2.4. Assays for Biochemistry, Organ Damage Markers, Inflammatory Mediators and Oxidative Stress

The concentrations of creatinine, blood urea nitrogen (BUN), derivatives of reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), cystatin-C, myoglobin, insulin, growth hormone, glucose, free fatty acid (FFA), uric acid, urine protein, albumin, N-acetyl-β-d-glucosaminidase (NAG) and osmolality were measured by Koutou-Biken Co. (Tsukuba, Japan). Plasma and urinary tumor necrosis factor (TNF)-α, IL-6 and granulocyte colony-stimulating factor (G-CSF) concentrations were measured using a Quantikine high-sensitivity enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). Plasma and urinary IL-1 receptor antagonist (IL-1ra), monocyte chemoattractant protein (MCP)-1, macrophage colony-stimulating factor (M-CSF), retinol binding protein 4 (RBP4), IL-18 and IL-18 binding protein α (IL-18BPa) concentrations were measured using a Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA). Plasma and urinary intestine-fatty acid binding protein (I-FABP) concentrations were measured using a Duoset ELISA kit (R&D Systems, Minneapolis, MN, USA). Plasma and urinary IL-2, IL-4, IL-10 and complement (C) 5a concentrations were measured using an OptEIA ELISA Kit (Beckton Dickinson Biosciences, San Diego, CA, USA). Plasma and urinary myeloperoxidase (MPO) and calprotectin concentrations and plasma lipopolysaccharide binding protein (LBP) concentrations were measured using ELISA kits from Hycult Biotech (Uden, The Netherlands). Plasma neutrophil gelatinase-associated lipocalin (NGAL) concentration was measured using a Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA), and urinary NGAL concentration was measured using BioPorto^® NGAL ELISA Kits (Enzo Life Sciences, Farmingdale, NY, USA). Plasma and urinary ileal-bile acid binding protein (I-BABP) concentrations were measured using a FABP6 ELISA kit (BioVendor Laboratory Medicine, Brno, Czech Republic). Plasma and urinary kidney injury molecule 1 (KIM-1) and cortisol concentrations were measured using an ELISA kit (Enzo Life Sciences). Plasma and urinary liver-fatty acid binding protein (L-FABP) were measured using a Human FABP1 Wide-range ELISA Kit (Uscn Life Science, Wuhan, China). Plasma aldosterone concentration was measured using an ELISA kit (Enzo Life sciences, Farmingdale, NY, USA), and urinary aldosterone concentration was measured using an Aldosterone Parameter Assay Kit (R&D Systems, Minneapolis, MN, USA). Plasma and urinary nitrotyrosine concentrations were measured using an ELISA Kit (StressMarq Biosciences, Victoria, BC, USA). Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) concentration was measured using an ELISA Kit (StressMarq Biosciences, Victoria, BC, USA). Plasma endotoxin concentration was measured via PYROGENT™-5000 Kinetic Turbidimetric LAL Assay test (LONZA, Walkersville, MD, USA). When we measured endotoxins, we used endotoxin-free tubes, tips, reservoirs and microplates. The absorbance was measured spectrophotometrically on a VersaMax Microplate Reader (Molecular Devices Inc., San Jose, CA, USA) or Spectra Max iD5 (Molecular Devices Inc., San Jose, CA, USA). Plasma and urinary thiobarbituric acid reactive substances (TBARS) concentrations were measured fluorescently using a TBARS Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA). The fluorescence was measured on a FLUOstar Optima plate reader (BMG Labtech Ltd., Ortenberg, Germany). The concentrations of each parameter were calculated by comparison with the standard curve established in the same measurement.

Plasma and serum parameters (except serum osmolality) were adjusted according to the percentage change in plasma volume calculated from hemoglobin and hematocrit levels [24]. Urinary parameters were adjusted as the gross amount per minute (raw concentration × urine volume/time) to correct urine condensation as previously described [7].