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*10-5 M 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.
Keywords: Choline oxidase; Enzyme; Fluorescence; Phospholipase D; Phospholipids; Platelet activation factor.
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