Research article Received: 31 July 2013
Revised: 22 October 2013
Accepted: 22 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3301
Photosensitized oxidation of phosphatidylethanolamines monitored by electrospray tandem mass spectrometry Tânia Melo,a Nuno Santos,a Diana Lopes,b Eliana Alves,b Elisabete Maciel,a Maria A. F. Faustino,c João P. C. Tomé,c Maria G. P. M. S. Neves,c Adelaide Almeida,b Pedro Domingues,a Marcela A. Segundod and M. Rosário M. Dominguesa* Photodynamic therapy combines visible light and a photosensitizer (PS) in the presence of molecular oxygen to generate reactive oxygen species able to modify biological structures such as phospholipids. Phosphatidylethanolamines (PEs), being major phospholipid constituents of mammalian cells and membranes of Gram-negative bacteria, are potential targets of photosensitization. In this work, the oxidative modifications induced by white light in combination with cationic porphyrins (Tri-Py+-Me-PF and Tetra-Py+-Me) were evaluated on PE standards. Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) were used to identify and characterize the oxidative modifications induced in PEs (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4). Photo-oxidation products of POPE, PLPE and PAPE as hydroxy, hydroperoxy and keteno derivatives and products due to oxidation in ethanolamine polar head were identified. Hydroperoxy-PEs were found to be the major photo-oxidation products. Quantification of hydroperoxides (PE-OOH) allowed differentiating the potential effect in photodamage of the two porphyrins. The highest amounts of PE-OOH were notorious in the presence of Tri-Py+-Me-PF, a highly efficient PS against bacteria. The identification of these modifications in PEs is an important key point in the understanding cell damage processes underlying photodynamic therapy approaches. Copyright © 2013 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: phosphatidylethanolamines; photodynamic therapy; photosensitizer; photo-oxidation; electrospray; tandem mass spectrometry
Introduction
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* Correspondence to: M. Rosário M. Domingues, Mass Spectrometry Centre, UI QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal. E-mail: [email protected] a Mass Spectrometry Centre, UI QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal b Centre for Environmental and Marine Studies, Department of Biology, University of Aveiro, Aveiro, Portugal c Organic Chemistry Research Unit, QOPNA & Department of Chemistry, University of Aveiro, Aveiro, Portugal d REQUIMTE, Department of Chemistry, Faculty of Pharmacy, University of Porto, Portugal
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Photodynamic therapy (PDT) is a well-established photochemotherapeutic approach in cancer treatment[1–4] and with prospective applications as antimicrobial therapy.[5–12] It is based on the combination of visible light and a photosensitizer (PS), as porphyrins derivatives, mediated by the presence of molecular oxygen (3O2) to form reactive oxygen species (ROS), which are able to oxidize several biomolecules (proteins, lipids and nucleic acids) leading to cytotoxicity and cell death.[13–19] The hydrophobic nature of certain PS allows their location in membrane bilayers. Thus, membrane lipids and phospholipids are key targets for the harmful attack of ROS produced during the photodynamic process.[20,21] It is well known that under oxidative conditions phospholipids undergo structural modifications, and the wide range of oxidized products generated are structurally different and can lose function or have biological activities distinct from the native phospholipids.[22–25] While some studies are focused on the effect of the photooxidation of phosphatidylcholine, the information on phosphatidylethanolamines (PEs) photodamage is scarce.[26–29] However, PEs are the second most abundant lipid constituents in eukaryotic cell membranes and of lipoproteins.[30,31] In addition, in bacterial membranes namely in the Gram-negative Escherichia coli, PEs are the most abundant component, generally reported
to be 70–75% of total phospholipid content.[30,32–34] The high amounts of PEs in membranes make them potential targets to undergo oxidation namely during the photosensitization process. Oxidative modifications in PEs were already reported during in vitro oxidation induced by several oxidative stimuli as well as in vivo systems as reviewed recently.[35,36] Domingues and co-authors reported the in vitro oxidation of 1-palmitoyl-2linoleoyl-sn-glycero-3-phosphoethanolamine by Fenton reaction and identified long- and short-chain oxidation products.[37] Gugiu and co-authors reported the in vitro oxidation of different
T. Melo et al. PEs using distinct oxidation systems including myeloperoxidase, Fenton reaction and UV, with formation of truncated PEs, and also the detection of these oxidation products in rat retina.[38] More recently, the first group reported the in vitro oxidation of PEs bearing oleic, linoleic and arachidonic fatty acyl chains induced by UV-A irradiation with formation of long-chain oxidation products and phosphatidic acid derivatives but no shortchain derivatives.[39] Nevertheless, there are no studies that state the effects of photodynamic oxidation of PEs. Recent work in our lab allowed to verify that PE in E. coli can be affected by photooxidation in the presence of porphyrinic PSs[40]. However, since E. coli possesses mainly monounsaturated fatty acids, there are no reports on the effect of the sensitization in PE bearing polyunsaturated fatty acid, which are the major PEs components in lipid membranes in mammalians. Additionally, as PDT is nowadays used in the treatment of cancer, particularly skin cancer, and in ocular diseases such as age-related macular degeneration and myopia,[41,42] and considering that PEs are particularly abundant in the skin and in the retina, it will be very valuable to understand at what extent PDT affects PEs. In this work, three different PE standards, 1-palmitoyl-2-oleoyl-sn-g lycero-3-phosphoethanolamine (POPE; C16:0/C18:1), 1-palmitoyl-2-li noleoyl-sn-glycero-3-phosphoethanolamine (PLPE; C16:0/C18:2) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PAPE; C16:0/C20:4) (Fig. 1), were submitted to photo-oxidation induced by visible light in the presence of two distinct cationic PSs: 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide (Tri-Py+-Me-PF) and 5,10,15,20-tetrakis(1-methy lpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) (Fig. 1B). The selection of the three molecular species of PEs (POPE, PLPE and PAPE) was based on the different unsaturation degree of acyl chains
R1
O O
O
O P O -O H O
NH2
O R2
R2
and consequently on their different susceptibility to be oxidized. The two porphyrinic derivatives were chosen in order to assess how the structure of PS affects the nature and the extension of induced modifications on the selected PEs. Both derivatives already showed ability to inactivate and destroy efficiently several microorganisms including Gram-negative and Gram-positive bacteria, bacterial endospores, fungi and viruses[5,6,10,11,43–47] after photodynamic treatment, without recovery of their viability and development of resistance mechanisms against the photodynamic process, as reported before.[8,48] Oxidation was monitored by electrospray ionization mass spectrometry (ESI-MS) and oxidation products were structurally characterized by tandem mass spectrometry (MS/MS) in negative mode.
Experimental PSs The 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl) porphyrin tri-iodide (Tri-Py+-Me-PF) and 5,10,15,20-tetrakis(1-meth ylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) (Fig. 1) were prepared according to the literature.[47,49] Their purity was confirmed by thin-layer chromatography and by 1H NMR spectroscopy. An appropriate amount of PSs was dissolved in dimethyl sulfoxide (500 μM work solution) and sonicated for 30 min before use at room temperature.
Reagents/Chemicals Phospholipid standards 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho ethanolamine (POPE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosp hoethanolamine (PLPE) and 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphoethanolamine (PAPE) were purchased from Avanti® Polar Lipids, Inc. (Alabaster, AL, USA) and used without further purification. HPLC grade chloroform and methanol were purchased from Fisher Scientific Ltd. (Leicestershire, UK). Milli-Q water (Synergy®, Millipore Corporation, Billerica, MA, USA) was used and all other reagents were purchased from major commercial sources.
POPE
Photodynamic treatment of PE/Photosensitization protocol PLPE PAPE
IN+
IN+
IN+
IN+
N
N NH
NH
HN F
N
HN N
F I- N+
I- N+
F F
N+ I
F
Tri-Py+-Me-PF
Tetra-Py+-Me
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Figure 1. Representative structures of POPE, PLPE and PAPE and structures of the 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin + tri-iodide (Tri-Py -Me-PF) and 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)por+ phyrin tetra-iodide (Tetra-Py -Me).
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PE standards were dissolved in 1 ml of chloroform (1 mg ml 1). Samples of 250 μl (250 μg) were transferred to 50 ml glass beakers, dried with nitrogen and ressuspended in 2 ml of ammonium hydrogen carbonate buffer (5 mM, pH 7.4). The solution was vortex-mixed and sonicated for the formation of vesicles. Then, the beakers were exposed to light in the presence of 2.5, 5.0 and 10 μM of Tri-Py+-Me-PF or Tetra-Py+-Me (treated standards). The sample was then incubated in the dark for 10 min under 100 rpm stirring, at 25 °C, to promote the porphyrin binding to PEs. Then, the samples underwent a 30, 90 and 270 min period of irradiation (total light doses of 7.2, 21.6 and 64.8 J cm 2, respectively) with artificial white light (PAR radiation, 380–700 nm, 13 OSRAM 21 lamps of 18 W each) with an irradiance rate of 4 mW cm 2 (measured with a radiometer Li-COR Model LI-250), stirred at 100 rpm. After the photo-treatment, PEs were harvested by centrifugation (10 min, 13 000 × g, 4 °C), washed three times with Milli-Q water at 4 °C and the pellet was kept on ice until lipid extraction. The controls underwent the same experimental conditions without irradiation. This procedure was done in triplicate for each standard.
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ESI-MS of photosensitized phosphatidylethanolamines
Results and discussion
The PEs (irradiated and controls) were extracted from reaction mixture using the Fölch’s method.[50] A mixture of chloroform: methanol:water (8:4:3, V/V/V) was added to the samples, in PYREX® glass centrifuge tubes, followed by homogenization with a vortex. Then, samples were centrifuged at 2000 rpm for 10 min (Mixtasel, JP Selecta S.A., Barcelona, Spain) at room temperature to resolve a two-phase system: an aqueous upper phase and an organic lower phase from which lipids were obtained. The organic phase was separated and transferred to a clean tube. Extracts were dried under nitrogen flow, dissolved in 250 μl of chloroform and stored at 20 °C in 2 ml amber glass vials. No oxidation was detected during sample preparation in control samples.
Photodynamic oxidation of PE standards was studied using ESIMS in negative ion mode. The unmodified PEs were identified in the ESI-MS spectra as [M-H] ions at m/z 716.4 (POPE), 714.4 (PLPE) and 738.4 (PAPE) (Fig. 2A1, B1 and C1, respectively). Comparison of the ESI-MS spectra of all PEs, before and after photo-irradiation in the presence of both porphyrins (Fig. 2), allowed the identification of new ions with higher m/z values than the [M-H] ions observed after the oxidation of POPE, PLPE and PAPE. These new ions were identified as long-chain oxidation products of PEs as ketene, hydroxy and hydroperoxy derivatives ([M-H + nO] , n = 1–4 for POPE; n = 1–6 for PLPE and n = 1–8 for PAPE), as summarized in Table 1. The oxidation products at m/z 748.4 observed for POPE, at m/z 746.4 and 778.2 observed for PLPE and at m/z 770.3 and 802.3 observed for PAPE, are the main products observed during PEs photosensitization (Fig. 2). These products were assigned as hydroperoxy (mass shift of 32 Da from non-modified PE) and dihydroperoxy (mass shift of 64 Da from non-modified PE) derivatives of PEs. These results are in agreement with the ones reported during the photosensitized oxidation of cardiolipin in the presence of a silicon phthalocyanine where hydroperoxy and dihydroperoxy of cardiolipin were identified as the major products.[52] Interestingly, short chain oxidation products formed by cleavage of sn-2 acyl moiety were not detected after photooxidation. Although this type of oxidation products was detected during Fenton oxidation of PLPE,[37,53] they were absent in UVA-induced photo-oxidation of PEs and glycated PEs.[39] Nevertheless, during UV-A-induced photo-oxidation of PEs and glycated PEs[39], some phosphatidic acid derivatives were found, but not for Fenton-induced oxidation of PLPE[37,53] neither in our present work. We also assessed how the irradiation time and the PS concentration affect the nature and the extent of PEs oxidation (Figs 3 and 4). The results showed that the type of oxidation products was independent of the PS concentration and irradiation time used. However, their amount showed to be concentration and time dependent, i.e. lower abundance of oxidation products when lower concentration and lower irradiation time were tested and higher abundance for higher time of exposure and concentration. To have some knowledge about the structure of the new products formed, we performed ESI-MS/MS analysis. From the analysis of the MS/MS spectra of [M-H] ions of the non-oxidized PEs, the RCOO carboxylate anions (R1COO /R2COO ) were identified and it was possible to assign the modified carboxylate anions (R′2COO ) in the spectra of the oxidized PE species. The results of MS/MS analysis of [M-H] ions of PEs oxidation products will be expressed and explained in detail for POPE, since the fragmentation pattern of oxidation products identified was similar for the others PEs (PLPE and PAPE). Figure 5 A1 shows the MS/MS spectrum of the [M-H] ions at m/z 716.5 corresponding to the non-oxidized POPE. The product ions corresponding to the loss of sn-2 fatty acyl chain as R2COOH, at m/z 434.3, and as ketene derivatives (loss of R2 = C = O), at m/z 452.2 can be detected from this spectrum. Ions formed by the loss of sn-1 fatty acyl chain as R1 = C = O are also present at m/z 478.3. The carboxylate anions of sn-1 and sn-2 fatty acyl chains were also identified at m/z 255.2 and 281.2, respectively.
Quantification of hydroperoxides by FOX 2 assay FOX 2 assay was performed according to the procedure described by Wolff[51] with some modifications. The reagent (100 ml) was prepared as follows: 250 μM (NH4)2Fe(SO4).6H2O (9.8 mg) and 25 mM H2SO4 (139 μl) were dissolved in 5 ml of water, mixed with 4 mM 2,6-di-tert-butyl-p-hydroxytoluene (BHT) (88.2 mg), 100 μM xylenol orange (7.2 mg) and 45 ml of methanol. Then, another 45 ml of methanol and 5 ml of water were added. Aliquots of 50 μg (50 μl) of PEs (control and samples) were added to FOX2 reagent (950 μl) in microtubes, homogenized in a vortex mixer and left to react for 30 min at room temperature, in the dark. H2O2 standards with concentrations ranging from 0.0 to 0.4 mM were also prepared and underwent the same treatment of the studied samples. After incubation, the absorbance of samples was read at 560 nm against the H2O2 standards. Data are reported as H2O2 equivalents. Mass spectrometry The extent of photooxidative modifications and the identification of the new products formed were monitored by ESI-MS in negative mode, in a linear ion trap mass spectrometer LXQ (Thermo Finnigan, San Jose, CA, USA). The sample was introduced through direct infusion, and the ESI conditions in negative mode were as follows: flow rate of 8 μl min 1; electrospray voltage of 4.7 kV; capillary temperature of 275 °C and the sheath gas flow of 25 units. An isolation width of 0.5 Da was used with a 30 ms activation time for MS/MS experiments. Full scan MS spectra and MS/ MS spectra were acquired with a 50 ms and 200 ms maximum ion injection time, respectively. Normalized collision energy TM (CE) was varied between 17 and 20 (arbitrary units) for MS/MS. Data acquisition and treatment of results were carried out on an Xcalibur Data System (version 2.0).
Statistical analysis Quantification of hydroperoxides was performed independently and in different days, three times (n = 3), both for control and for samples. Results were analyzed using one-way analysis of variance with the Bonferroni post-hoc test to determine significant differences among samples, and were expressed as the mean ± standard error. A value of p < 0.05 was considered significant. Statistics was done using PRISMW GraphPad Software, Inc (GraphPad Prism 5.0).
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Lipid extraction
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Figure 2. ESI-MS spectra in negative mode showing the [M-H] ions of POPE (A), PLPE (B) and PAPE (C) before (A1–C1) and after 90 min of photo-irradiation in + + the presence of 5 μM of Tri-Py -Me-PF (A2-C2) or Tetra-Py -Me (A3–C3).
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Figures 5 A2 and A3 depict the MS/MS spectra of the oxidation products of POPE at m/z 730.5 (keto-PE) and 732.5 (hydroxyl PE). The ions at m/z 434.3 and 452.3 can be justified due to the loss of modified sn-2 acyl chains as acid and ketene derivatives, respectively; also, the fragment ions at m/z 295.2 (281 + 14 Da) and 297.2 (281 + 16 Da) are consistent to the loss of modified sn-2 carboxylate anion. These fragments confirm the occurrence of oxidation in the oleoyl fatty acyl chain. The MS/MS of the hydroperoxy-POPE at m/z 748.5 (Fig. 5 A4) showed modified sn-2 carboxylate anions, namely the hydroperoxy oleoyl carboxylate anion at m/z 313.2 (281 + 32 Da) and also another product ions at m/z 295.2 (281 + 32-18 Da) due to further loss of a water molecule. An important point observed in the MS/MS spectra is the differences in the relative abundance of the modified R’2COOanions. For POPE plus 14 Da, identified as the keto derivative (Fig. 5 A2) and POPE plus 16 Da identified as the hydroxy derivative (Fig. 5 A3), the corresponding modified R′2COO anions are the highest abundant peaks. A different situation is observed for POPE plus 32 Da identified as the hydroperoxy derivative (Fig. 5 A4) where the most abundant ion observed in MS/MS formed is due to the loss of 18 Da from the modified R′2COO anion. This feature can be justified due the higher instability of the hydroperoxy species. The hydroxy- and epoxy- molecular species, as well as the hydroperoxy and dihydroxy-molecular species, have identical m/z values. However, since they are isobaric species with the same molecular weight, they generate the same [M-H] ion. It is believed that the primary photoxidation product is the hydroperoxide (as confirmed by FOX2 assay) that can suffer decomposition affording hydroxy and ketone derivatives. If the hydroperoxide is formed next to a double bond, it can lead to epoxides that are isobaric with ketones (loss of 2H). On the other hand, when epoxides are isobaric with hydroxy lipids (no loss of 2H), the epoxidation must occur in a double bond via a preformed hydroperoxide (as a side reaction).
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However, the distinction among these species is very hard by tandem mass spectrometry, even with MS3 analysis. In ESI-MS spectra of oxidized POPE and PLPE (Fig. 2), other new ions with odd and higher m/z values when compared to each unmodified PE were present. These ions were proposed to be PE oxidation products with modifications in the polar head group formed due to an oxidative deamination of the ethanolamine polar head with loss of the amino group follow by the formation of a terminal aldehyde (Fig. 6, Table 2). Oxidative modifications in polar head was also found during Fenton-induced oxidation of glycated PLPE as reported by Simões and co-authors,[53] but not during UV-A-induced photo-oxidation of PEs and glycated PEs.[39] This type of oxidation products was not detected in PAPE probably due to the fact that the arachidonic induces extensive oxidation in the fatty acyl chain. Corroboration of the proposed structure for these ions was achieved by MS/MS that showed the modified carboxylate anions (R2′COO ) as depicted in Fig. 6. Product ions due to cleavage in the polar head, as reported in previous works, were not detected in these studies. This fact can be justified by the low abundance of oxidation products with modified polar head suggesting that the oxidative deamination of polar head is a minor pathway compared with the lateral chain oxidation. Hydroperoxide derivatives were the major products formed during photosensitization of PEs, similarly with what was previously reported for photosensitization of cardiolipin with a phthalocyanine PS.[52] Due to the variety of the oxidation products seen in the MS spectra of the photo-oxidized mixtures (Fig. 2), and in order to assess the differences observed in the MS spectra between both PSs, namely in the relative abundance of the hydroperoxy species versus the non-modified PE, the quantification of hydroperoxides was used to evaluate the different efficacy in the photosensitization of phospholipids. The amount of lipid hydroperoxides (LOOH) generated from PE oxidation after photosensitization in the presence of the two different PSs was
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ESI-MS of photosensitized phosphatidylethanolamines Table 1. Oxidation products observed in the negative ESI-MS spectra from each oxidized PE (POPE, PLPE and PAPE) with the identification and the indication of the calculated m/z values of the [M-H] ions Modification
--+14 (+O-2 Da) +16 (+O) +28 (+2O-4 Da) +30 (+2O-2 Da) +32 (+2O) +46 (+3O-2 Da) +48 (+3O) +60 (+4O-4 Da) +62 (+4O-2 Da) +64 (+4O) +76 (+5O-4 Da) +78 (+5O-2 Da) +80 (+5O) +92 (+6O-4 Da) +94 (+6O-2 Da) +96 (+6O) +108 (+7O-4 Da) +110 (+7O-2 Da) +112 (+7O) +124 (+8O-4 Da) +126 (+8O-2 Da) +128 (+8O)
[M-H]
Polar head
POPE
PLPE
PAPE
716.52 730.50 732.52 744.48 746.50 748.51 762.49 764.51 776.47 778.49 780.50
714.51 728.49 730.50 742.47 744.48 746.50 760.48 762.50 774.46 776.47 778.49 790.45 792.47 794.48 806.45 808.46 810.48
738.51 752.49 754.50 766.47 768.48 770.50 784.48 786.50 798.50 800.47 802.49 814.45 816.47 818.48 830.45 832.46 834.48 846.44 848.46 850.47 862.44 864.45 866.47
O O P O OH
NH2
Figure 3. ESI-MS spectra in negative mode showing the [M-H] ions of POPE after 30 (A1, B1 and C1), 90 (A2, B2 and C2) and 270 min (A3, B3 and C3) of + photo-irradiation in the presence of 2.5 μM (A), 5 μM (B) and 10 μM (C) of Tri-Py -Me-PF.
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be more efficient in inducing photoinactivation of bacteria than Tetra-Py+-Me.[46,54] The reorganization of the relationship between the chemical structure and the charge of the PS in the photoinactivation of biological targets is needed to understand the effect of the
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evaluated using the modified ferrous iron/xylenol orange II (FOX 2) assay and the results are summarized in Fig. 7. In the presence of Tri-Py+-Me-PF (gray bar), the amounts of LOOH, for all the PEs, are higher than in the presence of Tetra-Py+-Me. It is worth to refer that this tricationic derivative (Tri-Py+-Me-PF) showed to
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Figure 4. ESI-MS spectra in negative mode showing the [M-H] ions of POPE after 30 (A1, B1 and C1), 90 (A2, B2 and C2) and 270 min (A3, B3 and C3) of + photo-irradiation in the presence of 2.5 μM (A), 5 μM (B) and 10 μM (C) of Tetra-Py -Me.
Figure 5. ESI-MS/MS spectra of the [M-H] ions of non-oxidized POPE (A1), and of the correspondent photo-oxidation products keto (A2), hydroxy (A3), + and hydroperoxy (A4) derivatives formed after 90 min of photo-irradiation in the presence of 5 μM of Tri-Py -Me-PF. The proposed structures for each modified PE are also represented.
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photosensitization process, to contribute to design improved PSs and to decide about the best conditions for photodynamic inactivation.[55] During irradiation, the PS is energized by absorption of a photon from visible light with appropriate wavelength, passing from the ground state to an unstable electronically excited state. From
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this state, the PS can lose its energy and return again to the ground state by radiative (fluorescence emission) or non-radiative (internal conversion) transitions or it can undergo intersystem crossing process to pass into the extremely reactive triplet excited state. In this excited state, the PS can follow one or both competitive mechanisms, either type I or II, proposed to be involved in photodynamic
Copyright © 2013 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2013, 48, 1357–1365
ESI-MS of photosensitized phosphatidylethanolamines
Figure 6. ESI-MS/MS spectra of the [M-H] ions at m/z 731.5 (A1) and 747.5 (A2) of POPE and at m/z 745.4 (A3) and 761.5 (A4) of PLPE oxidation product with modifications in polar head. The proposed structure of deprotonated molecule is also represented.
Table 2. POPE and PLPE oxidation products with modifications in polar head formed by oxidative deamination of ethanolamine polar head observed in ESI-MS spectra. The table shows the m/z value of the [M-H] ions, their most probable identification, including the modified polar head and the indication of the calculated m/z values of the [M-H] ions Modification
+16 +32 +48 +64 +80 +96
(+O) (+2O) (+3O) (+4O) (+5O) (+6O)
[M-H]
Polar head
POPE
PLPE
731.49 747.48 763.48 779.47
729.47 745.47 761.46 777.46 793.45 809.45
O
O
O P O OH
Figure 7. Lipid hydroperoxides (LOOH) quantification after 90 min of ir2 radiation with artificial white light (total light dose 21.6 J cm ) in the + + presence of 5 μM of Tri-Py -Me-PF (gray bars) and Tetra-Py -Me (black bars) which were directly compared to each other. Values were the mean ± SD. ***, significantly different from control group (P < 0.0001). No hydroperoxides were detected in control samples.
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inactivation. In type I mechanism, the PS can interact with biological targets (lipids, proteins, DNA), by hydrogen-atom abstraction or electron-transfer reactions, to produce free radicals that can
afterwards react with the ground state 3O2 to yield ROS. On the other hand, the PS can transfer the excitation energy directly to 3O2 to form singlet oxygen (1O2, type II mechanism).[14,56–58] This highly reactive ROS can lead to the formation of further oxygen radicals or directly react with closer molecules in neighborhood. After transfer of the excitation energy from the triplet state, the PS returns to the lowest excited singlet state.[15,56,58] The effectiveness of the process depends on several key points: the structural features and charge of the PS, the concentrations of PS, 3O2 and substrate and the properties of the light used (e.g. wavelength, type, dose and fluence rate).[14–16,57,43,48,44] Lipid photo-oxidation can also be classified as type I or type II. In the first mechanism, lipid peroxidation proceeds by free radical-mediated chain reactions, similar to ordinary lipid autoxidation widely described.[23–25] The latter is a non-free radical process that involves 1 O2,[20] which reacts directly with the double bond of unsaturated lipids mainly by ‘ene’ addition reactions to give hydroperoxides with concomitant double bond migration[25] and without generating radicals. However, the accurate mechanism behind the lipid peroxidation during photosensitization is still unknown. This work showed that phospholipids, namely membrane phospholipids, are potential targets for modifications during the photodynamic process and that hydroperoxides are the main products of photo-oxidation. In this way, it is important to study which mechanism is involved in lipid peroxidation mediated by each PS and the oxidized species formed as a consequence of lipid oxidation. Studies focused on oxidative modifications induced in phospholipids standards during in vitro oxidative stress conditions induced by several oxidative systems that mimic the ones that occur in vivo are highly important since the products formed by each may be different and may have distinct effects. Despite their analysis may be a hard challenge, mass spectrometry is a helpful tool that has been widely used to monitor, identify and characterize oxidized phospholipids[24,36,37,59–63] contributing to a better understanding of the alterations that may occur during the photodynamic inactivation of bacteria or in the general photodynamic process.
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Conclusions In this study, three PE standards (POPE, PLPE and PAPE) were submitted to photosensitization in the presence of two porphyrin derivatives: Tri-Py+-Me-PF and Tetra-Py+-Me. The products formed were identified and characterized by ESI-MS and MS/ MS. Long-chain oxidation products due to oxidation in unsaturated fatty acyl chain, namely keto, hydroxyl and hydroperoxy derivatives were identified. Products with modifications both in fatty acyl chain and in ethanolamine polar head with formation of a terminal aldehyde were also identified. This topic may have an important role during the photodynamic process in cells and tissues. Phospholipid hydroperoxides were found to be the most abundant products formed during photosensitization with the cationic porphyrins used. The higher amounts of hydroperoxy derivatives were found upon photosensitization with Tri-Py+Me-PF that has been reported to be more efficient in bacteria inactivation than Tetra-Py+-Me. Since phospholipids are targets of photosensitization, they can be used to evaluate and monitor photodamage in biological systems. Acknowledgements The authors are thankful to the University of Aveiro, Fundação para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, COMPETE and FEDER for funding the project PTDC/QUI-BIQ/ 104968/2008, REDE/1504/REM/2005 (that concerns the Portuguese Mass Spectrometry Network), Centre for Environmental and Marine Studies (CESAM) unit (project Pest-C/MAR/LA0017/2013) and the QOPNA research unit (project PEst-C/QUI/UI0062/2013, FCOMP-010124-FEDER-037296). Tânia Melo (SFRH/BD/84691/2012), Eliana Alves (SFRH/BD/41806/2007) and Elisabete Maciel (SFRH/BD/ 73203/2010) are grateful to FCT for doctoral grants.
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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Copyright © 2013 John Wiley & Sons, Ltd.
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Revised: 22 October 2013
Accepted: 22 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3301
Photosensitized oxidation of phosphatidylethanolamines monitored by electrospray tandem mass spectrometry Tânia Melo,a Nuno Santos,a Diana Lopes,b Eliana Alves,b Elisabete Maciel,a Maria A. F. Faustino,c João P. C. Tomé,c Maria G. P. M. S. Neves,c Adelaide Almeida,b Pedro Domingues,a Marcela A. Segundod and M. Rosário M. Dominguesa* Photodynamic therapy combines visible light and a photosensitizer (PS) in the presence of molecular oxygen to generate reactive oxygen species able to modify biological structures such as phospholipids. Phosphatidylethanolamines (PEs), being major phospholipid constituents of mammalian cells and membranes of Gram-negative bacteria, are potential targets of photosensitization. In this work, the oxidative modifications induced by white light in combination with cationic porphyrins (Tri-Py+-Me-PF and Tetra-Py+-Me) were evaluated on PE standards. Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) were used to identify and characterize the oxidative modifications induced in PEs (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4). Photo-oxidation products of POPE, PLPE and PAPE as hydroxy, hydroperoxy and keteno derivatives and products due to oxidation in ethanolamine polar head were identified. Hydroperoxy-PEs were found to be the major photo-oxidation products. Quantification of hydroperoxides (PE-OOH) allowed differentiating the potential effect in photodamage of the two porphyrins. The highest amounts of PE-OOH were notorious in the presence of Tri-Py+-Me-PF, a highly efficient PS against bacteria. The identification of these modifications in PEs is an important key point in the understanding cell damage processes underlying photodynamic therapy approaches. Copyright © 2013 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: phosphatidylethanolamines; photodynamic therapy; photosensitizer; photo-oxidation; electrospray; tandem mass spectrometry
Introduction
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* Correspondence to: M. Rosário M. Domingues, Mass Spectrometry Centre, UI QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal. E-mail: [email protected] a Mass Spectrometry Centre, UI QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal b Centre for Environmental and Marine Studies, Department of Biology, University of Aveiro, Aveiro, Portugal c Organic Chemistry Research Unit, QOPNA & Department of Chemistry, University of Aveiro, Aveiro, Portugal d REQUIMTE, Department of Chemistry, Faculty of Pharmacy, University of Porto, Portugal
Copyright © 2013 John Wiley & Sons, Ltd.
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Photodynamic therapy (PDT) is a well-established photochemotherapeutic approach in cancer treatment[1–4] and with prospective applications as antimicrobial therapy.[5–12] It is based on the combination of visible light and a photosensitizer (PS), as porphyrins derivatives, mediated by the presence of molecular oxygen (3O2) to form reactive oxygen species (ROS), which are able to oxidize several biomolecules (proteins, lipids and nucleic acids) leading to cytotoxicity and cell death.[13–19] The hydrophobic nature of certain PS allows their location in membrane bilayers. Thus, membrane lipids and phospholipids are key targets for the harmful attack of ROS produced during the photodynamic process.[20,21] It is well known that under oxidative conditions phospholipids undergo structural modifications, and the wide range of oxidized products generated are structurally different and can lose function or have biological activities distinct from the native phospholipids.[22–25] While some studies are focused on the effect of the photooxidation of phosphatidylcholine, the information on phosphatidylethanolamines (PEs) photodamage is scarce.[26–29] However, PEs are the second most abundant lipid constituents in eukaryotic cell membranes and of lipoproteins.[30,31] In addition, in bacterial membranes namely in the Gram-negative Escherichia coli, PEs are the most abundant component, generally reported
to be 70–75% of total phospholipid content.[30,32–34] The high amounts of PEs in membranes make them potential targets to undergo oxidation namely during the photosensitization process. Oxidative modifications in PEs were already reported during in vitro oxidation induced by several oxidative stimuli as well as in vivo systems as reviewed recently.[35,36] Domingues and co-authors reported the in vitro oxidation of 1-palmitoyl-2linoleoyl-sn-glycero-3-phosphoethanolamine by Fenton reaction and identified long- and short-chain oxidation products.[37] Gugiu and co-authors reported the in vitro oxidation of different
T. Melo et al. PEs using distinct oxidation systems including myeloperoxidase, Fenton reaction and UV, with formation of truncated PEs, and also the detection of these oxidation products in rat retina.[38] More recently, the first group reported the in vitro oxidation of PEs bearing oleic, linoleic and arachidonic fatty acyl chains induced by UV-A irradiation with formation of long-chain oxidation products and phosphatidic acid derivatives but no shortchain derivatives.[39] Nevertheless, there are no studies that state the effects of photodynamic oxidation of PEs. Recent work in our lab allowed to verify that PE in E. coli can be affected by photooxidation in the presence of porphyrinic PSs[40]. However, since E. coli possesses mainly monounsaturated fatty acids, there are no reports on the effect of the sensitization in PE bearing polyunsaturated fatty acid, which are the major PEs components in lipid membranes in mammalians. Additionally, as PDT is nowadays used in the treatment of cancer, particularly skin cancer, and in ocular diseases such as age-related macular degeneration and myopia,[41,42] and considering that PEs are particularly abundant in the skin and in the retina, it will be very valuable to understand at what extent PDT affects PEs. In this work, three different PE standards, 1-palmitoyl-2-oleoyl-sn-g lycero-3-phosphoethanolamine (POPE; C16:0/C18:1), 1-palmitoyl-2-li noleoyl-sn-glycero-3-phosphoethanolamine (PLPE; C16:0/C18:2) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PAPE; C16:0/C20:4) (Fig. 1), were submitted to photo-oxidation induced by visible light in the presence of two distinct cationic PSs: 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide (Tri-Py+-Me-PF) and 5,10,15,20-tetrakis(1-methy lpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) (Fig. 1B). The selection of the three molecular species of PEs (POPE, PLPE and PAPE) was based on the different unsaturation degree of acyl chains
R1
O O
O
O P O -O H O
NH2
O R2
R2
and consequently on their different susceptibility to be oxidized. The two porphyrinic derivatives were chosen in order to assess how the structure of PS affects the nature and the extension of induced modifications on the selected PEs. Both derivatives already showed ability to inactivate and destroy efficiently several microorganisms including Gram-negative and Gram-positive bacteria, bacterial endospores, fungi and viruses[5,6,10,11,43–47] after photodynamic treatment, without recovery of their viability and development of resistance mechanisms against the photodynamic process, as reported before.[8,48] Oxidation was monitored by electrospray ionization mass spectrometry (ESI-MS) and oxidation products were structurally characterized by tandem mass spectrometry (MS/MS) in negative mode.
Experimental PSs The 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl) porphyrin tri-iodide (Tri-Py+-Me-PF) and 5,10,15,20-tetrakis(1-meth ylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) (Fig. 1) were prepared according to the literature.[47,49] Their purity was confirmed by thin-layer chromatography and by 1H NMR spectroscopy. An appropriate amount of PSs was dissolved in dimethyl sulfoxide (500 μM work solution) and sonicated for 30 min before use at room temperature.
Reagents/Chemicals Phospholipid standards 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho ethanolamine (POPE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosp hoethanolamine (PLPE) and 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphoethanolamine (PAPE) were purchased from Avanti® Polar Lipids, Inc. (Alabaster, AL, USA) and used without further purification. HPLC grade chloroform and methanol were purchased from Fisher Scientific Ltd. (Leicestershire, UK). Milli-Q water (Synergy®, Millipore Corporation, Billerica, MA, USA) was used and all other reagents were purchased from major commercial sources.
POPE
Photodynamic treatment of PE/Photosensitization protocol PLPE PAPE
IN+
IN+
IN+
IN+
N
N NH
NH
HN F
N
HN N
F I- N+
I- N+
F F
N+ I
F
Tri-Py+-Me-PF
Tetra-Py+-Me
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Figure 1. Representative structures of POPE, PLPE and PAPE and structures of the 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin + tri-iodide (Tri-Py -Me-PF) and 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)por+ phyrin tetra-iodide (Tetra-Py -Me).
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PE standards were dissolved in 1 ml of chloroform (1 mg ml 1). Samples of 250 μl (250 μg) were transferred to 50 ml glass beakers, dried with nitrogen and ressuspended in 2 ml of ammonium hydrogen carbonate buffer (5 mM, pH 7.4). The solution was vortex-mixed and sonicated for the formation of vesicles. Then, the beakers were exposed to light in the presence of 2.5, 5.0 and 10 μM of Tri-Py+-Me-PF or Tetra-Py+-Me (treated standards). The sample was then incubated in the dark for 10 min under 100 rpm stirring, at 25 °C, to promote the porphyrin binding to PEs. Then, the samples underwent a 30, 90 and 270 min period of irradiation (total light doses of 7.2, 21.6 and 64.8 J cm 2, respectively) with artificial white light (PAR radiation, 380–700 nm, 13 OSRAM 21 lamps of 18 W each) with an irradiance rate of 4 mW cm 2 (measured with a radiometer Li-COR Model LI-250), stirred at 100 rpm. After the photo-treatment, PEs were harvested by centrifugation (10 min, 13 000 × g, 4 °C), washed three times with Milli-Q water at 4 °C and the pellet was kept on ice until lipid extraction. The controls underwent the same experimental conditions without irradiation. This procedure was done in triplicate for each standard.
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ESI-MS of photosensitized phosphatidylethanolamines
Results and discussion
The PEs (irradiated and controls) were extracted from reaction mixture using the Fölch’s method.[50] A mixture of chloroform: methanol:water (8:4:3, V/V/V) was added to the samples, in PYREX® glass centrifuge tubes, followed by homogenization with a vortex. Then, samples were centrifuged at 2000 rpm for 10 min (Mixtasel, JP Selecta S.A., Barcelona, Spain) at room temperature to resolve a two-phase system: an aqueous upper phase and an organic lower phase from which lipids were obtained. The organic phase was separated and transferred to a clean tube. Extracts were dried under nitrogen flow, dissolved in 250 μl of chloroform and stored at 20 °C in 2 ml amber glass vials. No oxidation was detected during sample preparation in control samples.
Photodynamic oxidation of PE standards was studied using ESIMS in negative ion mode. The unmodified PEs were identified in the ESI-MS spectra as [M-H] ions at m/z 716.4 (POPE), 714.4 (PLPE) and 738.4 (PAPE) (Fig. 2A1, B1 and C1, respectively). Comparison of the ESI-MS spectra of all PEs, before and after photo-irradiation in the presence of both porphyrins (Fig. 2), allowed the identification of new ions with higher m/z values than the [M-H] ions observed after the oxidation of POPE, PLPE and PAPE. These new ions were identified as long-chain oxidation products of PEs as ketene, hydroxy and hydroperoxy derivatives ([M-H + nO] , n = 1–4 for POPE; n = 1–6 for PLPE and n = 1–8 for PAPE), as summarized in Table 1. The oxidation products at m/z 748.4 observed for POPE, at m/z 746.4 and 778.2 observed for PLPE and at m/z 770.3 and 802.3 observed for PAPE, are the main products observed during PEs photosensitization (Fig. 2). These products were assigned as hydroperoxy (mass shift of 32 Da from non-modified PE) and dihydroperoxy (mass shift of 64 Da from non-modified PE) derivatives of PEs. These results are in agreement with the ones reported during the photosensitized oxidation of cardiolipin in the presence of a silicon phthalocyanine where hydroperoxy and dihydroperoxy of cardiolipin were identified as the major products.[52] Interestingly, short chain oxidation products formed by cleavage of sn-2 acyl moiety were not detected after photooxidation. Although this type of oxidation products was detected during Fenton oxidation of PLPE,[37,53] they were absent in UVA-induced photo-oxidation of PEs and glycated PEs.[39] Nevertheless, during UV-A-induced photo-oxidation of PEs and glycated PEs[39], some phosphatidic acid derivatives were found, but not for Fenton-induced oxidation of PLPE[37,53] neither in our present work. We also assessed how the irradiation time and the PS concentration affect the nature and the extent of PEs oxidation (Figs 3 and 4). The results showed that the type of oxidation products was independent of the PS concentration and irradiation time used. However, their amount showed to be concentration and time dependent, i.e. lower abundance of oxidation products when lower concentration and lower irradiation time were tested and higher abundance for higher time of exposure and concentration. To have some knowledge about the structure of the new products formed, we performed ESI-MS/MS analysis. From the analysis of the MS/MS spectra of [M-H] ions of the non-oxidized PEs, the RCOO carboxylate anions (R1COO /R2COO ) were identified and it was possible to assign the modified carboxylate anions (R′2COO ) in the spectra of the oxidized PE species. The results of MS/MS analysis of [M-H] ions of PEs oxidation products will be expressed and explained in detail for POPE, since the fragmentation pattern of oxidation products identified was similar for the others PEs (PLPE and PAPE). Figure 5 A1 shows the MS/MS spectrum of the [M-H] ions at m/z 716.5 corresponding to the non-oxidized POPE. The product ions corresponding to the loss of sn-2 fatty acyl chain as R2COOH, at m/z 434.3, and as ketene derivatives (loss of R2 = C = O), at m/z 452.2 can be detected from this spectrum. Ions formed by the loss of sn-1 fatty acyl chain as R1 = C = O are also present at m/z 478.3. The carboxylate anions of sn-1 and sn-2 fatty acyl chains were also identified at m/z 255.2 and 281.2, respectively.
Quantification of hydroperoxides by FOX 2 assay FOX 2 assay was performed according to the procedure described by Wolff[51] with some modifications. The reagent (100 ml) was prepared as follows: 250 μM (NH4)2Fe(SO4).6H2O (9.8 mg) and 25 mM H2SO4 (139 μl) were dissolved in 5 ml of water, mixed with 4 mM 2,6-di-tert-butyl-p-hydroxytoluene (BHT) (88.2 mg), 100 μM xylenol orange (7.2 mg) and 45 ml of methanol. Then, another 45 ml of methanol and 5 ml of water were added. Aliquots of 50 μg (50 μl) of PEs (control and samples) were added to FOX2 reagent (950 μl) in microtubes, homogenized in a vortex mixer and left to react for 30 min at room temperature, in the dark. H2O2 standards with concentrations ranging from 0.0 to 0.4 mM were also prepared and underwent the same treatment of the studied samples. After incubation, the absorbance of samples was read at 560 nm against the H2O2 standards. Data are reported as H2O2 equivalents. Mass spectrometry The extent of photooxidative modifications and the identification of the new products formed were monitored by ESI-MS in negative mode, in a linear ion trap mass spectrometer LXQ (Thermo Finnigan, San Jose, CA, USA). The sample was introduced through direct infusion, and the ESI conditions in negative mode were as follows: flow rate of 8 μl min 1; electrospray voltage of 4.7 kV; capillary temperature of 275 °C and the sheath gas flow of 25 units. An isolation width of 0.5 Da was used with a 30 ms activation time for MS/MS experiments. Full scan MS spectra and MS/ MS spectra were acquired with a 50 ms and 200 ms maximum ion injection time, respectively. Normalized collision energy TM (CE) was varied between 17 and 20 (arbitrary units) for MS/MS. Data acquisition and treatment of results were carried out on an Xcalibur Data System (version 2.0).
Statistical analysis Quantification of hydroperoxides was performed independently and in different days, three times (n = 3), both for control and for samples. Results were analyzed using one-way analysis of variance with the Bonferroni post-hoc test to determine significant differences among samples, and were expressed as the mean ± standard error. A value of p < 0.05 was considered significant. Statistics was done using PRISMW GraphPad Software, Inc (GraphPad Prism 5.0).
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Lipid extraction
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Figure 2. ESI-MS spectra in negative mode showing the [M-H] ions of POPE (A), PLPE (B) and PAPE (C) before (A1–C1) and after 90 min of photo-irradiation in + + the presence of 5 μM of Tri-Py -Me-PF (A2-C2) or Tetra-Py -Me (A3–C3).
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Figures 5 A2 and A3 depict the MS/MS spectra of the oxidation products of POPE at m/z 730.5 (keto-PE) and 732.5 (hydroxyl PE). The ions at m/z 434.3 and 452.3 can be justified due to the loss of modified sn-2 acyl chains as acid and ketene derivatives, respectively; also, the fragment ions at m/z 295.2 (281 + 14 Da) and 297.2 (281 + 16 Da) are consistent to the loss of modified sn-2 carboxylate anion. These fragments confirm the occurrence of oxidation in the oleoyl fatty acyl chain. The MS/MS of the hydroperoxy-POPE at m/z 748.5 (Fig. 5 A4) showed modified sn-2 carboxylate anions, namely the hydroperoxy oleoyl carboxylate anion at m/z 313.2 (281 + 32 Da) and also another product ions at m/z 295.2 (281 + 32-18 Da) due to further loss of a water molecule. An important point observed in the MS/MS spectra is the differences in the relative abundance of the modified R’2COOanions. For POPE plus 14 Da, identified as the keto derivative (Fig. 5 A2) and POPE plus 16 Da identified as the hydroxy derivative (Fig. 5 A3), the corresponding modified R′2COO anions are the highest abundant peaks. A different situation is observed for POPE plus 32 Da identified as the hydroperoxy derivative (Fig. 5 A4) where the most abundant ion observed in MS/MS formed is due to the loss of 18 Da from the modified R′2COO anion. This feature can be justified due the higher instability of the hydroperoxy species. The hydroxy- and epoxy- molecular species, as well as the hydroperoxy and dihydroxy-molecular species, have identical m/z values. However, since they are isobaric species with the same molecular weight, they generate the same [M-H] ion. It is believed that the primary photoxidation product is the hydroperoxide (as confirmed by FOX2 assay) that can suffer decomposition affording hydroxy and ketone derivatives. If the hydroperoxide is formed next to a double bond, it can lead to epoxides that are isobaric with ketones (loss of 2H). On the other hand, when epoxides are isobaric with hydroxy lipids (no loss of 2H), the epoxidation must occur in a double bond via a preformed hydroperoxide (as a side reaction).
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However, the distinction among these species is very hard by tandem mass spectrometry, even with MS3 analysis. In ESI-MS spectra of oxidized POPE and PLPE (Fig. 2), other new ions with odd and higher m/z values when compared to each unmodified PE were present. These ions were proposed to be PE oxidation products with modifications in the polar head group formed due to an oxidative deamination of the ethanolamine polar head with loss of the amino group follow by the formation of a terminal aldehyde (Fig. 6, Table 2). Oxidative modifications in polar head was also found during Fenton-induced oxidation of glycated PLPE as reported by Simões and co-authors,[53] but not during UV-A-induced photo-oxidation of PEs and glycated PEs.[39] This type of oxidation products was not detected in PAPE probably due to the fact that the arachidonic induces extensive oxidation in the fatty acyl chain. Corroboration of the proposed structure for these ions was achieved by MS/MS that showed the modified carboxylate anions (R2′COO ) as depicted in Fig. 6. Product ions due to cleavage in the polar head, as reported in previous works, were not detected in these studies. This fact can be justified by the low abundance of oxidation products with modified polar head suggesting that the oxidative deamination of polar head is a minor pathway compared with the lateral chain oxidation. Hydroperoxide derivatives were the major products formed during photosensitization of PEs, similarly with what was previously reported for photosensitization of cardiolipin with a phthalocyanine PS.[52] Due to the variety of the oxidation products seen in the MS spectra of the photo-oxidized mixtures (Fig. 2), and in order to assess the differences observed in the MS spectra between both PSs, namely in the relative abundance of the hydroperoxy species versus the non-modified PE, the quantification of hydroperoxides was used to evaluate the different efficacy in the photosensitization of phospholipids. The amount of lipid hydroperoxides (LOOH) generated from PE oxidation after photosensitization in the presence of the two different PSs was
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ESI-MS of photosensitized phosphatidylethanolamines Table 1. Oxidation products observed in the negative ESI-MS spectra from each oxidized PE (POPE, PLPE and PAPE) with the identification and the indication of the calculated m/z values of the [M-H] ions Modification
--+14 (+O-2 Da) +16 (+O) +28 (+2O-4 Da) +30 (+2O-2 Da) +32 (+2O) +46 (+3O-2 Da) +48 (+3O) +60 (+4O-4 Da) +62 (+4O-2 Da) +64 (+4O) +76 (+5O-4 Da) +78 (+5O-2 Da) +80 (+5O) +92 (+6O-4 Da) +94 (+6O-2 Da) +96 (+6O) +108 (+7O-4 Da) +110 (+7O-2 Da) +112 (+7O) +124 (+8O-4 Da) +126 (+8O-2 Da) +128 (+8O)
[M-H]
Polar head
POPE
PLPE
PAPE
716.52 730.50 732.52 744.48 746.50 748.51 762.49 764.51 776.47 778.49 780.50
714.51 728.49 730.50 742.47 744.48 746.50 760.48 762.50 774.46 776.47 778.49 790.45 792.47 794.48 806.45 808.46 810.48
738.51 752.49 754.50 766.47 768.48 770.50 784.48 786.50 798.50 800.47 802.49 814.45 816.47 818.48 830.45 832.46 834.48 846.44 848.46 850.47 862.44 864.45 866.47
O O P O OH
NH2
Figure 3. ESI-MS spectra in negative mode showing the [M-H] ions of POPE after 30 (A1, B1 and C1), 90 (A2, B2 and C2) and 270 min (A3, B3 and C3) of + photo-irradiation in the presence of 2.5 μM (A), 5 μM (B) and 10 μM (C) of Tri-Py -Me-PF.
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be more efficient in inducing photoinactivation of bacteria than Tetra-Py+-Me.[46,54] The reorganization of the relationship between the chemical structure and the charge of the PS in the photoinactivation of biological targets is needed to understand the effect of the
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evaluated using the modified ferrous iron/xylenol orange II (FOX 2) assay and the results are summarized in Fig. 7. In the presence of Tri-Py+-Me-PF (gray bar), the amounts of LOOH, for all the PEs, are higher than in the presence of Tetra-Py+-Me. It is worth to refer that this tricationic derivative (Tri-Py+-Me-PF) showed to
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Figure 4. ESI-MS spectra in negative mode showing the [M-H] ions of POPE after 30 (A1, B1 and C1), 90 (A2, B2 and C2) and 270 min (A3, B3 and C3) of + photo-irradiation in the presence of 2.5 μM (A), 5 μM (B) and 10 μM (C) of Tetra-Py -Me.
Figure 5. ESI-MS/MS spectra of the [M-H] ions of non-oxidized POPE (A1), and of the correspondent photo-oxidation products keto (A2), hydroxy (A3), + and hydroperoxy (A4) derivatives formed after 90 min of photo-irradiation in the presence of 5 μM of Tri-Py -Me-PF. The proposed structures for each modified PE are also represented.
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photosensitization process, to contribute to design improved PSs and to decide about the best conditions for photodynamic inactivation.[55] During irradiation, the PS is energized by absorption of a photon from visible light with appropriate wavelength, passing from the ground state to an unstable electronically excited state. From
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this state, the PS can lose its energy and return again to the ground state by radiative (fluorescence emission) or non-radiative (internal conversion) transitions or it can undergo intersystem crossing process to pass into the extremely reactive triplet excited state. In this excited state, the PS can follow one or both competitive mechanisms, either type I or II, proposed to be involved in photodynamic
Copyright © 2013 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2013, 48, 1357–1365
ESI-MS of photosensitized phosphatidylethanolamines
Figure 6. ESI-MS/MS spectra of the [M-H] ions at m/z 731.5 (A1) and 747.5 (A2) of POPE and at m/z 745.4 (A3) and 761.5 (A4) of PLPE oxidation product with modifications in polar head. The proposed structure of deprotonated molecule is also represented.
Table 2. POPE and PLPE oxidation products with modifications in polar head formed by oxidative deamination of ethanolamine polar head observed in ESI-MS spectra. The table shows the m/z value of the [M-H] ions, their most probable identification, including the modified polar head and the indication of the calculated m/z values of the [M-H] ions Modification
+16 +32 +48 +64 +80 +96
(+O) (+2O) (+3O) (+4O) (+5O) (+6O)
[M-H]
Polar head
POPE
PLPE
731.49 747.48 763.48 779.47
729.47 745.47 761.46 777.46 793.45 809.45
O
O
O P O OH
Figure 7. Lipid hydroperoxides (LOOH) quantification after 90 min of ir2 radiation with artificial white light (total light dose 21.6 J cm ) in the + + presence of 5 μM of Tri-Py -Me-PF (gray bars) and Tetra-Py -Me (black bars) which were directly compared to each other. Values were the mean ± SD. ***, significantly different from control group (P < 0.0001). No hydroperoxides were detected in control samples.
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inactivation. In type I mechanism, the PS can interact with biological targets (lipids, proteins, DNA), by hydrogen-atom abstraction or electron-transfer reactions, to produce free radicals that can
afterwards react with the ground state 3O2 to yield ROS. On the other hand, the PS can transfer the excitation energy directly to 3O2 to form singlet oxygen (1O2, type II mechanism).[14,56–58] This highly reactive ROS can lead to the formation of further oxygen radicals or directly react with closer molecules in neighborhood. After transfer of the excitation energy from the triplet state, the PS returns to the lowest excited singlet state.[15,56,58] The effectiveness of the process depends on several key points: the structural features and charge of the PS, the concentrations of PS, 3O2 and substrate and the properties of the light used (e.g. wavelength, type, dose and fluence rate).[14–16,57,43,48,44] Lipid photo-oxidation can also be classified as type I or type II. In the first mechanism, lipid peroxidation proceeds by free radical-mediated chain reactions, similar to ordinary lipid autoxidation widely described.[23–25] The latter is a non-free radical process that involves 1 O2,[20] which reacts directly with the double bond of unsaturated lipids mainly by ‘ene’ addition reactions to give hydroperoxides with concomitant double bond migration[25] and without generating radicals. However, the accurate mechanism behind the lipid peroxidation during photosensitization is still unknown. This work showed that phospholipids, namely membrane phospholipids, are potential targets for modifications during the photodynamic process and that hydroperoxides are the main products of photo-oxidation. In this way, it is important to study which mechanism is involved in lipid peroxidation mediated by each PS and the oxidized species formed as a consequence of lipid oxidation. Studies focused on oxidative modifications induced in phospholipids standards during in vitro oxidative stress conditions induced by several oxidative systems that mimic the ones that occur in vivo are highly important since the products formed by each may be different and may have distinct effects. Despite their analysis may be a hard challenge, mass spectrometry is a helpful tool that has been widely used to monitor, identify and characterize oxidized phospholipids[24,36,37,59–63] contributing to a better understanding of the alterations that may occur during the photodynamic inactivation of bacteria or in the general photodynamic process.
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Conclusions In this study, three PE standards (POPE, PLPE and PAPE) were submitted to photosensitization in the presence of two porphyrin derivatives: Tri-Py+-Me-PF and Tetra-Py+-Me. The products formed were identified and characterized by ESI-MS and MS/ MS. Long-chain oxidation products due to oxidation in unsaturated fatty acyl chain, namely keto, hydroxyl and hydroperoxy derivatives were identified. Products with modifications both in fatty acyl chain and in ethanolamine polar head with formation of a terminal aldehyde were also identified. This topic may have an important role during the photodynamic process in cells and tissues. Phospholipid hydroperoxides were found to be the most abundant products formed during photosensitization with the cationic porphyrins used. The higher amounts of hydroperoxy derivatives were found upon photosensitization with Tri-Py+Me-PF that has been reported to be more efficient in bacteria inactivation than Tetra-Py+-Me. Since phospholipids are targets of photosensitization, they can be used to evaluate and monitor photodamage in biological systems. Acknowledgements The authors are thankful to the University of Aveiro, Fundação para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, COMPETE and FEDER for funding the project PTDC/QUI-BIQ/ 104968/2008, REDE/1504/REM/2005 (that concerns the Portuguese Mass Spectrometry Network), Centre for Environmental and Marine Studies (CESAM) unit (project Pest-C/MAR/LA0017/2013) and the QOPNA research unit (project PEst-C/QUI/UI0062/2013, FCOMP-010124-FEDER-037296). Tânia Melo (SFRH/BD/84691/2012), Eliana Alves (SFRH/BD/41806/2007) and Elisabete Maciel (SFRH/BD/ 73203/2010) are grateful to FCT for doctoral grants.
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