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 (Ptd2Gro) - 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 Ptd2Gro > PtdEtn ≫ PtdCho ≫ PtdSer > PtdIns > CerPCho. The number of polyunsaturated oxidizable species decreased in the order Ptd2Gro ≫ PtdEtn > PtdCho > PtdSer > PtdIns > CerPCho. Thus a relatively minor class of phospholipids, Ptd2Gro, 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 staurosporine (STS) revealed that three anionic phospholipids — Ptd2Gro ≫ 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 Ptd2Gro, PtdSer, and PtdIns. Experiments in model systems where total cortex Ptd2Gro and PtdSer fractions were incubated in the presence of cytochrome c (cyt c) and H2O2, 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 Ptd2Gro and PtdSer suggest that cyt c acts as a catalyst of selective peroxidation of anionic phospholipids yielding Ptd2Gro 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.

Structural Characterization of Oxidized Glycerophosphatidylserine: Evidence of Polar Head Oxidation

“…These oxidation products will be briefly discussed in this work since they have already been described and fully characterized [13,23,25].…”

“…PS hydroperoxy and hydroxy species were identified by Kagan and co-authors using electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) in negative mode. Oxidized PS has been identified in several pathological conditions: Tyurina et al identified mono-hydroperoxy derivatives of PS after intestinal injury induced by γ-irradiation, [22,23]; Bayir et al also proposed the presence of PS hydroperoxides derivatives in traumatic brain injury after controlled cortical impact [24]; hydroperoxyl and hydroxyl PS derivates were also identified by Tyurin et al during apoptosis induced in neurons by staurosporine [25] and in cells and tissues after pro-apoptotic and pro-inflammatory stimuli [26]; the same group showed that the pattern of phospholipid oxidation during apoptosis is non-random and that PS is one of the preferred peroxidation substrates [8].…”

Alterations in phospholipidomic profile in the brain of mouse model of depression induced by chronic unpredictable stress

“…Furthermore, CL peroxidation may lead to an overall loss of detectable CL content, either by preferential hydrolysis of peroxidized acyl chains by PLA 2 , direct decomposition of lipid peroxides, or the formation of CL-protein complexes (Chicco and Sparagna, 2007). Moreover, the loss of CL content and CL oxidation are related with the development of many diseases .Several studies have correlated oxidative changes in CL with neuronal diseases and brain injuries (Adibhatla et al, 2006, Adibhatla and Hatcher, 2007, Bayir et al, 2007, Adibhatla and Hatcher, 2008, Tyurin et al, 2008, Tyurin et al, 2009). The reduction of CL level in the brain with aging has been reported and associated with an increase of lipid peroxidation in rat brain mitochondria exposed to oxidative stress (Paradies et al, 2011).…”

“…Lipid hydroperoxides are the primary products of lipid oxidation and have been used in several studies to evaluate the oxidation status of cells and/or tissues (Tyurin et al, 2008, Sparvero et al, 2010.…”

Detection of phosphatidylserine with a modified polar head group in human keratinocytes exposed to the radical generator AAPH

“…Several studies showed that oxidized PS is preferentially recognized by macrophage scavenger receptors over non-oxidized PS [4][5][6][7]. The oxidation products of PS identified in vivo consisted of oxidative modifications in the fatty acyl chains, such as PS hydroxide and hydroperoxide derivatives and truncated sn-2 fatty acyl species [4,8,9]. These types of oxidation products retained an intact PS head-group and are usually identified by the typical fragmentation pathways under MS/MS conditions, which involved the loss of the serine group [8,10,11].…”