Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

Publisher Summary This chapter discusses microsomal lipid peroxidation Lipid peroxidation is a complex process known to occur in both plants and animals It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipids, and the eventual destruction of membrane lipids, producing a variety of breakdown products, including alcohols, ketones, aldehydes, and ethers Biological membranes are often rich in unsaturated fatty acids and bathed in an oxygen-rich, metal-containing fluid Lipid peroxidation begins with the abstraction of a hydrogen atom from an unsaturated fatty acid, resulting in the formation of a lipid radical The formation of lipid endoperoxides in unsaturated fatty acids containing at least 3 methylene interrupted double bonds can lead to the formation of malondialdehyde as a breakdown product Nonenzymic peroxidation of microsomal membranes also occurs and is probably mediated in part by endogenous hemoproteins and transition metals The direct measurement of lipid hydroperoxides has an advantage over the thiobarbituric acid assay in that it permits a more accurate comparison of lipid peroxide levels in dissimilar lipid membranes

Microsomal lipid peroxidation.

Publisher Summary The classification of cell death can be based on morphological or biochemical criteria or on the circumstances of its occurrence. Currently, irreversible structural alteration provides the only unequivocal evidence of death; biochemical indicators of cell death that are universally applicable have to be precisely defined and studies of cell function or of reproductive capacity do not necessarily differentiate between death and dormant states from which recovery may be possible. It has also proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances. One of these patterns is the swelling proceeding to rupture of plasma and organelle membranes and dissolution of organized structure—termed “coagulative necrosis.” It results from injury by agents, such as toxins and ischemia, affects cells in groups rather than singly, and evokes exudative inflammation when it develops in vivo. The other morphological pattern is characterized by condensation of the cell with maintenance of organelle integrity and the formation of surface protuberances that separate as membrane-bounded globules; in tissues, these are phagocytosed and digested by resident cells, there being no associated inflammation.

Cell death : the significance of apoptosis

When hemolyzates from erythrocytes of selenium-deficient rats were incubated in vitro in the presence of ascorbate or H(2)O(2), added glutathione failed to protect the hemoglobin from oxidative damage. This occurred because the erythrocytes were practically devoid of glutathione-peroxidase activity. Extensively purified preparations of glutathione peroxidase contained a large part of the (75)Se of erythrocytes labeled in vivo. Many of the nutritional effects of selenium can be explained by its role in glutathione peroxidase.

https://science.sciencemag.org/content/sci/179/4073/588.full.pdf

Selenium: Biochemical Role as a Component of Glutathione Peroxidase

Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.

Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes.

Several broad lines of scientific evidence showing that vitamin E functions as a lipid antioxidant, inhibiting the destructive peroxidation of polyunsaturated lipids, have been presented in recent reviews.'-Q Further, the function of vitamin E is a very broad topic, study of which can be based in part on knowledge of other lipid antioxidant reactions and interrelationships. Also, the reactions of and the damage caused by free radical peroxidation of lipids are complex topics, and in a relative sense the mechanisms of damage are not well understood, although they appear to have counterparts in radiation biochemistry. In view of the breadth of this topic, it is appropriate in the first section of this paper to direct attention to the major review sources in which information on the subject is summarized, and to some recently published papers. TABLE 1 lists some of the major factors in the suppression of lipid peroxidation. On the basis of the chemical properties of a-tocopherol and the mechanisms of antioxidation, a-tocopherol would be expected to react as a chain-breaking antioxidant in the inhibition of the free radical peroxidation of lipids. The propagation reaction in which the polyunsaturated lipid LH is peroxidized, L' + OZ'LOZ' LOZ. + LH+ LOOH + L' 1

Vitamin e and free radical peroxidation of lipids

A procedure was developed to isolate 75Se-labeled rat liver glutathione peroxidase (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) at 30--50% purity with 20--30% yields in 4--5 days. Using these preparations of glutathione peroxidase, the selenium moiety in the enzyme was identified as selenocysteine by derivatizing the seleno group with either iodoacetate or ethylenimine in the intact protein, hydrolyzing the protein with 6 N HCl, and cochromatographing the 75Se-labeled products with known standards. Techniques employed were anion-exchange chromatography, cation-exchange chromatography, gel-permeation chromatography, two-dimensional thin-layer chromatography, and automated amino acid analysis. The selenocysteine moiety was identified as the catalytic site in glutathione peroxidase by specifically labeling the enzyme with [14C]iodoacetate on the 75Se-labeled selenium atom and fractionating the 14C, 75Se-labeled derivative after acid hydrolysis. It was concluded that the reduced form of glutathione peroxidase contains the selenocysteine selenol (-SeH) at the catalytic site.

Al L. Tappel

Papers

Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes.

Vitamin e and free radical peroxidation of lipids

Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine

The Inhibition of Mitochondrial Peroxidation by Ubiquinone and Ubiquinol

Measurement of fluorescent lipid peroxidation products in biological systems and tissues.