| Levuglandins (LGs) and isolevuglandins (isoLGs), formed by rearrangement of endoperoxide intermediates generated through the cyclooxygenase and free radical induced oxidation of arachidonates, are extraordinarily reactive, forming covalent adducts incorporating protein lysyl epsilon-amino groups. Because they accumulate, these adducts provide a dosimeter of oxidative injury.;The thesis reports the detection in vivo and quantitative analysis of LG/isoLG adducts that incorporate the amino group of phosphatidylethanolamines (PEs) into LG/isoLG-hydroxylactams. Notably, liquid chromatography-tandem mass spectrometry detection of these hydroxylactams is achieved with samples that are an order of magnitude smaller and sample processing is much simpler and less time-consuming than required for measuring protein-derived LG/isoLG-lysyl-lactams. A key feature of our protocol is treatment of biological phospholipid extracts with phospholipase A2 to generate mainly 1-palmitoyl-2-lysoPE-hydroxylactams from heterogeneous mixtures of phospholipids with a variety of acyl groups on the 2-position. LG/isoLG-PE-hydroxylactam levels in plasma from patients with age-related macular degeneration (N = 15, 5.2 +/- 1.4 ng/ml) were significantly elevated (P < 0.0001) compared to healthy volunteers (N = 15, 3.4 +/- 0.5 ng/ml). Two-fold higher levels ( P = 0.00021) were detected in liver from chronic ethanol-fed (27% of calories for 4 weeks) mice (C57BL/6 female, N = 6, 32.4 +/- 6.3 ng/g) compared to pair-fed animals (N = 4, 12.1 +/- 1.5 ng/g).;C22-11-isolevuglandin E4 (10-acetyl-11-formyl-14-hydroxynonadeca-4(Z), 7(Z),12(E),16(Z)-tetraenoic acid ) is one of 128 structurally isomeric levulinaldehyde derivatives that can be generated during the free radical-induced oxidation of docosahexaenoic acid (DHA). Its synthesis was accomplished by conjugate addition of a higher order vinyl cyanocuprate to a gamma-alkoxyenone to construct the carbon skeleton. A lower side chain synthon was prepared by nucleophilic addition of a beta-stannyl vinyllithium to a volatile and chemically sensitive beta, gamma-ethylenic aldehyde. Basic and acidic hydrolysis in a sequence of functional and protecting group manipulations delivered C22-11-isoLGE4 if precautions were taken to avoid free-radical induced oxidation of this air sensitive product. The synthesis of C22-11-isoLGE4 provides an authentic standard for characterization of biological samples and products of DHA oxidation in vitro. |
