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Oxidative Lipidomics of Apoptosis: Quantitative Assessment of Phospholipid Hydroperoxides in Cells and Tissues

  • Vladimir A. Tyurin
  • Yulia Y. Tyurina
  • Vladimir B. Ritov
  • Andriy Lysytsya
  • Andrew A. Amoscato
  • Patrick M. Kochanek
  • Ronald Hamilton
  • Steven T. DeKosky
  • Joel S. Greenberger
  • Hülya Bayir
  • Valerian E. Kagan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 610)

Abstract

Oxidized phospholipids play essential roles in execution of mitochondrial stage of apoptosis and clearance of apoptotic cells. The identification and quantification of oxidized phospholipids generated during apoptosis can be successfully achieved by oxidative lipidomics. With this approach, diverse molecular species of phospholipids and their hydroperoxides are identified and characterized by soft-ionization mass-spectrometry techniques such as electrospray ionization (ESI). Quantitative assessment of lipid hydroperoxides is performed by fluorescence HPLC-based protocol. The protocol is based on separation of phospholipids using two-dimensional-high-performance thin-layer chromatography (2-D-HPTLC). Phospholipids are hydrolyzed using phospholipase A2. The fatty acid hydroperoxides (FA-OOH) released is quantified by a fluorometric assay using Amplex red reagent and microperoxidase-11 (MP-11). Detection limit of this protocol is 1–2 pmol of lipid hydroperoxides. Lipid arrays vs. oxidized lipid arrays can be performed by comparing the abundance of phospholipids with the abundance of oxidized phospholipids. Using oxidative lipidomics approach we show that the pattern of phospholipid oxidation during apoptosis is nonrandom and does not follow their abundance in several types of cells undergoing apoptosis and a variety of disease states. This has important implications for evaluation of apoptosis in vivo. The anionic phospholipids, cardiolipin (CL) and phosphatidylserine (PS), are the preferred peroxidation substrates.

Key words

Phospholipid hydroperoxides cardiolipin phosphatidylserine ESI-MS fluorescence HPLC apoptosis cytochrome c controlled cortical impact gamma-irradiation Alzheimer’s disease 

Notes

Acknowledgments

Supported by grants from NIH HL70755, NIH U19 AIO68021, NIOSH OH008282, HD057587, AHA0535365N, Pennsylvania Department of Health SAP 4100027294, Human Frontier Science Program.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Vladimir A. Tyurin
    • 1
  • Yulia Y. Tyurina
    • 1
  • Vladimir B. Ritov
    • 2
  • Andriy Lysytsya
    • 1
  • Andrew A. Amoscato
    • 3
  • Patrick M. Kochanek
    • 4
  • Ronald Hamilton
    • 3
  • Steven T. DeKosky
    • 5
  • Joel S. Greenberger
    • 6
  • Hülya Bayir
    • 1
  • Valerian E. Kagan
    • 1
  1. 1.Center for Free Radical and Antioxidant Health,Department of Environmental & Occupational Health and Critical Care MedicineUniversity of PittsburghPittsburghUSA
  2. 2.Department of MedicineUniversity of PittsburghPittsburghUSA
  3. 3.Department of PathologyUniversity of PittsburghPittsburghUSA
  4. 4.Department of Critical Care MedicineUniversity of PittsburghPittsburghUSA
  5. 5.Department of NeurologyUniversity of PittsburghPittsburghUSA
  6. 6.Department of Radiation OncologyUniversity of PittsburghPittsburghUSA

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