Hemileaflet susceptibility to oxidative damage in the intestinal brush-border membrane

Oxidation of biological membranes is characteristic of many types of tissue injury, including those observed with inflammatory bowel disease. The lipid compositions of the inner and outer leaflets of biological membranes differ significantly, making one leaflet theoretically more susceptible to oxidative stress than the other. In this study, we evaluated the susceptibility of each membrane hemileaflet for peroxyl radical-mediated oxidation. In vitro peroxidation of intestinal brush-border membrane was initiated with the peroxyl radical-generator 2,2'-azobis-(2-amidinopropane)hydrochloric acid (AAPH). Oxidation events were monitored by following the oxidation-sensitive degradation of the lipid-soluble fluorescent probe cis-parinaric acid (PnA). The degradation patterns were clearly distinct in the inner and outer hemileaflet. PnA degradation in the inner hemileaflet was consistent with a slow first-order reaction, whereas degradation in the outer leaflet appeared as two first-order processes delayed in time. The results suggest that the sum of available antioxidants and endogenous substrates for oxidation are consumed more rapidly in the outer membrane hemileaflet, making this leaflet more susceptible to peroxidation compared with the cytofacial leaflet.