Urinary Biomarkers in Cardiac Bypass

Urine samples were collected 2.5–4 h after the end of cardiac bypass and immediately frozen (−20°C) before subsampling and refrozen for long-term conservation (−80°C). Urinary peptidome analysis was performed as previously described [8 (link)]. For urinary neutrophil gelatinase-associated lipocalin (uNGAL) measurement, urine samples were centrifuged for 10 min at 2500 rpm. NGAL was measured using an enzyme-linked immunosorbent assay (Human Lipocalin-2/NGAL Duoset ELISA; DY1757; R&D Systems, Minneapolis, MN, USA) in diluted supernatant (1:10 or 1:100) according to the manufacturer's protocol. Creatinine was measured using the QuantiChrom Creatinine Assay Kit (DICT 500; BioAssay Systems, Hayward, CA, USA) according to the manufacturer's protocol. Creatinine-normalized uNGAL (μg/g) was used for performance evaluations. [TIMP-2] and [IGFBP7] were measured in urine supernatants using the VITROS NephroCheck immunoassay on a VITROS 5600 Integrated System (Ortho Clinical Diagnostics, Raritan, NJ, USA) according to the manufacturer's instructions. The VITROS NephroCheck test result is a single number, which is a product of the measured concentrations of the two analytes in the sample divided by 1000. Certified laboratory technicians blinded to clinical data performed the analyses.