Au400

The blood samples were collected by trained nurses in the morning after 12 h of fasting and 48 h after the last training session. The variables were determined immediately after blood collection (without freezing) by standard methods performed in an accredited laboratory of clinical analysis (Laboratório UÁLIA – Análises Clínicas). Blood serum (centrifugate at 3500 RPM) were used for LDL-C, HDL-C, total cholesterol (TC), TG, FBG and high-sensitivity C-reactive protein (hs-CRP) quantification using Beckman Coulter AU 400 (Brea, United States) commercial kits. Further, blood serum samples were used to quantify insulin and C-peptide (CP) using the Roche Cobas (Mannheim, Germany) commercial kits. Whole blood was used for glycated hemoglobin (HbA1c) using Menarini–Arkray HA 8140 (Kyoto, Japan) commercial kit. Error analysis was less than 1 standard deviation for all variables (LDL-C, HDL-C, TC, TG, hs-CRP, CP, FBG and HbA1C). The evaluation of the homeostasis model of insulin resistance (HOMA-IR) was also used to determine insulin resistance and was calculated by a free online calculator (HOMA Calculator, Version 2.2.3, University of Oxford, Oxford, United Kingdom).

Since there is no gold standard for Lp-PLA₂ activity to assess assay accuracy, the Bland-Altman plot was used to perform method comparison between the Dimension Vista^® 1500 and the analytical platform used by the manufacturer. Lp-PLA₂ activity was measured in duplicates in a single assay run on 40 blind serum samples spanning an activity range of 5 - 365 U/L, which were provided by diaDexus. Our results were then compared to the activity values previously measured by diaDexus on the same samples using a Beckman Coulter AU400^® analyser (Beckman Coulter Inc., Brea, USA).

Anthropometric and performance measurements were performed at the same time, and blood was collected in the following 4 months. The blood samples were drawn after a 12 hours fast, and creatinine, cystatine C, hemoglobin, albumin, serum 25(OH) vitamin D and intact PTH levels were measured. Serum FGF23 was analyzed by a second generation C-terminal FGF23 two-site Enzyme Linked Immunonosorbent Assay (ELISA; Immutopics, San Clemente, CA, USA). This assay detects two epitopes in the C-terminus of FGF23, and has a sensitivity of 1.5 relative units per mL (RU/mL), and inter- and intra-assay coefficients of variations of less than 5%. Serum creatinine was measured using a modified kinetic Jaffé colorimetric method on an autoanalyzer (AU 400, Beckman Coulter, CA. USA), which was calibrated to isotope dilution mass spectrometry, using a standard reference material (914a) traceable to the National Institutes of Standards and Technology (NIST). Plasma cystatin C levels were determined by an automated particle-enhanced immunoturbidimetric method using a Beckman AU 400 analyzer (Beckman Coulter), and reagents (code number LX002. s2361. X0973. X0974) obtained from DakoCytomation (Glostrup, Denmark), following the procedures recommended by the reagent producer. We estimated glomerular filtration rate (eGFR) by the CKD-EPI creatinine-cystatin C equation. All samples were frozen at −80°C.

Statistical analysis was performed using MedCalc for Windows, version 13.0 (MedCalc Software, Ostend, Belgium). Method comparison between the Dimension Vista^® 1500 and the Beckman AU400^® was performed by Bland-Altman plot and Passing-Bablok regression analysis. The Kolmogorov-Smirnov test was used to assess the normality of distribution of Lp-PLA₂ activity in the whole study population, and in males and females separately. Reference intervals were defined according to CLSI document C28-A3 using the nonparametric method (minimum sample size required for each partition group: N = 120) and central 95^th percentile of reference values (23 ). Differences between genders were tested by unpaired t-test. Spearman’s and Pearson’s correlation test was used to test the association between serum Lp-PLA₂ activity and serum concentrations of total cholesterol, LDL-cholesterol (directly measured), HDL-cholesterol, and triglycerides and the association between serum Lp-PLA₂ activity and age in the study population. The values P < 0.05 were considered statistically significant.

Starting at 8 weeks of age, animals were weighed and albuminuria was measured weekly. Daily observations included monitoring animals for physical condition and for clinical signs of disease which included rough coat, edema, labored breathing and respiratory distress. In order to alleviate suffering, moribund animals were euthanized by CO2 inhalation after two consecutive readings of ≥ 1000 mg/dl of albuminuria and/or when they exhibited respiratory distress. Using these criteria animals were euthanized before they became severely ill. On two occasions, an animal was found dead without having clinical signs of disease or elevated albuminuria. Kidney disease was evaluated using a number of serum and urine markers of glomerular/tubular leakage and kidney function. Urine was analyzed for total protein which was used as a primary end point. Serum was analyzed for blood urea nitrogen (BUN), creatinine, and cholesterol. All measurements were done using the Beckman Coulter AU400.

The activity of Lp‐PLA2, also known as platelet‐activating factor acetylhydrolase (PAF‐AH) was measured at the Biomarker Core Laboratory of the Atlanta VA, using a PAF‐AH activity assay kit, which produces results based on a colorimetric shift (Biovision, Milpitas, CA), according to the manufacturer's instructions. Performance characteristics for the Lp‐PLA2 test were established using the enzymatic method Beckman Coulter AU400, a dual monoclonal antibody immunoassay standardized to recombinant Lp‐PLA2 (PLAC test, diaDexus, Inc) (Dada et al. 2002). To assess intra‐assay precision and total variability for Lp‐PLA2 measurement, five human plasma samples and two buffer controls with Lp‐PLA2 activity distributed throughout the calibration range of the assay were measured in 40 separate assays to determine the intra‐assay coefficient of variation (Le et al. 2015).