Studies on phospholipids. 3. Determination of choline

Abstract

A colorimetric method for the determination of choline in lipids has been developed in which the choline in a hydrolysate is precipitated by phosphomolybdic acid. A sample (0.5 ml) of hydrolysate containing 0.3-3.0 [mu]moles of choline was mixed in a 15 ml centrifuge tube with 0.1 ml of a saturated aqueous solution of phosphomolybdic acid. The mixture was kept for 30 minutes at 5[degree] and then centrifuged; the supernatant was decanted and the tube was inverted and allowed to drain for 15 minutes. The precipitate was then suspended in 1 ml of isobutanol and centrifuged, the supernatant was discarded and the process was repeated. The washed precipitate was therrdissolved in 3 ml of acetone. To this solution was added 1 ml of 10 N H2SO4 and 0.05 ml of SuCl2 csolution and the mixture made up to 10 ml with ethanol. The optical density of the blue solution was measured in a Hilger Spekker absorptiometer with an IIford 608 filter. The term "molecular-extinction coefficient" as used in this paper refers to the value obtained with the optical density thus measured and is approximately 87% of the true value at 630 mu. The plot of concentration (0.3-3.0 moles/0.5 ml) against the optical density was linear and passed through the origin. The value of the "molecular-extinction coefficient" was 4810 [plus or minus] 170 (mean of 24 determinations). Choline added to a lipid hydrolysate was quantitatively recovered, as was also the choline in mixtures of lecithin and kephalin. The method agreed with the re-ineckate metho''d but was not affected by light and is simpler to use.