A new thiobarbituric acid (TBA) method for determining free malondialdehyde (MDA) and hydroperoxides selectively as a measure of lipid peroxidation

Abstract

A new thiobarbituric acid (TBA) method for selectively determining free malondialdehyde (MDA) and lipid hydroperoxides is described. Partitioning of MDA and hydroperoxides in a Bligh and Dyer extraction was studied with pure substances. It was shown that MDA was present in the methanol-water phase exclusively, and the hydroperoxides (polar as well as nonpolar) were found in the chloroform phase. Therefore, the TBA test on these phases determines MDA directly present in the sample and the MDA formed during a ferric ion.catalyzed cleavage of hydroperoxides, respectively. The MDA present in the methanol-water phase was not hound to amino groups. Hydroperoxide values obtained with the present method corresponded well with a eolarimetric determination of peroxides and showed better linearity for higher amounts of hydroperoxides. The possibility of using an iron chelator for preventing hydroperoxide eleavage during the TBA reaction, making the determination selective for MDA without Bligh and Dyer extraction, was investigated. The iron chelator did not completely inhibit peroxide cleavage; therefore, it is necessary to perform the extraction berate the TBA determination. The method is suitable for samples with low fat content such as cod mince.The most common method for measuring oxidative changes in biological samples and food products is the thiobarbituric acid (TBA} test based on a spectrophotometric quantitation of a red-violet complex formed with malondialdehyde (MDA) (1}. This method determines both the MDA already formed naturally from hydroperoxide cleavage, and the secondary release due to the heating step in the TBA reaction. Therefore, the TBA values obtained are dependent on methodology and not easily reproduced between laboratories. The TBA test is often used for determination of lipid peroxides (2-4), but the present methods measure both free MDA and lipid peroxides. Several investigators have reported selective methods for determining free MDA by HPLC (5-9). Furthermore, HPLC has often been used for determining the MDA-TBA adduct selectively (10-12). However, the adduct measured can arise from both peroxides and free MDA.In samples with low lipid content the TBA determination on tipids isolated by a Bligh and Dyer extraction (13) has been suggested to increase the sensitivity (14), but the authors did not elucidate whether the reaction was due to MDA, hydroperoxides or both. Neither was the possible loss of MDA by evaporation accounted for.Albro et at. (] 5) described a method for measuring free MDA and "labile lipid peroxidation products" selectively. This method, however, is based on the fact that lipids Presented in part at the AOCS Annual Meeting in New Orleans, Louisiana, in May 1987. co-precipitate when proteins are precipitated with trichloroacetic acid (TCA) in a microsomal system. The method, therefore, is not applicable for most food products and oils.A selective TBA determination of free MDA and hydroperoxides, measured as MDA formed during the TBA reaction, has not been reported previously. T...

Methods for Evaluation of the Oxidative Stability of Lipid-Containing Foods.

“…Modifications of the original TBA test have been reported by Marcuse & Johansson (1973), Ke & Woyewoda (1979), Robles-Martinez et a/ (1982), Pokorny et al (1985), Shahidi et al ( 1987), Tomas & Funes ( 1987) and Schmedes & Holmer ( 1989). However, it has been suggested TBARS provide an excellent means for evaluating the relative oxidative status of a system as affected by storage or processing variables (Gray & Pearson, 1987 (Gray, 1978) and canola (Hawrysh, 1990).…”

Antioxidative Effect of Rice Bran Oil and Medium Chain Fatty Acid Rich Rice Bran Oil in Arsenite Induced Oxidative Stress in Rats

“…Then, 10 ml of chloroform was added and homogenization continued for another 30 s. Finally, 10 ml of redistilled water were added and the mixture was homogenised for 30 s. The tubes were then centrifuged for 20 min at 4000 rpm, and the chloroform layer was separated 27 . Thiobarbituric acidreactive substances were measured according to the method described by Schmedes & Hølmer 28 .…”

A randomized double blind comparison of short-term duodenally administrated whale and seal blubber oils in patients with inflammatory bowel disease and joint pain

“…TBARS was determined in marine oils by a modified in vitro method, measuring mainly malondialdehyde and other aldehydes, secondary lipid oxidation parameters [40,41].…”