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

Corinne M. Spickett, Nicola Rennie, Helen Winter, Laura Zambonin, Laura Landi, Andreas Jerlich, R. Jörg Schaur, Andrew R. Pitt

Research output: Contribution to journalArticle

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 Fe2+, 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.

Original languageEnglish
Pages (from-to)449-457
Number of pages9
JournalBiochemical Journal
Volume355
Issue number2
DOIs
Publication statusPublished - 15 Apr 2001

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Electrospray ionization
Phospholipids
High Pressure Liquid Chromatography
Chlorohydrins
Lipids
Oxidation
U937 Cells
Oxidative stress
Direct injection
Phosphatidylcholines
Hydrogen Peroxide
Oxidative Stress
Cells
tert-Butylhydroperoxide
Injections
HL-60 Cells
Hydrophobicity
Membrane Lipids
Complex Mixtures
Hydrophobic and Hydrophilic Interactions

Keywords

  • chlorohydrin
  • U937 cells
  • lipid peroxidation
  • electrospray ionization
  • HL60 cells
  • Pharmacy and materia medica

Cite this

Spickett, Corinne M. ; Rennie, Nicola ; Winter, Helen ; Zambonin, Laura ; Landi, Laura ; Jerlich, Andreas ; Schaur, R. Jörg ; Pitt, Andrew R. / Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS. In: Biochemical Journal. 2001 ; Vol. 355, No. 2. pp. 449-457.
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Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS. / Spickett, Corinne M.; Rennie, Nicola; Winter, Helen; Zambonin, Laura; Landi, Laura ; Jerlich, Andreas; Schaur, R. Jörg; Pitt, Andrew R.

In: Biochemical Journal, Vol. 355, No. 2, 15.04.2001, p. 449-457.

Research output: Contribution to journalArticle

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T1 - Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS

AU - Spickett, Corinne M.

AU - Rennie, Nicola

AU - Winter, Helen

AU - Zambonin, Laura

AU - Landi, Laura

AU - Jerlich, Andreas

AU - Schaur, R. Jörg

AU - Pitt, Andrew R.

PY - 2001/4/15

Y1 - 2001/4/15

N2 - 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 Fe2+, 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.

AB - 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 Fe2+, 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.

KW - chlorohydrin

KW - U937 cells

KW - lipid peroxidation

KW - electrospray ionization

KW - HL60 cells

KW - Pharmacy and materia medica

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DO - 10.1042/0264-6021:3550449

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SP - 449

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JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

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