Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological fluids using fluorescent substrates.
The purpose of the present study was the development and evaluation of a fluorimetric method for the screening and differential determination of phospholipase A(2) and PAF-acetylhydrolase in bronchoalveolar lavage (BAL) fluid and serum. Phospholipase A(2) was determined using C(12)-NBD-PC in the presence of Ca(2+), from the slope of the fluorescence enhancement due to the formation of C(12)-NBD-fatty acid. PAF-acetylhydrolase was determined using C(6)-NBD-PC, from the slope of the curve due to C(6)-NBD-fatty acid formation in the absence of Ca(2+). The results were confirmed after TLC analysis. The method's selectivity was evaluated by comparing to radiometric measurements. Light scattering did not interfere and inner filter effects was not observed under our experimental conditions. The effects of pH, temperature, and Ca(2+) were investigated. Protein caused an increase in the background fluorescence of both NBD-PCs. The standard curves of both NBD-fatty acids exhibited the same slope. Linearity extended at least up to 4. 5 nmoles per ml of reaction mixture at the normal pH 7.4. The fluorescence of the NBD-fatty acids remained stable for increasing concentrations of BAL fluid and serum and for BSA up to 100 microg/ml of reaction mixture. Porcine pancreatic PLase A(2) showed preference for C(12)-NBD-PC in the presence of Ca(2+), while without Ca(2+), serum PAF-AcH hydrolyzed only C(6)-NBD-PC. The method is highly sensitive, accurate, and reproducible and can be applied for the differential determination of phospholipase A(2) and PAF-acetylhydrolase activities in BAL fluid and serum.
Kitsiouli
E I
EI
Chemistry Department, Laboratory of Biochemistry, University of Ioannina, 451 10 Ioannina, Greece.
Nakos
G
G
Lekka
M E
ME
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001826:Body Fluids / Q000201:enzymology*; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology; D005227:Fatty Acids / Q000302:isolation & purification; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D055370:Lung Injury; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000032:analysis* / Q000097:blood; D010972:Platelet Activating Factor; D012015:Reference Standards; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013010:Sonication; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature
D005227:Fatty Acids; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D010972:Platelet Activating Factor; C067460:1-oleoyl-2-(12-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D012710:Serum Albumin, Bovine; C044745:1-palmitoyl-2-(6-(N-(7-nitrobenz)-2-oxa-1,3-diazol-4-yl)aminocaproyl)phosphatidylcholine; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D009327:4-Chloro-7-nitrobenzofurazan; D002118:Calcium