HPLC-fluorimetric assay of phospholipase A(2). Application to biological samples with high protein content and various reaction conditions

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dc.contributor.authorKarkabounas, A.en
dc.contributor.authorKitsiouli, E. I.en
dc.contributor.authorNakos, G.en
dc.contributor.authorLekka, M. E.en
dc.date.accessioned2015-11-24T16:39:09Z
dc.date.available2015-11-24T16:39:09Z
dc.identifier.issn1570-0232-
dc.identifier.urihttps://olympias.lib.uoi.gr/jspui/handle/123456789/8124
dc.rightsDefault Licence-
dc.subjectenzymic assayen
dc.subjectphospholipase a(2)en
dc.subjectnbd lipidsen
dc.subjecthplcen
dc.subjectfluorescenceen
dc.subjecthuman pancreatic phospholipase-a2en
dc.subjectactivating-factor acetylhydrolaseen
dc.subjectrespiratory-distress-syndromeen
dc.subjectacute lung injuryen
dc.subjecttetrahymena-pyriformisen
dc.subjectserumen
dc.subjectlipidsen
dc.subjectaciden
dc.subjectsuperfamilyen
dc.subjectmacrophagesen
dc.titleHPLC-fluorimetric assay of phospholipase A(2). Application to biological samples with high protein content and various reaction conditionsen
heal.abstractPhospholipase A(2) (PLA(2)) quantitation in real-time, using (7-nitro-2-1,3-benzoxadiazol-4-yl)aminoderivatives of phosphatidylcholine (NBD-PCs) as substrates, is influenced by high protein content, color or turbidity. The purpose of the study was to overcome such limitations by a complementary reversed-phase HPLC step to separate the substrates from the products of the reaction. Plasma and bronchoalveolar lavage (BAL) fluid were applied as enzymic sources, while standard porcine PLA(2) and human plasma PAF-acetylhydrolase (PAF-AH) were employed as positive controls. The method was validated with a radiometric assay and compared with the real-time fluorimetric assay. Regarding PLA(2) and PAF-AH determination, the isocratic elution systems CH(3)OH-H(2)O (80:20, v/v) and CH(3)OH-H(2)O-CH(3)COOH (60:40:0.2, v/v/v) separated efficiently C(12)-NBD-FA/C(12)-NBD-PC and C(6)-NBD-FA/C(6)-NBD-PC, with 4.4 and 2.2 resolution, respectively. Analysis time was similar to 15 min/injection. The reproducibility, expressed as relative standard deviation, was <= 13% for PLA(2) and <= 16% for PAF-AH, respectively. The assay was linear for PLA(2) activities extending from 1 pmol up to at least 250 nmol FA/h/mL sample, similar to the radiometric assay. It was appropriate for samples with high protein content, where the real-time fluorimetric assay was insufficient. The HPLC method was also evaluated under elevated temperatures, various pH values and Ca(2+) concentrations. (C) 2011 Elsevier B.V. All rights reserved.en
heal.accesscampus-
heal.fullTextAvailabilityTRUE-
heal.identifier.primaryDOI 10.1016/j.jchromb.2011.03.047-
heal.identifier.secondary<Go to ISI>://000291330600010-
heal.identifier.secondaryhttp://ac.els-cdn.com/S1570023211002169/1-s2.0-S1570023211002169-main.pdf?_tid=a0d565bebb4e9eeb38d024d7b9fcf588&acdnat=1333033667_287e4ab46604e27285c3b2892db75c5b-
heal.journalNameJournal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciencesen
heal.journalTypepeer reviewed-
heal.languageen-
heal.publicationDate2011-
heal.publisherElsevieren
heal.recordProviderΠανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείαςel
heal.typejournalArticle-
heal.type.elΆρθρο Περιοδικούel
heal.type.enJournal articleen

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