Rapid isolation of microsomes for studies of lipid peroxidation.
Conventional isolation of microsomes by high-speed centrifugation from isotonic sucrose requires exposure to air for several hours, leading to the formation of low levels of lipid peroxidation products. Sucrose interferes in protein and malondialdehyde assays and provides no protection against lipid peroxidation during workup. A new procedure for the purification of microsomes from rat liver substitutes mannitol (a hydroxyl radical scavenger) for sucrose and takes advantage of the properties of morpholinopropane sulfonic acid (MOPS) buffer and triethylenetetramine to provide protection against lipid peroxidation during the rapid (less than one hour) workup and subsequent low-temperature storage. The microsomal fractions prepared by the proposed method are free of detectable mitochondrial contamination and at least as pure overall as those prepared by the conventional method, but they have higher glucose-6-phosphatase and laurate hydroxylase activities and significantly less malondialdehyde than conventional microsomes at the time isolation is complete. Laurate hydroxylase activity is more stable during frozen storage in mannitol medium. The kinetics of lipid peroxidation in vitro are quite different for microsomes prepared by the two methods.
D000818:Animals; D002499:Centrifugation, Density Gradient; D005227:Fatty Acids / Q000302:isolation & purification; D066298:In Vitro Techniques; D008054:Lipid Peroxides / Q000096:biosynthesis* / Q000302:isolation & purification; D008353:Mannitol / Q000494:pharmacology; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000378:metabolism / Q000648:ultrastructure; D010084:Oxidation-Reduction; D051381:Rats; D013048:Specimen Handling; D013696:Temperature
D005227:Fatty Acids; D008054:Lipid Peroxides; D008353:Mannitol