A Simple Stabilization Method of Reduced Albumin in Blood and Plasma for the Reduced/Oxidized Albumin Ratio Measurement

Albumin (Alb) is mixture of reduced and oxidized forms. It is physiologically significant to determine Alb(red)%, which is the proportion of reduced Alb in the sum of Alb. However, reduced Alb in both blood and plasma samples is easily converted to oxidized Alb. Accordingly, the stabilization of Alb in samples is necessary to determine precise Alb(red)% values. Alb stabilization in blood or plasma was achieved by pH control and buffer dilution. At least a 50-fold dilution with 50 mmol/l phosphate buffer (pH 6.0) was required for human plasma. For human blood, a 10-fold dilution with 0.5 mol/l sodium citrate buffer (pH 4.3) was required. To measure Alb(red)%, treated samples were applied to HPLC or LC-ESI-TOFMS. We also developed a “pre-incubation method”, to accelerate the oxidative reaction in plasma by heating at 37°C. Alb(red)% values were maintained around the initial value for 48 h after stabilizing human plasma and 72 h after stabilizing human blood. Accelerating the oxidative reaction in plasma produced large differences in the Alb(red)% values between normal and model disease samples. Precise Alb(red)% values were routinely obtained under the stabilization control. Additionally, pre-incubation of the plasma before measurement is useful to enhance the difference between normal and disease samples.