Oxidative lipidomics of apoptosis: quantitative assessment of phospholipid hydroperoxides in cells and tissues.
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 A(2). 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.
Tyurin
Vladimir A
VA
Center for Free Radical and Antioxidant Health, Department of Environmental & Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA.
Tyurina
Yulia Y
YY
Ritov
Vladimir B
VB
Lysytsya
Andriy
A
Amoscato
Andrew A
AA
Kochanek
Patrick M
PM
Hamilton
Ronald
R
Dekosky
Steven T
ST
Greenberger
Joel S
JS
Bayir
Hülya
H
Kagan
Valerian E
VE
D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008054:Lipid Peroxides / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D005227:Fatty Acids; D008054:Lipid Peroxides; D010743:Phospholipids