Levuglandin E<INF>2</INF>−Protein Adducts in Human Plasma and Vasculature

The prostaglandin endoperoxide PGH<INF>2</INF> rearranges nonenzymatically to generate prostaglandins and secoprostanoic acid levulinaldehyde derivatives such as PGE<INF>2</INF> and levuglandin (LG) E<INF>2</INF>, respectively. Direct detection of LGE<INF>2</INF> in biological samples is complicated because it is rapidly sequestered by covalent adduction to endogenous nucleophiles including proteins, which produces LGE<INF>2</INF>-derived protein-bound pyrroles. Therefore, to detect LGE<INF>2</INF>−protein adducts in vivo, antibodies were raised against a covalent adduct of LGE<INF>2</INF> with keyhole limpet hemocyanin (KLH). This antigen enabled the production of high-titer antibodies that exhibit minimal cross-specificity and are sensitive for detecting LGE<INF>2</INF>-derived pyrroles. Although pyrrole yields are low at LG/protein ratios found in vivo, an enzyme-linked immunosorbent assay with the LGE<INF>2</INF>−KLH antibodies detects LGE<INF>2</INF>-derived protein-bound pyrrole immunoreactivity in human plasma from specific patient populations. Furthermore, prominent immunocytochemical staining of human brain thin sections revealed the presence of LGE<INF>2</INF>-derived pyrrole immunoreactivity, especially in the meningeal vessels of some patients. This demonstration of LG−protein adducts in human plasma and vasculature provides the first evidence for the biological occurrence of levuglandins in vivo and further suggests that these antibodies might prove useful in diagnostic and mechanistic studies of various disease conditions.