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Immobilization of Lipid Substrates: Application on Phospholipase A2 Determination

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Lipids

Abstract

The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A2 (PLA2) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C12-NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn-2 acyl chain of the phospholipid stands out and is accessible by PLA2. Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA2. Mass spectrometry showed that only C12-NBD-FA, the PLA2 hydrolysis product, was detected in the reaction mixture, indicating that PLA2 recognizes PVA-SbQ/C12-NBD-PtdCho as a surface to perform catalysis.

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Abbreviations

AFM:

Atomic force microscopy

ARDS:

Acute respiratory distress syndrome

BAL fluid:

Bronchoalveolar lavage fluid

BSA:

Bovine serum albumin

C12-NBD-FA:

12-[(7-Nitro-2-1,3-benzoxadiazol-4-yl)amino}dodecanoic acid

C12-NBD-PtdCho:

1-Palmitoyl-2-{12-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphocholine

C6-NBD-PtdCho:

1-Palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine

EDTA:

Ethylenediaminetetraacetic acid

FA:

Fatty acid

HPLC:

High performance liquid chromatography

MS:

Mass spectrometry

PAF:

Platelet activating factor

PLA2 :

Phospholipase A2

PVA-SbQ:

Photocrosslinkable polyvinyl alcohol with steryl pyridinium residues

RSD:

Relative standard deviation

XRD:

X-ray diffraction analysis

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Acknowledgments

The authors would like to thank the Unit of Environmental, Organic and Biochemical high resolution analysis-ORBITRAP-LC–MS of the University of Ioannina for providing access to the facilities. The authors would like to thank the Confocal Laser Scanning Microscopy Unit of the University of Ioannina for providing access to the facilities.

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Authors and Affiliations

  1. Chemistry Department, University of Ioannina, 45110, Ioannina, Greece

    Athanassios Karkabounas, Agni M. Kosmas & Marilena E. Lekka

  2. Institute of Microelectronics, NCSR Demokritos, 15310, Aghia Paraskevi, Greece

    Dimitra G. Georgiadou & Panagiotis Argitis

  3. Institute of Advanced Materials Physicochemical Processes Nanotechnology and Microsystems, NCSR Demokritos, 15310, Aghia Paraskevi, Greece

    Vassilios Psycharis

  4. Intensive Care, Faculty of Medicine, University of Ioannina, 45110, Ioannina, Greece

    George Nakos

Authors
  1. Athanassios Karkabounas
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  2. Dimitra G. Georgiadou
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  3. Panagiotis Argitis
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  4. Vassilios Psycharis
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  5. George Nakos
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  7. Marilena E. Lekka
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Corresponding author

Correspondence to Marilena E. Lekka.

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Karkabounas, A., Georgiadou, D.G., Argitis, P. et al. Immobilization of Lipid Substrates: Application on Phospholipase A2 Determination. Lipids 50, 1259–1271 (2015). https://doi.org/10.1007/s11745-015-4076-y

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  • DOI: https://doi.org/10.1007/s11745-015-4076-y

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