Membrane Lipid Fluidity Affects the Nitroxide Radical Decay of 5-Doxyl Stearic Acids in Isolated Rat Hepatocytes

Abstract

We investigated the effect of membrane fluidity on the nitroxide radical decay rate of 5-doxyl stearic acid in spin-labeled rat hepatocytes. The half-time (t_1/2) for the EPR signal decay of 5-doxyl stearic acids incorporated into the membranes of isolated rat hepatocytes was 12 min (mean value). When spin-labeled hepatocytes were separated into membrane and cytosol fractions, the t_1/2 of the membrane fraction was prolonged by more than 2 hrs. However, when the cytosolic fraction was added to the membrane fraction, the radical decay reaction recovered (t_1/2 was 27 min). Incubation of hepatocytes with a stream of 95% O₂ at 37°C for 2 hrs prolonged t_1/2 by 106% and was associated with a 18% decrease in water-soluble antioxidant content. When the measurement temperature was changed from 24 degrees C to 37 degrees C, t_1/2 was shortened with a decrease in the order parameter (S). The t_1/2 and S in hepatocytes treated with phosphatidylcholine (PC) were reduced by 14% and 0.008, respectively. Conversely, after treatment with phosphatidylethanolamine (PE), PC+cholesterol and PE+cholesterol, t_1/2 and S increased by 14% and 0.014, 20% and 0.018 and 29% and 0.040, respectively. These findings suggest that the nitroxide radical decay of 5-doxyl stearic acids incorporated into hepatocyte membranes is mediated by the antioxidants in the cytosol fraction, and that the nitroxide radical decay rate is affected not only by water-soluble antioxidant content but also by the membrane lipid fluidity of the hepatocytes.

Key Words:

• nitroxide radical decay
• 5-doxyl stearic acid
• hepatocytes
• antioxidant content
• membrane fluidity
• EPR
• spin label