title: Immobilization of Lipid Substrates: Application on Phospholipase A2 Determination.
abstract: The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A(2) (PLA(2)) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C(12)-NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn-2 acyl chain of the phospholipid stands out and is accessible by PLA(2). Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA(2). Mass spectrometry showed that only C(12)-NBD-FA, the PLA(2 )hydrolysis product, was detected in the reaction mixture, indicating that PLA(2) recognizes PVA-SbQ/C(12)-NBD-PtdCho as a surface to perform catalysis.
authors.affiliation: Chemistry Department, University of Ioannina, 45110, Ioannina, Greece.
authors.forename: Athanassios
authors.initials: A
authors.lastname: Karkabounas
authors.affiliation: Institute of Microelectronics, NCSR Demokritos, 15310, Aghia Paraskevi, Greece.
authors.forename: Dimitra G
authors.initials: DG
authors.lastname: Georgiadou
authors.affiliation: Institute of Microelectronics, NCSR Demokritos, 15310, Aghia Paraskevi, Greece.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Argitis
authors.affiliation: Institute of Advanced Materials Physicochemical Processes Nanotechnology and Microsystems, NCSR Demokritos, 15310, Aghia Paraskevi, Greece.
authors.forename: Vassilios
authors.initials: V
authors.lastname: Psycharis
authors.affiliation: Intensive Care, Faculty of Medicine, University of Ioannina, 45110, Ioannina, Greece.
authors.forename: George
authors.initials: G
authors.lastname: Nakos
authors.affiliation: Chemistry Department, University of Ioannina, 45110, Ioannina, Greece.
authors.forename: Agni M
authors.initials: AM
authors.lastname: Kosmas
authors.affiliation: Chemistry Department, University of Ioannina, 45110, Ioannina, Greece. mlekka@cc.uoi.gr.
authors.forename: Marilena E
authors.initials: ME
authors.lastname: Lekka
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019295:Computational Biology; D005240:Feasibility Studies; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D008567:Membranes, Artificial; D018625:Microscopy, Atomic Force; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2 / Q000097:blood / Q000378:metabolism*; D010865:Pilot Projects; D011142:Polyvinyl Alcohol / Q000031:analogs & derivatives / Q000737:chemistry; D011650:Pulmonary Alveoli / Q000201:enzymology*; D011726:Pyridinium Compounds / Q000737:chemistry; D015203:Reproducibility of Results; D020545:Respiratory Mucosa / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D013379:Substrate Specificity; D034421:Sus scrofa
chemical_list: C000610521:1-palmitoyl-2-(12-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino)dodecanoyl)glycero-3-phosphocholine; D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D011726:Pyridinium Compounds; D011142:Polyvinyl Alcohol; D054467:Phospholipases A2; D009327:4-Chloro-7-nitrobenzofurazan
keywords: Fluorescence; HPLC; Mass spectrometry; Phospholipase A2; Phospholipases; Phospholipids