Determination of Malonaldehyde Precursor in Tissues by Thiobarbituric Acid Test

A thiobarbituric acid (TBA) test procedure with reasonable reproducibility applicable to the assay of lipoperoxides in various animal tissue homogenates is described. It was concluded that the deproteinization of homogenate prior to coloration is not needed, but double wavelength measurement is necessary to avoid interference and the reaction should be performed with phosphoric acid at a definite pH near 2.0. The most reproducible procedure is as follows: To 0.5 ml of 10% homogenate of the tissue sample, add 3 ml of 1% H3PO4 and 1 ml of 0.6% TBA aqueous solution; stir and heat the mixture on a boiling water bath for 45 min. After cooling, add 4 ml of n-butanol, shake, and separate the butanol layer by centrifugation; determine the optical density of the butanol layer at 535 and 520 nm; and calculate the difference of optical density between the two determinations to be taken as the TBA value.