Densitometry of phosphatidylcholine and sphingomyelin in high-density lipoproteins

We describe the quantitative densitometric determination of phosphatidylcholine (PC) and sphingomyelin (SP) in human serum after precipitation with phosphotungstic acid/MgCl2 and use of thin-layer chromatography. After development, chromatographic plates were charred with methanolic sulfuric acid and MnCl2 and scanned by direct reflectance densitometry in an automated densitometric system interfaced to a basic programmable computing integrator. The method is sensitive enough to detect abnormally low concentrations of PC and SP in high-density lipoproteins. The accuracy of the method was tested either with the Bartlett phosphorus assay or with enzymatic methods for PC and SP; correlations of the described method with the enzymatic determinations were r = 0.93 and 0.88, respectively. Day-to-day precision (CV) for the phospholipid determination was 8.6% for PC and 12.2% for SP. The major advantage of this inexpensive technique is that native plasma or serum or the serum supernate after precipitation can be used without prior delipidation. With this technique serum high-density lipoproteins had PC values of 1.08 (SD 0.32) mmol/L in men (n = 158) and 1.12 (SD 0.37) mmol/L in women (n = 192); similarly, SP values were 0.23 (SD 0.07) mmol/L in the men and 0.23 (SD 0.08) mmol/L in the women. The differences by sex are not significant.