Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electroscopy MS

Abstract

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe(2+), lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.

Induction by β‐bungarotoxin of apoptosis in cultured hippocampal neurons is mediated by Ca2+‐dependent formation of reactive oxygen species

“…The increase in ROS is consistent with the general role of PLA2 as an inducer of lipid peroxidation in neurodegeneration (Farooqui et al 1997). Hippocampal cells accumulated a lipid peroxide characterized by a signal in MALDI-TOF mass spectrometry at 818 m/z, consistent with the formation of the monohydroxyperoxide of phosphatidylcholine (PC18:0,18:2; Spickett et al 2001). Appearance of this signal was accompanied by a dramatic loss of the 786 m/z signal, corresponding to native phosphatidylcholine.…”

“…In lipids extracted from cells treated with b-BuTx for 4 h, a new mass signal appeared at 818 m/z, which was not detectable in controls (Figs 5a and b). This mass signal is characteristic of hydroxyperoxides of stearoyl-linoleoyl phosphatidylcholines (PC18:0,18:2; Spickett et al 2001). In contrast, the ion peak at 786 m/z indicative of native phosphatidylcholine (18:0,18:2) was dramatically depleted as compared to untreated cells.…”

“…The concentration of malonyl dialdehyde (MDA) was calculated using an extinction coefficient of 1.56 · 10 )3 l · mol )1 · cm )1 (Hermann et al 1999). For analysis of lipid hydroxyperoxides (Spickett et al 2001), harvested cells were sonicated in 0.3 mL 20 mM Tris-HCl (pH 7.4). Cell lipids were extracted by chloroform : methanol 2 : 1 (v/v), chloroform layers were transferred to a clean tube and dried.…”

“…Generally, apoptotic cell death is triggered by intracellular signalling pathways, including a rise in intracellular free Ca 2+ (Kruman et al 1998), the generation of reactive oxygen species (ROS) and peroxidation products of membrane lipids (Pettmann and Henderson 1998;Guegan and Przedborski 2003). Formation of peroxidation products comprizes an early phase, characterized by the accumulation of phospholipid hydroxyperoxides (Spickett et al 2001) and generation of the end product malonyl dialdehyde (Girotti 1998;Hermann et al 1999). Similar events are observed regardless of the agent employed to induce neuronal apoptosis, including glutamate (Tan et al 1998), NMDA (Tenneti et al 1998) and staurosporine (Kruman et al 1998).…”

Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox

“…Reverse phase liquid chromatography-MS (LC-MS) was carried out essentially as described previously (Spickett et al 2001), but using a Thermo-Finnigan Surveyor system and a Luna C8 column (5 μm RP-Sct, 1 mm inner diameter × 150 mm; Phenomenex, UK). This column was operated at a flow rate of 100 μl/min with an isocratic solvent system of 71 : 4 : 8 (by vol.)…”