Srage

Blood samples were obtained within 6 h of admission to the ICU. The plasma was separated and then stored at − 80 °C until further processing. Previously collected and frozen serum samples were used to determine the levels of sRAGE (R&D Systems, Minneapolis, MN) and esRAGE (B-Bridge International, Sunnyvale, CA) by a commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit. During the ELISAs, all measurements were performed according to the manufacturer’s instructions.

Blood samples were collected at enrollment and serum was obtained by centrifuging blood at 3000 rpm at 4°C for 10 minutes. Specimens were stored at –80°C. Plasma electrolytes were measured on a Hitachi 911 automatic analyzer (Roche Diagnostics, Mannheim, Germany). The eGFR was calculated using the four-component abbreviated Modification of Diet in Renal Disease equation. Glycated hemoglobin (HbA1c) was measured by automated high-performance liquid chromatography (Biorad Diamat, CA, USA), and fasting lipids by enzymatic colorimetry. Total cholesterol was measured by enzymatic colorimetric methods and low-density lipoprotein cholesterol was calculated using the Friedewald equation. Serum C-reactive protein levels were measured using the Synchron LX® system on a Beckman Coulter analyser.
Serum sRAGE (R&D Systems, Minneapolis, MN) and esRAGE (B-Bridge International, Cupertino, CA) were measured using a sandwich ELISA according to the manufacturer’s instructions. The limit of detection was 4.1 pg/ml and 25 pg/ml respectively. The intra-assay and inter-assay coefficients of variation values for sRAGE were 6.0% and 7.2% respectively and for esRAGE were 1.5% and 10.0% respectively. Samples were run in duplicate and the mean values used for analysis.

Blood was sampled from all patients and sera were separated from whole blood after complete coagulation, by centrifugation at 3000 rpm for 10 min. Sera were stored at −70 °C until ELISA assay analyses.
Levels of soluble RAGE, C-Terminal FGF23, Leptin, Adiponectin and Visfatin in serum were determined by commercial assays, according to the manufacturers’ instructions (sRAGE: R&D Systems, Minneapolis, Minnesota, USA; C-Terminal FGF23: Imuunotopiscs, San Clemente, CA, USA; Leptin: Enzo Life Sciences, Farmingdale, New York, USA; Adiponectin and Visfatin: AdipoGen AG, Liestal, Switzerland).
For the sRAGE assay, the sensitivity was 4.44 pg/mL, and intra- and inter-assay coefficients of variation were 2.4% and 4.7%, respectively. For the FGF23 assay, the sensitivity was 1.5 RU /mL, and intra- and inter-assay coefficients of variation were2.4% and 4.7%, respectively. According to manufacturer (, Imuunotopiscs, San Clemente, CA, USA) 1RU roughly equates to 2 pg/mL. For the Leptin assay, the sensitivity was 23.4. pg/mL, and intra- and inter-assay coefficients of variation were 4.4% and 3.7%, respectively. For the Adiponectin assay, the sensitivity was 1 ng/mL, and intra- and inter-assay coefficients of variation were 3.3% and 2.75%, respectively. For the Visfatin assay, the sensitivity was 30.0 pg/mL, and intra- and inter-assay coefficients of variation were 2.3% and 4.6%, respectively.

Concentrations of RAGE were determined in serum and sputum using ELISA kits sRAGE (R&D Systems, USA), esRAGE (B-Bridge International, USA) and the RAGE ligands: enRAGE (CircuLex, USA) and AGEs (Cell Biolab, USA), using standard protocols.

Levels of soluble RAGE (Receptor of Activated Glycoslation Endproducts) in plasma were determined by ELISA commercial assays, according to the manufacturers’ instructions (sRAGE: R&D Systems, Minneapolis, Minnesota, USA). For the sRAGE assay, the sensitivity was 4.44 pg/mL, and intra- and inter-assay coefficients of variation were 2.4 and 4.7%, respectively.

For each patient, 2 mL of fresh blood was drawn into a vacuum tube containing EDTA at the following time points: 20 minutes after the induction of anesthesia in the supine position without pneumoperitoneum (T1), 40 minutes after the 30° Trendelenburg position with pneumoperitoneum (T2), at skin closure in the supine position (T3), and 24 hours after the operation (T4). After centrifugation at 3000 rpm for 15 minutes at 4 °C, the plasma was divided into aliquots and frozen at −80 °C until assay. sRAGE and S100A12 levels were measured using commercially available ELISA kits (sRAGE: R&D Systems, Minneapolis, MN, USA; S100A12: Cirulex; Cyclex Co. Ltd., Nagano, Japan), according to the manufacturer’s instructions. The total levels of plasma protein were determined with an autobiochemistry analyzer and used for sRAGE and S100A12 normalization (sRAGEN, sRAGE normalized for total protein; S100A12N, S100A12 normalized for total protein). Laboratory staff was blinded to postoperative respiratory complications, and investigators involved in the interpretation of postoperative respiratory complications were blinded to sRAGE and S100A12 levels.

Blood was collected in an evacuated tube containing EDTA (Sherwood Medical, St Louis, Missouri, USA) and immediately centrifuged at 800 rpm for 10 min at 4°C. The plasma samples were subsequently stored at -80°C until analysis. Plasma esRAGE (B-Bridge international Inc., UK) and sRAGE (R&D systems, Minneapolis, USA) were measured by ELISA. Plasma protein-bound CML and CEL were measured by liquid chromatography tandem mass spectrometry [18 (link)] and pentosidine was measured by HPLC with fluorescence detection [19 (link)] and expressed per lysine concentrations. Plasma levels of high density lipoproteins (HDL), triglycerides, glucose, C–reactive protein (CRP) and creatinine were measured in an auto-analyzer (ABX Pentra 400, HORIBA ABX S.A.S, France). Glomerular filtration rate (GFR) was calculated using the Cockcroft-Gault formula [20 (link)].