出版社：American Society for Biochemistry and Molecular Biology
摘要：Sphingomyelin (SM) and phosphatidylcholine (PC) are two majorphospholipids on plasma lipoproteins. Their concentration isclassically 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 measurementsfor both phospholipids. Plasma was incubated with bacterialsphingomyelinase (for SM measurement) or bacterial PC-specificphospholipase 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 optimalabsorption at 595 nm, was generated. PC levels did not influenceSM measurement and vice versa. The linear range for the SM measurementwas 0.5–5 µg, and that for PC was 2.5–20 µg.The mean percentage recovery was 98.0 ± 5.2% for SM and96.6 ± 3.8% for PC. The interassay coefficient of variationof the assay was 1.7 ± 0.05% for SM and 3.1 ±0.13% for PC. These two new methods are amenable to automationand can be adapted for large-scale, high-throughput assays.Supplementary key words phospholipid measurement • large-scale and high-throughput assays • enzymatic measurements
Abbreviations: DAOS, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline; PC, phosphatidylcholine; SM, sphingomyelin; SMase, sphingomyelinase
