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.
Luquain
C
C
Laboratoire de Biochimie and Pharmacologie, INSERM U352-INSA de Lyon, 20 avenue A. Einstein, Villeurbanne Cedex, 69621, France.
Laugier
C
C
Lagarde
M
M
Pageaux
J F
JF
D000818:Animals; D002478:Cells, Cultured / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004281:Docosahexaenoic Acids / Q000032:analysis; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D008246:Lysophospholipids / Q000032:analysis* / Q000737:chemistry; D050178:Monoglycerides; D009288:Naproxen / Q000032:analysis / Q000737:chemistry*; D010715:Phosphatidylglycerols / Q000378:metabolism; D051381:Rats; D017154:Stromal Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D014599:Uterus / Q000166:cytology
D005456:Fluorescent Dyes; D008246:Lysophospholipids; D050178:Monoglycerides; D010715:Phosphatidylglycerols; C012786:bis(monoacylglyceryl)phosphate; D004281:Docosahexaenoic Acids; D009288:Naproxen