Measured Plasma Aldosterone Research Articles

5042 The Prevalence of Primary Aldosteronism in Incidentally Discovered Adrenocortical Adenomas Irrespective of Blood Pressure: A Prospective Cohort Study

Abstract Disclosure: T. Uslar: None. A. Burnier: None. B. Sanfuentes: None. P. Böhm: None. M. Orellana: None. F.J. Guarda: None. R.I. Olmos: None. Á. Huete: None. N. Mertens: None. C. Besa: None. A. Majerson: None. J. Cartes: None. C.E. Fardella: None. F. Allende: None. S. Solari: None. A. Vaidya: Consulting Fee; Self; Corcept Therapeutics, HRA Pharmaceuticals. R. Baudrand: None. Background: Incidental adrenocortical adenomas are commonly found on imaging. Guidelines recommend screening for primary aldosteronism (PA) only when adrenocortical adenomas are accompanied by hypertension or unexplained hypokalemia. Objective: To prospectively evaluate the prevalence of PA in consecutive patients with incidentally discovered adrenocortical adenomas. Design: Prospective, longitudinal, single-center study. Methods: 254 consecutive patients with incidentally discovered adrenocortical adenomas were studied, without any evidence of autonomous cortisol secretion, excluded via 1mg dexamethasone suppression test. A structured protocol was followed, which involved measurement of plasma aldosterone (PAC), 24h-urinary aldosterone and sodium, plasma renin activity (PRA) and/or direct renin (DRC). Samples were collected in the morning, in a seated posture, after withdrawing interfering medications. Possible PA was defined across a continuum by having a low renin (DRC <10 uIU/mL or PRA < 1ng/mL/hr) in addition to one of the following 3 criteria reflecting the severity of PA: 1) overt PA: aldosterone/renin ratio (ARR) >30, PA >15 ng/dL, and/or 24h urinary aldosterone >10 ug/24h; 2) moderate PA: ARR 20-30 and PAC 10-15 ng/dL; 3) mild PA: ARR <20 and PAC 5-10ng/dL. Adjusted linear regressions were performed. Patients were followed longitudinally to evaluate treatment outcomes with paired T-test. Results: Among patients with incidentally discovered adrenocortical adenomas, 35% (n=89/254) had possible PA. Overt, moderate, and mild PA were identified in 10%, 12%, and 13% of the overall sample, respectively. Notably, one in five of these individuals (20%) were normotensive. There were trends across the 3 phenotypes of PA indicating a continuum of severity, including: systolic blood pressure (152 ±19, 144 ±13, 142 ±16 mmHg, p-trend=0.02), resistant hypertension (56%, 38%, 33% p-trend=0.034), daily defined dose of antihypertensives (DDD) (2.9 ±1.8, 2.1 ±1.5, 1.0 ±0.8 p-trend=0.001), higher PAC (30.7 ±29.4, 10.3 ± 2.7, 7.9 ±2.5ng/dL p-trend<0.001) and urinary aldosterone (18.2 ±12, 5.6 ±3.6, 5.5 ±3.9 ug/24h p-trend<0.001). In longitudinal treatment follow-up (mean 28-months), 83% had a favorable response to treatment (84% medical, 16% adrenalectomy). After treatment, there was a decrease in SBP of -31.3 ±23 mmHg in overt PA, -13.2 ±19 mmHg in moderate PA, and -9.4 ±18 mmHg in mild PA (p<0.001 in all groups). Similar trends were observed for DDD, with a significant increase in renin in all groups. Conclusions: Overt PA, as well as a continuum of PA, was frequently seen in up to 35% of incidentally discovered adrenocortical adenomas. These findings underscore the importance of screening for PA in all patients with adrenocortical adenomas, irrespective of blood pressure and/or hypokalemia. Presentation: 6/1/2024

Abstract P428: Angiotensin II vascular abnormalities in 32.5% of 3322 patients with essential hypertension(EH): Use of angiotensin II analogue, saralasin.

Introduction: Renin-AII abnormalities in EH were documented by infusing saralasin , the AII analogue:patients with a abnormal fall in mean BP(<=-8mmHg) indicating a “angiotensinogenic” component to their hypertension and those with a abnormal rise in mean BP(>=8mmHg) indicating, as previously reported, an increased AII vascular sensitivity. Methods: Pa tients tested for secondary HBP in a 1-day BP study with recumbent BP by Arteriosonde, infusion of Lasix, recumbent for 1 hr, stand for 2 hours, measure renin(ng/ml/hr), recumbent for 1/2 hr, infusion of saralasin for 1/2 hr, infusion of 2 l of 0.9%NS over 4 hr and measurement of plasma aldosterone and cortisol. Patients with secondary causes of hypertension, those age<18, those with renal insufficiency, vascular abnormalities (eg diabetes, ASHD) were excluded. Data is now stored using excel 365. Linear regression was calculated using GraphPad Prism version 10.0.0 for Windows, www.graphpad.com. Results: 3691 adults(mean age = 43.4±0.2, 46.3% female) with EH of whom 3322 received saralasin: “Angiotensinogenic” HBP occurred in 12.9% of EH and they were younger[p=.0012,t=3.2]than the EH group, and had a high renin(nl:1.7-6.6). The saralasin-induced fall in BP was -15.6±.6/-13.0±.3. Saralasin increased the % of SBP that was <=130 from 28.1% to 54.7%. Saralasin induced an abnormal BP rise in 19.6% of patients who were older with low renin. There was a significant linear correlation with log 10 of renin for 3322 patients with EH for Δ Mean BP with saralasin[ΔMBP = -9.3*(log renin) + 4.3] (p<.0001,f=1056, r=-.50). Results were similar in 1463 patients on no BP meds. [ΔMBP=-9.5*(log renin) +4.46](p<0.0001,f=470,r=-.50). 12.5% were "angiotensinogenic", 16.7% pressor responders. Conclusions: EH has a significant “angiotensingenic” component (12.9%) and increased AII vascular sensitivity(19.6%), both of which may contribute to their HBP. table: three saralasin groups(BP fall, rise,no Δ) as in abstract(data are mean+se) Change in mean BP to ------------ #(%) -------- initial BP ------- post lasix BP ---- saralasin BP -- Age(yr) - renin i nfusion of saralasin (1)<= -8 mmHg ---- 430(12.9%) -- 160±2*/99±1* --- 147±1*/96±1* ---- 132±1*/83±1* --- 41.1±.7* ---8.1±.4* (2)>=+8 mmHg---- 651(19.6%) -- 170±1 & /103+1 & - 142±1 & /90±1 ns1 160±1 & /101±1 & - 50.2±.5 & --- 1.5±.1 & (3)>8,<-8 mmHg-2241(67.5%) - 155±1 @ /98±1 ! --- 137±1 @ / 90±1 @ - 138±1 @ /90±1 @ - 41.3±.9 ns2 - 3.4±.1 @ ___________________________________________ *p<.00001(1v2), & p<.0001(2v3), @ p<.0001(1v3), ! p=ns(1v3), ns1 (2v3), ns2 (1v3)

Screening Cutoff Values for the Detection of Aldosterone-Producing Adenoma by LC-MS/MS and a Novel Noncompetitive CLEIA.

Detecting patients with surgically curable aldosterone-producing adenoma (APA) among hypertensive individuals is clinically pivotal. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the ideal method of measuring plasma aldosterone concentration (PAC) because of the inaccuracy of conventional chemiluminescent enzyme immunoassay (CLEIA). However, LC-MS/MS is expensive and requires expertise. We have developed a novel noncompetitive CLEIA (NC-CLEIA) for measuring PAC in 30 minutes. This work aimed to validate NC-CLEIA PAC measurements by comparing them with LC-MS/MS measurements and determining screening cutoffs for both measurements detecting APA. We retrospectively measured PAC using LC-MS/MS and NC-CLEIA in 133 patients with APA, 100 with bilateral hyperaldosteronism, and 111 with essential hypertension to explore the accuracy of NC-CLEIA PAC measurements by comparing with LC-MS/MS measurements and determined the cutoffs for detecting APA. Passing-Bablok analysis revealed that the values by NC-CLEIA (the regression slope, intercept, and correlation coefficient were 0.962, -0.043, and 0.994, respectively) were significantly correlated and equivalent to those by LC-MS/MS. Bland-Altman plot analysis of NC-CLEIA and LC-MS/MS also demonstrated smaller systemic errors (a bias of -0.348 ng/dL with limits of agreement of -4.390 and 3.694 within a 95% CI) in NC-CLEIA than LC-MS/MS. The receiver operating characteristic analysis demonstrated that cutoff values for aldosterone/renin activity ratio obtained by LC-MS/MS and NC-CLEIA were 31.2 and 31.5 (ng/dL per ng/mL/hour), with a sensitivity of 91.0% and 90.2% and specificity of 75.4% and 76.8%, respectively, to differentiate APA from non-APA. This newly developed NC-CLEIA for measuring PAC could serve as a clinically reliable alternative to LC-MS/MS.

Impact of the transition from radioimmunoassay (RIA) to chemiluminescent enzyme immunoassay (CLEIA) for the measurement of plasma aldosterone concentration (PAC) on the diagnosis of primary aldosteronism (PA) via retrospective analyses in Okinawa, Japan.

In Japan, the traditional method for measuring plasma aldosterone concentration (PAC) was radioimmunoassay (RIA), which had several challenges, including poor traceability of certified reference materials and reduced detection sensitivity at low concentrations. To overcome these issues, a chemiluminescent enzyme immunoassay (CLEIA) for PAC measurement was introduced in April 2021 and the Japan Endocrine Society published new guidelines for primary aldosteronism (PA). This study aimed to evaluate the impact of the transition from RIA to CLEIA for PAC measurement on PA diagnosis. Data from 190 patients admitted to the Second Department of Internal Medicine, University of the Ryukyus Hospital, between April 2012 and March 2021 were analyzed. Patients who were diagnosed with PA underwent adrenal venous sampling. The PAC measured by RIA (PAC(RIA)) was converted to the estimated PAC measured by CLEIA (ePAC(CLEIA)) using a conversion formula. The present study evaluated the discordance rates in diagnoses based on screening (SC), captopril challenge test (CCT), saline infusion test (SIT), and diagnosis of PA between results judged by PAC(RIA) according to the previous guidelines and those judged by ePAC(CLEIA) according to the new guidelines. The results revealed discordant diagnosis rates of 6.4% for SC and 10.1% for CCT, with no discordance for SIT. The discordant diagnosis rate for PA was 3.7%. Our study reveals the challenges in establishing appropriate diagnostic criteria for PA using PAC(CLEIA) and highlights the demand for further research on provisionally positive categories.

Plasma aldosterone concentration / plasma renin activity ratio in patients with essential hypertension before treatment

Abstract Background The renin–angiotensin–aldosterone-sodium system, an important player in maintaining blood pressure, is influenced by antihypertensive drugs. However, the analysis of the renin-aldosterone-sodium system in hypertensive patients without adrenal adenoma without antihypertensive drugs is not still enough. Purpose We analyzed plasma aldosterone level, renin activity, serum potassium level, and serum sodium level in hypertensive patients without adrenal adenoma before treatment. Methods We measured plasma aldosterone concentration (PAC; pg/mL), plasma renin activity (PRA; ng/mL/h), serum potassium and sodium levels (mEq/L) in 456 consecutive new hypertensive patients untreated with antihypertensive drugs. We sampled all blood after 30 minutes bedrest. We excluded 28 cases with blood pressures less than 135/85 mmHg at rest, 4 patients with adrenal adenoma, and 3 patients with renovascular hypertension. We analyzed 421 patients with essential hypertension before treatment (232 males, mean age 56.5±12.8 years, and 189 females, mean age 61.0±13.5 years). Results There was a significant positive correlation between PRA and PAC (ρ=0.2445, p<0.001). There was a significant positive correlation between PAC/PRA ratio and serum sodium levels (ρ=0.1604, p=0.0027); however, no correlation was observed between PAC/PRA ratio and serum potassium levels. There were significant negative correlations between PRA and serum sodium levels (ρ=-0.1925, p<0.001). However, there was no correlation between PAC and serum sodium levels. On univariate analyses, a high PAC/PRA ratio (>200) was significantly associated with female (p<0.001), high serum sodium level (p=0.003), and low pulse rate (p=0.02). Multiple logistic analyses revealed that the most predictive independent factor for high PAC/PRA was female (p<0.001). On analyses of gender differences, the incidence rates of high PAC/PRA (>200) were 33.9% in females and 16.4% in males. There was a significant positive correlation between PRA and PAC in both genders (males: ρ=0.2731, p<0.001; females: ρ=0.1976, p=0.0064). In females, there was a significant positive correlation between PAC/PRA and serum sodium levels (ρ=0.2221, p=0.0043); however, there were no significant correlations between PAC/PRA and serum sodium levels in males. In females, there was a significant negative correlation between PRA and serum sodium levels (ρ=-0.2668, p<0.001); however, there were no significant correlations between PRA and serum sodium levels in males. Serum sodium levels were significantly higher in females than in males (p=0.02). Conclusion We revealed gender differences in the renin-aldosterone-sodium system in patients with essential hypertension before treatment.

FRI134 Transcriptomic Profiling Of Monocytes To Seek A Potential Biomarker For Primary Aldosteronism

Abstract Disclosure: R. Libianto: None. N. Karthigan: None. J. Morgan: None. D. Powell: None. P.J. Fuller: None. M.J. Young: None. J. Yang: None. Primary aldosteronism (PA), the most common endocrine cause of hypertension, is associated with increased cardiovascular risk compared with essential hypertension. The diagnosis of PA currently relies on measurements of plasma aldosterone and renin levels which can be compromised by patient- or assay-related issues and relies on arbitrary thresholds. A cellular marker that reflects excess endogenous aldosterone activity could enhance the diagnostic process. Mineralocorticoid receptor (MR) activation in macrophages has previously been shown to play a key role in mediating cardiovascular injury. Macrophage precursors, the monocytes, also express MR and can be easily isolated from circulation. We therefore sought to identify unique transcriptomic markers of aldosterone excess in peripheral blood monocytes. Monocytes were isolated from nine male patients with unilateral PA before and 3 months after curative adrenalectomy. Each patient’s post-adrenalectomy samples served as the control for comparison with the pre-adrenalectomy samples. RNA sequencing (RNA-seq) was performed on monocyte RNA samples to identify differentially expressed genes (DEGs), followed by evaluation using a second method by real-time quantitative PCR (RT-qPCR). Distinct clustering of gene expression was observed between the “before” and “after-surgery” samples with 1679 significantly differentially expressed (false discovery rate <0.05). The top 26 DEGs were selected for evaluation by RT-qPCR. Differences were observed in the relative expression of phosphoglycerate mutase 1 (PGAM1), ferrochelatase (FECH), and nuclear enriched abundant transcript 1 (NEAT1) genes pre- and post-surgery. The ratios of the relative expression of these genes accentuated the difference between the before- and after-surgery samples. In conclusion, differential gene expression was found between monocytes of patients with unilateral PA before and after adrenalectomy. Further validation studies are needed in female patients and those with bilateral PA and essential hypertension. These transcriptomic markers or their ratios have the potential to serve as novel biomarkers of aldosterone excess. Presentation: Friday, June 16, 2023

B-192 An LC-MS/MS Method using the Xevo TQ Absolute Mass Spectrometer for the Combined Analysis of Aldosterone and Plasma Renin Activity in Plasma for Clinical Research

Abstract Background Primary aldosteronism (PA) is a common cause of hypertension, whereby uncontrollable amounts of aldosterone are produced by a benign tumour or hyperplasia of the adrenal glands. Excess aldosterone results in significant sodium reabsorption in the kidneys, increasing water retention and blood volume, thereby causing hypertension. The renin-angiotensin-aldosterone system (RAAS) regulates the production of aldosterone and in this system, renin and aldosterone should move in synchronicity with each other throughout the day. Therefore, these two components are used to assess the status of the RAAS, particularly in the evaluation of new therapies in clinical research studies. Historically, the assessments of aldosterone and plasma renin activity (PRA) have been performed using separate methods using immunoassay or more recently liquid chromatography - tandem mass spectrometry (LC-MS/MS) platforms. One of the benefits of using LC-MS/MS for clinical research is the ability to measure multiple analytes across the proteome and metabolome using the same system and even in the same analysis to provide more information in less time and save costs. Here we evaluate a single LC-MS/MS method for the combined measurement of plasma aldosterone and renin activity for clinical research purposes. Methods Aldosterone certified reference material (Merck, UK) and Angiotensin I (Cambridge BioScience, UK) were used to create calibrators in 2% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS). In-house QC material prepared in both 2% BSA in PBS and K2EDTA plasma (BioIVT, UK), were used to evaluate method precision. Plasma samples were analyzed using the newly developed method and the quantified results were compared to separate independent LC-MS/MS methods for aldosterone and plasma renin activity. Plasma samples were treated with generation buffer (Sodium acetate, EDTA, acetic acid, SBTI and PMSF) and mixed for three hours at 37 °C. Samples were precipitated, diluted and centrifuged prior to SPE. Sample supernatant was transferred to a Waters Oasis™ µElution 96 Well Plate, followed by a wash and elution. Using an ACQUITY™ UPLC™ I-Class System, samples were injected onto a Waters XBridge™ C8, 2.5 µm, 2.1 × 50 mm Column using a water/methanol/ammonium fluoride gradient elution profile and quantified with a Waters Xevo™ TQ Absolute Mass Spectrometer. Results The method demonstrated no significant carryover or matrix effects and was shown to be linear from 10 - 2500 pg/mL for aldosterone and 0.1–25 ng/mL/h for PRA. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (<20%) at 10 pg/mL and 0.1 ng/mL/h, for aldosterone and PRA, respectively. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all <10% (n = 25) for aldosterone and PRA. Comparison with samples previously analyzed by an independent LC-MS/MS method demonstrated good agreement for aldosterone and PRA. Conclusions We have successfully quantified aldosterone and renin activity in plasma in a single method using an SPE protocol with LC-MS/MS analysis, for clinical research purposes. The method demonstrates excellent linearity and precision, with minimal matrix effects. For Research Use Only. Not for Use in Diagnostic Procedures.

PS-BPB04-9: DEVELOPMENT AND VALIDATION OF NEW METHOD TO MEASURE PLASMA ALDOSTERONE AND RENIN ACTIVITY USING LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETER (LC-MS/MS)

Objective: Measurement of aldosterone, renin, and aldosterone-renin ratio (ARR), are used to diagnose primary aldosteronism (PA). Immunoassays used to measure plasma aldosterone yield higher results compared to tandem mass spectrometry (LC-MS/MS). This can lead to false positive diagnosis of PA, and potential for unnecessary adrenal vein sampling. We report our development and validation of a novel test using LC-MS/MS, with reduced run time, for measurement of plasma aldosterone concentration (PAC) and plasma renin activity (PRA). Design and method: The detection and quantification of plasma aldosterone and angiotensin 1 (product for the calculation of renin activity) in the mass spectrometer was based on the multiple reaction monitoring of precursor to fragment ions under negative and positive ionization modes, respectively. Validation of the tests were based on the CLSI (Clinical & Laboratory Standards Institute) guidelines. Concurrent measurement for 190 samples were done on our instrument, and on a commercial lab using LC-MS/MS (Mayo Laboratories, USA) for comparison. Results: Our measurements of PAC and PRA was sensitive with a limit of quantification at 3 ng/dL and 0.25 ng/ml/h, respectively, and analytical run times were 6.5 min and 3.5 min, respectively. Both aldosterone and angiotensin 1 calibration curves had excellent linearity in the ranges of 3 - 300 ng/dL and 0.25 - 25 ng/ml/h, respectively, with coefficient of correlation, r2 > 0.994. The intra and inter day accuracy of aldosterone and angiotensin 1 were in the range of 95–110% with CV of < 13%. Reference ranges were established for our local population. Compared with the other LC-MS/MS laboratory, there was good correlation for PAC, r2 = 0.90, 95%CI: 0.86 - 0.92, and PRA, r2 = 0.81, 95% CI: 0.75 - 0.86, using 190 samples. In addition, amongst those with low aldosterone < 6ng/dL, there was good agreement between both labs, with 95 of 122 (77.9%) samples having low aldosterone using both methods, which has been shown to be an ideal threshold to exclude the diagnosis of PA. Conclusions: Our laboratory has developed and validated a sensitive and accurate LC-MS/MS test for PAC and PRA with shortened run time, for the diagnosis PA.

Impact of a change to a novel chemiluminescent immunoassay for measuring plasma aldosterone on the diagnosis of primary aldosteronism.

In Japan, the standard method for measuring plasma aldosterone concentration (PAC) for primary aldosteronism (PA) diagnosis was changed from radioimmunoassay (RIA) to a novel chemiluminescent enzyme immunoassay (CLEIA). The purpose of this study is to simulate the possible impact of the change on PA diagnosis. This retrospective study assessed 2,289 PA patients. PACs measured by conventional RIA were transformed to estimated PACs (CLEIA) as follows: RIA (pg/mL) = 1.174 × CLEIA (pg/mL) + 42.3. We applied the estimated PAC (CLEIA) to the conventional cut-off of aldosterone-to-renin activity ratio ≥200 for screening and captopril challenge test (CCT) and PAC ≥60 pg/mL for saline infusion test (SIT). Application of the estimated PAC to screening and confirmatory tests decreased the number of PA diagnoses by 36% (743/2,065) on CCT and 52% (578/1,104) on SIT (discrepant cases). Among the discrepant cases, 87% (548/628) of CCT and 87% (452/522) of SIT were bilateral on adrenal venous sampling (AVS). Surgically treatable aldosterone-producing adenomas (APAs) were observed in 6% (36/579) and 5% (23/472) of discrepant cases on CCT and SIT, respectively; most were characterized by hypokalemia and/or adrenal nodule on CT imaging. Application of the PAC measured by the novel CLEIA to conventional cut-offs decreases the number of PA diagnoses. Although most discrepant cases were bilateral on AVS, there are some APA cases that were characterized by hypokalemia and/or adrenal tumor on CT. Further studies which evaluate PACs measured by both RIA and CLEIA for each patient are needed to identify new cut-offs for PAC measured by CLEIA.

Role of α1-blockers in the current management of hypertension.

There is emerging evidence that α1‐blockers can be safely used in the treatment of hypertension. These drugs can be used in almost all hypertensive patients for blood pressure control. However, there are several special indications. Benign prostatic hyperplasia is a compelling indication of α1‐blockers, because of the dual treatment effect on both high blood pressure and lower urinary tract symptoms. Many patients with resistant hypertension would require α1‐blockers as add‐on therapy. Primary aldosteronism screen is a rapidly increasing clinical demand in the management of hypertension, where α1‐blockers are useful for blood pressure control in the preparation for the measurement of plasma aldosterone and renin. Nonetheless, α1‐blockers have to be used under several considerations. Among the currently available agents, only long‐acting α1‐blockers, such as doxazosin gastrointestinal therapeutic system 4–8 mg daily and terazosin 2–4 mg daily, should be chosen. Orthostatic hypotension is a concern with the use of α1‐blockers especially in the elderly, and requires careful initial bedtime dosing and avoiding overdosing. Fluid retention is potentially also a concern, which may be overcome by combining an α1‐blocker with a diuretic.

Kidney and Gut Microbiome Crosstalk in Response to High Dietary Potassium

In this study, we tested the hypothesis that the gut microbiome influences the homeostatic response to a dietary K+ load. Male Specific Pathogen Free (SPF) and Germ‐Free (GF) mice (n = 5 per group) received 5% potassium citrate or K+alimentary matched control diets for 7 days. At the time of sacrifice, blood and urine parameters were compared, and the expression of relevant transporters and signaling molecules was assessed in the kidney by western blotting. No significant differences in whole blood K+ were observed between SPF and GF mice on control or high K+ diets. However, differences in other parameters were observed. Compared to control diet treated SPF mice, K+ loaded SPF mice exhibited a significant decrease in hematocrit (37 vs 39; p ≤ 0.0152) and hemoglobin (12.58 vs 13.26; p ≤ 0.0150), whereas GF mice exhibited no differences in these parameters regardless of dietary maneuver. Blood urea nitrogen (BUN) levels were significantly decreased in SPF mice (23.4mg/dL vs 27; p ≤ 0.0180) on High K+compared to SPF controls, while this was not the case for GF mice. These findings suggest that K+ loaded GF mice were less volume expanded than K+ loaded SPF mice. Consistent with this, plasma aldosterone measurements were higher in K+ loaded GF mice compared to K+ loaded SPF mice (966pg/mL vs 614; p ≤ 0.0155). In whole kidney homogenates, dietary K+ loading markedly increased the cleaved (active) form of the epithelial sodium channel gamma subunit (γENaC) relative to control diet treated mice in both the SPF and GF groups. However, cleaved γENaC was one‐fold higher in the K+ loaded GF mice compared to K+ loaded SPF mice. K+ citrate loading significantly upregulated pendrin in both GF and SPF groups (p ≤ 5.7146E‐05 and p ≤ 5.6654E‐06), likely an effect of the alkaline content of the diet. Interestingly, we also observed a significant 91% increase in Aquaporin 2 (AQP2) levels in the GF high K+ group (p ≤ 0.0168) relative to GF mice treated with control diet, an effect that was not observed in SPF mice (p= 0.165). These findings indicate that male germ‐free mice maintain plasma [K+] in response to a dietary alkaline potassium load, but develop reduced volume status compared to K+ loaded SPF controls. To compensate, germ free mice exhibit plasma elevated aldosterone concentrations, and upregulate cleaved γENaC and AQP2 abundance in the kidney. This suggests that an intact gut microbiome helps to defend against excessive volume depletion during the homeostatic response to alkaline K+ loading.

Preoperative Plasma Aldosterone Predicts Complete Remission of Type 2 Diabetes after Bariatric Surgery

Introduction: Bariatric surgery (BS) has beneficial effects on body weight and type 2 diabetes. However, 44–52%, 20–40%, and 19–25% of patients with type 2 diabetes who undergo sleeve gastrectomy, sleeve gastrectomy with duodenal-jejunal bypass, and Roux-en-Y gastric bypass, respectively, show insufficient improvement 1 year after BS. It is thus important to predict the improvement in type 2 diabetes before BS. Many hormones are related to hyperglycemia. However, the relationship between hormones and improvement in type 2 diabetes after BS has not been studied. We aimed to evaluate the relationship between the improvement in type 2 diabetes and hormones in patients with obesity and type 2 diabetes who underwent BS. Methods: We retrospectively reviewed 79 patients with obesity and type 2 diabetes who underwent BS, with a follow-up period of 12 months. We analyzed the relationship between some clinical parameters and complete remission (CR) of type 2 diabetes after BS. Patients were divided into two groups (type 2 diabetes CR and non-CR). Multiple regression analysis was performed to determine the parameters associated with type 2 diabetes resolution after BS. Results: BS significantly improved body weight and glucose metabolism. Preoperative liver function, glycated hemoglobin (HbA1c), insulin secretion (homeostatic model assessment [HOMA]2-%B), renin activity, plasma aldosterone level, and duration of type 2 diabetes were significantly different between the CR and non-CR groups. Multiple regression analysis showed that preoperative HbA1c, HOMA2-%B, aldosterone concentration, and duration of type 2 diabetes were predictors of CR of type 2 diabetes after BS. Plasma aldosterone was the strongest predictor. Discussion/Conclusion: Preoperative plasma aldosterone levels were related to the CR of type 2 diabetes after BS. Measuring plasma aldosterone levels preoperatively is useful for predicting the CR of type 2 diabetes after BS.

The Saline Infusion Test for Primary Aldosteronism: Implications of Immunoassay Inaccuracy.

ContextDiagnosis of primary aldosteronism (PA) for many patients depends on positive results for the saline infusion test (SIT). Plasma aldosterone is often measured by immunoassays, which can return inaccurate results.ObjectiveThis study aimed to establish whether differences in aldosterone measurements by immunoassay versus mass spectrometry (MS) might impact confirmatory testing for PA.MethodsThis study, involving 240 patients tested using the SIT at 5 tertiary care centers, assessed discordance between immunoassay and MS-based measurements of plasma aldosterone.ResultsPlasma aldosterone measured by Liaison and iSYS immunoassays were respectively 86% and 58% higher than determined by MS. With an immunoassay-based SIT cutoff for aldosterone of 170 pmol/L, 78 and 162 patients had, respectivel, negative and positive results. All former patients had MS-based measurements of aldosterone < 117 pmol/L, below MS-based cutoffs of 162 pmol/L. Among the 162 patients with pathogenic SIT results, MS returned nonpathologic results in 62, including 32 under 117 pmol/L. Repeat measurements by an independent MS method confirmed nonpathogenic results in 53 patients with discordant results. Patients with discordant results showed a higher (P < 0.0001) prevalence of nonlateralized than lateralized adrenal aldosterone production than patients with concordant results (83% vs 28%). Among patients with nonlateralized aldosterone production, 66% had discordant results. Discordance was more prevalent for the Liaison than iSYS immunoassay (32% vs 16%; P = 0.0065) and was eliminated by plasma purification to remove interferents.ConclusionThese findings raise concerns about the validity of immunoassay-based diagnosis of PA in over 60% of patients with presumed bilateral disease. We provide a simple solution to minimize immunoassay inaccuracy-associated misdiagnosis of PA.

Developing an ultra-performance liquid chromatography-tandem mass spectrometry for detecting aldosterone in human plasma.

BackgroundAccurately measuring plasma aldosterone concentration is difficult but meaningful for primary aldosteronism (PA) diagnosis.MethodsIn this study, we developed an ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method for plasma aldosterone detection, evaluated its performance according to guidelines issued by CLSI, including detection limit, linearity, precision, and compared it with chemiluminescence immunoassay. Then, a reference range of plasma aldosterone in young people was established by using this method.ResultsThe lower limit of quantitation (LOQ) was 10 pg/ml. The mean recovery rates of analyte added to serum were 100.07–102.05% in different concentrations. The linearity range was 20–2000 pg/ml. Inter‐assay CVs were 2.20–3.97% at aldosterone concentrations of 65.66–854.75 pg/ml. The regression equation of UPLC‐MS/MS (x) and chemiluminescence immunoassay (y) was y = 1.002x + 65.854 (r = 0.9456, n = 237). The reference range of plasma aldosterone detected by UPLC‐MS/MS was 11.30–363.82 pg/ml in young people in South China, and there was no statistically significant difference in plasma aldosterone concentration between two genders.ConclusionIn conclusion, UPLC‐MS/MS can rapidly and accurately detect plasma aldosterone and is appropriate for clinical application.

DEVELOPING A MOST EQUIVALENT ALDOSTERONE MEASUREMENT TO LC-MS/MS BY A NOVEL HIGH-QUANTITATIVE NONCOMPETITIVE CHEMILUMINESCENCE IMMUNOASSAY

Objective: Measurement of plasma aldosterone and renin concentration, or activity, is useful for selecting antihypertensive agents and detecting hyperaldosteronism in hypertensive patients. However, it takes several days to get results even if measured by inaccurate radioimmunoassay, or we must accept high-cost LC/MS, and development of a more rapid and accurate substitute has been long hoped. We have developed a novel, fully-automated, high-quantitative noncompetitive chemiluminescence immunoassay (NC-CLEIA) for detecting aldosterone in serum and plasma, and its performance is evaluated as compared to LC/MS measurement. Design and method: Recently a unique anti-metatype antibody, which recognizes the immunocomplex of aldosterone and its monoclonal antibody, was established. Using this antibody for sensing permitted the construction of non-competitive assay for the detection of aldosterone. The reaction protocol of novel aldosterone assay is the following. In the 1st reaction, aldosterone in patient's sample is captured on anti-body coated magnetic particles. Alkaline phosphatase-conjugated anti-metatype antibody is added and incubated as 2nd reaction following a wash. Then substrate solution is added after washing immunocomplex. The resulting reaction signals are proportional to the amount of aldosterone in the sample allowing quantitative determination of in serum or plasma sample. The overall reaction is completed within 30 min. Results: Limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ) of our NC-CLEIA aldosterone assay were 0.09 ng/dL, 0.21 ng/dL and 0.57 ng/dL, respectively. NC-CLEIA aldosterone measurements were linearly well correlated with LC/MS aldosterone measurements (N = 130, y = 1.027x - 0.23 ng/dL, Spearman's p = 0.996, p &lt; 0.0001). Bland-Altman plot analysis between NC-CLEIA and LC-MS/MS of aldosterone revealed a bias of 0.40 ng/dL with the limits of agreement of -4.60 and 5.41 ng/dL with 95% confidence interval. Conclusions: Our novel NC-CLEIA aldosterone assay was well-correlated and had only a very low bias with LC-MS/MS method and also was able to accurately quantify low level samples even in essential hypertension patients. This assay may be useful for the routine analysis of aldosterone and for the screening and diagnosis of PA from large number of hypertensive patients.

THE EFFECT OF AGE, BMI, AND SEX ON ALDOSTERONE, RENIN AND THE ALDOSTERONE-TO-RENIN RATIO IN ESSENTIAL HYPERTENSIVES

Abstract Objective: The aldosterone-to-renin ratio (ARR) is widely used as a screening test for primary aldosteronism (PA) in patients with hypertension. The purpose of the present investigation is to identify the impact of age, body mass index (BMI) and sex on renin, aldosterone, and the ARR, when measured under strictly standardised conditions. Design and method: We measured plasma aldosterone, plasma renin concentration and the ARR in 190 essential hypertensive patients (120 males, 70 females). All measurements were taken in the supine position at 9am. A low sodium diet (55mmol/day) was in place for no less than two weeks prior to screening. In those who were receiving antihypertensive medications, all medications were discontinued at least 2 week before testing. Results: Renin and aldosterone were significantly higher in men, and remained so when controlling for BMI and age. However, the ARR was not significantly affected by sex. Aldosterone was not significantly influenced by age but renin correlated inversely with age, with and without controlling for BMI (r = -0.285, p &lt; 0.001). The ARR was positively correlated with age (r = 0.236, p &lt; 0.001), with and without controlling for BMI. Subgroup analysis demonstrated a significant effect on the ARR in males only, with and without controlling for BMI (p &lt; 0.001 and p0.004, respectively). Although renin and aldosterone individually were not significantly influenced by BMI, the ARR showed a positive correlation (p = 0.022). However, this effect was not seen when age was controlled for nor was it seen in the subgroup analysis of males and females (p = 0.125, p = 0.515, p = 0.207, respectively) Conclusions: Under strictly standardized conditions, advanced age is associated with an increased ARR in male essential hypertensives. BMI when controlling for age does not influence the ARR. The impact of sex on the ARR appears negligible.

BNP LEVELS AND LEFT VENTRICULAR MORPHOLOGY AND FUNCTION IN HYPERTENSIVE PATIENTS BEFORE AND AFTER AN INTRAVENOUS SALINE LOAD

Objective: Previous studies have reported a relationship between BNP levels and left ventricular (LV) morphology and function in essential hypertension (EH), but results are conflicting. The aim of the study was to investigate the relationships between BNP levels measured before and after a standard saline load (SLT), plasma aldosterone (PA), active renin (AR), and LV variables in EH patients without heart failure or valvular disease. Design and method: In 155 EH patients (age 48 ± 12 years, 92 males, 64 treatment-naïve, the remaining 91 after drug wash-out), a SLT (0.9% saline, 2 L in 4 hours) was performed with baseline and post-SLT measurement of PA, AR, BNP pre- and post-test (pre-BNP, post-BNP). Echocardiography with Tissue Doppler Imaging (TDI) was performed, with measurement of indexed LV mass (LVMI), ejection fraction (EF), TDI-S velocity, E/A and E/e’ ratios, and TDI-e’ velocity. Results: Pre-BNP level was higher in females than males, and in patients with previous antihypertensive treatment than in naive patients. Pre-BNP level was significantly and directly related to age, LVMI, E/e’, and inversely related to glomerular filtration (GFR), AR, TDI-S, E/A, and TDI-e’. Post-BNP levels was higher in females and in previously treated patients, and it was positively related to age, E/e’ ratio, and inversely related to GFR, AR, TDI-S, E/A, and TDI-e’. At multivariate analysis pre-BNP level was independently associated with age, female gender, GFR, and AR, while post-BNP level was independently associated with age, AR, and previous antihypertensive treatment. Conclusions: In EH patients, plasma BNP is related with LVMI and LV systolic and diastolic function before and after a SLT, but this relationship seems to be limited to female sex and older subjects. Measurement of BNP might be useful for identification and follow-up of subclinical LV abnormalities in these patients.

SHORT-TERM VARIABILITY OF THE ALDOSTERONE-TO-RENIN RATIO IN AN ESSENTIAL HYPERTENSIVE POPULATION

Objective: The aldosterone-to-renin ratio (ARR) is a commonly used metric in the screening of primary aldosteronism. However, insufficient high-quality research evaluating its precision and accuracy is available. In particular, limited information is known with regard to intra-patient short-term variability in the ARR. Highly variable intra-patient values could severely hamper the ability of the ARR to effectively screen for primary aldosteronism. Design and method: 68 unmedicated, essential hypertensive participants (female n = 26, male n = 42), had two blood samples taken in the morning measuring plasma aldosterone and active plasma renin concentration within a 2-hour time window under strictly standardised conditions and while consuming a regulated sodium intake (3 g/day). Bland-Altman plots, mountain plots and correlation analysis were performed, comparing the mean values per participant with the difference between their measurements. Results: Using Bland-Altman plots, we found that renin, aldosterone and the ARR become increasingly variable as their mean values increase. Correlation analysis of the absolute difference in aldosterone-to-renin ratio values between measurements and their mean resulted in an R-squared value of 34.7% (p-value &lt; 0.000)). This trend was also noted for plasma aldosterone (R-squared 9.6%; p-value 0.010) and active plasma renin concentration (R-squared 7.9%; p-value 0.021) separately. Taking an ARR value above 30 as abnormally high, 29.4% of the participants with a mean value above 30, had one measurement below 30. Conversely, 3.9% of those with mean values below 30 had one measurement above 30. Conclusions: Based on our findings, it can be concluded that spot measurements of the aldosterone-to-renin ratio have acceptable short-term variability in the lower ranges, but have increasingly doubtful reliability as aldosterone-to-renin values rise. Thus, in a clinical context, great caution should be taken in interpreting spot measurements of moderate to high aldosterone-to-renin ratio values, in particular when values are mildly elevated.

High prevalence and a long delay in the diagnosis of primary aldosteronism among patients with young-onset hypertension.

Despite being the most common cause of secondary hypertension, prevalence of primary aldosteronism (PA) among patients with young-onset hypertension (YH - age of hypertension onset <40 years) remains poorly studied. We assessed the prevalence of PA in patients with YH referred for evaluation of secondary hypertension. In this prospective, cross-sectional study, 202 patients with YH, visiting endocrine and cardiology clinics of All India Institute of Medical Sciences, India, were evaluated. Primary aldosteronism was screened by measuring plasma aldosterone concentration (PAC) and direct renin concentration (DRC) and calculating aldosterone-to-renin ratio (ARR), followed by confirmatory saline infusion test (SIT) according to Endocrine Society Guideline. Those confirmed with post-SIT PAC >5ng/dl underwent adrenal computed tomography (CT), followed by adrenal venous sampling (AVS). Of 202 YH patients, 38 (18.8%) screened positive, and PA was confirmed in 36 (17.8%). The mean age was 43.9±10.9years, and median duration of hypertension was 10.5 (3.5-18) years. The prevalence of PA increased with grade of hypertension (8.1% in grade 1 to 37.1% in grade 3), number of antihypertensive medications (2.5% in those taking ≤1 to 50% in those taking ≥4 medications) and severity of hypokalaemia (0% in potassium >5 to 85.7% in potassium <3.5mmol/L). The prevalence of PA by age of hypertension onset was highest in age group 30-39years (31.3%). There is a high prevalence and a long delay in diagnosis of PA among patients with YH, and YH should be considered as a separate high-risk category in PA screening algorithm.

Novel chemiluminescent immunoassay to measure plasma aldosterone and plasma active renin concentrations for the diagnosis of primary aldosteronism

Determination of plasma aldosterone concentrations (PAC) and plasma active renin concentrations (ARC) is essential for the diagnosis of primary aldosteronism (PA). In Japan, although PAC and ARC are measured by radioimmunoassay and immunoradiometric assay, respectively, non-radioisotopic methods with better detection sensitivity, measurement accuracy, and technical simplicity are needed. We developed two-site sandwich chemiluminescent enzyme immunoassays (CLEIAs) to measure both PAC and ARC using monoclonal antibodies immobilized onto ferrite particles. The results of both assays are obtained simultaneously from a single plasma sample within 30 min using a fully automated system. The novel CLEIAs were validated using plasma samples from patients with PA (n = 52) and essential hypertension (n = 23). The PAC determined by the CLEIA was significantly correlated with that measured by liquid chromatography/mass spectrometry or conventional radioimmunoassay. The ARC determined by the CLEIA was significantly correlated with that measured by immunoradiometric assay. The limits of detection of the CLEIAs for PAC and ARC were 0.1 ng/dl and 0.04 pg/ml, respectively, which were better than those of conventional methods (PAC: 2.5 ng/dl; ARC: 5 pg/ml). The PAC and PAC/ARC ratio (ARR) were significantly higher, and the ARC significantly lower, in patients with PA than in those with essential hypertension. An ARR cut-off of 1.31 ng/dl per pg/ml showed a sensitivity of 96.2% and specificity of 78.3% for PA screening. The newly developed CLEIAs for measuring PAC and ARC could provide a clinically powerful alternative to conventional methods used for hypertension screening in clinical practice.