APPLICATION OF HPLC ESI TANDEM MASS SPECTROMETRY IN THE STUDY OF EICOSANOID SIGNALING DURING THE WOUND HEALING PROCESS

摘要

Eicosanoids are crucial early signaling lipid molecules released at a wound site. The initial rate limiting step for this process is the liberation of arachidonic acid from the sn-2 position of phospholipids by phospholipase A2 enzymes. The liberated arachidonic acid is then converted to any number of nearly 150 biologically active eicosanoids by enzymatic on non-enzymatic means. These eicosanoids exert biological events such as early stage vasoconstriction at a wound site to prevent blood loss, later stage vasodilation to allow neutrophil migration, as chemotactic agents for leukocyte migration etc. The eicosanoid profile changes during the course of wound healing. Deviations from these specific profiles can lead to complications in wound healing. 2 X 6 mm full thickness wounds created on the dorsal surface of BalbC mice. For short term eicosanoid measurement, the wound was washed 3 times with 20 microliters of PBS 10 minutes post wounding. For late term eicosanoid measurement, the wound was allowed to heal for 12 days followed by debridement of the wound bed and homogenization of the debrided wound bed material in PBS. The PBS solutions thus obtained was diluted with an equal volume of ethanol and centrifuged at 17,000 g for 20 minutes. 10 microliters of the extract thus obtained was separated via reverse phase HPLC and analyzed for eicosanoids by ESI tandem mass spectrometry in operating the negative ion mode. Significantly elevated levels of PGE2, PGD2 and PGF2&alpha were observed within the first ten minutes of surface wounding. Also a measurable level of LTE4 was also observed indicating that the secreted leukotrienes are already being converted to the cystenyl leukotrienes. Additionally other lipoxygenase products like 5HETE, 11HETE and 12 HETE were also detected in significant quantities. This profile changed significantly by day 12. While the PGE2, PGD2 and PGF2&alpha were still detectable, there presence was much less prominent compared to that of the HETE's. The cystenyl leukotrienes were not detectable by this stage while the levels of the HETE's were much more elevated compared to day 1. Also, in addition to 5, 11 and 12 HETE, 8 and 15 HETE were also detectable at the wound site. In addition to these, multiple other eicosanoid species also demonstrated significant profile changes. We have demonstrated the applicability of HPLC tandem mass spectrometry towards the study of eicosanoid signaling during wound healing process. For the first time a mass spectrometric approach confirms the importance of CERK in the homeostasis of the cellular eicosadome lipid mediated signaling events during the wound healing process.