Effect of uni-adrenalectomy on blood pressure in a patient with excessive adrenal 18-hydroxy-11-deoxycorticosterone production bilaterally.

A 46-year-old woman was presented with mineralocorticoid excess syndrome and a large mass originating from the right adrenal gland. Clinical examination before right adrenalectomy revealed elevated serum concentrations of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) both systemically and in the adrenal veins bilaterally. Histopathological and immunohistochemical analyses of the surgical specimen demonstrated adrenal hyperplasia of outer fasciculata cells, and the presence of cystic mass. The adrenalectomy ameliorated her blood pressure (BP) from 156/96 mmHg to 148/87 mmHg with a concomitant increase of serum potassium concentration from 3.1 mEq/l to 3.5 mEq/l. These results suggest that uni-adrenalectomy is, at least in part, effective in ameliorating not only BP but also potassium concentration in a patient of adrenal hyperplasia with excessive bilateral 18-OH-DOC production.

Preclinical Cushing's syndrome due to ACTH-independent bilateral macronodular adrenocortical hyperplasia with excessive secretion of 18-hydroxydeoxycorticosterone and corticosterone.

A 64-year-old woman developed hypertension and hypokalemia, due to ACTH-independent bilateral macronodular adrenocortical hyperplasia (AIMAH) with excessive secretion of 18-hydroxydeoxycorticosterone and corticosterone. Plasma cortisol did not show a diurnal rhythm, and was not suppressed by dexamethasone (8 mg). Plasma cortisol responded to ACTH and was increased by hypoglycemia without modifying ACTH levels. Radiological studies demonstrated that adrenal glands were enlarged with macronodules. Although the patient exhibited a low plasma renin activity and aldosterone levels, hypokalemia and hypertension were observed. Hormonal findings would support the hypothesis that the tumor of AIMAH originated from cells of the upper zona fasciculata.

A two cell type theory for aldosterone biosynthesis: the roles of 11 beta-hydroxylase and aldosterone synthase, and a high capacity tightly binding steroid carrier for 18-hydroxydeoxycorticosterone in rat adrenals.

Several lines of experimentation suggest that a tissue-sequestered pool of 18-hydroxydeoxycorticosterone (18-OH-DOC) in the rat adrenal may be mobilized as an aldosterone precursor. We show here that this steroid is maintained in a non-extractable form in the membranes of collagenase-dispersed fasciculata/reticularis cells. Because of this stability, the complex can be identified by immunocytochemistry and also, in IEF gels of solubilized inner adrenocortical zone membrane preparations, by immunoblotting. However, the complexed steroid cannot be extracted from the gels into organic solvent unless first treated with trypsin. Preincubation of viable whole glandular tissue with trypsin significantly enhanced aldosterone output and eliminated the trypsin-releasable 18-OH-DOC pool in IEF gels of solubilized inner zone membranes. Both prior sodium depletion and acute trypsin stimulation of whole glands enhanced extractable 18-OH-DOC in glomerulosa tissue membranes. Other experiments using in situ hybridization show that mRNA coding for 11 beta-hydroxylase (which generates 18-OH-DOC) is confined to the inner adrenocortical zones, whereas aldosterone synthase (which does not) is transcribed exclusively in the glomerulosa. The data suggest that a pool of 18-OH-DOC in inner zone membranes can be mobilized for utilization as an aldosterone precursor in the glomerulosa. The results also indicate the existence of an entirely novel tightly binding steroid carrier from which steroid cannot be extracted by organic solvent unless first subjected to proteolytic degradation.

Cortisol: a tool to study aldosterone biosynthesis in rats.

Corticosterone (B) and 18-hydroxy-11-deoxycorticosterone (18OHDOC) but not 11-deoxycorticosterone (DOC) displaced cortisol (F) specifically bound to rat adrenal mitochondria. F. competitively inhibited aldosterone formation from B, 18OHB and 18OHDOC but did not inhibit conversions of DOC to B or 18OHDOC. High concentrations of DOC increased its conversion to 18OHDOC rather than B.

Thyrotropin-releasing hormone inhibits glucocorticoid secretion of rat adrenal cortex: in vivo and in vitro studies.

The bolus iv administration of TRH dose-dependently decreased ACTH-enhanced plasma corticosterone (B) concentration in rats, without affecting the basal one. The effects of TRH on steroid secretion of dispersed rat inner adrenocortical cells were investigated by HPLC. TRH significantly decreased both basal and ACTH-stimulated post-11-deoxycorticosterone (DOC) secretion (i.e. 18-hydroxy-DOC and B) and concomitantly raised DOC and progesterone release, so that the total postpregnenolone yield of our preparations was unaffected. TRH did not alter either basal or ACTH-stimulated pregnenolone production by isolated rat adrenocortical cells. It was concluded that TRH is an inhibitor of glucocorticoid secretion in rats, which electively impairs the late steps of B synthesis (i.e. 11- and 18-hydroxylation) without affecting the earlier steps, including the rate-limiting one of this process.

In vitro glucocorticosteroid and mineralocorticosteroid biosynthesis in Conn's adenoma tissues.

The in vitro metabolism of [1,2-3H] deoxycorticosterone (DOC), [1,2-3H] 18-hydroxy-11-deoxycorticosterone (18-OHDOC) and [1,2-3H] 11-deoxycortisol (S) was studied in adrenal adenoma homogenates from patients with primary hyperaldosteronism. Tumor tissues actively converted deoxycorticosterone and 18-hydroxy-11-deoxycorticosterone to 18-hydroxycorticosterone and aldosterone. Yields of cortisol and cortisone were also large showing that the tissues did not lack the zona fasciculata-like 11 beta-hydroxylation ability.

A case of weak mineralocorticoid-producing benign adrenal tumor.

Tumors producing weak mineralocorticoids are exceedingly rare and most of them predominantly produce 11-deoxycorticosterone. We present a case of adrenocortical adenoma in which the overproduction of 18-hydroxydeoxycorticosterone exceeded that of 11-deoxycorticosterone.

Origin of aldosterone in trypsin-stimulated rat adrenal zona glomerulosa incubations.

The time-course for the in-vitro secretion of aldosterone and 18-hydroxycorticosterone (18-OH-B) by rat adrenal whole capsular tissue (largely zona glomerulosa) was studied under control and stimulated conditions. The stimulatory effect of trypsin was relatively delayed, and the steroids were significantly enhanced only after 1 h, in contrast to the actions of ACTH, which produced effects after 15 or 30 min. Tissue-sequestered 18-hydroxydeoxycorticosterone (t-18-OH-DOC), which is not affected by ACTH, was significantly depleted by trypsin, but secreted 18-OH-DOC was not consistently affected by either stimulant. In contrast to the apparent mobilization of t-18-OH-DOC, the conversion of exogenously added [3H]18-OH-DOC to [3H]18-OH-B was inhibited by trypsin, and aldosterone was unaffected. When trilostane was added to inhibit de-novo steroidogenesis, under conditions in which the steroid secretory response to ACTH is completely inhibited, aldosterone and 18-OH-B secretion was still stimulated by trypsin although yields were lower. Compared with controls, trilostane reduced t-18-OH-DOC concentrations, and trypsin caused a further depletion. In other studies, glomerulosa plasma membrane enriched preparations were homogenized and centrifuged, and the supernatants were dialysed and added to incubations of dispersed zona glomerulosa cells in the presence or absence of stimulators of aldosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

[18-Hydroxycorticosterone, 18-hydroxydesoxycorticosterone. Why, when, how to determine plasma levels in some adrenal pathologies]. / 18-Hydroxycorticostérone, 18-hydroxydésoxycorticostérone. Pourquoi, quand, comment en déterminer les taux plasmatiques dans certaines pathologies surrénaliennes.

The authors review some current ideas concerning the role of 18-hydroxylated corticosteroids as mineralocorticoids themselves and as possible precursors of the principal mineralocorticoid, aldosterone. In particular, the physiological and pharmacological agents affecting their secretion are discussed together with a description of the methods used for their analysis in plasma in the department of Clinical Biochemistry Pitié-Salpétrière. Finally, the value of these assays in the differential diagnosis of mineralocorticoid hypertension and inborn errors of corticosteroid biosynthesis is assessed and the constraints on sampling technique listed.

Regulation of mineralocorticoid secretion by the superfused fetal monkey adrenal gland: lack of stimulation of aldosterone by ACTH.

The rhesus monkey fetal adrenal secretion of mineralocorticoids was studied in vitro. Superfusion of fetal adrenal minces (n = 6) demonstrated that the fetal adrenal secretes aldosterone as well as desoxycorticosterone, 18 hydroxydesoxy corticosterone, and 18 hydroxycorticosterone. Addition of 250 ng/ml ACTH to the superfusion medium did not result in stimulation of aldosterone, but did increase these other mineralocorticoids. These data indicate that aldosterone production is not readily stimulated by ACTH in the fetal rhesus monkey, although other steroids in the mineralocorticoid pathway are.

Studies on the mechanisms of ACTH-induced inhibition of aldosterone biosynthesis in the rat adrenal cortex.

In rats, chronic treatment with high doses of ACTH (10-40 micrograms/100 g body weight per day) leads to a marked reduction in aldosterone synthesis by adrenal capsules. The possibility that this inhibition is secondary to a decrease in plasma potassium levels or in renin angiotensin system (RAS) activity has been explored. The effects of chronic ACTH treatment were compared in (I) animals in which the endogenous RAS activity was stimulated by restricting dietary sodium intake, (II) animals in which plasma angiotensin II was increased by infusion from implanted osmotic minipumps and (III) animals which received supplementary potassium and in which hypokalaemia was prevented. In all cases, rates of aldosterone biosynthesis in vitro by adrenal capsules were decreased in ACTH-treated animals to an extent similar to those in untreated controls. In addition, ACTH treatment of hypophysectomized rats resulted in a similar inhibition of aldosterone biosynthesis to that found in sham-operated controls. It may be concluded that the ACTH-induced reduction of aldosterone biosynthesis is independent of the secretion of other pituitary hormones, and cannot be simply ascribed to either a reduction in RAS activity or in plasma potassium levels. The results are consistent with the view that the effects of chronic ACTH treatment are mediated by a direct action on the zona glomerulosa cell, which leads to its transformation into a zona fasciculata-like form.

Suppression of adrenal renin in Dahl salt-sensitive rats.

We previously showed that adrenal renin is highest in the rat zona glomerulosa (ZG) and that low sodium or high potassium and nephrectomy increase adrenal ZG renin and aldosterone. Dahl salt-sensitive rats (S) have been shown to have lower plasma renin activity and plasma aldosterone and higher plasma 18-hydroxy-11-deoxycorticosterone than Dahl salt-resistant rats (R). In this study we assess the possible role of adrenal ZG renin in the suppression of aldosterone in S rats. Adrenal ZG renin was significantly decreased in S as compared with R rats even at 6 weeks of age, when both S and R rats are still normotensive (S = 7.2 +/- 0.2, R = 18.0 +/- 1.6 ng angiotensin I/mg protein/hr). Adrenal ZG aldosterone was also significantly lower in S than in R rats (S = 21.1 +/- 4.3, R = 39.5 +/- 3.6 ng/mg protein). Furthermore, the rise in adrenal ZG renin and aldosterone after nephrectomy in S rats was significantly less than that in R rats. To determine if the suppressed adrenal ZG renin of S rats is due to volume expansion, we studied the effect of a sodium-deficient diet on adrenal ZG renin in S and R rats. After 2 weeks of a sodium-deficient diet S rats had significantly lower basal adrenal ZG renin than did R rats (S = 7.6 +/- 0.4, R = 21.7 +/- 1.9 ng angiotensin I/mg protein/hr) and a marked blunting of the adrenal ZG renin response to nephrectomy (S = 13.6 +/- 1.1, DR = 167 +/- 16.1 ng angiotensin I/mg protein/hr).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of 18,19-dihydroxycorticosterone.

A four-step synthesis of 18,19-dihydroxycorticosterone 5c, starting with 19,21-dihydroxy-3,20-dioxopregn-5-ene-18,11 beta-lactone-di-(ethylene ketal) 2, is presented. Reduction of 2 with sodium aluminum bis-(methoxyethoxy)hydride gave 11 beta,18,19,21-tetrahydroxy-pregn-5-ene-3,20-dione-di-(ethylene ketal) 3a. Acetylation furnished the corresponding 18,19,21-triacetate 3b, which on treatment with a mixture of perchloric and acetic acids gave 18,19-dihydroxycorticosterone 18,19,21-triacetate 4b. Mild saponification yielded the title compound which, on the basis of ir and nmr spectra, exists as one C-20 isomer of the hemiacetal structure 5c. Periodate oxidation of 5c gave the expected 11 beta, 19-dihydroxy-3-oxoandrost-4-ene-17 beta, 18-carbolactone 6b.

Regulation of 11 beta/18-steroid hydroxylation in newborn rat adrenal cells in primary culture.

In newborn rat adrenal cells in primary culture, the level of activity of the 11 beta/18-steroid hydroxylase system involved in the last step of the corticosteroid biosynthesis is increased by ACTH. A parallel study of 11 beta- and 18-hydroxylation showed the same apparent Km values (64 microM) for both hydroxylations. The Vmax values differed: 11.5 micrograms/10(6) cells/h for corticosterone and 6.9 micrograms/10(6) cells/h for 18-hydroxyDOC. A dose response study of the ACTH effect, measured by the bioconversion of deoxycorticosterone to corticosterone and 18-hydroxyDOC, showed maximum hydroxylation with a dose of 2.2 mU of ACTH/ml. Addition of ACTH after several weeks in culture produced a smaller increase in 11 beta/18-hydroxylation. Removal of ACTH after several weeks of treatment produced an immediate decrease in corticosteroid production; readdition of ACTH produced an increase to the previous level in the case of the 22 mU/ml dose, but not in the case of the 2.2 mU/ml dose. The use of actinomycin D demonstrated that ACTH affects mainly the biosynthesis of protein which must be renewed approximately every 24 h. Finally, the effect of pretreatment or co-treatment with various concentrations of the end products of the reaction showed no inhibition or destruction of the 11 beta/18-hydroxylating enzyme system. Therefore, the regulation of the 11 beta/18-steroid hydroxylase system in these cell cultures seems to be accomplished through the induction by ACTH of the transcription involved in the biosynthesis of cytochrome P450(11) beta and the amount of available precursor furnished by endogenous steroidogenesis.

The role of ACTH in determining the metabolic pathways of deoxycorticosterone by newborn rat adrenal cells in primary culture.

The metabolism of deoxycorticosterone (DOC) by newborn rat adrenal cells in primary culture at various times after culture, with and without ACTH, was studied. After 5 days in culture before addition of ACTH, the main products of the metabolism of DOC were corticosterone and 18-hydroxy-11-deoxycorticosterone in a 2:1 ratio. Smaller amounts of 20 alpha-dihydrocorticosterone and 18-hydroxycorticosterone were also found. No reduced metabolites of DOC were detected. Without ACTH the conversion of DOC to corticosterone and 18-hydroxyDOC declined rapidly. After 13 days in culture, this conversion accounted for only half the metabolites. The reductive metabolism of DOC which yields products reduced at 20 alpha and/or 3 alpha/beta and 5 alpha accounted for the other half. When ACTH (22 mU/ml) was added to the culture daily for several weeks, the primary metabolism of DOC remained that of 11 beta- and 18-hydroxylation yielding corticosterone and 18-hydroxyDOC. A minor reductive metabolism was found. Both cultures produced 6 beta-hydroxyDOC. These results demonstrate that ACTH is needed to maintain the efficiency of the 11 beta/18-hydroxylating system. They also show that ACTH controls the type of metabolism predominant in the rat adrenal cell and may be responsible for the balance between the biosynthesis of glucocorticoids and their reductive catabolism in the fasciculata zone of the adrenal gland.

Mineralocorticoid properties of potential metabolites of 18-hydroxydeoxycorticosterone and 18-hydroxyprogesterone.

The high secretion rate of 18-hydroxydeoxycorticosterone in hypertensives and the steroids implication as a mineralocorticoid has led to the synthesis of potential di-, tetra-, and hexahydro metabolites of it and 18-hydroxy-progesterone. These potential metabolites have been synthesized by reduction of the double bond and the 3- and 20-ketones, singly or in combination. They have been evaluated for pro- and antimineralocorticoid activity and their affinity for the renal aldosterone receptor. All except one of the potential metabolites either lack or have reduced mineralocorticoid activity and aldosterone receptor binding affinity. The exception is the 3-ketopregn-4-ene-18,20-diol which has high receptor affinity but functions as an aldosterone antagonist.

19-Hydroxylation of 18-hydroxy-11-deoxycorticosterone by adrenal mitochondria prepared from various animal species.

We have recently reported that bovine adrenocortical cytochrome P-45011 beta catalyzes 19-hydroxylation of 18-hydroxy-11-deoxycorticosterone (18(OH)DOC) in addition to 11 beta-hydroxylation of the steroid. In this report, we examine the presence of these two activities in 18(OH)DOC and 11 beta- and 18-hydroxylation activities on deoxycorticosterone (DOC) among the adrenal mitochondria prepared from man, ox, pig, rabbit, guinea-pig and rat. The results indicate that these animals could be classified into three groups with respect of these hydroxylation activities. Mitochondria of the first group comprising ox and pig showed rather high 19- and 11 beta-hydroxylation activities on 18(OH)DOC compared to the hydroxylation activities on DOC. Mitochondria prepared from the second group which comprised rabbit, guinea-pig and man showed low 19-hydroxylation activity on 18(OH)DOC, whereas the 11 beta-hydroxylation of 18(OH)DOC well occurred in these species. The last group comprising rat had very low activity both of 11 beta- and 19-hydroxylations when 18(OH)DOC was used as the substrate, whereas both 11 beta- and 18-hydroxylations of DOC were high in rat adrenal mitochondria. No significant difference of these activities could be found between zona glomerulosa cells and zonae fasciculata-reticularis cells of bovine adrenal cortex, and between adrenal mitochondria from spontaneously hypertensive rat and those from WKY normotensive rat.

18,19-Dihydroxydeoxycorticosterone, a new metabolite produced from 18-hydroxydeoxycorticosterone by cytochrome P-450(11) beta. Chemical synthesis and structural analysis by 1H NMR.

A new metabolite was produced from 18-hydroxydeoxycorticosterone by the cytochrome P-450(11) beta linked hydroxylase system purified from bovine adrenocortical mitochondria. It was identified as 18,19-dihydroxydeoxycorticosterone by chemical synthesis on the basis of high-performance liquid chromatography, gas chromatography-mass spectrometry, and proton nuclear magnetic resonance (1H NMR) spectroscopy, and detailed structural analysis of it was performed by 1H NMR spectroscopy. The methylene protons at the C-19 position of the steroid were nonequivalent and coupled with each other, having a coupling constant of 10.6 Hz. These protons had different coupling constants, 6.7 and 3.4 Hz, for the hydroxy proton at the C-19 position. Due to these couplings, the signals of the methylene protons were observed around 3.9 ppm as two double doublets. The methylene protons at the C-21 position were also nonequivalent, having a coupling constant of 11.1 Hz. Coupling constants between these methylene protons and the hydroxy proton at the C-21 position were 8.2 and 4.2 Hz, respectively. These results indicate that both hydroxymethyl groups at the C-19 and C-21 positions do not freely rotate in chloroform solution. The signals of hydroxy protons at the C-19 and C-21 positions were found at 1.25 and 1.87 ppm, respectively, by means of decoupling of the corresponding methylene protons. The hydroxy proton at the C-18 position was found to scarcely couple with any proton. This fact suggests that this hydroxy group is linked to the C-20 position, making a hemiketal bridge between the C-18 and the C-20.