Assessment of bis(monoacylglycerol)phosphate isomers by thin layer chromatography using a modified solvent system

Bis(monoacylglycerol)phosphates <b>(</b>BMPs) are biologically functional acidic lipids present in acidic cell compartments. BMPs are deacylated by LPLA2, with distinct substrate specificity for positional BMP isomers. The conversion of S,S-(2,2'-diacyl)-BMP to other BMP isomers may be crucial for LPLA2-mediated BMP degradation. Recently, we have shown that the isomerization of S,S-(2,2'-diacyl)-BMP to S,S-(2,3'-diacyl)- and S,S-(3,3'-diacyl)-BMPs is enhanced by various factors, including intracellular alkalinization, albumin, and specific metal ions. Here we present a convenient method to distinguish S,S-(2,2'-diacyl)-BMP from other BMP isomers using thin layer chromatography (TLC). The conventional acidic solvent system consisting of chloroform/methanol/acetone/acetic acid/water used to distinguish BMP isomers was replaced with chloroform/methanol/acetic acid/water (65:15:10:2, v/v), providing a better separation and shortening the development time for BMP isomers in one-dimensional TLC. Using this new TLC system, isomerization-promoting activity was found and characterized in the 150,000<i>g</i> supernatant of RAW 264.7 cell homogenate. Employing the new system, it was observed that the BMP band corresponding to S,S-(2,2'-diacyl)-BMP in the same cell homogenates almost completely disappeared after incubating the homogenates for 15 h at 37 °C under neutral conditions, but not at acidic pH. Two-dimensional TLC performed using the same new acidic solvent system in the first dimension revealed that the disappearance of the band observed above was mainly due to isomerization rather than decomposition of BMP. This suggests that the cells possess an endogenous promoter for the isomerization of S,S-(2,2'-diacyl)-BMPs into other isomers. This TLC assay using the new acidic solvent system may be useful in assessing the isomerization of BMPs.