Lipid alterations occurring in microsomes during the enzymic oxidation of TPNH

Abstract

Received January 15, 1965 Hochstein and Ernstes (1963) have recently reported that when microsomes were incubated in the presence of TPNH, the addition of ADP resulted in a marked increase in TPNH oxidation and oxygen uptake. In addition, there was formation of a chromogen reacting with thiobarbituric acid (TBA) and designated as malonaldehyde. The high oxygen uptake, in excess of that required for TPNH oxidation, and the formation of chromogen were attributed to peroxidation of microsomal lipids. We have analyzed the lipid from rat liver microsomes which were incubated at hyperbaric psessures of oxygen in the presence (EQerimental) and absence (Control) of TPNH and ADP. Marked changes in the chromatographic behavior and the chemical characteristics were observed only in the lipids extracted from the experimental system. Materials: Washed microsomes were prepared by differential centrifugation of rat liver homogenates in 0.15 M phosphate buffer, pH 7.5, and stored in the pellet form at -2O'C. Just before use, the microsomes were suspended in 0.1 M Tris, pH 7.5, so that 0.1 ml of the suspension contained the quantity of microsomes from 100 mg of liver. Results and Discussion: -It was previously observed (McCay et al., 1960) that oxygen pressure above one atmosphere activates gulonolactone oxidase, another microsomal enzyme also producing a TBA-reacting chromogen which is dependent on the enzymic oxidation of gulonolactone. It was of interest, therefore, to investigate the effect of hyperbaric oxygen pressures on the TPNH-ADP-dependent TBA chromogen formation in microsomes