Elsevier

Analytical Biochemistry

Volume 519, 15 February 2017, Pages 30-37
Analytical Biochemistry

Enzymatic methods for choline-containing water soluble phospholipids based on fluorescence of choline oxidase: Application to lyso-PAF

https://doi.org/10.1016/j.ab.2016.12.005Get rights and content

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*10−5 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.

Introduction

Glycerophospholipids (PLs) constitute a class of lipids with very important biological roles in human health and nutrition. One of the most interesting PLs families is that containing choline (Ch-PLs). In fact, the most common biological PLs are phosphatidylcholines (PCs) [1], produced by PLs esterification with a Ch molecule. PC is the major constituents of the cell membrane. It has many common applications in the pharmaceutical industry (as a drug, in drug delivery systems [2] or simply as an excipient [1]), in the food industry [3], [4] (as part of food preparations and as emulsifiers) and many others.

Since PC has two fatty chains (C16 or C18), it is a water insoluble compound, which limits some of its applications. However, there are other Ch-PLs which are fairly soluble (wCh-PLs), especially those having only one large chain (-lyso compounds) or those having short chains.

The Platelet Activating Factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a wCh-PL having only one chain. It is a potent bioactive mediator related with platelet aggregation, inflammatory response [5] and other processes [6]. Lyso-PAF (1-O-alkyl-sn-glycero-3-phosphocholine) is a precursor [7] and a metabolite [8] of PAF. Since PAF is quickly deacetylized by the enzyme acetylhydrolase yielding Lyso-PAF, the concentration level of the former in blood or biological samples [9] is about three orders of magnitude lower than that of the latter (blood [10], [11] and organ [12] levels are in the part-per-million range). It was believed that the lyso-PAF lacks bioactivity, but in recent years it has been attributed a biological role balancing some of the bioactivities of PAF [13] and it has acquired relevance as a biological PAF marker.

Short chain wCh-PLs refer to those PLs having one or two C6 to C9 chains. These are increasingly being used as biological surfactants, because they are less aggressive against protein denaturation [14], and also in new chemical drug delivery systems (virosomes) [15].

From the analytical point of view, there is no well-established methodology for short-chain wCh-PLs determination. wCh-PLs have been used as substrates for determining the activity of phospholipase D (PLD) [16]. For lyso-PAF determination, methods based on the capability of PAF as a platelet aggregation (lyso-PAF is previously acetylated) have been used [9], but for the selective determination of the analyte, HPLC [12], [17] (reverse phase and detection by fluorescence) or GC (with MS detector) [10] are more common.

Enzymatic methods [18], [19], [20] are an interesting alternative for the analysis of Ch-PLs. They involve a hydrolysis step catalyzed by PLDCh-PL + H2O ← PLD → Ch + phosphatidic acid

and an oxidation step of the Ch to Glycine Betaine (GB) using Choline Oxidase (ChOx)Ch + 2O2 ← ChOx → GB + 2H2O2

In the indicating step, in the presence of peroxidase enzyme (HRP), the hydrogen peroxide reacts with a dye precursor to generate a fluorophore or chromophore. Several commercial kits have been developed. These methods give good results, but they are time-consuming and not fully reversible. The optical properties of proteins have been widely used in bioanalysis [21] and these problems can be partially overcome by exploiting the spectroscopic properties of the enzymes, especially the fluorescence of flavoenzymes (but not only this [22]) or the absorption of metalloenzymes [23]. This methodology is better understood if the mechanism by which flavoenzymes catalyse the oxidation of substrates (i.e. ChOx in (2)) is considered:

As noted, the flavin group of the enzyme oxidizes the substrate giving the product and it becomes reduced to FAD.H2; then, the oxygen regenerates the FAD and H2O2 is simultaneously formed. This reaction can be followed using three alternatives [24], [25], [26]:

  • A)

    tryptophan (Trp) fluorescence. Due to an energy transfer mechanism with FAD, the fluorescence of the Trp also changes during the enzymatic reaction; depending on the Ch concentration, the fluorescence intensity changes during the reaction as indicated in Fig. 1.

  • B)

    flavin fluorescence; since both FAD and FAD.H2 have different fluorescence properties (the oxidized forms being more fluorescent than the reduced) the fluorescence intensity changes during the reaction as a mirror image to that shown in Fig. 1.

  • C)

    probe fluorescence; some fluorophores linked to the enzyme, such as fluorescein (FS), also modify their fluorescence during the reaction; signals similar to those given in Fig. 1 are obtained. Option A requires a lower enzyme concentration than B. Option C is recommended when a high background is observed.

This paper proposes a methodology based on the combination of PLD and ChOx for wCh-PLs determination, which can also be extended to the determination of the PLD activity. Two configurations have been tested. In the first one, reactions (1) and (2) are carried out sequentially, and the tryptophan fluorescence is used (method A). In the second one, both enzymatic processes are carried out simultaneously and the indicating method C (using a Ru(II) chelate as probe) is optimized and used. Both methods have been evaluated with 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8PC), a water soluble short alkyl chain Ch-PC, and successfully applied to the determination of this compound and lyso-PAF. Since a preconcentration step is not applied, the proposed method does not improve the sensitivity of some previously reported HPLC and GC methods, but it is faster and easier to apply. The application of this method to PLD activity determination is also discussed.

Section snippets

Reagents

Acetate buffer solution (0.1 M, pH 5.5) prepared from CH3COOH (Poch, 568760114) and solid NaOH (Scharlau, SO0469). Carbonate solution (0.1 M and 0.01 M, pH 8.0, 9.0 and 10.0) prepared from solid NaHCO3 and Na2CO3 (Sigma S5761 and 222321). Phosphate buffer solutions (0.1 M and 0.01M, pH 6.0, 7.0 and 8.0) prepared from Na2HPO4 and NaH2PO4 solids (Sigma S9638 and S9763). TRIS buffer solution (0.1 M, pH 9.0) prepared from TRIS (Biorad 161-0719) and HCl (Poch 575283115).

For the choline (Ch) stock

Analytical signal

As indicated above, the method is intended to determine wC-PLs, represented here by C8PC, based on measuring the variation of fluorescence of the enzyme ChOx when catalyzing the oxidation of the Ch formed in the previous hydrolysis reaction with PLD. In the Supplementary Material the spectra of ChOx are shown (SM1). 286 and 340 nm were chosen as the excitation and the fluorescence wavelengths, respectively.

Both reactions (oxidation (1) and hydrolysis (2)) can be combined in two different ways,

CONCLUSIONS

The determination of phospholipids containing choline can be carried out using a methodology based on the combination of PLD and ChOx, and measuring the fluorescence of the latter (intrinsic and extrinsic).

The method has been tested with a short-chain phospholipid (soluble in water) and applied to the determination of lyso-PAF. It could be extended to all phospholipids containing choline.

The direct method is fast and very interesting for the further development of biosensors, while the two-step

Acknowledgements

This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) (CTQ 2012-34774) and by the Regional Government of Aragón (DGA-FEDER) (Support to Research Group E-74).

References (34)

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