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• W2885283225 abstract "Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species. Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species. The maintenance of optimal pulmonary surfactant function and metabolism is critical in minimizing surface tension forces within the lungs and preventing alveolar collapse at the end of expiration. Primary insufficiency of the immature lungs of preterm infants is the principal cause of neonatal respiratory distress syndrome (1.Avery M.E. Mead J. Surface properties in relation to atelectasis and hyaline membrane disease.AMA J. Dis. Child. 1959; 97: 517-523PubMed Google Scholar). Secondary surfactant dysfunction also contributes to the severe lung pathology of adult patients ventilated for acute respiratory distress syndrome due to a combination of increased concentrations of inhibitory edema proteins (2.Robertson B. Surfactant inactivation and surfactant therapy in acute respiratory distress syndrome (ARDS).Monaldi Arch. Chest Dis. 1998; 53: 64-69PubMed Google Scholar, 3.Lopez-Rodriguez E. Perez-Gil J. Structure-function relationships in pulmonary surfactant membranes: from biophysics to therapy.Biochim. Biophys. Acta. 2014; 1838: 1568-1585Crossref PubMed Scopus (177) Google Scholar, 4.Echaide M. Autilio C. Arroyo R. Perez-Gil J. Restoring pulmonary surfactant membranes and films at the respiratory surface.Biochim. Biophys. Acta. 2017; 1859: 1725-1739Crossref PubMed Scopus (61) Google Scholar), phospholipase-mediated hydrolysis of surfactant phospholipids (5.Arbibe L. Koumanov K. Vial D. Rougeot C. Faure G. Havet N. Longacre S. Vargaftig B.B. Bereziat G. Voelker D.R. et al.Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction.J. Clin. Invest. 1998; 102: 1152-1160Crossref PubMed Scopus (155) Google Scholar), oxidant and antioxidant imbalance (6.Lang J.D. McArdle P.J. O'Reilly P.J Matalon S. Oxidant-antioxidant balance in acute lung injury.Chest. 2002; 122: 314S-320SAbstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), and damage to the alveolar type II (ATII) epithelial cell responsible for surfactant synthesis and secretion (7.Crim C. Longmore W.J. Sublethal hydrogen peroxide inhibits alveolar type II cell surfactant phospholipid biosynthetic enzymes.Am. J. Physiol. 1995; 268: L129-L135PubMed Google Scholar). Phospholipids represent the major surface-active components of surfactant, with cholesterol and surfactant proteins B and C regulating their fluidity (8.Nahak P. Nag K. Hillier A. Devraj R. Thompson D.W. Manna K. Makino K. Ohshima H. Nakahara H. Shibata O. et al.Effect of serum, cholesterol and low density lipoprotein on the functionality and structure of lung surfactant films.J. Oleo Sci. 2014; 63: 1333-1349Crossref PubMed Scopus (4) Google Scholar) and rapid adsorption to the alveolar air-liquid interface (9.Hobi N. Giolai M. Olmeda B. Miklavc P. Felder E. Walther P. Dietl P. Frick M. Perez-Gil J. Haller T. A small key unlocks a heavy door: the essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies.Biochim. Biophys. Acta. 2016; 1863: 2124-2134Crossref PubMed Scopus (30) Google Scholar, 10.Parra E. Perez-Gil J. Composition, structure and mechanical properties define performance of pulmonary surfactant membranes and films.Chem. Phys. Lipids. 2015; 185: 153-175Crossref PubMed Scopus (179) Google Scholar). Phospholipid composition is dominated by phosphatidylcholine (PC), with a high content of the disaturated species dipalmitoylphosphatidylcholine (DPPC) compared with typical mammalian cell membranes (11.Postle A.D. Heeley E.L. Wilton D.C. A comparison of the molecular species compositions of mammalian lung surfactant phospholipids.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 65-73Crossref PubMed Scopus (146) Google Scholar). This elevated DPPC, the major surface-active agent, is maintained by a complex intra-alveolar metabolism. PC is synthesized on the endoplasmic reticulum of the ATII cell with a lower content of DPPC (12.Post M. Schuurmans E.A.J.M. Batenburg J.J. Van Golde L.M.G. Mechanisms involved in the synthesis of disaturated phosphatidylcholine by alveolar type 2 cells isolated from adult rat lung.Biochim. Biophys. Acta. 1983; 750: 68-77Crossref PubMed Scopus (62) Google Scholar, 13.Burdge G.C. Kelly F.J. Postle A.D. Synthesis of phosphatidylcholine in guinea-pig fetal lung involves acyl remodelling and differential turnover of individual molecular species.Biochim. Biophys. Acta. 1993; 1166: 251-257Crossref PubMed Scopus (26) Google Scholar) and is then subjected to selective ABCA3-mediated transport into anhydrous lamellar storage organelles (14.Matsumura Y. Sakai H. Sasaki M. Ban N. Inagaki N. ABCA3-mediated choline-phospholipids uptake into intracellular vesicles in A549 cells.FEBS Lett. 2007; 581: 3139-3144Crossref PubMed Scopus (53) Google Scholar, 15.Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. ABCA3 is a lamellar body membrane protein in human lung alveolar type ii cells.FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (234) Google Scholar) combined with a process of acyl remodeling catalyzed by sequential phospholipase A2 and lysophosphatidylcholine (lysoPC) acyltransferase activities (13.Burdge G.C. Kelly F.J. Postle A.D. Synthesis of phosphatidylcholine in guinea-pig fetal lung involves acyl remodelling and differential turnover of individual molecular species.Biochim. Biophys. Acta. 1993; 1166: 251-257Crossref PubMed Scopus (26) Google Scholar, 16.Bridges J.P. Ikegami M. Brilli L.L. Chen X. Mason R.J. Shannon J.M. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice.J. Clin. Invest. 2010; 120: 1736-1748Crossref PubMed Scopus (101) Google Scholar). Surfactant enriched in DPPC is then secreted into the alveolar lining fluid by exocytosis of lamellar bodies, followed by rapid adsorption to the air-liquid interface. Surfactant is subsequently catabolized by ATII cell endocytosis (17.Jain D. Dodia C. Fisher A.B. Bates S.R. Pathways for clearance of surfactant protein A from the lung.Am. J. Physiol. 2005; 289: L1011-L1018Crossref PubMed Scopus (19) Google Scholar), metabolism by alveolar macrophages (18.Gräbner R. Meerbach W. Phagocytosis of surfactant by alveolar macrophages in vitro.Am. J. Physiol. 1991; 261: L472-L477Crossref PubMed Google Scholar), and loss up the bronchial tree (19.Wright S.M. Hockey P.M. Enhorning G. Strong P. Reid K.B. Holgate S.T. Djukanovic R. Postle A.D. Altered airway surfactant phospholipid composition and reduced lung function in asthma.J. Appl. Physiol. 2000; 89: 1283-1292Crossref PubMed Scopus (118) Google Scholar). A proportion of the surfactants taken up by ATII cells is subsequently recycled into lamellar bodies for resecretion into the alveolus (20.Gross N.J. Barnes E. Narine K.R. Recycling of surfactant in black and beige mice, pool sizes and kinetics.J. Appl. Physiol. 1988; 64: 2017-2025Crossref PubMed Scopus (30) Google Scholar, 21.Wright J.R. Clearance and recycling of pulmonary surfactant.Am. J. Physiol. 1990; 259: L1-L12PubMed Google Scholar). Studies in rabbits indicate that surfactant recycling is more active in neonatal compared with adult animals (22.Jacobs H. Jobe A. Ikegami M. Jones S. Surfactant phosphatidylcholine source, fluxes and turnover times in 3-day-old, 10-day-old and adult rabbits.J. Biol. Chem. 1982; 257: 1805-1810Abstract Full Text PDF PubMed Google Scholar), leading to slower apparent alveolar turnover and increased half-life. While these metabolic pathways have been well-defined by elegant studies that monitored the incorporation into PC of substrates labeled with radioactive isotopes (12.Post M. Schuurmans E.A.J.M. Batenburg J.J. Van Golde L.M.G. Mechanisms involved in the synthesis of disaturated phosphatidylcholine by alveolar type 2 cells isolated from adult rat lung.Biochim. Biophys. Acta. 1983; 750: 68-77Crossref PubMed Scopus (62) Google Scholar, 13.Burdge G.C. Kelly F.J. Postle A.D. Synthesis of phosphatidylcholine in guinea-pig fetal lung involves acyl remodelling and differential turnover of individual molecular species.Biochim. Biophys. Acta. 1993; 1166: 251-257Crossref PubMed Scopus (26) Google Scholar), there is a paucity of information available regarding their regulation in molecular terms, which are the biologically relevant molecules defined by their combination of esterified fatty acids. Many studies have relied on the quantification of total disaturated PC species after OsO4 oxidation of unsaturated species (23.Mason R.J. Nellenbogen J. Clements J.A. Isolation of disaturated phosphatidylcholine with osmium tetroxide.J. Lipid Res. 1976; 17: 281-284Abstract Full Text PDF PubMed Google Scholar), but this analytical approach provides minimal information at the level of individual PC molecular species. Second, analyzing radioactive-labeled PC species is laborious and time-consuming (13.Burdge G.C. Kelly F.J. Postle A.D. Synthesis of phosphatidylcholine in guinea-pig fetal lung involves acyl remodelling and differential turnover of individual molecular species.Biochim. Biophys. Acta. 1993; 1166: 251-257Crossref PubMed Scopus (26) Google Scholar), and the use of radioactivity is precluded in clinical studies. Our group has developed methodologies to probe the metabolism of surfactant molecular species in greater detail (24.Bernhard W. Pynn C.J. Jaworski A. Rau G.A. Hohlfeld J.M. Freihorst J. Poets C.F. Stoll D. Postle A.D. Mass spectrometric analysis of surfactant metabolism in human volunteers using deuteriated choline.Am. J. Respir. Crit. Care Med. 2004; 170: 54-58Crossref PubMed Google Scholar, 25.Postle A.D. Henderson N.G. Koster G. Clark H.W. Hunt A.N. Analysis of lung surfactant phosphatidylcholine metabolism in transgenic mice using stable isotopes.Chem. Phys. Lipids. 2011; 164: 549-555Crossref PubMed Scopus (23) Google Scholar). These protocols monitor the incorporation of stable isotope-labeled substrates into PC molecular species, which are then resolved by electrospray ionization MS/MS. The incorporation of deuterated methyl-D9-choline is common to the de novo synthesis of all PC molecular species by the CDP-choline pathway, and we have established mechanisms of acyl remodeling of surfactant PC synthesis by animal models (25.Postle A.D. Henderson N.G. Koster G. Clark H.W. Hunt A.N. Analysis of lung surfactant phosphatidylcholine metabolism in transgenic mice using stable isotopes.Chem. Phys. Lipids. 2011; 164: 549-555Crossref PubMed Scopus (23) Google Scholar) and adult volunteers and acute respiratory distress syndrome patients (26.Dushianthan A. Goss V. Cusack R. Grocott M.P. Postle A.D. Phospholipid composition and kinetics in different endobronchial fractions from healthy volunteers.BMC Pulm. Med. 2014; 14: 10Crossref PubMed Scopus (21) Google Scholar, 27.Dushianthan A. Goss V. Cusack R. Grocott M.P. Postle A.D. Altered molecular specificity of surfactant phosphatidycholine synthesis in patients with acute respiratory distress syndrome.Respir. Res. 2014; 15: 128Crossref PubMed Scopus (17) Google Scholar). In this study, we aimed to explore the differences between the catabolism, turnover, and metabolism of two exogenous pulmonary surfactants: CHF5633, which is a new synthetic surfactant produced by Chiesi Farmaceutici (Parma, Italy) currently under phase-II clinical investigations for neonatal respiratory distress syndrome treatment (28.Sweet D.G. Turner M.A. Stranak Z. Plavka R. Clarke P. Stenson B.J. Singer D. Goelz R. Fabbri L. Varoli G. et al.A first-in-human clinical study of a new SP-B and SP-C enriched synthetic surfactant (CHF5633) in preterm babies with respiratory distress syndrome.Arch. Dis. Child. Fetal Neonatal Ed. 2017; 102: F497-F503Crossref PubMed Scopus (47) Google Scholar), and poractant alfa (Curosurf; Chiesi Farmaceutici), an animal-derived surfactant preparation commonly used in clinical practice (29.Singh N. Halliday H.L. Stevens T.P. Suresh G. Soll R. Rojas-Reyes M.X. Comparison of animal-derived surfactants for the prevention and treatment of respiratory distress syndrome in preterm infants.Cochrane Database Syst. Rev. 2015; : CD010249PubMed Google Scholar). CHF5633 contains DPPC and 1-palmitoyl-2-oleoylglycero-3-phospho-1-glycerol (1:1) and incorporates analogs of both surfactant proteins B and C. This synthetic preparation has already been subjected to several translational in vivo acute studies that showed an efficacy profile similar to poractant alfa (30.Ricci F. Murgia X. Razzetti R. Pelizzi N. Salomone F. In vitro and in vivo comparison between poractant alfa and the new generation synthetic surfactant CHF5633.Pediatr. Res. 2017; 81: 369-375Crossref PubMed Scopus (37) Google Scholar, 31.Seehase M. Collins J.J. Kuypers E. Jellema R.K. Ophelders D.R. Ospina O.L. Perez-Gil J. Bianco F. Garzia R. Razzetti R. et al.New surfactant with SP-B and C analogs gives survival benefit after inactivation in preterm lambs.PLoS One. 2012; 7: e47631Crossref PubMed Scopus (66) Google Scholar). Nevertheless, while poractant alfa metabolism has been investigated in animal studies (32.Alberti A. Pettenazzo A. Enzi G.B. Palamidese A. Mapp C. Ventura P. Baritussio A. Uptake and degradation of curosurf after tracheal administration to newborn and adult rabbits.Eur. Respir. J. 1998; 12: 294-300Crossref PubMed Scopus (10) Google Scholar), CHF5633 metabolism needs to be explored in order to evaluate whether its synthetic origin and molecular composition differentiate its long-term fate following administration compared with animal-derived surfactants. Indeed, CHF5633 does not contain any polyunsaturated phospholipids, and its surfactant protein B and C analogs do not contain any amino acids (such as methionine, which is on the contrary present in the natural surfactant proteins) that is sensitive to oxidation (30.Ricci F. Murgia X. Razzetti R. Pelizzi N. Salomone F. In vitro and in vivo comparison between poractant alfa and the new generation synthetic surfactant CHF5633.Pediatr. Res. 2017; 81: 369-375Crossref PubMed Scopus (37) Google Scholar). In this study, we investigated the catabolism and metabolism of poractant alfa and CHF5633 containing as a tracer DPPC, where all 40 carbon atoms were replaced by the stable carbon-13 isotope [U13C]DPPC. The use of [U13C]DPPC has several significant advantages compared with partially labeled DPPC. First, it is very unlikely that hydrolysis products will be recycled back into fully labeled DPPC, and consequently the loss of [U13C]DPPC will represent catabolism in the absence of recycling. Second, as all hydrolysis products will also be fully 13C-labeled, the fate of their incorporation into all potential metabolic products can be readily assessed. The study was undertaken by administering [U13C]DPPC-labeled surfactants to adult mice. We also studied endogenous surfactant production and turnover in adult mice by giving them methyl-D9-choline as a precursor for the synthesis of lung PC to establish any potential for the inhibition of endogenous PC synthesis by exogenous surfactants. [U13C]DPPC (48.4 mg) was dissolved in 4 ml dichloromethane and transferred to a 500 ml round-bottom Quickfit flask to which total lipid extracts of four vials of poractant alfa (960 mg total weight), prepared using dichloromethane and methanol, were added. The organic solvent was removed by rotary evaporation under vacuum for 30 min at 40°C to provide a thin film of dried lipid, followed by purging with nitrogen gas to remove any residual solvent. PBS (10 ml) was then added to the flask, and the dried lipid extract was solvated by rotary evaporation without a vacuum for 30 min at 40°C. The resultant labeled surfactant emulsion was then stored in 1 ml aliquots at 4°C at a final DPPC concentration of 34 mg/ml. [U13C]DPPC was formulated into the fully synthetic surfactant during the production phase, replacing an equivalent amount of unlabeled DPPC (Chiesi Farmaceutici). The functional performance of both U13C-labeled surfactant preparations was judged to be fully comparable to their corresponding nonlabeled counterparts as assayed in the captive bubble surfactometer (31.Seehase M. Collins J.J. Kuypers E. Jellema R.K. Ophelders D.R. Ospina O.L. Perez-Gil J. Bianco F. Garzia R. Razzetti R. et al.New surfactant with SP-B and C analogs gives survival benefit after inactivation in preterm lambs.PLoS One. 2012; 7: e47631Crossref PubMed Scopus (66) Google Scholar, 33.Hidalgo A. Salomone F. Fresno N. Orellana G. Cruz A. Perez-Gil J. Efficient interfacially driven vehiculization of corticosteroids by pulmonary surfactant.Langmuir. 2017; 33: 7929-7939Crossref PubMed Scopus (30) Google Scholar) (see supplemental Fig. S1). Labeled and unlabeled preparations had comparable ability to adsorb rapidly into the air-water interface to form films capable of reproducibly reaching surface tensions <5 mN/m upon repetitive compression-expansion cycling. Dynamic compression-expansion isotherms of poractant alfa and CHF5633 had distinctive features that were fully mirrored by each corresponding labeled preparation. All animal procedures were approved internally by the University of Southampton Animal Welfare and Ethical Review Body and externally by the Home Office Animals in Science Regulation Unit. Male C57BL/6 wild-type mice aged 8–12 weeks were used for this study. The mice were bred in-house and kept under a normal 12 h dark/light cycle with free access to pelleted food and water. All animals in this study received appropriate care according to the criteria outlined in Kilkenny et al. (34.Kilkenny C. Browne W.J. Cuthill I.C. Emerson M. Altman D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.PLoS Biol. 2010; 8: e1000412Crossref PubMed Scopus (4729) Google Scholar). Each mouse was intranasally instilled with 50 µl (4 mg; equivalent to 200 mg/kg body weight) of either poractant alfa or the synthetic surfactant CHF5633 containing [U13C]DPPC as described previously and successfully found to deliver material to the alveoli of the lung (35.Knudsen L. Ochs M. Mackay R. Townsend P. Deb R. Muhlfeld C. Richter J. Gilbert F. Hawgood S. Reid K. et al.Truncated recombinant human SP-D attenuates emphysema and type II cell changes in SP-D deficient mice.Respir. Res. 2007; 8: 70Crossref PubMed Scopus (71) Google Scholar). At the same time, each mouse also received a 100 µl intraperitoneal injection of methyl-D9-choline chloride (10 mg/ml in water). After labeling, the mice were euthanized by carbon dioxide asphyxia at 0, 1.5, 3, 6, 12, 18, 24, 48, 72, and 96 h (n = 10–21 mice per time point/group). Bronchoalveolar lavage was performed in situ with 4 × 0.9 ml PBS, and the recovered bronchoalveolar lavage fluid (BALF) aliquots were combined. BALF was centrifuged at 300 g for 10 min at 4°C to pellet cells, and the supernatants were then transferred to new vials and stored at −80°C until extraction. Lung parenchyma was quickly dissected from the main bronchi, placed in cryotubes, and snap-frozen in liquid nitrogen. Right and left lung lobes were stored separately at −80°C until further analysis. Lavaged right lung lobes were weighed and homogenized in 1.6 ml of 0.9% saline using a Heidolph Silent Crusher S. Total lipid extraction was performed by the Bligh and Dyer method on 800 µl aliquots of lung homogenates or BALF supernatants after the addition of a dimyristoyl PC (10 nmol) internal standard to each sample (36.Bligh E.G. Dyer W.J.A. A rapid and sensitive method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42799) Google Scholar). Dichloromethane (2 ml), methanol (2 ml), and water (1 ml) were added to each sample, mixed well to allow for the formation of a biphasic mixture, and then centrifuged at 1,500 g for 10 min at 20°C. The dichloromethane-rich lower phase was recovered, dried under a stream of nitrogen gas, and stored at −20°C until analysis by MS. MS analysis was performed on a Waters XEVO TQ-MS instrument using electrospray ionization. Dried samples were dissolved in a 1 ml mixture of methanol-dichloromethane-concentrated ammonium acetate (300 mM) in water (66:30:4 [v/v]). The sample solution was infused into the instrument without chromatography using the loop injection method. Different diagnostic precursor scans were performed to detect the different head group species. The use of different MS approaches was successfully applied for the characterization of phospholipid classes of poractant alfa (37.Pelizzi N. Catinella S. Barboso S. Zanol M. Different electrospray tandem mass spectrometric approaches for rapid characterization of phospholipid classes of curosurf, a natural pulmonary surfactant.Rapid Commun. Mass Spectrom. 2002; 16: 2215-2220Crossref PubMed Scopus (19) Google Scholar). In this study, the various diagnostic MS/MS scans used for characterizing PC metabolism are summarized in Table 1. Unlabeled PC and newly synthesized PC labeled with [D9]choline were calculated from precursor ion scans of phosphorylcholine fragment ions at m/z 184 and m/z 193, respectively. Precursor ion scans of m/z 189 detected the PC species containing five labeled 13C atoms in their choline head group. The various neutral loss scans all detected DPPC species with a variety of labeled components. Neutral loss scans of 551 and 586 detected unlabeled DPPC and [U13C]DPPC, respectively, while the ions at 737 and 771 were their respective M+3+ and M-3+ isotopomers. All other neutral loss scans detected DPPC species incorporating the various 13C-metabolic products of [U13C]DPPC hydrolysis.TABLE 1Diagnostic MS/MS scans for the analysis of PC metabolismDiagnostic MS/MS Scans for Choline Head GroupsMS/MS ModeLipidsRange (m/z)CharacterizationPrecursor m/z 184PC400–900PC species containing phosphorylcholinePrecursor m/z 189[513C]PC400–900PC species containing [513C]cholinePrecursor m/z 193[D9]PC400–900PC species containing [D9]cholineDiagnostic MS/MS Scans for DPPC with Different Labeled Diacylglycerol ComponentsMS/MS ModeLipidsRange (m/z)Ion (m/z)CharacterizationNeutral loss 551DPPC732–745734.5Unlabeled DPPCNeutral loss 554[313C]DPPC735–748737.5DPPC–[313C]glycerol or DPPC M+3+Neutral loss 567[1613C]DPPC748–761750.8DPPC–one 1613C palmitateNeutral loss 570[1913C]DPPC751–764753.5DPPC–one 1613C palmitate + [313C]glycerol[2413C]DPPC758.5DPPC–[2413C]lysoPC16:0 + [1612C]palmitateNeutral loss 583[3213C]DPPC764–777766.5DPPC–two 1613C palmitates[3713C]DPPC771.5[U13C]DPPC M-3+Neutral loss 586[U13C]DPPC767–780774.5[U13C]DPPCDiagnostic MS/MS Precursor Ion Scans in Negative IonizationMS/MS ModeLipidsRangeCharacterizationPrecursor 255PC400–900PC species containing unlabeled palmitatePrecursor 271[1613C]PC400–900PC species containing [1613C]palmitate Open table in a new tab Spectra were processed using a visual basic macro program developed in-house. Initially, MS spectra were smoothed, baseline-subtracted, and exported to individual Excel files. These files were then imported into the macro program and corrected for 12C- or 13C-isotopic effects. Enrichment values for individual molecular species were calculated from the percentage ratio of the corrected abundances of the stable isotope-labeled isotopomer to the sum of all isotopomers. For [U13C]DPPC, as an example, the calculation would be as follows: These calculated enrichment values were then normalized using the enrichments of [U13C]DPPC in the labeled CHF5633 and poractant alfa preparations according to the following formula: The composition and enrichment of [U13C]DPPC surfactants were also determined by electrospray ionization MS using the same phospholipid extraction protocol as mentioned above for samples. Enrichment values were determined using the same precursor ions, giving values for two batches of poractant alfa of 4.54% and 7.55% and one for CHF5633 of 2.87%. Diagnostic precursor scans of the phosphocholine ion fragment readily distinguished 13C-labeled PC from unlabeled PC. Precursor ion scans of m/z 184 (P184) detected the unlabeled PC composition of the mouse surfactant, poractant alfa, and CHF5633 (Fig. 1A–C). MS/MS fragmentation of [U13C]DPPC generated an ion product of m/z 189 that contained five 13C atoms and, consequently, a precursor ion scan of m/z 189 (P189) detected [U13C]DPPC (Fig. 1D). The P189 scan was then used to quantify exogenous surfactant catabolism. While the mouse surfactant (Fig. 1A) and poractant alfa (Fig. 1B) contain similar molecular species of PC, they have different compositions, with the mouse surfactant containing double the percentage distribution of the monounsaturated species PC32:1 (PC16:0/16:1; m/z 732) and CHF5633 containing only PC32:0 (PC16:0/16:0; m/z 734). As shown in supplemental Fig. S2, these differences in composition are important for providing an independent verification of the turnover of the labeled surfactants. As expected from a fully labeled molecule, the isotopomer pattern for [U13C]DPPC was M-1, -2, -3 instead of M+1, +2, +3, demonstrating isotopomers with one, two, or three 12C atoms. An analysis of this isotopomer pattern indicated that [U13C]DPPC was isotopically ∼99% enriched with the label (Fig. 2D), but it also contained minor amounts of labeled contaminants (e.g., m/z 804). These contaminants, however, did not significantly impair the characterization of any PC species that contained 13C-labeled moieties derived from the metabolism of [U13C]DPPC (see below). Compositions of phosphatidylglycerol (precursor scan m/z 153) and phosphatidylinositol (precursor scan m/z 241) were very similar for the mouse surfactant and poractant alfa and, as expected, CHF5633 contained only PG16:0/18:1 (m/z 747) (results not shown). We had two potential concerns about the use of this heavily labeled DPPC as a tracer of surfactant metabolism. First, would the presence of the labeled material significantly impair the physical surface tension-reducing properties of either surfactant and, second, would exogenous labeled and unlabeled surfactants be metabolized at equivalent rates? Captive bubble surfactometer analysis (supplemental Fig. S1) confirmed that both poractant alfa and CHF5633 retained entirely comparable surface properties after formulation with their nonlabeled counterparts. Initial adsorption isotherms are typically different, as exhibited by CHF5633 and poractant alfa, and the ones shown in supplemental Fig. S1 are representative of such a difference. Poractant alfa always adsorbs to the interface very rapidly, reaching close to equilibrium surface tensions in a few seconds. There are no significant differences as a consequence of the reconstitution of an isotopically labeled version of poractant alfa with respect to the original formulation. In contrast, CHF5633 typically exhibits a somewhat heterogeneous behavior at initial adsorption, with a sharp decay in surface tension to the equilibrium values occurring at variable times (from a few to tens of seconds) after injection. This is the reason why initial adsorption isotherms of CHF5633 exhibit frequently large standard deviations, as a consequence of averaging isotherms with the surface tension decay occurring at different times. Still, the behavior in terms of initial adsorption is again" @default.
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• W2885283225 title "Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice" @default.
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