Gas chromatographic-mass spectrometric analysis of lipoxygenase products in post-ischemic rabbit myocardium

Leukotriene B4 (LTB4) is a potent chemotactic compound for neutrophils and is thought to be an important mediator of myocardial ischemia-reflow injury. We have measured LTB4 in rabbit cardiac tissue following ischemia-reflow using a sensitive and specific gas chromatographic-mass spectrometric (GC-MS) assay. The concentration of LTB4 in rabbit myocardium following 45 min ischemia and 3 h reflow was 48.7 +/- 12.5 pg/g, significantly higher than in non-ischemic tissue from the same animal (17.5 +/- 3.9 pg/g). These concentrations were at least an order of magnitude lower than previously reported values assessed by radioimmunoassay (RIA). Compared with the GC-MS method, RIA greatly overestimated LTB4 concentrations in cardiac tissue. The capacity of post-ischemic myocardium to produce lipoxygenase products, LTB4, 5-, 12- and 15-HETEs was also assessed following incubation of myocardium ex vivo with calcium ionophore. In all animals ischemic cardiac tissue produced greater amounts of LTB4, 5-, and 12-HETEs than non-ischemic myocardium and 12-HETE was the major product. Neutrophils that have accumulated in the injured tissue may be a major source of these products. However, in contrast to cardiac tissue, isolated rabbit neutrophils stimulated with A23187 produced 5-HETE as the major product with very little 12-HETE formed. These latter findings suggest that cells other than neutrophils may contribute to the production of lipoxygenase products during myocardial ischemia-reflow injury.