J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.D500040-JLR200 on December 21, 2005

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Journal of Lipid Research, Vol. 47, 673-676, March 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Methods

Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine

Mohammad Reza Hojjati and Xian-Cheng Jiang1

Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203

Published, JLR Papers in Press, December 21, 2005.

1 To whom correspondence should be addressed. e-mail: xjiang{at}downstate.edu

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 µg, and that for PC was 2.5–20 µg. 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.

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


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