The amino acid substitutions Ser288Gly and Val320Ala convert the cortisol producing enzyme, CYP11B1, into an aldosterone producing enzyme.

Transfection studies with cDNAs encoding hybrids between the highly similar cytochrome P450 enzymes, CYP11B1 (steroid 11 beta-hydroxylase) and CYP11B2 (aldosterone synthase) have identified which amino acids determine the different activities of the enzymes.

[Dysfunction of the aldosterone synthesis pathway as a marker of malignity in symptomatic and asymptomatic adrenal masses]. / Du dysfonctionnement de la voie de synthèse de l'aldostérone comme marqueur de malignité des masses surrénaliennes symptomatiques et asymptomatiques.

Fundamental research performed in the author's laboratory led to the understanding of mechanisms of the mineralocorticoid biosynthetic pathway. Sensitive assays were then developed to allow measurement of the different mineralocorticoid metabolites in several biological fluids. Using these methods biological markers that contribute to the differential diagnosis between benign and malignant adrenal tumors were identified. In the present paper we report that the exploration of the entire mineralocorticoid pathway in the plasma of patients during basal state and after stimulation and/or inhibition test is a powerful tool to predict or validate diagnosis of adrenal malignancy. Moreover, mineralocorticoid exploration can help differentiate between two different types of malignancy, ie malignant cortical adrenaloma and metastases of other cancer. The biochemical mechanisms leading to the atypical mineralocorticoid metabolism in the case of malignant cortical adrenaloma are now under study.

Hypoaldosteronism accompanied by normal or elevated mineralocorticosteroid pathway steroid: a marker of adrenal carcinoma.

In order to find a biochemical marker to assist the physician in the difficult differential diagnosis between malignant and nonmalignant adrenal tumors, plasma levels of the mineralocorticosteroids (deoxycorticosterone, 18-hydroxydeoxycorticosterone, corticosterone, 18-hydroxycorticosterone, and aldosterone) were determined. The same method (RIA which is preceded by a crucial separation step) was used to measure all these steroids including aldosterone. The subjects included 15 adults presenting various clinical signs of adrenocortical tumors (histopathologically: 6 with adrenal carcinoma, 1 with a history of adrenal carcinoma, 1 with adrenal metastasis from other forms of cancer, 6 with adenoma, and 1 with hyperplasia). The results show that both presurgery and during a recurrence of adrenal carcinoma, hypoaldosteronism occurs which contrasts with the normal or even elevated levels of some aldosterone precursors. In the 7 cases of adrenal cortical carcinoma, this dysfunction of the aldosterone pathway was detected regardless of the impairment of the other steroidogenesis pathways, whereas it was never found with a nonmalignant tumor. Despite the limited number of cases so far available, these findings suggest that detection of abnormalities of the aldosterone pathway, and particularly the detection of hypoaldosteronism by an assay method involving a crucial steroid separating step, could contribute to a differential diagnosis between benign and malignant adrenocortical tumor and between adrenal metastasis and other forms of cancer.

Cortisol and testosterone: inhibitory agents of the mineralocorticoid pathway. Is there a physiopathological meaning in adrenal tumors?

The authors incubated adrenal mitochondria to study the in vitro action of cortisol and testosterone on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that cortisol at concentrations of 5 x 10(-6) and 10(-4) M inhibit the conversion of corticosterone into aldosterone by 23.6 to 90%; testosterone 5 x 10(-5) and 10(-4) M inhibit the reaction by 78.4 and 87.2%, respectively. The inhibition of the conversion of 18-hydroxycorticosterone into aldosterone is 12.5 to 91% by cortisol with concentrations ranging from 5 x 10(-7) to 5 x 10(-5) M and testosterone 5 x 10(-5) and 10(-4) M inhibits the reaction by 87.3 and 91%, respectively. Aldosterone (10(-8) and 10(-6) M) does not inhibit aldosterone biosynthesis from corticosterone or 18-hydroxycorticosterone. It thus appears that cortisol and testosterone have an effect on the aldosterone biosynthesis pathways in mitochondria. This action may be located at the binding site of the cytochrome P450 11 beta, which catalyzes all hydroxylation steps in the mineralocorticoid biosynthesis pathway. Because cortisol and testosterone may interfere with aldosterone biosynthesis, and since functional zonation is expected in adrenal carcinomas, the presence of these steroids in substantial amounts could explain the very low plasma aldosterone level usually observed, in adrenal carcinomas studied in our laboratory.

[Organ transplantation. One of the biochemical reasons for the race against the clock]. / Transplantations d'organes. Une des raisons biochimiques de la course contre la montre.

The authors have studied the subcellular functioning of human adrenal glands removed from subjects in a stage IV coma. The present study has a two-fold interest: on the one hand, it offers biochemical information on a key element in the intermediate metabolism (namely, the mitochondrial energetic metabolism, occurring in a fragile tissue which, under a state of shock, is primarily affected; the results obtained on such type of tissue may therefore be inferred to other organs); on the other hand, it allows a wider approach of the adrenal biochemical mechanisms during a stage IV coma. The mitochondrial fraction was obtained by differential centrifugation carried out immediately after organ removal. The steroid synthesis, studied using radioactive precursors, turned out to be similar to that found in other mammals. Respiratory characteristics, determined by polarography with a Clark oxygen electrode, at 37 degrees C, were satisfactory: respiratory intensity was 77.25 +/- 12.16 nanomoles O2/min/mg mitochondrial protein in the presence of succinate 15 mM and respiratory control was 1.93 +/- 0.15 in the presence of ADP 37 microM. The respiratory chain functioned in a classical manner: rotenone 25 microM did not inhibit respiration in the presence of succinate 15 mM, while it did with L-malate 15 mM. In the presence of succinate 15 mM, the respiratory intensity was inhibited at 87.4 percent and 76.7 percent by KCN 0.01 microM and antimycin A 0.09 microM respectively; with DNP 85 microM, it was multiplied by 5. However, the value of the P/O ratio was low (0.24 +/- 0.04). Under the present conditions, this may highlight the difficulty to synthetize ATP whenever neither the functioning nor the regulation of the multi-enzymatic complex accounting for oxygen consumption are affected. This result clearly confirms that the shortest possible delay between organ removal and transplantation is crucial, as the renewal of cell structures requires energy. These fundamental research studies account for the major concerns of reanimation teams. This also raises the issue of the role of fundamental researchers within a transplant surgery team.

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria.

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c1. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we notices a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.