Nonchromatographic Assay for Malondialdehyde–Thiobarbituric Acid Adduct with HPLC Equivalence

Abstract

Reaction of malondialdehyde (MDA) with thiobarbituric acid (TBA) or its diethyl derivative has been applied widely to assess lipid peroxidation in biological material (1). The reaction yields a red MDA-TBA adduct, the product of 2 mol of TBA plus 1 mol of MDA (2). The colored complex can be quantified spectrophotometrically from its visible absorbance (λmax 532 nm) (3)(4) or by spectrofluorometry (exc 532 nm, em 553 nm) (5)(6) and is readily extractable into organic solvents such as butanol (7). Preanalytical factors aside (8)(9)(10), interfering chromogens include several other ordinary side-products of lipid autooxidation, alkanals, alkenals, and alkadienals as well as bile pigments, cyclic peroxides, carbohydrates, and amino acids (11)(12)(13)(14)(15)(16)(17)(18). Isolation and quantification of the MDA-TBA adduct by HPLC reportedly largely eliminates the interfering chromogens (19)(20). HPLC methods generally yield lower values than those based on direct measurements, and this would seem to reinforce their specificity. However, mean reference values for lipoperoxide concentrations in plasma of healthy adults (0.60 ± 0.13 μmol/L; n = 41) were found by Wong et al. (21), who used a specific HPLC method, to be consistent with those of Francesco et al. (22) (0.61 ± 0.11 μmol/L; n = 20), without chromatographic separation. Similarly, Fukunaga et al. (23) reported but failed to explain a close correlation ( r = 0.972, n = 14) between their specific HPLC assay (2.51 ± 0.75 μmol/L; MDA values taken from correlation plot) and the traditional direct Yagi method (5) (2.21 ± 0.54 μmol/L; similarly calculated). In a discipline where published reference values can vary 100-fold (24), these examples represent remarkable concordance and question the ability of HPLC to reduce spectrophotometric interference to the MDA-TBA …