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Background: Although estimates of suboptimal adherence to oral corticosteroids in asthma range from 30% to 50%, no ideal method for measurement exists; the impact of poor adherence in severe asthma is likely to be particularly high. Research Questions: What is the prevalence of suboptimal adherence detected by self-reporting and direct measures? Is suboptimal adherence associated with disease activity? Study Design and Methods: Data were included from individuals with severe asthma taking part in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study and prescribed daily oral corticosteroids. Participants completed the Medication Adherence Report Scale, a five-item questionnaire used to grade adherence on a scale from 1 to 5, and provided a urine sample for analysis of prednisolone and metabolites by liquid chromatography-mass spectrometry. Results: Data from 166 participants were included in this study: mean (SD) age, 54.2 (± 11.9) years; FEV 1, 65.1% (± 20.5%) predicted; female, 58%; 37% completing the Medication Adherence Report Scale reported suboptimal adherence; and 43% with urinary corticosteroid data did not have detectable prednisolone or metabolites in their urine. Good adherence by both methods was detected in 49 of the 142 (35%) of participants in whom both methods were performed; adherence detection did not match between methods in 53%. Self-reported high adherers had better asthma control and quality of life, whereas directly measured high adherers had lower blood eosinophil levels. Interpretation: Low adherence is a common problem in severe asthma, whether measured directly or self-reported. We report poor agreement between the two methods, suggesting some disassociation between self-assessment of medication adherence and regular oral corticosteroid use, which suggests that each approach may provide complementary information in clinical practice.

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 β subunit (GMCSFRβ) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRβ 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.

Climate change-induced global warming threatens the survival of key ecosystems including shallow water coral reefs. Elevated temperatures can disrupt the normal physiological functioning of photosynthetic organisms by altering the fluidity and permeability of chloroplast membranes that is defined and regulated by their lipid composition. Since the habitat-forming reef corals rely on the obligatory symbiosis with dinoflagellates of the family Symbiodiniaceae, their heat stress response can be expected to be strongly influenced by the symbiont's lipid metabolism. However, in contrast to the steady increase in the knowledge of the functioning of coral symbionts at the genomic and transcriptomic level, the understanding of their membrane lipid composition and regulation in response to temperature stress is lagging behind. We have utilised mass spectrometry-based lipidomic analyses to identify the key polar lipids that form the biological membranes of reef coral symbionts, comparing the thermotolerant species Durusdinium trenchii with the thermosensitive taxon Cladocopium C3, both hosted by Acropora valida. Our results indicate that the superior thermotolerance D. trenchii inside the host corals could be achieved through (1) the amount and saturation of sulfoquinovosyldiacylglycerols, in particular through putative photosystem II interactions, (2) the increased digalactosyldiacylglycerol to monogalactosyldiacylglycerol ratio with the potential to stabilise thylakoid membranes and integrated proteins, and (3) the chaperone-like function of lyso-lipids. Thereby, our study provides novel insights into the heat tolerance of coral symbionts, contributing to the understanding of the potential of coral reef ecosystems to respond and adjust to heat stress events that are becoming more frequent due to climate change. Finally, our identification of multiple mechanisms of heat tolerance in Symbiodiniaceae furthers the knowledge of the general stress physiology of photosynthetic organisms.

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

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero) phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipidcomposition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D proteinin BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS IIIA. usc Refereed/Peer-reviewed

Optical spectroscopy offers a potential non-invasive, label free and rapid method to assist clinicians to diagnose diseases for which biomarkers are known. Neonatal respiratory distress syndrome (nRDS) diagnosis in preterm infants is known to be correlated with the lecithin/sphingomyelin ratio (LS ratio) in gastric aspirates, with a ratio less than 2.2 indicating that surfactant replacement therapy is needed. Currently no widespread method exists that can give clinically relevant answers in less than 2 hours from the point of sample collection as it is difficult to identify those who could benefit from prompt surfactant treatment. Various LS ratios were generated using pure dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin (SM) dissolved in dichloromethane and infrared spectra generated using Attenuated Total Reflection (ATR) assisted Fourier Transform InfraRed spectrometry (FTIR). Subsequent analysis obtained the LS ratio using the spectra alone. Further, we demonstrate the application of principal component regression (PCR) and partial least squares (PLS) fits to measured spectra to assist in the determination of the LS ratio using a model trained with multiple runs of the different batches of the same concentration.

Unsaturated membrane phospholipids are susceptible to oxidation, either by reactive oxygen species or enzymatically, to generate a complex mixture of peroxy and hydroxyl species. They can then spontaneously decompose to truncated oxidised phospholipids composed of aldehyde, carboxyl and hydroxyl species of five to nine carbon atoms chain length, many of which exhibit potent biological activities. In addition, aldehydes can form Schiff's base reactions with protein lysines to form oxidised lipid:protein adducts. While a selection of oxidised phospholipids have been characterised in detail by a range of mass spectrometry techniques, including direct infusion and liquid chromatography mass spectrometry, there are relatively few reports of comprehensive analyses of oxidised phospholipids in disease states. Oxidised phospholipid species are widely thought to be central to the pathology of many diseases, but there is relatively little direct evidence to confirm this in vivo. This review provides an overview of the various analytical methodologies and then summarises their application to examples of chronic and acute disease, cardiovascular disease and acute respiratory distress syndrome, respectively. It highlights the gaps in information and indicates directions for future research.

Mass spectrometry imaging (MSI) visualises molecular distributions throughout tissues but is blind to dynamic metabolic processes. Here, MSI with high mass resolution together with multiple stable isotope labelling 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, universally13C-labelled dipalmitoyl PC (U[13C]DPPC). The parenchymal distributions of newly synthesised PC species were also imaged from incorporations ofmethyl-D9-choline. This dual labelling strategy demonstrated both lack of inhibition of endogenous PC synthesis by exogenous surfactant and location of acyl chain remodelling processes acting on the U[13C]DPPC-labelled surfactant, leading to formation of polyunsaturated PC lipids. This ability to visualise discrete metabolic events will greatly enhance our understanding of lipid metabolism in diverse tissues, and has potential application to both clinical and experimental studies.

Objectives SARS-Cov-2 virus preferentially binds to the Angiotensin Converting Enzyme 2 (ACE2) on alveolar epithelial type II cells, initiating an inflammatory response and tissue damage which may impair surfactant synthesis contributing to alveolar collapse, worsening hypoxia and leading to respiratory failure. The objective of this study is to evaluate the feasibility, safety and efficacy of nebulised surfactant in COVID-19 adult patients requiring mechanical ventilation for respiratory failure. Trial design This study is a dose-escalating randomized open-label clinical trial of 20 COVID-19 patients. Participants This study is conducted in two centres: University Hospital Southampton and University College London Hospitals. Eligible participants are aged ≥18, hospitalised with COVID-19 (confirmed by PCR), who require endotracheal intubation and are enrolled within 24 hours of mechanical ventilation. For patients unable to consent, assent is obtained from a personal legal representative (PerLR) or professional legal representative (ProfLR) prior to enrolment. The following are exclusion criteria: imminent expected death within 24 hours; specific contraindications to surfactant administration (e.g. known allergy, pneumothorax, pulmonary hemorrhage); known or suspected pregnancy; stage 4 chronic kidney disease or requiring dialysis (i.e., eGFR < 30); liver failure (Child-Pugh Class C); anticipated transfer to another hospital, which is not a study site, within 72 hours; current or recent (within 1 month) participation in another study that, in the opinion of the investigator, would prevent enrollment for safety reasons; and declined consent or assent. Intervention and comparator Intervention: The study is based on an investigational drug/device combination product. The surfactant product is Bovactant (Alveofact®), a natural animal derived (bovine) lung surfactant formulated as a lyophilized powder in 108 mg vials and reconstituted to 45 mg/mL in buffer supplied in a prefilled syringe. It is isolated by lung lavage and, by weight, is a mixture of: phospholipid (75% phosphatidylcholine, 13% phosphatidylglycerol, 3% phosphatidylethanolamine, 1% phosphatidylinositol and 1% sphingomyelin), 5% cholesterol, 1% lipid-soluble surfactant-associated proteins (SP-B and SP-C), very low levels of free fatty acid, lyso-phosphatidylcholine, water and 0.3% calcium. The Drug Delivery Device is the AeroFact-COVID™ nebulizer, an investigational device based on the Aerogen® Solo vibrating mesh nebulizer. The timing and escalation dosing plans for the surfactant are as follows. Cohort 1: Three patients will receive 10 vials (1080 mg) each of surfactant at dosing times of 0 hours, 8 hours and 24 hours. 2 controls with no placebo intervention. Cohort 2: Three patients will receive 10 vials (1080 mg) of surfactant at dosing times of 0 hours and 8 hours, and 30 vials (3240 mg) at a dosing time of 24 hours. 2 controls with no placebo intervention. Cohort 3: Three patients will receive 10 vials (1080 mg) of surfactant at a dosing time of 0 hours, and 30 vials (3240 mg) at dosing times of 8 hours and 24 hours. 2 controls with no placebo intervention. Cohort 4: Three patients will receive 30 (3240 mg) vials each of surfactant at dosing times of 0 hours, 8 hours and 24 hours. 2 controls. 2 controls with no placebo intervention. The trial steering committee, advised by the data monitoring committee, will review trial progression and dose escalation/maintenance/reduction after each cohort is completed (48-hour primary outcome timepoint reached) based on available feasibility, adverse event, safety and efficacy data. The trial will not be discontinued on the basis of lack of efficacy. The trial may be stopped early on the basis of safety or feasibility concerns. Comparator: No placebo intervention. All participants will receive usual standard of care in accordance with the local policies for mechanically ventilated patients and all other treatments will be left to the discretion of the attending physician. Main outcomes The co-primary outcome is the improvement in oxygenation (PaO2/FiO2 ratio) and pulmonary ventilation (Ventilation Index (VI), where VI = [RR x (PIP − PEEP) × PaCO2]/1000) at 48 hours after study initiation. The secondary outcomes include frequency and severity of adverse events (AEs), Adverse Device Effects (ADEs), Serious Adverse Events (SAEs) and Serious Adverse Device Events (SADEs), change in pulmonary compliance, change in positive end-expiratory pressure (PEEP) requirement of ventilatory support at 24 and 48 hours after study initiation, clinical improvement defined by time to one improvement point on the ordinal scale described in the WHO master protocol (2020) recorded while hospitalised, days of mechanical ventilation, mechanical ventilator free days (VFD) at day 21, length of intensive care unit stay, number of days hospitalised and mortality at day 28. Exploratory end points will include quantification of SARS-CoV-2 viral load from tracheal aspirates using PCR, surfactant dynamics (synthesis and turnover) and function (surface tension reduction) from deep tracheal aspirate samples (DTAS), surfactant phospholipid concentrations in plasma and DTAS, inflammatory markers (cellular and cytokine) in plasma and DTAS, and blood oxidative stress markers. Randomisation After informed assent, patients fulfilling inclusion criteria will be randomised to 3:2 for the treatment and control arms using an internet-based block randomization service (ALEA tool for clinical trials, FormsVision BV) in combination with electronic data collection. Randomisation will be done by the recruiting centre with a unique subject identifier specific to that centre. Blinding (masking) This is an open-labelled unblinded study. Numbers to be randomised (sample size) The total sample size is 20 COVID-19 mechanically ventilated patients (12 intervention; 8 control). Trial Status Current protocol version is V2 dated 5th of June 2020. The recruitment is currently ongoing and started on the 14th of October 2020. The anticipated study completion date is November 2021. Trial registration ClinicalTrials.gov: NCT04362059 (Registered 24 April 2020), EUDAMED number: CIV-GB-20-06-033328, EudraCT number: 2020-001886-35 (Registered 11 May 2020) Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

BACKGROUND Lipid metabolism in pregnancy delivers PUFAs from maternal liver to the developing fetus. The transition at birth to diets less enriched in PUFA is especially challenging for immature, extremely preterm infants who are typically supported by total parenteral nutrition. OBJECTIVE The aim was to characterize phosphatidylcholine (PC) and choline metabolism in preterm infants and demonstrate the molecular specificity of PC synthesis by the immature preterm liver in vivo. METHODS This MS-based lipidomic study quantified the postnatal adaptations to plasma PC molecular composition in 31 preterm infants

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.

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.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

BackgroundHeart surgery with cardio-pulmonary bypass (CPB) is associated with lung ischemia leading to injury and inflammation. It has been suggested this is a result of the lungs being kept deflated throughout the duration of CPB. Low frequency ventilation (LFV) during CPB has been proposed to reduce lung dysfunction.MethodsWe used a semi-biased multi-omic approach to analyse lung biopsies taken before and after CPB from 37 patients undergoing coronary artery bypass surgery randomised to both lungs left collapsed or using LFV for the duration of CPB. We also examined inflammatory and oxidative stress markers from blood samples from the same patients.Results30 genes were induced when the lungs were left collapsed and 80 by LFV. Post-surgery 26 genes were significantly higher in the LFV vs. lungs left collapsed, including genes associated with inflammation (e.g. IL6 and IL8) and hypoxia/ischemia (e.g. HIF1A, IER3 and FOS). Relatively few changes in protein levels were detected, perhaps reflecting the early time point or the importance of post-translational modifications. However, pathway analysis of proteomic data indicated that LFV was associated with increased “cellular component morphogenesis” and a decrease in “blood circulation”. Lipidomic analysis did not identify any lipids significantly altered by either intervention.DiscussionTaken together these data indicate the keeping both lungs collapsed during CPB significantly induces lung damage, oxidative stress and inflammation. LFV during CPB increases these deleterious effects, potentially through prolonged surgery time, further decreasing blood flow to the lungs and enhancing hypoxia/ischemia.

Background Asthma prevalence is 339 million globally. ‘Severe asthma’ (SA) comprises subjects with uncontrolled asthma despite proper management. Objectives To compare asthma from diverse ethnicities and environments. Methods A cross-sectional analysis of two adult cohorts, a Brazilian (ProAR) and a European (U-BIOPRED). U-BIOPRED comprised of 311 non-smoking with Severe Asthma (SAn), 110 smokers or ex-smokers with SA (SAs) and 88 mild to moderate asthmatics (MMA) while ProAR included 544 SA and 452 MMA. Although these projects were independent, there were similarities in objectives and methodology, with ProAR adopting operating procedures of U-BIOPRED. Results Among SA subjects, age, weight, proportion of former smokers and FEV1 pre-bronchodilator were similar. The proportion of SA with a positive skin prick tests (SPT) to aeroallergens, the scores of sino-nasal symptoms and quality of life were comparable. In addition, blood eosinophil counts (EOS) and the % of subjects with EOS > 300 cells/μl were not different. The Europeans with SA however, were more severe with a greater proportion of continuous oral corticosteroids (OCS), worse symptoms and more frequent exacerbations. FEV1/FVC pre- and post-bronchodilator were lower among the Europeans. The MMA cohorts were less comparable in control and treatment, but similar in the proportion of allergic rhinitis, gastroesophageal reflux disease and EOS >3%. Conclusions ProAR and U-BIOPRED cohorts, with varying severity, ethnicity and environment have similarities, which provide the basis for global external validation of asthma phenotypes. This should stimulate collaboration between asthma consortia with the aim of understanding SA, which will lead to better management.

Although the distribution of cellular membrane phospholipid composition is well characterised in human erythrocytes, in-vivo turnover and dynamic flux of phospholipids between plasma and erythrocytes in physiological and in particular during disease states are mostly unknown. Erythrocyte mass primarily consisted of lipids and phosphatidylcholine (PC) contributes to the significant proportion of phospholipid membrane composition. Esterified membrane PC can be utilised during pathological processes to generate pro and anti-inflammatory lipid mediators, which can contribute to the pathogenesis of acute respiratory distress syndrome (ARDS). In this study, utilising isotope labelling of choline and analytical methods with electrospray mass spectrometry (ESI-MS/MS), we characterised individual molecular composition and dynamic exchange of PC, sphingomyelins (SM) and lysopho-sphatidylcholines (LPC) between plasma and erythrocytes. In ARDS patients, there were significant alterations in PC molecular composition, coupled with a continuous loss of arachidonoyl-PC species over time. Infusion of methyl-D9-choline chloride resulted in enrichment of labelled choline into plasma PC and LPC via CDP-choline pathway with subsequent incorporation into erythrocyte PC. As expected, erythrocyte methyl-D9 PC enrichment is much slower than plasma. Patients had much faster and higher fractional enrichment of all PC and LPC molecules suggesting increased flux between plasma and erythrocytes. There was a particular pattern of incorporation, where the arachidonoyl-PC species achieved equilibrium with plasma rapidly and retained highest concentrations of enrichment compared to the other PC species. Increased enrichment of arachidonoyl-PC coupled with virtually no increase or depletion of its concentrations suggests the possibility of substrate donation for other cell types for the participation of eicosanoid biosynthesis during inflammatory conditions like ARDS. In summary, this study revealed an alerted pattern erythrocyte molecular phospholipid composition and flux in patients with acute respiratory distress syndrome and the pathological consequences of these changes needs further exploration.

BackgroundSystemic inflammation is a marker of ill health and has prognostic implications in multiple health settings. Urinary neopterin is an excellent candidate as a nonspecific marker of systemic inflammation. Expression as urinary neopterin-to-creatinine ratio (UNCR) normalizes for urinary hydration status. Major attractions include (a) urine vs blood sampling, (b) integration of inflammation over a longer period compared with serum sampling, and (c) high stability of neopterin and creatinine.MethodsA high-throughput ultraperformance LC-MS method was developed to measure neopterin and creatinine together from the same urine sample. The assay was applied in several clinical scenarios: healthy controls, symptomatic infections, and multiple sclerosis. Area under the curve was compared between weekly and monthly sampling scenarios. Analysis of a single pooled sample was compared with averaging results from analysis of individual samples.ResultsThe assay has excellent intraassay and interassay precision, linearity of dilution, and spike and recovery. Higher UNCR was demonstrated in female vs male individuals, older age, inflammatory disease (multiple sclerosis), and symptomatic infections. In healthy controls, fluctuations in inflammatory state also occurred in the absence of symptomatic infection or other inflammatory triggers. Analysis of a single pooled sample, made up from weekly urine samples, integrates inflammatory activity over time.ConclusionsUNCR is a useful biomarker of systemic inflammation. The method presented offers simplicity, speed, robustness, reproducibility, efficiency, and proven utility in clinical scenarios. UNCR fluctuations underline the importance of longitudinal monitoring, vs a single time point, to capture a more representative estimate of an individual's inflammatory state over time.

Lipid metabolism is one of the cornerstones of biochemistry, and these chemically diverse biomolecules play key roles in molecular physiology and mediate interactions between microbes and their environment that play out on cell ular to ecosystem scales. Marine phytoplankton fix in the range of 1 billion tonnes of carbon as lipid biomass each year, which goes on to fuel higher trophic levels or ends up in the marine dissolved organic matter pool. Yet despite the importance of the vast marine lipidome for global biogeochemistry, surprisingly little is known about its diverse inventory of molecular structures, or the influence that dynamic environmental conditions exert on microbial lipid synthesis, remodelling and turnover. To aid in this research, a high-throughput platform for comprehensive analysis of phytoplankton lipids was developed using Ultra-High Performance Supercritical Fluid Chromatography (UHPSFC). This recently developed technology combines a primary supercritical fluid (CO2) mobile phase with an organic co-solvent of choice. Using a simple 10 minute gradient and a sub-2 μm particle column, UHPSFC efficiently separates all of the major neutral and polar lipid classes encountered in phytoplankton in a single analysis. These can then be measured by tandem mass spectrometry using established precursor and neutral mass loss scans. To demonstrate the analytical power of this novel platform the lipid compositions of a diverse range of phytoplankton species grown in culture, as well as phytoplankton community samples from the Western Antarctic Peninsula, were analysed. With higher chromatographic resolution and a much shorter analysis time than current liquid chromatography methods, the application of U HPSFC has considerable potential to benefit large-scale lipidomic studies, including in the field of environmental microbiology.

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is, Highlights • The thiol redox metabolome comprises free and bound thiols, disulfides, and sulfide. • We developed a LC-MS/MS platform for detection of the thiol redox metabolome in biological samples. • Thiols in blood, urine and saliva are stabilized by derivatization with N-ethylmaleimide, also preventing oxidation. • Total and acid-labile thiols are determined following addition of DTT and acid. • Accuracy was investigated by comparison with an established method to assess GSH and GSSG. • Applicability was shown for plasma of 10 healthy volunteers, blood cells, urine and saliva.

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.

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalised management approach can be provided.OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinico-physiological parameters and performed an -omics analysis of sputum.METHODS: Partition-around-medoid clustering was applied to a training set of 266 asthma participants from the European U-BIOPRED adult cohort using 8 pre-specified clinic-physiological variables. This was repeated in a separate validation set of 152 asthmatics. The clusters were compared based on sputum proteomic and transcriptomic data.RESULTS: Four reproducible and stable clusters of asthmatics were identified. The training set cluster T1 consists of well-controlled moderate-to-severe asthmatics, while cluster T2 is a group of late-onset severe asthmatics with history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of non-smokers. Cluster T4 is predominantly composed of obese female uncontrolled severe asthmatics with increased exacerbations, but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters, T2, T3 and T4, had higher sputum eosinophilia than T1 with no differences in sputum neutrophil counts, exhaled nitric oxide and serum IgE levels.CONCLUSION: Clustering based on clinico-physiological parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.CLINICAL IMPLICATIONS: The definition of four distinct clusters of asthma linked to different pathobiological pathways provides a better template for the phenotyping and personalised treatment of severe asthma, where high unmet needs remain.CAPSULE SUMMARY: Unsupervised clustering of asthma on clinical features alone has led to the definition of four phenotypes. Sputum 'omics' analysis has revealed different biological pathways pointing towards potential new treatments.

Introduction: placental glutamine synthesis has been demonstrated in animals and is thought to increase the availability of this metabolically important amino acid to the fetus. Glutamine is of fundamental importance for cellular replication, cellular function and inter-organ nitrogen transfer. The objective of this study was to investigate the role of glutamate/glutamine metabolism by the isolated perfused human placenta in the provision of glutamine to the fetus.Methods: glutamate metabolism was investigated in the isolated dually perfused human placental cotyledon. U–13C-glutamate was used to investigate the movement of carbon and 15N-leucine to study movement of amino-nitrogen. Labelled amino acids were perfused via maternal or fetal arteries at defined flow rates. The enrichment and concentration of amino acids in the maternal and fetal veins were measured following 5 h of perfusion.Results: glutamate taken up from the maternal and fetal circulations was primarily converted into glutamine the majority of which was released into the maternal circulation. The glutamine transporter SNAT5 was localised to the maternal-facing membrane of the syncytiotrophoblast. Enrichment of 13C or 15N glutamine in placental tissue was lower than in either the maternal or fetal circulation, suggesting metabolic compartmentalisation within the syncytiotrophoblast.Discussion: placental glutamine synthesis may help ensure the placenta's ability to supply this amino acid to the fetus does not become limiting to fetal growth. Glutamine synthesis may also influence placental transport of other amino acids, metabolism, nitrogen flux and cellular regulation.Conclusions: placental glutamine synthesis may therefore be a central mechanism in ensuring that the human fetus receives adequate nutrition and is able to maintain growth

One of the most developed theories of phospholipid homeostasis is the intrinsic curvature hypothesis, which, in broad terms, postulates that cells regulate their lipid composition so as to keep constant the membrane stored curvature elastic energy. The implication of this hypothesis is that lipid composition is determined by a ratio control function consisting of the weighted sum of concentrations of type II lipids in the numerator and the weighted sum of concentrations of Type 0 lipids in the denominator. In previous work we used a data-driven approach, based on lipidomic data from asynchronous cell cultures, to determine a criterion that allows the different lipid species to be assigned to the set of type 0 or of type II lipids, and hence construct a ratio control function that serves as a proxy for the lipid contribution to total membrane stored curvature elastic energy in vivo. Here we apply the curvature elastic energy proxy to the analysis of lipid composition data from synchronous HeLa cells as they traverse the cell cycle. Our analysis suggests HeLa cells modify their membrane stored elastic energy through the cell cycle. In S-phase type 0 lipids are the most abundant, whilst in G2 type II lipids are most abundant. Changes in our proxy for membrane stored elastic energy correlate with membrane curvature dependent processes in the HeLa cell around division, providing some insights into the interplay between the individual lipid and protein contributions to membrane free energy.

The pathways and mechanisms that regulate pulmonary surfactant synthesis, processing, secretion and catabolism have been extensively characterised using classical biochemical and analytical approaches. These have constructed a model, largely in experimental animals, for surfactant phospholipid metabolism in the alveolar epithelial cell whereby phospholipid synthesised on the endoplasmic reticulum is selectively transported to lamellar body storage vesicles, where it is subsequently processed before secretion into the alveolus. Surfactant phospholipid is a complex mixture of individual molecular species defined by the combination of esterified fatty acid groups and a comprehensive description of surfactant phospholipid metabolism requires consideration of the interactions between such molecular species. However, until recently, lipid analytical techniques have not kept pace with the considerable advances in understanding of the enzymology and molecular biology of surfactant metabolism. Refinements in electrospray ionisation mass spectrometry (ESI-MS) can now provide very sensitive platforms for the rapid characterisation of surfactant phospholipid composition in molecular detail. The combination of ESI-MS and administration of phospholipid substrates labelled with stable isotopes extends this analytical approach to the quantification of synthesis and turnover of individual molecular species of surfactant phospholipid. As this methodology does not involve radioactivity, it is ideally suited to application in clinical studies. This review will provide an overview of the metabolic processes that regulate the molecular specificity of surfactant phosphatidylcholine together with examples of how the application of stable isotope technologies in vivo has, for the first time, begun to explore regulation of the molecular specificity of surfactant synthesis in human subjects.

Aim: Asthma has an increased prevalence of GORD. Our aim was to identify lipid biomarkers by unbiased lipidomics in sputum that predict clinically active GORD in asthmatic participants. Methods: This was a study of 157 asthmatics classified clinically into 3 groups: 82 severe non-smoking, 37 severe smoking and 38 mild/moderate, as defined by U-BIOPRED criteria: 57 with active GORD and 100 without GORD. Lipid profiles of sputum supernatants were obtained by high resolution shotgun mass spectrometry. Lipids with ≥20% abundance across the sample set were selected for analysis. Clinical and lipid data were analysed by non-parametric tests, univariate (ULR) and multiple logistic regression (MLR) analyses with stepwise backward selection for best predictors of active GORD. Results: Of all tested species, 123 differentially present lipids were selected by ULR (p≤0.10) and screened for contaminants. Further MLR was performed on 33 species (p≤0.05): 11 were found to be independent predictors by backward MLR, adjusted for cohort and gender (p m/z= 778.5802 and negative ESI m/z= 568.3601. The best clinical predictors were smoking severe asthma (p Conclusions: This study confirms disease severity as an independent predictor of active GORD and shows, for the first time, a set of lipid biomarkers that differentially predict active GORD, implying an effect of GORD on the airways. The role of these lipids remain to be elucidated.

The role of neutrophils in acute oxidative lung injury in preterm babies is presently unclear, with some investigators maintaining they contribute to tissue injury while others believe they do not. The aim of the present study was to determine whether neutropenia, induced by a specific neutrophil antibody, influenced the time course or extent of oxygen-induced injury of the immature lung. Preterm guinea pigs, delivered by caesarean section at 65 days' gestation (term=68 days), were injected intraperitoneally with either control serum (CS) or neutrophil antiserum (NAS; 200 μl/100 g body weight) once daily for 5 days. Pups were exposed to 95% oxygen for the first 72 h, and then allowed to recover in 21% oxygen for the subsequent 48 h. Groups of treated animals were also maintained in 21% oxygen for 5 days. Lungs were examined by bronchoalveolar lavage (BAL) at 72 h or 120 h. In CS-treated pups, exposure to 95% oxygen increased both the number of circulating neutrophils and those recovered by BAL at both 72 h and 120 h. Protein concentration in BAL fluid, an index of lung microvascular permeability, and BAL elastase and β-glucuronidase activities, indices of neutrophil activation, were significantly increased in pups exposed to 95% oxygen. Pups exposed to 95% oxygen and treated with NAS showed a decrease in numbers of circulating neutrophils (72 h, 9.53 vs 0.66 x 10(5)/ml, P0.0005; 120 h, 4.9 vs 0.08 x 10(5)/ml, P0.0005) and BAL fluid neutrophils (72 h, 3.1 vs 0.7 x 10(5)/ml, P0.05; 120 h, 12.4 vs 3.8 x 10(5)/ml, P0.05). BAL protein concentration, neutrophil elastase and β-glucuronidase activities in hyperoxia-exposed pups were similar following treatment with either CS or NAS. Although the number of circulating neutrophils were markedly depleted and expansion of the alveolar neutrophil pool was restricted in NAS-treated pups, the neutrophils recruited to the lung were activated and could have contributed to the increase in microvascular permeability in hyperoxia-exposed pups.

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 mu L 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. Funding Agencies|European Unions Horizon research and innovation programme