Empower 2 system software

The redox state of albumin was determined by high-performance liquid chromatography (HPLC). The HPLC system consisted of an M-504F autosampler (Eicom, Kyoto, Japan; injection volume, 20 μL of plasma diluted by 10-fold with 50 mM sodium phosphate, 150 mM sodium chloride buffer) and a 1525 binary pump (Waters, Milford, MA, USA) as well as Empower 2 system software (Waters). The chromatograph was obtained using a 2996 photodiode array detector (detection area, 210–400 nm with 1 nm step; Waters). A Shodex-Asahipak ES-502N 7C column (10 × 0.76 cm I.D., DEAE-form for ion-exchange HPLC; Showa Denko, Tokyo, Japan; column temperature, 35.0 ± 0.5°C) was used in this study. Linear gradient elution was performed with varying ethanol concentrations (0-1 min, 0%; 1–50 min, 0→10%; 50–55 min, 10→0%; and 55–60 min, 0%) for serum in 0.05 M sodium acetate and 0.40 M sodium sulfate mixture (pH 4.85) at a flow rate of 1.0 mL/min. Deaeration of the buffer solution was performed with an inline degasser AF (Waters).
HPLC profiles obtained from these procedures were subjected to numerical curve fitting with PeakFit version 4.05 simulation software (SPSS Inc., Chicago, IL, USA), and each peak shape was approximated by a Gaussian function (Figure 1). Next, the levels for the HMA, HNA-1, and HNA-2 compared to total HSA were calculated (ƒ(HMA), ƒ(HNA-1), and ƒ(HNA-2), resp.).