Steroid metabolome analysis reveals prevalent glucocorticoid excess in primary aldosteronism.

BACKGROUND: Adrenal aldosterone excess is the most common cause of secondary hypertension and is associated with increased cardiovascular morbidity. However, adverse metabolic risk in primary aldosteronism extends beyond hypertension, with increased rates of insulin resistance, type 2 diabetes, and osteoporosis, which cannot be easily explained by aldosterone excess. METHODS: We performed mass spectrometry-based analysis of a 24-hour urine steroid metabolome in 174 newly diagnosed patients with primary aldosteronism (103 unilateral adenomas, 71 bilateral adrenal hyperplasias) in comparison to 162 healthy controls, 56 patients with endocrine inactive adrenal adenoma, 104 patients with mild subclinical, and 47 with clinically overt adrenal cortisol excess. We also analyzed the expression of cortisol-producing CYP11B1 and aldosterone-producing CYP11B2 enzymes in adenoma tissue from 57 patients with aldosterone-producing adenoma, employing immunohistochemistry with digital image analysis. RESULTS: Primary aldosteronism patients had significantly increased cortisol and total glucocorticoid metabolite excretion (all P < 0.001), only exceeded by glucocorticoid output in patients with clinically overt adrenal Cushing syndrome. Several surrogate parameters of metabolic risk correlated significantly with glucocorticoid but not mineralocorticoid output. Intratumoral CYP11B1 expression was significantly associated with the corresponding in vivo glucocorticoid excretion. Unilateral adrenalectomy resolved both mineralocorticoid and glucocorticoid excess. Postoperative evidence of adrenal insufficiency was found in 13 (29%) of 45 consecutively tested patients. CONCLUSION: Our data indicate that glucocorticoid cosecretion is frequently found in primary aldosteronism and contributes to associated metabolic risk. Mineralocorticoid receptor antagonist therapy alone may not be sufficient to counteract adverse metabolic risk in medically treated patients with primary aldosteronism. FUNDING: Medical Research Council UK, Wellcome Trust, European Commission.

Mass Spectrometry-Based Adrenal and Peripheral Venous Steroid Profiling for Subtyping Primary Aldosteronism.

BACKGROUND: Differentiating patients with primary aldosteronism caused by aldosterone-producing adenomas (APAs) from those with bilateral adrenal hyperplasia (BAH), which is essential for choice of therapeutic intervention, relies on adrenal venous sampling (AVS)-based measurements of aldosterone and cortisol. We assessed the utility of LC-MS/MS-based steroid profiling to stratify patients with primary aldosteronism. METHODS: Fifteen adrenal steroids were measured by LC-MS/MS in peripheral and adrenal venous plasma from AVS studies for 216 patients with primary aldosteronism at 3 tertiary referral centers. Ninety patients were diagnosed with BAH and 126 with APAs on the basis of immunoassay-derived adrenal venous aldosterone lateralization ratios. RESULTS: Among 119 patients confirmed to have APAs at follow-up, LC-MS/MS-derived lateralization ratios of aldosterone normalized to cortisol, dehydroepiandrosterone, and androstenedione were all higher (P < 0.0001) than immunoassay-derived ratios. The hybrid steroids, 18-oxocortisol and 18-hydroxycortisol, also showed lateralized secretion in 76% and 35% of patients with APAs. Adrenal venous concentrations of glucocorticoids and androgens were bilaterally higher in patients with BAH than in those with APAs. Consequently, peripheral plasma concentrations of 18-oxocortisol were 8.5-fold higher, whereas concentrations of cortisol, corticosterone, and dehydroepiandrosterone were lower in patients with APAs than in those with BAH. Correct classification of 80% of cases of APAs vs BAH was thereby possible by use of a combination of steroids in peripheral plasma. CONCLUSIONS: LC-MS/MS-based steroid profiling during AVS achieves higher aldosterone lateralization ratios in patients with APAs than immunoassay. LC-MS/MS also enables multiple measures for discriminating unilateral from bilateral aldosterone excess, with potential use of peripheral plasma for subtype classification.

Clinical Management and Outcomes of Adrenal Hemorrhage Following Adrenal Vein Sampling in Primary Aldosteronism.

Aldosterone-producing adenoma and bilateral adrenal hyperplasia account for >90% of all primary aldosteronism cases. Distinguishing between bilateral and unilateral disease is of fundamental importance because it allows targeted therapy. Adrenal vein sampling (AVS) is the only reliable means to preoperatively differentiate between unilateral and bilateral subtypes. A rare but serious complication of AVS is an adrenal hemorrhage (AH). We retrospectively examined in detail 24 cases of AH during AVS in 6 different referral hypertension centers. AH more often affected the right adrenal (n=18) than the left (n=5, P<0.001); 1 bilateral. Median duration of experience of the radiologist in AVS at the time of AH was 5.0 years (0.6-7.8) and AH occurred with both highly experienced (>10 years) and less experienced radiologists. Of 9 patients who suffered AH in the gland contralateral to an aldosterone-producing adenoma and who underwent complete (n=6) or partial (n=3) unilateral adrenalectomy, only one required long-term corticosteroid replacement for adrenal insufficiency. No reduction in blood pressure or biochemical resolution of primary aldosteronism occurred in any of those patients who experienced AH in the gland ipsilateral to an aldosterone-producing adenoma (n=6) or who had bilateral adrenal hyperplasia (n=9). No patient required invasive treatments to control bleeding or blood transfusion. In conclusion, AH usually has a positive outcome causing either no or minor effects on adrenal function, and AVS should remain the best approach to primary aldosteronism subtype differentiation.

Genotype-Specific Steroid Profiles Associated With Aldosterone-Producing Adenomas.

Primary aldosteronism comprises 2 main subtypes: unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia. Somatic KCNJ5 mutations are found in APA at a prevalence of around 40% that drive and sustain aldosterone excess. Somatic APA mutations have been described in other genes (CACNA1D, ATP1A1, and ATP2B3) albeit at a lower frequency. Our objective was to identify genotype-specific steroid profiles in adrenal venous (AV) and peripheral venous (PV) plasma in patients with APAs. We measured the concentrations of 15 steroids in AV and PV plasma samples by liquid chromatography-tandem mass spectrometry from 79 patients with confirmed unilateral primary aldosteronism. AV sampling lateralization ratios of steroids normalized either to cortisol or to DHEA+androstenedione were also calculated. The hybrid steroid 18-oxocortisol exhibited 18- and 16-fold higher concentrations in lateralized AV and PV plasma, respectively, from APA with KCNJ5 mutations compared with all other APA combined together (P<0.001). Lateralization ratios for the KCNJ5 group were also generally higher. Strikingly, we demonstrate that a distinct steroid signature can differentiate APA genotype in AV and PV plasma. Notably, a 7-steroid fingerprint in PV plasma correctly classified 92% of the APA according to genotype. Prospective studies are necessary to translate these findings into clinical practice and determine if steroid fingerprinting could be of value to select patients with primary aldosteronism who are particularly suitable candidates for adrenal venous sampling because of a high probability of having an APA.

Coexisting Prolactinoma and Primary Aldosteronism: Is There a Pathophysiological Link?

CONTEXT: Coexisting prolactinoma-primary aldosteronism (PA) is infrequently reported. OBJECTIVE: The objective of the study was to identify patients with prolactinoma-PA and test the hypothesis that elevated prolactin (PRL) concentrations play a role in PA pathogenesis. SETTING AND DESIGN: Hyperprolactinemia/prolactinoma was diagnosed in PA patients from two referral centers (Munich, Germany, and Turin, Italy) and in essential hypertensive (EH) patients from one center (Turin). PRL receptor (PRLR) gene expression was determined by microarrays on aldosterone-producing adenomas and normal adrenals and validated by real-time PCR. H295R adrenal cells were incubated with 100 nM PRL, and gene expression levels were determined by real-time PCR and aldosterone production was quantified. RESULTS: Seven patients with prolactinoma-PA were identified: four of 584 and three of 442 patients from the Munich and Turin PA cohorts, respectively. A disproportionate number presented with macroprolactinomas (five of seven). There were five cases of hyperprolactinemia with no cases of macroprolactinoma of 14 790 patients in a general EH cohort. In a population of PA patients case-control matched 1:3 with EH patients there were two cases of hyperprolactinemia of 270 PA patients and no cases in the EH cohort (n = 810). PRLR gene expression was significantly up-regulated in the aldosterone-producing adenomas compared with normal adrenals (1.7-fold and 1.5-fold by microarray and real-time PCR, respectively). In H295R cells, PRL treatment resulted in 1.3-fold increases in CYP11B2 expression and aldosterone production. CONCLUSION: Elevated PRL caused by systemic hyperprolactinemia may contribute to the development of PA in those cases in which the two entities coexist.

Drosophila TRPM channel is essential for the control of extracellular magnesium levels.

The TRPM group of cation channels plays diverse roles ranging from sensory signaling to Mg2+ homeostasis. In most metazoan organisms the TRPM subfamily is comprised of multiple members, including eight in humans. However, the Drosophila TRPM subfamily is unusual in that it consists of a single member. Currently, the functional requirements for this channel have not been reported. Here, we found that the Drosophila TRPM protein was expressed in the fly counterpart of mammalian kidneys, the Malpighian tubules, which function in the removal of electrolytes and toxic components from the hemolymph. We generated mutations in trpm and found that this resulted in shortening of the Malpighian tubules. In contrast to all other Drosophila trp mutations, loss of trpm was essential for viability, as trpm mutations resulted in pupal lethality. Supplementation of the diet with a high concentration of Mg2+ exacerbated the phenotype, resulting in growth arrest during the larval period. Feeding high Mg2+ also resulted in elevated Mg2+ in the hemolymph, but had relatively little effect on cellular Mg2+. We conclude that loss of Drosophila trpm leads to hypermagnesemia due to a defect in removal of Mg2+ from the hemolymph. These data provide the first evidence for a role for a Drosophila TRP channel in Mg2+ homeostasis, and underscore a broad and evolutionarily conserved role for TRPM channels in Mg2+ homeostasis.