Interactions of plasmalogens and their diacyl analogs with singlet oxygen in selected model systems

Abstract

Plasmalogens are phospholipids containing a vinyl-ether linkage at the sn-1 position of the glycerophospholipid backbone. Despite being quite abundant in humans, the biological role of plasmalogens remains speculative. It has been postulated that plasmalogens are physiological antioxidants with the vinyl-ether functionality serving as a sacrificial trap for free radicals and singlet oxygen. However, no quantitative data on the efficiency of plasmalogens at scavenging these reactive species are available. In this study, rate constants of quenching of singlet oxygen, generated by photosensitized energy transfer, by several plasmalogens and, for comparison, by their diacyl analogs were determined by time-resolved detection of phosphorescence at 1270nm. Relative rates of the interactions of singlet oxygen with plasmalogens and other lipids, in solution and in liposomal membranes, were measured by electron paramagnetic resonance oximetry and product analysis using HPLC-EC detection of cholesterol hydroperoxides and iodometric assay of lipid hydroperoxides. The results show that singlet oxygen interacts with plasmalogens significantly faster than with the other lipids, with the corresponding rate constants being 1 to 2 orders of magnitude greater. The quenching of singlet oxygen by plasmalogens is mostly reactive in nature and results from its preferential interaction with the vinyl-ether bond. The data suggest that plasmalogens could protect unsaturated membrane lipids against oxidation induced by singlet oxygen, providing that the oxidation products are not excessively cytotoxic.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Fig.1

Oxygen uptake monitored by EPR oximetry in liposomes composed of DMPC-Chol (A) or DMPC- PlgPC 16:0/16:0 (B) photosensitized with rose Bengal. 1mM DMPC concentration was held constant whereas Chol or PlgPC concentration was varied. A. Cholesterol concentration are as follows: 0.4 mM (triangles down), 0.6 mM (triangles up), 0.8 mM ( rectangle),1 mM (circle). B. Plasmalogen concentration are as follows: 0.1 mM (triangles up), 0.3 mM (triangles down), 0.4 mM (squares).

Fig.1

Oxygen uptake monitored by EPR oximetry in liposomes composed of DMPC-Chol (A) or DMPC- PlgPC 16:0/16:0 (B) photosensitized with rose Bengal. 1mM DMPC concentration was held constant whereas Chol or PlgPC concentration was varied. A. Cholesterol concentration are as follows: 0.4 mM (triangles down), 0.6 mM (triangles up), 0.8 mM ( rectangle),1 mM (circle). B. Plasmalogen concentration are as follows: 0.1 mM (triangles up), 0.3 mM (triangles down), 0.4 mM (squares).

Fig.2

Oxygen uptake monitored by EPR oximetry in large unilamellar liposomes photosensitized with MC 540. 4.4 mM SUV were composed of DMPC and one of additional lipid (Chol, SOPC, POPC, PlgPC 18:0/18:1 or PlgPC 16:0/16:0 designated as “+” in Fig.2B) in molar ratio 3:2. A. The kinetics of oxygen decay in LUV composed of: DMPC-PlgPC 16:0/16:0 dark control (circles), control DMPC (diagonal cross), DMPC-POPC (triangles down), DMPC-PlgPC 16:0/16:0 (triangles up). B. Calculated oxygen uptake [mM/min] in LUV of different lipid composition. DMPC is control of pure DMPC liposomes.

Fig.3

Oxygen uptake monitored by EPR oximetry in TX-100 micelles photosensitized with MC 540. 5mM lipid mixtures were composed of DMPC and one of additional lipid (Chol, POPC, or PlgPC 16:0/16:0 designated as “+”) in molar ratio 3:2. Additional controls are as follows: “control MC” is a control of pure photosensitizer in buffer, “DMPC” is a control of pure 5 mM DMPC micelles and “control TX-100” is a photosensitizer in micelles.

Fig.4

HPLC-EC(Hg) chromatograms of lipids extracted from photosensitised DMPC-Chol-PlgPC 16:0/16:0 liposomes with RB (A) or MC 540 (B). Lipid mol ratio was 5:8:3 of DMPC: Chol: PlgPC 16:0/16:0. The peak identities are as follows: (1) 7α/β-cholesterol hydroperoxide (7α/β-OOH), (2) 5α-cholesterol hydroperoxide (5α-OOH), (3) 6α-cholesterol hydroperoxide (6α-OOH), (4) 6β-cholesterol hydroperoxide (6β-OOH)

Fig.4

HPLC-EC(Hg) chromatograms of lipids extracted from photosensitised DMPC-Chol-PlgPC 16:0/16:0 liposomes with RB (A) or MC 540 (B). Lipid mol ratio was 5:8:3 of DMPC: Chol: PlgPC 16:0/16:0. The peak identities are as follows: (1) 7α/β-cholesterol hydroperoxide (7α/β-OOH), (2) 5α-cholesterol hydroperoxide (5α-OOH), (3) 6α-cholesterol hydroperoxide (6α-OOH), (4) 6β-cholesterol hydroperoxide (6β-OOH)

Fig.5

A. Kinetics of 5α-OOH accumulation detected with HPLC-EC(Hg) system in CS2 solution of either pure 0.5 mM Chol (circles) or 0.5 mM Chol with 0.2 mM PlgPC 16:0/16:0 (triangles) photosensitized with TPP. B. Effect of different concentration of PlgPC 16:0/16:0 on 5α-OOH accumulation after 8 minutes irradiation with TPP.

Fig.6

Kinetics of lipid hydroperoxides accumulation determined by iodometric assay in CCl4 solution of either 0.1 mM PlgPC 16:0/16:0 (circles) or 2 mM Chol (triangles). TPP was used as a photosensitizer.

Fig.7

Singlet oxygen phosphorescence decay at 1270 nm in an irradiated samples of TPP dissolved in CCl4 (solid line) and after quenching with 0.1 mM PlgPC 16:0/18:1 (doted line) or 1.1 mM PlgPC 16:0/18:1 (dashed line). Inset shows a typical dependence of pseudo-first order rate constant [s−1] of singlet oxygen quenching on concentration of PlgPC 18:0/18:1. From the straight-line slope, the actual bi-molecular rate constants are calculated. Such data are summarized in Table1.