TABLE 2

Comparison of maximal rates of oxygenation and product profiles for AA, EPA, and DHA incubated with huPGHS-2 and aspirin-acetylated huPGHS-2

Rates were determined by measuring O₂ consumption using an O₂ electrode as described under Materials and Methods. Values for rates of O₂ consumption were converted to values for rates of FA consumption based on the percentages of mono-oxygenated and bis-oxygenated (endoperoxide) products. The latter percentages were determined by radioactive thin-layer chromatography and autoradiography with [1-¹⁴C]AA or [1-¹⁴C]EPA as substrates or by thin-layer chromatography and visualizing the products by staining in the case of DHA; only mono-oxygenated cyclooxygenase products of DHA were observed. Errors are standard deviations from at least three kinetic measurements for each substrate. Values for AA- and EPA-derived products were determined by measuring the percentage of radioactivity from [1-¹⁴C]AA and [1-¹⁴C]EPA, respectively, that cochromatographed with monohydroxy acid (15-HETE) and PGH₂ upon radio thin-layer chromatography and using the ratio of hydroxy acids as determined by LC-MS/MS. Values for DHA were determined by LC-MS/MS directly because only mono-oxygenated cyclooxygenase products were observed by thin-layer chromatography.