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

In rat uterine stromal cells (U_III cells), docosahexaenoic acid (DHA) was esterified extensively in alkenylacyl-glycerophosphoethanolamine and in an unknown phospholipid accounting for only 0.7% of the total phospholipid. The latter was identified as a bis(monoacylglycerol) phosphate (BMP) using MS. Incorporation studies using C_18:3n-3 and C_20:5n-3 demonstrated that BMP had a high specificity to incorporate DHA and C₂₂ 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_III cells with 5µM DHA for 24h increased the DHA content of BMP from 36 to 71% of the total acyl chains. [³H]DHA-labelled BMP purified as a single TLC spot was resolved into three peaks using HPLC. These peaks were also observed when cells were labelled with [³H]phosphatidylglycerol, an exogenous BMP precursor, and with [³³P]P_i. Electrospray MS of BMP from control cells showed that the first two peaks contained the same molecular species (mainly C_22:6n-3/C_22:6n-3 and C_18:1n-9/C_22:6n-3) while the third peak mainly contained the C_18:1n-9/C_18:1n-9 species. The stereoconfiguration analysis of the compounds revealed an sn-glycero-3-phospho-1′-sn-glycerol configuration for the first peak and sn-glycero-1-phospho-1′-sn-glycerol configurations for the other two. BMP from rat testis was used to establish the positions of the acyl groups. More than 70% of its acyl chains were C_22:5n-6. It was separated on HPLC into three peaks that co-migrated with the three peaks of BMP from U_III cells. Lipase activity and NMR analysis of the second peak showed that fatty acids esterified the primary alcohol group on each glycerol moiety. We conclude that the three peaks are stereoisomeric compounds with different acyl-chain locations and may be the result of different metabolic fates depending on subcellular localization.