Fatty acids in phospholipids isolated from human red cells

Total lipids of packed erythrocytes from healthy men 22 to 25 years old were extracted with chloroform-methanol mixture. Phospholipid classes were separated from neutral lipids and pigments on a silicic acid column. Phosphatidyl inositol (PI) was freed of its contaminants phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) on an aluminum oxide column. Additional silicic acid columns with modified solvent systems were needed for complete separation of other overlapped phospholipid classes. The identification of phospholipids in each eluted fraction was accomplished by TLC, using the appropriate spray tests and reference compounds, and confirmed on each of the isolated phospholipids by IR spectrophotometry.The total content of phospholipids as determined by phosphorus analysis was found to be 2.63 mg/ml of packed cells. These phospholipids were found to have the following composition (in per cent of total phospholipid): PI, 2.3; PE, 13.4; ethanolamine plasmalogen (EP), 14.5; PS, 3.9; lecithin (L), 34.2; choline plasmalogen (CP), 1.4; sphingomyelin (Sph), 28.4 and lysolecithin (LL), 1.7. The fatty acid composition of each phospholipid was determined by GLC. The average number of double bonds per fatty acid in the isolated phospholipids was found to be as follows: PI, 1.5; PE, 1.9; EP, 3.6; PS, 2.1; L, 1.0; CP, 2.0; Sph, 0.2 and LL, 0.5. The positional distribution of fatty acids in both L and PE was ascertained by selective enzymatic hydrolysis with phospholipase A. Saturated fatty acids of L were esterified predominantly in the alpha'-position, whereas in PE only 63.9 mole per cent of the saturated fatty acids were found in this position.