Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury.

A rapid aqueous acid extraction thiobarbituric acid method for measuring malondialdehyde as a marker of lipid peroxidation in animal tissue, food, and feedstuff samples has been developed. Sample is homogenized with aqueous trichloroacetic acid in the presence of hexane and butylated hydroxytoluene, and the homogenate is centrifuged. Following reaction with thiobarbituric acid reagent, malondialdehyde is directly quantified on the basis of the third-derivative absorption spectrum of the pink complex formed. Further purification is not required because the derivative transformation of the conventional analytical band at around 532 nm virtually eliminates spectral interferences arising from other compounds. The effect of antioxidants and the optimum conditions for the reaction have been established, and the analytical performance of the new method has been evaluated. The applicability of the method on various animal tissue, food, and feedstuff samples has been also tested. Owing to its simplicity and increased sensitivity and specificity, the method may be preferred over other methods for estimating the extent of lipid peroxidation.

Rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food, and feedstuff samples

Publisher Summary Malonaldehyde (MA) is of interest primarily as a product of lipid peroxidation in vivo and as an index of oxidative rancidity in foods. In biological materials, it exists in its free form and as a complex with various tissue constituents. It reacts with a variety of compounds to form derivatives, which can be estimated spectrophotometricaily. It has also been identified among the products of the oxidative decomposition of amino acids, complex carbohydrates, pentoses, and hexoses formed in the presence of a metal catalyst, as a product of free radicals generated by ionizing radiation in vivo, and as a byproduct of prostaglandin biosynthesis. However, peroxidation of fatty acids with three or more double bonds (notably arachidonic acid) is believed to be its major source. Because of its interest as an indicator of lipid peroxidation, various methods have been proposed for its estimation. The most widely employed method for the determination of MA in biological materials is based on its reaction with thiobarbituric acid (TBA).

Comparative studies on different methods of malonaldehyde determination.

The wide diffusion of 2-thiobarbituric acid (TBA) in the scientific literature is due to the TBA assay, or TBA test, which has been employed in the determination of autoxidative alterations of fats and oils. Two processes occur in autoxidation, generally: the free radical and the photo-oxidation mechanisms. The better studied is the free radical mechanism. The hydroperoxiepidioxides and bicycloendoperoxides are malonaldehyde (MDA) precursors. The absorption spectrum obtained with oxidized fatty foods is like the spectrum obtained when TBA and MDA react. However, during the secondary phase of the autoxidation process other aldehydes (alkanals, 2-alkenals, dienals) are formed which react with TBA, and they are responsible for off-flavors. Three kinds of pigments (yellow, orange, red adducts) are involved. Also, aromatic aldehydes, which constitute the flavor profile of diverse fruits and essential oils, form with TBA the characteristic arylidene-2-TBA acids. Other substances, such as ketones, ketosteroids, acids, esters, sugars, imides and amides, amino acids, oxidized proteins, pyridines, pyrimidines, and vitamins can react with TBA; they are named TBARS (substances that react with TBA), and form principally in meats and meat derivatives. Several organic or bio-organic acids, as shikimic and sorbic acids, react photometrically with TBA if a Malaprade reaction takes place before. A structural study of the red adduct TBA-MDA has been carried out.

The thiobarbituric acid (TBA) reaction in foods: a review.

Storage of frozen fish brings about a decrease of extractability of myofibrillar proteins. There is also deterioration of the texture and functional properties of the flesh. In model systems, aggregation of myosin, actin, tropomyosin, and whole myofibrils have been described. These changes are caused by concurrent action of partial dehydration due to the freezing out of water, exposure of the proteins to inorganic salts which are concentrated in the remaining nonfrozen fluid, interactions with free fatty acids liberated from phospholipids and with lipid oxidation products, and cross-linking by formaldehyde produced in some species of fish as a result of enzymic decomposition of trimethylamine oxide. The extent of protein alterations increases with time and temperature of storage as well as with advanced disintegration of the tissues and intermixing of their components. The role played by the individual factors and the significance of different types of bonds, i.e., hydrophobic adherences, ionic bonds, and covalent cross-links in particular cases are not yet fully disclosed. Retardation of the deteriorative changes of proteins in frozen fish is possible by avoiding high storage temperatures and oxidation of lipids, removing hematin compounds and other constituents promoting cross-linking reactions, and by adding cryoprotectors like sugars, several organic acids, amino acids, or peptides.

Protein changes in frozen fish

Different malonaldehyde-amine condensation products were tested for their relative color contribution to the thiobarbituric acid (TBA) test, an indicator for oxidative rancidity of polyunsaturated lipids The open chain mono- and disubstituted malonaldehyde (M) addition products (R-N=CH-CH=CHOH and R-N=CH-CH=CH-NH-R) gave complete (100%) recovery ofM on a mole basis When theM residue was incorporated into cyclic products which formed between the ureido- or guanidino- substituents of α-amino acids such as citrulline or arginine andM, recovery ofM by the TBA color test was 30% and 6%, respectively Products, from imine-amine type interactions, containing theM residue as in pyrazoline or pyrazole ring systems, released from 4% to noM

H. Buttkus

Papers

Amine-malonaldehyde condensation products and their relative color contribution in the thiobarbituric acid test