Elsevier

Experimental Eye Research

Volume 89, Issue 6, December 2009, Pages 840-853
Experimental Eye Research

Red blood cell plasmalogens and docosahexaenoic acid are independently reduced in primary open-angle glaucoma

https://doi.org/10.1016/j.exer.2009.07.008Get rights and content

Abstract

Among several theories involved in the pathogenesis of primary open-angle glaucoma (POAG), the vascular theory considers the disease to be a consequence of reduced ocular blood flow associated with red blood cell abnormalities. Red blood cell membrane structure and function are influenced by their phospholipid composition. We investigated whether specific lipid entities that may affect the membrane physiology, namely, polyunsaturated fatty acids (PUFAs) and plasmalogens, are modified in POAG and whether these potential variations are related to the stage of glaucoma. Blood samples were collected from 31 POAG patients and 10 healthy individuals. The stage of glaucoma was determined according to the Hodapp and Parrish classification. Lipids were extracted from red blood cell membranes and individual phospholipid species were quantified by liquid chromatography combined with mass spectrometry using triple quadrupole technology. POAG patients had reduced erythrocyte levels of phosphatidyl-choline (PC) carrying docosahexaenoic acid (DHA). POAG patients also displayed lower levels of choline plasmalogens (PlsC) carrying PUFAs other than DHA. These differences were greater as the severity of the disease increased. Linear regressions predicted that red blood cell PlsC levels would decrease years before clinical symptoms, whereas the levels of PC carrying DHA were linearly correlated to visual field loss. Our data demonstrate the selective loss of some individual phospholipid species in red blood cell membranes, which may partly explain their loss of flexibility in POAG.

Introduction

Glaucomatous optic neuropathy is the second leading cause of blindness worldwide and will affect more than 60 million people in 2010 (Quigley and Broman, 2006). Despite extensive research, the pathogenesis of glaucoma is still not well understood. The term “glaucoma” encompasses a variety of pathophysiologic processes, all sharing the common definition of an optic neuropathy with characteristic optic nerve head remodeling and visual field defects. Primary open-angle glaucoma (POAG) is the most common form of glaucoma. Even if elevated intraocular pressure (IOP) is recognized as a major risk factor for POAG (Anderson, 1989), observations showing that elevated IOP does not lead to glaucoma and that glaucoma can develop under normal IOP conditions (Sommer et al., 1991, Drance, 1992) strongly suggest that factors other than IOP are likely to play a role in the pathogenesis of glaucomatous optic neuropathy (Pache and Flammer, 2006, Shields, 2008).

Different factors are suspected of influencing the onset and/or the progression of POAG, and it has been shown that systemic findings related to vascular disorders and blood flow occur in POAG patients more often than in healthy individuals (Flammer et al., 2002, Pache and Flammer, 2006). In addition to vasospastic factors (Flammer et al., 2001), it appears from several studies that patients with POAG have an altered hemorheology, since their red blood cells display increased aggregability and rigidity (Mary et al., 1993, Hamard et al., 1994, Vetrugno et al., 2004). Together with these data, the finding of increased erythrocyte acetylcholinesterase activity in POAG patients – which is considered an index of the alteration of membrane integrity – confirms that membrane abnormalities in red blood cells may be responsible for their altered function and deformability (Zabala et al., 1999).

Since the early 1960s, cell membranes, and particularly erythrocyte cell membranes, have been known to contain principally phospholipids (Horwitt et al., 1959, Phillips and Roome, 1959, Ways et al., 1963). These are lipidic entities consisting of a glycerol connected to two fatty acid radicals at the sn-1 and sn-2 positions and to a polar head group at the sn-3 position. Depending on the nature of the polar head, the two main classes of phospholipids are phosphatidyl-ethanolamine (PE) and phosphatidyl-choline (PC). Like other tissues or cell types, red blood cell phospholipids also consist of other phospholipids called plasmalogens, which have a fatty alcohol radical and a vinyl-ether bond at the sn-1 position of glycerol instead of a fatty acid radical (Nagan and Zoeller, 2001). As with conventional phospholipids, plasmalogens are classified according to their sn-3 position, with the most abundant plasmalogens being plasmenyl-ethanolamine (PlsE) and plasmenyl-choline (PlsC). Although the exact functions of plasmalogens remain unclear, their involvement in membrane dynamics has been largely documented by biophysical and biochemical research (Hermetter et al., 1989, Glaser and Gross, 1995). Plasmalogens facilitate membrane fluidity because of the presence of the vinyl-ether bond at the sn-1 position as well as the preferential esterification of polyunsaturated fatty acids (PUFAs) at their sn-2 position (Sugiura et al., 1983, Ford and Gross, 1989b, Ford and Gross, 1989a). Indeed, it is clearly established that PUFAs and particularly those from the omega-3 family, such as docosahexaenoic acid (DHA, 22:6ω3), enhance cell membrane flexibility owing to their multiple double bonds (Stillwell and Wassall, 2003).

One study has shown reduced levels of omega-3 PUFAs in the erythrocyte membranes of patients with POAG, which may partly explain their increased rigidity and aggregability (Ren et al., 2006). However, this study did not consider plasmalogens that may also affect erythrocyte membrane structure and function, or whether these modifications were associated with glaucoma stage. Consequently, the purpose of the present investigation was to determine the complete phospholipid composition of erythrocyte membranes in POAG patients. The second objective of this study was to see whether the changes were related to glaucoma stage.

Section snippets

Glaucoma subject selection

The data for this study were obtained under research protocols approved by the Ethics Committee of Burgundy (France). Informed consent was obtained from all subjects before participation. The procedures adhered to the tenets of the Declaration of Helsinki for human experimentation.

Consecutive POAG patients (n = 31) and control subjects (n = 10) were enrolled in the study at the Department of Ophthalmology of the University Hospital in Dijon (France) (Table 1). None of the subjects was related

Determination of the sn-positions of phospholipids by ESI-MSn

The sn-1 and sn-2 radicals of phospholipids were determined in an MSn experiment using ESI in the negative mode. We used commercially asymmetrical PE and PC standards where fatty acids in the sn-1 and sn-2 positions were identified. When operated in the single-stage MS mode using ESI in the negative mode, PE and PC molecular species produced abundant [M − H] and [M − CH3] ions, respectively. Using trap ion mass spectrometer in the MSn mode, the PE species have a characteristic fragmentation.

Discussion

Two major theories have been postulated to explain the pathophysiology of glaucomatous optic neuropathy: the mechanical theory and the vascular theory (Fechtner and Weinreb, 1994). The mechanical theory considers glaucomatous optic neuropathy a direct consequence of increased IOP, which leads to physical damage of the lamina cribrosa and retinal neurons (Burgoyne et al., 2005). The vascular theory assumes that glaucomatous optic neuropathy is a consequence of abnormal blood supply caused by

Funding

This study was supported by a grant from University Hospital in Dijon, France (Programme Hospitalier de Recherche Clinique, Direction de la Recherche Clinique).

Commercial relationships

None.

Acknowledgements

The authors are highly indebted to Prof. Balwantray Chauhan for his helpful advice in preparing this manuscript. They gratefully acknowledge Françoise Candé for excellent management of human sample preparation and Linda Northrup for editing the manuscript.

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