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• W2895550979 abstract "Despite improvements in supportive care, mortality from acute respiratory distress syndrome (ARDS) remains high (approximately 30% to 40%).1Famous K.R. Delucchi K. Ware L.B. Kangelaris K.N. Liu K.D. Thompson B.T. et al.Acute respiratory distress syndrome subphenotypes respond differently to randomized fluid management strategy.Am J Respir Crit Care Med. 2017; 195: 331-338Crossref PubMed Scopus (395) Google Scholar In recent years, several biomarkers of inflammation, epithelial damage, endothelial damage, and coagulation have been identified that show promise for identifying ARDS endotypes with different prognosis and therapeutic responsiveness.1Famous K.R. Delucchi K. Ware L.B. Kangelaris K.N. Liu K.D. Thompson B.T. et al.Acute respiratory distress syndrome subphenotypes respond differently to randomized fluid management strategy.Am J Respir Crit Care Med. 2017; 195: 331-338Crossref PubMed Scopus (395) Google Scholar Unfortunately, few lung-specific biomarkers have been validated, and none have been adopted into routine clinical practice. Cholestenoic acid (CA; 3β-hydroxy-5-cholestenoic acid) is an oxysterol produced by the action of the mitochondrial-localized enzyme sterol 27-hydroxylase (CYP27A1) on cholesterol.2Babiker A. Andersson O. Lund E. Xiu R.J. Deeb S. Reshef A. et al.Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport.J Biol Chem. 1997; 272: 26253-26261Crossref PubMed Scopus (202) Google Scholar In human subjects the plasma concentration of CA is reported to derive virtually quantitatively from biosynthesis in the lung.3Babiker A. Andersson O. Lindblom D. van der Linden J. Wiklund B. Lutjohann D. et al.Elimination of cholesterol as cholestenoic acid in human lung by sterol 27-hydroxylase: evidence that most of this steroid in the circulation is of pulmonary origin.J Lipid Res. 1999; 40: 1417-1425Abstract Full Text Full Text PDF PubMed Google Scholar Remarkably, plasma CA levels decrease by approximately 50% after pneumonectomy and by 31% within 60 minutes of cardiopulmonary bypass.3Babiker A. Andersson O. Lindblom D. van der Linden J. Wiklund B. Lutjohann D. et al.Elimination of cholesterol as cholestenoic acid in human lung by sterol 27-hydroxylase: evidence that most of this steroid in the circulation is of pulmonary origin.J Lipid Res. 1999; 40: 1417-1425Abstract Full Text Full Text PDF PubMed Google Scholar Alveolar macrophages (AMs) express much greater CYP27A1 and secrete much greater CA than all other cells surveyed2Babiker A. Andersson O. Lund E. Xiu R.J. Deeb S. Reshef A. et al.Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport.J Biol Chem. 1997; 272: 26253-26261Crossref PubMed Scopus (202) Google Scholar and are thought to be the major source of circulating CA.2Babiker A. Andersson O. Lund E. Xiu R.J. Deeb S. Reshef A. et al.Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport.J Biol Chem. 1997; 272: 26253-26261Crossref PubMed Scopus (202) Google Scholar, 3Babiker A. Andersson O. Lindblom D. van der Linden J. Wiklund B. Lutjohann D. et al.Elimination of cholesterol as cholestenoic acid in human lung by sterol 27-hydroxylase: evidence that most of this steroid in the circulation is of pulmonary origin.J Lipid Res. 1999; 40: 1417-1425Abstract Full Text Full Text PDF PubMed Google Scholar Delivery of cholesterol substrate to CYP27A1 at the inner mitochondrial membrane, the rate-limiting step for CYP27A1 function, requires respiring mitochondria.4Allen A.M. Taylor J.M. Graham A. Mitochondrial (dys)function and regulation of macrophage cholesterol efflux.Clin Sci (Lond). 2013; 124: 509-515Crossref PubMed Scopus (22) Google Scholar We hypothesized that plasma CA is a biomarker of AM functional integrity and that it would decrease with increasing disease severity in patients with ARDS, perhaps because of mitochondrial dysfunction, other cytotoxicity, or both. Reported methods for CA quantitation in plasma have generally used a starting volume of 0.5 mL or greater and have required reverse-phase and ion-exchange solid-phase extractions, methylation, and trimethylsilylation before chromatographic separation.3Babiker A. Andersson O. Lindblom D. van der Linden J. Wiklund B. Lutjohann D. et al.Elimination of cholesterol as cholestenoic acid in human lung by sterol 27-hydroxylase: evidence that most of this steroid in the circulation is of pulmonary origin.J Lipid Res. 1999; 40: 1417-1425Abstract Full Text Full Text PDF PubMed Google Scholar Here we developed a simplified and robust methodology to measure CA from 4 μL of plasma, as detailed in the Methods section, Fig E1, and Tables E1 and E2 in this article's Online Repository at www.jacionline.org. First, we confirmed that AMs freshly collected from healthy volunteers (n = 5) by means of bronchoalveolar lavage (BAL) had robust expression of CYP27A1 (Fig 1, A). AM lysates had a mean CA content of approximately 6.4 ng (SD, 1.6 ng) per million cells but no substantial quantity of the CA metabolites 3β,7α-dihydroxy-5-cholestenoic acid or 7α-hydroxy-3-oxo-4-cholestenoic acid, suggesting that CA is the primary C27 carboxylic acid synthesized by human AMs. CA was also detected in BAL fluid (n = 7; mean, 0.90 ± 0.73 ng/mL). Ex vivo treatment of human AMs with LPS for 4 hours did not modify CYP27A1 expression (Fig 1, B). These data rule out neither a change in CYP27A1 function nor changes in CYP27A1 expression at later time points. Indeed, a more prolonged time course of CYP27A1 expression in mice after in vivo LPS inhalation revealed a transient downregulation on day 3 in CD64+F4/80+CD11chighCD11blow resident AMs, whereas CD64+F4/80+CD11clowCD11bhigh recruited (ie, blood monocyte–derived) AMs exhibited a progressive and time-dependent upregulation (Fig 1, C).E1Mould K.J. Barthel L. Mohning M.P. Thomas S.M. McCubbrey A.L. Danhorn T. et al.Cell origin dictates programming of resident versus recruited macrophages during acute lung injury.Am J Respir Cell Mol Biol. 2017; 57: 294-306Crossref PubMed Scopus (96) Google Scholar Next, we measured plasma CA levels on day 1 of study enrollment in 144 patients with ARDS with sepsis who participated in the National Heart, Lung, and Blood Institute's ARDS Network ALVEOLI trial of low versus high positive end-expiratory pressure (PEEP; see Table E3 in this article's Online Repository at www.jacionline.org).5Brower R.G. Lanken P.N. MacIntyre N. Matthay M.A. Morris A. Ancukiewicz M. et al.Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome.N Engl J Med. 2004; 351: 327-336Crossref PubMed Scopus (1437) Google Scholar Regression analyses adjusted for body mass index, sex, race/ethnicity, Acute Physiology and Chronic Health Evaluation (APACHE) III score, ARDS risk factor, diabetes, plasma bilirubin, and plasma albumin revealed that each 1 ng/mL increase in plasma CA level was associated with a nearly 1% reduction in 90-day mortality (Table I). Albumin and bilirubin were included in the model given that CA is carried on serum albumin and likely hepatically cleared.3Babiker A. Andersson O. Lindblom D. van der Linden J. Wiklund B. Lutjohann D. et al.Elimination of cholesterol as cholestenoic acid in human lung by sterol 27-hydroxylase: evidence that most of this steroid in the circulation is of pulmonary origin.J Lipid Res. 1999; 40: 1417-1425Abstract Full Text Full Text PDF PubMed Google Scholar, 6Babiker A. Diczfalusy U. Transport of side-chain oxidized oxysterols in the human circulation.Biochim Biophys Acta. 1998; 1392: 333-339Crossref PubMed Scopus (115) Google Scholar Each 1 ng/mL increase in plasma CA level was also associated with an increase in adjusted ventilator-free days (VFDs) of 0.016 days (P = .04) and with an increase in adjusted organ failure–free days of 0.025 days (P < .001). On stratification, plasma CA levels were associated with statistically significant increases in organ failure–free days within multiple individual organ systems.Table IRelationship of plasma CA to mortality and adverse clinical outcomes in patients with ARDS with sepsisNo.‡The number is smaller than in the Table E3 study population because only those subjects with data for all model variables were included.Odds ratio∗Logistic regression of plasma CA concentration to death at 90 days. Odds ratios are adjusted for body mass index, sex, race/ethnicity, APACHE III score, ARDS risk factor, diabetes mellitus, plasma bilirubin, and plasma albumin./β†Linear regression of plasma CA concentration to VFDs, organ failure–free days, and organ system–specific and organ failure–free days adjusted for the same covariates used in the logistic regression for mortality. The β coefficient designates the mean change in VFDs or organ failure–free days per unit (1 ng/mL) increase in plasma CA concentration.95% CIP valueMortality∗Logistic regression of plasma CA concentration to death at 90 days. Odds ratios are adjusted for body mass index, sex, race/ethnicity, APACHE III score, ARDS risk factor, diabetes mellitus, plasma bilirubin, and plasma albumin.1240.9940.989-0.998.01VFDs†Linear regression of plasma CA concentration to VFDs, organ failure–free days, and organ system–specific and organ failure–free days adjusted for the same covariates used in the logistic regression for mortality. The β coefficient designates the mean change in VFDs or organ failure–free days per unit (1 ng/mL) increase in plasma CA concentration.1240.0160.001-0.030.04Organ failure–free days†Linear regression of plasma CA concentration to VFDs, organ failure–free days, and organ system–specific and organ failure–free days adjusted for the same covariates used in the logistic regression for mortality. The β coefficient designates the mean change in VFDs or organ failure–free days per unit (1 ng/mL) increase in plasma CA concentration.1230.0250.012-0.039<.001 Cardiovascular1230.0220.009-0.034.001 Respiratory1230.0200.004-0.037.014 Coagulation1230.0130.001-0.026.03 Renal1230.0250.010-0.039.001 Hepatic1210.0200.005-0.036.012∗ Logistic regression of plasma CA concentration to death at 90 days. Odds ratios are adjusted for body mass index, sex, race/ethnicity, APACHE III score, ARDS risk factor, diabetes mellitus, plasma bilirubin, and plasma albumin.† Linear regression of plasma CA concentration to VFDs, organ failure–free days, and organ system–specific and organ failure–free days adjusted for the same covariates used in the logistic regression for mortality. The β coefficient designates the mean change in VFDs or organ failure–free days per unit (1 ng/mL) increase in plasma CA concentration.‡ The number is smaller than in the Table E3 study population because only those subjects with data for all model variables were included. Open table in a new tab We examined CYP27A1 expression in AMs (n = 30 patients) and peripheral blood monocytes (n = 29 patients) purified within 48 hours of ARDS diagnosis from patients in a Phase II trial of enteral fish oil versus saline placebo treatment in ARDS (see Table E4 in this article's Online Repository at www.jacionline.org).7Stapleton R.D. Martin T.R. Weiss N.S. Crowley J.J. Gundel S.J. Nathens A.B. et al.A phase II randomized placebo-controlled trial of omega-3 fatty acids for the treatment of acute lung injury.Crit Care Med. 2011; 39: 1655-1662Crossref PubMed Scopus (104) Google Scholar Lower CYP27A1 mRNA expression in AMs and monocytes was observed in patients with lower VFDs (ie, higher illness severity; Fig 1, D). This suggests that the reduced plasma CA levels in patients with severe ARDS might derive, at least in part, from CYP27A1 downregulation. An inverse relationship was also observed between AM CYP27A1 mRNA expression and BAL fluid cytokine levels (Fig 1, E). There was no relationship of Pao2/fraction of inspired oxygen (Fio2) ratio to either AM or monocyte CYP27A1 levels (not depicted), suggesting that CYP27A1 expression does not correlate with oxygenation. Similar to other oxysterols, CA is an agonist of liver X receptor, a nuclear receptor that induces cholesterol efflux genes and also suppresses nuclear factor κB–dependent proinflammatory genes.8Fessler M.B. A new frontier in immunometabolism. Cholesterol in lung health and disease.Ann Am Thorac Soc. 2017; 14: S399-S405Crossref PubMed Scopus (37) Google Scholar Given this, we speculated that CA might attenuate proinflammatory gene expression in human AMs. However, at concentrations that induced liver X receptor target genes in human AMs ex vivo, CA had no effect on LPS-induced cytokines (see Fig E2 in this article's Online Repository at www.jacionline.org). This suggests that although decreasing CA levels might serve as a readout of AM dysfunction, a local reduction in CA levels in the alveolus is unlikely itself to feedback on AM proinflammatory functions during ARDS. Within AMs and other cells, CYP27A1-dependent synthesis of CA is rate limited by delivery of cholesterol substrate to the inner mitochondrial membrane, a pathway that requires functional polarized mitochondria.4Allen A.M. Taylor J.M. Graham A. Mitochondrial (dys)function and regulation of macrophage cholesterol efflux.Clin Sci (Lond). 2013; 124: 509-515Crossref PubMed Scopus (22) Google Scholar Mitotoxic stressors can set a vicious cycle in motion given that CYP27A1 plays a role in detoxification of oxidized lipids and prevention of mitotoxic cholesterol overload in macrophages.4Allen A.M. Taylor J.M. Graham A. Mitochondrial (dys)function and regulation of macrophage cholesterol efflux.Clin Sci (Lond). 2013; 124: 509-515Crossref PubMed Scopus (22) Google Scholar Because increased numbers of cholesterol-laden macrophage “foam” cells are found in patients with a wide range of lung diseases,8Fessler M.B. A new frontier in immunometabolism. Cholesterol in lung health and disease.Ann Am Thorac Soc. 2017; 14: S399-S405Crossref PubMed Scopus (37) Google Scholar it is intriguing to consider that insufficient CYP27A1 product flux, perhaps arising from mitochondrial dysfunction, could be a unifying and perhaps targetable event in human lung disease. We propose plasma CA as a novel biomarker of AM mitochondrial function during ARDS. However, future studies will be required to verify this postulate. CYP27A1 product flux is expected to also be sensitive to the expression and function of the intracellular proteins that transfer cholesterol into mitochondria. In addition, given that macrophage efflux of sterols other than CA might be favored in the presence of extracellular lipoprotein acceptors,2Babiker A. Andersson O. Lund E. Xiu R.J. Deeb S. Reshef A. et al.Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport.J Biol Chem. 1997; 272: 26253-26261Crossref PubMed Scopus (202) Google Scholar it is conceivable that leakage of plasma lipoproteins into the airspace during lung injury could modify CA flux from AMs. CA has several features of a robust biomarker. Plasma CA concentrations display little diurnal variation,9Lund E. Andersson O. Zhang J. Babiker A. Ahlborg G. Diczfalusy U. et al.Importance of a novel oxidative mechanism for elimination of intracellular cholesterol in humans.Arterioscler Thromb Vasc Biol. 1996; 16: 208-212Crossref PubMed Scopus (146) Google Scholar and unlike other oxysterols, CA is not substantially produced by artefactual autoxidation.6Babiker A. Diczfalusy U. Transport of side-chain oxidized oxysterols in the human circulation.Biochim Biophys Acta. 1998; 1392: 333-339Crossref PubMed Scopus (115) Google Scholar Given that the plasma samples we analyzed had been stored at −80°C for approximately 10 years, it appears that CA analysis is robust to long-term storage. Unlike other sterols, CA is not carried on plasma lipoproteins and has no significant correlation with plasma cholesterol levels,3Babiker A. Andersson O. Lindblom D. van der Linden J. Wiklund B. Lutjohann D. et al.Elimination of cholesterol as cholestenoic acid in human lung by sterol 27-hydroxylase: evidence that most of this steroid in the circulation is of pulmonary origin.J Lipid Res. 1999; 40: 1417-1425Abstract Full Text Full Text PDF PubMed Google Scholar suggesting it is not substantially confounded by dyslipidemia. CA also has little to no renal excretion under normal conditions3Babiker A. Andersson O. Lindblom D. van der Linden J. Wiklund B. Lutjohann D. et al.Elimination of cholesterol as cholestenoic acid in human lung by sterol 27-hydroxylase: evidence that most of this steroid in the circulation is of pulmonary origin.J Lipid Res. 1999; 40: 1417-1425Abstract Full Text Full Text PDF PubMed Google Scholar and thus might be little affected during renal failure. Glucocorticoids reportedly upregulate CYP27A1 activity in cell culture,E2Araya Z. Tang W. Wikvall K. Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1.Biochem J. 2003; 372: 529-534Crossref PubMed Google Scholar whereas the sedative dexmedetomidine inhibits itE3Mast N. Lin J.B. Pikuleva I.A. Marketed drugs can inhibit cytochrome P450 27A1, a potential new target for breast cancer adjuvant therapy.Mol Pharmacol. 2015; 88: 428-436Crossref PubMed Scopus (37) Google Scholar; whether this translates to changes in plasma CA levels in vivo is uncertain. Given that ARDS is commonly associated with multiple comorbidities and therapeutic exposures, future studies will need to carefully address the specificity of changes in plasma CA levels to ARDS pathophysiology. Taken together, we identify CA as a novel ARDS biomarker. We speculate that plasma CA might represent a valuable new peripheral blood indicator of AM mitochondrial function and propose that it should now be measured in patients with a wider range of lung diseases and potentially in emerging studies of cell-based mitochondrial rescue in the lung. The ARDS Clinical Trials Network's ALVEOLI trial was a randomized trial of lower versus higher PEEP in patients with acute lung injury (ALI)/ARDS and was approved by a protocol review committee convened by the National Heart, Lung, and Blood Institute and by the institutional review board at all participating institutions.E4Brower R.G. Lanken P.N. MacIntyre N. Matthay M.A. Morris A. Ancukiewicz M. et al.Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome.N Engl J Med. 2004; 351: 327-336Crossref PubMed Scopus (1848) Google Scholar As previously described,E4Brower R.G. Lanken P.N. MacIntyre N. Matthay M.A. Morris A. Ancukiewicz M. et al.Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome.N Engl J Med. 2004; 351: 327-336Crossref PubMed Scopus (1848) Google Scholar patients with ALI/ARDS were randomly assigned to receive mechanical ventilation with either lower or higher PEEP levels, which were set according to tables of predetermined combinations of PEEP and inspired oxygen. At enrollment, APACHE III scores were calculated, and the primary risk factor for development of ALI/ARDS was identified.E5Eisner M.D. Thompson T. Hudson L.D. Luce J.M. Hayden D. Schoenfeld D. et al.Efficacy of low tidal volume ventilation in patients with different clinical risk factors for acute lung injury and the acute respiratory distress syndrome.Am J Respir Crit Care Med. 2001; 164: 231-236Crossref PubMed Scopus (262) Google Scholar For sepsis classification, the Society of Critical Care Medicine clinical definition was used, which includes a known or suspected source of systemic infection and at least 2 of the following: (1) temperature of less than 36°C or greater than 38°C; (2) heart rate of greater than 90 beats/min; (3) respiratory rate of greater than 20 breaths/min or Paco2 of less than 32 mm Hg; and (4) white blood cell count of greater than 12,000/mm3, less than 4000/mm3, or greater than 10% bands. Known infection is defined as a documented source of infection (ie, positive blood cultures). Suspected infection is defined as 1 or more of the following: leukocytes in a normally sterile body fluid, perforated viscus, radiographic evidence of pneumonia plus purulent sputum, or a syndrome associated with a high infection risk (eg, ascending cholangitis).E6Suratt B.T. Eisner M.D. Calfee C.S. Allard J.B. Whittaker L.A. Engelken D.T. et al.Plasma granulocyte colony-stimulating factor levels correlate with clinical outcomes in patients with acute lung injury.Crit Care Med. 2009; 37: 1322-1328Crossref PubMed Scopus (21) Google Scholar Plasma was collected from patients on day 1 of the study. VFDs was defined as the number of days of unassisted breathing after initiating spontaneous breathing to day 28 after randomization. Organ failure–free days was defined as the number of days from randomization to day 28 free from failure in renal, hepatic, central nervous system, coagulation, and/or circulatory systems. The Fish Oil Phase-II Randomized Placebo-Controlled TrialE7Stapleton R.D. Martin T.R. Weiss N.S. Crowley J.J. Gundel S.J. Nathens A.B. et al.A phase II randomized placebo-controlled trial of omega-3 fatty acids for the treatment of acute lung injury.Crit Care Med. 2011; 39: 1655-1662Crossref PubMed Scopus (129) Google Scholar was a study of enteral fish oil versus saline placebo treatment for up to 14 days in adult mechanically ventilated patients with ALI, as defined by the American-European Consensus Conference.E8Bernard G.R. Artigas A. Brigham K.L. Carlet J. Falke K. Hudson L. et al.The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination.Am J Respir Crit Care Med. 1994; 149: 818-824Crossref PubMed Scopus (5301) Google Scholar The trial was conducted at 5 North American centers and was approved by human subject research committees at all sites. As previously described,E9Morrell E.D. Radella Ii F. Manicone A.M. Mikacenic C. Stapleton R.D. Gharib S.A. et al.Peripheral and alveolar cell transcriptional programs are distinct in acute respiratory distress syndrome.Am J Respir Crit Care Med. 2018; 197: 528-532Crossref PubMed Scopus (36) Google Scholar AMs (from BALF) and peripheral blood monocytes were isolated from patients by means of negative antibody selection within 48 hours of diagnosis of ARDS. CYP27A1 expression was analyzed by using the Illumina HumanRef-8 BeadChip (Illumina, San Diego, Calif). BAL fluid concentrations of IL-8 and IL-6 were determined by using the Luminex bead-based immunoassay (R&D Systems, Minneapolis, Minn). Healthy nonsmoking adult subjects (age, 23-51 years) were recruited to the National Institute of Environmental Health Sciences Clinical Research Unit and underwent informed consent in accordance with an National Institute of Environmental Health Sciences institutional review board–approved protocol (#11-E-0006). After topical anesthesia to the airway, subjects underwent a 250-mL saline BAL of the right middle lobe. BAL cells were pelleted (at 800g for 10 minutes), plated in tissue culture plates, and gently washed with sterile 1 × PBS, pH 7.4, after 1 to 2 hours to enrich for a pure macrophage population. 3β-Hydroxy-5-cholestenoic acid (CA), 3β,7α-dihydroxy-5-cholestenoic acid, and 7α-hydroxy-3-oxo-4-cholestenoic acid were purchased from Avanti Polar Lipids (Alabaster, Ala). 5β-Cholanic acid-3α-ol-2,2,4,4-d4 (lithocolic acid-d4) and 5β-cholanic acid-3α, 7α-diol-2,2,4,4-d4 (chenodeoxycholic acid-d4) were purchased from Steraloids (Newport, RI). HybridSPE-Phospholipid Ultra 30 mg/1 mL SPE tubes were purchased from Sigma-Aldrich (St Louis, Mo). Escherichia coli 0111:B4 LPS was from List Biological Laboratories (Campbell, Calif). Plasma samples were diluted 10-fold with 0.9% NaCl and stored at −80°C before analysis. Four hundred microliters of acetonitrile chilled on ice and containing 1% formic acid and 0.01% butylated hydroxy toluene was placed in HybridSPE reservoirs. Forty microliters of thawed diluted plasma solution was added to the acetonitrile, followed by internal standard solution (10 μL, 2 pg/μL acetonitrile). Samples were then placed on a shaker plate for 10 minutes at 4°C before elution under vacuum. SPE tubes were rinsed with 100 μL of 90% acetonitrile and 1% formic acid. Samples were then dried under a vacuum and reconstituted in 100 μL of 60% acetonitrile for analysis. A Thermo Fisher Ultimate 3000 UHPLC system (Thermo Fisher, Waltham, Mass) was used for chromatographic separation. Samples were injected onto a Halo C18 Fused Core column (Advanced Materials Technology, Wilmington, Del), 2.1 × 100 mm, with 2.7 μm held at 50°C in a column compartment. Mobile phase A was 0.1% acetic acid in water, and mobile phase B was 0.1% acetic acid in acetonitrile. The initial condition of 40% B and flow rate of 500 μL/min was held for 1 minute. A linear gradient was applied to 50% B at 7 minutes, 70% B at 12 minutes, and 100% B at 15 minutes. These conditions were maintained until 16 minutes to remove matrix components. The solvent composition was then returned to 40% B, and the column was re-equilibrated for 2.5 minutes. The column flow between 4 and 13 minutes was directed to a Thermo Fisher Quantiva triple quadrupole mass spectrometer for tandem mass spectral detection. Negative electrospray ionization was performed with a spray voltage of −2750 V. Sheath gas, auxiliary gas, and sweep gas were set to 30, 15, and 2 arbitrary units, respectively. The vaporizer temperature was 400°C, and the ion transfer tube temperature was 350°C. The collision cell pressure was 2 mTorr. Further parameters are summarized in Table E1. A lack of fragment ions necessitated the use of parent-to-parent selected reaction monitoring. To increase specificity, Q1 was set to 0.2 full width at half-maximum resolution, whereas Q3 was set to 0.7 full width at half-maximum. Collision energy (CE) was set to produce approximately 90% of maximum signal. The result was a 2-fold increase in CE, attenuating the chemical background with minimal loss of analyte intensity. A second higher CE, which yielded 50% to 60% of the first, was used as a secondary measurement and allowed for ion ratio calculations. For 3β,7α-dihydroxy-5-cholestenoic acid, the higher CE resulted in better baseline definition of the analytic peak and therefore was used for quantitation. Matrix-matched calibrants displayed R2 values of greater than 0.99. Technical reproducibility of the full protocol was assessed in pooled human plasma (5 subjects) by performing 4 replicate extractions, analyzing them by using liquid chromatography–tandem mass spectrometry 5 replicate times, and then repeating mass spectrometric analysis 7 days later. Acceptable variance was observed (Table E2), indicating good reproducibility of the full protocol. Human AMs were plated in Dulbecco minimal essential medium supplemented with 10% FBS and cultured for 16 to 18 hours (37°C in a 5% CO2 atmosphere) in dimethyl sulfoxide vehicle or 10 μmol/L CA or 3β,7α-dihydroxy-5-cholestenoic acid (Avanti Polar Lipids). Cells were then left untreated or exposed to 10 ng/mL E coli 0111:B4 LPS for 4 hours. RNA was isolated from AMs and reverse transcribed by using the cDNA reverse transcription kit (Applied Biosystems, Waltham, Mass) and used as a template for TaqMan expression assays, according to the manufacturer's guidelines (Applied Biosystems). Primer/probe sets were as follows: CYP27A1 (Hs01017992_g1), ITGAM (Hs00167304_m1), SFTPC (Hs00951326_g1), TNF (Hs00174128_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), IL10 (Hs00961622_m1), ABCA1 (Hs01059137_m1), ABCG1 (Hs00245154_m1), and MYLIP (Hs00982312_m1). C57BL/6 mice (male and 10-12 weeks of age) were exposed intratracheally to 20 μg of E coli 0555:B5 LPS. All experiments were performed in accordance with the Animal Welfare Act and the US Public Health Service Policy on Humane Care and Use of Laboratory Animals after review by the Animal Care and Use Committee of the National Jewish Hospital Animal Care and Use Committee. At various postexposure time points, resident (CD64+F4/80+CD11chighCD11blow) and recruited (CD64+F4/80+CD11clowCD11bhigh) AMs were purified from BAL fluid by using fluorescence-activated cell sorting after excluding dead and contaminating cells with the use of forward and side scatter, 4′-6-diamidino-2-phenylindol dihydrochloride, and antibodies against CD3, NK1.1, B220, and Ly6G, as previously described.E1Mould K.J. Barthel L. Mohning M.P. Thomas S.M. McCubbrey A.L. Danhorn T. et al.Cell origin dictates programming of resident versus recruited macrophages during acute lung injury.Am J Respir Cell Mol Biol. 2017; 57: 294-306Crossref PubMed Scopus (96) Google Scholar RNA was prepared, and CYP27A1 mRNA was quantified by means of RNA sequencing, as previously described.E1Mould K.J. Barthel L. Mohning M.P. Thomas S.M. McCubbrey A.L. Danhorn T. et al.Cell origin dictates programming of resident versus recruited macrophages during acute lung injury.Am J Respir Cell Mol Biol. 2017; 57: 294-306Crossref PubMed Scopus (96) Google Scholar For analysis of patients with ARDS with sepsis, logistic regression of plasma CA concentration to death at 90 days was performed, adjusting for body mass index, sex, race/ethnicity, APACHE III score, ARDS risk factor, diabetes mellitus, plasma bilirubin, and plasma albumin. For race/ethnicity, the ALVEOLI study designated patients as non-Hispanic white, non-Hispanic black, Hispanic, Asian/Pacific Islander, American Indian/Alaskan Native, and other. Because of the very small number of subjects in the last 4 categories, they were pooled into a single “other” category for means of analysis. Linear regression of plasma CA concentration to VFDs and organ failure–free days was performed, adjusting for the same covariates. STATA software, version 14.2 (StataCorp, College Station, Tex) was used. The relationship between cellular CYP27A1 gene expression (log2 probe intensity) and VFD tertiles was tested by using the Kruskal-Wallis test with post hoc Dunn correction. The relationship between AM CYP27A1 expression and BAL fluid IL-6 and IL-8 concentrations was tested with linear regression. Analyses were performed with GraphPad Prism statistical software (GraphPad Software, San Diego, Calif) for cell treatment, monocyte/AM gene expression, and animal experiments. The 2-tailed student t test was applied for comparisons of 2 groups, and ANOVA was applied for analyses of 3 or more groups. For all tests, a P value of less than .05 was considered significant.Fig E2Effect of CA on proinflammatory macrophage functions. A, Human AMs were treated for 16 to 18 hours in vehicle, 10 μmol/L CA, or 10 μmol/L 3β,7α-diOH-cholestenoic acid, a downstream metabolite of CA. Cells were then left nonstimulated (NS) or treated with LPS (10 ng/mL, 4 hours), after which mRNA for the indicated cytokine targets was quantified by using quantitative RT-PCR. AU, Arbitrary units normalized to GUSB. B, Human AMs were treated for 16 to 18 hours as shown, and then mRNA for the indicated liver X receptor target genes was quantified by using quantitative RT-PCR. Inducible degrader of low-density lipoprotein receptor (IDOL) is encoded by the MYLIP gene. ABCA1, ATP binding cassette transporter A1; ABCG1, ATP binding cassette transporter G1. Data derive from triplicate wells and are representative of 2 independent experiments. Similar results were obtained in AMs from a third donor treated with 5 μmol/L of the CA species. Data are depicted as means ± SDs. **P < .01 and ***P < .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Analyte parameter tableFull nameAbbreviationQ1 (m/z)Q3 (m/z)RF lens (V)CE (V)RT (min)Internal standard5β-Cholanic acid-3α,7α-diol-2,2,4,4-d4CDCA-d4395.3395.4130194.683β,7α-Dihydroxy-5-cholestenoic acid431.3431.4125306.54CDCA-d47α-Hydroxy-3-oxo-4-cholestenoic acid429.3411.4120307.52CDCA-d45β-Cholanic acid-3α-ol-2,2,4,4-d4LCA-d4379.3379.4120199.573β-Hydroxy-5-cholestenoic acidCA415.3415.41252011.97LCA-d4CDCA-d4, Chenodeoxycholic acid-d4; LCA-d4, lithocolic acid-d4; RF, radiofrequency; RT, retention time. Open table in a new tab Table E2Technical reproducibility of analyte measurementsAnalyteDay 1 averageDay 1 SDDay 1 %RSDDay 8 averageDay 8 SDDay 8 %RSDDay 1/Day 83β-Hydroxy-5-cholestenoic acid158.86.23.9151.922.214.61.053β,7α-Dihydroxy-5-cholestenoic acid73.41.41.973.72.73.61.007α-Hydroxy-3-oxo-4-cholestenoic acid58.32.23.846.14.49.61.26Pooled plasma from 5 volunteers of varied sex and race was analyzed on 2 days (days 1 and 8) to assess technical reproducibility. Four extraction replicates were performed on each day, and each sample was analyzed 5 times. Concentrations are expressed as nanograms per milliliter of plasma.%RSD, Percentage relative SD. Open table in a new tab Table E3Demographic characteristics and unadjusted outcomes (n = 144)Age (y), mean ± SD52.7 ± 17.2Sex (no. [% women])68 (47.2)Race/ethnicity, no. (%) Non-Hispanic white106 (74) Non-Hispanic black23 (16) Other∗Other includes Hispanic (n = 10), Asian/Pacific Islander (n = 3), American Indian/Alaskan Native (n = 1), and other (n = 1).15 (10)Pao2/Fio2, mean ± SD126.2 ± 58.1APACHE III score, mean ± SD99.4 ± 33.0Prerandomization tidal volume/kg predicted body weight, mean ± SD8.2 ± 2.1Diabetes (%)28 (19.6)ALI risk factor Trauma, no. (%)1 (0.7) Sepsis, no. (%)89 (61.8) Pneumonia, no. (%)42 (29.2) Other, no. (%)12 (8.3)90-d Mortality, no. (%)39 (27.1)VFDs13.8 ± 10.8Organ failure–free days (not including CNS)14.1 ± 10.6CNS, Central nervous system.∗ Other includes Hispanic (n = 10), Asian/Pacific Islander (n = 3), American Indian/Alaskan Native (n = 1), and other (n = 1). Open table in a new tab Table E4Characteristics of subjects from AM and peripheral blood monocyte CYP27A1 analyses∗Subjects are those from the Fish Oil studyE7 who had the relationship of AM or peripheral blood monocyte CYP27A1 expression to clinical outcomes tested.CharacteristicAM (n = 30)Monocyte (n = 29)Patient age (y), mean ± SD44 ± 1742 ± 16Male patients, no. (%)18 (60)18 (62)White, no. (%)26 (87)25 (86)Comorbidities, no. (%) Diabetes4 (13)5 (17) Cirrhosis2 (7)1 (3)ARDS risk factor, no. (%)†Risk factors for ARDS are not mutually exclusive. Sepsis17 (57)15 (52) Trauma14 (47)14 (48) Pneumonia9 (30)7 (24) Other4 (13)4 (14)APACHE II score, mean ± SD21 ± 620 ± 6Pao2/Fio2 ratio, mean ± SD199 ± 61193 ± 62VFDs, median (IQR)13 (0-23)19 (0-23)28-d Mortality, no. (%)4, (13)3, (10)∗ Subjects are those from the Fish Oil studyE7Stapleton R.D. Martin T.R. Weiss N.S. Crowley J.J. Gundel S.J. Nathens A.B. et al.A phase II randomized placebo-controlled trial of omega-3 fatty acids for the treatment of acute lung injury.Crit Care Med. 2011; 39: 1655-1662Crossref PubMed Scopus (129) Google Scholar who had the relationship of AM or peripheral blood monocyte CYP27A1 expression to clinical outcomes tested.† Risk factors for ARDS are not mutually exclusive. Open table in a new tab CDCA-d4, Chenodeoxycholic acid-d4; LCA-d4, lithocolic acid-d4; RF, radiofrequency; RT, retention time. Pooled plasma from 5 volunteers of varied sex and race was analyzed on 2 days (days 1 and 8) to assess technical reproducibility. Four extraction replicates were performed on each day, and each sample was analyzed 5 times. Concentrations are expressed as nanograms per milliliter of plasma. %RSD, Percentage relative SD. CNS, Central nervous system." @default.
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• W2895550979 date "2019-01-01" @default.
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• W2895550979 title "Cholestenoic acid is a prognostic biomarker in acute respiratory distress syndrome" @default.
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