Elecsys 2010 platform

Blood samples were collected in plastic vials containing ETDA. Vials were centrifuged at 3100g for 20 minutes and then frozen at −70°C. No sample was thawed more than twice.
Concentrations of proBNP 1‐76 (NT‐proBNP) were analyzed on the Elecsys 2010 platform (Roche Diagnostics, Mannheim, Germany). The total coefficient of variation (CV) was 4.6% at 426.5 pg/mL (n=487) and 3.2% at 2308 pg/mL (n=495). Plasma concentrations of MR‐proADM, copeptin, and MR‐proANP were analyzed on the Kryptor platform (Brahms AG, Hennigsdorf, Germany). Total CV for copeptin was 4% at a concentration of 15 pmol/L (n=18) and 3.5% at concentrations of 100 pmol/L (n=18). For MR‐proADM, the intraassay CV, according to the manufacturer, was ≤10% for concentrations between 0.2 and 0.5 nmol/L, <4% for concentrations between 0.5 and 2 nmol/L, <2% for concentrations between 2 and 6 nmol/L, and <3.5% for concentrations over 6 nmol/L. The intraassay CV for MR‐proANP according to the manufacturer was ≤5% for concentrations between 10 and 20 pmol/L, <3.5% for concentrations between 20 and 1000 pmol/L, and <3.5% for concentrations over 1000 pmol/L.

Plasma leptin concentrations were determined by RIA (Linco Research) with a detection limit of 0.25 ng/mL. The intra-assay coefficients of variation were 5.6% at 3.5 ng/mL and 6.0% at 21.1 ng/mL. The corresponding inter-assay coefficients of variation were 7.9 and 7.0% respectively. E was assayed using a Roche Diagnostics Cobas e411 automatic clinical platform assay instrument. The intra-assay and inter-assay coefficients of variation were all less than 10% and the assay detection limit was 5 pg/mL. Cortisol was assayed by electro-chemiluminescence using the Elecsys 2010 Platform (Roche Diagnostics). DHEAS was assayed using RIA with a highly specific antibody for DHEAS-17-(O-carboxymethyl)oxime-BSA (Endocrine Services, Tarzana, CA, USA) and [3H]DHEAS (SA, 22 Ci/mmol). For the cortisol and DHEAS assays, the intra-assay and inter-assay coefficients of variation were less than 10% and the assay detection limits were 3 ng/mL.

Baseline demographic characteristics, blood pressure, body mass index (BMI), medication use, prevalent risk factors and cardiovascular disease, and fasting laboratory values were assessed using standardized protocols, as previously reported.¹⁶ Cardiac biomarkers, including high‐sensitivity cardiac troponin T and NT‐proBNP (N‐terminal pro‐B‐type natriuretic peptide), were measured using the Elecsys 2010 platform (Roche Diagnostics, Indianapolis, IN), as previously reported.¹⁷ Insulin resistance was measured using the Homeostatic Model Assessment of Insulin Resistance, and was calculated as follows: [fasting glucose (mmol/L)×level of fasting insulin (μU/mL)]/22.5.¹⁸ Whole body total fat percentage and whole body total lean mass were measured using dual‐energy x‐ray absorptiometry (Delphi W scanner [Hologic Inc, Bedford, MA] and Discovery software, version 12.2), as reported previously.¹⁹

Plasma determinations were performed at the laboratory of Nephrology at the Gómez‐Ulla hospital and at the Biochemistry Laboratory at Fundación Jiménez Díaz. The investigators who performed the laboratory studies were unaware of clinical data. Plasma calcidiol levels were quantified by chemiluminescent immunoassay (CLIA) on the LIAISON XL analyser (LIAISON 25OH‐Vitamin D total Assay DiaSorin, Saluggia, Italy), FGF23 was measured by an enzyme‐linked immunosorbent assay which recognizes epitopes within the carboxyl‐terminal portion of FGF‐23 (Human FGF23, C‐Term, Immutopics Inc, San Clemente, CA), klotho levels by ELISA (Human soluble alpha klotho assay kit, Immuno‐Biological Laboratories Co., Japan), intact parathormone was analysed by a second‐generation automated chemiluminescent method (Elecsys 2010 platform, Roche Diagnostics, Mannheim, Germany), phosphate was determined by an enzymatic method (Integra 400 analyser, Roche Diagnostics, Mannheim, Germany), and high‐sensitivity C‐reactive (hs‐CRP) protein was assessed by latex‐enhanced immunoturbidimetry (ADVIA 2400 Chemistry System, Siemens, Germany). Lipids, glucose, and creatinine determinations were performed by standard methods (ADVIA 2400 Chemistry System, Siemens, Germany). The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) equation.