F 1050

Chromatographic analysis of serum albumin in haemodialysis patients was performed as described previously24 (link). High-performance liquid chromatography (HPLC) analysis of 5 μL aliquots of each serum sample was performed using a Shodex Asahipak ES-502N column (Showa Denko Co., Ltd., Tokyo, Japan; column temperature, 35 ± 0.5 °C). The HPLC system was composed of an intelligent pump (L-6200) equipped with a gradient programmer and an F-1050 fluorescence detector (Hitachi Co., Ltd., Tokyo, Japan). Elution was performed using a linear gradient with increasing ethanol concentrations from 0% to 5% for serum in 0.05 mol/L of sodium acetate and 0.40 mol/L of sodium sulfate mixture (pH 4.85) at a flow rate of 1.0 mL/min. From the HPLC profiles of serum albumin, the value of oxidized albumin content was estimated by dividing the area of oxidized albumin by the total area corresponding to albumin. The serum level of 8-isoprostane was measured using an enzyme-linked immunosorbent assay kit (Detroit R&D Inc. Detroit, MI, USA).

Plasma albumin was measured by HPLC, as described previously [14 (link)]. The HPLC system was composed of an intelligent pump L-6200 equipped with a gradient programmer and an F-1050 fluorescence detector (Hitachi Co., Ltd., Tokyo, Japan). Shodex Asahipak ES-502N column (Showa Denko Co., Ltd., Tokyo, Japan) was used as the stationary phase. The mobile phase consisted of (A) 0.05 mol/L sodium acetate and 0.40 mol/L sodium sulphate mixture (pH 4.85) and (B) 100% ethanol. The gradient step started with 0% of solvent B and finally reached 5% in 30 min. The flow rate was 1.0 mL/min. The excitation/emission wavelengths were 280/340 nm, respectively. From the HPLC profiles, the content of human mercaptalbumin (HMA) and human non-mercaptalbumin (HNA) was estimated by dividing the area of each fraction by the total area corresponding to HSA, as described previously [14 (link)].