Abstract
Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins. Their concentration is classically measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots. Here, we describe two rapid, specific, and sensitive enzymatic measurements for both phospholipids. Plasma was incubated with bacterial sphingomyelinase (for SM measurement) or bacterial PC-specific phospholipase D (for PC measurement), alkaline phosphatase, choline oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, and 4-aminoantipyrine for 45 min. A blue dye, with an optimal absorption at 595 nm, was generated. PC levels did not influence SM measurement and vice versa. The linear range for the SM measurement was 0.5-5 microg, and that for PC was 2.5-20 microg. The mean percentage recovery was 98.0 +/- 5.2% for SM and 96.6 +/- 3.8% for PC. The interassay coefficient of variation of the assay was 1.7 +/- 0.05% for SM and 3.1 +/- 0.13% for PC. These two new methods are amenable to automation and can be adapted for large-scale, high-throughput assays.
Highlights
Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins
We and others demonstrated for the first time that administration of myriocin into apolipoprotein E knockout mice dramatically decreased SM, increased PC, decreasing the SM/PC ratio in the plasma, and significantly decreased the atherosclerotic lesion area [10, 11]
These data suggest that SM might play a promoting role, whereas PC might play a preventive role, in the development of atherosclerosis, and their measurement might provide new insights into atherogenesis in humans and in various mouse models as well
Summary
Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins Their concentration is classically measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots. We and others demonstrated for the first time that administration of myriocin (an inhibitor of SM synthesis) into apolipoprotein E knockout mice dramatically decreased SM, increased PC, decreasing the SM/PC ratio in the plasma, and significantly decreased the atherosclerotic lesion area [10, 11] These data suggest that SM might play a promoting role, whereas PC might play a preventive role, in the development of atherosclerosis, and their measurement might provide new insights into atherogenesis in humans and in various mouse models as well. Plasma SM and PC were measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots This method is time-consuming and not sensitive.
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