Journal of Lipid Research, Vol 34, 853-858, Copyright © 1993 by Lipid Research, Inc.
Simultaneous determination of hydroxyeicosanoid (HETE) binding to cells and its cellular metabolism
BM Vonakis and JY Vanderhoek
Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC 20037.
In order to study the mechanism(s) through which certain biologically
active lipids, such as the hydroxyeicosanoids (HETEs), exert their effects,
it is necessary to distinguish between binding of these lipids to cells and
their cellular metabolism. A novel and simple method is described for the
simultaneous determination of [3H]15- hydroxyeicosanoid (15-HETE) binding
to cells and cellular [3H]15-HETE metabolism. The method involves initial
separation of radiolabeled cells by filtration, filter extraction of
cellular lipids by methanol, and thin-layer chromatography (or high
performance liquid chromatography) determination of both nonesterified
15-HETE bound to cells and 15-HETE incorporation into cellular
phospholipids. The method was applied to both PT-18 mast/basophil cells and
rat basophilic leukemia (RBL-1) cells and should be applicable to other
cells as well as other metabolizable hydroxy fatty acids or lipids.
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