Document Detail


Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological fluids using fluorescent substrates.
MedLine Citation:
PMID:  10588961     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
E I Kitsiouli; G Nakos; M E Lekka
Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of lipid research     Volume:  40     ISSN:  0022-2275     ISO Abbreviation:  J. Lipid Res.     Publication Date:  1999 Dec 
Date Detail:
Created Date:  2000-02-10     Completed Date:  2000-02-10     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0376606     Medline TA:  J Lipid Res     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  2346-56     Citation Subset:  IM    
Affiliation:
Chemistry Department, Laboratory of Biochemistry, University of Ioannina, 451 10 Ioannina, Greece.
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MeSH Terms
Descriptor/Qualifier:
1-Alkyl-2-acetylglycerophosphocholine Esterase
4-Chloro-7-nitrobenzofurazan / analogs & derivatives,  metabolism
Animals
Body Fluids / enzymology*
Bronchoalveolar Lavage Fluid / chemistry
Calcium / pharmacology
Fatty Acids / isolation & purification
Fluorescent Dyes
Humans
Hydrogen-Ion Concentration
Kinetics
Lung / metabolism
Lung Injury
Pancreas / enzymology
Phosphatidylcholines / metabolism
Phospholipases A / analysis*,  blood
Platelet Activating Factor
Reference Standards
Serum Albumin, Bovine / pharmacology
Sonication
Spectrometry, Fluorescence
Substrate Specificity
Swine
Temperature
Chemical
Reg. No./Substance:
0/Fatty Acids; 0/Fluorescent Dyes; 0/Phosphatidylcholines; 0/Platelet Activating Factor; 0/Serum Albumin, Bovine; 10199-89-0/4-Chloro-7-nitrobenzofurazan; 131479-28-2/1-oleoyl-2-(12-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)dodecanoyl)phosphatidylcholine; 7440-70-2/Calcium; 81005-34-7/1-palmitoyl-2-(6-(N-(7-nitrobenz)-2-oxa-1,3-diazol-4-yl)aminocaproyl)phosphatidylcholine; EC 3.1.1.-/Phospholipases A; EC 3.1.1.47/1-Alkyl-2-acetylglycerophosphocholine Esterase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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