Lipid peroxide formation in microsomes. General considerations

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 (Fe(2+) or Fe(3+)), which activates, inhibit both ascorbate-induced and NADPH-induced peroxidation. Mg(2+) increases the rate of peroxidation whereas Ca(2+) 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.