scholarly journals Lipid peroxide formation in microsomes. General considerations

1969 ◽  
Vol 113 (2) ◽  
pp. 315-324 ◽  
Author(s):  
E. D. Wills
Keyword(s):  
Liver Microsomes ◽  
Lipid Peroxide ◽  
Maximum Activity ◽  
Specific Rate ◽  
Peroxide Formation ◽  
Unsaturated Lipids ◽  
Low Concentrations ◽  
Transport Chain ◽  
Maximum Specific Rate ◽  
Lipid Peroxide Formation

1. Liver microsomes form lipid peroxide when incubated with ascorbate or NADPH, but not with NADH. Increasing the concentration of ascorbate beyond the optimum (0·5mm) decreases the rate of lipid peroxide formation, but this effect does not occur with NADPH. Other reducing agents such as p-phenylenediamine or ferricyanide were not able to replace ascorbate and induce lipid peroxide formation. 2. The rate of ascorbate-induced peroxidation is optimum at pH6·0 whereas the rate of the NADPH system is optimum at pH7·0. Both systems require phosphate for maximum activity. 3. Lipid peroxide formation occurs at the maximum specific rate in very dilute microsome suspensions (0·15mg. of protein/ml.). 4. Treatment of microsomes with deoxycholate and other detergents causes membrane disintegration and inhibits lipid peroxide formation. 5. Lipid peroxide formation is accompanied by a rapid uptake of oxygen and there is a large excess of oxygen utilized for each molecule of malonaldehyde measured in the peroxide method. 6. Boiled microsomes form lipid peroxide in the presence of ascorbate, but not if NADPH is added. 7. Lipid peroxide formation induced by NADPH is strongly inhibited by p-chloromercuribenzoate, weakly inhibited by N-ethylmaleimide and unaffected by iodoacetamide. Ascorbate-induced peroxidation in untreated microsomes is unaffected by p-chloromercuribenzoate, but inhibited if boiled microsomes are used. These experiments may be interpreted on the basis that a ferredoxin-type protein forms part of the system in which NADPH induces lipid peroxide formation. 8. Most heavy-metal ions, with the exception of inorganic iron (Fe2+ or Fe3+), which activates, inhibit both ascorbate-induced and NADPH-induced peroxidation. Mg2+ increases the rate of peroxidation whereas Ca2+ inhibits it. 9. Lipid peroxide formation is inhibited strongly by GSH and weakly by cysteine. Ascorbate-induced peroxidation is much more sensitive than NADPH-induced peroxidation. 10. Peroxidation is strongly inhibited by addition of low concentrations (0·01–0·1mm) of cytochrome c or of haemoglobin. 11. It is considered that lipid peroxide formation occurs as a result of the operation of the microsomal electron-transport chain switching from hydroxylation to oxidize unsaturated lipids of the endoplasmic reticulum.

scholarly journals Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation

1969 ◽  
Vol 113 (2) ◽  
pp. 333-341 ◽  
Author(s):  
E. D. Wills
Keyword(s):  
Liver Microsomes ◽  
Lipid Peroxide ◽  
Rat Liver Microsomes ◽  
Concentrated Solutions ◽  
Peroxide Formation ◽  
Transport Chain ◽  
Site Of Action ◽  
Relationship Of ◽  
Iron Chelating Agents ◽  
Lipid Peroxide Formation

1. Aminopyrine strongly inhibits NADPH-induced lipid peroxide formation in rat liver microsomes, but ascorbate-induced peroxidation is inhibited to a smaller extent. 2. Aminopyrine oxidation is stimulated by Mg2+ but inhibited by Ca2+. Concentrated solutions (10mm) of iron-chelating agents inhibit aminopyrine oxidation, but the more dilute solutions (0·5mm) of chelators that block lipid peroxide formation do not inhibit aminopyrine oxidation. Microsomes prepared from sucrose–EDTA homogenates rapidly oxidize aminopyrine, but do not form lipid peroxide when incubated with ascorbate or NADPH. 3. Aminopyrine oxidation is strongly inhibited by p-chloromercuribenzoate, less by iodoacetamide and weakly by N-ethylmaleimide. The site of action of these compounds is considered to be a ferredoxin-type protein. GSH and cysteine also inhibit. 4. Other drugs oxidized by microsomes such as caffeine, phenobarbitone and hexobarbitone had either no or little effect on lipid peroxide formation, but codeine inhibited. 5. Most aliphatic hydrocarbons, alcohols, ketones and aldehydes did not affect lipid peroxide formation, but chloroform and carbon tetrachloride inhibited. 6. Many aromatic compounds inhibited lipid peroxide formation. Only aromatic acids were without any effect and phenols and amines were very strong inhibitors. 7. Induction of lipid peroxide formation in microsomes by incubation with ascorbate or NADPH or by treatment with ionizing radiation leads to a sharp decline in the ability of microsomes to oxidize aminopyrine or hydroxylate aniline. 8. It is considered that the two processes of hydroxylation and lipid peroxide formation are closely linked in microsomes. They probably depend on the same electron-transport chain, and peroxide formation, which involves membrane disintegration, may be part of the normal membrane remodelling process.

scholarly journals Lipid peroxide formation in microsomes. The role of non-haem iron

1969 ◽  
Vol 113 (2) ◽  
pp. 325-332 ◽  
Author(s):  
E. D. Wills
Keyword(s):  
Metal Ion ◽  
Chelating Agents ◽  
Adenine Nucleotides ◽  
Liver Microsomes ◽  
Lipid Peroxide ◽  
Rat Liver Microsomes ◽  
Peroxide Formation ◽  
Haem Iron ◽  
Lipid Peroxide Formation

1. Metal ion-chelating agents such as EDTA, o-phenanthroline or desferrioxamine inhibit lipid peroxide formation when rat liver microsomes prepared from homogenates made in pure sucrose are incubated with ascorbate or NADPH. 2. Microsomes treated with metal ion-chelating agents do not form peroxide on incubation unless inorganic iron (Fe2+ or Fe3+) in a low concentration is added subsequently. No other metal ion can replace inorganic iron adequately. 3. Microsomes prepared from sucrose homogenates containing EDTA (1mm) do not form lipid peroxide on incubation with ascorbate or NADPH unless Fe2+ is added. Washing the microsomes with sucrose after preparation restores most of the capacity to form lipid peroxide. 4. Lipid peroxide formation in microsomes prepared from sucrose is stimulated to a small extent by inorganic iron but to a greater extent if adenine nucleotides, containing iron compounds as a contaminant, are added. 5. The iron contained in normal microsome preparations exists in haem and in non-haem forms. One non-haem component in which the iron may be linked to phosphate is considered to be essential for both the ascorbate system and NADPH system that catalyse lipid peroxidation in microsomes.

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