Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila.

Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts.

Stratification of asthma by lipidomic profiling of induced sputum supernatant.

BACKGROUND: Asthma is a chronic respiratory disease with significant heterogeneity in its clinical presentation and pathobiology. There is need for improved understanding of respiratory lipid metabolism in asthma patients and its relation to observable clinical features. OBJECTIVE: We performed a comprehensive, prospective, cross-sectional analysis of the lipid composition of induced sputum supernatant obtained from asthma patients with a range of disease severities, as well as from healthy controls. METHODS: Induced sputum supernatant was collected from 211 adults with asthma and 41 healthy individuals enrolled onto the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study. Sputum lipidomes were characterized by semiquantitative shotgun mass spectrometry and clustered using topologic data analysis to identify lipid phenotypes. RESULTS: Shotgun lipidomics of induced sputum supernatant revealed a spectrum of 9 molecular phenotypes, highlighting not just significant differences between the sputum lipidomes of asthma patients and healthy controls, but also within the asthma patient population. Matching clinical, pathobiologic, proteomic, and transcriptomic data helped inform the underlying disease processes. Sputum lipid phenotypes with higher levels of nonendogenous, cell-derived lipids were associated with significantly worse asthma severity, worse lung function, and elevated granulocyte counts. CONCLUSION: We propose a novel mechanism of increased lipid loading in the epithelial lining fluid of asthma patients resulting from the secretion of extracellular vesicles by granulocytic inflammatory cells, which could reduce the ability of pulmonary surfactant to lower surface tension in asthmatic small airways, as well as compromise its role as an immune regulator.

Prediction of Neonatal Respiratory Distress Biomarker Concentration by Application of Machine Learning to Mid-Infrared Spectra.

The authors of this study developed the use of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) combined with machine learning as a point-of-care (POC) diagnostic platform, considering neonatal respiratory distress syndrome (nRDS), for which no POC currently exists, as an example. nRDS can be diagnosed by a ratio of less than 2.2 of two nRDS biomarkers, lecithin and sphingomyelin (L/S ratio), and in this study, ATR-FTIR spectra were recorded from L/S ratios of between 1.0 and 3.4, which were generated using purified reagents. The calibration of principal component (PCR) and partial least squares (PLSR) regression models was performed using 155 raw baselined and second derivative spectra prior to predicting the concentration of a further 104 spectra. A three-factor PLSR model of second derivative spectra best predicted L/S ratios across the full range (R2: 0.967; MSE: 0.014). The L/S ratios from 1.0 to 3.4 were predicted with a prediction interval of +0.29, -0.37 when using a second derivative spectra PLSR model and had a mean prediction interval of +0.26, -0.34 around the L/S 2.2 region. These results support the validity of combining ATR-FTIR with machine learning to develop a point-of-care device for detecting and quantifying any biomarker with an interpretable mid-infrared spectrum.

Mass spectrometry imaging of phosphatidylcholine metabolism in lungs administered with therapeutic surfactants and isotopic tracers.

Mass spectrometry imaging (MSI) visualizes molecular distributions throughout tissues but is blind to dynamic metabolic processes. Here, MSI with high mass resolution together with multiple stable isotope labeling provided spatial analyses of phosphatidylcholine (PC) metabolism in mouse lungs. Dysregulated surfactant metabolism is central to many respiratory diseases. Metabolism and turnover of therapeutic pulmonary surfactants were imaged from distributions of intact and metabolic products of an added tracer, universally 13C-labeled dipalmitoyl PC (U13C-DPPC). The parenchymal distributions of newly synthesized PC species were also imaged from incorporations of methyl-D9-choline. This dual labeling strategy demonstrated both lack of inhibition of endogenous PC synthesis by exogenous surfactant and location of acyl chain remodeling processes acting on the U13C-DPPC-labeled surfactant, leading to formation of polyunsaturated PC lipids. This ability to visualize discrete metabolic events will greatly enhance our understanding of lipid metabolism in diverse tissues and has potential application to both clinical and experimental studies.

Chronic pharmacological antagonism of the GM-CSF receptor in mice does not replicate the pulmonary alveolar proteinosis phenotype but does alter lung surfactant turnover.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.

Increased surfactant protein-D levels in the airways of preterm neonates with sepsis indicated responses to infectious challenges.

AIM: Sepsis is multifactorial and potentially devastating for preterm neonates. Changes in surfactant protein-D (SP-D), phosphatidylcholine (PC) and PC molecular species during infection may indicate innate immunity or inflammation during sepsis. We aimed to compare these important pulmonary molecules in ventilated neonates without or with sepsis. METHODS: Endotracheal aspirates were collected from preterm neonates born at 23-35 weeks and admitted to the neonatal intensive care unit at the John Radcliffe Hospital, Oxford, UK, from October 2000 to March 2002. Samples were collected at one day to 30 days and analysed for SP-D, total PC and PC molecular species concentrations using enzyme-linked immunosorbent assay and mass spectrometry. RESULTS: We found that 8/54 (14.8%) neonates developed sepsis. SP-D (p < 0.0001), mono- and di-unsaturated PC were significantly increased (p = 0.05), and polyunsaturated PC was significantly decreased (p < 0.01) during sepsis compared to controls. SP-D:PC ratios were significantly increased during sepsis (p < 0.001), and SP-D concentrations were directly related to gestational age in neonates with sepsis (r2  = 0.389, p < 0.01). CONCLUSION: Increased SP-D levels and changes in PC molecular species during sepsis were consistent with direct or indirect pulmonary inflammatory processes. Very preterm neonates we able to mount an acute inflammatory innate immune response to infectious challenges, despite low levels of surfactant proteins at birth.

Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells.

Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors.

Abnormal liver phosphatidylcholine synthesis revealed in patients with acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with a severe pro-inflammatory response; although decreased plasma cholesterol concentration has been linked to systemic inflammation, any association of phospholipid metabolic pathways with ARDS has not been characterized. Plasma phosphatidylcholine (PC), the major phospholipid of circulating lipoproteins, is synthesized in human liver by two biologically diverse pathways: the cytidine diphosphocholine (CDP):choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways. Here, we used ESI-MS/MS both to characterize plasma PC compositions and to quantify metabolic fluxes of both pathways using stable isotopes in patients with severe ARDS and in healthy controls. Direct incorporation of methyl-D9-choline estimated CDP:choline pathway flux, while PEMT flux was determined from incorporations of one and two methyl-D3 groups derived from methyl-D9-choline. The results of MS/MS analysis showed significant alterations in plasma PC composition in patients with ARDS versus healthy controls. In particular, the increased overall methyl-D9-PC enrichment and, most importantly, the much lower methyl-D3-PC and methyl-D6-PC enrichments suggest increased flux through the CDP:choline pathway and reduced flux through the PEMT pathway in ARDS. To our knowledge, this study is the first to demonstrate significant plasma PC molecular compositional changes combined with associated alterations in the dynamics of PC synthetic pathways in patients with ARDS.

Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice.

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.

Lipidomics of Thalassiosira pseudonana under Phosphorus Stress Reveal Underlying Phospholipid Substitution Dynamics and Novel Diglycosylceramide Substitutes.

Phytoplankton replace phosphorus-containing lipids (P-lipids) with non-P analogues, boosting growth in P-limited oceans. In the model diatom Thalassiosira pseudonana, the substitution dynamics of lipid headgroups are well described, but those of the individual lipids, differing in fatty acid composition, are unknown. Moreover, the behavior of lipids outside the common headgroup classes and the relationship between lipid substitution and cellular particulate organic P (POP) have yet to be reported. We investigated these through the mass spectrometric lipidomics of P-replete (P+) and P-depleted (P-) T. pseudonana cultures. Nonlipidic POP was depleted rapidly by the initiation of P stress, followed by the cessation of P-lipid biosynthesis and per-cell reductions in the P-lipid levels of successive generations. Minor P-lipid degradative breakdown was observed, releasing P for other processes, but most P-lipids remained intact. This may confer an advantage on efficient heterotrophic lipid consumers in P-limited oceans. Glycerophosphatidylcholine (PC), the predominant P-lipid, was similar in composition to its betaine substitute lipid. During substitution, PC was less abundant per cell and was more highly unsaturated in composition. This may reflect underlying biosynthetic processes or the regulation of membrane biophysical properties subject to lipid substitution. Finally, levels of several diglycosylceramide lipids increased as much as 10-fold under P stress. These represent novel substitute lipids and potential biomarkers for the study of P limitation in situ, contributing to growing evidence highlighting the importance of sphingolipids in phycology. These findings contribute much to our understanding of P-lipid substitution, a powerful and widespread adaptation to P limitation in the oligotrophic ocean.IMPORTANCE Unicellular organisms replace phosphorus (P)-containing membrane lipids with non-P substitutes when P is scarce, allowing greater growth of populations. Previous research with the model diatom species Thalassiosira pseudonana grouped lipids by polar headgroups in their chemical structures. The significance of the research reported here is threefold. (i) We described the individual lipids within the headgroups during P-lipid substitution, revealing the relationships between lipid headgroups and hinting at the underlying biochemical processes. (ii) We measured total cellular P, placing P-lipid substitution in the context of the broader response to P stress and yielding insight into the implications of substitution in the marine environment. (iii) We identified lipids previously unknown in this system, revealing a new type of non-P substitute lipid, which is potentially useful as a biomarker for the investigation of P limitation in the ocean.

Lipid phenotyping of lung epithelial lining fluid in healthy human volunteers.

BACKGROUND: Lung epithelial lining fluid (ELF)-sampled through sputum induction-is a medium rich in cells, proteins and lipids. However, despite its key role in maintaining lung function, homeostasis and defences, the composition and biology of ELF, especially in respect of lipids, remain incompletely understood. OBJECTIVES: To characterise the induced sputum lipidome of healthy adult individuals, and to examine associations between different ELF lipid phenotypes and the demographic characteristics within the study cohort. METHODS: Induced sputum samples were obtained from 41 healthy non-smoking adults, and their lipid compositions analysed using a combination of untargeted shotgun and liquid chromatography mass spectrometry methods. Topological data analysis (TDA) was used to group subjects with comparable sputum lipidomes in order to identify distinct ELF phenotypes. RESULTS: The induced sputum lipidome was diverse, comprising a range of different molecular classes, including at least 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids. TDA identified two distinct phenotypes differentiated by a higher total lipid content and specific enrichments of diacyl-glycerophosphocholines, -inositols and -glycerols in one group, with enrichments of sterols, glycolipids and sphingolipids in the other. Subjects presenting the lipid-rich ELF phenotype also had significantly higher BMI, but did not differ in respect of other demographic characteristics such as age or gender. CONCLUSIONS: We provide the first evidence that the ELF lipidome varies significantly between healthy individuals and propose that such differences are related to weight status, highlighting the potential impact of (over)nutrition on lung lipid metabolism.

Hepatic Steatosis Accompanies Pulmonary Alveolar Proteinosis.

Maintenance of tissue-specific organ lipid compositions characterizes mammalian lipid homeostasis. The lungs and liver synthesize mixed phosphatidylcholine (PC) molecular species that are subsequently tailored for function. The lungs progressively enrich disaturated PC directed to lamellar body surfactant stores before secretion. The liver accumulates polyunsaturated PC directed to very-low-density lipoprotein assembly and secretion, or to triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested for potential coordination between these spatially separated but possibly complementary phenomena under a major derangement of lung PC metabolism, pulmonary alveolar proteinosis (PAP), which overwhelms homeostasis and leads to excessive surfactant accumulation. Using static and dynamic lipidomics techniques, we compared (1) tissue PC compositions and contents, and (2) in lungs, the absolute rates of synthesis in both control mice and the granulocyte-macrophage colony-stimulating factor knockout model of PAP. Significant disaturated PC accumulation in bronchoalveolar lavage fluid, alveolar macrophage, and lavaged lung tissue occurred alongside increased PC synthesis, consistent with reported defects in alveolar macrophage surfactant turnover. However, microscopy using oil red O staining, coherent anti-Stokes Raman scattering, second harmonic generation, and transmission electron microscopy also revealed neutral-lipid droplet accumulations in alveolar lipofibroblasts of granular macrophage colony-stimulating factor knockout animals, suggesting that lipid homeostasis deficits extend beyond alveolar macrophages. PAP plasma PC composition was significantly polyunsaturated fatty acid enriched, but the content was unchanged and hepatic polyunsaturated fatty acid-enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with nonalcoholic fatty liver disease. These data suggest a hepatopulmonary axis of PC metabolism coordination, with wider implications for understanding and managing lipid pathologies in which compromise of one organ has unexpected consequences for another.

Stable isotope analysis of dynamic lipidomics.

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Rapid test for lung maturity, based on spectroscopy of gastric aspirate, predicted respiratory distress syndrome with high sensitivity.

AIM: Respiratory distress syndrome (RDS) is a major cause of mortality and morbidity in premature infants. By the time symptoms appear, it may already be too late to prevent a severe course, with bronchopulmonary dysplasia or mortality. We aimed to develop a rapid test of lung maturity for targeting surfactant supplementation. METHODS: Concentrations of the most surface-active lung phospholipid dipalmitoylphosphatidylcholine and sphingomyelin in gastric aspirates from premature infants were measured by mass spectrometry and expressed as the lecithin/sphingomyelin ratio (L/S). The same aspirates were analysed with mid-infrared spectroscopy. Subsequently, L/S was measured in gastric aspirates and oropharyngeal secretions from another group of premature infants using spectroscopy and the results were compared with RDS development. The 10-minute analysis required 10 µL of aspirate. RESULTS: An L/S algorithm was developed based on 89 aspirates. Subsequently, gastric aspirates were sampled in 136 infants of 24-31 weeks of gestation and 61 (45%) developed RDS. The cut-off value of L/S was 2.2, sensitivity was 92%, and specificity was 73%. In 59 cases, the oropharyngeal secretions had less valid L/S than gastric aspirate results. CONCLUSION: Our rapid test for lung maturity, based on spectroscopy of gastric aspirate, predicted RDS with high sensitivity.

Ready-to-use therapeutic food with elevated n-3 polyunsaturated fatty acid content, with or without fish oil, to treat severe acute malnutrition: a randomized controlled trial.

BACKGROUND: Ready-to-use therapeutic foods (RUTF) are lipid-based pastes widely used in the treatment of acute malnutrition. Current specifications for RUTF permit a high n-6 polyunsaturated fatty acid (PUFA) content and low n-3 PUFA, with no stipulated requirements for preformed long-chain n-3 PUFA. The objective of this study was to develop an RUTF with elevated short-chain n-3 PUFA and measure its impact, with and without fish oil supplementation, on children's PUFA status during treatment of severe acute malnutrition. METHODS: This randomized controlled trial in children with severe acute malnutrition in rural Kenya included 60 children aged 6 to 50 months who were randomized to receive i) RUTF with standard composition; ii) RUTF with elevated short chain n-3 PUFA; or iii) RUTF with elevated short chain n-3 PUFA plus fish oil capsules. Participants were followed-up for 3 months. The primary outcome was erythrocyte PUFA composition. RESULTS: Erythrocyte docosahexaenoic acid (DHA) content declined from baseline in the two arms not receiving fish oil. Erythrocyte long-chain n-3 PUFA content following treatment was significantly higher for participants in the arm receiving fish oil than for those in the arms receiving RUTF with elevated short chain n-3 PUFA or standard RUTF alone: 3 months after enrollment, DHA content was 6.3% (interquartile range 6.0-7.3), 4.5% (3.9-4.9), and 3.9% (2.4-5.7) of total erythrocyte fatty acids (P <0.001), respectively, while eicosapentaenoic acid (EPA) content was 2.0% (1.5-2.6), 0.7% (0.6-0.8), and 0.4% (0.3-0.5) (P <0.001). RUTF with elevated short chain n-3 PUFA and fish oil capsules were acceptable to participants and carers, and there were no significant differences in safety outcomes. CONCLUSIONS: PUFA requirements of children with SAM are not met by current formulations of RUTF, or by an RUTF with elevated short-chain n-3 PUFA without additional preformed long-chain n-3 PUFA. Clinical and growth implications of revised formulations need to be addressed in large clinical trials. TRIAL REGISTRATION: Clinicaltrials.gov NCT01593969. Registered 4 May 2012.

Regulation of lung surfactant phospholipid synthesis and metabolism.

The alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states.

Altered molecular specificity of surfactant phosphatidycholine synthesis in patients with acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening critical illness, characterised by qualitative and quantitative surfactant compositional changes associated with premature airway collapse, gas-exchange abnormalities and acute hypoxic respiratory failure. The underlying mechanisms for this dysregulation in surfactant metabolisms are not fully explored. Lack of therapeutic benefits from clinical trials, highlight the importance of detailed in-vivo analysis and characterisation of ARDS patients according to patterns of surfactant synthesis and metabolism. METHODS: Ten patients with moderate to severe ARDS were recruited. Most (90%) suffered from pneumonia. They had an infusion of methyl-D9-choline chloride and small volume bronchoalveolar lavage fluid (BALF) was obtained at 0,6,12,24,48,72 and 96 hours. Controls were healthy volunteers, who had BALF at 24 and 48 hours after methyl-D9-choline infusion. Compositional analysis and enrichment patterns of stable isotope labelling of surfactant phosphatidylcholine (PC) was determined by electrospray ionisation mass spectrometry. RESULTS: BALF of patients with ARDS consisted of diminished total PC and fractional PC16:0/16:0 concentrations compared to healthy controls. Compositional analysis revealed, reductions in fractional compositions of saturated PC species with elevated levels of longer acyl chain unsaturated PC species. Molecular specificity of newly synthesised PC fraction showed time course variation, with lower PC16:0/16:0 composition at earlier time points, but achieved near equilibrium with endogenous composition at 48 hours after methyl-D9-choline infusion. The enrichment of methyl-D9-choline into surfactant total PC is nearly doubled in patients, with considerable variation between individuals. CONCLUSIONS: This study demonstrate significant alterations in composition and kinetics of surfactant PC extracted from ARDS patients. This novel approach may facilitate biochemical phenotyping of ARDS patients according to surfactant synthesis and metabolism, enabling individualised treatment approaches for the management of ARDS patients in the future.

Phospholipid composition and kinetics in different endobronchial fractions from healthy volunteers.

BACKGROUND: Alterations in surfactant phospholipid compositions are a recognized feature of many acute and chronic lung diseases. Investigation of underlying mechanisms requires assessment of surfactant phospholipid molecular composition and kinetics of synthesis and turnover. Such studies have recently become possible in humans due to the development of stable isotope labelling combined with advances in analytical methods in lipidomics. The objectives of this study are to compare phospholipid molecular species composition and phosphatidylcholine synthesis and turnover in surfactant isolated from various endobronchial compartments in healthy adults. METHODS: Healthy adults (N = 10) were infused with methyl-D9-choline chloride and samples of induced sputum, tracheal wash and small volume bronchoalveolar lavage fluid were obtained subsequently at intervals up to 96 hours. Surfactant phospholipid composition and incorporation of stable isotope into surfactant phosphatidylcholine were determined by electrospray ionisation mass spectrometry. RESULTS: While molecular species compositions of phospholipids were similar for all three sample types, dipalmitoylphosphatidylcholine content was highest in lavage, intermediate in tracheal wash and lowest in sputum. Methyl-D9-choline incorporation into surfactant phosphatidylcholine was lower for sputum at 24 hours but reached equilibrium with other sample types by 48 hours. Fractional methyl-D9-dipalmitoylphosphatidylcholine incorporation for all sample types was about 0.5% of the endogenous composition. Lysophosphatidylcholine enrichment was twice than that of phosphatidylcholine. CONCLUSIONS: Tracheal secretions may be of value as a surrogate to assess bronchoalveolar lavage fluid surfactant molecular composition and metabolism in healthy people. Despite minor differences, the phospholipid molecular composition of induced sputum also showed similarities to that of bronchoalveolar lavage fluid. Detailed analysis of newly synthesized individual phosphatidylcholine species provided novel insights into mechanisms of surfactant synthesis and acyl remodelling. Lysophosphatidylcholine methyl-D9 incorporation patterns suggest that these species are secreted together with other surfactant phospholipids and are not generated in the air spaces by hydrolysis of secreted surfactant phosphatidylcholine. Application into patient populations may elucidate potential underlying pathophysiological mechanisms that lead to surfactant alterations in disease states.

In Vivo Cellular Phosphatidylcholine Kinetics of CD15+ Leucocytes and CD3+ T-Lymphocytes in Adults with Acute Respiratory Distress Syndrome.

Mammalian cell membranes composed of a mixture of glycerophospholipids, the relative composition of individual phospholipids and the dynamic flux vary between cells. In addition to their structural role, membrane phospholipids are involved in cellular signalling and immunomodulatory functions. In this study, we investigate the molecular membrane composition and dynamic flux of phosphatidylcholines in CD15+ leucocytes and CD3+ lymphocytes extracted from patients with acute respiratory distress syndrome (ARDS). We identified compositional variations between these cell types, where CD15+ cells had relatively higher quantities of alkyl-acyl PC species and CD3+ cells contained more arachidonoyl-PC species. There was a significant loss of arachidonoyl-PC in CD3+ cells in ARDS patients. Moreover, there were significant changes in PC composition and the methyl-D9 enrichment of individual molecular species in CD15+ cells from ARDS patients. This is the first study to perform an in vivo assessment of membrane composition and dynamic changes in immunological cells from ARDS patients.