title: Mass-spectrometric characterization of phospholipids and their primary peroxidation products in rat cortical neurons during staurosporine-induced apoptosis.
abstract: The molecular diversity of phospholipids is essential for their structural and signaling functions in cell membranes. In the current work, we present, the results of mass spectrometric characterization of individual molecular species in major classes of phospholipids -- phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), sphingomyelin (CerPCho), and cardiolipin (Ptd(2)Gro) -- and their oxidation products during apoptosis induced in neurons by staurosporine (STS). The diversity of molecular species of phospholipids in rat cortical neurons followed the order Ptd(2)Gro > PtdEtn >> PtdCho >> PtdSer > PtdIns > CerPCho. The number of polyunsaturated oxidizable species decreased in the order Ptd(2)Gro >> PtdEtn > PtdCho > PtdSer > PtdIns > CerPCho. Thus a relatively minor class of phospholipids, Ptd(2)Gro, was represented in cortical neurons by the greatest variety of both total and peroxidizable molecular species. Quantitative fluorescence HPLC analysis employed to assess the oxidation of different classes of phospholipids in neuronal cells during intrinsic apoptosis induced by STS revealed that three anionic phospholipids -- Ptd(2)Gro >> PtdSer > PtdIns -- underwent robust oxidation. No significant oxidation in the most dominant phospholipid classes -- PtdCho and PtdEtn -- was detected. MS-studies revealed the presence of hydroxy-, hydroperoxy- as well as hydroxy-/hydroperoxy-species of Ptd(2)Gro, PtdSer, and PtdIns. Experiments in model systems where total cortex Ptd(2)Gro and PtdSer fractions were incubated in the presence of cytochrome c (cyt c) and H(2)O(2), confirmed that molecular identities of the products formed were similar to the ones generated during STS-induced neuronal apoptosis. The temporal sequence of biomarkers of STS-induced apoptosis and phospholipid peroxidation combined with recently demonstrated redox catalytic properties of cyt c realized through its interactions with Ptd(2)Gro and PtdSer suggest that cyt c acts as a catalyst of selective peroxidation of anionic phospholipids yielding Ptd(2)Gro and PtdSer peroxidation products. These oxidation products participate in mitochondrial membrane permeability transition and in PtdSer externalization leading to recognition and uptake of apoptotic cells by professional phagocytes.
authors.affiliation: Center for Free Radical and Antioxidant Health, Pittsburgh, PA, USA. vtyurin@pitt.edu
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Tyurin
authors.forename: Yulia Y
authors.initials: YY
authors.lastname: Tyurina
authors.forename: Weihong
authors.initials: W
authors.lastname: Feng
authors.forename: Alexandra
authors.initials: A
authors.lastname: Mnuskin
authors.forename: Jianfei
authors.initials: J
authors.lastname: Jiang
authors.forename: Minke
authors.initials: M
authors.lastname: Tang
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Zhang
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhao
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Kochanek
authors.forename: Robert S B
authors.initials: RS
authors.lastname: Clark
authors.forename: Hülya
authors.initials: H
authors.lastname: Bayir
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D045304:Cytochromes c / Q000378:metabolism; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015227:Lipid Peroxidation / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019311:Staurosporine / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D010743:Phospholipids; D045304:Cytochromes c; D053148:Caspase 3; D019311:Staurosporine