AbstractsBiology & Animal Science

Oxidation of phospholipids containing docosahexaenoate leads to the formation of 4-hydroxy-7-oxo-hept-5-enoyl phospholipids (HOHA-PLs) that were previously shown to react with biological primary amines to deliver 2-¿-carboxyethylpyrroles (CEPs), which are novel factors that induce angiogenesis. A spontaneous deacylation product of HOHA-PLs, 4-hydroxy-7-oxo-5-heptenoic acid (HOHA)-lactone, was now found to react with proteins to generate CEPs. An in vitro study found that HOHA-PC forms CEP modifications primarily through initial release of HOHA-lactone followed by reaction of this intermediate with the epsilon-amino group of lysyl residues. These observations, along with the fact that HOHA-lactone was directly produced during lipid oxidation, suggest a novel mechanism of CEP generation in vivo that involves HOHA-lactone as a major intermediate.The metabolism of HOHA-lactone in cells suggests a cellular self-defense system against CEP generation. In a noncytotoxic dose, HOHA-lactone was found to diffuse rapidly into cultured a human retinal pigmented epithelium cell line (ARPE-19) and was metabolized to glutathione adducts and a carboxylic acid derivative, which were then secreted by the cells to extracellular medium. Our study showing that, although it is cytotoxic at high concentration levels, low levels of HOHA-lactone induce RPE cell proliferation and stimulate the secretion of vascular endothelial growth factor (VEGF). These findings suggest that HOHA-lactone plays a role in oxidative stress-induced cell signaling.CEP-derivatives of phosphatidylethanolamines (CEP-PEs) were also found to have proangiogenic effects similar to CEP-protein derivatives. A mass spectrometric assay was developed involving phospholipase D-promoted conversion to a CEP-ethanolamine derivative. The method was applied to the analysis of CEP-PEs in phospholipid extracts of human plasma. Elevated CEP-PE levels were found in plasma from AMD patients compared to plasma from healthy controls.In vivo, CEP-derived proteins were postulated to be degraded by proteasomes and cellular proteases to CEP-lysine. In an in vitro model experiment, CEP-lysine and its isotopomers were synthesized and then perfused to rat livers. Two major metabolites of CEP-lysine were identified and characterized by mass spectrometric analysis. CEP-BSA protein perfused in rat liver was found to be degraded to produce CEP-lysine and CEP-lysine-derived metabolites. Overall, this thesis provided a new understanding of the mechanism for CEP generation and metabolism.