QUANTITATION OF THE DIACYL, ALKYLACYL, AND ALK-1-ENYLACYL SUBCLASSES OF CHOLINE GLYCEROPHOSPHOLIPIDS BY CHEMICAL DEPHOSPHORYLATION AND BENZOYLATION

A method for the determination of the relative amounts of the diacyl, alkylacyl, and alk-1-enylacyl subclasses of choline glycerophospholipi ds by benzoolysis is described. The procedure consists of simply heati ng the phospholipid with benzoic anhydride in the presence of boric ac id for 5 h at 100 degrees C, followed by reaction with 4-dimethylamino pyridine for 2 h at room temperature and HPLC analysis of the lipidic products formed. With model compounds of the three subclasses it is sh own that diacyl- and alkylacylglycerophosphocholines are completely de phosphorylated by this procedure, yielding quantitatively the correspo nding diacyl-and alkylacylglycerobenzoates. The same procedure applied to a model alk-1-enylacylglycerophosphocholine gives only 53.5% of de phosphorylation, while the dephosphorylated products in turn are quant itatively converted into the corresponding acylglycerodibenzoates. The latter figure is shown to be fairly reproducible. The reduced dephosp horylation rate of plasmalogens appears to be due to complete disrupti on of the vinyl ether bond. The liberated fatty aldehyde gives rise to an addition product with benzoic anhydride, which was identified by g as chromatography-mass spectrometry. It is demonstrated that acyl migr ation occurring during the benzoolysis does not interfere with the HPL C separation of the glycerobenzoates and dibenzoates derived from the three distinct subclasses. Results of subclass determinations by benzo olysis of several natural diradylglycerophosphocholines are in good ac cordance with literature values. The agreement between the plasmalogen contents, determined by benzoolysis and by phosphorus determination f ollowing exposure to HCl and separation by thin-layer chromatography, is satisfactory. The reliability of the benzoolysismethod is generally discussed. (C) 1994 Academic Press, Inc.