Cholesterol, Not Polyunsaturated Fatty Acids, is Target Molecule in Oxidation Induced by Reactive Oxygen Species in Membrane of Human Erythrocytes

Abstract

The oxidation of polyunsaturated fatty acids (PUFAs) by reactive oxygen species (ROS) is linked to aging and to many diseases. We herein employ initiating peroxyl radical (ROO(.-)) derived from the decomposition of 2,2\'- azobis(2-amidinopropane dihydrochloride), hydroxyl radical generated by the Fenton reaction and peroxyl radical (ROO(.-)) and alkoxyl radical (LO(.-)) derived from PUFAs by addition of Cu(2+) as ROS sources to oxidize human erythrocytes in vitro. The fatty acids in the erythrocyte membrane are transesterified from phosphoglycerides under alkaline conditions in the presence of methanol instead of being treated traditionally by diazomethane (CH(2)N(2)) under acidic conditions (pH = 2.0), to obtain corresponding methyl esters for the combination of gas chromatography with mass spectrometry determination. It was found that all the PUFAs in the membrane are perfectly preserved after oxidation by ROS, even though sufficient time is available for the interaction between human erythrocytes and ROS. This indicates that ROS do not damage PUFAs during reaction time. However, three products (cholesta-4,6-dien-3-ol, cholesta-4,6-dien-3-one, and cholesta-3,5-dien-7-one) are produced from the oxidation of cholesterol within this time frame. This qualitative finding suggests that the cholesterol in the membrane of human erythrocytes is more susceptible to ROS-induced oxidation than are PUFAs, and compels us to re-evaluate the physiological roles of cholesterol and PUFAs in the human erythrocyte membrane.