Structural Characterization of Oxidized Glycerophosphatidylserine: Evidence of Polar Head Oxidation

Abstract

Non-oxidized phosphatidylserine (PS) is known to play a key role in apoptosis but there is considerable research evidence suggesting that oxidized PS also plays a role in this event, leading to the increasing interest in studying PS oxidative modifications. In this work, different PS (1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) were oxidized in vitro by hydroxyl radical, generated under Fenton reaction conditions, and the reactions were monitored by ESI-MS in negative mode. Oxidation products were then fractionated by thin layer chromatography (TLC) and characterized by tandem mass spectrometry (MS/MS). This approach allowed the identification of hydroxyl, peroxy, and keto derivatives due to oxidation of unsaturated fatty acyl chains. Oxidation products due to oxidation of serine polar head were also identified. These products, with lower molecular weight than the non-modified PS, were identified ashydroxyacetaldehyde plus hydroxy fatty acyl chain). Phosphatidic acid was also formed in these conditions. These findings confirm the oxidation of the serine polar head induced by the hydroxyl radical. The identification of these modifications may be a valuable tool to evaluate phosphatidylserine alteration under physiopathologic conditions and also to help understand the biological role of phosphatidylserine oxidation in the apoptotic process and other biological functions.

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

“…confirming that the PS modified at the polar head-group can be formed during AAPH-induced radical attack, as previously reported during the oxidation of PS induced by hydroxyl radical [10]. Oxidation products at the serine polar head-group and the fatty acyl chain (m/z 745.6 (GPAA+14 Da), 747.6 (GPAA+16 Da) and 763.6 (GPAA+32 Da) were also identified ( Figure 6).…”

“…To observe whether PS polar head-group oxidation occurred in HaCaT cells after AAPH incubation, as observed after radical oxidation of PS by hydroxyl radical [10,25], cells were subjected to oxidation with AAPH (30 and 50 mM). The oxidative stimuli used were the same as reported in a previous study that mimicked oxidative stress injury in keratinocytes as a model of inflammation [18].…”

“…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]. However, in a recent study of oxidation of PS standards using the Fenton reagent, it was observed that the PS head-group can also undergo oxidative modifications leading to the formation of modified polar head-groups [10,12].…”

“…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]. However, in a recent study of oxidation of PS standards using the Fenton reagent, it was observed that the PS head-group can also undergo oxidative modifications leading to the formation of modified polar head-groups [10,12]. Among these oxidation products, the derivatives with a polar head-group containing an acetic acid linked to the phosphate group, called glycerophosphoacetic acid (GPAA), were found to be the most abundant [10].…”

“…However, in a recent study of oxidation of PS standards using the Fenton reagent, it was observed that the PS head-group can also undergo oxidative modifications leading to the formation of modified polar head-groups [10,12]. Among these oxidation products, the derivatives with a polar head-group containing an acetic acid linked to the phosphate group, called glycerophosphoacetic acid (GPAA), were found to be the most abundant [10]. These products showed a distinct fragmentation and neutral loss under MS/MS conditions and the typical loss of the serine group is absent.…”