[Aldosterone precursors and hypertension with hypokalemia and adrenal module non caused by primary hyperaldosteronism]. / Précurseurs de l'aldostérone et hypertension artérielle avec hypokaliémie et nodule surrénalien, non attribuée à un hyperaldostéronisme primaire.

The purpose of the study was to evaluate the interest of aldosterone precursors assays in arterial hypertension with hypokaliemia and adrenal nodules non due to aldosterone. Seven hypertensive patients, 3 men and 4 women, aged 59.5 +/- 10.1 years were included in the study. After drug withdrawal, kaliemia was 3.1 +/- 0.3 mmol/l (2.7-3.6), active renin 2.9 +/- 1.4 ng/l, plasma aldosterone (aldo) 108 +/- 49.4 pg/ml, cortisol 13 +/- 3.1 micrograms/100 ml, and [S] 0.47 +/- 0.5 micrograms/100 ml. Adrenal CT scan showed an adenoma in 3 patients (30.5 +/- 5 mm) and an unilateral nodular hyperplasia in 4 patients. In all patients, the plasma levels (RIA, chomatographic step) of the following steroids in the mineralocorticoid (MC) pathway were determined: DOC, 18 OH-DOC, B, 18 OH-B and aldosterone. Two from 7 (28%) exerted aldosterone precursors excess, 1 with DOC-producing adenoma (DOC-PA) (table), and 1 with a partial 11 beta hydroxylase deficiency (DOC: 211 pg/ml; S: 1 mu/100 ml). Aldosterone/DOC + 18 OH-DOC ratio proposed as a malignancy index was decreased in the patient with DOC-PA (8.1). No dysfunction in the MC pathway was identified in the 5 other patients. [table: see text] The study suggests the relevance of aldosterone precursors assays in low renin hypertension non due to aldosterone and in incidentally discovered adrenal masses.

Plasma levels of aldosterone versus aldosterone precursors: a way to estimate the malignancy of asymptomatic and nonsecretory adrenal tumors: a French Retrospective Multicentric Study.

The aim of this study was to find out whether the dysfunction of aldosterone pathway, previously proposed as a marker of secretory adrenal carcinoma, is also found in nonsecretory adrenal carcinomas, which pose even more difficult diagnostic problems even for patients with hypertension accompanied or not by hypokalemia. The exploration consisted of using the same method (RIA preceded by a chromatographic step) to determine the plasma levels of the following steroids in the mineral corticosteroid pathway: deoxycorticosterone (DOC), 18-hydroxydeoxycorticosterone (18-OHDOC), corticosterone (B), 18 hydroxycorticosterone (18 OH B), and aldosterone. The subjects included 16 adults, each presenting with an endocrinologically asymptomatic adrenal mass associated for some patients with hypokalemia and hypertension (8 with adrenal carcinoma, 2 with adrenal metastasis from other forms of cancer, and 6 adenomas). These results show that even in nonsecretory adrenal carcinoma, there is a dysfunction of the aldosterone pathway, which can be evaluated from the ratio between aldosterone and the substrate of 11 beta hydroxylase (DOC) and its derivative (18-OH DOC). This study suggests that exploration of mineralocorticosteroid pathway can be used as a hormonal marker of adrenal carcinoma for both secretory and non-secretory malignant masses.

Suramin: an inhibitor of the final steps of the mineralocorticoid pathway?

The authors used incubated adrenal mitochondria to study the in vitro effect of suramin, an antiparasitic drug, on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that, under conditions preserving membrane integrity, the "impermeance" of suramin meant that concentrations similar to the plasma-levels reached in treated patients induced only slight inhibition of the final intramitochondrial steps in aldosterone synthesis. However, suramin strongly inhibited mitochondrial respiration. The inhibition of two intramitochondrial mechanisms (respiration and steroid synthesis) suggests that the effect of suramin involves partial inhibition of metabolic intermediate carriers. The inhibition of the activity of various extramitochondrial enzymes involved in intermediate metabolism, suggests that the inhibition of steroid biosynthesis can be explained only on the basis of an extramitochondrial action of suramin. The action of suramin must, therefore, primarily and directly affect extramitochondrial steroid synthesis and only indirectly affect intramitochondrial steroid synthesis as a result of an impact on the reducing equivalent supply. However, even if suramin does not bind to cytochrome P450 11 beta which catalyzes the final steps of aldosterone biosynthesis pathway, this does not imply that suramin has no direct effect on steroid synthesis within the mitochondria, in addition to its toxic effects, particularly if the cell structure is disrupted (as is often the case in tumor tissues).

The reoxidation of cytochrome P-450 by paraquat inhibits aldosterone biosynthesis from 18-hydroxycorticosterone.

Paraquat is an artificial electron carrier that captures electrons from reduced cytochrome P-450 instead of the natural acceptors, thus decreasing the concentration of reduced mitochondrial cytochrome P-450. In the present study, paraquat inhibited the biosynthesis of aldosterone from 18-hydroxycorticosterone by mitochondria from duck adult adrenal gland, under aerobic conditions. Since paraquat did not induce any change in the absorption spectrum of highly purified cytochrome P-450 11 beta, the possibility of a displacement of steroid by the drug is ruled out. Moreover, paraquat did not affect oxidative phosphorylating chain nor did it alter by itself the chemical structure of 18-hydroxycorticosterone. In our conditions, the inhibitory role of paraquat seems restricted to a capture of electrons from reduced cytochrome P-450. Under the same conditions metopirone and spironolactone, known to bind cytochrome P-450 11 beta at the steroid binding site, also inhibited the reaction. Altogether these results show that for aldosterone synthesis from 18-hydroxycorticosterone to take place, the steroid binding site on cytochrome P-450 must be accessible to 18-hydroxycorticosterone and that the cytochrome P-450 must be the direct donor of reducing equivalents. Hence, cytochrome P-450 appears as the final linking point between 18-hydroxycorticosterone and the reducing equivalents provided by NADPH.

Verapamil directly inhibits aldosterone synthesis by adrenal mitochondria in vitro.

The action of verapamil, a calcium channel blocker, on the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) was studied using duck adrenal mitochondria in the absence of regulatory factors. Results show that 10(-5) M verapamil inhibits the transformation of 18-hydroxycorticosterone into aldosterone by 52.8%. Moreover, our findings show that verapamil induces only a slight inhibition of respiratory capacity without action on respiratory control and does not displace 18-hydroxycorticosterone from cytochrome P450 11 beta which catalyses the reaction. Thus, this study does not explain the mechanism of inhibition induced by verapamil on the last step of aldosterone synthesis but it is of interest to note, for clinical use, that this inhibition is not linked to regulatory factors of aldosterone production. Since primary hyperaldosteronisms are characterized by their independence vis-á-vis regulatory factors, administration of verapamil may be particularly interesting for treatment of primary hyperaldosteronisms.

Stimulation of oxygen consumption at the cytochrome A3 level inhibits aldosterone biosynthesis from 18-hydroxycorticosterone.

A mitochondrial preparation from duck adrenal gland was used, under aerobic conditions, to show that the oxygen requirement for the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) is at the cytochrome P-450 level only. Vitamin C and tetramethyl-p-phenylene-diamine (TMPD) were used to increase oxygen consumption at the cytochrome a3 level, thereby decreasing its availability to cytochrome P-450. The vitamin C plus TMPD system acts as an 'oxygen trap'. Results show that despite reducing equivalents provided by L-malate, vitamin C plus TMPD strongly inhibits aldosterone biosynthesis from 18-hydroxycorticosterone (89%). Moreover, we used KCN in order to block oxygen consumption, even in the presence of vitamin C plus TMPD. Under these conditions, the inhibition of aldosterone biosynthesis from 18-hydroxycorticosterone is reduced by 51%. The reversal of this inhibition by KCN was evident but only partial. According to polarographic and electron microscopy studies, the reversal of inhibition can only be explained by an increased availability of oxygen at the cytochrome P-450 level. Experiments performed under aerobic conditions, without a nitrogen atmosphere, show that oxygen is required in the transformation of 18-hydroxycorticosterone into aldosterone, at the cytochrome P-450 level. This suggests that a classical hydroxylating mechanism is involved.

Action of different nucleotides on the last step of aldosterone synthesis.

The effect of various nucleotides on the last step of aldosterone biosynthesis, the so-called "18 oxidation" (transformation of 18-hydroxycorticosterone to aldosterone), was studied by incubation of tritiated 18-hydroxycorticosterone with untreated duck adrenal mitochondria in vitro. The study was carried out in the absence or in the presence of antimycin A which blocks the respiratory chain. Results show that, when oxidative phosphorylation chain functions normally, GTP and CTP had no effect, UTP stimulated this reaction but ADP and ATP inhibited the transformation of 18-hydroxycorticosterone into aldosterone to the same extent. For this reason ATP is included in all controls for experiments studying the effect of ATP when "18 oxidation" is inhibited by antimycin A. When oxidative phosphorylation chain is inhibited by antimycin A, ATP is able to reverse the inhibition of "18 oxidation" induced by antimycin A, in the presence of succinate. Under these conditions UTP is not able to reverse the inhibition induced by antimycin A; GTP and CTP had no effect. Effects of ATP and UTP on the last step of aldosterone biosynthesis are related to different mechanisms. ATP clearly acts as an energy source for "18 oxidation" in the presence of succinate. The role of UTP must still be determined.

The final step of aldosterone biosynthesis requires reducing power. It is not a dehydrogenation.

A mitochondrial preparation from adult duck adrenal gland was used to study the mechanism (dehydrogenation or other) of the last step of aldosterone biosynthesis (18-oxidation) by incubation of tritiated 18-hydroxycorticosterone. Results show that the role of citric acid cycle metabolites is to provide reducing power. When reducing power is provided by malate, which yields NADH or NADPH directly, the reoxidation of reduced coenzymes in the oxidative phosphorylation chain is not necessary. In the presence of succinate, the oxidative phosphorylation chain is required, but only to provide ATP. Stimulation of the reaction by low concentrations of KCN in the presence of malate shows that the reducing power is not used in the oxidative phosphorylation chain. These data suggest that the reaction is not a dehydrogenation and that the reducing power is used in a pathway competing with the respiratory chain, most probably a hydroxylating pathway, in mitochondria.