HPLC-fluorimetric assay of phospholipase A(2). Application to biological samples with high protein content and various reaction conditions
Φόρτωση...
Ημερομηνία
Συγγραφείς
Karkabounas, A.
Kitsiouli, E. I.
Nakos, G.
Lekka, M. E.
Τίτλος Εφημερίδας
Περιοδικό ISSN
Τίτλος τόμου
Εκδότης
Elsevier
Περίληψη
Τύπος
Είδος δημοσίευσης σε συνέδριο
Είδος περιοδικού
peer reviewed
Είδος εκπαιδευτικού υλικού
Όνομα συνεδρίου
Όνομα περιοδικού
Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences
Όνομα βιβλίου
Σειρά βιβλίου
Έκδοση βιβλίου
Συμπληρωματικός/δευτερεύων τίτλος
Περιγραφή
Phospholipase 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.
Περιγραφή
Λέξεις-κλειδιά
enzymic assay, phospholipase a(2), nbd lipids, hplc, fluorescence, human pancreatic phospholipase-a2, activating-factor acetylhydrolase, respiratory-distress-syndrome, acute lung injury, tetrahymena-pyriformis, serum, lipids, acid, superfamily, macrophages
Θεματική κατηγορία
Παραπομπή
Σύνδεσμος
<Go to ISI>://000291330600010
http://ac.els-cdn.com/S1570023211002169/1-s2.0-S1570023211002169-main.pdf?_tid=a0d565bebb4e9eeb38d024d7b9fcf588&acdnat=1333033667_287e4ab46604e27285c3b2892db75c5b
http://ac.els-cdn.com/S1570023211002169/1-s2.0-S1570023211002169-main.pdf?_tid=a0d565bebb4e9eeb38d024d7b9fcf588&acdnat=1333033667_287e4ab46604e27285c3b2892db75c5b
Γλώσσα
en
Εκδίδον τμήμα/τομέας
Όνομα επιβλέποντος
Εξεταστική επιτροπή
Γενική Περιγραφή / Σχόλια
Ίδρυμα και Σχολή/Τμήμα του υποβάλλοντος
Πανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείας