Direct Method for Quantification of Free Malondialdehyde with High-Performance Capillary Electrophoresis in Biological Samples

Abstract

Lipid peroxidation, a general mechanism of tissue damage by free radicals, damages cells and may induce pathological events (1). Analytical tests to assess lipid peroxidation include the following techniques: fluorometry of lipofuscin-like substances in serum (2); spectrophotometry of conjugated dienes in lipid extracts of plasma and microsomes (3)(4); gas chromatography of ethane or pentane in exhaled breath (5); hydroperoxide determination (6); malondialdehyde (MDA) evaluation with or without thiobarbituric acid (TBA) reaction and spectrophotometry (7), fluorometry (8), and high-performance liquid (9)(10) or gas–liquid (11)(12) chromatographies; measurements of other saturated and unsaturated aldehydes (13); and oxygen uptake during lipid peroxidation (14). The most widely used method is the TBA reaction with MDA. This method is of particular interest because of its procedural simplicity and nanomolar sensitivity. However, its specificity has been questioned (10)(11)(12)(13)(14)(15)(16), and it overestimates MDA (17)(18)(19)(20)(21). We describe a method to measure free MDA with use of high-performance capillary electrophoresis (HPCE). We used a Waters Quanta-4000 Capillary System with UV detection at 280 nm, AUFD 0.002, mercury lamp, phosphor window and cElect-P175 (Supelco) neutral hydrophilic phase capillary (75 μm x 60 cm). Buffer with 10 mmol/L sodium tetraborate, 10 mmol/L sodium monophosphate (Sigma Chemical Co.), pH 8.0, with 100 mL/L acetonitrile (analytical grade, Fisher Scientific) was filtered with a 0.45-μm filter; running voltage was +18 kV. Samples were injected hydrostatically in 10 s. Data were recorded on a Waters-746 integrator. In …