Formation of sk-1,2-diradyl glycerol following eicosapentaenoic and docosahexaenoic ethyl ester supplementation w vivo

The formation of diradylgtycarol (DG) that contains n-3 polyunsaturated tatty acid (n-3PUFA) in the sn-2 positron was investigated in peritoneal macrophages of mice fed purified diets for three weeks. Basal diet mix was reconstituted with a 3.0% lipid component of either 3.0% corn oil ethyl ester (COEE) (control diet) or one of various mixtures totalling 2.0% of (n-3) eicosapentaenoic ethyl ester (EPEE) and (n-3) docosahexaenoic ethyl ester (DHEE) pius 1.0% COEE: 0%EPEE and 2.0% DHEE; 0.5% EPEE and 1.5% DHEE; 1.0% EPEE and 1.0% DHEE; 1.5% EPEE and 0.5% DHEE; or 2.0% EPEE and 0% DHEE. COEE contained primarily [18:2n6], [I8:1n9], [16:0] and [18:3n3[ EPEE contained predominantly [20:5n3] and [22:6n3]. DHEE contain ed principally [22:6n3] and [20:4n3J. All experimental diets were supplemented with SOmgAg DL-a-tocopheryl acetate. All diets and solvents were prepared and stored under NThree days after i.p. thiogry coll ate, peritoneal macrophages were adhered, washed and incubated for 16 h in RPMI 1640 medium, then washed and stimulated for 10 min with 0.1 \iM PAF, 0.1 ng/ml PMA, 10 \iM ionomycin or control. Separate wells were used for (i) separation of basal phospholipid classes and analysis of their molecular species compositions by Ag-TLC (ii) analysis ol DG molecular species composition by Ag-TLC and (iii) analysis of totaf DG mass. Each experiment was performed in duplicate and each condition was plated in duplicate or triplicate. The proportion of EPA incorporated into the sn-2 position of membrane phospholipids is significantly greater than that of DHEE when equivalent amounts of the ethyl esters are present in the diet. Phospholipid incorporation of EPEE and DHEE also produced significant dose-dependent changes in the molecular species profiles of PC, PI, PS and PE. The proportion of DG molecular species with EPA or DHA in the sn-2 position is significantly elevated under basal conditions and after stimulation ol macrophages with PAF, PMA and ionomycin, when compared to control diet. The proportion ol DG molecular species formation with EPA at the sn-2 position is considerably greater than that of species with DHA under conditions of equtmolar dietary content. Quantitatively, the Incorporation of EPA into DG demonstrated a greater capacity to attenuate total DG mass compared to DHA. Furthermore, a dose-dependent decrease in total DG mass paralleled the decrease in DHEE component and increase in EPEE component of the diet. These results demonstrate that incorporation of EPA and DHA into phospholipids and into DG vary dose-dependently with dietary content and that EPA is incorporated preferentially over DHA. Furthermore, we suggest that physiologically and therapeutically relevant differences may exist between EPA and DHA. Understanding the changes in cellular processes attributable to the incorporation of n-3PUFA into diradylglyc erol and the resulting changes in signal transduction could further contribute to our understanding of the pathological mechanisms underlying atherogenic syndromes, inflammatory diseases and neoplasia.