Biosynthesis, pharmacology and metabolism of 5-oxo-eicosatetraenoic acid and FOG(7)

The use of mass spectrometry to analyze eicosanoids has been critical for structural determination and quantitation of these potently biologically active molecules. In this thesis, a potent eosinophil chemotactic agent, 5-oxo-6,8,11,14-eicosatetraenoic acid, was identified in vivo for the first time. A quantitative mass spectrometric assay of this potently active molecule was developed and used to study whether or not this molecule was present in lung samples of patients with pulmonary hypertension and a murine model of asthma. Elevated levels of 5-oxo-ETE were observed in patients with pulmonary hypertension and decreased when pulmonary hypertensive patients were treated with prostacyclin.; A second use for mass spectrometry used in these thesis studies was for structural determination of 5-oxo-ETE metabolites. 5-Oxo-ETE was metabolized quite slowly and this molecule may possibly be used as a marker of such diseases as pulmonary hypertension in vivo. Metabolites of 5-oxo-ETE were eventually found and many novel compounds were discovered, including a glutathionyl adduct of 5-oxo-ETE, called FOG₇.; Inactivation of eicosanoids usually occurs through metabolic transformation. Interestingly, FOG₇ retained the activity of 5-oxo-ETE to elicit actin polymerization and chemotaxis of both neutrophils and eosinophils. However, FOG₇ was unable to induce calcium mobilization. Though it has been known that glutathione addition to α,β-unsaturated ketones can reduce the chemical reactivity of the molecule, the only other known biologically active glutathione-adducted lipid, besides FOG₇, was LTC₄ . The oxidative metabolic processes resulted in the formation of ω-1 and ω-2 hydroxylated metabolites of 5-oxo-ETE and formed compounds that were not biologically active as determined by the calcium mobilization assay in neutrophils.; Mass spectrometry was also used to study metabolism of FOG₇ in both in vitro and ex vivo systems. The in vitro metabolism by γ-glutamyl transpeptidase and leucine aminopeptidase resulted in the loss of the glutamate and glycine portions of the glutathione. The metabolism of FOG₇ in whole human blood resulted in the formation of 5,18-diHETrE, a compound also identified as a product of 5-oxo-ETE.