Dca2000 hba1c reagent kit

The blood HbA1c level was measured using the DCA2000 HbA1c reagent kit (SIEMENS Healthcare Diagnostics, Inc., Tarrytown, NY, USA). The blood was centrifuged at 900 g for 15 min at 4 °C, and the supernatant was collected. The levels of plasma ALT, cholesterols, triglycerides, and free fatty acids were measured using an EnzyChrom™ colorimetric assay kit (BioAssay Systems, Hayward, CA, USA). The levels of plasma insulin were measured using ELISA kits (Millipore Corporation, Billerica, MA, USA) according to the manufacturer’s protocol. The plasma and the liver levels of malondialdehyde, as detected by a reaction with thiobarbituric acid, were used as an index of the level of LPO. Plasma and the livers were mixed with 0.8% (w/v) thiobarbituric acid in acetic acid and incubated for 1 h in boiling water. After cooling, the absorbance of malondialdehyde was measured at an excitation of 555 nm and an emission at 515 nm. The levels of malondialdehyde were determined based on a standard curve generated with 1,1,3,3-tetraethoxypropane as the standard. TGF-β1 protein expression in the livers was assessed with commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s recommendations. The intra-assay coefficient of variation (CV) was 2.8%, and the inter-assay CV was 7.5%.

Blood samples were collected with a heparinized syringe before mice were sacrificed. Blood glucose level was determined using the glucose oxidase method. HbA1c level was determined using the DCA2000 HbA1c reagent kit (SIEMENS Healthcare Diagnostics, Inc., Tarrytown, NY, USA). Plasma creatinine level was determined using a Detect X Serum Creatinine Detection Kit (Arbor Assays, Ann Arbor, MI, USA). Cystatin C level was measured using a cystatin C Elisa kit (W Systems, Minneapolis, MN, USA). LPO level was measured by reacting with thiobarbituric acid (TBA) as described previously [33 (link)].