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David A Ocon
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NEW METHOD FOR MEASURING ANTHRANILOYL-BUFODIENOLIDES IN PLASMA BY HPLC David A. Ocon*1, Cassandra Moreno *2, and Daniel N. Darlington, PhD*2. 1*Summer Intern, Pittsburgh Tissue Engineering Initiative, Undergraduate Student at University of South Florida 2*US Army Institute of Surgical Research, Fort Sam Houston, Texas. A critical issue for both the military and civilian population is hemorrhagic shock. Recent studies show that normal restoration of plasma volume fails after inhibition of Na/K ATPase and turn a normally non-lethal hemorrhage into a lethal one. Bufodienolides, a large family of powerful Na/K ATPase inhibitors, originally isolated from amphibia, have recently been found in human, mouse, and rat plasma. The purpose of this study was to develop a method to measure plasma levels of bufodienolides in hemorrhagic shock. The method began by extracting the bufodienolides out of plasma, conjugating the bufodienolides to a fluorescent probe, and then measuring the bufodienolide-fluorescent conjugate on High Pressure Liquid Chromatography (HPLC). As a result, Bufalin, Cinobufagin, and Resibufogenin were successfully extracted from pig plasma. We were also able to successfully conjugate NMIA to these bufodienolides. The bufodienolide-conjugates were then separated and measured on HPLC. Varying concentrations of the bufodienolides led to a dose-dependent rise in the peak height on the HPLC. Standard curves were developed for each bufodienolide which allowed us to accurately measure bufodienolides in mammalian plasma. This method can be extended to measure bufodienolides in plasma of patients with hemorrhagic or other forms of shock.