U 32012 centrifuge

Whole blood and urine were collected at four time points: baseline (precontrast) and 24 hours, 48 hours, and 5 to 7 days after contrast administration. Blood and urine samples were centrifuged at 5000 rpm at 4°C for 10 and 2 minutes, respectively (U-32012 Centrifuge, Boeco, Germany), and the sera and urine were stored at −80°C until assayed. Concentrations of IL10, IL18, TNFα, NGAL, KIM1, and cystatin C were determined using Magnetic Luminex® Screening Assays (#LXSAHM-3, R&D Systems, Inc., Minneapolis, USA) in accordance with the manufacturer's instructions on the BioPlex™ 200 system (Bio-Rad, Texas, USA). The Bio-Plex manager software, version 5, was used for the determination of concentrations. Serum concentrations of β₂M were determined by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc.). Serum creatinine was determined using the Jaffe method.

Plasma samples were immediately centrifuged at 3000× g for 15 min at 4 °C (U-32012 Centrifuge, Boeco^®, Hamburg, Germany) upon collection and stored at −80 °C. Plasma Fetuin-A and adiponectin concentrations were determined by using enzyme-linked immunosorbent assays (Fetuin-A, Human Fetuin-A Immunoassay, R&D Systems^®, Minneapolis, MN, USA; Adiponectin, Human Total Adiponectin/Acrp30 Immunoassay, R&D Systems^®, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. The manufactural dilution factor was used while each sample ran in duplicate. The average coefficients of variation for plasma intra-assay (inter-assay) precision were 4.3% (8.0%) and 3.5% (6.5%) for Fetuin-A and adiponectin, respectively. The FAR value was derived from fasting Fetuin-A (ng/mL) divided by adiponectin (ng/mL).

Upon collection, blood and urine samples were immediately (within 30 min) centrifuged at 3000× g at 4 °C for 15 min (U-32012 Centrifuge, Boeco^®, Hamburg, Germany) and stored at −80 °C until assayed. The concentrations of plasma and urinary NGAL and KIM-1 were determined using commercially available enzyme-linked immunosorbent assays (NGAL, Human Lipocalin-2/NGAL Immunoassay, R&D Systems^®, Minneapolis, MN, USA; KIM-1, Human TIM-1/KIM-1/HAVCR Immunoassay, R&D Systems^®, USA) in accordance with the manufacturer’s instructions and the pre-test recommended dilution factor provided by the individual assay kits. A microplate reader (SpectraMax 190 and SoftMax Pro7.0.2, Molecular Devices^®, San Jose, USA) was used for the determination of concentrations and all samples were run in duplicate. The average coefficients of variation for intra-assay (inter-assay) precisions were 4.0% (6.7%), 4.0% (6.7%), 2.9% (6.4%) and 3.0% (6.2%) for plasma NGAL, urinary NGAL, plasma KIM-1 and urinary KIM-1, respectively. A few of the urinary NGAL and KIM-1 concentrations were under the detectable range and were therefore approximated to the lowest measurement of the population. Although not completely within the standard range, several concentrations were predicted based on the trend of the standard curve. The urinary levels of NGAL and KIM-1 were adjusted to the urinary Cr concentration.