Acyl coa oxidase based colorimetric kit

Determination of triglycerides in liver, heart, skeletal muscle and measuring of cholesterol in liver were performed as described previously (Qi et al. 2002) . Briefly, tissues were powdered under liquid N 2 and extracted in the mixture of chloroform: methanol (2:1). Afterwards, the solution of 2 % potassium dihydrogenphosphate was added and an organic phase formed in the mixture was taken and evaporated under N 2 . The residue was dissolved in isopropyl alcohol. Triglycerides (TAG) and cholesterol concentrations were determined by enzymatic assay (TG L 250S, Erba-Lachema, Brno, Czech Republic). Blood glucose concentrations were measured by the glucose oxidase assay (Erba-Lachema, Brno, Czech Republic) using tail vein blood drawn into 5 % trichloracetic acid and then centrifuged. Serum triglycerides and cholesterol concentrations were determined by standard enzymatic methods (Erba-Lachema, Brno, Czech Republic). Serum nonesterified fatty acids (NEFA) levels were analyzed using an acyl-CoA oxidase-based colorimetric kit (Roche diagnostics, Mannheim, Germany). Serum insulin concentrations were measured using a rat insulin ELISA kit (Mercodia, Uppsala, Sweden).

Rat serum CRP and human serum CRP were measured using ELISA kits (Alpha Diagnostics International, San Antonio, USA). Blood glucose levels were measured by the glucose oxidase assay (Erba-Lachema, Brno, Czech Republic) using tail vein blood drawn into 5 % trichloracetic acid and promptly centrifuged. NEFA levels were determined using an acyl-CoA oxidase-based colorimetric kit (Roche Diagnostics GmbH, Mannheim, Germany). Serum triglyceride and cholesterol concentrations were measured using standard enzymatic methods (Erba-Lachema, Brno, Czech Republic). Serum insulin concentrations were determined using a rat insulin ELISA kit (Mercodia, Uppsala, Sweden). Serum IL6 was measured using rat ELISA kits (BioSource International, Inc., Camarillo, USA). Rat MCP-1 was measured by ELISA kits (Instant ELISA eBioscience, Austria). ALT, AST, and ALP enzyme activities were determined spectrophotometrically by routine clinical biochemistry methods using a Roche kit. HDL-cholesterol was measured after double precipitation with dextran and MgCl 2 by spectrophotometric methods using commercially available kits (Roche Diagnostics GmbH, Mannheim, Germany).

Folate levels in serum and urine were determined by the Folate III Assay Kit (Roche GmbH, Basel, Switzerland) (the coefficient of variation for the assays for folate is <5 %). Concentrations of total homocysteine, cysteine, glutathione (GSH), GSH precursor gamma-glutamylcysteine and GSH degradation product cysteineylglycine in plasma were determined by reversed-phase HPLC with fluorescent detection after derivatization with ammonium 7-fluorobenzo-2-oxa-1,3diazole-4-sulfonate. The reduction of disulfides and protein bound homocysteine and cysteine was performed with tris(2-carboxyethyl)phosphine as described previously (the coefficients of variation for the assays for homocysteine and cysteine are <3 %).
Blood glucose levels were measured by the glucose oxidase assay (Erba-Lachema, Brno, Czech Republic) using tail vein blood drawn into 5 % trichloroacetic acid and promptly centrifuged. NEFA levels were determined using an acyl-CoA oxidase-based colorimetric kit (Roche Diagnostics GmbH, Mannheim, Germany). Serum triglyceride concentrations were measured by standard enzymatic methods (Erba-Lachema, Brno, Czech Republic). Serum insulin concentrations were determined using a rat insulin ELISA kit (Mercodia, Uppsala, Sweden).