed characterization of the enzymatic reactions involved in SL activation and for localization of the sites of metabolism. However, studies with isolated adrenal cells can provide information on the impact of competing metabolic pathways, cofactor limitations, detoxification reactions, and other factors relevant to the whole cell. In addition, the use of intact cells allows for determination of the consequences of SL-induced enzymatic changes on overall steroidogenic activity, i. e. Cortisol synthesis.
Cortisol was measured with a highly specific radioimmunoassay. The Cortisol antiserum was obtained from Radioassay Systems Laboratories, Inc., Carson, CA. Cross reaction of the antiserum with spironolactone, 7a-thiospironolactone, aldosterone or androstenedione was less than 0.01 %. Data are presented as the mean values of 4 experiments + SE. Statistical analyses were done with the Student's t-test or the Newman-Keuls multiple range test, as appropriate. Results and Discussion Preincubation of adrenal cells for up to 60 minutes in the absence of spironolactone (SL) did not affect subsequent Cortisol production in response to ACTH stimulation (Figure 1). Addition of 100 jiM SL to the cell suspensions, but without preincubation at 37 °C, similarly did not affect the amount of Cortisol subsequently produced. However, preincubation of adrenal cells with 100 (J.M SL caused a time-dependent decrease in Cortisol production (Figure 1), suggesting that SL metabolism might be required for the inhibition of steroidogenesis. 7a-Thio-SL, which is believed to be an obligatory intermediate in the actions of SL on adrenal cytochromes P-450 {Sherry et al. 1986), also inhibited Cortisol production by adrenal cells (Figure 2). The extent of inhibition of steroidogenesis by 7a-thio-SL, like that by SL, was dependent upon the duration of the preincubation with adrenal cells. In the absence of preincubation, 7a-thio-SL did not affect Cortisol levels (data not shown). The effects of 7a-thio-SL resulting from preincubation were far greater than those of SL (Figure 2). The concentrations of SL and 7a-thio-SL effecting approximately 50 % decreases in subsequent Cortisol production were 20 u,M and 2 u,M respectively.
The inhibition of Cortisol production by SL suggests that the drug-induced degradation of microsomal cytochrome^) P-450 previously demonstrated has functional consequences. The preincubation requirement for SL inhibition of Cortisol synthesis is consistent with the formation of an active metabolite within adrenocortical cells (Greiner et al. 1976; Menard et al. 1979). In addition, the far greater potency of 7a-thio-SL than SL lends additional support to its role as an intermediate in the activation pathway (Sherry et al. 1986), but 7a-thio-SL must apparently be further metabolized for inhibition of steroidogenesis to occur. Thus, these findings suggest that the results obtained previously with adrenal subcellular fractions are relevant to hormone production by intact adrenocortical cells. Further investigations on the actions and metabolism of SL in whole cell preparations may help to determine the specific mechanism(s) responsible for the inhibition of steroidogenesis. Acknowledgements These investigations were supported by USPHS research grants CA 43604 and GM 30261. Spironolactone and 7 athiospironolactone were generously provided by G. D. Searle and Co., Chicago, Illinois. References Abshagen, V., S. Sporl, M. Schoneshofer, W. Oelkers: Increased plasma 11-deoxycorticosterone during spironolactone medication. J. Clin. Endocrinol. 44:1190-1193 (1977) Eacho, P. I., H. D. Colby: Regional distribution of adrenal xenobiotic and steroid metabolism. Life Sci. 32:1119 -1127 (1983)
Downloaded by: National University of Singapore. Copyrighted material.
20
O •
D. Colby
Horm. metab. Res. 22 (1990)
Requests for reprints should be addressed to: Givens, J. R.: Treatment of hirsutism with spironolactone. Fertil. Steril. 43:841-843(1985) Howard D. Colby, Ph. D. Greiner, J. W., R. E. Kramer, J. Jarrell, H D. Colby: Mechanism of action of spironolactone on adrenocortical function in guinea pigs. J. Department of Pharmacology and Toxicology Pharmacol. Exp.Ther. 198:709-715(1976) Philadelphia College of Pharmacy and Science Hall, P. F.: Role of cytochromes P-450 in the biosynthesis of steroid Woodland Avenue and 43rd Street hormones. Vitam. Horm. 42:315-368 (1985) Philadelphia, PA 19104 (U. S. A.) Martin, K. O., V. H. Black: Delta 4-hydrogenase in guinea pig adrenal: Evidence of localization in zona reticularis and age-related change. Endocrinology 110:1749-1757(1982) Menard, R. J., T. M. Guenthner, H. Kon, J. R. Gillette: Studies on the destruction of adrenal and testicular cytochrome P-450 by spironolactone. J. Biol. Chem. 254:1726-1733 (1979) Nishikawa, T., C. A. Strott: Cortisol production by cells isolated from the outer and inner zones of the adrenal cortex of the guinea pig. Endocrinology 114; 486-491 (1984) Saunders, F. J., R. L. Alberti: Aldactone; Spironolactone: A Comprehensive Review. Searle, Inc., New York (1978) Serafini, P., R. A. Lobo: The effects of spironolactone on adrenal steroidogenesis in hirsute women. Fertil. Steril. 44: 595—599 (1985) Sherry, J. H., J. P. O'Donnell, L. Flowers, L. B. LaCagnin, H. D. Colby: Metabolism of spironolactone by adrenocrotical and hepatic microsomes: Relationship to cytochrome P-450 destruction. J. Pharmacol. Exp.Ther. 236:675-680 (1986) Tuck, M. L., J. R. Sowers, D. B. Fittingoff, J. S. Fisher, G. J. Berg, N. D. Asp, D. M. Mayes: Plasma corticosteroid concentrations during spironolactone administration: Evidence for adrenal biosynthetic blockade in man. J. Clin. Endocrinol. 52:1057-1061 (1981)
575
Downloaded by: National University of Singapore. Copyrighted material.
Spironolactone and Adrenocortical Function
Summary Prior investigations with adrenal subcellular fractions demonstrated that the diuretic, spironolactone (SL), was converted to a reactive metabolite by adrenal microsomes, resulting in the degradation of microsomal cytochrome(s) P-450. Studies were done to evaluate the effects of SL and 7a-thio-SL, a putative intermediate in the activation pathway, on Cortisol production by intact guinea pig adrenocortical cells. Preincubation of adrenal cells with SL or 7a-thio-SL caused time-dependent and concentration-dependent decreases in subsequent ACTH-stimulated Cortisol production. 7a-Thio-SL was a far more potent inhibitor than SL. In the absence of a preincubation period, neither SL nor 7a-thio-SL affected Cortisol production. The results indicate that the effects of SL on adrenal microsomal cytochrome(s) P-450 compromise steroid synthesis by intact adrenal cells and lend support to the hypothesis that metabolism of the drug is required for the inhibition of steroidogenesis. Key words Spironolactone — Adrenal Cortex — Cortisol — Steroidogenesis — Cytochrome P-450
Introduction Spironolactone (SL) is a potassium sparing diuretic used widely in the treatment of congestive heart failure, cirrhosis and other edematous states (Saunders and Alberti 1978). In recent years, SL has also been commonly employed to treat hirsutism (Givens 1985). The side effects of SL include destruction of adrenal and testicular cytochromes P-450 (Greiner, Kramer, Jarrell and Colby 1976; Menard, Guenthner, Kon and Gillette 1979), the terminal oxidases for several steroidogenic enzymes (Hall 1985). The actions of SL on cytochromes P-450 are apparently mediated by a reactive metabolite produced by adrenal and testicular microsomal enzymes. Recent studies indicate that the deacetylation of SL to 7a-thioSL is an obligatory first step in the activation pathway, but the further metabolism of 7a-thio-SL is required for P-450 degradation (Sherry, O'Donnell, Flowers, LaCagnin and Colby 1986). Horm. metab. Res. 22(1990) 573-575 ©Georg Thieme Verlag Stuttgart-New York
Although the decline in adrenal cytochrome(s) P-450 caused by SL decreases the maximal activities of several steroidogenic enzymes (Greiner et al. 1976; Menard et al. 1979), little is known about the extent to which these changes impact on adrenal steroid synthesis and/or secretion. The rate-limiting step for adrenal steroidogenesis is cholesterol sidechain cleavage, a mitochondrial enzyme (Hall 1985), but it is microsomal cytochromes P-450 that are most dramatically affected by SL (Greiner et al. 1976; Menard et al. 1979). Changes in plasma corticosteroid levels as a result of spironolactone treatment have been noted by several investigators (Abshagen, Sporl, Schoneshofer and Oelkers 1977; Serafini and Lobo 1985; Tuck, Sowers, Fittingoff, Fisher, Berg, Asp and Mayes 1981). However, studies concerning the actions of SL in vivo on adrenal steroid secretion are complicated by the potential for extraadrenal effects which can secondarily influence plasma steroid levels. Prior in vitro investigations have been done with adrenal subcellular fractions which are useful for mechanistic studies but which are unphysiological preparations and their use precludes the possibility of evaluating changes in overall steroid synthesis from normal endogenous precursors. Therefore, the following studies were done to evaluate the direct effects of SL and 7a-thio-SL on Cortisol production by intact guinea pig adrenocortical cells. Methods Adult, male English Short Hair guinea pigs weighing approximately 800-1000 g were obtained from Camm Research Institute, Wayne, NJ and used in all experiments. Animals were housed under standardized conditions of light (0600—1800) and temperature (22 °C) and received food and water ad lib. All animals were killed by decapitation between 0800—0900. Adrenal glands were quickly removed, trimmed free of fat, and placed in cold Minimal Essential Medium (MEM). Adrenals were then bisected longitudinally and the tan outer zone, consisting of zona glomerulosa and zona fasciculata, was gently dissected from the dark inner zone, consisting of zona reticularis, as described previously (Martin and Black 1982; Eacho and Colby 1983). Isolated adrenal cells were prepared from the outer zone preparations by collagenase dispersion (Nishikawa and Strott 1984). We previously demonstrated that the outer zone is the major source of Cortisol in the guinea pig adrenal cortex (Eacho and Colby 1983). The final cell pellet was resuspended in MEM containing 2% bovine serum albumin and 25 mM Hepes buffer (pH 7.4). Cell numbers were determined with a hemocytometer and cell viability by trypan blue exclusion (Eacho and Colby 1983). Viability was usually about 85—90 %. For time-course and concentration-dependence studies on the effects of SL or 7a-thio-SL, the isolated adrenal cells were Received: 9 Nov. 1989
Accepted: 21 March 1990 after revision
Downloaded by: National University of Singapore. Copyrighted material.
Kelly A. Rourke and H. D. Colby Department of Pharmacology and Toxicology, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania, U. S. A.
Horm. metab. Res. 22 (1990)
Kelly A. RourkeandH.
O •
40
Time (min)
20
Spironolactone 7a-Thio-SL
40
60
80
100
Drug Concentration (/iM)
Fig. 1 Time-course for the preincubation effects of spironolactone (SL) on Cortisol production by isolated adrenal cells in response to ACTH (10 ng). All flasks were preincubated with SL or ethanol vehicle and then incubated with ACTH as described in the Methods. Cortisol was measured by radioimmunoassay. Values are means + the SE of 4 experiments.
Fig. 2 Effects of preincubating isolated adrenal cells with varying concentrations of spironolactone (SL) or 7a-thio-SL for 60 minutes on subsequent Cortisol production in response to ACTH (10 ng). Control flasks received ethanol vehicle only. Values are expressed as mean % of control ±SE of 4 experiments. Control values (100%) were 7.2±0.4 ng cortisol/105 cells/90 min.
resuspended to a final concentration of 1 x 10 cells/ml. SL or 7athio-SL (in 5 or 10 JJ,1 of ethanol) was preincubated with aliquots of the cell suspension in a rotary shaker bath at 37 °C under air for 60 minutes unless otherwise indicated. Control flasks received equal volumes of vehicle only. After the preincubation period, cells were pelleted by centrifugation for 5 minutes at 100 xg. Pellets were washed twice with MEM-BSA medium and aliquots (1 x 105 cells) were then incubated with and without 10 ng adrenocorticotropic hormone (ACTH 1—24 fragment, Sigma) for 90 minutes at 37 °C under air in a total volume of 2 ml. The results of preliminary experiments indicated that 10 ng ACTH caused a maximal Cortisol secretory response irrespective of the preincubation conditions. After incubation, steroids were extracted from the medium with 5 ml methylene dichloride and reconstituted in 50 u.1 ethanol after evaporation of the methylene dichloride.
These observations confirm and extend earlier studies on SL actions in broken adrenal cell preparations {Greiner et al. 1976; Menard et al. 1979). The use of adrenal subcellular fractions has been necessary for a detailed characterization of the enzymatic reactions involved in SL activation and for localization of the sites of metabolism. However, studies with isolated adrenal cells can provide information on the impact of competing metabolic pathways, cofactor limitations, detoxification reactions, and other factors relevant to the whole cell. In addition, the use of intact cells allows for determination of the consequences of SL-induced enzymatic changes on overall steroidogenic activity, i. e. Cortisol synthesis.
Cortisol was measured with a highly specific radioimmunoassay. The Cortisol antiserum was obtained from Radioassay Systems Laboratories, Inc., Carson, CA. Cross reaction of the antiserum with spironolactone, 7a-thiospironolactone, aldosterone or androstenedione was less than 0.01 %. Data are presented as the mean values of 4 experiments + SE. Statistical analyses were done with the Student's t-test or the Newman-Keuls multiple range test, as appropriate. Results and Discussion Preincubation of adrenal cells for up to 60 minutes in the absence of spironolactone (SL) did not affect subsequent Cortisol production in response to ACTH stimulation (Figure 1). Addition of 100 jiM SL to the cell suspensions, but without preincubation at 37 °C, similarly did not affect the amount of Cortisol subsequently produced. However, preincubation of adrenal cells with 100 (J.M SL caused a time-dependent decrease in Cortisol production (Figure 1), suggesting that SL metabolism might be required for the inhibition of steroidogenesis. 7a-Thio-SL, which is believed to be an obligatory intermediate in the actions of SL on adrenal cytochromes P-450 {Sherry et al. 1986), also inhibited Cortisol production by adrenal cells (Figure 2). The extent of inhibition of steroidogenesis by 7a-thio-SL, like that by SL, was dependent upon the duration of the preincubation with adrenal cells. In the absence of preincubation, 7a-thio-SL did not affect Cortisol levels (data not shown). The effects of 7a-thio-SL resulting from preincubation were far greater than those of SL (Figure 2). The concentrations of SL and 7a-thio-SL effecting approximately 50 % decreases in subsequent Cortisol production were 20 u,M and 2 u,M respectively.
The inhibition of Cortisol production by SL suggests that the drug-induced degradation of microsomal cytochrome^) P-450 previously demonstrated has functional consequences. The preincubation requirement for SL inhibition of Cortisol synthesis is consistent with the formation of an active metabolite within adrenocortical cells (Greiner et al. 1976; Menard et al. 1979). In addition, the far greater potency of 7a-thio-SL than SL lends additional support to its role as an intermediate in the activation pathway (Sherry et al. 1986), but 7a-thio-SL must apparently be further metabolized for inhibition of steroidogenesis to occur. Thus, these findings suggest that the results obtained previously with adrenal subcellular fractions are relevant to hormone production by intact adrenocortical cells. Further investigations on the actions and metabolism of SL in whole cell preparations may help to determine the specific mechanism(s) responsible for the inhibition of steroidogenesis. Acknowledgements These investigations were supported by USPHS research grants CA 43604 and GM 30261. Spironolactone and 7 athiospironolactone were generously provided by G. D. Searle and Co., Chicago, Illinois. References Abshagen, V., S. Sporl, M. Schoneshofer, W. Oelkers: Increased plasma 11-deoxycorticosterone during spironolactone medication. J. Clin. Endocrinol. 44:1190-1193 (1977) Eacho, P. I., H. D. Colby: Regional distribution of adrenal xenobiotic and steroid metabolism. Life Sci. 32:1119 -1127 (1983)
Downloaded by: National University of Singapore. Copyrighted material.
20
O •
D. Colby
Horm. metab. Res. 22 (1990)
Requests for reprints should be addressed to: Givens, J. R.: Treatment of hirsutism with spironolactone. Fertil. Steril. 43:841-843(1985) Howard D. Colby, Ph. D. Greiner, J. W., R. E. Kramer, J. Jarrell, H D. Colby: Mechanism of action of spironolactone on adrenocortical function in guinea pigs. J. Department of Pharmacology and Toxicology Pharmacol. Exp.Ther. 198:709-715(1976) Philadelphia College of Pharmacy and Science Hall, P. F.: Role of cytochromes P-450 in the biosynthesis of steroid Woodland Avenue and 43rd Street hormones. Vitam. Horm. 42:315-368 (1985) Philadelphia, PA 19104 (U. S. A.) Martin, K. O., V. H. Black: Delta 4-hydrogenase in guinea pig adrenal: Evidence of localization in zona reticularis and age-related change. Endocrinology 110:1749-1757(1982) Menard, R. J., T. M. Guenthner, H. Kon, J. R. Gillette: Studies on the destruction of adrenal and testicular cytochrome P-450 by spironolactone. J. Biol. Chem. 254:1726-1733 (1979) Nishikawa, T., C. A. Strott: Cortisol production by cells isolated from the outer and inner zones of the adrenal cortex of the guinea pig. Endocrinology 114; 486-491 (1984) Saunders, F. J., R. L. Alberti: Aldactone; Spironolactone: A Comprehensive Review. Searle, Inc., New York (1978) Serafini, P., R. A. Lobo: The effects of spironolactone on adrenal steroidogenesis in hirsute women. Fertil. Steril. 44: 595—599 (1985) Sherry, J. H., J. P. O'Donnell, L. Flowers, L. B. LaCagnin, H. D. Colby: Metabolism of spironolactone by adrenocrotical and hepatic microsomes: Relationship to cytochrome P-450 destruction. J. Pharmacol. Exp.Ther. 236:675-680 (1986) Tuck, M. L., J. R. Sowers, D. B. Fittingoff, J. S. Fisher, G. J. Berg, N. D. Asp, D. M. Mayes: Plasma corticosteroid concentrations during spironolactone administration: Evidence for adrenal biosynthetic blockade in man. J. Clin. Endocrinol. 52:1057-1061 (1981)
575
Downloaded by: National University of Singapore. Copyrighted material.
Spironolactone and Adrenocortical Function
