LIPIDOMIC ANALYSIS OF EICOSANOID SIGNALING DURING CHORIOALLANTOIC MEMBRANE DEVELOPMENT IN AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS)
Theresa Cantu1, John Bowdenes2, Louis Guillette1.
1Medical University of South Carolina, Charleston, SC, 2National Institute of Standards and Technology, Charleston, SC.
Eicosanoids are important signaling molecules commonly associated with inflammation and immune response. To date, few methods have explored the presence and role of eicosanoids with respect to embryonic development. Traditionally, inflammation is studied in disease etiology; however inflammatory processes occur during normal development and the role of these compounds is poorly understood. We hypothesize that pro-inflammatory lipids play a significant role during the earlier stages of alligator chorioallantoic membrane (CAM) development, and that anti-inflammatory signals increase before hatch-out. We have developed an LC-ESI-MS/MS method capable of quantifying 40 eicosanoids in biological tissues. Here, we examine the chorioallantoic membrane (CAM) of the American alligator. Eggs were collected from Lake Woodruff National Wildlife Refuge and incubated in the laboratory at several temperatures. CAM tissue was collected at different stages of development, flash frozen, cryo-homogenized, spiked with internal standards, and extracted. The resultant extract was analyzed on an Agilent 1100 LC system equipped with a C18 column (4.6 x 250 mm, 5 mm i.d.). Measurement and quantification of each eicosanoid species uses standard calibration curves and the concentrations are expressed as pmol eicosanoid per mg of protein. Initial results identified the amount of tissue needed for extraction of these compounds and detected 32 of 40 compounds in CAM tissue. Future work will ascertain changes in these compounds during embryonic development and provide insight on the role of eicosanoids in the developing chorioallantoic membrane of the American alligator. This is the first study to investigate eicosanoids analytically in the CAM tissue of any wildlife species.