High-performance liquid chromatography determination of bis(monoacylglycerol) phosphate and other lysophospholipids

Bis(monoacylglycerol) phosphate (BMP) is a very minor component of the phospholipid (PL) fraction in rat uterine stromal cell cultures (U(III) cells). Under several culture conditions, including the addition of (n-3) or (n-6) polyunsaturated fatty acids, BMP selectively accumulates docosahexaenoic acid (DHA). We have recently described the structure of this PL, but its biological function is still largely unknown, except for a role in late endosomes trafficking. In order to further investigate this function, we have developed a sensitive assay for accurate determination of BMP in small biological samples. Total PL from cells, labeled or not with trace amount of [3H]DHA, were extracted and PL classes separated by thin-layer chromatography. After extraction of the gel corresponding to the BMP area, a known amount of an internal standard was added. The free hydroxyl groups of PL were totally derivatized with naproxen. Derivatized PL were separated by normal-phase high-pressure liquid chromatography and quantified using UV absorption at 231 nm. Since the sensitivity of the proposed method was about 0.1 nmol for BMP, samples of only 3 x 10(5) cells were required. The BMP level was found to be 616 +/- 46 pmol for 10(6) control cells. It was increased threefold in starved cells and significantly increased in cells cultured in the presence of exogenous phosphatidylglycerol.