Elsevier

Clinical Biochemistry

Volume 38, Issue 10, October 2005, Pages 925-933
Clinical Biochemistry

The phosphatidylcholine/lysophosphatidylcholine ratio in human plasma is an indicator of the severity of rheumatoid arthritis: Investigations by 31P NMR and MALDI-TOF MS

https://doi.org/10.1016/j.clinbiochem.2005.06.006Get rights and content

Abstract

Objectives:

Lipid second messengers, e.g. lysophosphatidylcholines (LPC) are involved in the pathogenesis of inflammatory diseases, for instance, rheumatoid arthritis (RA). Unfortunately, the analysis of LPC in complex mixtures as present in body fluids is still challenging.

Design and methods:

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for phospholipid (PL) analysis of organic extracts of synovial fluids from patients with RA as well as the corresponding plasma. These data were compared with results obtained by high resolution 31P NMR spectroscopy.

Results:

Synovial fluids may be replaced by plasma since the analysis of both body fluids gives very similar results. Patients undergoing treatment with TNF-α inhibitors (ADALIMUMAB (HUMIRA®)) were examined in order to investigate whether the clinically-significant attenuation of disease activity is accompanied by changes of the PL composition of plasma. It will be shown that especially the PC/LPC ratios of plasma represent a reliable measure of inflammation and increase upon therapy.

Conclusions:

Since plasma samples are readily available, our approach might be useful to draw conclusions before puncture of the affected joints is necessary and the PC/LPC ratio detected in plasma may serve as an indicator of RA in early stages.

Introduction

Rheumatoid arthritis (RA), an autoimmune disease of widely unknown etiology characterized by chronic synovitis and cartilage destruction, affects about 1% of the total population. In arthritic joints, the polymeric components of cartilage and the synovial (joint) fluid are degraded [1], leading to functional loss of the joint. A more detailed understanding of these processes is a necessary prerequisite of the development of therapeutic and preventive methodologies to control disease progression.

Recent investigations of the degradation pathways involved in RA implicate the significant contribution of neutrophilic granulocytes (neutrophils) which are found in large numbers in the synovial fluids of RA patients [2]. Neutrophils are able to release a mixture of proteolytic enzymes combined with different reactive oxygen species (ROS) which may trigger the destruction of the cartilage tissue [3], [4]. Among others, phospholipids (PL) and enzymes involved in lipid metabolism were demonstrated to affect the regulation of the signaling steps leading to neutrophil activation [5], [6], [7].

One of these messenger molecules is lysophosphatidylcholine (LPC) that plays an important role in inflammation [8]. LPC is generated by the hydrolysis of membrane phosphatidylcholine (PC) by phospholipase A2 (PLA2), acts as a chemoattractant at the sites of inflammation and promotes the inflammatory reaction. PLA2 has originally been isolated from inflamed synovial fluids [9], [10] and its role in inflammatory processes like RA was intensively studied [11], [12], [13].

For PL analysis, primarily chromatographic techniques are established. However, chromatographic methods have the disadvantage that a differentiation between individual acyl chain residues of a given PL class is hardly possible [14]. Additionally, quantitative determination of LPC is still a challenge. Therefore, two new approaches–matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 31P NMR–are applied in this paper for PL composition analysis of synovial fluids and plasma from patients suffering from RA.

Our group has shown recently that the quantitative determination of LPC can be performed directly from the MALDI-TOF mass spectra without prior separation of the sample into the individual lipid classes [15], [16]. A more comprehensive review on MALDI-TOF MS of lipids is available in [17] and detailed comments on reproducibility and accuracy of MALDI-TOF MS in order to determine PC/LPC ratios are provided in [16]. In order to have an additional, independent method, PL analysis was also performed by high resolution 31P NMR. By using sodium cholate as detergent, the formation of PL bilayers is prevented and highly resolved spectra are obtained [18], [19], [20]. In that way, individual PL classes can be easily differentiated and quantitative information about the relative moieties of PL is readily available by integration. An overview of this approach is given in [21].

In the present investigation, 31P NMR spectroscopy in combination with MALDI-TOF mass spectrometry were applied to investigate the organic extracts of the joint fluids and plasma from patients suffering from RA. Additionally, plasma from patients undergoing treatment with TNF-α inhibitors (Adalimumab (Humira®)) were examined to find out whether the therapeutic success can be monitored by measuring the PC/LPC ratio. It will be shown that this ratio is a reliable measure of the therapeutic success of TNF-α inhibitors and can be determined in the extracts of plasma. This is an important result because plasma samples are more readily and in higher amounts available than joint fluids.

Section snippets

Chemicals

All chemicals for NMR spectroscopy (sodium cholate, EDTA and deuterated water with an isotopic purity of 99.6%), buffer preparation (NaCl, KH2PO4, Na2HPO4 and TRIS) and mass spectrometry (2,5-dihydroxybenzoic acid (DHB) and trifluoroacetic acid (TFA)) as well as all solvents (chloroform and methanol) were obtained in highest commercially available purity from Fluka Feinchemikalien GmbH (Neu-Ulm, Germany).

Phosphatidylcholine (PC) from egg yolk used for means of comparison (PLA2 digestion) was

31P NMR and MALDI-TOF MS of isolated PL

First, the special patterns of MALDI-TOF mass spectra of phospholipids will be discussed on the hand of an artificial sample. Fig. 1 shows selected positive ion MALDI-TOF mass spectra indicative of the effect of phospolipase A2 (PLA2) digestion of PC. The upper spectrum (a) represents commercially-available PC from egg yolk and the lower spectrum (b) the same sample after digestion with pancreatic PLA2. The most intense peaks occurring in trace (a) represent the proton adducts of PC 16:0, 18:2 (

Conclusions

The most important result of this study is that synovial fluid may be replaced by plasma since both body fluids provide very similar results for the PC/LPC ratio. Prior to therapy, this ratio is 9.7 ± 5 and is nearly doubled to 19 ± 5 during therapy. This indicates a marked decrease of the LPC concentration and/or a further conversion of the LPC into glycerophosphatidylcholine (GPC) that lacks both acyl chain residues. Two enzymes in plasma can convert PC into LPC: LCAT (lecithin cholesterol

Acknowledgments

This work was supported by the Interdisziplinäre Zentrum für Klinische Forschung (IZKF) Leipzig at the Faculty of Medicine of the Universität Leipzig (Project A17). We thank Dr. M. Petković, J. Leßig and H. Spalteholz from the Institute of Medical Physics and Biophysics, Medical Department of the University of Leipzig for helpful discussions.

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