Specific, rapid, and sensitive enzymatic measurement of sphingomyelin, phosphatidylcholine and lysophosphatidylcholine in serum and lipid extracts.
OBJECTIVE
Human serum sphingomyelin (SM) and phosphatidylcholine (PC) play important roles in the development of atherosclerosis. However, there are no rapid and sensitive methods for SM and PC measurement. The present report describes a novel enzymatic method for measuring SM, PC and lysophosphatidylcholine (lyso-PC) levels in plasma and lipid extracts.


METHODS
The total choline-containing phospholipids (total PL), SM and PC were measured using a two-reagent system involving specific enzymes for choline-based phospholipids. The procedure was performed using either microplate or automatic analyzer technology. The concentration of lyso-PC was calculated by subtracting the concentration of SM plus PC from the total PL concentration.


RESULTS
Assay results showed linear correlations between sample concentration and absorbance. The within-run and between-run coefficients of variation for PC, SM, and lyso-PC concentrations were 2.0-4.4% for the microplate analyzer and 0.9-2.9% for the automatic analyzer. Analysis of normal human serum showed that the total PL concentration strongly correlated with the SM plus PC concentration (r=0.9850). There were moderate correlations between serum PC and SM levels (r=0.6228) and between serum PC and lyso-PC levels (r=0.7806). SM, PC, and lyso-PC levels in normal human serum (n=50) were 0.54+/-0.07, 1.99+/-0.22 and 0.60+/-0.15 mmol/L, respectively.


CONCLUSIONS
The present enzymatic method allowed for rapid, simple, and accurate measurement of SM, PC, and lyso-PC levels in lipid extracts and in serum. The method is suitable for both microplate and automatic analyzer assays.
Hidaka
Hiroya
H
Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Nagano, 390-8621, Japan. hiroyan@hsp.md.shinshu-u.ac.jp
Yamauchi
Kazuyoshi
K
Ohta
Hiroyashi
H
Akamatsu
Taiji
T
Honda
Takayuki
T
Katsuyama
Tsutomu
T
D001671:Biochemistry / Q000379:methods*; D002138:Calibration; D006262:Health; D006801:Humans; D006868:Hydrolysis; D008055:Lipids / Q000097:blood*; D008075:Lipoproteins, HDL / Q000097:blood; D008244:Lysophosphatidylcholines / Q000097:blood*; D013058:Mass Spectrometry; D010713:Phosphatidylcholines / Q000097:blood*; D010739:Phospholipase D / Q000378:metabolism*; D015203:Reproducibility of Results; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000097:blood*; D013379:Substrate Specificity; D014020:Tissue Extracts
D008055:Lipids; D008075:Lipoproteins, HDL; D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D014020:Tissue Extracts; D013108:Sphingomyelin Phosphodiesterase; D010739:Phospholipase D