Oxidative Lipidomics of γ-Radiation-Induced Lung Injury: Mass Spectrometric Characterization of Cardiolipin and Phosphatidylserine Peroxidation

Total-Body Irradiation (TBI)

Groups of C57BL/6NHsd female mice were irradiated with 0, 10 or 15 Gy TBI using a J. L. Shepherd Mark 1 Model 68 cesium irradiator at a dose rate of 80 cGy/min. Mice were euthanized 24 h later by CO₂ inhalation. Lungs were perfused, removed, washed in PBS and frozen in liquid nitrogen. All procedures were approved and performed according to the protocols established by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

Isolation of Murine Lung Endothelial Cells (MLECs)

Mice were euthanized by CO₂ inhalation and the chest was opened. Lungs were flushed with HBSS containing 10 U/ml heparin and excised lungs were finely minced and digested in type I collagenase. The mixture was filtered, centrifuged, resuspended and incubated with magnetic beads coated with antibody (rat anti-mouse) to PECAM-1 (BD Pharmingen). Magnetic beads were removed gently by a series of rinses (trypsin/EDTA) and the cells were isolated for subculture. At approximately passage 2, cells were incubated with fluorescently labeled diacetylated LDL (diI-LDL) and sorted to homogeneity by FACS. The enriched PECAM and diI-LDL population was subcultured on a collagen/gelatin matrix in 2% O₂, 5% CO₂, 93% N₂ in a Coy Hypoxic Glove Box/Chamber in Opti-MEM (Gibco), 10% FBS, 2 mM glutamine, 0.2% retinal derived growth factor (Vec Technologies), 10 U/ml heparin, 0.1 mM non-essential amino acid supplement (Gibco), and 55 μM β-mercaptoethanol.

Exposure of MLECs to γ Radiation

Cultured MLECs were plated in full MLEC medium and grown in a hypoxic chamber (2% O₂, 5% CO₂, balance N₂) for 2 days before irradiation. Before irradiation the medium was replaced with OptiMEM + 10% FBS. We used a XRad 160 irradiator (Precision X-Ray, Inc) calibrated with a PTW Unidos E dosimeter TN 30010 Farmer Chamber that allows for irradiation of cells in the same flasks in which they were grown. Irradiation with 15 Gy (160 kVp, 2 mm aluminum, 4.4 Gy/min) was completed within 200 s. During irradiation, flasks with cells (in 2% oxygen) were tightly sealed with parafilm. Thus cells were irradiated in an atmosphere of 2% oxygen. Forty-eight hours after irradiation, cells were trypsinized, washed with PBS, counted and used for assessment of caspase 3/7 activity and lipid analysis.

Caspase 3/7 activity in lung homogenates as well as in MLECs was measured using a luminescence Caspase-Glo™ assay kit obtained from Promega (Madison, WI). Luminescence was determined at time zero and after 1 h incubation at 25°C using an ML1000 luminescence plate reader (Dynatech). Caspase 3 activity was expressed as the luminescence produced within 1 h incubation per μg of protein.

Lipid Extraction and 2D-HPTLC Analysis

Total lipids were extracted from lung homogenates and MLEC using the procedure of Folch et al. (40) with some modifications that allowed highly reproducible quantitative analysis of all lipid fractions, including anionic phospholipids and oxidatively modified phospholipids (41-43). The modified Folch procedure included a 20-fold excess of solvent vs tissue and cells (v/v) and a longer extraction time (overnight at 4°C). After this, an additional re-extraction of phospholipids from the methanol/aqueous phase was performed by adding chloroform (in the amount equal to that of the lower chloroform-enriched phase). We have validated this extraction procedure using different tissue samples and results in the literature (32, 33, 44-48). Almost 99% of phospholipids were recovered by this procedure. Addition of an antioxidant such as butylated hydroxyltoluene (BHT) to samples during lipid extraction interferes with the phospholipid hydroperoxide assay. Therefore, instead of addition of an antioxidant, the procedure of phospholipid extraction and analysis was performed under N₂. The lipidomics analysis was conducted immediately after the extraction. The sufficiency of these preventive strategies was confirmed by our results demonstrating non-random peroxidaton of CL and PS, whereas abundant and polyunsaturated phospholipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) remained non-oxidized and readily detectable in the lung or in lung endothelial cells. Lipid extracts were separated and analyzed by 2D-HPTLC (49). To prevent oxidative modification of phospholipids during separation, plates were treated with methanol containing 1 mM EDTA and 100 μM DTPA prior to application and separation of phospholipids by 2D-HPTLC. To minimize possible auto-oxidation of lipids during migration of lipids on the sorbent, we used chelators of metals, particularly of adventitious iron and copper, known to catalyze peroxidation of lipids. Because EDTA and DTPA are the two most widely used metal chelating agents, we pretreated HPTLC plates with methanol containing 1 mM EDTA and 100 μM DTPA to remove any metals present on the silica surface of HPTLC plates. EDTA binds divalent metals such as Ca²⁺ and Mg²⁺ with stability constants (log Ks) of 10.7 and 13.6, respectively. EDTA also effectively binds transition metals with stability constants (log Ks) of 25.1 and 18.8 for Fe³⁺ and Cu²⁺, respectively. However, these bound metals remain redox-active in the presence of reductants (50, 51). DTPA is a more effective chelator of Fe³⁺ and Cu²⁺with log Ks of 27.5 and 21.2, respectively (52). DTPA complexes of transition metals do not undergo redox cycling. Therefore, we used a combination of EDTA and DTPA to provide the most efficient binding and inhibition of possible redox activity of transition metals on silica plates (51). Total lipids (60 nmol) were applied onto plates under flow of N₂ and the plates were first developed with a solvent system consisting of chloroform:methanol:28% ammonium hydroxide (65:25:5 v/v). After the plates were dried with a forced N₂ blower to remove the solvent, they were developed in the second dimension with a solvent system consisting of chloroform:acetone:methanol:glacial acetic acid:water (50:20:10:10:5 v/v). The phospholipids were visualized by exposure to iodine vapors and identified by comparison with authentic phospholipid standards. For electrospray ionization mass spectrometry (ESI-MS) and analysis of phospholipid hydroperoxides (PL-OOH) by fluorescence HPLC using Amplex Red, the phospholipid spots on the silica plates were visualized by spraying the plates with deionized water. Then the spots were scraped from the silica plates and phospholipids were extracted in chloroform:methanol:water (10:5:1 v/v). Lipid phosphorus was determined by a micro-method (53).

Quantification of Lipid Hydroperoxides

PL-OOH were determined by fluorescence HPLC of resorufin formed in peroxidase-catalyzed reduction of specific PL-OOH with Amplex Red after hydrolysis by porcine pancreatic phospholipase A₂ (1 U/μl) in 25 mM phosphate buffer containing 1.0 mM Ca, 0.5 mM EGTA and 0.5 mM SDS (pH 8.0 at room temperature for 30 min). For the peroxidase reaction, 50 μM Amplex Red and microperoxidase-11 (1.0 μg/μl) were added to the hydrolyzed lipids, and the samples were incubated at 4°C for 40 min. The reaction was started by addition of microperoxidase-11 and terminated by a stop reagent (100 μl of a solution of 10 mM HCl and 4 mM butylated hydroxytoluene in ethanol). The samples were centrifuged at 10,000g for 5 min and the supernatant was used for HPLC analysis. Aliquots (5 μl) were injected into a C-18 reverse-phase column (Eclipse XDB-C18, 5 μm, 150 × 4.6 mm) and eluted using a mobile phase composed of 25 mM KH₂PO₄ (pH 7.0):methanol (60:40 v/v) at a flow rate of 1 ml/min. The resorufin fluorescence was measured at 590 nm after excitation at 560 nm. Shimadzu LC-100AT vp HPLC system equipped with a fluorescence detector (model RF-10Axl) and an autosampler (model SIL-10AD vp) was used (46).

ESI-MS

ESI-MS analysis was performed by direct infusion into linear ion-trap mass spectrometer LXQ™ with the Xcalibur operating system (Thermo Fisher Scientific, San Jose, CA) as described previously (46). Samples collected after 2D-HPTLC separation were evaporated under N₂, resuspended in chloroform:methanol 1:1 v/v (20 pmol/μl), and used for acquisition of negative-ion or positive-ion ESI mass spectra at a flow rate of 5 μl/min. The ESI probe was operated at a voltage differential of 3.5–5.0 kV in the negative- or positive-ion mode. Capillary temperature was maintained at 70 or 150°C. Using full-range zoom (200–2000 m/z) in positive- and negative-ion mode, the profile spectra were acquired. Tandem mass spectrometry (MS/MS analysis) of individual phospholipid species was employed to determine the fatty acid composition. The MS/MS spectra were acquired using an isolation width of 1.0 m/z. Singly charged ions were used for structural identification of CL as described by Hsu and coauthors (54). The scan time setting of ion trap for full MS (range 1400–1600 m/z) was 50 microscans with a maximum injection time of 1000 ms. MSⁿ analysis was performed using an isolation width of 1 m/z and 5 microscans with a maximum injection time of 1000 ms. Two ion activation techniques were used for MS analysis: collision-induced dissociation (CID, Q = 0.25, low mass cutoff at 28% of the precursor m/z) and pulsed-Q dissociation technique (PQD, with Q = 0.7 and no low mass cutoff for analysis of low-molecular-weight fragment ions). Based on the MS fragmentation data, the chemical structures of lipid molecular species were drawn using ChemDraw and confirmed by comparing with the fragmentation patterns presented in the Lipid Map Data Base (www.lipidmaps.org).

LC/ESI-MS of Phospholipids

To quantitatively assess different molecular species of CL and PS, LC/ESI-MS was performed using a Dionex Ultimate™ 3000 HPLC coupled online to ESI and a linear ion-trap mass spectrometer (LXQ Thermo-Fisher). The lipids were separated on a normal-phase column (Luna 3 μm Silica 100A, 150 × 2 mm, Phenomenex, Torrance, CA) with a flow rate of 0.2 ml/min using gradient solvents containing 5 mM CH₃COONH₄ [A, n-hexane:2-propanol:water, 43:57:1 (v/v/v); B, n-hexane:2-propanol:water, 43:57:10 (v/v/v)]. Analysis of phospholipid oxidized molecular species was performed as described previously (46). To account for isotopic interferences, we performed isotopic corrections by entering the chemical composition of each species into the Qual browser of Xcalibur (operating system) and using the simulation of the isotopic distribution to make adjustments for the major peaks. To minimize isotopic interferences between isolated masses M + 2, the MS/MS spectra were acquired using an isolation width of 1.0 m/z.

LC/ESI-MS of Oxygenated Fatty Acids

Oxygenated fatty acids were analyzed by LC/ESI-MS after hydrolysis of major classes of phospholipids with porcine pancreatic phospholipase A₂ (1 U/μl) in 25 mM phosphate buffer containing 1.0 mM Ca, 0.5 mM EGTA and 0.5 mM SDS (pH 8.0 at room temperature for 30 min). Aliquots of extracted lipids (5 μl) were injected into a C-18 reverse-phase column (Luna, 3 μm, 150 × 2 mm) and eluted using gradient solvents (A and B) containing 5 mM CH₃COONH₄ at a flow rate of 0.2 ml/min. Solvent A was 25:30:50:0.1 tetrahydrofuran:methanol:water:CH₃COOH (v/v/v/v). Solvent B was 90:10 methanol:water (v/v). The column was eluted during the first 3 min isocratically at 50% B, from 3 to 23 min with a linear gradient from 50% solvent B to 98% solvent B, then 23–40 min isocratically using 98% solvent B, 40–42 min with a linear gradient from 98% solvent B to 50% solvent B, 42–28 min isocratically using 50% solvent B for equilibration of the column. The hydroperoxy-fatty acids 13S-hydroperoxy-9Z,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid, and 15S-hydroperoxy-5Z,8Z,11Z13E-eicosatetraenoic acid from Cayman chemicals (Ann Arbor, MI) were used as standards.

Statistics

The results are presented as means ± SD from at least three experiments. Statistical analyses were performed with either a paired/unpaired Student’s t test or one-way ANOVA. Statistical significance was set at P < 0.05

Effect of TBI on Phospholipid Composition of Mouse Lung

The effects of radiation (10 Gy and 15 Gy) on the composition of major phospholipids in mouse lung were assessed by 2D-HPTLC (Fig. 1A inset). To quantify phospholipids, the silica spots were scraped off the plates and lipid phosphorus was determined. No significant changes in the composition of phospholipids were detected in mouse lung after TBI (Fig. 1A).

Effect of TBI on the Accumulation of Hydroperoxy Phospholipids in the Mouse Lung

The amounts of PL-OOH in major phospholipid fractions were detected using the HPLC Amplex Red protocol. Two phospholipids, CL and PS, underwent oxidation in the lung after TBI (Fig. 1B). CL-OOH and PS-OOH contents increased after exposure to 10 and 15 Gy compared to the control lung. Radiation-induced increments of CL-OOH and PS-OOH constituted 4.9 and 15.5 pmol/nmol CL and 8.0 and 6.9 pmol/nmol PS at 10 and 15 Gy, respectively. No significant oxidation in the other major phospholipid classes such as PC, PE and phosphatidylinositol (PI) was detected in lungs of irradiated animals.

ESI-MS Analysis of CL and PS Molecular Species of Lung Phospholipids

Molecular species of CL and PS were characterized by ESI-MS using the negative mode. Direct infusion experiments as well as LC/ESI-MS were employed in MS and MS/MS analyses for identification of the molecular species containing polyunsaturated fatty acid residues that are highly susceptible to oxidation. Typical full ESI-MS spectra of lung phospholipids are presented in Fig. 2A. All phospholipids contained highly unsaturated fatty acid residues with two-six double bonds. The results of the identification of phospholipids are presented as a lipid profile map in Fig. 2B.

ESI-MS Analysis of CL Molecular Species of Lung Phospholipids

Given that quantitative assessment of PL-OOH in irradiated lung showed that CL and PS underwent oxidative modification after TBI, we focused on identification of their oxidation products using both direct infusion ESI-MS and LC/ESI-MS. LC/MS analysis of CL molecular species from control lung revealed major CL molecular clusters at m/z 1449.9 and 1473.9 (Figs. 2A and 3A, a). Molecular clusters at m/z 1421.9, 1497.9 and 1521.9 were also detectable in the full MS spectrum but at relatively lower abundances. Fragmentation analysis showed that CL was enriched with molecular species containing linoleic acid (C_18:2). Arachidonic (C_20:4), docosapentaenoic (C_22:5) and docosahexaenoic (C_22:6) acyls were also present in CL molecules but at lower abundances (Fig. 2B).

ESI-MS Analysis of Oxidized CL Molecular Species of Lung Phospholipids

Quantitative assessments of radiation-induced changes in CL revealed a significant reduction of less abundant but more unsaturated species of CL with m/z 1495.9 and 1497.9 (Fig. 3B, a). The amount of these molecular species was decreased 2.1- and 1.7-fold and 2.0- and 1.4-fold, respectively, after TBI with doses of 10 Gy and 15 Gy compared to the control mice (Fig. 3B, a). The amounts of molecular species with m/z 1521.9, 1525.9 were also reduced 1.3- and 3.6-fold and 1.3- and 1.2-fold, respectively, after TBI with doses of 10 Gy and 15 Gy compared to control mice (Fig. 3B, b). No significant changes were found in clusters of less polyunsaturated CL molecules with m/z 1447.9–1451.8 and 1471.9–1479.9.

The loss of “oxidizable” CLs was accompanied by the appearance of their oxygenated species, as illustrated by a typical fragmentation pattern of the CL molecular ion with m/z 1529.9 (Supplementary Fig. 1). The MS/MS analysis showed that this oxidized molecular species originated from the molecular species with m/z 1497.9 (Supplementary Fig. 2) after addition of two oxygen atoms. Molecular ion with m/z 1497.9 was represented by three highly unsaturated molecular species: C_18:2/C_20:4/C_20:4/C_18:1, C_18:2/C_18:2/C_18:2/C_22:5 and C_18:1/C_18:2/C_18:2/C_22:6 (Supplementary Fig. 2). The molecular ion with m/z 1529.9 was identified as peroxidized CL molecular species containing mono-hydroperoxy-C_18:2 (C_18:1/C_18:2-OOH/C_18:2/C_22:6) and two mono-hydroxy-C_20:4 (C_18:2/C_20:4-OH/C_20:4-OH/C_18:1) (Supplementary Fig. 1). Oxidized molecular species of CL with m/z 1527.9, which originated from the molecular ion with m/z 1495.9 after the addition of two oxygens, were also detected (data not shown).

To further quantitatively assess and structurally characterize oxygenated fatty acids in the sn-2 position of oxidized CL species, lipids from control and irradiated lungs were separated by 2D-HPTLC; CL fractions were collected and treated with phospholipase A₂. The fatty acids obtained by hydrolysis were analyzed by LC/ESI-MS. Peaks with retention times of 9.44 min (m/z 311) and 12.28 min (m/z 325) were detected on LC/MS chromatograms of lipids from lungs of irradiated mice (Fig. 4A a, b). MS/MS analysis of the peak with m/z 311 revealed the presence of two overlapping isomers of mono-hydroperoxy-C_18:2 and two overlapping isomers of mono-hydroxy/mono-epoxy-C_18:2 that were identified as 13-hydroperoxy-C_18:2, 9-hydroperoxy-C_18:2 and 13-hydroxy,9-10-epoxy-C_18:2, 9-hydroxy,12-13-epoxy-C_18:2, respectively (Supplementary Fig. 3). Two other oxidation products of C_18:2 (13-oxo-,8-hydroxy-C_18:2; 9-oxo,14-hydroxy-C_18:2) were detected during fragmentation of molecular ions with m/z 325 (Supplementary Fig. 3). Quantitative assessment of oxygenated C_18:2 revealed a significant and dose-dependent increase of mono-hydroperoxy-, mono-hydroxy/mono-epoxy- and mono-hydroperoxy/mono-oxo- molecular species of C_18:2 in irradiated lungs (Fig. 4A, B). Molecular ions with m/z 309 (oxo-C_18:2) and m/z 319 (hydroxy-C_20:4) were also detected on LC chromatograms but at a lower abundance (data not shown). The levels of oxygenation of C_20:4 in the lung CL after TBI were significantly lower than those of C_18:2 and at 15 Gy did not exceed 1.4 pmol/nmol CL. Thus not only were less abundant molecular species of CL containing C_18:2, C_20:4, C_22:5 and C_22:6 fatty acids subject to radiation-induced oxidation but relatively less polyunsaturated C_18:2 fatty acid residues were also preferable oxidation substrates in the mouse lung. This demonstrates the specific, non-random nature of radiation-induced oxidation of CL in the lung.

ESI-MS Analysis of PS and its Oxidized Molecular Species in the Mouse Lung

In the mouse lung, PS was represented by three major molecular clusters with m/z 788.5, 810.5 and 834.5 containing C_18:0, C_18:1, C_20:4, C_22:5 and C_22:6 (Figs. 2A and 5A). A comparative LC/ESI-MS analysis of PS isolated from normal and irradiated lungs demonstrated a significant decrease in the intensity of less abundant molecular ions with m/z 786.5 corresponding to PS-C_18:0/C_18:2 (Fig. 5B). MS/MS analysis of this molecular species revealed the presence of daughter ions at m/z 283 and m/z 279 corresponding to C_18:0 and C_18:2 along with other ions typical for PS fragmentation (Supplementary Fig. 4A). We found that the amounts of PS-C_18:0/C_18:2 were reduced in the lungs of mice exposed to 10 Gy and 15 Gy compared to control lung (Fig. 5C). It is possible that these molecular species were oxidized in the mouse lung after TBI. Detailed analysis of oxidized PS molecular species with m/z 818.5 and 832.5 demonstrated that they originated from a molecular species of PS-C_18:0/C_18:2 (m/z 786.5) after the addition of 2 and 3 oxygens and corresponded to C_18:0/C_18:2+2O and C_18:0/C_18:2+3O, respectively (Supplementary Fig. 4B and C).

Similar to CL, molecular ions with m/z 311 (Fig. 6A, a) and 325 (Fig. 6B, a) were detectable on base-peak LC/MS chromatograms obtained after hydrolysis of PS by phospholipase A₂. MS/MS fragmentation showed that molecular ions with m/z 311 and 325 corresponded to 13-hydroperoxy-C_18:2, 9-hydroperoxy-C_18:2, 13-hydroxy, 9-10-epoxy-C_18:2, 9-hydroxy,12-13-epoxy-C_18:2, and 13- oxo-,8-hydroxy-C_18:2; 9-oxo,14-hydroxy-C_18:2, respectively (data not shown). Radiation caused a significant dose-dependent accumulation of mono-hydroperoxy-, mono-hydroxy/mono-epoxy- and mono-hydroperoxy/mono-oxo- molecular species of C_18:2 (Fig. 6A, b and B, b) in the lung. Small amounts of these oxygenated C_18:2 derivatives were detected in the control lung. No other oxygenated fatty acids were detected.

Radiation-Induced Apoptosis in Mouse Lung

Our previous studies established an association of selective peroxidation of CL and PS with the initiation and progression of apoptosis and clearance of apoptotic cells by professional phagocytes (22, 34). Therefore, we determined whether selective oxidation of CL and PS was accompanied by radiation-induced apoptosis. We found that caspase 3/7 activity was significantly increased in the lungs of mice after TBI compared to control nonirradiated mice (Fig. 7). A 2.5- and 4.5-fold increase in caspase 3/7 activity was detected after the exposure to 10 Gy and 15 Gy, respectively (Fig. 7).

ESI-MS Analysis of Phospholipid Molecular Species in MLECs Exposed to γ Radiation

We performed oxidative lipidomics studies of control MLECs and MLECs 48 h after γ irradiation since pulmonary endothelium is known to be a critical target of radiation-induced lung injury (23). Similar to the results obtained for whole lung, irradiation of MLECs resulted in oxidation of only two phospholipids: CL and PS. Decreased intensity of CL molecular ions with m/z 1423.9, m/z 1427.9, m/z 1447.9 and m/z 1473.9 corresponding to molecular species C_16:1/C_18:2/C_18:2/C_18:1, C_16:0/C_18:2/C_18:1/C_18:1, C_18:2/C_18:2/C_18:2/C_18:2 and C_18:1/C_18:2/C_18:2/C_20:4 was detected in LC/MS spectrum of CL from irradiated cells (Fig. 8A, a, b). In addition, a significant decrease of PS-C_18:0/C_18:2 molecular species was detected in irradiated cells (Fig. 8B, a and b). A 2-fold activation of caspase 3/7 occurred in MLECs 48 h after γ irradiation (Fig. 8C).