[Structure and function changes of oxidized human serum albumin: physiological significance of the biomarker and importance of sampling conditions for accurate measurement].

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases; however, little is known regarding the pathological and physiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both structural and functional differences between reduced and oxidized HSA. Using LC-ESITOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA, including protease susceptibility, ligand-binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions. In addition, we determined blood and plasma sampling conditions for accurate measurement of the oxidized albumin ratio in plasma using EST-TOFMS screening.

Identification and characterization of oxidized human serum albumin. A slight structural change impairs its ligand-binding and antioxidant functions.

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases. However, little is known regarding the pathophysiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both the structural and functional differences between reduced and oxidized HSA. Using LC-ESI-TOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA including protease susceptibility, ligand-binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions.

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.