Prostaglandin D2 (PGD2) is transformed predominantly via 11-ketoreductase pathway of metabolism to PGF-ring compounds. The initial product of 11- ketoreductase metabolism of PGD2 is 9 alpha, 11 beta-PGF2 which is biologically active. Recently, multiple isomeric forms of PGF2 compounds derived form 11-ketoreductase metabolism were identified in human plasma and urine. During the curse of developing a mass spectrometric method for analysis of these isomeric PGF2 compounds, we discovered that considerable (ng/ml) quantities of these compounds are present in human plasma in a sequestered form. Preliminary experiments suggest that they are not simply non-covalently bound to plasma proteins but are present in the form of a polar conjugate. The conjugate, however, avidly binds non-covalently to plasma proteins. PGF2 compounds can be liberated from the conjugate with mild alkaline hydrolysis. Possibilities regarding the nature of this PG conjugate(s) include conjugation with phospholipids, glucuronide, sulfate, amino acids, or carbohydrate. Preliminary evidence suggests the PGF2 compounds are not conjugated to protein. Experiments are proposed to further characterize the chemical characteristics of the conjugate(s) and determine whether specific hydrolytic enzymes will liberate PGF2 compounds. The possibility that other cyclooxygenase products are also present in plasma in the form of a conjugate will also be determined. Chromatographic methods will be developed for purification of the conjugate. Structural elucidation will then be accomplished primarily using fast atom bombardment mass spectrometry. Isolation and identification of urinary and biliary PG conjugates will then be performed following infusion of radiolabelled PGD2, 9 alpha, 11 beta,-PGF2 and 6-keto-PGF1 alpha in experimental animals. The discovery of the PG-conjugate has important analytical ramifications in that the conjugate is somewhat labile and PGF2 compounds are liberated by freezing and thawing plasma and during storage of plasma. In addition, the finding may have important physiological relevance in that formation of the conjugate and deconjugation could be regulated processes representing a mechanism by which eicosanoids can be inactivated and sorted intact and subsequently released in active form in the absence of de novo synthesis.
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