J Clin Endocrinol Metab 44: 799, 1977 AN ABNORMALITY IN STEROID REDUCTIVE METABOLISM IN A HYPERTENSIVE SYNDROME Stanley Ulick,* Leyla C. Ramirez, and Maria I. New *Medical Service, Veterans Administration Hospital, Bronx, New York 10468, Mount Sinai School of Medicine, New York, N.Y., and the Department of Pediatrics, The New York Hospital-Cornell Medical Center, New York, N.Y. ABSTRACT. Studies in a juvenile hypertensive syndrome associated with suppressed plasma renin activity and hypokalemic alkalosis failed to reveal overproduction of aldosterone or any other known steroid. There was however an abnormal increase in the fraction of unconjugated urinary steroids. Analysis of this fraction following the administration of labeled cortisol revealed that it was largely composed of dihydro metabolites reduced either at 4,5 or at C-20 and that the 4,5-dihydro fraction contained an abnormal increase in 5a- relative to 5(3-metabolites. There was, however, no absolute defect in the complete reduction of ring A to form tetrahydro derivatives. These findings, together with observations by Marver and Edelman that 5adihydrocortisol may be an effective mineralocorticoid, suggest the possibility of an etiologic relationship between the metabolic abnormality and the patient's hypertensive disorder. cortisol was added to urine, isolated chromatographically, reacted with •^C-acetic anhydride and purified chromatographically to constant specific activity.
We previously reported the features of a juvenile syndrome of hypertensive hypokalemic alkalosis associated with subnormal production of known corticosteroids (1) in which an exhaustive search failed to reveal overproduction of an unknown steroid factor (2). We report now on further studies in that patient which revealed an abnormality in steroid metabolism consisting of incomplete reductive metabolism of ring A. There was increased percentage excretion of unconjugated dihydro metabolites consisting of 20a-, 20(3-, 5a- and 5(3dihydrocortisols. Mineralocorticoid activity of the 5or-epimer may be an etiologic factor in the patient's disorder.
Preparation of free and conjugated steroid fractions. Each of the 4-14Q steroids (0.5 uCi each) listed in Table 1 were injected intravenously on different days and urine was collected for 24 hours. Unconjugated (free) steroids were extracted with methylene chloride and the extract washed with 5"/0 sodium carbonate and water, dried and evaporated. Unextracted urine plus washings from the free fraction was buffered to pH 5 with acetate and incubated with mammalian liver glucuronidase (500 units per ml) at 37 C for 48 hours. The glucuronide fraction consisted of an ethyl acetate extract that was washed neutral, dried and evaporated. The radioactivity in free and glucuronide fractions, determined by liquid scintillation counting, was expressed as a percentage of the total radioactivity in the urine.
METHODS General. Reference steroids were obtained from Steraloids (Wilton, N.H.) and labeled steroids from New England Nuclear Corp., and their purity verified chromatographically. 17-Hydroxycorticosteroids were measured by the Porter-Silber test (3) and urinary cortisol by a double isotopic method in which tritium-labeled
Measurement of urinary unconjugated metabolites of cortisol. The composition of the unconjugated or free
Submitted November 18, 1976.
799
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800
JCE & M • 1977 Vol 44 • No 4
RAPID COMMUNICATIONS
fraction was determined either by isomers, eluates of paper chromatodouble label isotope- or carrier digraphic systems (Table 1) containing lution. Tritium-labeled tetrahydro*C-cortisol metabolites and the apcortisone, tetrahydrocortisol, and propriate unlabeled carriers were cortisol, and carrier amounts (100 \xg) further chromatographed on a 30 cm of 5a-dihydrocortisol (11(3,17a, 21microBondapak C-18 column (Waters trihydroxy-5a-pregnane-3,20-dione), Associates) at a pressure of 1700 lb/ 20p-dihydrocortisol (lip, 17a, 20(3, 21in 2 and at 2 ml/min flow of the motetrahydroxy-4-pregnene-3,20-dione) , bile phase, acetonitrile:water (3:7). The column was monitored by refracand cortisone were added to the extract. Doubly-labeled steroids were tive index, fractions collected and their radioactivity determined. purified to constant tritium/^C ratio and others to constant specific activity using either the blue tetrazolium RESULTS reaction or ultraviolet absorption. Increased excretion of unconjugated TABLE 1. Chromatographic separation steroids. Table 2 shows the percent of the unconjugated urinary of unconjugated and glucuronide metabolites of cortisol. radioactivity for each of the four steroids. There was an abnormal inZone Steroid System crease in excretion of unconjugated I Tetrahydrocortisol steroid metabolites that was most striking in the case of the two 17a20(3-dihydrocortisol hydroxycorticosteroids, cortisol and Compound S, but there may also have been some increase in the excretion II Tetrahydrocortisone of unconjugated metabolites of aldosterone and corticosterone. III Cortisol IV
Cortisone 5a-dihydrocortisol
(1) Ethylene dichloride/ethylene glycol; (2) ethylene dichloride:toluene (1:1)/ethylene glycol; (3) silica gel thin layer; methanol:ethylene dichloride (1:9); (4) silica gel thin layer; methanol:ethylene dichloride (1:4). After the addition of tritiumlabeled or carrier steroids the extract was chromatographed first on the ethylene dichloride/ethylene glycol system for 18 hours and on the basis of radioactive scanning and ultraviolet absorption of the marker steroids divided into four zones. Table 1 shows the chromatographic sequence used to obtain radiochemical purity for each steroid. High pressure liquid chromatography. For the determination of the stereochemistry of the dihydrocortisol
TABLE 2. Distribution of radioactivity in free and conjugated fractions following intravenous injection of 4-l^C steroids. Steroid Aldosterone*Cortisol Corticosterone Compound S 2 NormalJ
Percent of urinary radioactivity Glucuronide Free
2.5 43 8.0 55
30 12
21 20
0.2-0 .6
pH 1-conjugate (18-glucuronide) accounted for 11.57O. 2ll-Deoxycortisol. ^Reference 4. Effect of the corticosteroid secretory rate on the relative excretion of unconjugated steroids. The patient had a very low secretory rate of the known corticosteroids but the secretion of cortisol rose, although somewhat sluggishly, in response to ACTH
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801
RAPID COMMUNICATIONS (1). To determine whether the relatively large fraction of unconjugated steroids was an adjustment to the low secretory rate itself, free and conjugated cortisol metabolites were measured at both low and high rates of cortisol secretion. As shown in Table 3, the unconjugated fraction was abnormally large at both high and low cortisol production rates. The free fraction normally represents less than 1% of the conjugated steroid fraction (4). TABLE 3. Distribution of urinary 17-hydroxycorticoids in free and conjugated fractions at low and high rates of cortisol secretion. Period Control ACTH
Porter-Silber chromogens mg/day Conjugated Free 0.25 0.18
0.10 0.22
1.15 1.1
5.8 6.3
Composition of the unconjugated steroid fraction. Table 4 shows that cortisol, normally the predominant component of this fraction, accounted for only 16.7%. There were in addition small amounts of the two tetrahydro metabolites which are normally excreted in much larger amounts as glucuronic acid conjugates. The striking abnormality was that more than one-half of the unconjugated metabolites of cortisol were dihydro derivatives formed by reduction either of the C-20 ketone or of the 4,5 double bond. The amount of each of the components of the unconjugated fraction was calculated from the cortisol excretory rate. Urinary free cortisol, determined by double isotopic analysis, in four 24-hour control period specimens was 8.6 + 2.3 jxg/day. Since the relative percentage excretion of the components was known, the excretion of each in (xg/day could be calculated.
The paper chromatographic systems used to obtain the quantitative data of Table 4 did not have sufficient resolving power to determine each pair of a- and (3-epimers specifically. For the separation and determination of each form, high pressure liquid chromatography was necessary. Fig. 1 shows that all relevant metabolites were well separated. Analysis of the 4,5-dihydrocortisol fraction showed that 77% of the radioactivity was associated with 5or-dihydrocortisol and 23% with 5(3-dihydrocortisol. High pressure liquid chromatographic analysis of the 20-dihydrocortisol fraction (not shown here) revealed equal amounts of the 20a- and 20(3hydroxy metabolites. TABLE 4. Radioactive metabolites of cortisol in free fraction of urine. p.g/day
Steroid Cortisone Tetrahydrocortisone Tetrahydrocortisol Cortisol 4,5-Dihydrocortisol 20-Dihydrocortisol
We previously reported the features of a juvenile syndrome of hypertensive hypokalemic alkalosis associated with subnormal production of known corticosteroids (1) in which an exhaustive search failed to reveal overproduction of an unknown steroid factor (2). We report now on further studies in that patient which revealed an abnormality in steroid metabolism consisting of incomplete reductive metabolism of ring A. There was increased percentage excretion of unconjugated dihydro metabolites consisting of 20a-, 20(3-, 5a- and 5(3dihydrocortisols. Mineralocorticoid activity of the 5or-epimer may be an etiologic factor in the patient's disorder.
Preparation of free and conjugated steroid fractions. Each of the 4-14Q steroids (0.5 uCi each) listed in Table 1 were injected intravenously on different days and urine was collected for 24 hours. Unconjugated (free) steroids were extracted with methylene chloride and the extract washed with 5"/0 sodium carbonate and water, dried and evaporated. Unextracted urine plus washings from the free fraction was buffered to pH 5 with acetate and incubated with mammalian liver glucuronidase (500 units per ml) at 37 C for 48 hours. The glucuronide fraction consisted of an ethyl acetate extract that was washed neutral, dried and evaporated. The radioactivity in free and glucuronide fractions, determined by liquid scintillation counting, was expressed as a percentage of the total radioactivity in the urine.
METHODS General. Reference steroids were obtained from Steraloids (Wilton, N.H.) and labeled steroids from New England Nuclear Corp., and their purity verified chromatographically. 17-Hydroxycorticosteroids were measured by the Porter-Silber test (3) and urinary cortisol by a double isotopic method in which tritium-labeled
Measurement of urinary unconjugated metabolites of cortisol. The composition of the unconjugated or free
Submitted November 18, 1976.
799
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 11:03 For personal use only. No other uses without permission. . All rights reserved.
800
JCE & M • 1977 Vol 44 • No 4
RAPID COMMUNICATIONS
fraction was determined either by isomers, eluates of paper chromatodouble label isotope- or carrier digraphic systems (Table 1) containing lution. Tritium-labeled tetrahydro*C-cortisol metabolites and the apcortisone, tetrahydrocortisol, and propriate unlabeled carriers were cortisol, and carrier amounts (100 \xg) further chromatographed on a 30 cm of 5a-dihydrocortisol (11(3,17a, 21microBondapak C-18 column (Waters trihydroxy-5a-pregnane-3,20-dione), Associates) at a pressure of 1700 lb/ 20p-dihydrocortisol (lip, 17a, 20(3, 21in 2 and at 2 ml/min flow of the motetrahydroxy-4-pregnene-3,20-dione) , bile phase, acetonitrile:water (3:7). The column was monitored by refracand cortisone were added to the extract. Doubly-labeled steroids were tive index, fractions collected and their radioactivity determined. purified to constant tritium/^C ratio and others to constant specific activity using either the blue tetrazolium RESULTS reaction or ultraviolet absorption. Increased excretion of unconjugated TABLE 1. Chromatographic separation steroids. Table 2 shows the percent of the unconjugated urinary of unconjugated and glucuronide metabolites of cortisol. radioactivity for each of the four steroids. There was an abnormal inZone Steroid System crease in excretion of unconjugated I Tetrahydrocortisol steroid metabolites that was most striking in the case of the two 17a20(3-dihydrocortisol hydroxycorticosteroids, cortisol and Compound S, but there may also have been some increase in the excretion II Tetrahydrocortisone of unconjugated metabolites of aldosterone and corticosterone. III Cortisol IV
Cortisone 5a-dihydrocortisol
(1) Ethylene dichloride/ethylene glycol; (2) ethylene dichloride:toluene (1:1)/ethylene glycol; (3) silica gel thin layer; methanol:ethylene dichloride (1:9); (4) silica gel thin layer; methanol:ethylene dichloride (1:4). After the addition of tritiumlabeled or carrier steroids the extract was chromatographed first on the ethylene dichloride/ethylene glycol system for 18 hours and on the basis of radioactive scanning and ultraviolet absorption of the marker steroids divided into four zones. Table 1 shows the chromatographic sequence used to obtain radiochemical purity for each steroid. High pressure liquid chromatography. For the determination of the stereochemistry of the dihydrocortisol
TABLE 2. Distribution of radioactivity in free and conjugated fractions following intravenous injection of 4-l^C steroids. Steroid Aldosterone*Cortisol Corticosterone Compound S 2 NormalJ
Percent of urinary radioactivity Glucuronide Free
2.5 43 8.0 55
30 12
21 20
0.2-0 .6
pH 1-conjugate (18-glucuronide) accounted for 11.57O. 2ll-Deoxycortisol. ^Reference 4. Effect of the corticosteroid secretory rate on the relative excretion of unconjugated steroids. The patient had a very low secretory rate of the known corticosteroids but the secretion of cortisol rose, although somewhat sluggishly, in response to ACTH
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 11:03 For personal use only. No other uses without permission. . All rights reserved.
801
RAPID COMMUNICATIONS (1). To determine whether the relatively large fraction of unconjugated steroids was an adjustment to the low secretory rate itself, free and conjugated cortisol metabolites were measured at both low and high rates of cortisol secretion. As shown in Table 3, the unconjugated fraction was abnormally large at both high and low cortisol production rates. The free fraction normally represents less than 1% of the conjugated steroid fraction (4). TABLE 3. Distribution of urinary 17-hydroxycorticoids in free and conjugated fractions at low and high rates of cortisol secretion. Period Control ACTH
Porter-Silber chromogens mg/day Conjugated Free 0.25 0.18
0.10 0.22
1.15 1.1
5.8 6.3
Composition of the unconjugated steroid fraction. Table 4 shows that cortisol, normally the predominant component of this fraction, accounted for only 16.7%. There were in addition small amounts of the two tetrahydro metabolites which are normally excreted in much larger amounts as glucuronic acid conjugates. The striking abnormality was that more than one-half of the unconjugated metabolites of cortisol were dihydro derivatives formed by reduction either of the C-20 ketone or of the 4,5 double bond. The amount of each of the components of the unconjugated fraction was calculated from the cortisol excretory rate. Urinary free cortisol, determined by double isotopic analysis, in four 24-hour control period specimens was 8.6 + 2.3 jxg/day. Since the relative percentage excretion of the components was known, the excretion of each in (xg/day could be calculated.
The paper chromatographic systems used to obtain the quantitative data of Table 4 did not have sufficient resolving power to determine each pair of a- and (3-epimers specifically. For the separation and determination of each form, high pressure liquid chromatography was necessary. Fig. 1 shows that all relevant metabolites were well separated. Analysis of the 4,5-dihydrocortisol fraction showed that 77% of the radioactivity was associated with 5or-dihydrocortisol and 23% with 5(3-dihydrocortisol. High pressure liquid chromatographic analysis of the 20-dihydrocortisol fraction (not shown here) revealed equal amounts of the 20a- and 20(3hydroxy metabolites. TABLE 4. Radioactive metabolites of cortisol in free fraction of urine. p.g/day
Steroid Cortisone Tetrahydrocortisone Tetrahydrocortisol Cortisol 4,5-Dihydrocortisol 20-Dihydrocortisol
