Red blood cells: reservoirs of cis- and trans-epoxyeicosatrienoic acids.

Epoxyeicosatrienoic acids (EETs) are candidate endothelium-derived hyperpolarizing factors that demonstrate a wide range of biological effects. The presence of both cis- and trans-EETs in rat plasma was identified with HPLC-electrospray ionization tandem mass spectrometry in this study. The total EETs in plasma are 38.2 ng/ml with cis-EETs representing 21.4 +/- 0.4 ng/ml and trans-EETs 16.8 +/- 0.4 ng/ml. EETs in RBCs were estimated to be 20.2 ng/10(9) RBCs, which corresponds to 200 ng in RBCs contained in 1 ml blood. RBC incubation with 10 mM tert-butyl hydroperoxide resulted in 4.4-fold increase of total cis-EETs (from 9.2 to 40.2 ng/10(9) RBCs) and 5.5-fold increase of total trans-EETs (from 11.0 to 60.8 ng/10(9) RBCs). EETs were released (2 ng/ml) from RBCs after incubation at 37 degrees C for 10 min even after being washed 3 times, indicating that RBCs are reservoirs of plasma EETs. The identification of cis- and trans-EETs in RBCs and in plasma as well as their release from RBCs suggest a vasoregulatory role of RBCs in view of their potent vasoactivity.

Identification of 5,6-trans-epoxyeicosatrienoic acid in the phospholipids of red blood cells.

A novel eicosanoid, 5,6-trans-epoxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-trans-EET), was identified in rat red blood cells. Characterization of 5,6-trans-EET in the sn-2 position of the phospholipids was accomplished by hydrolysis with phospholipase A(2) followed by gas chromatography/mass spectrometry as well as electrospray ionization-tandem mass spectrometry analyses. The electron ionization spectrum of 5,6-erythro-dihydroxyeicosatrienoic acid (5,6-erythro-DHET), converted from 5,6-trans-EET in the samples, matches that of the authentic standard. Hydrogenation of the extracted 5,6-erythro-DHET with platinum(IV) oxide/hydrogen resulted in an increase of the molecular mass by 6 daltons and the same retention time shift as an authentic standard in gas chromatography, suggesting the existence of three olefins as well as the 5,6-erythro-dihydroxyl structure in the metabolite. Match of retention times by chromatography indicated identity of the stereochemistry of the red blood cell 5,6-erythro-DHET vis à vis the synthetic standard. High pressure liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the phospholipase A(2)-hydrolyzed lipid extracts from red blood cells revealed match of the mass spectrum and retention time of the compound with the authentic 5,6-trans-EET standard, providing direct evidence of the existence of 5,6-trans-EET in red blood cells. The presence of other trans-EETs was also demonstrated. The ability of both 5,6-trans-EET and its product 5,6-erythro-DHET to relax preconstricted renal interlobar arteries was significantly greater than that of 5,6-cis-EET. In contrast, 5,6-cis-EET and 5,6-trans-EET were equipotent in their capacity to inhibit collagen-induced rat platelet aggregation, whereas 5,6-erythro-DHET was without effect. We propose that the red blood cells serve as a reservoir for epoxides which on release may act in a vasoregulatory capacity.