Determination of the catalytic activity of phospholipase A2: E. coli-based assay compared to a photometric micelle assay.
Phospholipase A2 activity in human sera was determined of the basis of the E. coli assay and compared to a photometric micelle assay. The E. coli assay is based on the hydrolysis of phospholipids from [1-14C]oleic acid-labelled E. coli biomembranes. In the photometric assay the phospholipase A2 acts on mixed phospholipid micelles. The amount of fatty acid produced is quantitated in a subsequent photometric assay by coupling in the reaction to the coenzyme A metabolism. The E. coli membranes are essentially resistant to other lipases in human sera, i.e. lipoprotein lipases, hepatic triacylglycerolipase or pancreatic lipase and thus a very specific substrate for the phospholipase A2 of human serum. The photometric assay, though, is susceptible to other lipases in human serum. The ratio of [1-14C]oleic acid to released total fatty acids served as the basis for the calculation of the true enzymatic activity. The assay closely correlated with the photometric assay based on mixed micelles in the higher ranges of phospholipase A2 activity, but not in the normal range. The sensitivity is higher by at least two powers of 10. The human serum phospholipase A2 strongly preferred E. coli membranes as substrate to the mixed micelles containing phosphatidylcholine/phosphatidylethanolamine. In conclusion, the modified phospholipase A2 assay based on E. coli membranes is a sensitive, specific, reliable, and convenient method for the measurement of phospholipase A2 activity in human sera. The photometric assay suffers from low sensitivity but has the advantage of practicability in a normal routine laboratory, including the amenability to automation.
Aufenanger
J
J
Institut für Klinische Chemie, Klinikum Mannheim, Fakultät für Klinische Medizin der Universität Heidelberg, Germany.
Zimmer
W
W
Püttmann
M
M
Ensenauer
R
R
D000209:Acute-Phase Proteins / Q000032:analysis; D002384:Catalysis; D004926:Escherichia coli; D005227:Fatty Acids / Q000032:analysis; D005260:Female; D006801:Humans; D008297:Male; D008823:Micelles; D010741:Phospholipases A / Q000097:blood*; D054467:Phospholipases A2; D010783:Photometry / Q000379:methods; D011874:Radiometry / Q000379:methods; D012016:Reference Values
D000209:Acute-Phase Proteins; D005227:Fatty Acids; D008823:Micelles; D010741:Phospholipases A; D054467:Phospholipases A2