title: Enzymatic methods for choline-containing water soluble phospholipids based on fluorescence of choline oxidase: Application to lyso-PAF.
abstract: In this paper we present methods to determine water soluble phospholipids containing choline (wCh-PL). The analytes were hydrolyzed by the enzyme phospholipase D and the choline formed was oxidized by the enzyme Choline Oxidase (ChOx); the fluorescence changes of the ChOx are followed during the enzymatic reaction, avoiding the necessity of an indicating step. Both reactions (hydrolysis and oxidation) can be combined in two different ways: 1) a two-step process (TSP) in which the hydrolysis reaction takes place during an incubation time and then the oxidation reaction is carried out, the analytical signal being provided by the intrinsic fluorescence of ChOx due to tryptophan; 2) a one-step process (OSP) in which both enzymatic reactions are carried out simultaneously in the same test; in this case the analytical signal is provided by the ChOx extrinsic fluorescence due to a fluorescent probe (Ru (II) chelate) linked to the enzyme (ChOx-RuC). The analytical capabilities of these methods were studied using 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8PC), a water soluble short alkyl chain Ch-PL as a substrate, and 1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF). The analytical features of merit for both analytes using both methods were obtained. The TSP gave a 10-fold sensitivity and lower quantification limit (1.0*10-5M for lyso-PAF), but OSP reduced the determination time and permitted to use the same enzyme aliquot for several measurements. Both methods gave similar precision (RSD 7%, n = 5). The TSP was applied to the determination of C8PC and lyso-PAF in spiked synthetic serum matrix using the standard addition method. The application of this methodology to PLD activity determination is also discussed.
authors.affiliation: Analytical Biosensors Group, Analytical Chemistry Department, Faculty of Sciences, Aragon Institute of Nanoscience, University of Zaragoza, 50009 Zaragoza, Spain. Electronic address: isasanz@unizar.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: Sanz-Vicente
authors.affiliation: Analytical Biosensors Group, Analytical Chemistry Department, Faculty of Sciences, Aragon Institute of Nanoscience, University of Zaragoza, 50009 Zaragoza, Spain.
authors.forename: Andrés
authors.initials: A
authors.lastname: Domínguez
authors.affiliation: Analytical Biosensors Group, Analytical Chemistry Department, Faculty of Sciences, Aragon Institute of Nanoscience, University of Zaragoza, 50009 Zaragoza, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Ferrández
authors.affiliation: Analytical Biosensors Group, Analytical Chemistry Department, Faculty of Sciences, Aragon Institute of Nanoscience, University of Zaragoza, 50009 Zaragoza, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Galbán
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010084:Oxidation-Reduction; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis*; D010972:Platelet Activating Factor / Q000031:analogs & derivatives* / Q000737:chemistry; D012995:Solubility; D014867:Water / Q000737:chemistry*
chemical_list: C029271:O-deacetyl platelet activating factor; D010743:Phospholipids; D010972:Platelet Activating Factor; D014867:Water; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D010739:Phospholipase D
keywords: Choline oxidase; Enzyme; Fluorescence; Phospholipase D; Phospholipids; Platelet activation factor