Abstract
Aspirin (ASA) triggers the formation of 15-epi-lipoxins (15-epi-LXs or ATL [ASA-triggered LX]), which are potent bioactive eicosanoids that may contribute to the therapeutic impact of ASA. To elucidate the role of these new compounds in vivo, it is essential to establish quick and sensitive detection methods. To this end, we prepared an enzyme-linked immunosorbent assay specific for 15-epi-LXA4that proved to be highly sensitive (IC50 ∼ 50 pg, minimum detection ∼ 3.5 pg) and stereoselective. The amounts of 15-epi-LXA4 generated by human neutrophils from peripheral blood of healthy volunteers using this enzyme-linked immunosorbent assay were in agreement with those values obtained by liquid chromatography. Formation of 15-epi-LXA4 was cell ratio-dependent during THP-1 (a monocytic leukemia cell line)-neutrophil interactions with ASA-treated cells, and 15-epi-LXA4 was not detected with either cell type alone. Generation of 15-epi-LXA4 was also examined in murine peritonitis with ASA administration. Exudates from ASA-treated mice showed increased production of 15-epi-LXA4 that was diminished by indomethacin, a blocker of ASA-dependent acetylation of prostaglandin G/H synthase. A cytochrome P450 inhibitor administered in the presence of ASA did not prevent 15-epi-LXA4 formation, which suggests that P450 does not significantly contribute to formation of 15-epi-LXA4 in this murine model. These results indicate that the new enzyme-linked immunosorbent assay is both sensitive and selective for 15-epi-LXA4 and that 15-epi-LXA4is produced by human leukocyte-leukocyte interactions. In addition, 15-epi-LXA4 is generated by inflammatory exudates when ASA is administered during murine peritonitis and when prostaglandin G/H synthase is upregulated and acetylated. This assay should provide rapid means to investigate 15-epi-LXA4 generation in both cellular and animal models.
Footnotes
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Send reprint requests to: Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115.
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↵1 This work was supported in part by grants no. GM38765 and DK50305 (C.N.S.) from the National Institutes of Health.
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↵2 Present address: Nephrology Division, Royal Victoria Hospital, 3775 University Street, Montreal, Quebec, Canada H3A 2B4.
- Abbreviations:
- AA
- arachidonic acid
- ASA
- aspirin (acetylsalicylic acid)
- ATL
- aspirin-triggered lipoxins
- ELISA
- enzyme-linked immunosorbent assay
- GM-CSF
- granulocyte monocyte colony-stimulating factor
- HETE
- hydroxy eicosatetraenoic acid
- 5S-HETE
- (5S)-5-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid
- 12S-HETE
- (12S)-12-hydroxy-5,8,14-cis-10-trans-eicosatetraenoic acid
- 15S-HETE
- (15S)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid
- 15R-HETE
- (15R)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid
- HRP
- horseradish peroxidase
- LC/MS/MS
- liquid chromatography tandem mass spectrometry-mass spectrometry
- LO
- lipoxygenase
- LPS
- lipopolysaccharide
- LTB4 (leukotriene B4)
- 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid
- LX
- lipoxin
- 15-epi-LXA4
- 5S,6R,15R-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid
- 15-epi-LXB4
- 5S, 14R,15R-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid
- 15-R/S-methyl-LXA4
- 5S,6R,15(R/S)-trihydroxy-methyl-7,9,13-trans-11-cis-eicosatetraenoic acid
- LXA4
- 5S, 6R,15S-trihydroxyl-7,9,13-trans-11-cis-eicosatetraenoic acid
- LXB4
- 5S, 14R,15S-trihydroxyl-6,10,12-trans-8-cis-eicosatetraenoic acid
- ODYA
- 17-octadecynoic acid
- PGHS
- prostaglandin G/H synthase
- PMN
- neutrophil(s)
- RP-HPLC
- reverse-phase high-pressure liquid chromatography
- KLH
- keyhole limpets hemocyanin
- THP-1
- human acute monocytic leukemia cell line. PVDF, polyvinylidine difluoride
- SDS-Page
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Received January 8, 1998.
- Accepted June 1, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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