γENaC/CD9 in urinary extracellular vesicles as a potential biomarker of MR activity.

Primary aldosteronism (PA) is caused by autonomous overproduction of aldosterone, which induces organ damage directly via activation of the mineralocorticoid receptor (MR); however, no specific or sensitive biomarkers are able to reflect MR activity. Recently, it is found that urinary extracellular vesicles (uEVs) are secreted by multiple cell types in the kidney and are an enriched source of kidney-specific proteins. Here, we evaluate sodium transporters in uEVs as candidates of biomarkers of MR activity in the clinical setting. Sixteen patients were examined to determine their plasma aldosterone concentration (PAC) and renin activity, and their morning urine was collected. The protein levels of two sodium transporters in uEVs, γ-epithelial sodium channel (γENaC) and thiazide-sensitive sodium chloride cotransporter (NCC), were quantified by Western blot analysis, and their clinical correlation with PAC was determined. Consequently, we found PAC was significantly correlated with the γENaC protein level adjusted by the CD9 protein level in uEVs (correlation coefficient = 0.71). PAC was also correlated with the NCC protein level adjusted by the CD9 protein level in uEVs (correlation coefficient = 0.61). In two PA patients, treatment with an MR antagonist or adrenalectomy reduced γENaC/CD9 in uEVs. In conclusion, γENaC/CD9 in uEVs is a valuable biomarker of MR activity in PA patients and may be a useful biomarker for other MR-associated diseases.

Serum Alpha-1-Acid Glycoprotein-1 and Urinary Extracellular Vesicle miR-21-5p as Potential Biomarkers of Primary Aldosteronism.

Primary aldosteronism (PA) is the most common cause of secondary hypertension and reaches a prevalence of 6-10%. PA is an endocrine disorder, currently identified as a broad-spectrum phenotype, spanning from normotension to hypertension. In this regard, several studies have made advances in the identification of mediators and novel biomarkers of PA as specific proteins, miRNAs, and lately, extracellular vesicles (EVs) and their cargo. Aim: To evaluate lipocalins LCN2 and AGP1, and specific urinary EV miR-21-5p and Let-7i-5p as novel biomarkers for PA. Subjects and Methods: A cross-sectional study was performed in 41 adult subjects classified as normotensive controls (CTL), essential hypertensives (EH), and primary aldosteronism (PA) subjects, who were similar in gender, age, and BMI. Systolic (SBP) and diastolic (DBP) blood pressure, aldosterone, plasma renin activity (PRA), and aldosterone to renin ratio (ARR) were determined. Inflammatory parameters were defined as hs-C-reactive protein (hs-CRP), PAI-1, MMP9, IL6, LCN2, LCN2-MMP9, and AGP1. We isolated urinary EVs (uEVs) and measured two miRNA cargo miR-21-5p and Let-7i-5p by Taqman-qPCR. Statistical analyses as group comparisons were performed by Kruskall-Wallis, and discriminatory analyses by ROC curves were performed with SPSS v21 and Graphpad-Prism v9. Results: PA and EH subjects have significantly higher SBP and DBP (p <0.05) than the control group. PA subjects have similar hs-CRP, PAI-1, IL-6, MMP9, LCN2, and LCN2-MMP9 but have higher levels of AGP1 (p <0.05) than the CTL&EH group. The concentration and size of uEVs and miRNA Let-7i-5p did not show any difference between groups. In PA, we found significantly lower levels of miR-21-5p than controls (p <0.05). AGP1 was associated with aldosterone, PRA, and ARR. ROC curves detected AUC for AGP1 of 0.90 (IC 95 [0.79 - 1.00], p <0.001), and combination of AGP1 and EV-miR-21-5p showed an AUC of 0.94 (IC 95 [0.85 - 1.00], p<0.001) to discriminate the PA condition from EH and controls. Conclusion: Serum AGP1 protein was found to be increased, and miR-21-5p in uEVs was decreased in subjects classified as PA. Association of AGP1 with aldosterone, renin activity, and ARR, besides the high discriminatory capacity of AGP1 and uEV-miR-21-5p to identify the PA condition, place both as potential biomarkers of PA.

Proteomic Profile of Urinary Extracellular Vesicles Identifies AGP1 as a Potential Biomarker of Primary Aldosteronism.

CONTEXT: Primary aldosteronism (PA) represents 6% to 10% of all essential hypertension patients and is diagnosed using the aldosterone-to-renin ratio (ARR) and confirmatory studies. The complexity of PA diagnosis encourages the identification of novel PA biomarkers. Urinary extracellular vesicles (uEVs) are a potential source of biomarkers, considering that their cargo reflects the content of the parent cell. OBJECTIVE: We aimed to evaluate the proteome of uEVs from PA patients and identify potential biomarker candidates for PA. METHODS: Second morning spot urine was collected from healthy controls (n = 8) and PA patients (n = 7). The uEVs were isolated by ultracentrifugation and characterized. Proteomic analysis on uEVs was performed using LC-MS Orbitrap. RESULTS: Isolated uEVs carried extracellular vesicle markers, showed a round shape and sizes between 50 and 150 nm. The concentration of uEVs showed a direct correlation with urinary creatinine (r = 0.6357; P = 0.0128). The uEV size mean (167 ±â€…6 vs 183 ±â€…4nm) and mode (137 ±â€…7 vs 171 ±â€…11nm) was significantly smaller in PA patients than in control subjects, but similar in concentration. Proteomic analysis of uEVs from PA patients identified an upregulation of alpha-1-acid glycoprotein 1 (AGP1) in PA uEVs, which was confirmed using immunoblot. A receiver operating characteristic curve analysis showed an area under the curve of 0.92 (0.82 to 1; P = 0.0055). CONCLUSION: Proteomic and further immunoblot analyses of uEVs highlights AGP1 as potential biomarker for PA.

Higher Blood Urea Nitrogen and Urinary Calcium: New Risk Factors for Diabetes Mellitus in Primary Aldosteronism Patients.

Purpose: The aim of the study was to investigate the prevalence and risk factors of diabetes mellitus (DM) in primary aldosteronism (PA) patients. Methods: This case-control study enrolled 259 PA patients in West China Hospital, China from January 2016 to January 2019. Patients were divided into three groups: PA group, PA + impaired fasting glucose (IFG)/impaired glucose tolerance (IGT) group and PA + DM group. Clinical characteristics (like age and sex) and laboratory variables (like plasma aldosterone concentration and plasma renin activity) were compared between three groups. Univariate and multivariate logistic regression analyses were performed to determine risk factors for DM in PA patients. The association of random blood glucose with the above-mentioned factors were also investigated by Pearson correlation analyses. Nomogram model was developed to predict the probability of DM in PA patients. Results: 49 (18.9%) patients were diagnosed with DM and 22 (8.5%) with IFG/IGT in 259 PA patients. Apart from older age, male, higher body mass index, higher triglycerides and lower cholesterol, we found that higher blood urea nitrogen (BUN) and higher 24 h urinary calcium (Ca) might be potential new risk factors for dysglycemia. The nomogram model for DM in PA patients had a good predictive accuracy, with the area under the curve of receiver operating characteristic of 0.839 (95% CI 0.784-0.893). Conclusions: PA patients were more likely to have DM compared with general population. Apart from older age, overweight and dyslipidemia, higher BUN and excessive excretion of urinary Ca may also be the new potential risk factors for DM in PA patients.

Adherence to consensus guidelines for screening of primary aldosteronism in an urban healthcare system.

BACKGROUND: Primary aldosteronism is a common cause of secondary hypertension. Treatment with adrenalectomy or mineralocorticoid receptor-blockers can prevent long-term adverse outcomes. This study aimed to determine primary aldosteronism screening rats in patients with hypertension in an underserved urban healthcare system. METHODS: We reviewed records of outpatient adults in an urban healthcare system from 2013 to 2017. Chart review along with International Statistical Classification of Diseases, 9th and 10th editions, diagnosis codes were used to identify patients meeting inclusion criteria for screening according to the 2016 Endocrine Society guidelines. The corresponding aldosterone, plasma renin activity, and 24-hour urine aldosterone values were identified. Multivariate logistic regression was performed to determine positive predictors of screening. RESULTS: Of 15,511 hypertensive patients seen, 6,809 (43.8%) met criteria for screening. Blacks were the most common racial group, and Medicare and Medicaid were the most frequent insurers. The aldosterone-to-renin ratio level was checked in 86 (1.3%) patients; 22 (25.6%) had an aldosterone-to-renin ratio >20. Of the 77 patients with hypertension and incidentaloma, 14 (18.2%) had an aldosterone-to-renin ratio checked. Additional positive predictors for being screened were hypertension and hypokalemia and sustained hypertension. CONCLUSION: Screening rates for primary aldosteronism in an underserved population are low. Proper identification of primary aldosteronism in those at risk could help ameliorate long-term effects of disease.

Elevated aldosterone and blood pressure in a mouse model of familial hyperaldosteronism with ClC-2 mutation.

Gain-of-function mutations in the chloride channel ClC-2 were recently described as a cause of familial hyperaldosteronism type II (FH-II). Here, we report the generation of a mouse model carrying a missense mutation homologous to the most common FH-II-associated CLCN2 mutation. In these Clcn2R180Q/+ mice, adrenal morphology is normal, but Cyp11b2 expression and plasma aldosterone levels are elevated. Male Clcn2R180Q/+ mice have increased aldosterone:renin ratios as well as elevated blood pressure levels. The counterpart knockout model (Clcn2-/-), in contrast, requires elevated renin levels to maintain normal aldosterone levels. Adrenal slices of Clcn2R180Q/+ mice show increased calcium oscillatory activity. Together, our work provides a knockin mouse model with a mild form of primary aldosteronism, likely due to increased chloride efflux and depolarization. We demonstrate a role of ClC-2 in normal aldosterone production beyond the observed pathophysiology.

Urinary Metabolic Signature of Primary Aldosteronism: Gender and Subtype-Specific Alterations.

PURPOSE: The current clinical investigation for primary aldosteronism (PA) diagnosis requires complex expensive tests from the initial suspicion to the final subtype classification, including invasive approaches; therefore, appropriate markers for subtype definition are greatly desirable. The present study performs a metabolomics analysis to further examine specific molecular signatures of PA urines EXPERIMENTAL DESIGN: The study considered PA subtype and gender-related differences using two orthogonal advanced UHPLC-MS metabolomics approaches. Patients with essential hypertension (n = 36) and PA (n = 50) who were referred to the outpatient hypertension clinic and matched healthy subjects (n = 10) are investigated. RESULTS: Statistically significant changes (p < 0.05 ANOVA, Fc > 1.5) of metabolites involved in central carbon, energy, and nitrogen metabolism are identified, especially purine and pyrimidine nucleosides and precursors, and free amino acids. PLS-DA interpretation provides strong evidence of a disease-specific metabolic pattern with dAMP, diiodothyronine, and 5-methoxytryptophan as leading factors, and a sex-specific metabolic pattern associated with orotidine 5-phosphate, N-acetylalanine, hydroxyproline, and cysteine. The results are verified using an independent sample set, which confirms the identification of specific signatures. CONCLUSIONS AND CLINICAL RELEVANCE: Metabolomics is used to identify low molecular weight molecular markers of PA, which paves the way for follow-up validation studies in larger cohorts.

Screening of primary aldosteronism by clinical features and daily laboratory tests: combination of urine pH, sex, and serum K.

OBJECTIVE: To develop and validate a scoring system for selection of patients who should proceed to endocrinologic examinations of primary aldosteronism in newly diagnosed hypertensive patients. METHODS: A multivariate logistic regression analysis for primary aldosteronism was undertaken by use of seven possible primary aldosteronism markers, age less than 40 years, female sex, moderate-to-severe hypertension, hypokalemia, serum Na minus Cl at least 40 mmol/l, serum uric acid 237.92 µmol/l or less (4.0 mg/dl), and urine pH (U-pH) at least 7.0, in consecutive outpatients newly diagnosed with hypertension. The diagnostic criteria of primary aldosteronism were plasma aldosterone concentration-to-plasma renin activity ratio [ARR, (ng/dl)/(ng/ml per h)] at least 20 and at least one positive result in four types of challenge tests. RESULTS: Of 130 patients, 24 were diagnosed with primary aldosteronism. The area under the receiver operating characteristic curve (AUC) for a logistic model incorporating all possible primary aldosteronism markers was 0.73 [95% confidence interval (CI): 0.61-0.85]. Removing high U-pH, female sex, and hypokalemia from the full model decreased the AUC by 0.059, 0.035, and 0.011, respectively. We devised pH of urine, female sex, low serum K (PFK) score, in which one point each was assigned to high U-pH, female sex, and hypokalemia. The prevalences of primary aldosteronism in patients with 0, 1, 2, and 3 points were 11, 14, 42, and 60%, respectively. In external validation datasets (n = 106), AUC of PFK score was significantly higher than that of hypokalemia alone (0.73, 95% CI: 0.63-0.83 vs. 0.53, 95% CI: 0.44-0.63, P < 0.01). CONCLUSION: PFK score may be a better parameter than hypokalemia alone for identifying patients with a high probability of having primary aldosteronism.

DIAGNOSIS OF ENDOCRINE DISEASE: 18-Oxocortisol and 18-hydroxycortisol: is there clinical utility of these steroids?

Since the early 1980s 18-hydroxycortisol and 18-oxocortisol have attracted attention when it was shown that the urinary excretion of these hybrid steroids was increased in primary aldosteronism. The development and more widespread use of specific assays has improved the understanding of their role in the (patho)physiology of adrenal disorders. The adrenal site of synthesis is not fully understood although it is clear that for the synthesis of 18-hydroxycortisol and 18-oxocortisol the action of both aldosterone synthase (zona glomerulosa) and 17α-hydroxylase (zona fasciculata) is required with cortisol as main substrate. The major physiological regulator is ACTH and the biological activity of both steroids is very low and therefore only very high concentrations might be effective in vivo In healthy subjects, the secretion of both steroids is low with 18-hydroxycortisol being substantially higher than that of 18-oxocortisol. The highest secretion of both steroids has been found in familial hyperaldosteronism type 1 (glucocorticoid-remediable aldosteronism) and in familial hyperaldosteronism type 3. Lower but yet substantially increased secretion is found in patients with aldosterone-producing adenomas in contrast to bilateral hyperplasia in whom the levels are similar to patients with hypertension. Several studies have attempted to show that these steroids, in particular, peripheral venous plasma 18-oxocortisol, might be a useful discriminatory biomarker for subtyping PA patients. The current available limited evidence precludes the use of these steroids for subtyping. We review the biosynthesis, regulation and function of 18-hydroxycortisol and 18-oxocortisol and their potential utility for the diagnosis and differential diagnosis of patients with primary aldosteronism.

Body mass index and contralateral ratio predict outcome following unilateral adrenalectomy in primary aldosteronism.

The effect of unilateral adrenalectomy on blood pressure (BP) outcome in primary aldosteronism (PA) is diverse. Therefore, we sought to investigate the preoperative factors contributing to postoperative BP outcome. Data for 96 PA patients who underwent unilateral adrenalectomy at our institution from January 2000 to February 2015 were retrospectively collected. Based on postoperative BP after a 12-month follow-up period, the patients were categorized into two groups: cured (C) (<140/90 mm Hg with no antihypertensive drug) and not cured (NC) (if not normotensive). Patient demographics, blood and urine data, data of loading tests and adrenal vein sampling were evaluated. In all, 46 patients were categorized as C and 50 patients as NC. There were significantly more males in the NC group. Age, body mass index (BMI), number of antihypertensive drugs prescribed, serum uric acid concentration and contralateral ratio (CR) after adrenocorticotropic hormone stimulation were significantly higher in the NC group. In the multivariate model, BMI and CR significantly correlated with resolution outcome. The optimal cutoff values were 23.3 kg m-2 for BMI and 0.5 for CR, and when both parameters were used as predictors, the most optimal cutoff values for BMI and CR were 25.2 kg m-2 and 0.1, respectively. BMI and CR significantly correlated with BP outcome after adrenalectomy. To our knowledge, this is the first report to show that in addition to BMI, CR is a factor in postoperative BP outcome and to determine the optimal cutoff values of BMI and CR and calculate their sensitivities and specificities.

Diagnostic value of potassium level in a spot urine sample as an index of 24-hour urinary potassium excretion in unselected patients hospitalized in a hypertension unit.

BACKGROUND: Primary hyperaldosteronism may be associated with elevated 24-hour urinary potassium excretion. We evaluated the diagnostic value of spot urine (SU) potassium as an index of 24-hour urinary potassium excretion. METHODS: We measured SU and 24-hour urinary collection potassium and creatinine in 382 patients. Correlations between SU and 24-hour collections were assessed for potassium levels and potassium/creatinine ratios. We used the PAHO formula to estimate 24-hour urinary potassium excretion based on SU potassium level. The agreement between estimated and measured 24-hour urinary potassium excretion was evaluated using the Bland-Altman method. To evaluate diagnostic performance of SU potassium, we calculated areas under the curve (AUC) for SU potassium/creatinine ratio and 24-hour urinary potassium excretion estimated using the PAHO formula. RESULTS: Strongest correlation between SU and 24-hour collection was found for potassium/creatinine ratio (r = 0.69, P<0.001). The PAHO formula underestimated 24-hour urinary potassium excretion by mean 8.3±18 mmol/d (95% limits of agreement -28 to +44 mmol/d). Diagnostic performance of SU potassium/creatinine ratio was borderline good only if 24-hour urinary potassium excretion was largely elevated (AUC 0.802 for 120 mmol K+/24 h) but poor with lower values (AUC 0.696 for 100 mmol K+/24 h, 0.636 for 80 mmol K+/24 h, 0.675 for 40 mmol K+/24 h). Diagnostic performance of 24-hour urinary potassium excretion estimated by the PAHO formula was excellent with values above 120 mmol/d and good with lower values (AUC 0.941 for 120 mmol K+/24 h, 0.819 for 100 mmol K+/24 h, 0.823 for 80 mmol K+/24 h, 0.836 for 40 mmol K+/24 h). CONCLUSIONS: Spot urine potassium/creatinine ratio might be a marker of increased 24-hour urinary potassium excretion and a potentially useful screening test when reliable 24-hour urine collection is not available. The PAHO formula allowed estimation of the 24-hour urinary potassium excretion based on SU measurements with reasonable clinical accuracy.

Increased urinary excretion of the epithelial Na channel activator prostasin in patients with primary aldosteronism.

OBJECTIVES: Prostasin is a glycosylphosphatidylinositol-anchored serine protease that is released in urine and is involved in epithelial Na channel activation. A direct association between urinary prostasin (u-prostasin) concentration and activation of the aldosterone-driven pathway has been suggested; however, in previous studies on primary aldosteronism, a semiquantitative evaluation, rather than a precise quantification, of prostasin was performed. We aim to investigate if u-prostasin concentrations are higher in patients with primary aldosteronism than in patients with essential hypertension and whether u-prostasin measurements could be a useful marker for diagnosing primary aldosteronism in hypertensive patients. METHODS: A total of 62 primary aldosteronism and 56 essential hypertension patients were enrolled. Biochemical and hormonal parameters were measured by applying routine laboratory methods, and u-prostasin levels were assessed by ELISA. RESULTS: Primary aldosteronism patients had higher u-prostasin levels than did essential hypertension patients. Prostasin levels were positively correlated with the aldosterone-to-renin ratio and inversely correlated with plasma K and urinary Na levels. In the highest concentration quartile, u-prostasin levels were associated with a several-fold higher probability of primary aldosteronism diagnosis in hypertensive patients. Receiver operating characteristic curve analysis showed that prostasin was specific but poorly sensitive as a diagnostic marker for primary aldosteronism. CONCLUSIONS: The study shows that an elevated u-prostasin concentration in humans is a specific marker for primary aldosteronism, which involves the classical model of epithelial Na channel activation. There was no statistically significant difference in prostasin concentrations among patients with different primary aldosteronism subtypes. Studies with a larger series of patients are necessary to clarify the clinical usefulness of the prostasin assay.

Body Mass Index Predicts 24-Hour Urinary Aldosterone Levels in Patients With Resistant Hypertension.

Prospective studies indicate that hyperaldosteronism is found in 20% of patients with resistant hypertension. A small number of observational studies in normotensive and hypertensive patients suggest a correlation between aldosterone levels and obesity while others could not confirm these findings. The correlation between aldosterone levels and body mass index (BMI) in patients with resistant hypertension has not been previously investigated. Our objective was to determine whether BMI is positively correlated with plasma aldosterone concentration, plasma renin activity, aldosterone:renin ratio, and 24-hour urinary aldosterone in black and white patients. We performed a cross-sectional analysis of a large diverse cohort (n=2170) with resistant hypertension. The relationship between plasma aldosterone concentration, plasma renin activity, aldosterone:renin ratio, 24-hour urinary aldosterone, and BMI was investigated for the entire cohort, by sex and race (65.3% white, 40.3% men). We demonstrate that plasma aldosterone concentration and aldosterone:renin ratio were significantly correlated to BMI (P<0.0001) across the first 3 quartiles, but not from the 3rd to 4th quartile of BMI. Plasma renin activity was not correlated with BMI. Twenty-four-hour urinary aldosterone was positively correlated across all quartiles of BMI for the cohort (P<0.0001) and when analyzed by sex (men P<0.0001; women P=0.0013) and race (P<0.05), and stronger for men compared with women (r=0.19, P<0.001 versus r=0.05, P=0.431, P=0.028) regardless of race. In both black and white patients, aldosterone levels were positively correlated to increasing BMI, with the correlation being more pronounced in black and white men. These findings suggest that obesity, particularly the abdominal obesity typical of men, contributes to excess aldosterone in patients with resistant hypertension.

Determination of urinary aldosterone using a plasma aldosterone 2D ID LC-MS/MS method.

AIM: In the diagnosis for primary aldosteronism, the measurement of urinary aldosterone is part of the confirmation test but diagnostic accuracy may be blunted due to poor immunoassay performance for urinary aldosterone. Nowadays, plasma aldosterone concentrations are measured preferably by LC-MS/MS yet such methods for urinary aldosterone are lacking. METHODS & RESULTS: We show that plasma and urinary aldosterone can be measured with the same 2D isotope dilution LC-MS/MS method. The accuracy of the method was tested against a certified reference material. The reference values for plasma and urinary aldosterone were established. DISCUSSION & CONCLUSION: With this method, urinary aldosterone concentrations can be measured precisely, simply and accurately together with plasma samples with one set of calibration standards.

Enhanced Soluble Serum CD40L and Serum P-Selectin Levels in Primary Aldosteronism.

Primary aldosteronism (PA) is one of the most frequent forms of secondary hypertension, associated with atherosclerosis and higher risk of cardiovascular events. Platelets play a key role in the atherosclerotic process. The aim of the study was to evaluate the platelet activation by measuring serum levels of soluble CD40L (sCD40L) and P-selectin (sP-selectin) in consecutive PA patients [subgroup: aldosterone-secreting adrenal adenoma (APA) and bilateral adrenal hyperplasia (IHA)], matched with essential hypertensive (EH) patients. The subgroup of APA patients was revaluated 6-months after unilateral adrenalectomy. In all PA group, we measured higher serum levels of both sP-selectin (14.29±9.33 pg/ml) and sCD40L (9.53±4.2 ng/ml) compared to EH patients (9.39±5.3 pg/ml and 3.54±0.94 ng/ml, respectively; p<0.001). After removal of APA, PA patients showed significant reduction of blood pressure (BP) values, plasma aldosterone (PAC) levels and ARR-ratio, associated with a significant reduction of sP-selectin (16.74±8.9 pg/ml vs. 8.1±3.8 pg/ml; p<0.01) and sCD40L (8.6±1 ng/ml vs. 5.24±0.94 ng/ml; p<0.001). In PA patients, we found a significant correlation between sP-selectin and sCD40L with PAC (r=0.52, p<0.01; r=0.50, p<0.01, respectively); this correlation was stronger in APA patients (r=0.54; p<0.01 r=0.63; p<0.01, respectively). Our results showed that PA is related to platelet activation, expressed as higher plasma values of sCD40L and sP-selectin values. Surgical treatment and consequent normalization of aldosterone secretion was associated with significant reduction of sCD40L and sP-selectin values in APA patients.

Sodium interference in the determination of urinary aldosterone.

OBJECTIVES: Primary hyperaldosteronism (PHA) is one of the most common endocrine forms of secondary hypertension. Among the most used confirmatory tests for PHA is urinary aldosterone determination after oral sodium loading test. The primary aim of our study was to investigate if sodium concentrations interfere with urinary aldosterone in an automated competitive immunoassay (Liaison®) as well as to verify the manufacturer's specifications. DESIGN AND METHODS: 24-hr urine samples were collected and stored frozen until assayed. Two pools at low and high aldosterone concentrations were prepared. Verification of performance for precision was tested according to Clinical and Laboratory Standards Institute (CLSI) document EP15-A2 and interference with increasing concentrations of NaCl according to CLSI EP7-A2. RESULTS: The assay met the quality specifications according to optimal biological variation. Our results show that sodium concentrations up to 200mmol/L do not interfere on urinary aldosterone quantification, but sodium concentrations above 486mmol/L negatively interfere with the test. CONCLUSIONS: The Liaison® automated method is useful for aldosterone determination in the PHA confirmatory test, but interferences with NaCl may occur. It is therefore recommended to determine urinary NaCl before measuring urinary aldosterone to avoid falsely low results.

Urinary Aldosterone/Creatinine Ratio After Fludrocortisone Suppression Consistent with PHA in a Cat.

A 9 yr old cat was presented with clinical signs and laboratory abnormalities attributed to arterial hypertension (mean systolic arterial pressure, 290 mm Hg). Plasma aldosterone concentration was increased at the time of admission (651 pmol/L), but serum creatinine and potassium concentrations were within the reference range. A second increased aldosterone (879 pmol/L) and normal plasma renin activity (1.85 ng/mL/hr) resulted in an increased aldosterone/renin ratio, which was suggestive of primary hyperaldosteronism (PHA). To further support the diagnosis of PHA, the urinary aldosterone/creatinine ratio was calculated both before and after oral administration of fludrocortisone acetate (0.05 mg/kg q 12 hr for 4 consecutive days). The urinary aldosterone/creatinine ratio was 92.6 × 10(-9) before fludrocortisone administration and 155.8 × 10(-9) 4 days later. Absence of suppression was typical of PHA. The cat had a limited response to antihypertensive medication and died before treatment for PHA could be instituted. A necropsy was not permitted by the owner.

Laboratory challenges in primary aldosteronism screening and diagnosis.

BACKGROUND AND OBJECTIVE: The laboratory has a critical role to play in the screening and diagnosis of primary aldosteronism. This review highlights some of the important analytical considerations and the new developments in the determination of aldosterone and renin. METHODS: The review considered the published literature and clinical practice guidelines in the area of primary aldosteronism. RESULTS: A brief introduction to primary aldosteronism is provided. A detailed description of the pre-analytical, analytical and post-analytical considerations for the laboratory determination of aldosterone, renin and the aldosterone to renin ratio follows. CONCLUSIONS: The lack of internationally accepted standardized methodologies and standard reference material has impeded screening and diagnosis of primary aldosteronism. The development of more accurate and sensitive methods by LC-MS/MS has improved the reliability of aldosterone and renin testing and the availability of commercial chemiluminescent assays may improve the standardization of reporting. Laboratorians need to understand the strengths and weaknesses of their analytical approach and ensure that their interpretative reports are appropriate to their assays.

Association between urine aldosterone and diastolic function in patients with primary aldosteronism and essential hypertension.

OBJECTIVE: To investigate the association between aldosterone and cardiac diastolic dysfunction. DESIGN AND METHODS: We prospectively enrolled 20 patients with primary aldosteronism (PA) and 22 patients with essential hypertension (EH). Plasma aldosterone concentration, plasma renin activity, and 24-h urine aldosterone level were measured. Echocardiography, including tissue Doppler image recordings, was performed. RESULTS: PA patients had a significantly higher left ventricular (LV) mass index and worse LV diastolic function than those in EH patients. Among various measures of aldosterone, log-transformed 24-h urine aldosterone level had the most consistent correlation with diastolic function. CONCLUSIONS: Aldosterone is strongly associated with LV diastolic dysfunction. Twenty-four hour urine aldosterone is a good indicator to evaluate the impact of aldosterone on LV diastolic function.

The role of urinary aldosterone for the diagnosis of primary aldosteronism.

When diagnosing primary aldosteronism, the measurement of urinary aldosterone after oral sodium loading is one of the currently recommended confirmatory tests. The aim of the study was to assess the repeatability and interpretation of urinary aldosterone in patients examined for suspected primary aldosteronism. Sixty-four hypertensive patients with suspected primary aldosteronism were prospectively enrolled and examined according to the study protocol. After antihypertensive medications interfering with renin-angiotensin-aldosterone system were withdrawn for at least 2 weeks, the confirmatory testing was performed: oral sodium loading preceded the collection of 24-h urine sample and subsequent saline infusion test. The identical procedures were repeated after 2 weeks. The concordant results of both saline infusion tests served for confirmation/exclusion of primary aldosteronism. Forty-nine patients were included in data analysis. Primary aldosteronism was excluded in 16, and confirmed in 33 individuals. The repeatability of urinary aldosterone was evaluated in 44 patients: the difference of urinary aldosterone levels ranged between 1 and 88% (median 31%). Ninety-three urine samples from 49 patients were used to validate the interpretation of urinary aldosterone in respect to the diagnosis of primary aldosteronism made by saline infusion testing; 96% sensitivity was characterized by urinary aldosterone ≥19 nmol/day, and 96% specificity was associated with urinary aldosterone ≥92 nmol/day. In 22 (45%) patients, urinary aldosterone remained in the "gray" zone between 19 and 92 nmol/day in all provided samples. The estimation of urinary aldosterone excretion after oral sodium loading is associated with marked intraindividual variability, and significant number of inconclusive results.