Estimation of Free and Bound MDA in Plant Extracts: Comparison Between Spectrophotometric and HPLC Methods

Abstract

Lipid peroxidation is a complex process in which polyunsaturated fatty acids (PUFAs) are subjected to attack by oxygen-derived free radicals resulting in the formation of lipid hydroperoxides. In biological tissues, these lipid hydroperoxides are broken down into a variety of products including aldehydes and ketones [1]. The extend of lipid peroxidation is considered to be an important parameter for the identification of the oxidative stress [2]. The improvement of a technique for the measurement of this lipid peroxidation in living tissues is, consequently, of importance. A large variety of methods are available for the detection of lipid peroxidation products. These include determination of diene conjugation, lipid hydroperoxides, chemiluminescence, hydroxy acids, ethane and Thiobarbituric acid (TBA)-reactive materials. The estimation of TBA reactive products is the most widely used. The success of the TBA reaction method depends on the accuracy of the determination of MDA content. MDA is a three carbon dialdehyde which is widely produced in organisms as a end product of polyunsaturated lipid peroxidation. This method is of particular interest because of its simplicity and relatively good sensitivity, but it lacks specificity and often leads to overestimation due to other interfering compounds ([3],[4]). Treatment of biological sample with TBA under appropriate conditions results in the formation of pinkcolored products (λmax = 532 nm) and many substances which are not related to lipid peroxidation (sugars, amino acids, biliverdin, chemicals, glyoxal,...) may also form a TBA complex with significant absorbance around 530–535 nm.