Metabolomics

Over 2.5 million neonatal dried blood spots (DBS) are stored at the Danish National Biobank. These samples offer extraordinary possibilities for metabolomics research, including prediction of disease and understanding of underlying molecular mechanisms of disease development. Nevertheless, Danish neonatal DBS have been little explored in metabolomics studies. One question that remains is the long-term stability of the large number of metabolites typically assessed in untargeted metabolomics over long time periods of storage. Here, we investigate temporal trends of metabolites measured in 200 neonatal DBS collected over a time course of 10 years, using an untargeted LC-MS/MS based metabolomics protocol. We found that a majority (79%) of the metabolome was stable during 10 years of storage at -20{degrees}C. However, we found decreasing trends for lipid-related metabolites, such as phosphocholines and acylcarnitines. A few metabolites, including glutathione and methionine, may be strongly influenced by storage, with changes in metabolite levels up to 0.1-0.2 standard deviation units per year. Our findings indicate that untargeted metabolomics of DBS samples, with long-term storage in biobanks, is suitable for retrospective epidemiological studies. We identify metabolites whose stability in DBS should be closely monitored in future studies of DBS samples with long-term storage.

Alcohol-related liver disease (ALD) is a major cause of mortality and disability adjusted life years. It is not fully understood why a small proportion of patients develop progressive forms of ALD (e.g. fibrosis, cirrhosis). Differences in the metabolic processes could be behind the individual progression of ALD. Our aim was to examine differences in serum metabolome between patients with non-progressive ALD and patients with an early form of progressive ALD. The study had three study groups: progressive ALD (alcohol-related steatohepatitis or early-stage fibrosis, n=50), non-progressive ALD (simple steatosis, n=50) and healthy controls (n=32). Both ALD groups took part in a voluntary alcohol rehabilitation program. A non-targeted metabolomics analysis and targeted analysis of short chain fatty acids was done to the serum samples taken on the day of admission. We found 111 significantly (p<0.0005) altered identified metabolites between the study groups. Our main finding was that levels of glycine conjugated bile acids, glutamic acid, 7-methylguanine and several phosphatidylcholines were elevated in the progressive ALD group in comparison to both the non-progressive ALD group and the controls. Glycine conjugated bile acid, glutamic acid and 7-methylguanine also positively correlated with increased levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, cell death biomarker M65, and liver stiffness. Our results indicate that the enterohepatic cycle of glycine conjugated bile acids as well as lipid and energy metabolism are altered in early forms of progressive ALD. These metabolic processes could be a target for preventing progression of ALD.

Background & AimsEmerging evidence suggest that abnormal activation of aldose reductase/the polyol pathway (Ar/PP) is associated with the pathogenesis or development of fatty liver, obesity and metabolic syndrome. However, the underlying mechanisms were unclear. In this study, we investigated the metabolic reprogramming following activation or inhibition of Ar, the first and the rate-limiting enzyme of PP. We also investigated the long-term effects of Ar/PP-mediated metabolic shift in vivo. MethodsMetabolomic analyses were performed with the AB-SCIE QTRAP-5500 LC-MS/MS System for control mouse hepatocytes and hepatocytes stably overexpressing Ar and exposed to 25 mM glucose. Glycolysis stress tests and mitochondrial stress tests were performed using the Seahorse Bioscience Extracellular Flux Analyzer. The in vivo long-term effects of Ar overexpression and inhibition were evaluated in either transgenic mice overexpressing AR or a line of double transgenic mice carrying an Ar-null mutation and an Agouti-yellow Ay mutation. ResultsAbnormal activation of Ar in hepatocytes was found to trigger and drive a drastic Warburg effect-like metabolic reprogramming, induce de novo lipogenesis, and alter insulin and AMP-activated protein kinase signaling. In glucose-fed AR-overexpressing transgenic mice, AR activation causes systemic alterations in physiological parameters and the development of overt phenotypes of insulin resistance, fatty liver, obesity. In the yellow obese syndrome mice, Ar deficiency greatly improves Agouti Ay mutation-induced abnormalities. ConclusionsCollectively, the results highlight the important contribution of Ar/PP or the putative pseudo-glycolysis in hepatic metabolic homeostasis and the development of metabolic diseases. These findings have profound implications for the development of therapeutic strategies or drugs against metabolic diseases and cancer. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/614395v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@de91ddorg.highwire.dtl.DTLVardef@3a801corg.highwire.dtl.DTLVardef@b0cf35org.highwire.dtl.DTLVardef@1f3c1c1_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIActivation of aldose reductase triggers and drives a Warburg effect-like metabolic eprogramming in hepatocytes. C_LIO_LILiver-specific activation of the polyol pathway leads to insulin resistance, fatty liver and obesity. C_LIO_LIInhibition of aldose reductase greatly ameliorates Agouti Ay-induced metabolic abnormalities. C_LI Impact and implicationsThis study reveals that abnormal activation of Ar/PP will trigger and drive a Warburg effect-like metabolic reprogramming in hepatocytes. In normal subjects, Ar/PP mediated metabolic reprogramming tends to promote lipogenesis, insulin resistance, fatty liver and obesity. In cancer cells, Ar/PP mediated metabolic reprogramming will be part of the Warburg effect to support the growth and proliferation of cancer cells. These findings imply that Ar and its down-stream metabolic enzymes are important therapeutic targets for cancers and metabolic diseases.

BackgroundAlcohol overuse disrupts liver function and alters microbial gut communities, with alcohol-related liver disease (ALD) accounting for half of all liver-related deaths worldwide. Bile acids (BAs) regulate liver and gut function, but their metabolism becomes disrupted in ALD. While it is known that gut microbes transform primary to secondary BAs, which are subsequently reabsorbed via the enterohepatic circulation, BA metabolism during ALD progression remains poorly understood. MethodsWe investigated BA co-metabolism in a cross-sectional cohort of individuals with ALD (n=462) and healthy controls (n=148). We validated key findings in two independent ALD cohorts (n=34 and n=52). We integrated BA concentrations, measured by targeted mass spectrometry in feces and plasma, with liver proteomics, and gut microbiome profiles derived from metagenomic and metatranscriptomic sequencing of fecal samples. ResultsAdvanced fibrosis was associated with decreased hepatic BA synthesis and impaired BA transport. Despite this, disease progression corresponded with increased levels of primary and secondary BAs in plasma and feces. The abundance of microbial secondary BA dehydroxylation and epimerization pathways in the gut microbiome changed with disease severity. Genes encoding early steps in the multi-step dehydroxylation pathway increased, whereas those involved in later steps were depleted, indicating a community-level microbial imbalance. In ALD, we identified Eggerthella lenta as a key mediator of BA dehydroxylation, while Mediterraneibacter torques and Bacteroides thetaiotaomicron facilitated most of the BA epimerization as a detoxification mechanism. ConclusionFibrotic ALD is characterized by disrupted primary BA synthesis and transport, leading to BA accumulation in the gut and blood circulation. Altered microbial secondary BA metabolism reflects a compensatory mechanism that becomes impaired at advanced fibrosis stages. Our findings highlight the gut-liver axis as an important factor influencing ALD progression. Impact and ImplicationsO_LIWith the progression of alcohol-related liver disease (ALD), levels of bile acids (BA) in serum and feces increase, but BA production and transport are impaired in the liver. C_LIO_LISecondary microbial BA metabolism, particularly epimerization and dehydroxylation, increases in ALD. However, a key enzyme, baiN, is depleted, illustrating a microbial community-level metabolic dysbiosis. C_LIO_LIThe main contributing microbial species were, among others, Mediterraneibacter torques, Bacteroides thetaiotaomicron, and Eggerthella lenta, which could serve as potential targets for future microbial-targeted interventions. C_LI Lay summaryAlcohol-related liver disease (ALD) from long-term alcohol overuse affects how the liver and gut interact, especially in handling bile acids (BAs), which are molecules produced by the liver and transformed by gut bacteria. Our study found that in people with ALD, the liver produces fewer BAs, but BAs accumulate in the gut and blood because their transport is impaired. We also observed that bacterial transformations of these BA change as the disease progresses, most likely due to an imbalance in the gut microbiome. For the first time, we identify specific bacterial species that strongly influence BA levels in ALD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/25332046v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@19966f0org.highwire.dtl.DTLVardef@352be6org.highwire.dtl.DTLVardef@d5146dorg.highwire.dtl.DTLVardef@1304022_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 0:C_FLOATNO Graphical Abstract C_FIG

Cyclophilin D (CypD) is a nuclear-encoded mitochondrial protein. Although best known as a regulator of the mitochondrial permeability transition pore (MPTP), it is also implicated in the regulation of cellular bioenergetics and extramitochondrial activities. In addition, being a peptidyl prolyl cis-trans isomerase (PPIase), deletion of CypD is likely to affect protein stability in the mitochondria; however, there is little direct evidence of this. In this study, untargeted 1H NMR metabolomics, coupled with multivariate analysis, was used to describe simultaneous changes in the metabolic system of CypD-deficient mice liver, heart, and pancreas, with data from the serum to identify systematic changes. Metabolomics Pathway Analyses (MetPA) revealed commonly perturbed metabolites in the different mouse tissues lacking CypD, with significantly enriched pathways that are related to amino acid, glucose and purine metabolisms, and bioenergetics. Serum from CypD-deficient mice confirmed changes in tissue urea cycle, lipid metabolism and ketogenesis. Overall this study reveals the role of CypD in maintaining protein homeostasis, concurring with its biochemical property as a peptidyl-prolyl isomerase and also demonstrates the wider metabolic adaptations induced by the deletion of the ppif gene, resulting in a CypD-deficient mice metabolome that is different from the wild-type system.

Nonalcoholic fatty liver disease (NAFLD) and Metabolic syndrome (MS) have become a global health concern as incidence of these metabolic disorders is growing rapidly in developing countries particularly in the Middle East, South America and Africa. Studies have shown that protein restriction is associated with increased risk of metabolic diseases, possibly through effects on fatty acid (FA) metabolism. In the present study, we investigated whether a low protein diet modulates FA metabolism and whether methyl donor supplementation can ameliorate these effects and improve metabolic health. Male C57BL/6 mice were fed either a low protein diet (LPD, 90 g/kg protein, n=8), a LPD supplemented with methyl donors (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12, n=8) or normal protein diet (NPD, 180 g/kg protein, n=8) for 7 weeks prior to analysis of serum fatty acid profiles by GC FID and MS and liver fatty acid synthesis and uptake gene expression by RT-qPCR. We observed significant depletion of serum C15:0 and C17:0 in LPD-fed males compared to NPD. Serum long chain saturated FAs C18:0 and C24:0 were increased in LPD male mice compared to NPD. Gene expression analysis revealed an upregulation of hepatic cluster of differentiation 36 (CD36) expression in LPD mice compared to NPD suggesting increased fat uptake in the liver. However, when LPD diet was supplemented with methyl donors, we observed either no change in serum C15: 0 and an increased serum C17:0 compared to LPD with no methyl donor supplementation. Again, methyl donor supplementation upregulated fatty acid desaturase 1 (FADS1), thioredoxin-1 (TRX1) and catalase (CAT) expression in the liver of MD-LPD fed mice compared to LPD mice. Altogether, our study revealed that odd chain fatty acids (OCFA)s are key early markers observed in a suboptimal diet-induced metabolic changes and may be potential targets to improve metabolic health outcomes.

Main objectives of this study were to analyse metabolomic profile features of patients with COVID-19 using mass spectrometry techniques while taking into account the clinical and laboratory history, and to study the relationship between the severity of COVID-19 symptoms and the concentration of primary metabolites, primarily amino acids. We used frozen blood serum samples of 935 COVID-19 patients from the City Hospital No. 40 biobank collection. Metabolomic profile was studied by HPLS-MS/MS method. R programming language was used for statistical data processing. The difference of metabolic profile of patients with COVID-19 depending on the severity of the disease was revealed based on the performed analysis - for 52 out of 84 detected compounds there were differences with reliability p<0,01. Statistically significant differences in concentration were recorded for organic acids, amino acids and their derivatives. Using samples from the biobank collection, a metabolomic study of the biomaterial of patients hospitalised with the diagnosis of COVID-19 was carried out. According to the results obtained, kynurenine, phenylalanine and acetylcarnitine were associated with the severity of COVID-19 infection.

Diurnal variation in biofluid metabolome as observed in a healthy human may alter in perturbed conditions. Biofluids like urine are rich in molecular constituents including metabolites, and infectious disease conditions like tuberculosis (TB) may influence diurnal differences for which limited reports are available in the literature. In this study, we present an optimized gas chromatography coupled to a quadrupole mass spectrometry (GC-MS) method to analyze processed and trimethylsilyl (TMS) derivatized urine metabolites. Urine samples were collected at four time points (0, 6, 12 and 24 hours) of study subjects [n=15; mean age 37 (24-70) in years] including controls [n=7; mean age 29.3 (24-35) years] and culture-confirmed active TB patients [ATB; n=8; mean age 43.7 (25-70) years] receiving treatment in the intensive phase. Global urine metabolite profiling was carried out using the optimized GC-MS method. Higher urine analyte diversity was observed in ATB patients (74) than in controls (36) during the day. Diurnal variations of the parent anti-TB drugs and their breakdown products (pyrazinamide, pyrazinoic acid, 5-hydroxy pyrazinoic acid, isonicotinic acid and alpha amino butyric acid) were observed with maximum abundance at 6 h. Interestingly, urine of ATB subjects at 6 h showed the highest metabolic diversity, whereas it was at 12 h in controls. Many analytes including glycine and alanine amino-acids showed diurnal variation in ATB and controls. These changes could be attributed to the altered host metabolic activities due to disease, treatment-associated decrease in total body bacterial burden and gut microbiota dysbiosis. And the optimized spiked-in internal standard, urine sample volume and GC-MS method could be used for global urine metabolome analysis in healthy and different perturbed conditions. "For Table of Contents Only" O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/21254606v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12dd33corg.highwire.dtl.DTLVardef@187464borg.highwire.dtl.DTLVardef@17fe4dorg.highwire.dtl.DTLVardef@1389f70_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDihydrosphingolipids are lipid molecules biosynthetically related to ceramides. An increase in ceramides is associated with enhanced fat storage in the liver and inhibition of their synthesis is reported to prevent the appearance of steatosis in animal models. However, the precise association of dihydrosphingolipids with non-alcoholic fatty liver disease (NAFLD) is yet to be established. We employed a diet-induced NAFLD mouse model to study the association between this class of compounds and disease progression. MethodsMice were fed a high-fat diet enriched in cholesterol and supplemented with glucose and fructose up to 40 weeks. A mouse subgroup was treated with carbon tetrachloride to accelerate fibrosis development. Animals were sacrificed at different time-points to reproduce the full spectrum of histological damage found in human disease, including steatosis (NAFL) and steatohepatitis (NASH) with and without significant fibrosis. Blood and liver tissue samples were obtained from patients (n=195) whose NAFLD severity was assessed histologically. Lipidomic analysis was performed using liquid chromatography-tandem mass spectrometry. ResultsTriglyceride, cholesterol ester and dihydrosphingolipid levels were increased in the liver of model mice in association with the degree of steatosis. Dihydroceramide concentrations increased with the histological severity of the disease in liver samples of mice (0.024 {+/-} 0.003 vs 0.049 {+/-} 0.005, non-NAFLD vs NASH-fibrosis, p<0.0001) and patients (0.105 {+/-} 0.011 vs 0.165 {+/-} 0.021, p=0.0221). Several dihydroceramide and dihydrosphingomyelin species were increased in plasma of NAFLD patients and correlated with accumulation of liver triglycerides. ConclusionsDihydrosphingolipids accumulate in the liver in response to increased free fatty acid overload and are correlated with progressive histological damage in NAFLD. The increase in dihydrosphingolipids is related to upregulation of hepatic expression of enzymes involved in de novo synthesis of ceramides. HIGHLIGHTSO_LINeutral lipids and dihydrosphingolipids accumulate in liver in correlation with the histological severity of NAFLD in both mice and humans. C_LIO_LIThe ceramide pathway is stimulated to alleviate the free fatty acid excess in liver of NAFLD models. C_LIO_LIAppearance of significant fibrosis is associated with reduced concentrations of neutral lipids but not dihydrosphingolipids in a mouse model of NAFLD. C_LI

Background and AimsActivating mutations in the CTNNB1 gene encoding {beta}-catenin are among the most frequently observed oncogenic alterations in hepatocellular carcinoma (HCC). HCC with CTNNB1 mutations show profound alterations in lipid metabolism including increases in fatty acid oxidation and transformation of the phospholipidome, but it is unclear how these changes arise and whether they contribute to the oncogenic program in HCC. MethodsWe employed untargeted lipidomics and targeted isotope tracing to quantify phospholipid production fluxes in an inducible human liver cell line expressing mutant {beta}-catenin, as well as in transgenic zebrafish with activated {beta}-catenin-driven HCC. ResultsIn both models, activated {beta}-catenin expression was associated with large changes in the lipidome including conserved increases in acylcarnitines and ceramides and decreases in triglycerides. Lipid flux analysis in human cells revealed a large reduction in phosphatidylcholine (PC) production rates as assayed by choline tracer incorporation. We developed isotope tracing lipid flux analysis for zebrafish and observed similar reductions in phosphatidylcholine synthesis flux accomplished by sex-specific mechanisms. ConclusionsThe integration of isotope tracing with lipid abundances highlights specific lipid class transformations downstream of {beta}-catenin signaling in HCC and suggests future HCC-specific lipid metabolic targets. SynopsisIn this work, we show by lipid specific isotope tracing that mutations in the oncogene CTNNB1 leads to conserved changes in lipid metabolism in hepatocellular carcinoma. These include the stimulation of fatty acid oxidation and a suppression of phosphorylcholine synthesis.

Metabolic dysfunction associated fatty liver disease (MAFLD) starts with increased fat deposition called steatosis progressing towards steatohepatitis, fibrosis, cirrhosis and end stage liver disease. Dysfunctional metabolism in type-2 diabetes or obesity is associated with gut dysbiosis, causing release of gut microbial endotoxins like lipopolysaccharides (LPS) into the liver via portal circulation also known as gut-liver axis. Endotoxins or LPS signals metabolic changes in the liver initiating steatosis via gut-liver axis. Various pre-clinical models have been studied to evaluate the role of fermented food derived bioactive compounds preventing the process of steatosis. Among them exopolysaccharides (EPS), as a part of fermented foods have been reported to produce metabolites by the gut microbial fermentation, providing liver with health benefits. In the present study, we have evaluated the role of a noble EPS or G (glucan) in the condition of liver steatosis by using cell culture model of gut-liver axis by co-culturing Caco-2 and HepG2 cells in transwell culture dishes. LPS was used to mimic gut dysbiosis, with or without pre-treatment of G to caco-2 cells in the upper chamber, wherease, HepG2 cells in lower chamber were extracted for metabolites to be analyzed by LC-Q-TOF. The global metabolomic profiles of LPS+G+ and LPS+ treated cells were compared. The primary target of G showed its role in cholesterol and primary bile acid metabolism along with changes in nucleotide, vitamins and branched chain aminoacid metabolites helpful in reducing fatty liver. Future studies with this glucan using targeted metabolomics could confirm biomarkers of therapeutic intervention of steatosis and could be translated into healthy food products.

BackgroundLipids are involved in the interaction between viral infection and the host metabolic and immunological response. Several studies comparing the lipidome of COVID-19-positive hospitalized patients vs. healthy subjects have already been reported. It is largely unknown, however, whether these differences are specific to this disease. The present study compared the lipidomic signature of hospitalized COVID-19-positive patients with that of healthy subjects, and with COVID-19-negative patients hospitalized for other infectious/inflammatory diseases. Potential COVID-19 biomarkers were identified. MethodsWe analyzed the lipidomic signature of 126 COVID-19-positive patients, 45 COVID-19-negative patients hospitalized with other infectious/inflammatory diseases and 50 healthy volunteers. Results were interpreted by machine learning. ResultsWe identified acylcarnitines, lysophosphatidylethanolamines, arachidonic acid and oxylipins as the most altered species in COVID-19-positive patients compared to healthy volunteers. However, we found similar alterations in COVID-19-negative patients. By contrast, we identified lysophosphatidylcholine 22:6-sn2, phosphatidylcholine 36:1 and secondary bile acids as the parameters that had the greatest capacity to discriminate between COVID-19-positive and COVID-19-negative patients. ConclusionThis study shows that COVID-19 infection shares many lipid alterations with other infectious/inflammatory diseases, but differentiating them from the healthy population. Also, we identified some lipid species the alterations of which distinguish COVID-19-positive from Covid-19-negative patients. Our results highlight the value of integrating lipidomics with machine learning algorithms to explore the pathophysiology of COVID-19 and, consequently, improve clinical decision making.

Background & Aims: Per- and polyfluoroalkyl substances (PFAS) are persistent endocrine-disrupting chemicals associated with metabolic dysfunction, including metabolic dysfunction-associated steatotic liver disease (MASLD). While PFAS perturb lipid and bile acid (BA) metabolism in a sex-specific manner, the underlying mechanisms remain unclear. We tested whether steroid hormones mediate PFAS-associated metabolic alterations. Methods: In 104 patients with biopsy-characterized MASLD, we performed sex-stratified analyses applied liquid chromatography coupled to mass spectrometry (LC-MS) for chemical analysis, integrating circulating steroids, PFAS exposure, hepatic lipidomics and BA profiles. Results: Steroid hormones were associated with MASLD severity in a sexually-dimorphic manner. Dihydrotestosterone showed consistent inverse associations with steatosis, fibrosis, necroinflammation and insulin resistance, particularly in females. PFAS exposure was associated with altered steroid profiles, predominantly indicating suppressed steroidogenesis in females. These PFAS-associated hormonal changes were linked to downstream alterations in hepatic lipids and BAs. Mediation analysis supported indirect effects of PFAS on metabolic pathways via steroids, including testosterone/epi-testosterone-mediated effects on ether phospholipids and estradiol-mediated effects on lithocholic acid. Females exhibited stronger PFAS-steroid-BA associations, whereas males showed weaker, lipid-centric effects. Conclusions: PFAS exposure is associated with sex-specific disruption of steroid hormone pathways that may link environmental exposure to lipid and BA dysregulation in MASLD. These findings identify steroid hormones as potential key mediators of PFAS-associated metabolic dysfunction and highlight sex as a critical determinant in environmental liver disease.

IntroductionIn-depth knowledge of metabolic dysregulations in colorectal cancer (CRC) (and other cancers as well) is essential for developing treatments that specifically kill neoplastic cells. It may also allow us to pinpoint metabolites or lipids with potential for development as tumor biomarkers for use in body-fluid or breath assays. CRC onset is preceded by an interval of [~]10 years characterized by the presence of precancerous lesions, and our previous studies have revealed epigenomic, transcriptomic, and proteomic evidence in these lesions of certain metabolic changes typical of CRC. These findings prompted us to conduct untargeted metabolomic and lipidomic analyses of CRCs and colorectal adenomas (the most common precancerous lesions of the gut). MethodsWe analyzed 29 endoscopically collected tumor tissue samples (29 adenomas [ADNs], 10 CRCs, each with a colon segment-matched sample of normal mucosa [i.e., 29 NM-ADN, 10 NM-CRC]). The freshly collected samples were promptly frozen in liquid nitrogen and later processed to obtain metabolite and lipid extracts. Each of the 78 samples was analyzed with nano-flow LC-MS/MS (liquid chromatography with mass spectrometry) to characterize its metabolome (using HILIC, Hydrophilic Interaction Liquid Chromatography) and lipidome (using RP, Reversed Phase chromatography). The data acquired were processed using Progenesis QI. For statistical and multivariate analysis of the resulting peak tables, we used basic R packages and the R package made4. ResultsUnsupervised between-group analysis based on the full set of detected metabolites (n=1830) and lipids (n=2365) clearly discriminated ADNs and CRCs from their matched samples of normal mucosa at both the metabolome and lipidome levels. Compared with the NM-ADN, the ADNs contained significantly different levels of 14.6% of the metabolites and 10.8% of the lipids. Fewer compounds (9.1% of metabolites, 6.2% of lipids) displayed differential abundance in CRCs (vs. NM-CRC). The metabolome and lipidome of the NM-ADN also differed from those of the NM-CRC, probably reflecting the presence of a field cancerization effect exerted by the invasive tumors. A substantial number of metabolites (n=340) and lipids (n=201) also displayed abundance differentials across the sequential tumorigenic stages represented by the NM-ADN (considered more representative of NM from a lesion-free colon) [-&gt;] ADN [-&gt;] CRC. In most cases, the trend consisted of progressive increases or progressive decreases in abundance as the tumorigenesis advanced. ConclusionsOur findings provide a preliminary picture of the progressive metabolomic and lipidomic changes occurring during the adenomatous phase of colorectal tumorigenesis. Once definitively annotated, the numerous differentially abundant compounds detected in this study may well shed valuable light on the metabolic dysregulations occurring during this process and provide useful clues for the development of novel tools for the diagnosis and treatment of colorectal tumors.

IntroductionChildhood is a critical period for the development of executive functioning skills, including selective attention and inhibitory control, which are essential for cognitive development. Optimal brain development during this time requires appropriate levels of macronutrient intake. Metabolomics can offer valuable insights into which metabolites cognitive functioning and the underlying gut-brain interactions. ObjectivesThis study aimed to explore to use of breathomics to investigate associations between exhaled metabolites and executive functioning in children. MethodsChildren (8-10 years; N = 31) were recruited via flyers at schools and after-school care. The assessment of executive functioning was done using Eriksen flanker task. Breath samples were collected in Tedlar(R) bags and analyzed with proton transfer reaction-mass spectrometry (PTR-MS). On-breath peaks were selected and subjected to partial least squares (PLS) regression. Significance multivariate correlation (sMC) was used afterwards to select metabolites bearing predictive power towards executive functioning. ResultsGut microbiome-related metabolites (methane, ethanol, and butyric acid) present in exhaled breath were associated with an improved executive functioning, whereas isoprene was linked to reduced executive functioning. Additionally, increased levels of inflammatory markers, ethylene and acetaldehyde, were associated with a higher compatibility effect in error rates, suggesting diminished cognitive control. These VOCs were putatively linked with specific gut microbial taxa; for instance, reduced Bacteroidetes abundance (associated with methane production) is associated with decreased inhibitory control, while Enterobacteriaceae were linked to lipopolysaccharide-induced inflammation which is also a process that causes increased ethylene production. ConclusionThis proof-of-concept study demonstrates that VOCs in exhaled breath could serve as a promising non-invasive tool for assessing gut-brain interactions related to executive functioning in children.

Hepatocyte phospholipase D1 (PLD1) knockout alleviated metabolic dysfunction-associated steatotic liver disease (MASLD) in mice, but the underlying mechanism is largely unknown. In this study, high-fat diet is fed to wild type (Con) and hepatocyte PLD1 knockout (Con_KO) mice to establish MASLD model (HFHC and HFHC_KO). Intestinal contents of mice are analyzed via metagenomics and metabolomics, the liver bile acids are assessed by mass spectrometry imaging. At phylum level, Bacillota in the intestines of MASLD model mice are significantly increased and Bacteroidota are significantly decreased. However, after the deletion of hepatocyte PLD1, Pseudomonadota and Candidatus Bathyarchaeota are significantly decreased in the MASLD model mice. Then at species level, compared with Con group, the Faecalibaculum rodentium is significantly increased in HFHC group, in which hepatocyte PLD1 knockout causes Desulfovibrionaceae bacterium LT0009 and Lachnospiraceae bacterium 10-1 to significantly decrease. As for intestinal bile acids, two bile acids (Hyodeoxycholic acid and Glycolithocholic acid) are found to be different between the HFHC_KO group and the HFHC group. Association analysis shows the Faecalibaculum co-occurs with DCA, {beta}MCA, {Omega}MCA and MCA, while probiotic Bacteroides uniformis is significantly correlated with UDCA, 12-KetoLCA, 7-KetoLCA. Finally, mass spectrometry imaging reveals that TCA and TDCA in liver are significantly decreased after hepatocyte PLD1 knockout. These findings demonstrate that hepatocyte PLD1 knockout alters gut microbiota and bile acids profiles, suggesting PLD1 deficiency may modulate MASLD progression by changing intestinal microbiota-bile acid homeostasis. HighlightsHere, we show that hepatocyte PLD1 knockout alters gut microbiota and bile acid profiles in metabolic fatty liver disease mouse by high-fat diet. O_LIWild type (Con) and hepatocyte PLD1 knockout (Con_KO) mice were used to establish HFHC and HFHC_KO models, respectively. C_LIO_LIIntestinal contents were collected for metagenomic and metabolomics analysis, and liver tissues were taken for mass spectrometry imaging to investigate gut microbiota-bile acid relationships. C_LIO_LIIn HFHC_KO mice, Desulfovibrionaceae bacterium LT0009 and Lachnospiraceae bacterium 10-1 were significantly reduced, accompanied by altered HDCA and GLCA. C_LIO_LIAssociation analysis revealed Faecalibaculum co-occurred with DCA, {beta}MCA, {Omega}MCA, and MCA, while Bacteroides uniformis was significantly associated with UDCA, 12-KetoLCA, and 7-KetoLCA. C_LIO_LIMass spectrometry imaging showed hepatocyte PLD1 knockout significantly decreased liver TCA and TDCA, suggesting PLD1 deficiency may modulates MASLD progression via microbiota-bile acid homeostasis. C_LI O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/663496v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1b5bfa8org.highwire.dtl.DTLVardef@139ed05org.highwire.dtl.DTLVardef@1f7e905org.highwire.dtl.DTLVardef@e34b59_HPS_FORMAT_FIGEXP M_FIG C_FIG

Metabolomic studies are important to understand microbial metabolism and interaction between the host and the gut microbiome. Although there have been efforts to standardize sample processing in metabolomic studies, infant samples are mostly disregarded. In birth cohort studies, the use of diaper liners is prevalent and its impact on fecal metabolic profile remains untested. In this study, we compared metabolite profiles of fecal samples collected as solid stool and those collected from stool saturated liner. One infants stool sample was collected in triplicate for solid stool and stool saturated liner. Comprehensive metabolomics analysis of the fecal samples was performed using NMR, UPLC and DI-MS. The total number, identities and concentrations of the metabolites were determined and compared between stool sample collection methods (stool vs. liner). The number and identity of metabolites did not differ between collection methods for NMR and DI-MS when excluding metabolites with a coefficient of variation (CV) > 40%. NMR analysis demonstrated lowest bias between collection methods, and lowest technical precision between triplicates of the same method followed by DI-MS then UPLC. Concentrations of many metabolites from stool and stool saturated liner differed significantly as revealed by Bland-Altman plots and t-tests. Overall, a mean bias of 10.2% in the Bland-Altman analysis was acceptable for some metabolites confirming mutual agreement but not for others with a wide range of bias (-97-117%). Consequently, stool and stool-saturated liner could be used interchangeably only for some select metabolite classes e.g. amino acids. Differences between the metabolomic profiles of solid stool samples and stool saturated liner samples for some important molecules e.g., ethanol, fumarate, short chain fatty acids and bile acids, indicate the need for standardization in stool collection method for metabolomic studies performed in infants.

BackgroundGut microbiota alteration is implicated in the pathogenesis of alcoholic liver disease (ALD) and HCC. No study has characterized the dysbiosis associated with ALD by microbial culturomics, an approach that certifies viability and allows the characterization of pathobiont strain candidates. MethodsA case-control study was conducted on patients with ALD without HCC (ALD-NoHCC) (n=16), ALD with HCC (ALD-HCC) (n=19), and controls (n=24). 16S rRNA amplicon sequencing and microbial culturomics were used as complementary methods for gut microbiome profiling. ResultsBy microbial culturomics, Thomasclavelia ramosa was the most enriched and detected in all ALD samples (100%), while it was cultivated in only a small proportion of controls (20%, p < 0.001). By 16S rRNA amplicon sequencing and 3-groups linear discriminant analysis, T. ramosa was increased explicitly in the ALD-HCC group (LDA-score > 5, p < 0.05). ConclusionsT. ramosa, identified by culturomics and 16 rRNA sequencing, is associated with ALD and ALD-HCC. Alongside the recently reported in vitro genotoxicity of this species in colorectal cancer, this species has been identified as a candidate oncobiont in ALD-HCC. HighlightsO_LIThe gut microbiota signature of ALD and ALD-HCC was explored by microbial culturomics and 16S amplicon sequencing C_LIO_LIBy culturomics, T. ramosa was the most enriched and cultured from all included ALD patients, but in only 20% of controls (p < 0.05). C_LIO_LIT. ramosa was significantly associated with alcohol-related HCC by 16S sequencing. C_LIO_LIT. ramosa is identified as a putative oncobiont associated with ALD-HCC, thus opening new avenues for diagnosis and treatment. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/24312231v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@7db628org.highwire.dtl.DTLVardef@1420dc8org.highwire.dtl.DTLVardef@196fc7borg.highwire.dtl.DTLVardef@19a0ed4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Brazil has the second highest COVID-19 death rate while Rio de Janeiro is among the states with the highest rate in the country. Although effective vaccines have been developed, it is anticipated that the ongoing COVID-19 pandemic will transition into an endemic state. Under this scenario, it is worrisome that the underlying molecular mechanisms associated with the disease clinical evolution from mild to severe, as well as the mechanisms leading to long COVID are not yet fully understood. In this study, 1H Nuclear Magnetic Resonance spectroscopy and Liquid Chromatography-Mass spectrometry-based metabolomics were used to identify potential pathways and metabolites involved in COVID-19 pathophysiology and disease outcome. We prospectively enrolled 35 severe RT-PCR confirmed COVID-19 cases within 72 hours from intensive care unit admission, between April and July 2020 from two reference centers in Rio de Janeiro, and 12 samples from non-infected control subjects. Of the 35 samples from COVID-19 patients, 18 were from survivors and 17 from non-survivors. We observed that patients with severe COVID-19 had their plasma metabolome significantly changed if compared to control subjects. We observed lower levels of glycerophosphocholine and other choline-related metabolites, serine, glycine, and betaine, indicating a dysregulation in methyl donors and one-carbon metabolism. Importantly, non-survivors had higher levels of creatine/creatinine, 4-hydroxyproline, gluconic acid and N-acetylserine compared to survivors and controls, reflecting uncontrolled inflammation, liver and kidney dysfunction, and insulin resistance in these patients. Lipoprotein dynamics and amino acid metabolism were also altered in severe COVID-19 subjects. Several changes were greater in women, thus patients sex should be considered in pandemic surveillance to achieve better disease stratification and improve outcomes. The incidence of severe outcome after hospital discharge is very high in Brazil, thus these metabolic alterations may be used to monitor patients organs and tissues and to understand the pathophysiology of long-post COVID-19.

BackgroundAging, Western diet (WD) intake, and bile acid (BA) receptor farnesoid X receptor (FXR) inactivation are risk factors for metabolic disease development including nonalcoholic fatty liver disease (NAFLD) and chronic inflammation-related health issues such as dementia. The progression of the metabolic disease can be escalated when those risks are combined. Inactivation of FXR is cancer prone in both humans and mice. The current study used omics data generated within the gut-liver axis to classify those risks using bioinformatics and machine learning approaches. MethodsDifferent ages (5, 10, and 15 months) of wild-type (WT) and FXR knockout (KO) male mice were fed with either a healthy control diet (CD) or a WD since weaning. Hepatic transcripts, liver, serum, and urine metabolites, hepatic bile acids (BAs), as well as gut microbiota were used for risk prediction. A linear support vector machine with K-fold cross-validation was used for classification and feature selection. ResultsIncreased urine sucrose alone achieved 91% accuracy in predicting WD intake. Hepatic lithocholic acid (LCA) and serum pyruvate had 100% and 95% accuracy, respectively to classify age. Association analyses showed hepatic LCA was positively associated with serum concentrations of acetone, a ketone body, and 1,3-dihydroxyacetone (DHA), but negatively correlated with serum pyruvate. Urine metabolites (decreased creatinine and taurine as well as increased succinate) or gut microbiota (increased Dorea, Dehalobacterium, and Oscillospira) could predict FXR functional status with greater than 90% accuracy. Integrated pathway analyses revealed that the predictors for diet and FXR expression were implicated in the central carbon metabolism in cancer. To assess the translational relevance, mouse hepatic transcripts were crosschecked with human NAFLD and hepatocellular carcinoma (HCC) datasets. WD-affected hepatic Cyp39a1 and Gramd1b expression were associated with human HCC and NAFLD, respectively. The metabolites and diseases interaction analyses uncovered that the identified features are implicated in human metabolic diseases, mental disorders, and cancer. ConclusionThe risk prediction using mouse models contributes to the identification of noninvasive biomarkers for early diagnosis of metabolic disease development.