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.
| Fatty Acid Substrate | huPGHS-2 |
Aspirin-Acetylated
huPGHS-2 |
||
|---|---|---|---|---|
| Rate of FA Oxygenation | Products | Rate of FA Oxygenation | Products | |
| μM FA/min/mg | % | μM FA/min/mg | % | |
| AA | 22 ± 0.2 | PGH2 (91) | 12 ± 0.5 | PGH2 (53) |
| HETEs (9) | 15-HETE (47) | |||
| EPA | 10.8 ± 0.5 | PGH3 (50) | 3.1 ± 0.2 | PGH3 (40) |
| 11-HEPE (32) | 11-HEPE (21) | |||
| 14-HEPE (18) | 14-HEPE (16) | |||
| 15-HEPE (16) | ||||
| 18-HEPE (7) | ||||
| DHA | 3.2 | 13-HDHA (100) | 0.9 | 13-HDHA (25) |
| 17-HDHA (75) | ||||