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Journal of Lipid Research, Vol 34, 1583-1591, Copyright © 1993 by Lipid Research, Inc.

ARTICLES

A systematic analytical chemistry/cell assay approach to isolate activators of orphan nuclear receptors from biological extracts: characterization of peroxisome proliferator-activated receptor activators in plasma

CD Banner, M Gottlicher, E Widmark, J Sjovall, JJ Rafter and JA Gustafsson
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.

Using a novel combination of analytical chemical and molecular biological techniques, lipophilic components of human plasma separated according to their physico-chemical properties were screened for their ability to activate the rat peroxisome proliferator-activated receptor (rPPAR). Activation of an rPPAR/glucocorticoid receptor chimera stably expressed in CHO cells by fractions in the initial screening guided further subfractionation. Characterization of an active subfraction by gas chromatography alone and in combination with mass spectrometry (GC- MS), indicated the presence of free fatty acids. Individual active components in this mixture were isolated by a final fractionation using high performance liquid chromatography (HPLC). GC-MS analyses of HPLC fractions able to activate the chimeric receptor identified palmitic acid, oleic acid, linoleic acid, and arachidonic acid as endogenous activators of rPPAR. No other activators were identified. This approach is able to specifically extract and identify endogenous activators of PPAR from a complex biological extract and as such may be valuable in the identification of activators of other orphan receptors in the steroid hormone receptor superfamily.
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