Integrative Plasma Metabolic and Lipidomic Modelling of SARS-CoV-2 Infection in Relation to Clinical Severity and Early Mortality Prediction.

An integrative multi-modal metabolic phenotyping model was developed to assess the systemic plasma sequelae of SARS-CoV-2 (rRT-PCR positive) induced COVID-19 disease in patients with different respiratory severity levels. Plasma samples from 306 unvaccinated COVID-19 patients were collected in 2020 and classified into four levels of severity ranging from mild symptoms to severe ventilated cases. These samples were investigated using a combination of quantitative Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry (MS) platforms to give broad lipoprotein, lipidomic and amino acid, tryptophan-kynurenine pathway, and biogenic amine pathway coverage. All platforms revealed highly significant differences in metabolite patterns between patients and controls (n = 89) that had been collected prior to the COVID-19 pandemic. The total number of significant metabolites increased with severity with 344 out of the 1034 quantitative variables being common to all severity classes. Metabolic signatures showed a continuum of changes across the respiratory severity levels with the most significant and extensive changes being in the most severely affected patients. Even mildly affected respiratory patients showed multiple highly significant abnormal biochemical signatures reflecting serious metabolic deficiencies of the type observed in Post-acute COVID-19 syndrome patients. The most severe respiratory patients had a high mortality (56.1%) and we found that we could predict mortality in this patient sub-group with high accuracy in some cases up to 61 days prior to death, based on a separate metabolic model, which highlighted a different set of metabolites to those defining the basic disease. Specifically, hexosylceramides (HCER 16:0, HCER 20:0, HCER 24:1, HCER 26:0, HCER 26:1) were markedly elevated in the non-surviving patient group (Cliff's delta 0.91-0.95) and two phosphoethanolamines (PE.O 18:0/18:1, Cliff's delta = -0.98 and PE.P 16:0/18:1, Cliff's delta = -0.93) were markedly lower in the non-survivors. These results indicate that patient morbidity to mortality trajectories is determined relatively soon after infection, opening the opportunity to select more intensive therapeutic interventions to these "high risk" patients in the early disease stages.

J-Edited DIffusional Proton Nuclear Magnetic Resonance Spectroscopic Measurement of Glycoprotein and Supramolecular Phospholipid Biomarkers of Inflammation in Human Serum.

Proton nuclear magnetic resonance (NMR) N-acetyl signals (Glyc) from glycoproteins and supramolecular phospholipids composite peak (SPC) from phospholipid quaternary nitrogen methyls in subcompartments of lipoprotein particles) can give important systemic metabolic information, but their absolute quantification is compromised by overlap with interfering resonances from lipoprotein lipids themselves. We present a J-Edited DIffusional (JEDI) proton NMR spectroscopic approach to selectively augment signals from the inflammatory marker peaks Glyc and SPCs in blood serum NMR spectra, which enables direct integration of peaks associated with molecules found in specific compartments. We explore a range of pulse sequences that allow editing based on peak J-modulation, translational diffusion, and T2 relaxation time and validate them for untreated blood serum samples from SARS-CoV-2 infected patients (n = 116) as well as samples from healthy controls and pregnant women with physiological inflammation and hyperlipidemia (n = 631). The data show that JEDI is an improved approach to selectively investigate inflammatory signals in serum and may have widespread diagnostic applicability to disease states associated with systemic inflammation.

Exploration of Human Serum Lipoprotein Supramolecular Phospholipids Using Statistical Heterospectroscopy in n-Dimensions (SHY-n): Identification of Potential Cardiovascular Risk Biomarkers Related to SARS-CoV-2 Infection.

SARS-CoV-2 infection causes a significant reduction in lipoprotein-bound serum phospholipids give rise to supramolecular phospholipid composite (SPC) signals observed in diffusion and relaxation edited 1H NMR spectra. To characterize the chemical structural components and compartmental location of SPC and to understand further its possible diagnostic properties, we applied a Statistical HeterospectroscopY in n-dimensions (SHY-n) approach. This involved statistically linking a series of orthogonal measurements made on the same samples, using independent analytical techniques and instruments, to identify the major individual phospholipid components giving rise to the SPC signals. Thus, an integrated model for SARS-CoV-2 positive and control adults is presented that relates three identified diagnostic subregions of the SPC signal envelope (SPC1, SPC2, and SPC3) generated using diffusion and relaxation edited (DIRE) NMR spectroscopy to lipoprotein and lipid measurements obtained by in vitro diagnostic NMR spectroscopy and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The SPC signals were then correlated sequentially with (a) total phospholipids in lipoprotein subfractions; (b) apolipoproteins B100, A1, and A2 in different lipoproteins and subcompartments; and (c) MS-measured total serum phosphatidylcholines present in the NMR detection range (i.e., PCs: 16.0,18.2; 18.0,18.1; 18.2,18.2; 16.0,18.1; 16.0,20.4; 18.0,18.2; 18.1,18.2), lysophosphatidylcholines (LPCs: 16.0 and 18.2), and sphingomyelin (SM 22.1). The SPC3/SPC2 ratio correlated strongly (r = 0.86) with the apolipoprotein B100/A1 ratio, a well-established marker of cardiovascular disease risk that is markedly elevated during acute SARS-CoV-2 infection. These data indicate the considerable potential of using a serum SPC measurement as a metric of cardiovascular risk based on a single NMR experiment. This is of specific interest in relation to understanding the potential for increased cardiovascular risk in COVID-19 patients and risk persistence in post-acute COVID-19 syndrome (PACS).

Low Volume in Vitro Diagnostic Proton NMR Spectroscopy of Human Blood Plasma for Lipoprotein and Metabolite Analysis: Application to SARS-CoV-2 Biomarkers.

The utility of low sample volume in vitro diagnostic (IVDr) proton nuclear magnetic resonance (1H NMR) spectroscopic experiments on blood plasma for information recovery from limited availability or high value samples was exemplified using plasma from patients with SARS-CoV-2 infection and normal controls. 1H NMR spectra were obtained using solvent-suppressed 1D, spin-echo (CPMG), and 2-dimensional J-resolved (JRES) spectroscopy using both 3 mm outer diameter SampleJet NMR tubes (100 µL plasma) and 5 mm SampleJet NMR tubes (300 µL plasma) under in vitro diagnostic conditions. We noted near identical diagnostic models in both standard and low volume IVDr lipoprotein analysis (measuring 112 lipoprotein parameters) with a comparison of the two tubes yielding R2 values ranging between 0.82 and 0.99 for the 40 paired lipoprotein parameters samples. Lipoprotein measurements for the 3 mm tubes were achieved without time penalty over the 5 mm tubes as defined by biomarker recovery for SARS-CoV-2. Overall, biomarker pattern recovery for the lipoproteins was extremely similar, but there were some small positive offsets in the linear equations for several variables due to small shimming artifacts, but there was minimal degradation of the biological information. For the standard untargeted 1D, CPMG, and JRES NMR experiments on the same samples, the reduced signal-to-noise was more constraining and required greater scanning times to achieve similar differential diagnostic performance (15 min per sample per experiment for 3 mm 1D and CPMG, compared to 4 min for the 5 mm tubes). We conclude that the 3 mm IVDr method is fit-for-purpose for quantitative lipoprotein measurements, allowing the preparation of smaller volumes for high value or limited volume samples that is common in clinical studies. If there are no analytical time constraints, the lower volume experiments are equally informative for untargeted profiling.

NMR Spectroscopic Windows on the Systemic Effects of SARS-CoV-2 Infection on Plasma Lipoproteins and Metabolites in Relation to Circulating Cytokines.

To investigate the systemic metabolic effects of SARS-CoV-2 infection, we analyzed 1H NMR spectroscopic data on human blood plasma and co-modeled with multiple plasma cytokines and chemokines (measured in parallel). Thus, 600 MHz 1H solvent-suppressed single-pulse, spin-echo, and 2D J-resolved spectra were collected on plasma recorded from SARS-CoV-2 rRT-PCR-positive patients (n = 15, with multiple sampling timepoints) and age-matched healthy controls (n = 34, confirmed rRT-PCR negative), together with patients with COVID-19/influenza-like clinical symptoms who tested SARS-CoV-2 negative (n = 35). We compared the single-pulse NMR spectral data with in vitro diagnostic research (IVDr) information on quantitative lipoprotein profiles (112 parameters) extracted from the raw 1D NMR data. All NMR methods gave highly significant discrimination of SARS-CoV-2 positive patients from controls and SARS-CoV-2 negative patients with individual NMR methods, giving different diagnostic information windows on disease-induced phenoconversion. Longitudinal trajectory analysis in selected patients indicated that metabolic recovery was incomplete in individuals without detectable virus in the recovery phase. We observed four plasma cytokine clusters that expressed complex differential statistical relationships with multiple lipoproteins and metabolites. These included the following: cluster 1, comprising MIP-1ß, SDF-1α, IL-22, and IL-1α, which correlated with multiple increased LDL and VLDL subfractions; cluster 2, including IL-10 and IL-17A, which was only weakly linked to the lipoprotein profile; cluster 3, which included IL-8 and MCP-1 and were inversely correlated with multiple lipoproteins. IL-18, IL-6, and IFN-γ together with IP-10 and RANTES exhibited strong positive correlations with LDL1-4 subfractions and negative correlations with multiple HDL subfractions. Collectively, these data show a distinct pattern indicative of a multilevel cellular immune response to SARS CoV-2 infection interacting with the plasma lipoproteome giving a strong and characteristic immunometabolic phenotype of the disease. We observed that some patients in the respiratory recovery phase and testing virus-free were still metabolically highly abnormal, which indicates a new role for these technologies in assessing full systemic recovery.

Integrative Modeling of Plasma Metabolic and Lipoprotein Biomarkers of SARS-CoV-2 Infection in Spanish and Australian COVID-19 Patient Cohorts.

Quantitative plasma lipoprotein and metabolite profiles were measured on an autonomous community of the Basque Country (Spain) cohort consisting of hospitalized COVID-19 patients (n = 72) and a matched control group (n = 75) and a Western Australian (WA) cohort consisting of (n = 17) SARS-CoV-2 positives and (n = 20) healthy controls using 600 MHz 1H nuclear magnetic resonance (NMR) spectroscopy. Spanish samples were measured in two laboratories using one-dimensional (1D) solvent-suppressed and T2-filtered methods with in vitro diagnostic quantification of lipoproteins and metabolites. SARS-CoV-2 positive patients and healthy controls from both populations were modeled and cross-projected to estimate the biological similarities and validate biomarkers. Using the top 15 most discriminatory variables enabled construction of a cross-predictive model with 100% sensitivity and specificity (within populations) and 100% sensitivity and 82% specificity (between populations). Minor differences were observed between the control metabolic variables in the two cohorts, but the lipoproteins were virtually indistinguishable. We observed highly significant infection-related reductions in high-density lipoprotein (HDL) subfraction 4 phospholipids, apolipoproteins A1 and A2,that have previously been associated with negative regulation of blood coagulation and fibrinolysis. The Spanish and Australian diagnostic SARS-CoV-2 biomarkers were mathematically and biologically equivalent, demonstrating that NMR-based technologies are suitable for the study of the comparative pathology of COVID-19 via plasma phenotyping.

Systemic Perturbations in Amine and Kynurenine Metabolism Associated with Acute SARS-CoV-2 Infection and Inflammatory Cytokine Responses.

We performed quantitative metabolic phenotyping of blood plasma in parallel with cytokine/chemokine analysis from participants who were either SARS-CoV-2 (+) (n = 10) or SARS-CoV-2 (-) (n = 49). SARS-CoV-2 positivity was associated with a unique metabolic phenotype and demonstrated a complex systemic response to infection, including severe perturbations in amino acid and kynurenine metabolic pathways. Nine metabolites were elevated in plasma and strongly associated with infection (quinolinic acid, glutamic acid, nicotinic acid, aspartic acid, neopterin, kynurenine, phenylalanine, 3-hydroxykynurenine, and taurine; p < 0.05), while four metabolites were lower in infection (tryptophan, histidine, indole-3-acetic acid, and citrulline; p < 0.05). This signature supports a systemic metabolic phenoconversion following infection, indicating possible neurotoxicity and neurological disruption (elevations of 3-hydroxykynurenine and quinolinic acid) and liver dysfunction (reduction in Fischer's ratio and elevation of taurine). Finally, we report correlations between the key metabolite changes observed in the disease with concentrations of proinflammatory cytokines and chemokines showing strong immunometabolic disorder in response to SARS-CoV-2 infection.

Incomplete Systemic Recovery and Metabolic Phenoreversion in Post-Acute-Phase Nonhospitalized COVID-19 Patients: Implications for Assessment of Post-Acute COVID-19 Syndrome.

We present a multivariate metabotyping approach to assess the functional recovery of nonhospitalized COVID-19 patients and the possible biochemical sequelae of "Post-Acute COVID-19 Syndrome", colloquially known as long-COVID. Blood samples were taken from patients ca. 3 months after acute COVID-19 infection with further assessment of symptoms at 6 months. Some 57% of the patients had one or more persistent symptoms including respiratory-related symptoms like cough, dyspnea, and rhinorrhea or other nonrespiratory symptoms including chronic fatigue, anosmia, myalgia, or joint pain. Plasma samples were quantitatively analyzed for lipoproteins, glycoproteins, amino acids, biogenic amines, and tryptophan pathway intermediates using Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Metabolic data for the follow-up patients (n = 27) were compared with controls (n = 41) and hospitalized severe acute respiratory syndrome SARS-CoV-2 positive patients (n = 18, with multiple time-points). Univariate and multivariate statistics revealed variable patterns of functional recovery with many patients exhibiting residual COVID-19 biomarker signatures. Several parameters were persistently perturbed, e.g., elevated taurine (p = 3.6 × 10-3 versus controls) and reduced glutamine/glutamate ratio (p = 6.95 × 10-8 versus controls), indicative of possible liver and muscle damage and a high energy demand linked to more generalized tissue repair or immune function. Some parameters showed near-complete normalization, e.g., the plasma apolipoprotein B100/A1 ratio was similar to that of healthy controls but significantly lower (p = 4.2 × 10-3) than post-acute COVID-19 patients, reflecting partial reversion of the metabolic phenotype (phenoreversion) toward the healthy metabolic state. Plasma neopterin was normalized in all follow-up patients, indicative of a reduction in the adaptive immune activity that has been previously detected in active SARS-CoV-2 infection. Other systemic inflammatory biomarkers such as GlycA and the kynurenine/tryptophan ratio remained elevated in some, but not all, patients. Correlation analysis, principal component analysis (PCA), and orthogonal-partial least-squares discriminant analysis (O-PLS-DA) showed that the follow-up patients were, as a group, metabolically distinct from controls and partially comapped with the acute-phase patients. Significant systematic metabolic differences between asymptomatic and symptomatic follow-up patients were also observed for multiple metabolites. The overall metabolic variance of the symptomatic patients was significantly greater than that of nonsymptomatic patients for multiple parameters (χ2p = 0.014). Thus, asymptomatic follow-up patients including those with post-acute COVID-19 Syndrome displayed a spectrum of multiple persistent biochemical pathophysiology, suggesting that the metabolic phenotyping approach may be deployed for multisystem functional assessment of individual post-acute COVID-19 patients.

Diffusion and Relaxation Edited Proton NMR Spectroscopy of Plasma Reveals a High-Fidelity Supramolecular Biomarker Signature of SARS-CoV-2 Infection.

We have applied nuclear magnetic resonance spectroscopy based plasma phenotyping to reveal diagnostic molecular signatures of SARS-CoV-2 infection via combined diffusional and relaxation editing (DIRE). We compared plasma from healthy age-matched controls (n = 26) with SARS-CoV-2 negative non-hospitalized respiratory patients and hospitalized respiratory patients (n = 23 and 11 respectively) with SARS-CoV-2 rRT-PCR positive respiratory patients (n = 17, with longitudinal sampling time-points). DIRE data were modelled using principal component analysis and orthogonal projections to latent structures discriminant analysis (O-PLS-DA), with statistical cross-validation indices indicating excellent model generalization for the classification of SARS-CoV-2 positivity for all comparator groups (area under the receiver operator characteristic curve = 1). DIRE spectra show biomarker signal combinations conferred by differential concentrations of metabolites with selected molecular mobility properties. These comprise the following: (a) composite N-acetyl signals from α-1-acid glycoprotein and other glycoproteins (designated GlycA and GlycB) that were elevated in SARS-CoV-2 positive patients [p = 2.52 × 10-10 (GlycA) and 1.25 × 10-9 (GlycB) vs controls], (b) two diagnostic supramolecular phospholipid composite signals that were identified (SPC-A and SPC-B) from the -+N-(CH3)3 choline headgroups of lysophosphatidylcholines carried on plasma glycoproteins and from phospholipids in high-density lipoprotein subfractions (SPC-A) together with a phospholipid component of low-density lipoprotein (SPC-B). The integrals of the summed SPC signals (SPCtotal) were reduced in SARS-CoV-2 positive patients relative to both controls (p = 1.40 × 10-7) and SARS-CoV-2 negative patients (p = 4.52 × 10-8) but were not significantly different between controls and SARS-CoV-2 negative patients. The identity of the SPC signal components was determined using one and two dimensional diffusional, relaxation, and statistical spectroscopic experiments. The SPCtotal/GlycA ratios were also significantly different for control versus SARS-CoV-2 positive patients (p = 1.23 × 10-10) and for SARS-CoV-2 negatives versus positives (p = 1.60 × 10-9). Thus, plasma SPCtotal and SPCtotal/GlycA are proposed as sensitive molecular markers for SARS-CoV-2 positivity that could effectively augment current COVID-19 diagnostics and may have value in functional assessment of the disease recovery process in patients with long-term symptoms.

Integrative Modeling of Quantitative Plasma Lipoprotein, Metabolic, and Amino Acid Data Reveals a Multiorgan Pathological Signature of SARS-CoV-2 Infection.

The metabolic effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on human blood plasma were characterized using multiplatform metabolic phenotyping with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). Quantitative measurements of lipoprotein subfractions, α-1-acid glycoprotein, glucose, and biogenic amines were made on samples from symptomatic coronavirus disease 19 (COVID-19) patients who had tested positive for the SARS-CoV-2 virus (n = 17) and from age- and gender-matched controls (n = 25). Data were analyzed using an orthogonal-projections to latent structures (OPLS) method and used to construct an exceptionally strong (AUROC = 1) hybrid NMR-MS model that enabled detailed metabolic discrimination between the groups and their biochemical relationships. Key discriminant metabolites included markers of inflammation including elevated α-1-acid glycoprotein and an increased kynurenine/tryptophan ratio. There was also an abnormal lipoprotein, glucose, and amino acid signature consistent with diabetes and coronary artery disease (low total and HDL Apolipoprotein A1, low HDL triglycerides, high LDL and VLDL triglycerides), plus multiple highly significant amino acid markers of liver dysfunction (including the elevated glutamine/glutamate and Fischer's ratios) that present themselves as part of a distinct SARS-CoV-2 infection pattern. A multivariate training-test set model was validated using independent samples from additional SARS-CoV-2 positive patients and controls. The predictive model showed a sensitivity of 100% for SARS-CoV-2 positivity. The breadth of the disturbed pathways indicates a systemic signature of SARS-CoV-2 positivity that includes elements of liver dysfunction, dyslipidemia, diabetes, and coronary heart disease risk that are consistent with recent reports that COVID-19 is a systemic disease affecting multiple organs and systems. Metabolights study reference: MTBLS2014.

Quantitative In-Vitro Diagnostic NMR Spectroscopy for Lipoprotein and Metabolite Measurements in Plasma and Serum: Recommendations for Analytical Artifact Minimization with Special Reference to COVID-19/SARS-CoV-2 Samples.

Quantitative nuclear magnetic resonance (NMR) spectroscopy of blood plasma is widely used to investigate perturbed metabolic processes in human diseases. The reliability of biochemical data derived from these measurements is dependent on the quality of the sample collection and exact preparation and analysis protocols. Here, we describe systematically, the impact of variations in sample collection and preparation on information recovery from quantitative proton (1H) NMR spectroscopy of human blood plasma and serum. The effects of variation of blood collection tube sizes and preservatives, successive freeze-thaw cycles, sample storage at -80 °C, and short-term storage at 4 and 20 °C on the quantitative lipoprotein and metabolite patterns were investigated. Storage of plasma samples at 4 °C for up to 48 h, freezing at -80 °C and blood sample collection tube choice have few and minor effects on quantitative lipoprotein profiles, and even storage at 4 °C for up to 168 h caused little information loss. In contrast, the impact of heat-treatment (56 °C for 30 min), which has been used for inactivation of SARS-CoV-2 and other viruses, that may be required prior to analytical measurements in low level biosecurity facilities induced marked changes in both lipoprotein and low molecular weight metabolite profiles. It was conclusively demonstrated that this heat inactivation procedure degrades lipoproteins and changes metabolic information in complex ways. Plasma from control individuals and SARS-CoV-2 infected patients are differentially altered resulting in the creation of artifactual pseudo-biomarkers and destruction of real biomarkers to the extent that data from heat-treated samples are largely uninterpretable. We also present several simple blood sample handling recommendations for optimal NMR-based biomarker discovery investigations in SARS CoV-2 studies and general clinical biomarker research.

Plasma lipoprotein subclass variation in middle-aged and older adults: Sex-stratified distributions and associations with health status and cardiometabolic risk factors.

BACKGROUND: Circulating lipids and lipoproteins mediate cardiovascular risk, however routine plasma lipid biochemistry provides limited information on pro-atherogenic remnant particles. OBJECTIVE: We analysed plasma lipoprotein subclasses including very low-density and intermediate-density lipoprotein (VLDL and IDL); and assessed their associations with health and cardiometabolic risk. METHODS: From 1,976 community-dwelling adults aged 45-67 years, 114/1071 women (10.6%) and 153/905 men (16.9%) were categorised as very healthy. Fasting plasma lipoprotein profiles comprising 112 parameters were measured using 1H nuclear magnetic resonance (NMR) spectroscopy, and associations with health status and cardiometabolic risk factors examined. RESULTS: HDL cholesterol was higher, and IDL and VLDL cholesterol and triglycerides lower, in very healthy women compared to other women, and women compared to men. IDL and VLDL cholesterol and triglyceride were lower in very healthy men compared to other men. HDL cholesterol and apolipoprotein (apo) A-I were inversely, and IDL and VLDL cholesterol, apoB-100, and apoB-100/apoA-I ratio directly associated with body mass index (BMI) in women and men. In women, LDL, IDL and VLDL cholesterol increased with age. Women with diabetes and cardiovascular disease had higher cholesterol, triglycerides, phospholipids and free cholesterol across IDL and VLDL fractions, with similar trends for men with diabetes. CONCLUSION: Lipoprotein subclasses and density fractions, and their lipid and apolipoprotein constituents, are differentially distributed by sex, health status and BMI. Very healthy women and men are distinguished by favorable lipoprotein profiles, particularly lower concentrations of VLDL and IDL, providing reference intervals for comparison with general populations and adults with cardiometabolic risk factors.

Targeted lipidomics coupled with machine learning for authenticating the provenance of chicken eggs.

Free-range eggs are ethically desirable but as with all high-value commercial products, the establishment of provenance can be problematic. Here, we compared a simple one-step isopropanol method to a two-step methyl-tert-butyl ether method for extracting lipid species in chicken egg yolks before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The isopropanol method extracted 937 lipid species from 20 major lipid subclasses with high reproducibility (CV < 30 %). Machine learning techniques could differentiate conventional cage, barn, and free-range eggs using an external test dataset with an accuracy of 0.94, 0.82, and 0.82, respectively. Lipid species that differentiated cage eggs were predominantly phosphocholines and phosphoethanolamines whilst the free-range egg lipidomes were dominated by acylglycerides with up to three fatty acids. The lipid profiles were found to be characteristic of the cage, barns, and free-range eggs. The lipidomic analysis together with the statistical modeling approach thus provides an efficient tool for verifying the provenance of conventional chicken eggs.

Cardiometabolic disease risk markers are increased following burn injury in children.

Introduction: Burn injury in children causes prolonged systemic effects on physiology and metabolism leading to increased morbidity and mortality, yet much remains undefined regarding the metabolic trajectory towards specific health outcomes. Methods: A multi-platform strategy was implemented to evaluate the long-term immuno-metabolic consequences of burn injury combining metabolite, lipoprotein, and cytokine panels. Plasma samples from 36 children aged 4-8 years were collected 3 years after a burn injury together with 21 samples from non-injured age and sex matched controls. Three different 1H Nuclear Magnetic Resonance spectroscopic experiments were applied to capture information on plasma low molecular weight metabolites, lipoproteins, and α-1-acid glycoprotein. Results: Burn injury was characterized by underlying signatures of hyperglycaemia, hypermetabolism and inflammation, suggesting disruption of multiple pathways relating to glycolysis, tricarboxylic acid cycle, amino acid metabolism and the urea cycle. In addition, very low-density lipoprotein sub-components were significantly reduced in participants with burn injury whereas small-dense low density lipoprotein particles were significantly elevated in the burn injured patient plasma compared to uninjured controls, potentially indicative of modified cardiometabolic risk after a burn. Weighted-node Metabolite Correlation Network Analysis was restricted to the significantly differential features (q <0.05) between the children with and without burn injury and demonstrated a striking disparity in the number of statistical correlations between cytokines, lipoproteins, and small molecular metabolites in the injured groups, with increased correlations between these groups. Discussion: These findings suggest a 'metabolic memory' of burn defined by a signature of interlinked and perturbed immune and metabolic function. Burn injury is associated with a series of adverse metabolic changes that persist chronically and are independent of burn severity and this study demonstrates increased risk of cardiovascular disease in the long-term. These findings highlight a crucial need for improved longer term monitoring of cardiometabolic health in a vulnerable population of children that have undergone burn injury.

Systemic long-term metabolic effects of acute non-severe paediatric burn injury.

A growing body of evidence supports the concept of a systemic response to non-severe thermal trauma. This provokes an immunosuppressed state that predisposes paediatric patients to poor recovery and increased risk of secondary morbidity. In this study, to understand the long-term systemic effects of non-severe burns in children, targeted mass spectrometry assays for biogenic amines and tryptophan metabolites were performed on plasma collected from child burn patients at least three years post injury and compared to age and sex matched non-burn (healthy) controls. A panel of 12 metabolites, including urea cycle intermediates, aromatic amino acids and quinolinic acid were present in significantly higher concentrations in children with previous burn injury. Correlation analysis of metabolite levels to previously measured cytokine levels indicated the presence of multiple cytokine-metabolite associations in the burn injury participants that were absent from the healthy controls. These data suggest that there is a sustained immunometabolic imprint of non-severe burn trauma, potentially linked to long-term immune changes that may contribute to the poor long-term health outcomes observed in children after burn injury.

Diagnostic Potential of the Plasma Lipidome in Infectious Disease: Application to Acute SARS-CoV-2 Infection.

Improved methods are required for investigating the systemic metabolic effects of SARS-CoV-2 infection and patient stratification for precision treatment. We aimed to develop an effective method using lipid profiles for discriminating between SARS-CoV-2 infection, healthy controls, and non-SARS-CoV-2 respiratory infections. Targeted liquid chromatography-mass spectrometry lipid profiling was performed on discovery (20 SARS-CoV-2-positive; 37 healthy controls; 22 COVID-19 symptoms but SARS-CoV-2negative) and validation (312 SARS-CoV-2-positive; 100 healthy controls) cohorts. Orthogonal projection to latent structure-discriminant analysis (OPLS-DA) and Kruskal-Wallis tests were applied to establish discriminant lipids, significance, and effect size, followed by logistic regression to evaluate classification performance. OPLS-DA reported separation of SARS-CoV-2 infection from healthy controls in the discovery cohort, with an area under the curve (AUC) of 1.000. A refined panel of discriminant features consisted of six lipids from different subclasses (PE, PC, LPC, HCER, CER, and DCER). Logistic regression in the discovery cohort returned a training ROC AUC of 1.000 (sensitivity = 1.000, specificity = 1.000) and a test ROC AUC of 1.000. The validation cohort produced a training ROC AUC of 0.977 (sensitivity = 0.855, specificity = 0.948) and a test ROC AUC of 0.978 (sensitivity = 0.948, specificity = 0.922). The lipid panel was also able to differentiate SARS-CoV-2-positive individuals from SARS-CoV-2-negative individuals with COVID-19-like symptoms (specificity = 0.818). Lipid profiling and multivariate modelling revealed a signature offering mechanistic insights into SARS-CoV-2, with strong predictive power, and the potential to facilitate effective diagnosis and clinical management.

Untargeted metabolomics of human plasma reveal lipid markers unique to chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

Chronic obstructive pulmonary disease (COPD) is characterised by airway inflammation and progressive airflow limitation, whereas idiopathic pulmonary fibrosis (IPF) is characterised by a restrictive pattern due to fibrosis and impaired gas exchange. We undertook metabolomic analysis of blood samples in IPF, COPD and healthy controls (HC) to determine differences in circulating molecules and identify novel pathogenic pathways. An untargeted metabolomics using an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) was performed to profile plasma of patients with COPD (n = 21), and IPF (n = 24) in comparison to plasma from healthy controls (HC; n = 20). The most significant features were identified using multiple database matching. One-way ANOVA and variable importance in projection (VIP) scores were also used to highlight metabolites that influence the specific disease groups. Non-polar metabolites such as fatty acids (FA) and membrane lipids were well resolved and a total of 4805 features were identified. The most prominent metabolite composition differences in lipid mediators identified at ∼2-3 fold higher in both diseases compared to HC were palmitoleic acid, oleic acid and linoleic acid; and dihydrotestosterone was lower in both diseases. We demonstrated that COPD and IPF were characterised by systemic changes in lipid constituents such as essential FA sampled from circulating plasma.

A simultaneous exploratory and quantitative amino acid and biogenic amine metabolic profiling platform for rapid disease phenotyping via UPLC-QToF-MS.

Metabolic phenotyping using mass spectrometry (MS) is being applied to ever increasing sample numbers in clinical and epidemiology studies. High-throughput and robust methods are being developed for the accurate measurement of metabolites associated with disease. Traditionally, quantitative assays have utilized triple quadrupole (QQQ) MS based methods; however, the use of such focused methods removes the ability to perform discovery-based metabolic phenotyping. An integrated workflow for the hybrid simultaneous quantification of 34 biogenic amines in combination with full scan high-resolution accurate mass (HRAM) exploratory metabolic phenotyping is presented. Primary and secondary amines are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate prior to revered-phase liquid chromatographic separation and mass spectrometric detection. Using the HRAM-MS data, retrospective phenotypic data mining could be performed, demonstrating the versatility of HRAM-MS instrumentation in a clinical and molecular epidemiological environment. Quantitative performance was assessed using two MS detector platforms: Waters TQ-XS (QQQ; n = 3) and Bruker Impact II QToF (HRAMS-MS; n = 2) and three human biofluids (plasma, serum and urine). Finally, each platform was assessed using a certified external reference sample (NIST SRM 1950 plasma). Intra- and inter-day accuracy and precision were comparable between the QQQ and QToF instruments (<15%), with excellent linearity (R2 > 0.99) over the quantification range of 1-400 µmol L-1. Quantitative values were comparable across all instruments for human plasma, serum and urine samples, and calculated concentrations were verified against certified reference values for NIST SRM 1950 plasma as an external reference. As a real-life biological exemplar, the method was applied to plasma samples obtained from SARS-CoV-2 positive patients versus healthy controls. Both the QQQ and QToF approaches were equivalent in being able to correctly classify SARS-CoV-2 positivity. Critically, the use of HRAM full scan data was also assessed for retrospective exploratory mining of data to extract additional biogenic amines of biomarker interest beyond the 34 quantified targets.

Correlative and dynamic imaging of the hatching biology of Schistosoma japonicum from eggs prepared by high pressure freezing.

BACKGROUND: Schistosome eggs must traverse tissues of the intestine or bladder to escape the human host and further the life cycle. Escape from host tissues is facilitated by secretion of immuno-reactive molecules by eggs and the formation of an intense strong granulomatous response by the host which acts to exclude the egg into gut or bladder lumens. Schistosome eggs hatch on contact with freshwater, but the mechanisms of activation and hatching are poorly understood. In view of the lack of knowledge of the behaviour of egg hatching in schistosomes, we undertook a detailed dynamic and correlative study of the hatching biology of Schistosoma japonicum. METHODOLOGY/PRINCIPAL FINDINGS: Hatching eggs of S. japonicum were studied using correlative light and electron microscopy (EM). The hatching behaviour was recorded by video microscopy. EM preparative methods incorporating high pressure freezing and cryo-substitution were used to investigate ultrastructural features of the miracidium and extra-embryonic envelopes in pre-activated and activated eggs, and immediately after eggshell rupture. Lectin cytochemistry was performed on egg tissues to investigate subcellular location of specific carbohydrate groups. CONCLUSIONS/SIGNIFICANCE: The hatching of S. japonicum eggs is a striking phenomenon, whereby the larva is liberated explosively while still encapsulated within its sub-shell envelopes. The major alterations that occur in the egg during activation are scission of the outer envelope-eggshell boundary, autolysis of the cellular inner envelope, and likely hydration of abundant complex and simple polysaccharides in the lacunal space between the miracidial larva and surrounding envelopes. These observations on hatching provide insight into the dynamic activity of the eggs and the biology of schistosomes within the host.