Free malondialdehyde determination by HPLC applied to microsomal studies.

Malondialdehyde (MDA) is a product of lipid peroxidation in vivo. The most widely employed method for determination of free MDA is based on its reaction with thiobarbituric acid (TBA) which produces a pink pigment with an absorption maximum at 532-535 nm. However, quantitation of MDA is limited by its lack of specificity and a high performance liquid chromatographic (HPLC) method was recently developed in several laboratories. In the present study, free MDA levels were measured, after TBA reaction, spectrophotometrically and by HPLC in microsomes of different tissues from rats fed a vitamin A-deficient diet or not for 8 weeks, and treated or not with carbon tetrachloride. Incubation in vitro with NADPH (0.25 mM) or ascorbate (0.50 mM) in the presence of Fe2+ (5 microM)-ADP (0.5 mM), allowed us to estimate the total amount of enzymatic or non enzymatic lipoperoxidation. The MDA amount determined by HPLC is significantly lower than the TBA-reactive substances (TBA-RS) calculated spectrophotometrically as MDA equivalents. Moreover, HPLC separations performed on a mu Bondapack C18 column with a mobile phase of methanol/water 45/55 (v/v), containing 1% cetrimide revealed that three chromogens are present in microsomes incubated with ascorbate or NADPH. The TBA-RS visible spectra of microsomes incubated with activator are complex with an absorption maximum at 533 nm, which is specific for the MDA-TBA chromogen, and one at 450 nm. Identification of these TBA-RS, different from the MDA-TBA complex, is under investigation in our laboratory.