Assessment of various techniques for the quantitative extraction of lysophospholipids from myocardial tissues.

ABSTRACT

Lipid extraction methods were evaluated for their effectiveness in extracting lysophosphatidylcholines and lysophosphatidylethanolamines from tissues and for subsequent recoveries during purification of crude extracts. The acid-butanol technique, although effective in complete extraction, resulted in partial hydrolysis (2-10%) of phospholipids containing 1-alk-1'-enyl-2-acylglycerophospholipids (plasmalogens) to produce artifactual lysophospholipids. This problem was avoided using a neutral butanol extraction or Bligh and Dyer techniques, but these resulted in only partial recoveries (60-72 and 75-80%, respectively) of these lipids. Tissue extracted with neutral chloroform-methanol mixtures provided virtually complete extraction (97-100%), but subsequent losses (up to 15%) occurred during purification of crude extracts with Folch synthetic upper phases. These losses could be circumvented by purification of the crude extract on Sephadex G-25 column. As an alternative, the Folch extraction technique was modified to achieve complete recoveries of lysophospholipids. This involved extraction of the tissue with a chloroform-methanol-saline biphasic system. After removal of the lower lipid phase, the upper phase containing residual tissue was reextracted twice more with Folch lower phase and once with lower phase containing HCl. This last extract was neutralized with NH3 vapor before pooling with the preceding extracts. This method (i) circumvents plasmalogenic hydrolysis, (ii) avoids use of time-consuming column chromatography, (iii) eliminates the losses of lipids during purification, and (iv) allows highly reproducible quantitative analyses of all lipid fractions including lysophospholipids and nonesterified fatty acids from myocardial tissue.