The Contribution of the Zona Fasciculata and Glomerulosa to Plasma 11-Deoxycorticosterone Levels in Man S. Y. TAN AND P. J. MULROW Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut ABSTRACT. Using a newly developed radioassay method, plasma 11-deoxycorticosterone (DOC) levels were studied in 6 human volunteers for diurnal variation and for response to ACTH, metyrapone, dexamethasone, and low or high dietary sodium. DOC reached its peak of 6.4 ± 1.2 ng/100 ml at 8:00 AM, and its nadir of less than 1 ng/100 ml at midnight. Corresponding plasma cortisol values were 14.1 ± 1.4 /ig/100 ml and 5.9 ± 1.3 /ig/100 ml respectively. After intramuscular ACTH (Cortrosyn 0.25 mg), DOC rose to 28.7 ± 1.8 ng/100 ml in 1 h. Dexamethasone treatment for 3 days reduced DOC to less than 1 ng/100 ml in all 6 subjects. Oral metyrapone for 24 h resulted in dramatically elevated DOC levels of 1568 ± 183 ng/100 ml. High dietary sodium did not affect DOC levels which averaged 5.6 ± 0.7 ng/100 ml. After 3 days of sodium restriction, DOC levels
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1-DEOXYCORTICOSTERONE (DOC) is a well-known intermediate in the adrenal biosynthesis of corticosterone and aldosterone (1). The secretion rate and urinary excretion of DOC have been investigated but plasma levels and physiological regulation in humans have been less well studied. We have recently developed a simple yet highly sensitive and reliable radioassay for plasma DOC in humans using dog transcortin as the binding protein and Sephadex LH-20 column chromatography (2). This paper describes the use of this method in the study of the relative roles of the zona fasciculata and glomerulosa in contributing to the levels of plasma DOC. Materials and Methods All radioactive steroids were 3H-labelled (SA 40-60 Ci/mmol), and purchased from New England Nuclear Corp. They were purified on a Sephadex LH-20 column before use. Non radioactive Received February 6, 1975. Supported by USPHS grant PHHL 12758-13, and a Connecticut Heart Foundation Research Fellowship. Presented in part at the 56th Annual Meeting of The Endocrine Society, Atlanta, Georgia, June, 1974.
were unchanged at 4.8 ± 0.5 ng/100 ml (P > 0.9) despite high plasma renin activity and elevated plasma and urinary aldosterone. Dexamethasone was then added, and the diet continued for a further 2 days. In contrast to the effect of dexamethasone during ad lib sodium intake, DOC was not suppressed but slightly elevated to 8.6 ± 1.4 ng/100 ml (P = 0.01), whereas plasma aldosterone decreased from 32.9 ± 1.5 to 22.1 ±2.1 ng/100 ml. Seven additional subjects underwent the same diet for 5 days without the addition of dexamethasone. There was no change in their DOC values. It is concluded that the zona fasciculata is the main source of DOC, but in the presence of dexamethasone a contribution from the zona glomerulosa during sodium depletion is uncovered. (J Clin Endocrinol Metab 41: 126, 1975)
steroids were purchased from Sigma Chemicals Co. or Calbiochem. Corp. Sephadex LH-20 was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden, and used as received. RTU disposable culture tubes 12 x 75 mm (BectonDickinson, Rutherford, New Jersey) were used in the radioassays. All organic solvents were reagent grade and were distilled once before use. Radioactive counting was performed using a Packard Tri-Carb liquid scintillation spectrometer with efficiencies of 45% in PPO-POPOPtoluene and 30% in Aquasol (New England Nuclear Corp.). The details of the competitive protein binding radioassy for DOC have been published elsewhere (2). The method employs dog transcortin as the binding protein and Sephadex LH20 column chromatography for isolation of the steroid, and has a sensitivity of at least 50 pg. Urinary aldosterone (acid labile conjugate) was measured by a modification of the radioimmunoassay of Sealy et al. (3). Plasma aldosterone was measured by the method of Underwood and Williams (4). Plasma renin activity (PRA) was measured by radioimmunoassay following the generation of angiotensin I (5). The pH was stabilized at 7.4 during the incubation procedure with 0.3M Tris buffer. Cortisol and 11-desoxycortisol (compound S) were measured by the method of Murphy (6,7).
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ZONA FASCICULATA AND DOC Six healthy, normotensive, human volunteers (5 men, 1 woman) ages 20-30, comprised the study group. Blood was withdrawn by venipuncture, centrifuged in the cold, and the plasma immediately processed or kept frozen until needed. For the study of diurnal variation, blood sampling was done every 4 h on the first day of the protocol. The effects of ACTH (0.25 mg cortrosyn intramuscularly), dexamethasone suppression (0.5 mg orally every 6 h for 3 days) and metyrapone administration (750 mg orally every 4 h for 24 h) were then studied. DOC and cortisol measurements were made V2 h and 1 h after ACTH and after dexamethasone. DOC and compound S were measured after metyrapone. The subjects were then placed on a 10 meq sodium 75 meq potassium diet for 3 days preceded by 40 mg furosemide orally. Dexamethasone, 0.5 mg orally every 6 h was then added, and the diet continued for a further 2 days. Seven additional subjects underwent the same diet for 5 days without the addition of dexamethasone. Finally the subjects were placed for 3 days on a 200 meq sodium diet. Measurements for DOC,
PRA, aldosterone, cortisol and urinary sodium were made at the beginning and end of each diet. Results Plasma DOC levels were highest at 0800 h, averaging 6.4 ± 1.2 (SE) ng% and fell progressively over the course of the day, reaching their nadir of less than 1 ng/100 ml at 0000 h. This diurnal variation paralleled that of cortisol, and is shown in Fig. 1. After metyrapone administration, DOC rose dramatically to 1568 ± 183 ng/100 ml. Compound S rose from less than 0.5 to 11.5 ± 1.0 /Ltg/100 ml during this period (Fig. 2). Plasma DOC rose from a mean of 5.7 ± 0.9 to 23.5 ± 1.6 and 28.7 ± 1.8 ng/100 ml half and one hour respectively after the intramuscular administration of ACTH. Plasma cortisol rose from 14.1 ± 1.4 to 24.8 ± 1.9 and 28.2 ± 2.4 jiig/lOO ml respectively (Fig. 3).
DOC (mean ± S E )
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Cortisol
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1
1-DEOXYCORTICOSTERONE (DOC) is a well-known intermediate in the adrenal biosynthesis of corticosterone and aldosterone (1). The secretion rate and urinary excretion of DOC have been investigated but plasma levels and physiological regulation in humans have been less well studied. We have recently developed a simple yet highly sensitive and reliable radioassay for plasma DOC in humans using dog transcortin as the binding protein and Sephadex LH-20 column chromatography (2). This paper describes the use of this method in the study of the relative roles of the zona fasciculata and glomerulosa in contributing to the levels of plasma DOC. Materials and Methods All radioactive steroids were 3H-labelled (SA 40-60 Ci/mmol), and purchased from New England Nuclear Corp. They were purified on a Sephadex LH-20 column before use. Non radioactive Received February 6, 1975. Supported by USPHS grant PHHL 12758-13, and a Connecticut Heart Foundation Research Fellowship. Presented in part at the 56th Annual Meeting of The Endocrine Society, Atlanta, Georgia, June, 1974.
were unchanged at 4.8 ± 0.5 ng/100 ml (P > 0.9) despite high plasma renin activity and elevated plasma and urinary aldosterone. Dexamethasone was then added, and the diet continued for a further 2 days. In contrast to the effect of dexamethasone during ad lib sodium intake, DOC was not suppressed but slightly elevated to 8.6 ± 1.4 ng/100 ml (P = 0.01), whereas plasma aldosterone decreased from 32.9 ± 1.5 to 22.1 ±2.1 ng/100 ml. Seven additional subjects underwent the same diet for 5 days without the addition of dexamethasone. There was no change in their DOC values. It is concluded that the zona fasciculata is the main source of DOC, but in the presence of dexamethasone a contribution from the zona glomerulosa during sodium depletion is uncovered. (J Clin Endocrinol Metab 41: 126, 1975)
steroids were purchased from Sigma Chemicals Co. or Calbiochem. Corp. Sephadex LH-20 was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden, and used as received. RTU disposable culture tubes 12 x 75 mm (BectonDickinson, Rutherford, New Jersey) were used in the radioassays. All organic solvents were reagent grade and were distilled once before use. Radioactive counting was performed using a Packard Tri-Carb liquid scintillation spectrometer with efficiencies of 45% in PPO-POPOPtoluene and 30% in Aquasol (New England Nuclear Corp.). The details of the competitive protein binding radioassy for DOC have been published elsewhere (2). The method employs dog transcortin as the binding protein and Sephadex LH20 column chromatography for isolation of the steroid, and has a sensitivity of at least 50 pg. Urinary aldosterone (acid labile conjugate) was measured by a modification of the radioimmunoassay of Sealy et al. (3). Plasma aldosterone was measured by the method of Underwood and Williams (4). Plasma renin activity (PRA) was measured by radioimmunoassay following the generation of angiotensin I (5). The pH was stabilized at 7.4 during the incubation procedure with 0.3M Tris buffer. Cortisol and 11-desoxycortisol (compound S) were measured by the method of Murphy (6,7).
126
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 September 2015. at 02:14 For personal use only. No other uses without permission. . All rights reserved.
127
ZONA FASCICULATA AND DOC Six healthy, normotensive, human volunteers (5 men, 1 woman) ages 20-30, comprised the study group. Blood was withdrawn by venipuncture, centrifuged in the cold, and the plasma immediately processed or kept frozen until needed. For the study of diurnal variation, blood sampling was done every 4 h on the first day of the protocol. The effects of ACTH (0.25 mg cortrosyn intramuscularly), dexamethasone suppression (0.5 mg orally every 6 h for 3 days) and metyrapone administration (750 mg orally every 4 h for 24 h) were then studied. DOC and cortisol measurements were made V2 h and 1 h after ACTH and after dexamethasone. DOC and compound S were measured after metyrapone. The subjects were then placed on a 10 meq sodium 75 meq potassium diet for 3 days preceded by 40 mg furosemide orally. Dexamethasone, 0.5 mg orally every 6 h was then added, and the diet continued for a further 2 days. Seven additional subjects underwent the same diet for 5 days without the addition of dexamethasone. Finally the subjects were placed for 3 days on a 200 meq sodium diet. Measurements for DOC,
PRA, aldosterone, cortisol and urinary sodium were made at the beginning and end of each diet. Results Plasma DOC levels were highest at 0800 h, averaging 6.4 ± 1.2 (SE) ng% and fell progressively over the course of the day, reaching their nadir of less than 1 ng/100 ml at 0000 h. This diurnal variation paralleled that of cortisol, and is shown in Fig. 1. After metyrapone administration, DOC rose dramatically to 1568 ± 183 ng/100 ml. Compound S rose from less than 0.5 to 11.5 ± 1.0 /Ltg/100 ml during this period (Fig. 2). Plasma DOC rose from a mean of 5.7 ± 0.9 to 23.5 ± 1.6 and 28.7 ± 1.8 ng/100 ml half and one hour respectively after the intramuscular administration of ACTH. Plasma cortisol rose from 14.1 ± 1.4 to 24.8 ± 1.9 and 28.2 ± 2.4 jiig/lOO ml respectively (Fig. 3).
DOC (mean ± S E )
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Cortisol
12 O CO
o o a < 4
ft ° 8 O
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