SUPPLEMENTAL DIGITAL CONTENT 1, METHODS
Materials. All chemicals, unless otherwise specified, were purchased from Sigma Chemical Corporation (St. Louis, MO). Solutions and buffers were prepared from sterile water for human injection or normal saline (NS) (Baxter Healthcare Corp, Deerfield, NY) for intravenous administration, followed by sterile-filtration by NalgeneTM MF75 series disposable sterilization filter units from Fisher Scientific (Hampton, NH). Propionylcarnitine, S- adenosylhomocysteine, and glycerol-3-phosphate were purchased from Cayman Chemical (Ann Arbor, MI). N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2- oxoethyl]]-, bis[(acetyloxy)methyl] ester (BAPTA-AM) was purchased from Molecular Probes (Eugene, OR). 3-[1-[3-(Dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)- 1H-pyrrole-2,5-dione (Gö 6983) was purchased from Enzo Life Sciences (Farmingdale, NY).
Rabbit anti-rat PMN antibody and the isotype control (Accurate Chemical, Westbury, NY) were prepared using 2:1 dilution with NS and sterile filtration with 0.2 μM filters. PE50 tubing was obtained from Fisher Scientific.
ARDS post-injury. From April 2014 to March 2017 144 patients were enrolled.1 Eligibility was determined at the injury scene. The inclusion criteria were injured adults (age
>18 years), with either a systolic blood pressure (SBP) ≤70 mmHg or 71-90 mmHg with heart rate ≥108 bpm presumably due to acute blood loss, based upon a Resuscitation Outcomes Consortium’s similarly structured trial.2 Exclusion criteria were known prisoner status, known pregnancy, isolated gunshot to the head with GCS<5, field asystole/cardiopulmonary resuscitation prior to randomization, known objection to blood products, opt-out bracelet or necklace, or family objection to patients’ enrollment.1 The trial, which investigated the potential benefit of transfusion of fresh frozen plasma initiated in the field, was stopped due to futility to
produce a difference in 28-day mortality following enrollment of 144 out of the 150 planned population size. There were no statistical differences between the groups in safety outcomes and adverse events with the mortality in both the plasma-transfused and the saline-infused controls
≥15%.1
Platelet-poor plasma samples (centrifuged first at 5,000g for 10 min at 4oC followed by a spin at 12,600g for 6 min at 4oC to remove platelets and acellular debris, aliquoted, snap frozen, and stored at -80oC) from 67 critically injured patients (ISS >15) enrolled in the COMBAT study at the Ernest E Moore Shock Trauma Center, DHMC were analyzed. Clinical data collected are in Table 1. These plasma samples were from whole blood collected in the field (76%) or in the emergency room within 4 hours post-arrival (24%). PaO2/FiO2 (PaO2: partial pressure of oxygen in arterial circulation (mm Hg)/FiO2, fraction of inspired oxygen (%)) ratios were calculated and corrected for altitude (Denver, CO: 5,280 feet using the following equation P/F * (measured barometric pressure mmHg/760 mmHg).3 The median barometric pressure in Denver over the study interval was 667 mm Hg; thus, as an example in Denver mild ARDS <263, moderate ARDS <175, and severe <89, rather than <300, <200, and <100 at sea level, respectively.3 We excluded ABG data within 8 hours of admission to eliminate patients undergoing operative intervention or active resuscitation. Patients were then stratified by the presence or absence of ARDS using the Berlin Criteria.3 Ventilator free days (VFDs) were calculated as described,4 If there were >5% missing values for critical variables, these patients were excluded.
Untargeted Metabolomics. Citrated whole blood was collected, the plasma isolated, and metabolomics analyses completed as previously described.5 In brief, whole blood was collected in sodium citrate, platelet free plasma was obtained by sequential centrifugation, aliquotted, snap-frozen, and stored at -80°C. Metabolites were extracted as described, and the supernatant
was analyzed on a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive high resolution mass spectrometer. Sample preparation and data acquisition and analysis were performed as previously described.5,6 Metabolites were assigned and peak areas integrated using Maven (Princeton University) in conjunction with the KEGG database. A list of metabolites identified appears in Table 2.
Metabolite Quantification. Metabolomic analyses were performed with absolute quantification using spiked-in stable isotope-labeled standards for 2/4 metabolites that significantly changed in the initial patient plasma (spiked at concentrations as previously described).5,6 Absolute concentration measurements were determined using the ratio of unlabeled (endogenous) metabolite to labeled standard according to the following equation:
[light] = (peak area light)/(peak area standard)*[standard]*DF
where DF = dilution factor, in this case 25 (1 part plasma and 24 parts extraction buffer) This manually selected, targeted strategy was completed for metabolites involved in energy and redox metabolism, which were validated against multiple quality control measures and quantified by spike-in stable isotope-labeled internal standards.5,6 From the many thousands of metabolites 138 were chosen and appear in Supplemental Digital Content 3, Table. Spermine and spermidine were not measured in these patients; however, a previous data set of 500 patients has direct measurements of these from the first sample in the Emergency Department.7 These data guided the concentrations for testing these metabolites (Table 3).
PMN Isolation and Priming of the Oxidase. PMNs were isolated from heparinized whole blood as described 8. Isolated PMNs were incubated with NS or metabolites of interest at a range of concentrations (Table 3) for 5 minutes (pH 7.35), followed by activation with 1M N-formyl-
Met-Leu-Phe (fMLF).8 The maximal rate of O2- production was measured as previously reported.8
Succinate Receptor Expression. PMNs were incubated with NS or succinate (500 and- 1000 M) for 1 or 5 minutes, fixed with 4% paraformaldehyde, and smeared onto slides. The slides were washed, fixed, permeabilized PMNs, air dried, and blocked with 10% normal donkey serum in PBS. The slides were then incubated with rabbit polyclonal anti-GPR91 (Novus Biological, Littleton, CO), with a rabbit isotypic antibody control at equal concentrations to control for nonspecific binding (Santa Cruz Biotech, Santa Cruz, CA), and then incubated with a species-specific fluorescent antibody to Alexa-Fluor-555. Alexa Fluor-633 conjugated wheat germ agglutinin (WGA) (Life Technologies, Carlsbad, CA) was used to identify membrane glycoproteins and DAPI (Prolong Gold Antifade Mount with DAPI, Life Technology, Carlsbad, CA) to stain the nuclei.9-12 Images were obtained with a Zeiss Axio Observer Z1 microscope using Chroma Multiple Bandpass filter wheel controlled by Slidebook v 6.0 (Intelligent Imaging Innovations, Denver CO).9-12 All images were captured at 100x magnification (n=3). GPR91 mean fluorescence intensity was measured for each image by a blinded observer and compared between groups.
GPR91 Inhibitor Synthesis. An inhibitor to GPR91, GPR91-2c, was synthesized by the Peptide and protein Chemistry Core at the University of Colorado School of Medicine, Anschutz Medical Campus.13 The compound was HPLC purified and 16.9 mg 2c-hydrochloride compound (C26H25N3O.HCl: 395.5+36.46 = 431.96) was lyophilized from 3 ml of acetonitrile:
water = 1:1. This HCl salt is hygroscopic and soluble in NS with higher concentrations soluble in DMSO. The GPR91-2c inhibitor was solubilized in DMSO with further dilutions in NS (DMSO-NS) or 1.25% fatty acid free human serum albumin (HSA), with DMSO-NS or DMSO-
HSA used as the vehicle controls for all in vitro and in vivo studies.
Inhibition of Succinate-induced PMN priming. PMNs (n=7) were pre-incubated with vehicle control (0.1% DMSO in NS) or the GPR91-2c inhibitor (30 nM) for 5 minutes. These PMNs were then primed with DMSO/NS or 500 µM succinate for 5 minutes, activated by 1 M fMLF, and the maximal rate of O2- production was measured.8
Inhibition of Calcium- and PKC-Mediated Signaling. PMNs were pre-incubated with DMSO, the intracellular Ca2+ chelator BAPTA-AM [50 M], or the classical PKC inhibitor Gö6983 [50 nM] for 10 min as described.14,15 PMNs were then primed with NS or 1000 M succinate for 5 min, activated by 1 M fMLF, and the maximal rate of O2- production was measured.8,14,15
Two-event in vivo model of ALI. Animal experiments were performed under a protocol
approved by the University of Colorado Denver Institute for Animal Care and Use Committee and maintained in the accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats underwent an established two-event in-vivo model of ARDS.9,16 Briefly, depending on the model, rats (330-430g) were given an IP injection of LPS (Salmonella enteritides, 2 mg/kg) 2 hours prior to being anesthetized with sodium Pentobarbital and the femoral artery and vein cannulated. The second event was then given consisting of an infusion of NS (control) or succinate [25-50 µM]FINAL via the femoral vein followed by an infusion of Evans Blue Dye (EBD) (30 mg/kg) IV.9,16,17 When the events were reversed, the rats were anesthetized and given IV succinate 25-500 µM. After 6 hours of recovery, rats were transfused with LPS (100 g/kg, S.enteritides) or an equivalent volume of NS as the second event followed by EBD (30 mg/kg) IV.18 After an additional 2 hours, blood was drawn and a BAL performed as described.9,16 Blood and BAL fluid (BALF) were spun down
(5000g for 10 min at 4°C), and the cell-free supernatants stored at -80°C. Lung leak was measured as the % BALF EBD vs. the % plasma EBD.9,10,16,18
Histology and PMN sequestration. In selected experiments the histological changes were
identified using the identical two-event rat model (n=3-5 per group) without EBD or a BAL.
After completion of the two-event model and euthanasia, a median sternotomy was performed, the left lung was inflated with 1.5 mL of optimal cutting temperature (OCT) compound in 4%
sucrose, and the lungs were embedded in OCT and frozen. Sections were completed, stained with hematoxylin and eosin and read at 40X by a blinded observer.9,10,18 For evaluation of PMN sequestration animals underwent only the first event (NS or succinate), euthanized after 6 hours, and the pulmonary circulation flushed with PBS to remove non-adherent PMNs. Following clamping of the right main stem bronchus, the right lung was removed to measure myeloperoxidase (MPO) activity, while the left lung was inflated with OCT and sectioned as described above. Lung sections were fixed and permeabilized, blocked with 10% normal donkey serum in PBS, incubated with a rabbit antibody specific for rat PMNs in PBS with 1% BSA with negative controls consisting of normal rabbit serum (1:2450).9,10 Sections were incubated with species-specific fluorescent secondary antibodies (Alexa Fluor-555, Life Technologies, Carlsbad CA). Membranes were visualized with Alexa Fluor-488 conjugated WGA, and nuclei were stained with DAPI.9-12 Images were obtained and processed as described with all images were captured at 40X and deconvolved.9-12 In each image, the total PMN surface and total lung tissue surface were measured (area in square microns); the ratio of PMN/tissue was then calculated.
PMN Depletion. Rats were injected IP with a rabbit anti-rat PMN-antibody or an IgG isotypic control (400 µL of Ab/100g of rat weight, Accurate Chemical, Westbury, NY) 36 hours prior to experimentation.9,10 Heparinized blood was obtained from both treated and control rats
prior to the succinate infusion, smeared onto slides and stained with a modified Wright’s stain.9,10 Leukocyte differentials were completed by a blinded hematopathologist to ensure PMN depletion. Rats then underwent the described two-event model.
Inhibition of GPR91 in a Two-Event Model. Rats were infused with the GPR91 inhibitor (2c) ([30 nM] in 1.25% human serum albumin (HSA)) prior to the two-event model, described above, with 50-500 M succinate/LPS (n=6-7 per group) or the vehicle control (HSA). An infusion of vehicle control or inhibitor was started 15 minutes prior to the first event and continued for an additional 45 minutes to maintain a blood concentration of 30 nM.
Measurement of CINC- in the BALF and Lung MPO. Chemokine-induced neutrophil chemoattractant (CINC-1) was measured in the BALF using a commercial ELISA (R&D Systems, Minneapolis, MN). MPO activity in homogenized lungs was measured by reduction of o-dianisidine at 490 nM, as previously described.9
Measurement of Liver and Kidney Injury. Cell free plasma was obtained as described
and was assayed for Alanine Aminotransferase as a marker of liver injury. Plasma was assayed with the Vitros 5600 (Orthoclinical, Rochester, NY) per the manufacturer’s instructions.19 The urinary bladder was aspirated with a 25-gauge needle at completion of the experiment. The urine was flash frozen, and urinary neutrophil gelatinase-associated lipocalin (NGAL) was measured with a commercial ELISA (Abcam, Cambridge, MA).
Statistics. The metabolite data are not normally distributed and are expressed as the
median (interquartile range). Statistical differences were initially identified employing Mann- Whitney t-tests for non-parametric data between the ARDS cohort and the non-ARDS cohort with GraphPad (Version 7) or GB Stat (Version 8). Moreover, to identify potential confounders the analysis was done with and without exclusion of patients with underlying chronic lung
disease (n=3), severe TBI (defined as head AIS>3, n=9), and severe chest injury (defined as chest AIS>3, n=10). The statistical significance was initially set at a p<0.05 between groups and a ≥1.5-fold change for metabolites. Categorical variables were compared using Fisher’s exact test.1,20,21 Metabolomic analyses were performed with absolute quantification for 2/4 metabolites that significantly changed in the initial patient plasma.22
Normally distributed data from the in vitro and in vivo experiments described in this section and those that follow are reported as mean ± standard error of mean and compared for significance using a paired (in vitro) or nonparametric (in vivo) one-way analysis of variance (ANOVA) with post-hoc Tukey’s or Dunn’s comparisons depending upon the equality of variance in GraphPad (Version 7.0) or GB-Stat (Version 8).
Study Approval. The Control of Major Bleeding After Trauma (COMBAT) study was a pragmatic, randomized, placebo-controlled, clinical trial based at Denver Health Medical Center and was registered at ClinicalTrials.gov as NCT01838863.1 The experimental design and methods have been described.23,24 This protocol required exception from informed consent for emergency research, which was approved by the local institutional review board (COMIRB #12- 1349) and was conducted under FDA regulation (IND#15216) and monitored by the Department of Defense’s Human Research Protection Office (HRPO).24 An independent data and safety monitoring board (DSMB) oversaw the trial conduct and reviewed all suspected adverse events and the interim analyses.1
The trauma activation protocol (TAP) database is a prospective assessment of trauma- induced coagulopathy in all patients meeting criteria for trauma activation, the highest trauma designation, at the level 1 designated Rocky Mountain Regional Trauma Center at Denver Health Medical Center (DHMC).7 The TAP study was approved by the COMIRB (COMIRB#13-3087)
and completed under waiver of consent for emergency research. To date it consists of >800 patients, and plasma is obtained from each at presentation to the ED.7 Absolute quantification of spermine and spermidine was completed from the initial plasma samples from these patients.
Lastly, healthy human volunteers were consented under a protocol approved by the Colorado Multi-Institutional Review Board (COMIRB protocol # 12-1614).
References
1. Moore HB, Moore EE, Chapman MP et al. Plasma-first resuscitation to treat haemorrhagic shock during emergency ground transportation in an urban area: a randomised trial. Lancet 2018; 392:283-291.
2. Bulger EM, Jurkovich GJ, Nathens AB et al. Hypertonic resuscitation of hypovolemic shock after blunt trauma: a randomized controlled trial. Arch Surg 2008; 143:139-148.
3. Ranieri VM, Rubenfeld GD, Thompson BT et al. Acute respiratory distress syndrome: the Berlin Definition. JAMA 2012; 307:2526-2533.
4. Schoenfeld DA, Bernard GR. Statistical evaluation of ventilator-free days as an efficacy measure in clinical trials of treatments for acute respiratory distress syndrome. Crit Care Med 2002; 30:1772-1777.
5. Nemkov T, Reisz JA, Gehrke S et al. High-Throughput Metabolomics: Isocratic and Gradient Mass Spectrometry-Based Methods. Methods Mol Biol 2019; 1978:13-26.
6. D'Alessandro A, Reisz JA, Zhang Y et al. Effects of aged stored autologous red blood cells on human plasma metabolome. Blood Adv 2019; 3:884-896.
7. Samuels JM, Moore EE, Silliman CC et al. Severe traumatic brain injury is associated with a unique coagulopathy phenotype. J Trauma Acute Care Surg 2018.
8. Silliman CC, Clay KL, Thurman GW et al. Partial characterization of lipids that develop during the routine storage of blood and prime the neutrophil NADPH oxidase. J Lab Clin Med 1994; 124:684-694.
9. Kelher MR, Masuno T, Moore EE et al. Plasma from stored packed red blood cells and MHC class I antibodies causes acute lung injury in a 2-event in vivo rat model. Blood 2009; 113:2079-2087.
10. Kelher MR, Banerjee A, Gamboni F et al. Antibodies to major histocompatibility complex class II antigens directly prime neutrophils and cause acute lung injury in a two-event in vivo rat model. Transfusion 2016; 56:3004-3011.
11. McLaughlin NJ, Banerjee A, Kelher MR et al. Platelet-activating factor-induced clathrin- mediated endocytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation. J Immunol 2006;
176:7039-7050.
12. McLaughlin NJ, Banerjee A, Khan SY et al. Platelet-activating factor-mediated endosome formation causes membrane translocation of p67phox and p40phox that requires
recruitment and activation of p38 MAPK, Rab5a, and phosphatidylinositol 3-kinase in human neutrophils. J Immunol 2008; 180:8192-8203.
13. Bhuniya D, Umrani D, Dave B et al. Discovery of a potent and selective small molecule hGPR91 antagonist. Bioorg Med Chem Lett 2011; 21:3596-3602.
14. Kelher MR, McLaughlin NJ, Banerjee A et al. LysoPCs induce Hck- and PKCdelta- mediated activation of PKCgamma causing p47phox phosphorylation and membrane translocation in neutrophils. J Leukoc Biol 2017; 101:261-273.
15. Silliman CC, Elzi DJ, Ambruso DR et al. Lysophosphatidylcholines prime the NADPH oxidase and stimulate multiple neutrophil functions through changes in cytosolic calcium. J Leukoc Biol 2003; 73:511-524.
16. Masuno T, Moore EE, Cheng AM et al. Prehospital hemoglobin-based oxygen carrier resuscitation attenuates postinjury acute lung injury. Surgery 2005; 138.
17. Probst RJ, Lim JM, Bird DN et al. Gender differences in the blood volume of conscious Sprague-Dawley rats. J Am Assoc Lab Anim Sci 2006; 45:49-52.
18. Silliman CC, Kelher MR, Khan SY et al. Supernatants and lipids from stored red blood cells activate pulmonary microvascular endothelium through the BLT2 receptor and protein kinase C activation. Transfusion 2017; 57:2690-2700.
19. Akbas N, Gonzalez G, Edwards R et al. Assessment of liver function tests on Piccolo Xpress point of care chemistry analyzer in a pediatric hospital. Pract Lab Med 2015; 3:1-7.
20. Sauaia A, Moore EE, Johnson JL et al. Temporal trends of postinjury multiple-organ failure: still resource intensive, morbid, and lethal. J Trauma Acute Care Surg 2014;
76:582-92, discussion.
21. Sauaia A, Moore FA, Moore EE. Postinjury Inflammation and Organ Dysfunction. Crit Care Clin 2017; 33:167-191.
22. D'Alessandro A, Moore HB, Moore EE et al. Plasma succinate is a predictor of mortality in critically injured patients. J Trauma Acute Care Surg 2017; 83:491-495.
23. Chapman MP, Moore EE, Chin TL et al. Combat: Initial Experience with a Randomized Clinical Trial of Plasma-Based Resuscitation in the Field for Traumatic Hemorrhagic Shock. Shock 2015; 44 Suppl 1:63-70.
24. Chin TL, Moore EE, Coors ME et al. Exploring ethical conflicts in emergency trauma research: the COMBAT (Control of Major Bleeding after Trauma) study experience.
Surgery 2015; 157:10-19.
