A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method.
BACKGROUND
Several methods for measuring concentrations of lysophosphatidic acid (LPA), a lipid mediator, have been reported to date. However, these methods are not routinely used because most of them require specialized instrument and a complicated protocol.


METHODS
We developed a novel LPA assay using enzymatic cycling. LPA in a sample is hydrolyzed with lysophospholipase to glycerol-3-phosphate, followed by enzymatic cycling using glycerol-3-phosphate oxidase and glycerol-3-phosphate dehydrogenase. Amplified concentrations of hydrogen peroxides, a product of the enzymatic cycling, are then colorimetrically measured.


RESULTS
This method was specific for LPA, being insensitive to the presence of phosphatidic acid or lysophosphatidylcholine. The within-run and between-run CVs were 1.31-1.32% and 0.73-1.03%, respectively. The recoveries of exogenous LPA added to plasma were 100.3-101.6%. In males, LPA concentrations (mean+/-S.D.) of human serum and EDTA-plasma were 0.41+/-0.14 and 0.08+/-0.02 micromol/l, respectively. In females, they were 0.41+/-0.12 and 0.09+/-0.02 micromol/l, respectively.


CONCLUSIONS
This novel colorimetric assay for determination of LPA using enzymatic cycling is simple and highly sensitive. It can be used with an automatic analyzer. It may also be useful for further studies of the biological functions of LPA as well as clinical applications in various disorders.
Kishimoto
Tatsuya
T
Diagnostic Research and Development Department, R&D Division, Nesco Company, Azwell Inc., 2-24-3 Sho, Osaka 567-0806, Ibaraki, Japan. ta-kishimoto@azwell.co.jp
Matsuoka
Takeshi
T
Imamura
Shigeyuki
S
Mizuno
Koji
K
D000328:Adult; D002138:Calibration; D003124:Colorimetry / Q000379:methods; D004492:Edetic Acid; D005260:Female; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism; D005994:Glycerophosphates / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008245:Lysophospholipase / Q000378:metabolism; D008246:Lysophospholipids / Q000097:blood*; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D015219:Substrate Cycling; D013997:Time Factors
D005994:Glycerophosphates; D008246:Lysophospholipids; D004492:Edetic Acid; C029620:alpha-glycerophosphoric acid; D005993:Glycerolphosphate Dehydrogenase; C098831:glycerol-3-phosphate oxidase; D008245:Lysophospholipase