FREE AND BOUND MALONDIALDEHYDE MEASURED AS THIOBARBITURIC ACID ADDUCT BY HPLC IN SERUM AND PLASMA

Assay of free and total malondialdehyde (MDA) in human serum and plasma from healthy subjects and from patients with high risk of lipoperoxidation was performed as follows: (a) acidic (HClO4, pH 1, at 20-degrees-C) or basic (NaOH, pH 13, at 60-degrees-C) treatments for 30 min; (b) reaction of the protein-free extract (obtained by acid precipitation) with thiobarbituric acid (TBA); (c) HPLC separation on C-18 columns with an eluting solution of methanol/phosphate buffer, 10 mmol/L, pH 5.8 (40/60, by vol), at a flow rate of 1.5 mL/min. Free MDA averaged 0.042 (SEM 0.008) and 0.043 (SEM 0.007) mu-mol/L, respectively, in serum and plasma from healthy subjects. Free (+/- SEM) MDA increased significantly in the plasma from cancer patients (0.270 +/- 0.047-mu-mol/L) and from hemodialyzed patients (0.214 +/- 0.035-mu-mol/L). In serum of hemodialyzed patients, analyses for total MDA were unsuitable because of interfering peaks. MDA bound to NH2 groups constituted 83.2% and 83.5% of total MDA in serum and plasma of healthy subjects, respectively, and only 58% in plasma of hemodialyzed patients.