Effect of sphingomyelin versus dipalmitoylphosphatidylcholine on the extent of lipid oxidation

Abstract

Phospholipids with sites of unsaturation are targets of peroxidation. We investigated the effect of two types of lipids with identical headgroups, sphingomyelins (SMs) and dipalmitoylphosphatidylcholine (DPPC), on the extent of oxidation of stearoyl-arachidonoyl phosphatidylcholine (SAPC) with four double bonds. Peroxidation was induced with tert-butylhydroperoxide and FeCl₂ at 35 °C. The decrease of SAPC versus DPPC, or N-palmitoyl SM, or N-stearoyl SM, was monitored by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) at various reaction times. For corresponding molar ratios of DPPC:SAPC and SM:SAPC, SAPC oxidized faster and to a greater extent when DPPC, rather than N-palmitoyl SM or bovine brain SMs, was present. However, at 35 °C the hydrophobic tails in SM mixtures were more disordered than in those of DPPC. The slower oxidation of SAPC in SM-rich vesicles may result from the presence of a tight network of H-bonds that bridge neighboring SM molecules and poses a stronger interfacial barrier to the passage of oxidants.

Introduction

Oxidative damage of biomembrane lipids occurs during aging and leads to pathogenesis (Girotti, 1998, Spiteller, 2001). Sites of unsaturation in hydrocarbon chains are particularly susceptible to peroxidation. We are concerned about the deleterious effects of oxidation on membranes of mammalian lens fibers as they are exposed to photo- and metabolic oxidative processes throughout their lifetime (Babizhayev, 1996, Bender, 1994, Borchman et al., 2000, Borchman and Yappert, 1998, Borchman et al., 1997, Garland, 1990).

Sphingomyelins (SMs) and phosphatidylcholines (PCs) are the most abundant sphingophospholipids (SLs) and glycerophospholipids (GLs), respectively, in mammalian membranes (Voet and Voet, 1995). Both lipid types are preferentially enriched in the outer leaflet of plasma membranes (Gennis, 1989). SMs make up an unusually large fraction of the phospholipids in mammalian lenses (Broekhuyse et al., 1974, Greiner et al., 1994, Meneses et al., 1990, Roelfzema et al., 1976, Zelenka, 1984) and their relative content increases with age (Borchman et al., 1994, Merchant et al., 1991, Roelfzema et al., 1976). PCs are also present in mammalian lenses but their content decreases with age (Borchman et al., 1994). Both types of phospholipids possess phosphorylcholine as their head group. Their interfacial regions, however, are different. The presence of a hydroxyl and an amide group in the interfacial region of SLs, offers donating and accepting sites of H-bonds not present in GLs.

Regarding their hydrophobic chain composition, GLs contain, in general, saturated sn-1 hydrocarbon chains usually 16 or 18-carbon-long and sn-2 chains with 16–20 carbons and up to four cis double bonds (Voet and Voet, 1995). For mammalian SMs, the 18-carbon long sphingoid base (sphingosine) with a trans double bond between C4 and C5 is predominant and the amide-linked hydrocarbon chains are mainly saturated. Overall, the hydrophobic tails of SLs are more saturated than those of GLs (Brown, 1998). Although the level of order of the hydrocarbon chains is known to affect the extent of oxidation within a given type of phospholipids (Montfoort et al., 1987), little is known about the role of the interfacial region on the extent of lipid oxidation. For this reason, we set out to investigate possible differences in the extent of peroxidation of a highly unsaturated PC in the presence of a PC with saturated acyl chains or SMs. The peroxidation was induced chemically following well-documented approaches (Girotti, 1985, Girotti, 1998, Spiteller, 2001) based on the use of organic hydroperoxides in combination with Fe²⁺ salts (Lamba et al., 1994, Li et al., 2000a, Spickett et al., 1998, Spickett et al., 2001). Fe²⁺ has been proposed to promote the initiation of a radical-dependent lipid peroxidation chain reaction (Girotti, 1985, Ohyashiki et al., 2002) by producing OH radicals by Fenton-type chemistry. The OH and ROOH radicals are able to abstract a hydrogen atom from any CH bond; particularly efficient is the abstraction of hydrogens in allylic CH₂ groups. This abstraction and subsequent rearrangement lead to formation of conjugated diene radicals that react with oxygen to produce peroxyl radicals that are able to abstract hydrogens from other unsaturated hydrocarbon chains.

In this model study, the peroxidation of the highly unsaturated stearoyl-arachidonoyl PC (SAPC) was performed in large multilamellar vesicles prepared with either dipalmitoylphosphatidylcholine (DPPC) or SMs from semisynthetic or natural sources. To follow the extent of SAPC oxidation we applied matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI (Benard et al., 1999, Petkovic et al., 2001, Schiller et al., 1999, Schiller et al., 2000, Schiller and Arnold, 2000, Schiller et al., 2001), as well as electrospray ionization (Brugger et al., 1997, Kerwin et al., 1994, Spickett et al., 1998, Spickett et al., 2001) provide relatively soft means of ionization and, therefore, minimize fragmentation of analytes. MALDI-TOF MS has been shown to be rapid and sensitive to perform analytical measurements on ionizable phospholipids, such as PCs and SMs (Harvey, 1995a, Harvey, 1995b).