Determination of Malondialdehyde as Dithiobarbituric Acid Adduct in Biological Samples by HPLC with Fluorescence Detection: Comparison with Ultraviolet-Visible Spectrophotometry

Abstract

Living cells are constantly exposed to reactive oxygen species, some of which are capable of initiating lipid peroxidation by abstraction of an allylic proton from a polyunsaturated fatty acid. This process, by multiple stages leading to the formation of lipid hydroperoxides, is a known contributor to the development of atherosclerosis (1). The thiobarbituric acid (TBA) test is an easy and quick assay for the assessment of lipid peroxidation in which malondialdehyde (MDA) is derivatized. The rationale and methodology have been discussed in detail elsewhere (2)(3) and have rightfully been criticized for low specificity and artifact formation because only a fraction of the MDA measured was generated in vivo (1)(3). Furthermore, the TBA derivatization procedure itself leads to the formation of several MDA-unrelated ultraviolet (UV)-absorbing and fluorescent species, the latter as shown in Fig. 1⇓ , A–C. Despite this fact, the method remains one of the most useful and commonly used measurements of oxidative damage because of its simplicity. In recent years, several HPLC-based TBA assays have evolved with increased specificity (4)(5)(6)(7), but nevertheless the spectrophotometric methods remain commonly used. In the present report, MDA was quantified in plasma, erythrocytes, and liver homogenates from 3-month-old guinea pigs by either our “modern” TBA test based on HPLC with fluorescence detection, which quantifies only the genuine MDA(TBA)2 adduct, or by the original less specific and less sensitive spectrophotometric method, which measures the total absorbance of several species. The purpose was to evaluate the use of the original TBA test by today’s standards by assessing the overestimation and sensitivity in the various applications that are routinely performed in several laboratories. All compounds were of the highest quality available. Specifically, butylated hydroxytoluene (BHT), tetramethoxypropane, TBA, and phosphotungstic acid were from Fluka. Sample …