Primary Aldosteronism: Changing Definitions and New Concepts of Physiology and Pathophysiology Both Inside and Outside the Kidney.

In the 60 years that have passed since the discovery of the mineralocorticoid hormone aldosterone, much has been learned about its synthesis (both adrenal and extra-adrenal), regulation (by renin-angiotensin II, potassium, adrenocorticotrophin, and other factors), and effects (on both epithelial and nonepithelial tissues). Once thought to be rare, primary aldosteronism (PA, in which aldosterone secretion by the adrenal is excessive and autonomous of its principal regulator, angiotensin II) is now known to be the most common specifically treatable and potentially curable form of hypertension, with most patients lacking the clinical feature of hypokalemia, the presence of which was previously considered to be necessary to warrant further efforts towards confirming a diagnosis of PA. This, and the appreciation that aldosterone excess leads to adverse cardiovascular, renal, central nervous, and psychological effects, that are at least partly independent of its effects on blood pressure, have had a profound influence on raising clinical and research interest in PA. Such research on patients with PA has, in turn, furthered knowledge regarding aldosterone synthesis, regulation, and effects. This review summarizes current progress in our understanding of the physiology of aldosterone, and towards defining the causes (including genetic bases), epidemiology, outcomes, and clinical approaches to diagnostic workup (including screening, diagnostic confirmation, and subtype differentiation) and treatment of PA.

Measurement of Equilibrium Angiotensin II in the Diagnosis of Primary Aldosteronism.

BACKGROUND: Many medications (including most antihypertensives) and physiological factors affect the aldosterone/renin ratio (ARR) when screening for primary aldosteronism (PA). We sought to validate a novel equilibrium angiotensin II (eqAngII) assay and compare correlations between the aldosterone/angiotensin II ratio (AA2R) and the current ARR under conditions affecting the renin-angiotensin system. METHODS: Among 78 patients recruited, PA was excluded in 22 and confirmed in 56 by fludrocortisone suppression testing (FST). Peripheral levels of eqAngII, plasma renin activity (PRA) and direct renin concentration (DRC) were measured. RESULTS: EqAngII showed good consistency with DRC and PRA independent of PA diagnosis, posture, and fludrocortisone administration. EqAngII showed close (P < 0.01) correlations with DRC (r = 0.691) and PRA (r = 0.754) during FST. DRC and PRA were below their assays' functional sensitivity in 43.9% and 15.1%, respectively, of the total 312 samples compared with only 7.4% for eqAngII (P < 0.01). Bland-Altman analysis revealed an overestimation of PRA and DRC compared with eqAngII in a subset of samples with low renin levels. The AA2R showed not only consistent changes with the ARR but also close (P < 0.01) correlations with the ARR, whether renin was measured by DRC (r = 0.878) or PRA (r = 0.880). CONCLUSIONS: Dynamic changes of eqAngII and the AA2R show good consistency and close correlations with renin and the ARR. The eqAngII assay shows better sensitivity than DRC and PRA assays, especially at low concentrations. Whether the AA2R can reduce the impact of some factors that influence the diagnostic power of the ARR warrants further study.

Pathogenesis of Familial Hyperaldosteronism Type II: New Concepts Involving Anion Channels.

PURPOSE OF REVIEW: The application of advanced genetic techniques has recently begun to unravel the genetic basis for familial primary aldosteronism type 2 (FH-II). RECENT FINDINGS: Whole-exome sequencing in a large family with FH-II revealed a shared rare damaging heterozygous variant in CLCN2 (chr.3: g.184075850C>T, p.Arg172Gln) in three severely affected members. The gene encodes a chloride channel, ClC-2. A cohort of 80 unrelated individuals diagnosed with early-onset primary aldosteronism was also examined for CLCN2 mutations finding three further occurrences of p.Arg172Gln mutations and four single cases of other potentially damaging heterozygous mutations for an overall prevalence of 9.9%. A concurrent report also found a different CLCN2 mutation (p.Gly24Asp) in a single severely affected patient from a cohort of 12 with early-onset PA for a prevalence of 8.3%. Cases of primary aldosteronism associated with CLCN2 mutations appear to be bilateral and respond well to medical treatment. In the adrenal, ClC-2 has been demonstrated to localize predominantly to the zona glomerulosa (ZG), and functional analysis suggests that mutations in ClC-2 predispose ZG cells to depolarization, thus leading to calcium influx via activation of voltage-gated calcium channels and increased aldosterone production. Germline CLCN2 mutations appear to account for a substantial proportion of early-onset primary aldosteronism cases, and genetic testing for mutations in this gene should be considered in appropriate cases.

Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

In Primary Aldosteronism, Mineralocorticoids Influence Exosomal Sodium-Chloride Cotransporter Abundance.

Distal tubular sodium retention is a potent driver of hypertension, and the thiazide-sensitive sodium-chloride cotransporter (NCC) has a key role in this process. In humans, factors regulating NCC are unclear, but in animal models, aldosterone is a potent regulator, possibly via effects on plasma potassium. We studied the effects of the mineralocorticoid fludrocortisone on the abundance of NCC and its phosphorylated form (pNCC) as well as WNK lysine deficient protein kinase 4 (WNK4) and STE20/SPS1-related, proline alanine-rich kinase (SPAK) in human urinary exosomes. We isolated exosomes from daily urine samples in 25 patients undergoing fludrocortisone suppression testing (100 µg every 6 hours for 4 days) to diagnose or exclude primary aldosteronism. Over the course of the test, NCC levels increased 3.68-fold (P<0.01) and pNCC levels increased 2.73-fold (P<0.01) relative to baseline. The ratio of pNCC/NCC dropped by 48% (P<0.01). The abundance of WNK4 increased 3.23-fold (P<0.01), but SPAK abundance did not change significantly (P=0.14). Plasma potassium concentration strongly and negatively correlated with pNCC, NCC, and WNK4 abundance (P<0.001 for all). This study shows that, in humans, mineralocorticoid administration is associated with a rapid increase in abundance of NCC and pNCC, possibly via the WNK pathway. These effects may be driven by changes in plasma potassium.

Does ACTH improve the diagnostic performance of adrenal vein sampling for subtyping primary aldosteronism?

OBJECTIVE: Adrenal vein sampling (AVS) is used for determining treatment options for primary aldosteronism (PA), but is a difficult procedure. Adrenocorticotropic hormone (ACTH) infusion or bolus has been reported to improve AVS success rates by increasing cortisol secretion, but effects on lateralization are controversial. We therefore assessed the effects of ACTH in regard to AVS success and lateralization in our unit, after a change in protocol to ACTH-stimulated AVS. SETTING: AVS was performed after overnight recumbency in patients with PA confirmed by fludrocortisone suppression testing. Bilateral sequential sampling was performed before and after an intravenous bolus of 250 mcg of ACTH. Lateralization was defined as an aldosterone/cortisol ratio in one adrenal vein at least twice peripheral, combined with a contralateral adrenal ratio no higher than peripheral (contralateral suppression). RESULTS: In 47 AVS procedures, the median adrenal/peripheral cortisol gradient increased on the left (11·6 vs 18·2 µg/100 ml, P < 0·001) and right (15·6 vs 31·5 µg/100 ml, P < 0·001) after ACTH. A total of 34 of 47 studies were diagnostic pre-ACTH (six failing because of low aldosterone levels bilaterally and seven failing to cannulate one or both sides) vs 44 of 47 (P = 0·011) studies diagnostic post-ACTH (failure to cannulate one or both sides in 3). Concordance between diagnostic studies pre- and post-ACTH was 91%, but two bilateral cases became unilateral after ACTH and one unilateral case before ACTH was bilateral afterwards. CONCLUSIONS: ACTH improved cortisol gradients and aldosterone secretion, resulting in a reduction in the proportion of nondiagnostic studies. There was a low proportion of discordance between pre- and post-ACTH diagnoses, the significance of which is unclear.

Does concomitant autonomous adrenal cortisol overproduction have the potential to confound the interpretation of adrenal venous sampling in primary aldosteronism?

OBJECTIVE: Demonstration of unilateral aldosterone production by adrenal venous sampling (AVS) is required to select appropriate candidates for adrenalectomy in patients with primary aldosteronism (PA). During AVS, aldosterone and cortisol levels are measured to assess successful cannulation and lateralization. In patients with aldosterone-producing adenoma (APA), concurrent autonomous cortisol secretion might confound AVS results. DESIGN AND PATIENTS: We retrospectively examined results in eight patients with cortisol-producing adenoma (CPA), but without PA, who underwent AVS. RESULTS: In all eight, cortisol was higher on the CPA side than contralateral (CL) (median 6·7-fold [range 2·4-27·2]; P = 0·012]). By cortisol criteria, CL catheter placement would have been labelled inadequate in six despite adrenal venous aldosterone levels markedly higher than peripheral (41·6-fold [7·2-510·5]; P < 0·001), suggesting successful cannulation. In all eight, adrenal venous aldosterone/cortisol (A/C) ratios on the CL side were indicative of increased aldosterone production (≥2 times peripheral), but in only three patients on the CPA side (difference CL side 44·5-fold [6·0-109·0] vs CPA side 1·65-fold [1·0-23·0]; P = 0·017). A/C ratios were higher on the CL vs the CPA side in seven (20·0-fold [4·7-76·0]). CONCLUSION: These results in patients with CPA suggest that in patients with APA, concurrent autonomous unilateral cortisol hypersecretion could confound AVS accuracy by increasing cortisol levels (reducing A/C ratio) on the CPA side, while reducing levels (increasing A/C ratio and suggesting failed cannulation) on the CL side. Misclassification of PA subtype or repeat AVS could result, underscoring the importance of adequately assessing cortisol production prior to AVS and the need to consider alternatives.

Should aldosterone suppression tests be conducted during a particular phase of the menstrual cycle, and, if so, which phase? Results of a preliminary study.

BACKGROUND: As renin and aldosterone levels vary during the menstrual cycle, and are critical criteria for interpretation of aldosterone suppression tests to confirm or exclude primary aldosteronism, outcome of testing may vary depending on the menstrual cycle phase. We assessed the effect of timing within the menstrual cycle on levels of renin, aldosterone and female sex steroids during fludrocortisone suppression testing (FST). METHODS: In 22 women undergoing FST who experienced regular menstrual cycles, renin (measured as both plasma renin activity and direct renin concentration), aldosterone (mass spectrometry) and cortisol, progesterone, oestradiol, LH and FSH (immunoassay) levels were compared, relative to phase of cycle. Aldosterone levels were compared to those in age-matched males undergoing FST. RESULTS: Progesterone (P < 0·0001) and aldosterone (P = 0·006) levels were higher in nine women (after one of 10 was excluded with anovulatory cycle) studied during the luteal phase than in the 12 studied during the follicular phase. All studied during the luteal phase had positive FST, and all three with negative FST were studied during the follicular phase. There were no significant differences in other parameters measured except FSH, which was higher (P = 0·02) during the follicular phase. Aldosterone was higher (P = 0·01) in women studied in the luteal (but not follicular) phase compared to men. CONCLUSION: The menstrual cycle may affect the outcome of FST and other suppression testing used to diagnose primary aldosteronism. Larger patient numbers and preferably restudy of the same patient in both phases should clarify this and determine the optimum time in the cycle for testing.

A systematic review and meta-analysis of thiazide-induced hyponatraemia: time to reconsider electrolyte monitoring regimens after thiazide initiation?

AIMS: Hyponatraemia is one of the major adverse effects of thiazide and thiazide-like diuretics and the leading cause of drug-induced hyponatraemia requiring hospital admission. We sought to review and analyze all published cases of this important condition. METHODS: Ovid Medline, Embase, Web of Science and PubMed electronic databases were searched to identify all relevant articles published before October 2013. A proportions meta-analysis was undertaken. RESULTS: One hundred and two articles were identified of which 49 were single patient case reports. Meta-analysis showed that mean age was 75 (95% CI 73, 77) years, 79% were women (95% CI 74, 82) and mean body mass index was 25 (95% CI 20, 30) kg m(-2) . Presentation with thiazide-induced hyponatraemia occurred a mean of 19 (95% CI 8, 30) days after starting treatment, with mean trough serum sodium concentration of 116 (95% CI 113, 120) mm and serum potassium of 3.3 (95% CI 3.0, 3.5) mm. Mean urinary sodium concentration was 64 mm (95% CI 47, 81). The most frequently reported drugs were hydrochlorothiazide, indapamide and bendroflumethiazide. CONCLUSIONS: Patients with thiazide-induced hyponatraemia were characterized by advanced age, female gender, inappropriate saliuresis and mild hypokalaemia. Low BMI was not found to be a significant risk factor, despite previous suggestions. The time from thiazide initiation to presentation with hyponatraemia suggests that the recommended practice of performing a single investigation of serum biochemistry 7-14 days after thiazide initiation may be insufficient or suboptimal. Further larger and more systematic studies of thiazide-induced hyponatraemia are required.

Reninoma: the importance of renal vein renin ratios for lateralisation and diagnosis.

BACKGROUND/AIM: Reninomas are rare juxtaglomerular tumours which can cause severe hypertension and hypokalaemia. Diagnosis can be problematic and these tumours can be difficult to locate on imaging. In this report we aim to demonstrate the value of carefully performed renal vein renin ratios (RVRRs) to assist in locating these tumours. METHOD/RESULTS: We report on 3 patients diagnosed with reninoma in our unit. The patients were all female, young (17, 16 and 30 years), severely hypertensive and hypokalaemic (2.5, 2.5 and 3.1 mmol/l). Plasma renin activity (PRA) was elevated (31.9, 274 and 175 ng/ml/h), and aldosterone was high-normal (19.9 ng/dl) or elevated (207 and 109.3 ng/dl). Renal artery stenosis was excluded by renal artery Doppler, DTPA scan and angiography. Renal CT detected the lesion in 2 patients, with one lesion visible on pre- and post-contrast CT and the other on post-contrast CT only. RVRRs were performed several weeks after withdrawing interfering medications, maintaining a <40 mmol/day low-sodium diet and maintaining recumbency overnight the night before and during the procedure. Ratios before and after captopril or enalaprilat administration were obtained and lateralised the tumours in all 3 cases (dominant/non-dominant ratios of 2.3, 4.3 and 3.8). All of the patients underwent nephrectomy yielding a typical juxtaglomerular tumour and resulting in cure of hypertension and hypokalaemia. CONCLUSIONS: Reninoma should be suspected in young hypertensives (especially females) with significant hypokalaemia and high PRA or direct renin concentration after renovascular hypertension has been excluded. CT imaging and carefully performed RVRRs provide the highest likelihood of locating these tumours.

Detection of mutations in KLHL3 and CUL3 in families with FHHt (familial hyperkalaemic hypertension or Gordon's syndrome).

The study of families with rare inherited forms of hypo- and hyper-tension has been one of the most successful strategies to probe the molecular pathophysiology of blood pressure control and has revealed dysregulation of distal nephron Na+ reabsorption to be a common mechanism. FHHt (familial hyperkalaemic hypertension; also known as Gordon's syndrome) is a salt-dependent form of hypertension caused by mutations in the regulators of the thiazide-sensitive Na+-Cl- co-transporter NCC [also known as SLC12A3 (solute carrier family 12 member 3)] and is effectively treated by thiazide diuretics and/or dietary salt restriction. Variation in at least four genes can cause FHHt, including WNK1 [With No lysine (=K) 1] and WNK4, KLHL3 (kelch-like family member 3), and CUL3 (cullin 3). In the present study we have identified novel disease-causing variants in CUL3 and KLHL3 segregating in 63% of the pedigrees with previously unexplained FHHt, confirming the importance of these recently described FHHt genes. We have demonstrated conclusively, in two unrelated affected individuals, that rare intronic variants in CUL3 cause the skipping of exon 9 as has been proposed previously. KLHL3 variants all occur in kelch-repeat domains and so probably disrupt WNK complex binding. We have found no evidence of any plausible disease-causing variants within SLC4A8 (an alternative thiazide-sensitive sodium transporter) in this population. The results of the present study support the existing evidence that the CUL3 and KLHL3 gene products are physiologically important regulators of thiazide-sensitive distal nephron NaCl reabsorption, and hence potentially interesting novel anti-hypertensive drug targets. As a third of our non-WNK FHHt families do not have plausible CUL3 or KLHL3 variants, there are probably additional, as yet undiscovered, regulators of the thiazide-sensitive pathways.

The Cl-/HCO3- exchanger pendrin is downregulated during oral co-administration of exogenous mineralocorticoid and KCl in patients with primary aldosteronism.

In primary aldosteronism (PA), the occurrence of K+ loss and hypertension suggest alterations in renal tubular transport, but the molecular basis of these alterations in humans is unclear. In this study, urinary extracellular vesicles (uEVs) isolated from patients undergoing fludrocortisone suppression testing (FST, as a means of confirming or excluding PA) were analyzed using mass spectrometry-based proteomics to determine the combined effects of an aldosterone analogue, NaCl and KCl supplementation on renal tubular protein abundance. Of quantified proteins, the Cl-/HCO3- exchanger pendrin decreased by a median 37% [-15, 57] (P < 0.01) and the potassium channel ROMK increased by a median 31% [-10, 85] (P < 0.01) during FST among 10 PA subjects. The trends remained, but to a lesser degree, in two subjects cured of PA by unilateral adrenalectomy. In PA subjects, plasma K+ increased from median 3.6 to 4.2 mM (P < 0.01) and 24 h urine K+ from 101 to 202 mmol (P < 0.01), while 24 h urine Na+/K+ decreased from 2.3 to 0.8 (P < 0.01). At baseline, pendrin negatively correlated with plasma K+ (P < 0.05) and positively correlated with plasma aldosterone (P < 0.01). There were no clear correlations between Δ pendrin (Δ = D4-D0) and changes in blood or urine variables, and no correlations between ROMK in any of the blood or urine variables either at baseline or during FST. We conclude that oral co-administration of mineralocorticoid and KCl in PA patients is associated with reduced pendrin and enhanced ROMK in uEVs. Pendrin reduction during FST suggests that the suppressive effects of oral KCl may outweigh pendrin upregulation by mineralocorticoids.

Evaluating the Diversity of Emergency Medicine Foundation (EMF) Grant Recipients in the Last Decade.

INTRODUCTION: To study diversity of researchers and barriers to success among Emergency Medicine Foundation (EMF) grant recipients in the last 10 years. METHODS: EMF grant awardees were approached to complete a brief survey, which included demographics, queries related to contributions to the literature, success in obtaining grants, and any perceived barriers they encountered. RESULTS: Of the 342 researchers contacted by email, a total of 147 completed the survey for a response rate of 43%. The respondents were predominately mid to late career white-male-heterosexual-Christian with an average age of 44 years (range 25-69 years of age). With regards to training and education, the majority of respondents (50%) were either Associate or Professor clinical rank (8% instructor/resident/fellow and 31% Assistant). Sixty-two percent of the respondents reported perceived barriers to career advancement since completion of residency. The largest perceived barrier to success was medical specialty (26%), followed by gender (21%) and age (16%). CONCLUSION: Our survey of EMF grant recipients in the last 10 years shows a considerable lack of diversity. The most commonly perceived barriers to career advancement by this cohort were medical specialty, gender, and age. An opportunity exists for further definition of barriers and development of mechanisms to overcome them, with a goal of increased success for those that are underrepresented.

Biochemical, Histopathological, and Genetic Characterization of Posture-Responsive and Unresponsive APAs.

CONTEXT AND OBJECTIVE: Posture-responsive and posture-unresponsive aldosterone-producing adenomas (APAs) account for approximately 40% and 60% of APAs, respectively. Somatic gene mutations have been recently reported to exist in approximately 90% of APAs. This study was designed to characterize the biochemical, histopathologic, and genetic properties of these 2 types of APA. METHODS: Plasma levels of aldosterone and hybrid steroids (18-oxocortisol and 18-hydroxycortisol) were measured by liquid chromatography-tandem mass spectrometry. Immunohistochemistry for CYP11B2 (aldosterone synthase) and CYP17A1 (17α-hydroxylase) and deoxyribonucleic acid sequencing (Sanger and next-generation sequencing) were performed on APA tissue collected from 23 posture-unresponsive and 17 posture-responsive APA patients. RESULTS: Patients with posture-unresponsive APA displayed higher (P < 0.01) levels of hybrid steroids, recumbent aldosterone and cortisol, larger (P < 0.01) zona fasciculata (ZF)-like tumors with higher (P < 0.01) expression of CYP17A1 (but not of CYP11B2) than patients with posture-responsive APA (most of which were not ZF-like). Of 40 studied APAs, 37 (92.5%) were found to harbor aldosterone-driving somatic mutations (KCNJ5 = 14 [35.0%], CACNA1D = 13 [32.5%], ATP1A1 = 8 [20.0%], and ATP2B3 = 2 [5.0%]), including 5 previously unreported mutations (3 in CACNA1D and 2 in ATP1A1). Notably, 64.7% (11/17) of posture-responsive APAs carried CACNA1D mutations, whereas 56.5% (13/23) of posture-unresponsive APAs harbored KCNJ5 mutations. CONCLUSIONS: The elevated production of hybrid steroids by posture-unresponsive APAs may relate to their ZF-like tumor cell composition, resulting in expression of CYP17A1 (in addition to somatic gene mutation-driven CYP11B2 expression), thereby allowing production of cortisol, which acts as the substrate for CYP11B2-generated hybrid steroids.

Diagnosis of Primary Aldosteronism by Seated Saline Suppression Test-Variability Between Immunoassay and HPLC-MS/MS.

BACKGROUND: In primary aldosteronism (PA), excessive, autonomous secretion of aldosterone is not suppressed by salt loading or fludrocortisone. For seated saline suppression testing (SSST), the recommended diagnostic cutoff 4-hour plasma aldosterone concentration (PAC) measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS is 162 pmol/L. Most diagnostic laboratories, however, use immunoassays to measure PAC. The cutoff for SSST using immunoassay is not known. We hypothesized that the cutoff is different between the assays. METHODS: We analyzed 80 of the 87 SSST tests that were performed during our recent study defining the HPLC-MS/MS cutoff. PA was confirmed in 65 by positive fludrocortisone suppression testing (FST) and/or lateralization on adrenal venous sampling and excluded in 15 by negative FST. PAC was measured by a chemiluminescence immunoassay (PACIA) in the SSST samples using the DiaSorin Liaison XL analyzer, and receiver operating characteristics (ROC) analysis was performed to identify the PACIA cutoff. RESULTS: ROC revealed good performance (area under the curve = 0.893; P < .001) of 4-hour postsaline PACIA for diagnosis of PA and an optimal diagnostic cutoff of 171 pmol/L, with sensitivity and specificity of 95.4% and 80.0%, respectively. A higher cutoff of 217 pmol/L improved specificity (86.7%) with lower sensitivity (86.2%). PACIA measurements strongly correlated with PAC measured by HPLC-MS (r = 0.94, P < .001). CONCLUSIONS: A higher diagnostic cutoff for SSST should be employed when PAC is measured by immunoassay rather than HPLC-MS/MS. The results suggest that (i) PA can be excluded if 4-hour PACIA is less than 171 pmol/L, and (ii) PA is highly likely if the PACIA is greater than 217 pmol/L by chemiluminescence immunoassay. A gray zone exists between the cutoffs of 171 and 217 pmol/L, likely reflecting a lower specificity of immunoassay.

Measurement of aldosterone in human plasma by semiautomated HPLC-tandem mass spectrometry.

BACKGROUND: Reliable measurement of aldosterone with less interlaboratory variation than RIA would help standardize testing for primary aldosteronism. We set out to validate a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for aldosterone in human plasma. METHODS: We prepared samples (EDTA plasma, lithium heparin plasma, and serum from separator and plain clot tubes) and measured aldosterone using online HPLC-MS/MS with d(7)-aldosterone as internal standard. We also analyzed EDTA plasma samples by immunoassay. We established a reference range for HPLC-MS/MS aldosterone by analyzing blood collected midmorning from 97 normotensive seated subjects. RESULTS: The linear range was 69.4-5548.0 pmol/L (2.5-200 ng/dL) (r(2) > 0.994, n = 14). Inter- and intraday analytical recovery and imprecision for quality control samples of 166.4, 1109.6, and 4161.0 pmol/L (6.0, 40.0, and 150.0 ng/dL) were 92.2%-102.0% and <6.3%, respectively (n = 5). The lower limit of quantification was 69.4 pmol/L (2.5 ng/dL), with inter- and intraday analytical recovery and imprecision of 91.4%-94.5% and <9.5% (n = 5). No interferences were observed in plasma from Addison's disease patients (n = 5). Comparison of collection tubes, using EDTA as the reference, revealed similar aldosterone results. Comparison of HPLC-MS/MS with immunoassay gave an acceptable mean bias (0.83%) but wide range (-44.8% to 39.7%) of differences. HPLC-MS/MS aldosterone concentrations in normotensive subjects ranged from <69.4 to 635.2 pmol/L (<2.5 to 22.9 ng/dL). CONCLUSIONS: This first reported aldosterone method using online HPLC-MS/MS is precise across the clinically relevant range, not influenced by collection tube type, and offers semiautomated sample preparation and high throughput.

Further evidence for linkage of familial hyperaldosteronism type II at chromosome 7p22 in Italian as well as Australian and South American families.

BACKGROUND: Familial hyperaldosteronism type II is a hereditary form of primary aldosteronism not attributable to the hybrid CYP11B1/CYP11B2 mutation that causes glucocorticoid remediable aldosteronism (or familial hyperaldosteronism type I). Although genetic defect(s) underlying familial hyperaldosteronism type II have not yet been elucidated, linkage to chromosome 7p22 was previously reported in two Australian families and a South American family with familial hyperaldosteronism type II. OBJECTIVE: To seek evidence of linkage to chromosome 7p22 in two Italian families with familial hyperaldosteronism type II based on markers that have already yielded evidence of linkage in one South American and two Australian familial hyperaldosteronism type II families and to assess the combined multipoint logarithm of odds score in these five families (two Australian, two Italian, and one South American). METHODS: Primary aldosteronism was diagnosed or excluded using widely accepted clinical and biochemical criteria. Genotypes of affected and unaffected Italian patients from two families were analysed using seven closely spaced microsatellite markers at 7p22, and multipoint logarithm of odds scores were calculated to assess linkage with familial hyperaldosteronism type II. RESULTS: All known affected individuals (four and two, respectively) from each of two Italian families shared identical haplotypes for the seven markers, consistent with linkage of the disease locus with the 7p22 region. The combined multipoint logarithm of odds score for five families showing linkage at 7p22 was highly significant at 5.22 (theta = 0) for markers D7S462 and D7S517. CONCLUSION: Linkage in two Italian families makes this the third geographical area to show linkage of familial hyperaldosteronism type II at 7p22, emphasizing the likely importance of this locus in identifying the causative mutation.

Aldosterone LC-MS/MS Assay-Specific Threshold Values in Screening and Confirmatory Testing for Primary Aldosteronism.

Context: Current threshold values for primary aldosteronism (PA) diagnostic testing are based on measuring aldosterone (PAC) using immunoassays. Quantification of PAC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) yields lower values. Objective: To compare aldosterone measurement by radioimmunoassay (RIA) with LC-MS/MS and evaluate performances of proposed LC-MS/MS-specific cutoffs for PA screening and confirmatory testing. Patients and Intervention: Forty-one patients underwent aldosterone/renin ratio (ARR) testing to screen for, and fludrocortisone suppression testing (FST) to confirm or exclude, PA. Renin (DRC) was measured by chemiluminescent immunoassay. Results: Median serum PACLC-MS/MS was 27.8% lower (P < 0.05) than plasma PACRIA in 164 pairs of FST samples. A positive correlation (Spearman coefficient, 0.894, P < 0.01; Pearson r coefficient, 0.861, P < 0.01) was observed between the two assays. Thirty-seven patients showed consistent FST diagnoses (29 positive, 8 negative), whereas four showed inconsistent FSTs by the two assays. Good agreement (κ coefficient, 0.736; P < 0.01) was observed between the current FST diagnostic PACRIA cutoff of 165 pmol/L and the proposed PACLC-MS/MS cutoff of 133 pmol/L. Among 37 patients with consistent FST results, no differences were observed in sensitivity (89.7% vs 93.1%) or specificity (87.5% vs 87.5%) for PA screening between the current ARR cutoff of 70 pmol/mU (PACRIA/DRC) and the proposed cutoff of 55 pmol/mU (PACLC-MS/MS/DRC). Conclusions: Adjustment of the current cutoffs for PA diagnostic testing is necessary if PAC is measured by LC-MS/MS. Our preliminary results suggest that the proposed LC-MS/MS cutoffs for ARR and FST perform as well as current RIA cutoffs.

Comparison of Seated With Recumbent Saline Suppression Testing for the Diagnosis of Primary Aldosteronism.

Context: Failure of plasma aldosterone suppression during fludrocortisone suppression testing (FST) or saline suppression testing (SST) confirms primary aldosteronism (PA). Aldosterone is often higher upright than recumbent in PA; upright levels are used during FST. In a pilot study (24 patients with PA), seated saline suppression testing (SSST) was more sensitive than recumbent saline suppression testing (RSST). Objective, Design, and Patients: The current validation study involved 100 patients who underwent FST, RSST, and SSST, eight before and after unilateral adrenalectomy. Of the 108 FSTs, 73 confirmed and 18 excluded PA. Four patients with inconclusive FST lateralized on adrenal venous sampling, making a total of 77 with PA. Results: The area under the receiver operating characteristic (ROC) curve was greater for SSST than RSST (0.96 vs. 0.80; P < 0.01). ROC analysis predicted optimal cutoff aldosterone levels of 162 pmol/L for SSST and 106 pmol/L for RSST. At these cutoffs, SSST showed high sensitivity for PA (87%) that markedly exceeded that for RSST (38%; P < 0.001) but similar specificity (94 vs. 94%; not significant). SSST was more sensitive than RSST in detecting both unilateral (n = 28, 93% vs. 68%, P < 0.05) and bilateral (n = 40, 85% vs. 20%, P < 0.001) forms of PA. Only three SSST (vs. 9 RSST and 17 FST) results were inconclusive. Conclusions: SSST is highly sensitive and superior to RSST in identifying both unilateral and bilateral forms of PA and has a low rate of false positives and inconclusive results. It therefore offers a reliable and much less complicated and expensive alternative to FST for confirming PA.

Effect of Moxonidine on the Aldosterone/Renin Ratio in Healthy Male Volunteers.

Background: The most popular screening test for primary aldosteronism is the plasma aldosterone/renin ratio (ARR). Medications, dietary sodium, posture, and time of day all affect renin and aldosterone levels and can result in false-negative or false-positive ARRs if not controlled. Most antihypertensive medications affect the ARR and can interfere with interpretation of results. To our knowledge, no study has been undertaken to evaluate the effects of moxonidine on the ARR. Methods: Normotensive, nonmedicated male volunteers (n = 20) underwent measurement (seated, midmorning) of plasma aldosterone (by high-performance liquid chromatography-tandem mass spectrometry), direct renin concentration (DRC), plasma renin activity (PRA), cortisol, electrolytes and creatinine; and urinary aldosterone, cortisol, electrolytes and creatinine at baseline and after 1 week of moxonidine at 0.2 mg/d and a further 5 weeks at 0.4 mg/d. Results: Compared with baseline, despite the expected significant falls in both systolic and diastolic blood pressure, levels of plasma aldosterone [median, 134 (range, 90 to 535) pmol/L], DRC [20 (10 to 37) mU/L], PRA [2.2 (1.0-3.8) ng/mL/h], and ARR using either DRC [8.0 (4.4 to 14.4)] or PRA [73 (36 to 218)] were not significantly changed after either 1 [135 (98-550) pmol/L, 20 (11-35) mU/L, 2.0 (1.2-4.1) ng/mL/h, 8.8 (4.2 to 15.9), and 73 (32-194), respectively] or 6 weeks [130 (90-500) pmol/L, 22 (8 to 40) mU/L, 2.1 (1.0 to 3.2) ng/mL/h, 7.7 (4.3 to 22.4), and 84 (32 to 192), respectively] of moxonidine. There were no changes in any urinary measurements. Conclusion: Moxonidine was associated with no significant change in the ARR and may therefore be a good option for maintaining control of hypertension when screening for primary aldosteronism.