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.

Intact spinach chloroplasts scavenge hydrogen peroxide with a peroxidase that uses a photoreductant as the electron donor, but the activity of ruptured chloroplasts is very low [Nakano and Asada (1980) Plant & Cell Physiol. 21: 1295]. Ruptured spinach chloroplasts recovered their ability to photoreduce hydrogen peroxide with the concomitant evolution of oxygen after the addition of glutathione and dehydroascorbate (DHA). In ruptured chloroplasts, DHA was photoreduced to ascorbate and oxygen was evolved in the process in the presence of glutathione. DHA reductase (EC 1.8.5.1) and a peroxidase whose electron donor is specific to L-ascorbate are localized in chloroplast stroma. These observations confirm that the electron donor for the scavenging of hydrogen peroxide in chloroplasts is L-ascorbate and that the L-ascorbate is regenerated from DHA by the system: photosystem I-*ferredoxin-*NADP^>glutathione. A preliminary characterization of the chloroplast peroxidase is given.

Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts

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

Hydroperoxide metabolism in mammalian organs.

During primate evolution, a major factor in lengthening life-span and decreasing age-specific cancer rates may have been improved protective mechanisms against oxygen radicals. We propose that one of these protective systems is plasma uric acid, the level of which increased markedly during primate evolution as a consequence of a series of mutations. Uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. We show that, at physiological concentrations, urate reduces the oxo-heme oxidant formed by peroxide reaction with hemoglobin, protects erythrocyte ghosts against lipid peroxidation, and protects erythrocytes from peroxidative damage leading to lysis. Urate is about as effective an antioxidant as ascorbate in these experiments. Urate is much more easily oxidized than deoxynucleosides by singlet oxygen and is destroyed by hydroxyl radicals at a comparable rate. The plasma urate levels in humans (about 300 microM) is considerably higher than the ascorbate level, making it one of the major antioxidants in humans. Previous work on urate reported in the literature supports our experiments and interpretations, although the findings were not discussed in a physiological context.

https://www.pnas.org/content/pnas/78/11/6858.full.pdf

Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis.

http://www.jbc.org/content/245/14/3632.full.pdf

Properties and regulation of glutathione peroxidase.

Cytochrome P-450 as a microsomal peroxidase utilizing a lipid peroxide substrate

The inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide has been investigated. The reaction obeyed two rate equations:



(a) In the absence of catalysts 








(b) In the presence of catalysts 






These Kinetics are identical to those previously observed during glutathione oxidation by peroxides.



Enzyme inactivation was shown to be strictly related to sulphydryl group modification and this modification can account for the inactivation. For total inactivation H2O2, like iodoacetate, modified about 3.5 sulphydryl groups in the enzyme. o-Iodosobenzoate, however, modified approximately 8.5 sulphydryl groups and it was concluded that H2O2, unlike o-iodosobenzoate, did not oxidize the “essential” sulphydryl groups of the enzyme to disulphide, but to sulphenic acid residues.



Peroxide-inactivated glyceraldehyde-3-phosphate dehydrogenase could be fully reactivated and sulphydryl oxidation fully reversed if the enzyme was treated immediately with excess small thiol. The reversibility of inactivation decreased progressively if thiol treatment was delayed. 4 hours after peroxide treatment, neither enzyme inactivation nor sulphydryl oxidation could be reversed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1969.tb00721.x

Mechanism of peroxide-inactivation of the sulphydryl enzyme glyceraldehyde-3-phosphate dehydrogenase.

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity.

Lipid peroxide formation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to a suspension of isolated hepatocytes. The reaction was monitored by malonaldehyde measurements. Upon the addition of iron, malonaldehyde production in the cells started immediately but ceased within 30–60 min, and the response was dose-related with iron concentrations ranging from 19 to 187 μM. Malonaldehyde formation was associated with increased oxygen uptake and conjugated diene production. The addition in vitro of N,N,N′;N′-tetramethyl-p-phenylenediamine, menadione or p-benzoquinone inhibited the iron-induced malonaldehyde production. It was also possible to demonstrate an apparent disappearance of malonaldehyde from fresh cells by addition of adequate amounts of N,N,N′,N′-tetramethyl-p-phenylenediamine (100 μM).



The attenuation of the iron-induced malonaldehyde production was found to be correlated with an increased binding of iron to an intracellular ferritin fraction. Further, malonaldehyde formation was also associated with a conversion of reduced glutathione to the oxidized form which, in turn, revealed a faster permeation out of the cells into the surrounding medium of the oxidized than of the reduced thiol. So, concomitant with the redox alterations, there was also an overall loss of glutathione from the cells.



Cumene hydroperoxide-induced malonaldehyde production could be initiated by the addition of this peroxide in concentrations ranging from 150 μM to the liver cell incubate. With concentrations below 150 μM, a lag phase was present which seemed to be glutathione-dependent.



It is concluded that iron enters the cell, then is probably reduced inside the cell by NADPH via the NADPH–cytochrome P-450 reductase, and in the reduced state initiates lipid peroxidation. The reaction is inhibited by intracellular mechanisms, the glutathione redox system being of principal importance, and possibly terminated by the iron-apoferritin complex formation.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1975.tb02473.x

Peter J. O'Brien

Papers

Properties and regulation of glutathione peroxidase.

Cytochrome P-450 as a microsomal peroxidase utilizing a lipid peroxide substrate

Mechanism of peroxide-inactivation of the sulphydryl enzyme glyceraldehyde-3-phosphate dehydrogenase.

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity.

Further Studies on Lipid-Peroxide Formation in Isolated Hepatocytes