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Regulation of 18-oxocortisol and 18-hydroxycortisol by the renin-angiotensin system and ACTH in man
J. Steroid Biochem. Molec. Biol. Vol. 46. No. 3, pp. 395-399, 1993
0960-0760-93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
R E G U L A T I O N OF 18-OXOCORTISOL A N D 18-HYDROXYCORTISOL BY THE R E N I N - A N G I O T E N S I N SYSTEM A N D ACTH IN M A N NORIYOSHI YAMAKITA,1. CELSO E. GOMEZ-SANCHEZ, 2 TOMOATSU MUNE, 3 HISASHI YOSHIDA,3 SEIJI MIYAZAKI, a KEIGO YASUDA3 a n d TOSHIAKI NAKAI ~ qnstitute of Clinical Medicine, University of Tsukuba, Tsukuba-City 305, Japan, 2Department of Internal Medicine, University of South Florida College of Medicine and James A. Haley Veterans' Hospital, Tampa, FL 33612-4798, U.S.A. and 3Department of Medicine, Gifu University School of Medicine, Gifu-City 500, Japan
(Received 17 March 1993; accepted 14 May 1993)
Summary--Based on urinary excretion studies the secretion of the cortisol derivatives, 18-oxocortisol and 18-hydroxycortisol are believed to be regulated by ACTH and to a lesser degree by the renin-angiotensin system. Plasma concentrations of 18-oxocortisol and 18hydroxycortisol were measured during the simultaneous activation of the renin-angiotensin system and inhibition of ACTH secretion. Five healthy male subjects consuming a sodium diet ad libitum were studied. Blood was drawn at 0800 h after 1 h in the supine position. In the first set of experiments, the subjects remained in the supine position from 0800 to 1000 h with or without the oral administration of 2 mg dexamethasone at 0800 h. In the second set of experiments the subjects were placed in the upright position after drawing the 0800 h sample. The subjects were studied with and without dexamethasone administered at 0800 h. Blood was drawn again at 1000 h. Plasma levels of 18-oxocortisol, 18-hydroxycortisol, ACTH, plasma renin activity (PRA), cortisol, aldosterone and 18-hydroxycorticosterone were measured by radioimmunoassay. None of these parameters changed during the 2 h in the supine position. 18-Oxocortisol, 18-hydroxycortisol, aldosterone, 18-hydroxycorticosterone and PRA increased, but ACTH and cortisol did not change when the subjects were placed in the upright position. After dexamethasone administration, 18-oxocortisol, 18-hydroxycortisol, cortisol, aldosterone and 18-hydroxycorticosterone decreased in the supine position and no increase occurred in 18-oxocortisol, 18-hydroxycortisol and 18-hydroxycorticosterone in the upright position. PRA and aldosterone increased and ACTH and cortisol decreased in these subjects. 18-Oxocortisol and 18-hydroxycortisol were more dependent on ACTH regulation and less on the renin-angiotensin system than aldosterone.
INTRODUCTION
The adrenal gland produces aldosterone in the zona glomerulosa and cortisol in the zona fasciculata and can also produce hybrid compounds which share structural characteristics of these steroids [1, 2]. Patients with aldosterone producing adenomas and glucocorticoid-suppressible aldosteronism excrete excessive amounts of 18-oxocortisol and 18-hydroxycortisol [2, 3]. Patients with primary aldosteronism due to bilateral zona glomerulosa hyperplasia excrete normal amounts of both steroids [3]. The glucocorticoid activity of 18-oxocortisol is only 3% that o f cortisol and the mineralocorticoid potency is only 1% that of aldosterone [4]. 18Hydroxycortisol is inactive [5]. Measurements *To whom correspondence should be addressed.
of 18-oxocortisol and 18-hydroxycortisol could be important in the differential diagnosis of primary aldosteronism and 18-oxocortisol could have pathophysiological significance. Aldosterone and 18-hydroxycorticosterone are regulated primarily by the renin-angiotensin system [6], but A C T H , potassium and other factors also play [6]. A C T H plays a greater role in the secretion of 18-hydroxycorticosterone than of aldosterone [7]. The urinary excretion of 18-hydroxycortisol and 18-oxocortisol are increased by A C T H administration and dietary sodium restriction [8, 9] and are inhibited by dexamethasone administration and by sodium loading [8, 9]. Plasma measurements for 18hydroxycortisol have been reported[10,11]. In the present study the effect of activation of the renin-angiotensin system with suppression of A C T H production was investigated using 395
NOPdYOSm YAMAKITAet al.
396
simultaneous measurement of plasma 18-oxocortisol and 18-hydroxycortisol. MATERIALS AND METHODS
Subjects
Five healthy Japanese male subjects (24.6_ 0.5 years) eating a salt diet ad libitum were studied after an overnight fast. Informed consent was obtained after the nature of the procedures was fully explained. Blood was drawn at 0800 h after 1 h in the supine position. The subjects remained in the supine position from 0800 to 1000 h and received placebo or 2 mg dexamethasone orally at 0800 h on different days. They were also similarly studied after standing from 0800 to 1000 h. At 1000 h a second blood sample was obtained. The studies were done with at least 4 days inbetween treatments. The blood samples were separated immediately and frozen at -20°C until assayed.
Hormone measurements Materials for buffers were purchased from Sigma Chemical Company (St Louis, MO, U.S.A.) or Wako Pure Chemical Industries Ltd (Osaka, Japan). Bond ElutTM columns, C-2 silica and celite were obtained from Analytichem International (Varian, Harbor City, CA, U.S.A.). All glass tubes used were silanized with dichlorodimethyl silane and extensively washed.
18-Oxocortisol radioimmunoassay The source of 18-oxocortisol, 18-hydroxycortisol, tritiated derivatives and antibodies have been reported previously [3, 8, 12]. The radioimmunoassay for 18-oxocortisol consisted in taking 1 ml plasma, adding 1500cpm freshly purified [1,23H]18-oxocortisol, mixing well and adding 10/zl 10% acetic acid. The sample was transferred to a column consisting of a 1 ml polypropylene syringe filled first with 100mg celite followed by 50 mg C-2 silica and washed with 1 ml methanol and 2 ml water. After addition of plasma, the column was washed with 1 ml water and 100/zl formamide in water was added. The column was eluted with 4 ml 40% ethyl acetate in hexane (containing 1% acetic acid). This eliminated over 98% of cortisol and aldosterone which crossreact with the antibody. The 18-oxocortisol is then eluted with 2 ml 80% ethyl acetate in hexane (containing 1% acetic acid) and collected in a silanized tube. The organic solvent was evaporated under air and
reconstituted in 250/~1 RIA buffer (borate 0.1 M, pH7.1 containing 0.5% bovine serum albumin and 1% sodium azide). An aliquot was used for assessment of recoveries and 50 #1 in triplicate for the assay using polypropylene tubes. A standard curve was prepared using 0, 2.5, 5, 10, 25, 50, 100, 250 and 500 pg 18-oxocortisol in polypropylene tubes. An antibody solution (1:6000), 2500 cpm [1,23H]18-oxocortisol and goat antirabbit gammaglobulin (1:100) were added in 250 #1. After an overnight incubation, 0.6ml 10% polyethylene glycol (Mw 6000) was added and centrifuged 120 min later. The samples were then decanted and counted in a liquid scintillation counter. The results were corrected for recovery. The recoveries from the column varied between 60-73%.
18-Hydroxycortisol radioimmunoassay The assay was performed as described above except that the plasma and solvents were not acidified and 18-hydroxycortisol was eluted with ethyl acetate. The recoveries from the column varied between 58-68%. 18-Hydroxycorticosterone was measured by RIA after extracting with dichloromethane [13]. Plasma cortisol was measured by Gamma Coat Cortisol KitTM (Incstar Co, Stillwater, MN, U.S.A.). Plasma aldosterone was measured by an Aldosterone-RIA KitTM (Shionogi Pharmaceutical Co., Osaka, Japan). ACTH and plasma renin activity (PRA) were measured by ACTHII-IRMA TM (Mitsubishi Petrochemical Co., Tokyo, Japan) and Renin RIA BeadTM (Dainabot RI Laboratory, Tokyo, Japan). Data were analyzed by Student's t-test for paired samples. RESULTS
The supine position for 2 h did not result in any significant change in the plasma concentrations of cortisol, aldosterone, 18-hydroxycorticosterone, 18-oxocortisol, 18-hydroxycortisol, PRA or ACTH. Plasma levels of aldosterone, 18-hydroxycorticosterone, 18-oxocortisol, 18hydroxycortisol and PRA but not ACTH and cortisol increased significantly after 2 h upright position. The ratios of plasma concentration of 18-oxocortisol (1.57 _+0.09), 18-hydroxycortisol (1.57__+ 0.21) and 18-hydroxycorticosterone (1.59 ___0.10) to that before the upright position were significantly smaller than that of aldosterone (2.87 ___0.20) (Fig. 1). Dexamethasone administration resulted in a significant decrease in
Regulation of 18-oxocortisol and 18-OH-cortisol
397
4'
Upright
Posture
• "
A
3"
4) m
18oxoF
[] 18OHF 18OHB aldosterone
2"
cortisol
.o tr
Fig. I. The ratios of plasma concentration of 18-oxocortisol (18oxoF), 18-hydroxycortisol (18OHF), 18-hydroxycorticosterone(18OHB), aldosterone and cortisol after the postural stimulation to those before the stimulation. *Significantly different, vs 18oxoF and 18OHF, P <0.02; vs 18OHB and cortisol, P < 0.01.
plasma A C T H and cortisol. Plasma aldosterone, 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol significantly decreased 2 h after taking dexamethasone in the supine position. In this test, the ratios of plasma levels of 18-oxocortisol ( 0 . 4 8 _ 0.09), 18-hydroxycortisol (0.52+0.13), and cortisol (0.56 _ 0.23) to those before taking dexamethasone were not significantly different from each other. 18-Hydroxycorticosterone and aldosterone ratios were significantly greater than those of 18-oxocortisol and 18-hydroxycortisol, respectively (Fig. 2). P R A and plasma aldosterone increased after taking dexamethasone in the upright position. 18-Oxocortisol also
increased, but the increase was not statistically significant. DISCUSSION The urinary excretion of 18-oxocortisol [9] and 18-hydroxycortisol [8] and plasma levels o f 18-hydroxycortisol[ll] were reported to increase in subjects given a low sodium diet, suggesting that activation of the reninangiotensin system played a role in their regulation. Infusion of angiotensin II did not increase plasma 18-hydroxycortisol, but as expected increased plasma aldosterone by 2.3- to 2.8-fold [11]. In the present study, plasma levels
1.0 Dex 0.8 4) m
[] 18oxoF 0.6
,< .0
[] 18OHF [] 18OHB
0.4
Illl aldosterone [] cortisol
0.2.
0.0 Fig. 2. The ratios of plasma concentration of 18oxoF, 18OHF, 18OHB, aldosterone and cortisol after dexamethasone administration to those before the administration. *Significantlydifferent, vs 18OHB and aldosterone, P < 0.001; **Significantlydifferent, vs 18OHB and aldosterone, P < 0.04.
NORIYOSHIYAMAKITAet al.
398
Table 1. Plasma levels of 18-oxocortisol (18oxoF), 18-hydroxycortisol (18OHF), 18-hydroxycorticosterone (18OHB), aldosterone, cortisol, ACTH and PRA Supine 0800 h
Uptight 1000 h
0800 h
Dex 1000 h
0800 h
Dex + Uptight 1000 h
0800 h
1000h
18oxoF 0.826 + 0.115 0.612+-0.161 0.675+ 0.092 1.022+ 0.106 0.862 + 0.105 0.407_+0.090 0.551 + 0.108 0.847 + 0.271 (nmol/l) ND P < 0.01 P < 0.02 ND 18OHF 3.32 +- 0.27 2.86 + 0.58 2.99 + 0.68 4.41 + 0.91 3.46 + 0.52 1.86 + 0.46 3.00 +- 0.40 3.03 + 0.51 (nmol/l) ND P < 0.03 P < 0.02 ND 18OHB 621+71 591+78 678+99 1076+_172 657+122 442_+70 594+57 653+-64 (pmol/l) ND P < 0.01 P < 0.05 ND Aldosterone 230 +- 50 174 ___20 269 + 10 766 + 29 234 + 11 170 + 26 266 + 87 612 + 211 (pmol/I) ND P < 0.0005 P < 0.02 P < 0.04 Cortisol 288 + 47 286 +- 35 299 + 49 336 +_ 18 336 + 52 187 + 43 341 + 42 240 +- 52 (nmol/l) ND ND P < 0.02 P < 0.04 ACTH 5.50+_ 1.11 4.90+-1.22 5.29+0.89 5.99+0.79 5.60+-1.44 2.54+-0.60 5.73_+1.01 2.37+-0.51 (pmol/l) ND ND P < 0.03 P < 0.01 PRA 0.135+-0.038 0.170+_0.047 0.123+-0.035 0.890+_0.247 0.349+_0.097 0.259+_0.072 0.176+_0.049 1.033+_0.287 (nmol/l • s) ND P < 0.005 ND P < 0.02 Mean + SE; Supine, supine position from 0800 to 1000 h without dexamethasone administration; Uptight, uptight position from 0800 to 1000h without dexamethasone administration; Dex, supine position from 0800 to 1000h with dexamethasone administration; Dex + Uptight, uptight position from 0800 to 1000h with dexamethasone administration. ND, not different.
of 18-hydroxycorticosterone, 18-oxocortisol and 18-hydroxycortisol increased by about 1.6 times when the subjects were placed in the upright position for 2 h. The increase in plasma aldosterone was greater than the other steroids. The studies suggest that the renin-angiotensin system plays a greater role in the stimulation of aldosterone secretion than it does in 18hydroxycorticosterone, 18-oxocortisol and 18hydroxycortisol secretion. The lesser response of 18-hydroxycorticosterone compared to aldosterone has also been reported by Kater et al. [14]. Orally administered dexamethasone is rapidly absorbed and serum concentration peaks at about 1 h[15]. Plasma cortisol gradually decreases from 1 h after the administration of dexamethasone [15]. ACTH, cortisol, aldosterone and 18-hydroxycorticosterone exhibit a diurnal rhythm, but the difference in time between 0800 and 1000 h was too close to show a significant change in plasma concentrations. The administration of dexamethasone decreased ACTH, cortisol, aldosterone, 18-hydroxycorticosterone, 18-oxocortisol and 18-hydroxycortisol. The degree of suppression of 18-oxocortisol and 18-hydroxycortisol was greater than that of 18-hydroxycorticosterone and aldosterone. The adrenal has two cytochrome P-450 1lflhydroxylases, one primarily located in the zona fasciculta-reticularis which converts deoxycorticosterone into corticosterone and 18hydroxydeoxycorticosterone. In the case of 17-hydroxylated steroids it converts 11-deoxycortisol into cortisol. It can also 18-hydroxylate ll-deoxycortisol to 18-hydroxydeoxycortisol, but this conversion has been poorly studied [16]. In the zona glomerulosa, deoxycorticosterone is
converted to corticosterone, 18-hydroxycorticosterone and aldosterone under the action of the aldosterone synthase [17]. Cortisol can act as a substrate and generate 18-oxocortisol and 18hydroxycortisol. Dexamethasone administration reduces the production of cortisol and as a consequence the generation of 18-oxocortisol and 18-hydroxycortisol. In conclusion, 18-hydroxycorticosterone, 18oxocortisol and 18-hydroxycortisol are less dependent on the renin-angiotensin system than aldosterone and 18-oxocortisol and 18-hydroxycortisol are more dependent on ACTH for stimulation. Acknowledgements--Dr Yamakita is supported by a grant from the University of Tsukuba Project Research. Dr Gomez-Sanchez is supported by medical research funds from the Department of Veterans Affairs and the National Institutes of Health (HL 27255).
REFERENCES 1. Chu M. D. and Ulick S.: Isolation and identification of 18-hydroxycortisol from the urine of patients with primary aldosteronism. J. Biol. Chem. 258 (1982) 2218-2224. 2. Ulick S., Chu M. D. and Land M.: Biosynthesis of 18-oxoeortisol by aldosterone producing adrenal tissue. J. Biol. Chem. 258 (1983) 5498-5502. 3. Gomoz-Sanchez C. E., Montgomery M., Ganguly A., Holland O. B., Gomez-Sanchez E. P., Grim C. E. and Weinbexger M. H.: Elevated urinary excretion of 18-oxoeortisol in glucoeorticoid suppressible aldosteronism. J. Clin. Endocr. Metab. 59 (1984) 1022-1024. 4. Gomoz-Sanchez C. E., Gomez-Sanchez E. P., Smith J. S., Fen'is M. W. and Foecking M. F.: Receptor binding and biological activity of 18-oxo-cortisol. Endocrinology 116 (1985) 6-10. 5. Gomez-Sanchez E. P., Gomez-Sanchez C. E., Smith J. S., Ferris M. W. and Foecking M. F.: Receptor binding and biological activity of 18-hydroxycortisol. Endocrinology 115 (1984) 462--466.
Regulation of 18-oxocortisol and 18-OH-cortisol 6. Muller J.: Regulation of AIdosterone Biosynthesis. Physiological and Clinical Aspects. Springer-Verlag, Berlin 2nd Edn. (1988) pp. 5-14. 7. Fraser R. and Lantos C. P.: 18-Hydroxycorticosterone: a review. J. Steroid Biochem. 9 (1978) 273-286. 8. Gomez-Sanchez C. E., Upcavage R. J., Zager P. G., Foecking M. F., Holland O. B. and Ganguly A.: Urinary 18-hydroxyeortisol and its relationship to the excretion of other adrenal steroids. J. Clin. Endocr. Metab. 65 (1987) 310-314. 9. Gomez-Sanchez C. E., Zager P. G., Foecking M. F., Holland O. B. and Ganguly A.: 18-oxocortisol: effect of dexamethasone, ACTH and sodium depletion. J. Steroid Biochem. 32 (1989) 409-412. 10. Corrie J. E. T., Edwards C. R. W. and Budd P. S.: A radioimmunoassay for 18-hydroxyeortisol in plasma and urine. Clin. Chem. 31 (1985) 849--852. 11. Corrie J. E. T., Edwards C. R. W., Jones D. B., Padfield P. L. and Budd P. S.: Factors affecting the secretion of 18-hydroxycortisol, a novel steroid of relevance to Conn's syndrome. Clin. Endocr. 23 (1985) 579-586. 12. Gomez-Sanchez C. E., Leon L. M. and Gomez-Sanchez E. P.: Biotin-hydrazide derivatives for the development of steroid enzyme-linked immunoassays. J. Steroid Biochem. Molec. BioL 43 (1992) 523-528.
399
13. Gomez-Sanchez C. E., Ferris M. W., Foecking M. F. and Gomez-Sanchez E. P.: Synthesis of 18-hydroxycortisol and 18-oxocortisol in bovine adrenal slices. J. Steroid Biochem. 33 (1989) 595-598. 14. Kater C. E., Biglieri E. G., Brust N., Chang B., Hirai J. and Irony I.: Stimulation and suppression of the mineralocorticoid hormones. Endocrine Rev. 10 (1989) 149-164. 15. Workman R. J., Vaughn W. K. and Stone W. J.: Dexamethasone testing in chronic renal failure: Pharmacokinetics of dexamethasone and demonstration of a normal hypothalamic-pituitary-adrenal axis. J. Clin. Endocr. Metab. 63 (1986) 741-746. 16. Gomez-Sanchez C. E., Foecking M. F., Shackleton C. H. L., Chavarri M. R. and Gomez-Sanchez E. P.: 18-hydroxy-ll-deoxycortisol: A new steroid isolated from incubations of the adrenal with 1 l-deoxycortisol. J. Steroid Biochem. 26 (1987) 105-111. 17. Kawamoto T., Mitsuuchi Y., Toda K., Yokoyama Y., Miyahara K., Miura S., Ohnishi T., Ichikawa Y., Nakao K., Imura H., Ulick S. and Shizuta Y.: Role of steroid llbeta-hydroxylase and steroid 18-hydroxylase in the biosynthesis of glucocorticoids and mineralocorticoids in humans. Proc. Natn. Acad. Sci. U.S.A. 89 (1992) 1458-1462.
0960-0760-93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
R E G U L A T I O N OF 18-OXOCORTISOL A N D 18-HYDROXYCORTISOL BY THE R E N I N - A N G I O T E N S I N SYSTEM A N D ACTH IN M A N NORIYOSHI YAMAKITA,1. CELSO E. GOMEZ-SANCHEZ, 2 TOMOATSU MUNE, 3 HISASHI YOSHIDA,3 SEIJI MIYAZAKI, a KEIGO YASUDA3 a n d TOSHIAKI NAKAI ~ qnstitute of Clinical Medicine, University of Tsukuba, Tsukuba-City 305, Japan, 2Department of Internal Medicine, University of South Florida College of Medicine and James A. Haley Veterans' Hospital, Tampa, FL 33612-4798, U.S.A. and 3Department of Medicine, Gifu University School of Medicine, Gifu-City 500, Japan
(Received 17 March 1993; accepted 14 May 1993)
Summary--Based on urinary excretion studies the secretion of the cortisol derivatives, 18-oxocortisol and 18-hydroxycortisol are believed to be regulated by ACTH and to a lesser degree by the renin-angiotensin system. Plasma concentrations of 18-oxocortisol and 18hydroxycortisol were measured during the simultaneous activation of the renin-angiotensin system and inhibition of ACTH secretion. Five healthy male subjects consuming a sodium diet ad libitum were studied. Blood was drawn at 0800 h after 1 h in the supine position. In the first set of experiments, the subjects remained in the supine position from 0800 to 1000 h with or without the oral administration of 2 mg dexamethasone at 0800 h. In the second set of experiments the subjects were placed in the upright position after drawing the 0800 h sample. The subjects were studied with and without dexamethasone administered at 0800 h. Blood was drawn again at 1000 h. Plasma levels of 18-oxocortisol, 18-hydroxycortisol, ACTH, plasma renin activity (PRA), cortisol, aldosterone and 18-hydroxycorticosterone were measured by radioimmunoassay. None of these parameters changed during the 2 h in the supine position. 18-Oxocortisol, 18-hydroxycortisol, aldosterone, 18-hydroxycorticosterone and PRA increased, but ACTH and cortisol did not change when the subjects were placed in the upright position. After dexamethasone administration, 18-oxocortisol, 18-hydroxycortisol, cortisol, aldosterone and 18-hydroxycorticosterone decreased in the supine position and no increase occurred in 18-oxocortisol, 18-hydroxycortisol and 18-hydroxycorticosterone in the upright position. PRA and aldosterone increased and ACTH and cortisol decreased in these subjects. 18-Oxocortisol and 18-hydroxycortisol were more dependent on ACTH regulation and less on the renin-angiotensin system than aldosterone.
INTRODUCTION
The adrenal gland produces aldosterone in the zona glomerulosa and cortisol in the zona fasciculata and can also produce hybrid compounds which share structural characteristics of these steroids [1, 2]. Patients with aldosterone producing adenomas and glucocorticoid-suppressible aldosteronism excrete excessive amounts of 18-oxocortisol and 18-hydroxycortisol [2, 3]. Patients with primary aldosteronism due to bilateral zona glomerulosa hyperplasia excrete normal amounts of both steroids [3]. The glucocorticoid activity of 18-oxocortisol is only 3% that o f cortisol and the mineralocorticoid potency is only 1% that of aldosterone [4]. 18Hydroxycortisol is inactive [5]. Measurements *To whom correspondence should be addressed.
of 18-oxocortisol and 18-hydroxycortisol could be important in the differential diagnosis of primary aldosteronism and 18-oxocortisol could have pathophysiological significance. Aldosterone and 18-hydroxycorticosterone are regulated primarily by the renin-angiotensin system [6], but A C T H , potassium and other factors also play [6]. A C T H plays a greater role in the secretion of 18-hydroxycorticosterone than of aldosterone [7]. The urinary excretion of 18-hydroxycortisol and 18-oxocortisol are increased by A C T H administration and dietary sodium restriction [8, 9] and are inhibited by dexamethasone administration and by sodium loading [8, 9]. Plasma measurements for 18hydroxycortisol have been reported[10,11]. In the present study the effect of activation of the renin-angiotensin system with suppression of A C T H production was investigated using 395
NOPdYOSm YAMAKITAet al.
396
simultaneous measurement of plasma 18-oxocortisol and 18-hydroxycortisol. MATERIALS AND METHODS
Subjects
Five healthy Japanese male subjects (24.6_ 0.5 years) eating a salt diet ad libitum were studied after an overnight fast. Informed consent was obtained after the nature of the procedures was fully explained. Blood was drawn at 0800 h after 1 h in the supine position. The subjects remained in the supine position from 0800 to 1000 h and received placebo or 2 mg dexamethasone orally at 0800 h on different days. They were also similarly studied after standing from 0800 to 1000 h. At 1000 h a second blood sample was obtained. The studies were done with at least 4 days inbetween treatments. The blood samples were separated immediately and frozen at -20°C until assayed.
Hormone measurements Materials for buffers were purchased from Sigma Chemical Company (St Louis, MO, U.S.A.) or Wako Pure Chemical Industries Ltd (Osaka, Japan). Bond ElutTM columns, C-2 silica and celite were obtained from Analytichem International (Varian, Harbor City, CA, U.S.A.). All glass tubes used were silanized with dichlorodimethyl silane and extensively washed.
18-Oxocortisol radioimmunoassay The source of 18-oxocortisol, 18-hydroxycortisol, tritiated derivatives and antibodies have been reported previously [3, 8, 12]. The radioimmunoassay for 18-oxocortisol consisted in taking 1 ml plasma, adding 1500cpm freshly purified [1,23H]18-oxocortisol, mixing well and adding 10/zl 10% acetic acid. The sample was transferred to a column consisting of a 1 ml polypropylene syringe filled first with 100mg celite followed by 50 mg C-2 silica and washed with 1 ml methanol and 2 ml water. After addition of plasma, the column was washed with 1 ml water and 100/zl formamide in water was added. The column was eluted with 4 ml 40% ethyl acetate in hexane (containing 1% acetic acid). This eliminated over 98% of cortisol and aldosterone which crossreact with the antibody. The 18-oxocortisol is then eluted with 2 ml 80% ethyl acetate in hexane (containing 1% acetic acid) and collected in a silanized tube. The organic solvent was evaporated under air and
reconstituted in 250/~1 RIA buffer (borate 0.1 M, pH7.1 containing 0.5% bovine serum albumin and 1% sodium azide). An aliquot was used for assessment of recoveries and 50 #1 in triplicate for the assay using polypropylene tubes. A standard curve was prepared using 0, 2.5, 5, 10, 25, 50, 100, 250 and 500 pg 18-oxocortisol in polypropylene tubes. An antibody solution (1:6000), 2500 cpm [1,23H]18-oxocortisol and goat antirabbit gammaglobulin (1:100) were added in 250 #1. After an overnight incubation, 0.6ml 10% polyethylene glycol (Mw 6000) was added and centrifuged 120 min later. The samples were then decanted and counted in a liquid scintillation counter. The results were corrected for recovery. The recoveries from the column varied between 60-73%.
18-Hydroxycortisol radioimmunoassay The assay was performed as described above except that the plasma and solvents were not acidified and 18-hydroxycortisol was eluted with ethyl acetate. The recoveries from the column varied between 58-68%. 18-Hydroxycorticosterone was measured by RIA after extracting with dichloromethane [13]. Plasma cortisol was measured by Gamma Coat Cortisol KitTM (Incstar Co, Stillwater, MN, U.S.A.). Plasma aldosterone was measured by an Aldosterone-RIA KitTM (Shionogi Pharmaceutical Co., Osaka, Japan). ACTH and plasma renin activity (PRA) were measured by ACTHII-IRMA TM (Mitsubishi Petrochemical Co., Tokyo, Japan) and Renin RIA BeadTM (Dainabot RI Laboratory, Tokyo, Japan). Data were analyzed by Student's t-test for paired samples. RESULTS
The supine position for 2 h did not result in any significant change in the plasma concentrations of cortisol, aldosterone, 18-hydroxycorticosterone, 18-oxocortisol, 18-hydroxycortisol, PRA or ACTH. Plasma levels of aldosterone, 18-hydroxycorticosterone, 18-oxocortisol, 18hydroxycortisol and PRA but not ACTH and cortisol increased significantly after 2 h upright position. The ratios of plasma concentration of 18-oxocortisol (1.57 _+0.09), 18-hydroxycortisol (1.57__+ 0.21) and 18-hydroxycorticosterone (1.59 ___0.10) to that before the upright position were significantly smaller than that of aldosterone (2.87 ___0.20) (Fig. 1). Dexamethasone administration resulted in a significant decrease in
Regulation of 18-oxocortisol and 18-OH-cortisol
397
4'
Upright
Posture
• "
A
3"
4) m
18oxoF
[] 18OHF 18OHB aldosterone
2"
cortisol
.o tr
Fig. I. The ratios of plasma concentration of 18-oxocortisol (18oxoF), 18-hydroxycortisol (18OHF), 18-hydroxycorticosterone(18OHB), aldosterone and cortisol after the postural stimulation to those before the stimulation. *Significantly different, vs 18oxoF and 18OHF, P <0.02; vs 18OHB and cortisol, P < 0.01.
plasma A C T H and cortisol. Plasma aldosterone, 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol significantly decreased 2 h after taking dexamethasone in the supine position. In this test, the ratios of plasma levels of 18-oxocortisol ( 0 . 4 8 _ 0.09), 18-hydroxycortisol (0.52+0.13), and cortisol (0.56 _ 0.23) to those before taking dexamethasone were not significantly different from each other. 18-Hydroxycorticosterone and aldosterone ratios were significantly greater than those of 18-oxocortisol and 18-hydroxycortisol, respectively (Fig. 2). P R A and plasma aldosterone increased after taking dexamethasone in the upright position. 18-Oxocortisol also
increased, but the increase was not statistically significant. DISCUSSION The urinary excretion of 18-oxocortisol [9] and 18-hydroxycortisol [8] and plasma levels o f 18-hydroxycortisol[ll] were reported to increase in subjects given a low sodium diet, suggesting that activation of the reninangiotensin system played a role in their regulation. Infusion of angiotensin II did not increase plasma 18-hydroxycortisol, but as expected increased plasma aldosterone by 2.3- to 2.8-fold [11]. In the present study, plasma levels
1.0 Dex 0.8 4) m
[] 18oxoF 0.6
,< .0
[] 18OHF [] 18OHB
0.4
Illl aldosterone [] cortisol
0.2.
0.0 Fig. 2. The ratios of plasma concentration of 18oxoF, 18OHF, 18OHB, aldosterone and cortisol after dexamethasone administration to those before the administration. *Significantlydifferent, vs 18OHB and aldosterone, P < 0.001; **Significantlydifferent, vs 18OHB and aldosterone, P < 0.04.
NORIYOSHIYAMAKITAet al.
398
Table 1. Plasma levels of 18-oxocortisol (18oxoF), 18-hydroxycortisol (18OHF), 18-hydroxycorticosterone (18OHB), aldosterone, cortisol, ACTH and PRA Supine 0800 h
Uptight 1000 h
0800 h
Dex 1000 h
0800 h
Dex + Uptight 1000 h
0800 h
1000h
18oxoF 0.826 + 0.115 0.612+-0.161 0.675+ 0.092 1.022+ 0.106 0.862 + 0.105 0.407_+0.090 0.551 + 0.108 0.847 + 0.271 (nmol/l) ND P < 0.01 P < 0.02 ND 18OHF 3.32 +- 0.27 2.86 + 0.58 2.99 + 0.68 4.41 + 0.91 3.46 + 0.52 1.86 + 0.46 3.00 +- 0.40 3.03 + 0.51 (nmol/l) ND P < 0.03 P < 0.02 ND 18OHB 621+71 591+78 678+99 1076+_172 657+122 442_+70 594+57 653+-64 (pmol/l) ND P < 0.01 P < 0.05 ND Aldosterone 230 +- 50 174 ___20 269 + 10 766 + 29 234 + 11 170 + 26 266 + 87 612 + 211 (pmol/I) ND P < 0.0005 P < 0.02 P < 0.04 Cortisol 288 + 47 286 +- 35 299 + 49 336 +_ 18 336 + 52 187 + 43 341 + 42 240 +- 52 (nmol/l) ND ND P < 0.02 P < 0.04 ACTH 5.50+_ 1.11 4.90+-1.22 5.29+0.89 5.99+0.79 5.60+-1.44 2.54+-0.60 5.73_+1.01 2.37+-0.51 (pmol/l) ND ND P < 0.03 P < 0.01 PRA 0.135+-0.038 0.170+_0.047 0.123+-0.035 0.890+_0.247 0.349+_0.097 0.259+_0.072 0.176+_0.049 1.033+_0.287 (nmol/l • s) ND P < 0.005 ND P < 0.02 Mean + SE; Supine, supine position from 0800 to 1000 h without dexamethasone administration; Uptight, uptight position from 0800 to 1000h without dexamethasone administration; Dex, supine position from 0800 to 1000h with dexamethasone administration; Dex + Uptight, uptight position from 0800 to 1000h with dexamethasone administration. ND, not different.
of 18-hydroxycorticosterone, 18-oxocortisol and 18-hydroxycortisol increased by about 1.6 times when the subjects were placed in the upright position for 2 h. The increase in plasma aldosterone was greater than the other steroids. The studies suggest that the renin-angiotensin system plays a greater role in the stimulation of aldosterone secretion than it does in 18hydroxycorticosterone, 18-oxocortisol and 18hydroxycortisol secretion. The lesser response of 18-hydroxycorticosterone compared to aldosterone has also been reported by Kater et al. [14]. Orally administered dexamethasone is rapidly absorbed and serum concentration peaks at about 1 h[15]. Plasma cortisol gradually decreases from 1 h after the administration of dexamethasone [15]. ACTH, cortisol, aldosterone and 18-hydroxycorticosterone exhibit a diurnal rhythm, but the difference in time between 0800 and 1000 h was too close to show a significant change in plasma concentrations. The administration of dexamethasone decreased ACTH, cortisol, aldosterone, 18-hydroxycorticosterone, 18-oxocortisol and 18-hydroxycortisol. The degree of suppression of 18-oxocortisol and 18-hydroxycortisol was greater than that of 18-hydroxycorticosterone and aldosterone. The adrenal has two cytochrome P-450 1lflhydroxylases, one primarily located in the zona fasciculta-reticularis which converts deoxycorticosterone into corticosterone and 18hydroxydeoxycorticosterone. In the case of 17-hydroxylated steroids it converts 11-deoxycortisol into cortisol. It can also 18-hydroxylate ll-deoxycortisol to 18-hydroxydeoxycortisol, but this conversion has been poorly studied [16]. In the zona glomerulosa, deoxycorticosterone is
converted to corticosterone, 18-hydroxycorticosterone and aldosterone under the action of the aldosterone synthase [17]. Cortisol can act as a substrate and generate 18-oxocortisol and 18hydroxycortisol. Dexamethasone administration reduces the production of cortisol and as a consequence the generation of 18-oxocortisol and 18-hydroxycortisol. In conclusion, 18-hydroxycorticosterone, 18oxocortisol and 18-hydroxycortisol are less dependent on the renin-angiotensin system than aldosterone and 18-oxocortisol and 18-hydroxycortisol are more dependent on ACTH for stimulation. Acknowledgements--Dr Yamakita is supported by a grant from the University of Tsukuba Project Research. Dr Gomez-Sanchez is supported by medical research funds from the Department of Veterans Affairs and the National Institutes of Health (HL 27255).
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