Unravelling inulin molecules in food sources using a matrix-assisted laser desorption/ionization magnetic resonance mass spectrometry (MALDI-MRMS) pipeline.

Inulin, a polysaccharide characterized by a ß-2,1 fructosyl-fructose structure terminating in a glucosyl moiety, is naturally present in plant roots and tubers. Current methods provide average degrees of polymerization (DP) but lack information on the distribution and absolute concentration of each DP. To address this limitation, a reproducible (CV < 10 %) high throughput (<2 min/sample) MALDI-MRMS approach capable of characterizing and quantifying inulin molecules in plants using matched-matrix consisting of α-cyano-4-hydroxycinnamic acid butylamine salt (CHCA-BA), chicory inulin-12C and inulin-13C was developed. The method identified variation in chain lengths and concentration of inulin across various plant species. Globe artichoke hearts, yacón and elephant garlic yielded similar concentrations at 15.6 g/100 g dry weight (DW), 16.8 g/100 g DW and 17.7 g/100 g DW, respectively, for DP range between 9 and 22. In contrast, Jerusalem artichoke demonstrated the highest concentration (53.4 g/100 g DW) within the same DP ranges. Jerusalem artichoke (DPs 9-32) and globe artichoke (DPs 9-36) showed similar DP distributions, while yacón and elephant garlic displayed the narrowest and broadest DP ranges (DPs 9-19 and DPs 9-45, respectively). Additionally, qualitative measurement for all inulin across all plant samples was feasible using the peak intensities normalized to Inulin-13C, and showed that the ratio of yacón, elephant garlic and Jerusalem was approximately one, two and three times that of globe artichoke. This MALDI-MRMS approach provides comprehensive insights into the structure of inulin molecules, opening avenues for in-depth investigations into how DP and concentration of inulin influence gut health and the modulation of noncommunicable diseases, as well as shedding light on refining cultivation practices to elevate the beneficial health properties associated with specific DPs.

Cross-Validation of Metabolic Phenotypes in SARS-CoV-2 Infected Subpopulations Using Targeted Liquid Chromatography-Mass Spectrometry (LC-MS).

To ensure biological validity in metabolic phenotyping, findings must be replicated in independent sample sets. Targeted workflows have long been heralded as ideal platforms for such validation due to their robust quantitative capability. We evaluated the capability of liquid chromatography-mass spectrometry (LC-MS) assays targeting organic acids and bile acids to validate metabolic phenotypes of SARS-CoV-2 infection. Two independent sample sets were collected: (1) Australia: plasma, SARS-CoV-2 positive (n = 20), noninfected healthy controls (n = 22) and COVID-19 disease-like symptoms but negative for SARS-CoV-2 infection (n = 22). (2) Spain: serum, SARS-CoV-2 positive (n = 33) and noninfected healthy controls (n = 39). Multivariate modeling using orthogonal projections to latent structures discriminant analyses (OPLS-DA) classified healthy controls from SARS-CoV-2 positive (Australia; R2 = 0.17, ROC-AUC = 1; Spain R2 = 0.20, ROC-AUC = 1). Univariate analyses revealed 23 significantly different (p < 0.05) metabolites between healthy controls and SARS-CoV-2 positive individuals across both cohorts. Significant metabolites revealed consistent perturbations in cellular energy metabolism (pyruvic acid, and 2-oxoglutaric acid), oxidative stress (lactic acid, 2-hydroxybutyric acid), hypoxia (2-hydroxyglutaric acid, 5-aminolevulinic acid), liver activity (primary bile acids), and host-gut microbial cometabolism (hippuric acid, phenylpropionic acid, indole-3-propionic acid). These data support targeted LC-MS metabolic phenotyping workflows for biological validation in independent sample sets.

Stratification of Sepsis Patients on Admission into the Intensive Care Unit According to Differential Plasma Metabolic Phenotypes.

Delayed diagnosis of patients with sepsis or septic shock is associated with increased mortality and morbidity. UPLC-MS and NMR spectroscopy were used to measure panels of lipoproteins, lipids, biogenic amines, amino acids, and tryptophan pathway metabolites in blood plasma samples collected from 152 patients within 48 h of admission into the Intensive Care Unit (ICU) where 62 patients had no sepsis, 71 patients had sepsis, and 19 patients had septic shock. Patients with sepsis or septic shock had higher concentrations of neopterin and lower levels of HDL cholesterol and phospholipid particles in comparison to nonsepsis patients. Septic shock could be differentiated from sepsis patients based on different concentrations of 10 lipids, including significantly lower concentrations of five phosphatidylcholine species, three cholesterol esters, one dihydroceramide, and one phosphatidylethanolamine. The Supramolecular Phospholipid Composite (SPC) was reduced in all ICU patients, while the composite markers of acute phase glycoproteins were increased in the sepsis and septic shock patients within 48 h admission into ICU. We show that the plasma metabolic phenotype obtained within 48 h of ICU admission is diagnostic for the presence of sepsis and that septic shock can be differentiated from sepsis based on the lipid profile.

Extracellular vesicles as a promising source of lipid biomarkers for breast cancer detection in blood plasma.

Extracellular vesicles (EVs), including exosomes and microvesicles, mediate intercellular communication in cancer, from development to metastasis. EV-based liquid biopsy is a promising strategy for cancer diagnosis as EVs can be found in cancer patients' body fluids. In this study, the lipid composition of breast cancer-derived EVs was studied as well as the potential of blood plasma EVs for the identification of lipid biomarkers for breast cancer detection. Initially, an untargeted lipidomic analysis was carried out for a panel of cancerous and non-cancerous mammary epithelial cells and their secreted EVs. We found that breast cancer-derived EVs are enriched in sphingolipids and glycerophospholipids compared to their parental cells. The initial in vitro study showed that EVs and their parental cells can be correctly classified (100% accuracy) between cancerous and non-cancerous, as well as into their respective breast cancer subtypes, based on their lipid composition. Subsequently, an untargeted lipidomic analysis was carried out for blood plasma EVs from women diagnosed with breast cancer (primary or progressive metastatic breast cancer) as well as healthy women. Correspondingly, when blood plasma EVs were analysed, breast cancer patients and healthy women were correctly classified with an overall accuracy of 93.1%, based on the EVs' lipid composition. Similarly, the analysis of patients with primary breast cancer and healthy women showed an overall accuracy of 95% for their correct classification. Furthermore, primary and metastatic breast cancers were correctly classified with an overall accuracy of 89.5%. This reveals that the blood plasma EVs' lipids may be a promising source of biomarkers for detection of breast cancer. Additionally, this study demonstrates the usefulness of untargeted lipidomics in the study of EV lipid composition and EV-associated biomarker discovery studies. This is a proof-of-concept study and a starting point for further analysis on the identification of EV-based biomarkers for breast cancer.

Rapid and Self-Administrable Capillary Blood Microsampling Demonstrates Statistical Equivalence with Standard Venous Collections in NMR-Based Lipoprotein Analysis.

We investigated plasma and serum blood derivatives from capillary blood microsamples (500 µL, MiniCollect tubes) and corresponding venous blood (10 mL vacutainers). Samples from 20 healthy participants were analyzed by 1H NMR, and 112 lipoprotein subfraction parameters; 3 supramolecular phospholipid composite (SPC) parameters from SPC1, SPC2, and SPC3 subfractions; 2 N-acetyl signals from α-1-acid glycoprotein (Glyc), GlycA, and GlycB; and 3 calculated parameters, SPC (total), SPC3/SPC2, and Glyc (total) were assessed. Using linear regression between capillary and venous collection sites, we explained that agreement (Adj. R2 ≥ 0.8, p < 0.001) was witnessed for 86% of plasma parameters (103/120) and 88% of serum parameters (106/120), indicating that capillary lipoprotein, SPC, and Glyc concentrations follow changes in venous concentrations. These results indicate that capillary blood microsamples are suitable for sampling in remote areas and for high-frequency longitudinal sampling of the majority of lipoproteins, SPCs, and Glycs.

Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Implementation of pure shift 1 H NMR in metabolic phenotyping for structural information recovery of biofluid metabolites with complex spin systems.

NMR spectroscopy is a mainstay of metabolic profiling approaches to investigation of physiological and pathological processes. The one-dimensional proton pulse sequences typically used in phenotyping large numbers of samples generate spectra that are rich in information but where metabolite identification is often compromised by peak overlap. Recently developed pure shift (PS) NMR spectroscopy, where all J-coupling multiplicities are removed from the spectra, has the potential to simplify the complex proton NMR spectra that arise from biosamples and hence to aid metabolite identification. Here we have evaluated two complementary approaches to spectral simplification: the HOBS (band-selective with real-time acquisition) and the PSYCHE (broadband with pseudo-2D interferogram acquisition) pulse sequences. We compare their relative sensitivities and robustness for deconvolving both urine and serum matrices. Both methods improve resolution of resonances ranging from doublets, triplets and quartets to more complex signals such as doublets of doublets and multiplets in highly overcrowded spectral regions. HOBS is the more sensitive method and takes less time to acquire in comparison with PSYCHE, but can introduce unavoidable artefacts from metabolites with strong couplings, whereas PSYCHE is more adaptable to these types of spin system, although at the expense of sensitivity. Both methods are robust and easy to implement. We also demonstrate that strong coupling artefacts contain latent connectivity information that can be used to enhance metabolite identification. Metabolite identification is a bottleneck in metabolic profiling studies. In the case of NMR, PS experiments can be included in metabolite identification workflows, providing additional capability for biomarker discovery.

Plasma lipidomics reveal systemic changes persistent throughout early life following a childhood burn injury.

Background: Non-severe paediatric burns can result in poor long-term health outcomes. This occurs even in cases with good acute burn-related outcomes, including minimal scarring. The mechanisms that underpin the transition from non-severe burn to sustained negative long-term health impacts are currently unknown. However, sustained metabolic and immune changes have been observed in paediatric burn studies, suggesting these changes may be important.The plasma lipidome consists of a rich pool of bioactive metabolites that play critical roles in systemic processes including molecular signalling and inflammation. We hypothesised that changes in the plasma lipidome may reflect underlying changes in health status and be linked to long-term health after burn trauma. Methods: This study analysed the lipidome in children who had previously experienced a non-severe burn, compared to non-injured controls. Thirty-three participants were recruited between the ages of 5 and 8 years who had experienced a non-severe burn between the ages of 1 and 3 years. Plasma samples were also collected from a non-injured, healthy, age and gender matched control group (n = 21). Plasma lipids were measured using reversed-phase liquid chromatographymass spectrometery (LC-MS). Results: In total 838 reproducible lipid species from 19 sub-classes passed quality control procedures and progressed to statistical analysis. Analysis of individual lipid metabolites showed significantly higher concentrations of lysophosphatidylethanolamines and phosphatidylethanolamines, and significantly lower concentrations in myristic, palmitic and palmitoleic acids in the plasma of those who had experienced burn injury compared to controls. Conclusion: Long-term changes in the lipid profile may give insight into the mechanisms underlying poor long-term health subsequent to non-severe burn injury. Further work to investigate the relationship between long-term pathology and lipidomic changes may lead to a better understanding of the causes of secondary morbidity post-burn and to clinical intervention to reduce the long-term health burden of burn trauma.

Nonsevere Burn Induces a Prolonged Systemic Metabolic Phenotype Indicative of a Persistent Inflammatory Response Postinjury.

Globally, burns are a significant cause of injury that can cause substantial acute trauma as well as lead to increased incidence of chronic comorbidity and disease. To date, research has primarily focused on the systemic response to severe injury, with little in the literature reported on the impact of nonsevere injuries (<15% total burn surface area; TBSA). To elucidate the metabolic consequences of a nonsevere burn injury, longitudinal plasma was collected from adults (n = 35) who presented at hospital with a nonsevere burn injury at admission, and at 6 week follow up. A cross-sectional baseline sample was also collected from nonburn control participants (n = 14). Samples underwent multiplatform metabolic phenotyping using 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry to quantify 112 lipoprotein and glycoprotein signatures and 852 lipid species from across 20 subclasses. Multivariate data modeling (orthogonal projections to latent structures-discriminate analysis; OPLS-DA) revealed alterations in lipoprotein and lipid metabolism when comparing the baseline control to hospital admission samples, with the phenotypic signature found to be sustained at follow up. Univariate (Mann-Whitney U) testing and OPLS-DA indicated specific increases in GlycB (p-value < 1.0e-4), low density lipoprotein-2 subfractions (variable importance in projection score; VIP > 6.83e-1) and monoacyglyceride (20:4) (p-value < 1.0e-4) and decreases in circulating anti-inflammatory high-density lipoprotein-4 subfractions (VIP > 7.75e-1), phosphatidylcholines, phosphatidylglycerols, phosphatidylinositols, and phosphatidylserines. The results indicate a persistent systemic metabolic phenotype that occurs even in cases of a nonsevere burn injury. The phenotype is indicative of an acute inflammatory profile that continues to be sustained postinjury, suggesting an impact on systems health beyond the site of injury. The phenotypes contained metabolic signatures consistent with chronic inflammatory states reported to have an elevated incidence postburn injury. Such phenotypic signatures may provide patient stratification opportunities, to identify individual responses to injury, personalize intervention strategies, and improve acute care, reducing the risk of chronic comorbidity.

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.

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.

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.

Comprehensive Lipidomic Workflow for Multicohort Population Phenotyping Using Stable Isotope Dilution Targeted Liquid Chromatography-Mass Spectrometry.

Dysregulated lipid metabolism underpins many chronic diseases including cardiometabolic diseases. Mass spectrometry-based lipidomics is an important tool for understanding mechanisms of lipid dysfunction and is widely applied in epidemiology and clinical studies. With ever-increasing sample numbers, single batch acquisition is often unfeasible, requiring advanced methods that are accurate and robust to batch-to-batch and interday analytical variation. Herein, an optimized comprehensive targeted workflow for plasma and serum lipid quantification is presented, combining stable isotope internal standard dilution, automated sample preparation, and ultrahigh performance liquid chromatography-tandem mass spectrometry with rapid polarity switching to target 1163 lipid species spanning 20 subclasses. The resultant method is robust to common sources of analytical variation including blood collection tubes, hemolysis, freeze-thaw cycles, storage stability, analyte extraction technique, interinstrument variation, and batch-to-batch variation with 820 lipids reporting a relative standard deviation of <30% in 1048 replicate quality control plasma samples acquired across 16 independent batches (total injection count = 6142). However, sample hemolysis of ≥0.4% impacted lipid concentrations, specifically for phosphatidylethanolamines (PEs). Low interinstrument variability across two identical LC-MS systems indicated feasibility for intra/inter-lab parallelization of the assay. In summary, we have optimized a comprehensive lipidomic protocol to support rigorous analysis for large-scale, multibatch applications in precision medicine. The mass spectrometry lipidomics data have been deposited to massIVE: data set identifiers MSV000090952 and 10.25345/C5NP1WQ4S.

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.

A patient-centric modeling framework captures recovery from SARS-CoV-2 infection.

The biology driving individual patient responses to severe acute respiratory syndrome coronavirus 2 infection remains ill understood. Here, we developed a patient-centric framework leveraging detailed longitudinal phenotyping data and covering a year after disease onset, from 215 infected individuals with differing disease severities. Our analyses revealed distinct 'systemic recovery' profiles, with specific progression and resolution of the inflammatory, immune cell, metabolic and clinical responses. In particular, we found a strong inter-patient and intra-patient temporal covariation of innate immune cell numbers, kynurenine metabolites and lipid metabolites, which highlighted candidate immunologic and metabolic pathways influencing the restoration of homeostasis, the risk of death and that of long COVID. Based on these data, we identified a composite signature predictive of systemic recovery, using a joint model on cellular and molecular parameters measured soon after disease onset. New predictions can be generated using the online tool http://shiny.mrc-bsu.cam.ac.uk/apps/covid-19-systemic-recovery-prediction-app , designed to test our findings prospectively.

An NMR-Based Model to Investigate the Metabolic Phenoreversion of COVID-19 Patients throughout a Longitudinal Study.

After SARS-CoV-2 infection, the molecular phenoreversion of the immunological response and its associated metabolic dysregulation are required for a full recovery of the patient. This process is patient-dependent due to the manifold possibilities induced by virus severity, its phylogenic evolution and the vaccination status of the population. We have here investigated the natural history of COVID-19 disease at the molecular level, characterizing the metabolic and immunological phenoreversion over time in large cohorts of hospitalized severe patients (n = 886) and non-hospitalized recovered patients that self-reported having passed the disease (n = 513). Non-hospitalized recovered patients do not show any metabolic fingerprint associated with the disease or immune alterations. Acute patients are characterized by the metabolic and lipidomic dysregulation that accompanies the exacerbated immunological response, resulting in a slow recovery time with a maximum probability of around 62 days. As a manifestation of the heterogeneity in the metabolic phenoreversion, age and severity become factors that modulate their normalization time which, in turn, correlates with changes in the atherogenesis-associated chemokine MCP-1. Our results are consistent with a model where the slow metabolic normalization in acute patients results in enhanced atherosclerotic risk, in line with the recent observation of an elevated number of cardiovascular episodes found in post-COVID-19 cohorts.

Characterizing the metabolic effects of the selective inhibition of gut microbial ß-glucuronidases in mice.

The hydrolysis of xenobiotic glucuronides by gut bacterial glucuronidases reactivates previously detoxified compounds resulting in severe gut toxicity for the host. Selective bacterial ß-glucuronidase inhibitors can mitigate this toxicity but their impact on wider host metabolic processes has not been studied. To investigate this the inhibitor 4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine (UNC10201652, Inh 9) was administered to mice to selectively inhibit a narrow range of bacterial ß-glucuronidases in the gut. The metabolomic profiles of the intestinal contents, biofluids, and several tissues involved in the enterohepatic circulation were measured and compared to control animals. No biochemical perturbations were observed in the plasma, liver or gall bladder. In contrast, the metabolite profiles of urine, colon contents, feces and gut wall were altered compared to the controls. Changes were largely restricted to compounds derived from gut microbial metabolism. This work establishes that inhibitors targeted towards bacterial ß-glucuronidases modulate the functionality of the intestinal microbiota without adversely impacting the host metabolic system.

Characterization of diet-dependent temporal changes in circulating short-chain fatty acid concentrations: A randomized crossover dietary trial.

BACKGROUND: Production of SCFAs from food is a complex and dynamic saccharolytic fermentation process mediated by both human and gut microbial factors. Knowledge of SCFA production and of the relation between SCFA profiles and dietary patterns is lacking. OBJECTIVES: Temporal changes in SCFA concentrations in response to 2 contrasting diets were investigated using a novel GC-MS method. METHODS: Samples were obtained from a randomized, controlled, crossover trial designed to characterize the metabolic response to 4 diets. Participants (n = 19) undertook these diets during an inpatient stay (of 72 h). Serum samples were collected 2 h after breakfast (AB), after lunch (AL), and after dinner (AD) on day 3, and a fasting sample (FA) was obtained on day 4. The 24-h urine samples were collected on day 3. In this substudy, samples from the 2 extreme diets representing a diet with high adherence to WHO healthy eating recommendations and a typical Western diet were analyzed using a bespoke GC-MS method developed to detect and quantify 10 SCFAs and precursors in serum and urine samples. RESULTS: Considerable interindividual variation in serum SCFA concentrations was observed across all time points, and temporal fluctuations were observed for both diets. Although the sample collection timing exerted a greater magnitude of effect on circulating SCFA concentrations, the unhealthy diet was associated with a lower concentration of acetic acid (FA: coefficient: -17.0; SE: 5.8; P-trend = 0.00615), 2-methylbutyric acid (AL: coefficient: -0.1; SE: 0.028; P-trend = 4.13 × 10-4 and AD: coefficient: -0.1; SE: 0.028; P-trend = 2.28 × 10-3), and 2-hydroxybutyric acid (FA: coefficient: -15.8; SE: 5.11; P-trend: 4.09 × 10-3). In contrast, lactic acid was significantly higher in the unhealthy diet (AL: coefficient: 750.2; SE: 315.2; P-trend = 0.024 and AD: coefficient: 1219.3; SE: 322.6; P-trend: 8.28 × 10-4). CONCLUSIONS: The GC-MS method allowed robust mapping of diurnal patterns in SCFA concentrations, which were affected by diet, and highlighted the importance of standardizing the timing of SCFA measurements in dietary studies. This trial was registered on the NIHR UK clinical trial gateway and with ISRCTN as ISRCTN43087333.

Enhancing the accuracy of surgical wound excision following burns trauma via application of Rapid Evaporative IonisationMass Spectrometry (REIMS).

BACKGROUND: Surgical wound excision is a necessary procedure for burn patients that require the removal of eschar. The extent of excision is currently guided by clinical judgement, with excessinto healthy tissue potentially leading to excessive scar, or inadequate debridement increasing risk of infection. Thus, an objective real-time measure to facilitate accurate excision could support clinical judgement and improve this surgical procedure. This study was designed to investigate the potential use of Rapid evaporative ionisation mass spectrometry (REIMS) as a tool to support data-driven objective tissue excision. METHODS: Data were acquired using a multi-platform approach that consisted of both Rapid Evaporative Ionisation Mass Spectrometry (REIMS) performed on intact skin, and comprehensive liquid chromatography-mass spectrometry (LC-MS/MS) lipidomics performed on homogenised skin tissue extracts. Data were analysed using principal components analysis (PCA) and multivariate orthogonal projections to latent squares discriminant analysis (OPLS-DA) and logistic regression to determine the predictability of the models. RESULTS: PCA and OPLS-DA models of the REIMS and LC-MS/MS lipidomics data reported separation of excised and healthy tissue. Molecular fingerprints generated from REIMS analysis of healthy skin tissue revealed a high degree of heterogeneity, however, intra-individual variance was smaller than inter-individual variance. Both platforms indicated high levels of skin classification accuracy. In addition, OPLS-DA of the LC-MS/MS lipidomic data revealed significant differences in specific lipid classes between healthy control and excised skin samples; including lower free fatty acids (FFA), monoacylglycerols (MAG), lysophosphatidylglycerol (LPG) and lysophosphatidylethanolamines (LPE) in excised tissue and higher lactosylceramides (LCER) and cholesterol esters (CE) compared to healthy control tissue. CONCLUSIONS: Having established the heterogeneity in the biochemical composition of healthy skin using REIMS and LC-MS/MS, our data show that REIMS has the potential to distinguish between excied and healthy skin tissue samples. This pilot study suggests that REIMS may be an effective tool to support accurate tissue excision during burn surgery.