Adrenal Androgens Versus Cortisol for Primary Aldosteronism Subtype Determination in Adrenal Venous Sampling

Marianna Viukari; Eeva Kokko; Ilkka Pörsti; Helena Leijon; Tiina Vesterinen; Tero Hinkka; Minna Soinio; Camilla Schalin-Jäntti; Niina Matikainen; Pasi I. Nevalainen


Clin Endocrinol. 2022;97(3):241-249. 

In This Article

Materials and Methods

Study Subjects

This prospective pre-specified secondary endpoint analysis included 49 patients with confirmed PA, who had plasma samples available from both AVs and IVC collected during the AVS procedure (Figure S1). The study design and main outcome have been reported previously.[7] All subjects were diagnosed according to the Endocrine Society guidelines[24] and underwent AVS at Tampere University Hospital as part of the diagnostic evaluation of PA. Hypercortisolism was excluded with clinical and biochemical assessments including 1 mg overnight dexamethasone test or measurement of 24-h urinary cortisol excretion (cut-points of 100 nmol/l and 144 nmol, respectively). Further inclusion and exclusion criteria were described by Soinio et al.[7] The study protocol was approved by the ethics committee of Turku University Hospital and was undertaken in accordance with the Declaration of Helsinki. Written consent was obtained from each patient after full explanation of the purpose and nature of all procedures used. The protocol was registered in the database (NCT01567111).

Mineralocorticoid receptor antagonists (MRAs) were discontinued at least 6 weeks before the AVS procedures. Hypokalaemia was corrected by oral potassium supplementation. AVS was performed under continuous cosyntropin stimulation of 50 μg/h as described earlier.[7] A portion of plasma was immediately used for routine cortisol analysis (electrochemiluminescence, ECLIA) and aldosterone was analysed by liquid chromatography with tandem mass spectrometry (LC-MS/MS), while another sample fraction was frozen at −80°C until androstenedione and DHEA were measured with LC-MS/MS and DHEAS with immunoassay (Abbott Architect). One subject had inadequate samples for both androstenedione and DHEA analysis, and 10 subjects for DHEAS analysis.

Catheterisation was considered successful when the selectivity index (SI = AV/IVC cortisol concentrations) on both sides was ≥5. PA was classified unilateral based on a lateralisation index (LI = aldosterone/cortisol ratio between the dominant and contralateral AVs) ≥4.[11] We also calculated the contralateral suppression index (CSI = aldosterone/cortisol ratio between the nondominant AV and IVC) where values <1 suggest unilateral disease. Figure S1 illustrates the division of subjects into adrenalectomy and medical therapy groups.

Because the cut-point for cortisol-based LI remains debated,[11,25] we did a post hoc evaluation of cases in which there was discrepancy between cortisol and other adrenal androgen derived indexes. All discrepancies between indexes fell into cortisol-derived LI range of 2.00–5.88, in whom we evaluated whether the use of additional adrenal androgens would have changed the diagnosis of the subtype of PA, and further in adrenalectomized patients, related this to immunohistochemical diagnosis and the cure after adrenalectomy.

Follow-up of the Patients

Among the adrenalectomy group, postoperative information was available at 3–6 months after surgery apart from the aldosterone and renin values, which were measured at variable times from a few days to 6 months postoperatively. Long-term follow-up data collected 4.3 ± 2 years after AVS were available in all subjects of the adrenalectomy group. Surgical cure was categorized as complete, partial or absent according to the Primary Aldosteronism Surgical Outcome (PASO) criteria.[26] Since aldosterone and renin measurements were not available at follow-up, biochemical cure was classified according to plasma potassium levels.

Histological and Immunohistochemical Analysis

Diagnostic hematoxylin-and-eosin-stained adrenal slides were reviewed centrally in the Helsinki University Hospital by a single pathologist with special expertise in endocrine pathology (Helena Leijon). One or two representative blocks per case were selected with following criteria: (a) adenoma coupled with normal adrenal cortex and (b) hyperplasia presenting with a dominant nodule. Immunohistochemical labelling was performed with previously described primary antibodies CYP11B1 (11β-hydroxylase, dilution 1:5) and CYP11B2 (aldosterone synthase, dilution 1:3000).[27] Each sample was categorized as APA or aldosterone-producing nodules (APN, also described as non-APA) based on immunohistochemistry (Figure S1) as described previously.[7,28,29] Immunoreactivity was semiquantitatively assessed with H-scoring system based on the staining intensity and the percentage of stained cells as described by Nakamura et al. and McCarty et al.[28,30] CYP11B1 H-score was analysed to investigate the role of subclinical cortisol co-secretion from APAs.[31]

Statistical Analysis

Results are expressed as either number and percentage, mean and standard deviation or median and interquartile range, as appropriate. Comparisons of independent samples were performed with either independent-samples t-test or Mann–Whitney U-test, as appropriate. Wilcoxon test was used for within-patient comparisons, as no transformation of skewed variables was applied. The relationships between the SIs and LIs with cortisol, androstenedione, DHEA, DHEAS and aldosterone were assessed using two-tailed Spearman's correlation coefficients. A receiver operating characteristics (ROC) curve was constructed from the pairs of sensitivity and specificity measured for LI calculated using cortisol, androstenedione, DHEA and DHEAS. The ROC analysis was conducted using allocation to operation or medical therapy as the standard. One patient chose not to have adrenalectomy despite having lateralisation in AVS and was therefore excluded from the ROC analyses. One patient in the medical treatment group had failed cannulation according to SI with androstenedione and DHEAS and was excluded from the ROC analysis of these two hormones. SPSS Statistics (version 27 for MAC) was used for statistical analyses.