Abstract Radiation is a common anticancer therapy for prostate cancer, which transforms tumor-associated normal fibroblasts to myofibroblasts, resulting in fibrosis. Oxidative stress caused by radiation-mediated mitochondrial damage is one of the major contributors to fibrosis. As diabetics are oxidatively stressed, radiation-mediated reactive oxygen species cause severe treatment failure, treatment-related side effects, and significantly reduced survival for diabetic prostate cancer patients as compared to non-diabetic prostate cancer patients. Hyperglycemia and enhanced mitochondrial damage significantly contribute to oxidative damage and disease progression after radiation therapy among diabetic prostate cancer patients. Therefore, reduction of mitochondrial damage in normal prostate fibroblasts after radiation should improve the overall clinical state of diabetic prostate cancer patients. We previously reported that MnTE-2-PyP, a manganese porphyrin, reduces oxidative damage in irradiated hyperglycemic prostate fibroblasts by scavenging superoxide and activating NRF2. In the current study, we have investigated the potential role of MnTE-2-PyP to protect mitochondrial health in irradiated hyperglycemic prostate fibroblasts. This study revealed that hyperglycemia and radiation increased mitochondrial ROS via blocking the mitochondrial electron transport chain, altered mitochondrial dynamics, and reduced mitochondrial biogenesis. Increased mitochondrial damage preceeded an increase in myofibroblast differentiation. MnTE-2-PyP reduced myofibroblast differentiation, improved mitochondrial health by releasing the block on the mitochondrial electron transport chain, enhanced ATP production efficiency, and restored mitochondrial dynamics and metabolism in the irradiated-hyperglycemic prostate fibroblasts. Therefore, we are proposing that one of the mechanisms that MnTE-2-PyP protects prostate fibroblasts from irradiation and hyperglycemia-mediated damage is by protecting the mitochondrial health in diabetic prostate cancer patients. Keywords: Radiation, Diabetes, Manganese porphyrin, Mitochondria, ROS, Fibroblast metabolism Graphical abstract A. In non-stressed conditions, PGC1α and NRF2 transcribe mitochondrial protein to supply new mitochondria in the cell. Oxidative phosphorylation (OXPHOS) produces ATP and physiological levels of ROS through the electron transport chain. Normal metabolic function is carried out by non-stressed mitochondria and there is no fibrosis.B. In hyperglycemia and radiation exposure, PGC1α and NRF2 levels are reduced, resulting in less mitochondrial biogenesis. Mitochondrial OXPHOS complexes are also inhibited, resulting in mitochondria consuming increased amount of oxygen to meet ATP demands and producing increased amount of ROS. PGAM5 was not released into the cytosol, and NRF2 was not localized to outer membrane of mitochondria; therefore, mitophagy was reduced. This accumulation of damaged mitochondria and ROS in the cells results in pathologic metabolism, increased lipid oxidation, and fibrosis.C. In MnTE-2-PyP treated, irradiated and hyperglycemic cells, PGC1α and NRF2 levels are maintained, resulting in increased mitochondrial biogenesis. Mitochondrial OXPHOS complexes are increased, which reduces ROS levels. Mitochondria consume less oxygen and produce higher amount of ATP. PGAM5 is released into the cytosol, and NRF2 localizes to the mitochondrial outer membrane, resulting in mitophagy. The elimination of damaged mitochondria and reduction of ROS, results in normal metabolism, reduced lipid peroxidation, and inhibition of fibrosis. [37]Image 1 [38]Open in a new tab Highlights * • MnTE-2-PyP protects mitochondria from radiation and hyperglycemia-induced stress. * • MnTE-2-PyP reduced mitochondrial ROS by restoring the levels of OXPHOS complexes. * • MnTE-2-PyP increased the number of healthy mitochondria and enhanced ATP production efficiency. * • Mitochondrial protection by MnTE-2-PyP inhibits myofibroblast differentiation. * • MnTE-2-PyP treatment partly restores radiation-mediated metabolic changes. 1. Introduction Radiation therapy (RT) is a common anticancer therapy for prostate cancer (PCa), but it also damages normal tissues surrounding the tumor. RT-mediated normal tissue damage not only causes severe side effects but also significantly contributes to enhanced radioresistant metastatic tumor growth and therapy resistance. More than 15% of all cancer patients are diabetic, which enhances the overall mortality rate and tumor recurrence by 42% and 21% respectively as compared to the non-diabetic cancer patients [[39]1,[40]2]. Specifically, diabetic prostate cancer (PCa) patients have increased therapy resistance, metastasis, and RT-mediated tissue damage, which results in 30% decreased survival in diabetic PCa patients as compared to non-diabetic PCa patients [[41]3,[42]4]. We and others have previously shown that radiation increases prostate tissue fibrosis [[43]5,[44]6], which is a significant cause for therapy resistance [[45]7]. In the irradiated tumor microenvironment, fibroblasts are the major regulators of post-RT fibrosis. Radiation exposure in diabetic PCa patients increases reactive oxygen species (ROS) [[46][8], [47][9], [48][10]], which can result in mitochondrial damage. Damaged mitochondria enhance cellular oxidative stress, which starts a vicious cycle of ROS-mediated tissue damage after RT. Therefore, in a diabetic irradiated environment, protection against mitochondrial damage in prostate fibroblasts may protect normal tissue after RT, maintain healthy metabolism, and increase the survival of the diabetic PCa patients. Radiation increases ROS-induced normal tissue damage via fibrosis, inflammation, and loss of antioxidant defenses and mitochondrial function. It is well reported that MnTE-2-PyP (T2E), a manganese porphyrin, scavenges ROS and acts as a potent radioprotector for normal cells during and after radiation. We have reported that T2E reduces normal tissue fibrosis, inflammation, and antioxidant damage via inhibition of NOX4-TGFβ-mediated pro-fibrotic signaling [[49]11], inhibition of the NFκB-p50- mediated proinflammatory response and upregulation of the NRF2-mediated antioxidant response [[50]12]. Previously, we have reported that T2E protects irradiated hyperglycemic prostate fibroblasts by increasing antioxidant defenses elicited by NRF2 [[51]12], a master transcriptional regulator of antioxidant signaling. T2E increased total NRF2 levels and nuclear localization of NRF2 both in hyperglycemic and normoglycemic conditions after radiation [[52]12]. Although DNA binding of NRF2 was significantly higher after T2E treatment in normoglycemia, it was not in hyperglycemic conditions. Rather, T2E increased AP1/NRF2-mediated secondary antioxidant response in hyperglycemic conditions by increasing DNA binding of AP1 [[53]12]. T2E increased cytoprotective effects against radiation both in normoglycemic and hyperglycemic conditions. Therefore, we hypothesized that DNA binding of NRF2 was not the only mechanism for T2E-mediated cytoprotection in hyperglycemia. One of the major cytoprotective roles of NRF2, besides transcriptional regulation of cytoprotective genes, is maintaining mitochondrial health. NRF2 is well reported as a vital regulator for ROS mediated mitochondrial damage protection [[54]13,[55]14]. NRF2 localization to the outer mitochondrial membrane was previously reported [[56]15,[57]16]. PGC1α, an important regulatory molecule for mitochondrial biogenesis [[58]17], also protects cells through NRF2 activation [[59]18]. Therefore, to determine the potential of T2E as a mitochondrial protector, we investigated mitochondrial health, function, and cellular metabolism in irradiated hyperglycemic prostate fibroblasts in the presence or absence of T2E in our current study. To mimic a diabetic irradiated environment, we have irradiated human prostate fibroblasts in a hyperglycemic environment in the presence or absence of T2E. This study revealed that in both radiated and non-radiated conditions, T2E increased mitochondrial NRF2 levels and total expression levels of PGC1α irrespective of glycemic levels. However, when NRF2 was knocked down, T2E was still able to protect mitochondrial health, which suggests that NRF2 is not directly involved in T2E-mediated mitochondrial protection. T2E increased mitochondrial ATP synthase levels, total mitochondrial number, and healthy mitochondrial number in the irradiated hyperglycemic cells. Mitochondrial efficiency, as measured by ATP production, was increased after T2E treatment. In addition, there was a large increase in mitochondrial ROS in irradiated and hyperglycemic fibroblasts, which was reduced significantly by T2E treatment. These results indicate that T2E protects from radiation and hyperglycemia-mediated mitochondrial damage. This study also revealed that mitochondrial damage preceeds the transformation of fibroblasts to myofibroblasts, which was prevented by T2E, in an irradiated, hyperglycemic environment. Thus, another way T2E inhibits fibrosis is by protecting mitochondrial health of fibroblasts. Finally, we have demonstrated the alterations of the metabolites, especially lipid changes, due to mitochondrial damage in irradiated and hyperglycemic fibroblasts. Specifically, radiation and hyperglycemia increased lipid oxidation, which was reduced to normal levels by T2E treatment. This is the first study to report that T2E restores healthy mitochondrial function and metabolic profiles in irradiated and hyperglycemic fibroblasts. 2. Materials and methods 2.1. Cell culture P3158 are normal human prostate fibroblasts that were immortalized by pBABE-hygro-hTERT plasmid (Addgene, plasmid cat #1773) and kindly gifted by Dr. J Tyson McDonald. RPMI-1640 (Hyclone, cat #: SH30027.01) media, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin were used to culture P3158 cells. Cells cultured in this media (containing 11.1 mM or 200 mg/dL glucose) are referred to as control or normo-glycemic condition in this study. To mimic hyperglycemia in diabetics, an extra 20 mM or 360 mg/dL glucose were added in the media, where the final glucose concentration was 560 (200 + 360) mg/dL. This condition is referred to as high glucose (HG) condition or hyperglycemia in this study. We have reported that the addition of 20 mM glucose was enough to induce hyperglycemia-associated oxidative stress without affecting osmotic pressure in P3158 cells [[60]12]. In the presence or absence of 20 mM glucose (for HG) and/or 30 μM MnTE-2-PyP (referred as T2E), P3158 cells were plated. After 24 h, half of the cells were irradiated with 3 Gy of X-ray using the Rad Source RS-2000 irradiator (referred as RAD). Our previous study showed that 3 Gy of radiation on the human prostate fibroblasts (P3158) increased ROS and decreases NRF2 activity, which was recovered by MnTE-2-PyP in normoglycemic and hyperglycemic conditions [[61]12]. Therefore, to investigate MnTE-2-PyP-mediated mitochondrial protection in irradiated and hyperglycemic prostate fibroblasts, we have selected 3 Gy of radiation. Cells were then incubated in 37 °C with 5% CO[2]. All the assays were executed on the fifth day after radiation exposure, if not otherwise mentioned. There were eight groups for each study: 1. Control (CON), 2. HG, 3. T2E, 4. T2E + HG (TG), 5. RAD, 6. RAD + HG (RG), 7. RAD + T2E (RT) and 8. RAD + HG + T2E (RGT), if not mentioned otherwise. 2.2. Western blot Whole cell, cytosolic, and mitochondrial protein extracts were prepared by plating 2.0 × 10^6 cells/T75 flask. Cells were treated with HG, T2E and RAD as mentioned above. Five days post-radiation, whole cell extracts, cytosolic and mitochondrial extracts were prepared. Cell pellets were lysed by cell lysis buffer [120 mM NaCl, 50 mM Tris- HCl, 5 mM EDTA, 1% NP-40 and complete protease inhibitor cocktail tablets (Roche, cat # 11697498001; 1 tablet/50 ml)] to prepare the whole cell extract. Mitochondrial and cytosolic extracts were prepared by following the manufacturer's instructions for the Mitochondria Isolation Kit for Mammalian Cells (Thermo Scientific, reference # 89874). Total protein concentrations were measured by Bradford reagent (Bio Rad, cat # 500-0006). Samples were electrophoresed on Bolt 4–12% Bis-Tris Plus gels (Thermo Fisher Scientific, cat # NW04120BOX) and transferred to nitrocellulose membranes (Life Technologies, cat # IB23002). Nonfat milk (5%) in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) was used to block the membrane for 2 h before incubation of the membrane with primary antibodies overnight. Membranes were then washed with TBST and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (1:10,000 dilution) for 1–2 h at room temperature. Blots were then developed with an ECL detection system (Thermo Fisher Scientific) and exposed to film after washing with TBST. ImageJ was used to analyze densitometry of the scanned images from the films. Ponceau staining of total proteins were performed as a loading control for each blot. In some cases, TOMM20 was used as a mitochondrial protein loading control. We have used the following primary antibodies in this study: α-SMA (Abcam, cat # ab5694, 1:3000 dilution), NRF2 (Abcam, cat # ab62352, 1:1000 dilution), PGC1α (Abcam, cat # ab54481, 1:1000 dilution), TOMM20 (Abcam, cat # ab56783, 1:1000 dilution), OXPHOS complexes cocktail (Abcam, cat # ab110413, 1:1000 dilution), DRP1 (Cell Signaling Technology, cat # 8570, 1:1000 dilution), MFN1 (Cell Signaling Technology, cat # 14739, 1:1000 dilution), MFN2 (Cell Signaling Technology, cat # 11925, 1:1000 dilution), OPA1 (Cell Signaling Technology, cat # 80471, 1:1000 dilution), PGAM5 (Abcam, cat # ab244218, 1:1000 dilution), and PINK1 (Cell Signaling Technology, cat # 6946, 1:1000 dilution). Secondary goat anti-rabbit antibody (Invitrogen, reference # A24537) and secondary goat anti-mouse antibody (Invitrogen, reference # A24524) were used with 1: 10,000 dilutions. 2.3. Immunofluorescence staining Cells were treated with HG, T2E and RAD and were incubated for five days post-radiation. Cells were then fixed in formalin for 10 min after washing with PBS once. Then cells were washed thrice with PBS and permeabilized with 0.5% Triton X in PBS for 8 min. Cells were blocked with 5% goat serum in PBS for 1 h after washing thrice with PBS. Then the primary antibodies were applied for 2 h at room temperature followed by washing with PBS. A fluorescence tagged secondary antibody was applied for 1 h at room temperature in the dark. Finally, cells were washed with PBS, mounted with ProLong Gold antifade reagent with DAPI (Invitrogen, cat #[62]P36931), coverslipped, and imaged using a Leica DM4000 B LED microscope. Mitochondria were stained with 150 nM of Mitotracker Red CMXRos (Invitrogen, Molecular probes, Cat #M7512) in serum free media for 20 min at 37 °C in 5% CO[2], in the dark, followed by washing with Hank's balanced salt solution (HBSS) buffer three times. For staining with another antibody along with Mitotracker Red CMXRos, the above-mentioned steps for fixation, permeabilization, blocking and immunostaining were followed after Mitotracker staining. Antibodies used for these experiments are as follows: ATP synthase Beta (Thermo Fisher Scientific, cat # A-21351, 1:250 dilution), NRF2 (Abcam, cat # ab62352, 1:250 dilution), α-SMA (Abcam, cat # ab5694, 1:500 dilution), Alexa fluor 488 goat anti-rabbit fluorescence secondary antibody (Life technology, reference # A11008, 1:500 dilution) and Alexa fluor 524 goat anti-rabbit fluorescence secondary antibody (Life technology, reference # A11008, 1:500 dilution). 2.4. Mitochondrial ROS measurement After treatment with HG, T2E and RAD, cells were incubated for five days after radiation, as indicated in the previous section. Then cells were trypsinized and washed with HBSS (with Ca^2+, Mg^2+) buffer. Cells were then incubated in the dark with 5 μM of MitoSOX Red (Invitrogen, Molecular probes, cat #[63]M36008, excitation/emission wavelength 510/580 nm) for 10 min at 37 °C in 5% CO[2]. After washing with HBSS (with Ca^2+, Mg^2+) buffer, cells were subjected to flow cytometry using a LSRII Green 532 Flow Cytometer (BD Biosciences, San Jose, CA) using the 488 excitation laser and a 575/26 with 550 long pass emission filter. 2.5. Mitochondrial oxygen consumption rate determination Cells (0.3 × 10^6) were plated in a T25 flask and treated with HG, T2E and RAD as mentioned before. Twenty-four hours after radiation, cells were trypsinized and reseeded on XFe96 seahorse assay cell culture plate at a density of 0.3 × 10 -^5 cells/well. The total media volume was 200 μl/well. HG and T2E were added in proper concentrations after reseeding. After 48 h of reseeding, cells were washed and replaced with fresh Mitostress assay medium. After 1 h incubation in a non-CO[2] incubator, mitochondrial oxygen consumption rate (OCR) was measured using the XFe96 analyzer (Seahorse Biosciences, Santa Clara, CA, USA). Oligomycin, carbonyl cyanide-4-phenyl hydrazone (FCCP), and Rotenone/Antimycin were used to measure basal, ATP linked, maximal, non-mitochondrial OCR and spare capacity. OCR data was normalized to total protein content of the cells, measured by Bradford reagent. 2.6. Measurement of ATP levels P3158 cells were treated with HG, RAD and T2E as previously described. After 5 days of radiation treatment, cells were trypsinized and counted. Sterile deionized water (1 ml), pre-heated to 135 °C, was added to 2.0 × 10^5 cells, and incubated in boiling water for 3 min. The cell suspension was then centrifuged at 12,000 g for 7 min at 4 °C. The supernatant (20 μl) was added to 180 μl of the ATP measurement reaction buffer, prepared by following the manufacture's protocol for ATP determination kit (Invitrogen Molecular probes, cat # A22066). Luminescence was measured to determine relative levels of ATP in experimental groups by using a Tecan Infinite M200 Pro plate reader. Deionized water (20 μl) was used to determine the background luminescence. Three technical replicates were used to read the luminescence for each sample for every biological replicate. 2.7. Measurement of total and healthy mitochondria levels Cells were treated with HG, T2E and RAD as described above. After 5 days post-radiation, cells were trypsinized and washed with PBS once. Cells were treated with 100 nM of both Mitotracker Red CMXRos (Invitrogen, Molecular probes, cat #M7512) and Mitotracker Green (Invitrogen, Molecular probes, cat #M7514) in serum free media for 20 min at 37 °C in 5% CO[2] in the dark, followed by washing with HBSS buffer three times. Cells were subjected to flow cytometry using a LSRII Green 532 Flow Cytometer (BD Biosciences, San Jose, CA) using the Y/G 605/15 laser for Mitotracker Red CMXRos (MTR) and blue 530/30 laser for Mitotracker Green (MTG). We have used carbonyl cyanide m-chlorophenylhydrazone (CCCP) as a positive control for altering mitochondrial membrane potential. We treated P3158 cells with 10 μM CCCP for 1 h before staining the cells with Mitotracker green and red. 2.7.1. siRNA treatment P3158 cells (75,000 cells/well) were seeded in a 6 well plate and transfected with 25 nM of control siRNA (Ambion, cat # 4390843) or NRF2 siRNA (Ambion, cat# 4392420). After 24 h, cells were treated with HG (20 mM) and T2E (30 μM) and incubated for another 24 h followed by irradiation (3 Gy). Cells were harvested and whole cell lysates were prepared for western blot analysis 4 days post-radiation. 2.8. Metabolite extraction protocol P3158 cells (1 × 10^6) were plated in 100 mm dishes and treated with HG, T2E and RAD, as described above. Six biological replicates for each experimental condition were collected 5 days post-radiation for the metabolomics study. The cells were washed with 1 ml of Nanopure water followed by pipet mixing and slow speed centrifugation. The cells were then centrifuged at 5000 g for 10 min at 4 ^°C and the cell wash was discarded. Quality control (QC) samples were prepared by combining equal amounts from each biological replicate. The same extraction procedure was applied to the QC and biological replicate samples. Cells were resuspended in 1 ml of 80% methanol and submitted to mechanical lysis with zirconia beads in a FastPrep® homogenizer (15 s at 1200 rpm repeated three times). The samples were placed in an ice bath for 30 s between cycles. The lysed sample was then centrifuged at 20,000 g for 20 min at 4 °C. The supernatant was collected and 1 ml of 50% methanol and 50% Nanopure water (v:v) was added to each cell pellet and vortexed for 10 s. The sample was centrifuged, the supernatant was collected and then combined with the previous supernatant. Each fibroblast cell sample was split 60:40 to prepare an NMR and mass spectrometry (MS) sample, respectively. Each sample was transferred to a rotary evaporator and then lyophilized to dryness. After extraction of the aqueous metabolites, 4 ml of 2:1:1 chloroform: methanol: water mixture was added to each cell pellet in a glass vial. The pellet was then vortexed for 30 s and centrifuged at 20,000 g for 20 min at 4 °C and the organic layer was collected. The process was repeated a total of three times and the organic layers were combined. Each sample was lyophilized to dryness on a rotary evaporator. The organic metabolome extraction was only used to prepare a MS sample. 2.9. Preparation of metabolomics samples for nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis After lyophilization, each dried aqueous cell extraction allocated for NMR analysis was resuspended in 0.3 ml of a 50 mM potassium phosphate buffer at pH 7.2 (uncorrected) in “100%” D[2]O containing 50 μM of 3-(trimethylsilyl) propionic-D4 (TMSP-D[4]) as a chemical shift reference. The sample was then transferred to a 3 mm NMR tube for data collection. After lyophilization, each dried aqueous cell extract allocated for MS analysis was resuspended in 100 μl of Nanopure water containing 0.1% of formic acid. After lyophilization, each dried organic cell extract was resuspended in 100 μl of isopropanol containing 0.1% formic acid. 2.10. One-dimensional (1D) ^1H NMR data acquisition 1D ^1H NMR spectra were collected on a Bruker Avance III-HD 700 MHz spectrometer equipped with a quadruple resonance QCI-P cryoprobe (^1H, ^13C, ^15N, ^31P) with z-axis gradients. A Bruker SampleJet sample changer with IconNMR and auto tune and match (ATM) were used to automate the NMR data collection. The 1D ^1H NMR data were collected at 298K with 32K data points, a spectrum width of 11 ppm, 64 scans and 4 dummy scans. The spectra were collected using excitation sculpting [[64]19] to remove the solvent and maintain a flat baseline. 2.11. Liquid chromatography-mass spectrometry (LC-MS) data acquisition LC-MS metabolomics was performed on a Waters Acquity ultra performance liquid chromatography (UPLC) system coupled to a Xevo G2-XS Q-TOF (Waters MS Technologies, Manchester, UK) equipped with an electrospray ionization (ESI) source operating in positive ionization mode. The metabolites were separated with an HSS T3 column (Waters, 1.0 mm × 50 mm, 1.8 μm) with a 31-min linear gradient from 0.1% to 85% mobile phase B. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The column and autosampler temperatures were set to 40 °C and 5 °C, respectively. The flow rate was set to 95 μl/min. The ionization source condition was set as follows: capillary voltage of 3.2 kV, sampling cone voltage of 40 V, and source offset voltage of 80 V. The source temperature was set to 120 °C and the desolvation temperature was set to 500 °C. The cone and desolvation gas flows were set to 50 and 800 L/h, respectively. Data acquisition was obtained in the MS^E mode, which simultaneously records exact mass precursor ion and fragment ion information. MS^E was performed with a low collision energy of 4 eV and a high collision energy ramped from 15 to 50 eV. The data was collected using an m/z range of 50 to 1200 with a scan time of 0.05 s. The data were acquired using an independent reference lock mass via the LockSpray interface to ensure accuracy and reproducibility during the MS analysis. Leucine Enkephalin was used as the reference compound ([M+H]^+ = 556.2771). LC-MS lipidomics was performed on a Waters Acquity UPLC system coupled to a Xevo G2-XS Q-TOF (Waters MS Technologies, Manchester, UK) equipped with an ESI source operating in either the positive or negative ionization mode. The lipids were separated on a C18 column (1.0 × 50 mm, 1.7 μm, Waters) with a 20-min linear gradient from 40% to 99% mobile phase B. Mobile phase A was an acetonitrile and water mixture (60:40, v/v) containing 10 mM ammonium formate and 0.1% formic acid. Mobile phase B was an isopropanol and acetonitrile mixture (90:10, v/v) containing 10 mM ammonium formate and 0.1% formic acid. The column and autosampler temperatures were set to 40 °C and 5 °C, respectively. The flow rate was set to 50 μl/min. The ionization source condition was set as follows: capillary voltage of 3.2 kV, sampling cone voltage of 40 V, and a source offset voltage of 80 V. The source and desolvation temperatures were set to 120 °C and 500 °C, respectively. The cone and desolvation gas flows were set to 50 and 800 L/h, respectively. MS^E was performed with a low collision energy of 4 eV, and the high collision energy was ramped from 15 to 40 eV. The data was collected using an m/z range of 100–2000, a scan time of 0.05 s, and an independent reference lock mass via the LockSpray interface to ensure accuracy and reproducibility during the MS analysis. Leucine Enkephalin was used as the reference compound ([M+H]^+ = 556.2771, [M − H]^-=554.2615). The EquiSplash® Lipidomics® mass spectrometry standards (Avanti polar lipids, Inc.) were used as a quality-control standard before collecting the experimental lipidomics samples. 2.12. NMR data processing and statistical analysis The 1D ^1H NMR spectra were processed with our MVAPACK metabolomics toolkit ([65]http://bionmr.unl.edu/mvapack.php) to generate a data matrix [[66]20]. The spectra were processed with a 1.0 Hz exponential apodization function, a single round of zero-filling, and a Fourier transformation. Spectra was referenced and aligned to TMSP-D[4] [[67]20]. Solvent signals and noise regions were excluded, and the spectra were either aligned using the Icoshift [[68]20] peak alignment algorithm or binned using an adaptive intelligent binning algorithm [[69]20]. The 1D ^1H NMR data matrix was pareto scaled and normalized with the median in order to generate a normal gaussian distribution using Metaboanalyst 5.0. The orthogonal projection to latent structures discriminant analysis (OPLS-DA) models was validated using permutation testing with n = 1000, R^2, Q^2, p-values were generated using Metaboanalyst 5.0 or MVAPACK. Discriminatory features were identified using Chenomx Suite 8.0 and Human Metabolome database (HMDB). A ^1H chemical shift uncertainty of 0.08 ppm was used to match experimental chemical shifts. 2.13. LC-MS data processing and statistical analysis The LC-MS datasets were imported into the Progenesis® QI metabolomics software (version 2.4, Nonlinear Dynamics, Newcastle, UK). The chromatographic peak alignment and peak picking were performed in an automatic manner with QC runs as references. Features were