Bis(monoacylglycerol) phosphate in rat uterine stromal cells: structural characterization and specific esterification of docosahexaenoic acid

In rat uterine stromal cells (U-111 cells), docosahexaenoic acid (DHA) was esterified extensively in alkenylacyl-glycerophosphoethanolamine and in an unknown phospholipid accounting for only 0.7% of the total phospholipid. Th e latter was identified as a bis(monoacylglycerol) phosphate (BMP) using MS . Incorporation studies using C(18:3)n-3 and C(20:5)n-3 demonstrated that B MP had a high specificity to incorporate DHA and C-23 polyunsaturated fatty acids of the (n-3) series. By contrast, polyunsaturated fatty acids of the (n-6) series were never incorporated into BMP. Incubation of U-111 cells w ith 5 muM DHA for 24 h increased the DHA content of BMP from 36 to 71% of t he total acyl chains. [H-3]DHA-labelled BMP purified as a single TLC spot w as resolved into three peaks using HPLC. These peaks were also observed whe n cells were labelled with [H-3]- phosphatidylglycerol, an exogenous BMP pr ecursor, and with [P-33]P-1. Electrospray MS of BMP from control cells show ed that the first two peaks contained the same molecular species (mainly C( 22:6)n-3/C(22:6)n-3 and C(18:1)n-9/C(22:6)n-3) while the third peak mainly contained the C(18:1)n-9/C(18:1)n-9 species. The stereoconfiguration analys is of the compounds revealed an sn-glycero-3-phospho-1'-sn-glycerol configu ration for the first peak and sn-glycero-1-phospho-1'-sn-glycerol configura tions for the other two. BMP from rat testis was used to establish the posi tions of the acyl groups. More than 70% of its acyl chains were C-22:5 n-6. It was separated on HPLC into three peaks that co-migrated with the three peaks of BMP from U-111 cells. Lipase activity and NMR analysis of the seco nd peak showed that fatty acids esterified the primary alcohol group on eac h glycerol moiety. We conclude that the three peaks are stereoisomeric comp ounds with different acyl-chain locations and may be the result of differen t metabolic fates depending on subcellular localization.