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Introduction Diabetic retinopathy (DR), diabetic kidney disease (DKD) and distal symmetric polyneuropathy (DSPN) share common pathophysiology and pose an additive risk of early mortality.Research design and methods In adults with type 1 diabetes, 49 metabolites previously associated with either DR or DKD were assessed in relation to presence of DSPN. Metabolites overlapping in significance with presence of all three complications were assessed in relation to microvascular burden severity (additive number of complications—ie, presence of DKD±DR±DSPN) using linear regression models. Subsequently, the same metabolites were assessed with progression to endpoints: soft microvascular events (progression in albuminuria grade, ≥30% estimated glomerular filtration rate (eGFR) decline, or any progression in DR grade), hard microvascular events (progression to proliferative DR, chronic kidney failure, or ≥40% eGFR decline), and hard microvascular or macrovascular events (hard microvascular events, cardiovascular events (myocardial infarction, stroke, or arterial interventions), or cardiovascular mortality), using Cox models. All models were adjusted for sex, baseline age, diabetes duration, systolic blood pressure, HbA1c, body mass index, total cholesterol, smoking, and statin treatment.Results The full cohort investigated consisted of 487 participants. Mean (SD) follow-up was 4.8 (2.9, 5.7) years. Baseline biothesiometry was available in 202 participants, comprising the cross-sectional cohort. Eight metabolites were significantly associated with presence of DR, DKD, and DSPN, and six with additive microvascular burden severity. In the full cohort longitudinal analysis, higher levels of 3,4-dihydroxybutanoic acid (DHBA), 2,4-DHBA, ribonic acid, glycine, and ribitol were associated with development of events in both crude and adjusted models. Adding 3,4-DHBA, ribonic acid, and glycine to a traditional risk factor model improved the discrimination of hard microvascular events.Conclusions While prospective studies directly assessing the predictive ability of these markers are needed, our results strengthen the role of clinical metabolomics in relation to risk assessment of diabetic complications in chronic type 1 diabetes.

Background & Aims: Infections are frequent in patients with cirrhosis and worsen prognosis. We evaluated the incidence of infections and their impact on decompensation and death in patients with early alcohol-related liver disease (ALD) during long-term follow-up. Methods: We performed a prospective cohort study of patients in secondary care with a history of excess alcohol intake, no prior decompensation, and with liver biopsies along with clinical investigations conducted at baseline. During follow-up, we reviewed the patients’ electronic healthcare records for cases of infections, hospitalizations, transient elastography measurements, decompensations, all-cause mortality, and alcohol intake. Results: We included 461 patients with a mean age of 56±10 years (76% males; fibrosis stage F0-1/F2/F3-4 = 259/107/93 [56%/23%/20%]). During a median follow-up of 4.5 years (IQR 2.9-6.3), 134 patients (29%) developed a total of 312 infections, most frequently pneumonia (106/312, 34%) and urinary tract infections (57/312, 18%). Excessive alcohol intake during follow-up, smoking ≥30 pack years, MELD score and elevated liver stiffness during follow-up were independent predictors of infections. Patients who developed at least one infection had a significantly increased risk of subsequent decompensation (hazard ratio 4.98, 95% CI 2.47-10.03) and death (hazard ratio 8.24, 95% CI 4.65-14.59). Infections increased the risk of decompensation and death independently of baseline fibrosis stage, age, gender, and MELD score. Conclusions: Almost one-third of patients with early ALD develop an infection, which worsens their prognosis by increasing the risk of decompensation and death. The risk of infections increases with liver disease severity and ongoing harmful use of alcohol. Impact and implications: This study reveals that infections significantly worsen the prognosis of patients with early alcohol-related liver disease (ALD), increasing the likelihood of decompensation and death by up to eight times. These findings, pertinent to healthcare providers, researchers, and policymakers, emphasize the importance of early prevention and management of infections in patients with ALD, even those in early stages who may be asymptomatic. It was observed that nearly one-third of patients with early-stage ALD developed infections over 4.5 years, with risk factors including alcohol overuse, smoking, and higher MELD scores. The research underscores the critical need to incorporate these insights into clinical practice and public health policies to improve patient outcomes and mitigate the impact of infections in patients with ALD.

Abstract Background Genome-wide association studies (GWAS) of Alzheimer’s disease (AD) have identified several risk loci, but many remain unknown. Cerebrospinal fluid (CSF) biomarkers may aid in gene discovery and we previously demonstrated that six CSF biomarkers (β-amyloid, total/phosphorylated tau, NfL, YKL-40, and neurogranin) cluster into five principal components (PC), each representing statistically independent biological processes. Here, we aimed to (1) identify common genetic variants associated with these CSF profiles, (2) assess the role of associated variants in AD pathophysiology, and (3) explore potential sex differences. Methods We performed GWAS for each of the five biomarker PCs in two multi-center studies (EMIF-AD and ADNI). In total, 973 participants (n = 205 controls, n = 546 mild cognitive impairment, n = 222 AD) were analyzed for 7,433,949 common SNPs and 19,511 protein-coding genes. Structural equation models tested whether biomarker PCs mediate genetic risk effects on AD, and stratified and interaction models probed for sex-specific effects. Results Five loci showed genome-wide significant association with CSF profiles, two were novel (rs145791381 [inflammation] and GRIN2D [synaptic functioning]) and three were previously described (APOE, TMEM106B, and CHI3L1). Follow-up analyses of the two novel signals in independent datasets only supported the GRIN2D locus, which contains several functionally interesting candidate genes. Mediation tests indicated that variants in APOE are associated with AD status via processes related to amyloid and tau pathology, while markers in TMEM106B and CHI3L1 are associated with AD only via neuronal injury/inflammation. Additionally, seven loci showed sex-specific associations with AD biomarkers. Conclusions These results suggest that pathway and sex-specific analyses can improve our understanding of AD genetics and may contribute to precision medicine.

Abstract Background Specific ceramides have been identified as risk markers for cardiovascular disease (CVD) years before onset of disease. Treatment with the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide has been shown to induce beneficial changes in the lipid profile and reduce the risk of CVD. Reducing lipotoxic lipids with an antidiabetic drug therapy could be a path towards precision medicine approaches for the treatment of complications to diabetes. In this post-hoc study, an investigation was carried out on the effect of liraglutide on CVD-risk associated ceramides in two randomized clinical trials including participants with type 2 diabetes (T2D). Methods This study analyzed plasma samples from two independent randomized placebo-controlled clinical trials. The first trial, Antiproteinuric Effects of Liraglutide Treatment (LirAlbu12) followed a crossover design where 27 participants were treated for 12 weeks with either liraglutide (1.8 mg/d) or placebo, followed by a four-week washout period, and then another 12 weeks of the other treatment. The second clinical trial, Effect of Liraglutide on Vascular Inflammation in Type-2 Diabetes (LiraFlame26), lasted for 26 weeks and followed a parallel design, where 102 participants were randomized 1:1 to either liraglutide or placebo. Heresix prespecified plasma ceramides were measured using liquid chromatography mass spectrometry and assessed their changes using linear mixed models. Possible confounders were assessed with mediation analyses. Results In the LiraFlame26 trial, 26-week treatment with liraglutide resulted in a significant reduction of two ceramides associated with CVD risk, C16 Cer and C24:1 Cer (p

IntroductionMicroglia and macrophages can influence the evolution of myelin lesions through the production of extracellular vesicles (EVs). While microglial EVs promote in vitro differentiation of oligodendrocyte precursor cells (OPCs), whether EVs derived from macrophages aid or limit OPC maturation is unknown.MethodsImmunofluorescence analysis for the myelin protein MBP was employed to evaluate the impact of EVs from primary rat macrophages on cultured OPC differentiation. Raman spectroscopy and liquid chromatography-mass spectrometry was used to define the promyelinating lipid components of myelin EVs obtained in vitro and isolated from human plasma.Results and discussionHere we show that macrophage-derived EVs do not promote OPC differentiation, and those released from macrophages polarized towards an inflammatory state inhibit OPC maturation. However, their lipid cargo promotes OPC maturation in a similar manner to microglial EVs. We identify the promyelinating endocannabinoids anandamide and 2-arachidonoylglycerol in EVs released by both macrophages and microglia in vitro and circulating in human plasma. Analysis of OPC differentiation in the presence of the endocannabinoid receptor antagonists SR141716A and AM630 reveals a key role of vesicular endocannabinoids in OPC maturation. From this study, EV-associated endocannabinoids emerge as important mediators in microglia/macrophage-oligodendrocyte crosstalk, which may be exploited to enhance myelin repair.

Abstract Background Identifying blood-based signatures of brain health and preclinical pathology may offer insights into early disease mechanisms and highlight avenues for intervention. Here, we systematically profiled associations between blood metabolites and whole-brain volume, hippocampal volume, and amyloid-β status among participants of Insight 46—the neuroscience sub-study of the National Survey of Health and Development (NSHD). We additionally explored whether key metabolites were associated with polygenic risk for Alzheimer’s disease (AD). Methods Following quality control, levels of 1019 metabolites—detected with liquid chromatography-mass spectrometry—were available for 1740 participants at age 60–64. Metabolite data were subsequently clustered into modules of co-expressed metabolites using weighted coexpression network analysis. Accompanying MRI and amyloid-PET imaging data were present for 437 participants (age 69–71). Regression analyses tested relationships between metabolite measures—modules and hub metabolites—and imaging outcomes. Hub metabolites were defined as metabolites that were highly connected within significant (p FDR

Abstract Background Cardiovascular disease remains the leading cause of mortality in individuals with diabetes and improved understanding of its pathophysiology is needed. We investigated the association of a large panel of metabolites and molecular lipid species with future cardiovascular events in type 1 diabetes. Methods The study included 669 individuals with type 1 diabetes. Non-targeted serum metabolomics and lipidomics analyses were performed using mass spectrometry. Data on cardiovascular events (cardiovascular mortality, coronary artery disease, stroke, and peripheral arterial interventions) were obtained from Danish Health registries and analyzed by Cox hazards models. Metabolites and molecular lipid species were analyzed in univariate models adjusted for false discovery rate (FDR). Metabolites and molecular lipid species fulfilling a pFDR

Introduction Hypoglycemia is a major limiting factor in achieving recommended glycemic targets for people with type 1 diabetes. Exposure to recurrent hypoglycemia results in blunted hormonal counter-regulatory and symptomatic responses to hypoglycemia. Limited data on metabolic adaptation to recurrent hypoglycemia are available. This study examined the acute metabolic responses to hypoglycemia and the effect of antecedent hypoglycemia on these responses in type 1 diabetes.Research design and methods Twenty-one outpatients with type 1 diabetes with normal or impaired awareness of hypoglycemia participated in a study assessing the response to hypoglycemia on 2 consecutive days by a hyperinsulinemic glucose clamp. Participants underwent a period of normoglycemia and a period of hypoglycemia during the hyperinsulinemic glucose clamp. Plasma samples were taken during normoglycemia and at the beginning and the end of the hypoglycemic period. Metabolomic analysis of the plasma samples was conducted using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry.Results In total, 68 metabolites were studied. On day 1, concentrations of the branched-chain amino acids, leucine (p=3.8×10−3) and isoleucine (p=2.2×10−3), decreased during hypoglycemia. On day 2, during hypoglycemia, five amino acids (including leucine and isoleucine) significantly decreased, and two fatty acids (tetradecanoic and oleic acids) significantly increased (p

Background: Education and cognition demonstrate consistent inverse associations with Alzheimer’s disease (AD). The biological underpinnings, however, remain unclear. Blood metabolites reflect the end point of biological processes and are accessible and malleable. Identifying metabolites with etiological relevance to AD and disentangling how these relate to cognitive factors along the AD causal pathway could, therefore, offer unique insights into underlying causal mechanisms. Methods: Using data from the largest metabolomics genome-wide association study (N ≈ 24,925) and three independent AD cohorts (N = 4725), cross-trait polygenic scores were generated and meta-analyzed. Metabolites genetically associated with AD were taken forward for causal analyses. Bidirectional two-sample Mendelian randomization interrogated univariable causal relationships between 1) metabolites and AD; 2) education and cognition; 3) metabolites, education, and cognition; and 4) education, cognition, and AD. Mediating relationships were computed using multivariable Mendelian randomization. Results: Thirty-four metabolites were genetically associated with AD at p < .05. Of these, glutamine and free cholesterol in extra-large high-density lipoproteins demonstrated a protective causal effect (glutamine: 95% confidence interval [CI], 0.70 to 0.92; free cholesterol in extra-large high-density lipoproteins: 95% CI, 0.75 to 0.92). An AD-protective effect was also observed for education (95% CI, 0.61 to 0.85) and cognition (95% CI, 0.60 to 0.89), with bidirectional mediation evident. Cognition as a mediator of the education-AD relationship was stronger than vice versa, however. No evidence of mediation via any metabolite was found. Conclusions: Glutamine and free cholesterol in extra-large high-density lipoproteins show protective causal effects on AD. Education and cognition also demonstrate protection, though education’s effect is almost entirely mediated by cognition. These insights provide key pieces of the AD causal puzzle, important for informing future multimodal work and progressing toward effective intervention strategies.

Aims/hypothesisTo identify novel pathophysiological signatures of longstanding type 1 diabetes (T1D) with and without albuminuria we investigated the gut microbiome and blood metabolome in individuals with T1D and healthy controls (HC). We also mapped the functional underpinnings of the microbiome in relation to its metabolic role.MethodsOne hundred and sixty-one individuals with T1D and 50 HC were recruited at the Steno Diabetes Center Copenhagen, Denmark. T1D cases were stratified based on levels of albuminuria into normoalbuminuria, moderate and severely increased albuminuria. Shotgun sequencing of bacterial and viral microbiome in stool samples and circulating metabolites and lipids profiling using mass spectroscopy in plasma of all participants were performed. Functional mapping of microbiome into Gut Metabolic Modules (GMMs) was done using EggNog and KEGG databases. Multiomics integration was performed using MOFA tool.ResultsMeasures of the gut bacterial beta diversity differed significantly between T1D and HC, either with moderately or severely increased albuminuria. Taxonomic analyses of the bacterial microbiota identified 51 species that differed in absolute abundance between T1D and HC (17 higher, 34 lower). Stratified on levels of albuminuria, 10 species were differentially abundant for the moderately increased albuminuria group, 63 for the severely increased albuminuria group while 25 were common and differentially abundant both for moderately and severely increased albuminuria groups, when compared to HC. Functional characterization of the bacteriome identified 23 differentially enriched GMMs between T1D and HC, mostly involved in sugar and amino acid metabolism. No differences in relation to albuminuria stratification was observed. Twenty-five phages were differentially abundant between T1D and HC groups. Six of these varied with albuminuria status. Plasma metabolomics indicated differences in the steroidogenesis and sugar metabolism and circulating sphingolipids in T1D individuals. We identified association between sphingolipid levels and Bacteroides sp. abundances. MOFA revealed reduced interactions between gut microbiome and plasma metabolome profiles albeit polar metabolite, lipids and bacteriome compositions contributed to the variance in albuminuria levels among T1D individuals.ConclusionsIndividuals with T1D and progressive kidney disease stratified on levels of albuminuria show distinct signatures in their gut microbiome and blood metabolome.

Background and objectiveBlood-based biomarkers represent a promising approach to help identify early Alzheimer’s disease (AD). Previous research has applied traditional machine learning (ML) to analyze plasma omics data and search for potential biomarkers, but the most modern ML methods based on deep learning has however been scarcely explored. In the current study, we aim to harness the power of state-of-the-art deep learning neural networks (NNs) to identify plasma proteins that predict amyloid, tau, and neurodegeneration (AT[N]) pathologies in AD.MethodsWe measured 3,635 proteins using SOMAscan in 881 participants from the European Medical Information Framework for AD Multimodal Biomarker Discovery study (EMIF-AD MBD). Participants underwent measurements of brain amyloid β (Aβ) burden, phosphorylated tau (p-tau) burden, and total tau (t-tau) burden to determine their AT(N) statuses. We ranked proteins by their association with Aβ, p-tau, t-tau, and AT(N), and fed the top 100 proteins along with age and apolipoprotein E (APOE) status into NN classifiers as input features to predict these four outcomes relevant to AD. We compared NN performance of using proteins, age, and APOE genotype with performance of using age and APOE status alone to identify protein panels that optimally improved the prediction over these main risk factors. Proteins that improved the prediction for each outcome were aggregated and nominated for pathway enrichment and protein–protein interaction enrichment analysis.ResultsAge and APOE alone predicted Aβ, p-tau, t-tau, and AT(N) burden with area under the curve (AUC) scores of 0.748, 0.662, 0.710, and 0.795. The addition of proteins significantly improved AUCs to 0.782, 0.674, 0.734, and 0.831, respectively. The identified proteins were enriched in five clusters of AD-associated pathways including human immunodeficiency virus 1 infection, p53 signaling pathway, and phosphoinositide-3-kinase–protein kinase B/Akt signaling pathway.ConclusionCombined with age and APOE genotype, the proteins identified have the potential to serve as blood-based biomarkers for AD and await validation in future studies. While the NNs did not achieve better scores than the support vector machine model used in our previous study, their performances were likely limited by small sample size.

Abstract The role of brain cholesterol metabolism in Alzheimer’s disease (AD) remains unclear. Peripheral and brain cholesterol levels are largely independent due to the impermeability of the blood brain barrier (BBB), highlighting the importance of studying the role of brain cholesterol homeostasis in AD. We first tested whether metabolite markers of brain cholesterol biosynthesis and catabolism were altered in AD and associated with AD pathology using linear mixed-effects models in two brain autopsy samples from the Baltimore Longitudinal Study of Aging (BLSA) and the Religious Orders Study (ROS). We next tested whether genetic regulators of brain cholesterol biosynthesis and catabolism were altered in AD using the ANOVA test in publicly available brain tissue transcriptomic datasets. Finally, using regional brain transcriptomic data, we performed genome-scale metabolic network modeling to assess alterations in cholesterol biosynthesis and catabolism reactions in AD. We show that AD is associated with pervasive abnormalities in cholesterol biosynthesis and catabolism. Using transcriptomic data from Parkinson’s disease (PD) brain tissue samples, we found that gene expression alterations identified in AD were not observed in PD, suggesting that these changes may be specific to AD. Our results suggest that reduced de novo cholesterol biosynthesis may occur in response to impaired enzymatic cholesterol catabolism and efflux to maintain brain cholesterol levels in AD. This is accompanied by the accumulation of nonenzymatically generated cytotoxic oxysterols. Our results set the stage for experimental studies to address whether abnormalities in cholesterol metabolism are plausible therapeutic targets in AD.

Summary: Background: Individuals with long standing diabetes duration can experience damage to small microvascular blood vessels leading to diabetes complications (DCs) and increased mortality. Precision diagnostic tailors a diagnosis to an individual by using biomedical information. Blood small molecule profiling coupled with machine learning (ML) can facilitate the goals of precision diagnostics, including earlier diagnosis and individualized risk scoring. Methods: Using data in a cohort of 537 adults with type 1 diabetes (T1D) we predicted five-year progression to DCs. Prediction models were computed first with clinical risk factors at baseline and then with clinical risk factors and blood-derived molecular data at baseline. Progression of diabetic kidney disease and diabetic retinopathy were predicted in two complication-specific models. Findings: The model predicts the progression to diabetic kidney disease with accuracy: 0.96 ± 0.25 and 0.96 ± 0.06 area under curve, AUC, with clinical measurements and with small molecule predictors respectively and highlighted main predictors to be albuminuria, glomerular filtration rate, retinopathy status at baseline, sugar derivatives and ketones. For diabetic retinopathy, AUC 0.75 ± 0.14 and 0.79 ± 0.16 with clinical measurements and with small molecule predictors respectively and highlighted key predictors, albuminuria, glomerular filtration rate and retinopathy status at baseline. Individual risk scores were built to visualize results. Interpretation: With further validation ML tools could facilitate the implementation of precision diagnosis in the clinic. It is envisaged that patients could be screened for complications, before these occur, thus preserving healthy life-years for persons with diabetes. Funding: This study has been financially supported by Novo Nordisk Foundation grant NNF14OC0013659.

Abstract Background Both serotonergic signalling disruption and systemic inflammation have been associated with the pathogenesis of Alzheimer’s disease (AD). The common denominator linking the two is the catabolism of the essential amino acid, tryptophan. Metabolism via tryptophan hydroxylase results in serotonin synthesis, whilst metabolism via indoleamine 2,3-dioxygenase (IDO) results in kynurenine and its downstream derivatives. IDO is reported to be activated in times of host systemic inflammation and therefore is thought to influence both pathways. To investigate metabolic alterations in AD, a large-scale metabolic phenotyping study was conducted on both urine and serum samples collected from a multi-centre clinical cohort, consisting of individuals clinically diagnosed with AD, mild cognitive impairment (MCI) and age-matched controls. Methods Metabolic phenotyping was applied to both urine (n = 560) and serum (n = 354) from the European-wide AddNeuroMed/Dementia Case Register (DCR) biobank repositories. Metabolite data were subsequently interrogated for inter-group differences; influence of gender and age; comparisons between two subgroups of MCI - versus those who remained cognitively stable at follow-up visits (sMCI); and those who underwent further cognitive decline (cMCI); and the impact of selective serotonin reuptake inhibitor (SSRI) medication on metabolite concentrations. Results Results revealed significantly lower metabolite concentrations of tryptophan pathway metabolites in the AD group: serotonin (urine, serum), 5-hydroxyindoleacetic acid (urine), kynurenine (serum), kynurenic acid (urine), tryptophan (urine, serum), xanthurenic acid (urine, serum), and kynurenine/tryptophan ratio (urine). For each listed metabolite, a decreasing trend in concentrations was observed in-line with clinical diagnosis: control > MCI > AD. There were no significant differences in the two MCI subgroups whilst SSRI medication status influenced observations in serum, but not urine. Conclusions Urine and serum serotonin concentrations were found to be significantly lower in AD compared with controls, suggesting the bioavailability of the neurotransmitter may be altered in the disease. A significant increase in the kynurenine/tryptophan ratio suggests that this may be a result of a shift to the kynurenine metabolic route due to increased IDO activity, potentially as a result of systemic inflammation. Modulation of the pathways could help improve serotonin bioavailability and signalling in AD patients.

Abstract Finding early disease markers using non-invasive and widely available methods is essential to develop a successful therapy for Alzheimer’s Disease. Few studies to date have examined urine, the most readily available biofluid. Here we report the largest study to date using comprehensive metabolic phenotyping platforms (NMR spectroscopy and UHPLC-MS) to probe the urinary metabolome in-depth in people with Alzheimer’s Disease and Mild Cognitive Impairment. Feature reduction was performed using metabolomic Quantitative Trait Loci, resulting in the list of metabolites associated with the genetic variants. This approach helps accuracy in identification of disease states and provides a route to a plausible mechanistic link to pathological processes. Using these mQTLs we built a Random Forests model, which not only correctly discriminates between people with Alzheimer’s Disease and age-matched controls, but also between individuals with Mild Cognitive Impairment who were later diagnosed with Alzheimer’s Disease and those who were not. Further annotation of top-ranking metabolic features nominated by the trained model revealed the involvement of cholesterol-derived metabolites and small-molecules that were linked to Alzheimer’s pathology in previous studies.

IntroductionDiabetic cardiovascular autonomic neuropathy (CAN) is associated with increased mortality and morbidity. To explore metabolic mechanisms associated with CAN we investigated associations between serum metabolites and CAN in persons with type 1 diabetes (T1D).Materials and MethodsCardiovascular reflex tests (CARTs) (heart rate response to: deep breathing; lying-to-standing test; and the Valsalva maneuver) were used to diagnose CAN in 302 persons with T1D. More than one pathological CARTs defined the CAN diagnosis. Serum metabolomics and lipidomic profiles were analyzed with two complementary non-targeted mass-spectrometry methods. Cross-sectional associations between metabolites and CAN were assessed by linear regression models adjusted for relevant confounders.ResultsParticipants were median (IQR) aged 55(49, 63) years, 48% males with diabetes duration 39(32, 47) years, HbA1c 63(55,69) mmol/mol and 34% had CAN. A total of 75 metabolites and 106 lipids were analyzed. In crude models, the CAN diagnosis was associated with higher levels of hydroxy fatty acids (2,4- and 3,4-dihydroxybutanoic acids, 4−deoxytetronic acid), creatinine, sugar derivates (ribitol, ribonic acid, myo-inositol), citric acid, glycerol, phenols, phosphatidylcholines and lower levels of free fatty acids and the amino acid methionine (p

BackgroundLiraglutide is a glucose-lowering medication used to treat type 2 diabetes and obesity. It is a GLP-1 receptor agonist with downstream metabolic changes beyond the incretin system, such as reducing the risk of cardiovascular complications. The understanding of these changes is critical for improving treatment outcomes. Herein, we present a post hoc experimental analysis using metabolomic phenotyping to discover molecular mecphanisms in response to liraglutide.MethodPlasma samples were obtained from The LiraFlame Study (ClinicalTrials.gov identifier: NCT03449654), a randomized double-blinded placebo-controlled clinical trial, including 102 participants with type 2 diabetes randomized to either liraglutide or placebo treatment for 26 weeks. Mass spectrometry-based metabolomics analyses were carried out on samples from baseline and the end of the trial. Metabolites (n=114) were categorized into pathways and linear mixed models were constructed to evaluate the association between changes in metabolites and liraglutide treatment.ResultsWe found the free fatty acid palmitoleate was significantly reduced in the liraglutide group compared to placebo (adjusted for multiple testing p-value = 0.04). The activity of stearoyl-CoA desaturase-1 (SCD1), the rate limiting enzyme for converting palmitate into palmitoleate, was found significantly downregulated by liraglutide treatment compared to placebo (p-value = 0.01). These metabolic changes have demonstrated to be linked to insulin sensitivity and cardiovascular health.

Alzheimer’s disease (AD) is the most frequent neurodegenerative disease with an increasing prevalence in industrialized, aging populations. AD susceptibility has an established genetic basis which has been the focus of a large number of genome-wide association studies (GWAS) published over the last decade. Most of these GWAS used dichotomized clinical diagnostic status, i.e., case vs. control classification, as outcome phenotypes, without the use of biomarkers. An alternative and potentially more powerful study design is afforded by using quantitative AD-related phenotypes as GWAS outcome traits, an analysis paradigm that we followed in this work. Specifically, we utilized genotype and phenotype data from n = 931 individuals collected under the auspices of the European Medical Information Framework for Alzheimer’s Disease Multimodal Biomarker Discovery (EMIF-AD MBD) study to perform a total of 19 separate GWAS analyses. As outcomes we used five magnetic resonance imaging (MRI) traits and seven cognitive performance traits. For the latter, longitudinal data from at least two timepoints were available in addition to cross-sectional assessments at baseline. Our GWAS analyses revealed several genome-wide significant associations for the neuropsychological performance measures, in particular those assayed longitudinally. Among the most noteworthy signals were associations in or near EHBP1 (EH domain binding protein 1; on chromosome 2p15) and CEP112 (centrosomal protein 112; 17q24.1) with delayed recall as well as SMOC2 (SPARC related modular calcium binding 2; 6p27) with immediate recall in a memory performance test. On the X chromosome, which is often excluded in other GWAS, we identified a genome-wide significant signal near IL1RAPL1 (interleukin 1 receptor accessory protein like 1; Xp21.3). While polygenic score (PGS) analyses showed the expected strong associations with SNPs highlighted in relevant previous GWAS on hippocampal volume and cognitive function, they did not show noteworthy associations with recent AD risk GWAS findings. In summary, our study highlights the power of using quantitative endophenotypes as outcome traits in AD-related GWAS analyses and nominates several new loci not previously implicated in cognitive decline.

Abstract Background There is an urgent need to understand the pathways and processes underlying Alzheimer’s disease (AD) for early diagnosis and development of effective treatments. This study was aimed to investigate Alzheimer’s dementia using an unsupervised lipid, protein and gene multi-omics integrative approach. Methods A lipidomics dataset comprising 185 AD patients, 40 mild cognitive impairment (MCI) individuals and 185 controls, and two proteomics datasets (295 AD, 159 MCI and 197 controls) were used for weighted gene co-expression network analyses (WGCNA). Correlations of modules created within each modality with clinical AD diagnosis, brain atrophy measures and disease progression, as well as their correlations with each other, were analyzed. Gene ontology enrichment analysis was employed to examine the biological processes and molecular and cellular functions of protein modules associated with AD phenotypes. Lipid species were annotated in the lipid modules associated with AD phenotypes. The associations between established AD risk loci and the lipid/protein modules that showed high correlation with AD phenotypes were also explored. Results Five of the 20 identified lipid modules and five of the 17 identified protein modules were correlated with clinical AD diagnosis, brain atrophy measures and disease progression. The lipid modules comprising phospholipids, triglycerides, sphingolipids and cholesterol esters were correlated with AD risk loci involved in immune response and lipid metabolism. The five protein modules involved in positive regulation of cytokine production, neutrophil-mediated immunity, and humoral immune responses were correlated with AD risk loci involved in immune and complement systems and in lipid metabolism (the APOE ε4 genotype). Conclusions Modules of tightly regulated lipids and proteins, drivers in lipid homeostasis and innate immunity, are strongly associated with AD phenotypes.

Background & Aims: In experimental models, alcohol induces acute changes in lipid metabolism that cause hepatocyte lipoapoptosis and inflammation. Here we study human hepatic lipid turnover during controlled alcohol intoxication. Methods: We studied 39 participants with 3 distinct hepatic phenotypes: alcohol-related liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), and healthy controls. Alcohol was administrated via nasogastric tube over 30 min. Hepatic and systemic venous blood was sampled simultaneously at 3 time points: baseline, 60, and 180 min after alcohol intervention. Liver biopsies were sampled 240 min after alcohol intervention. We used ultra-high performance liquid chromatography mass spectrometry to measure levels of more than 250 lipid species from the blood and liver samples. Results: After alcohol intervention, the levels of blood free fatty acid (FFA) and lysophosphatidylcholine (LPC) decreased, while triglyceride (TG) increased. FFA was the only lipid class to decrease in NAFLD after alcohol intervention, whereas LPC and FFA decreased and TG increased after intervention in ALD and healthy controls. Fatty acid chain uptake preference in FFAs and LPCs were oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid. Hepatic venous blood FFA and LPC levels were lower when compared with systemic venous blood levels throughout the intervention. After alcohol intoxication, liver lipidome in ALD was similar to that in NAFLD. Conclusions: Alcohol intoxication induces rapid changes in circulating lipids including hepatic turnaround from FFA and LPC, potentially leading to lipoapoptosis and steatohepatitis. TG clearance was suppressed in NAFLD, possibly explaining why alcohol and NAFLD are synergistic risk factors for disease progression. These effects may be central to the pathogenesis of ALD. Clinical Trials Registration: The study is registered at Clinicaltrials.gov (NCT03018990). Lay summary: We report that alcohol induces hepatic extraction of free unsaturated fatty acids and lysophosphatidylcholines, hepatotoxic lipids which have not been previously associated with alcohol-induced liver injury. We also found that individuals with non-alcoholic fatty liver disease have reduced lipid turnover during alcohol intoxication when compared with people with alcohol-related fatty liver disease. This may explain why alcohol is particularly more harmful in people with non-alcoholic fatty liver and why elevated BMI and alcohol have a synergistic effect on the risk of liver-related death.

Background and Objective: Plasma biomarkers for the diagnosis and stratification of Alzheimer’s disease (AD) are intensively sought. However, no plasma markers are well established so far for AD diagnosis. Our group has identified and validated various blood-based proteomic biomarkers relating to AD pathology in multiple cohorts. The study aims to conduct a meta-analysis based on our own studies to systematically assess the diagnostic performance of our previously identified blood biomarkers.Methods: To do this, we included seven studies that our group has conducted during the last decade. These studies used either Luminex xMAP or ELISA to measure proteomic biomarkers. As proteins measured in these studies differed, we selected protein based on the criteria that it must be measured in at least four studies. We then examined biomarker performance using random-effect meta-analyses based on the mean difference between biomarker concentrations in AD and controls (CTL), AD and mild cognitive impairment (MCI), MCI, and CTL as well as MCI converted to dementia (MCIc) and non-converted (MCInc) individuals.Results: An overall of 2,879 subjects were retrieved for meta-analysis including 1,053 CTL, 895 MCI, 882 AD, and 49 frontotemporal dementia (FTD) patients. Six proteins were measured in at least four studies and were chosen for meta-analyses for AD diagnosis. Of them, three proteins had significant difference between AD and controls, among which alpha-2-macroglobulin (A2M) and ficolin-2 (FCN2) increased in AD while fibrinogen gamma chain (FGG) decreased in AD compared to CTL. Furthermore, FGG significantly increased in FTD compared to AD. None of the proteins passed the significance between AD and MCI, or MCI and CTL, or MCIc and MCInc, although complement component 4 (CC4) tended to increase in MCIc individuals compared to MCInc.Conclusions: The results suggest that A2M, FCN2, and FGG are promising biomarkers to discriminate AD patients from controls, which are worthy of further validation.

BackgroundWhile Alzheimer disease (AD) and vascular dementia (VaD) may be accelerated by hypercholesterolemia, the mechanisms underlying this association are unclear. We tested whether dysregulation of cholesterol catabolism, through its conversion to primary bile acids (BAs), was associated with dementia pathogenesis.Methods and findingsWe used a 3-step study design to examine the role of the primary BAs, cholic acid (CA), and chenodeoxycholic acid (CDCA) as well as their principal biosynthetic precursor, 7α-hydroxycholesterol (7α-OHC), in dementia. In Step 1, we tested whether serum markers of cholesterol catabolism were associated with brain amyloid accumulation, white matter lesions (WMLs), and brain atrophy. In Step 2, we tested whether exposure to bile acid sequestrants (BAS) was associated with risk of dementia. In Step 3, we examined plausible mechanisms underlying these findings by testing whether brain levels of primary BAs and gene expression of their principal receptors are altered in AD. Step 1: We assayed serum concentrations CA, CDCA, and 7α-OHC and used linear regression and mixed effects models to test their associations with brain amyloid accumulation (N = 141), WMLs, and brain atrophy (N = 134) in the Baltimore Longitudinal Study of Aging (BLSA). The BLSA is an ongoing, community-based cohort study that began in 1958. Participants in the BLSA neuroimaging sample were approximately 46% male with a mean age of 76 years; longitudinal analyses included an average of 2.5 follow-up magnetic resonance imaging (MRI) visits. We used the Alzheimer's Disease Neuroimaging Initiative (ADNI) (N = 1,666) to validate longitudinal neuroimaging results in BLSA. ADNI is an ongoing, community-based cohort study that began in 2003. Participants were approximately 55% male with a mean age of 74 years; longitudinal analyses included an average of 5.2 follow-up MRI visits. Lower serum concentrations of 7α-OHC, CA, and CDCA were associated with higher brain amyloid deposition (p = 0.041), faster WML accumulation (p = 0.050), and faster brain atrophy mainly (false discovery rate [FDR] p = 4 million registrants from general practice clinics in the United Kingdom, we tested whether patients using BAS (BAS users; 3,208 with ≥2 prescriptions), which reduce circulating BAs and increase cholesterol catabolism, had altered dementia risk compared to those on non-statin lipid-modifying therapies (LMT users; 23,483 with ≥2 prescriptions). Patients in the study (BAS/LMT) were approximately 34%/38% male and with a mean age of 65/68 years; follow-up time was 4.7/5.7 years. We found that BAS use was not significantly associated with risk of all-cause dementia (hazard ratio (HR) = 1.03, 95% confidence interval (CI) = 0.72-1.46, p = 0.88) or its subtypes. We found a significant difference between the risk of VaD in males compared to females (p = 0.040) and a significant dose-response relationship between BAS use and risk of VaD (p-trend = 0.045) in males.Step 3: We assayed brain tissue concentrations of CA and CDCA comparing AD and control (CON) samples in the BLSA autopsy cohort (N = 29). Participants in the BLSA autopsy cohort (AD/CON) were approximately 50%/77% male with a mean age of 87/82 years. We analyzed single-cell RNA sequencing (scRNA-Seq) data to compare brain BA receptor gene expression between AD and CON samples from the Religious Orders Study and Memory and Aging Project (ROSMAP) cohort (N = 46). ROSMAP is an ongoing, community-based cohort study that began in 1994. Participants (AD/CON) were approximately 56%/36% male with a mean age of 85/85 years. In BLSA, we found that CA and CDCA were detectable in postmortem brain tissue samples and were marginally higher in AD samples compared to CON. In ROSMAP, we found sex-specific differences in altered neuronal gene expression of BA receptors in AD. Study limitations include the small sample sizes in the BLSA cohort and likely inaccuracies in the clinical diagnosis of dementia subtypes in primary care settings.ConclusionsWe combined targeted metabolomics in serum and amyloid positron emission tomography (PET) and MRI of the brain with pharmacoepidemiologic analysis to implicate dysregulation of cholesterol catabolism in dementia pathogenesis. We observed that lower serum BA concentration mainly in males is associated with neuroimaging markers of dementia, and pharmacological lowering of BA levels may be associated with higher risk of VaD in males. We hypothesize that dysregulation of BA signaling pathways in the brain may represent a plausible biologic mechanism underlying these results. Together, our observations suggest a novel mechanism relating abnormalities in cholesterol catabolism to risk of dementia.

Abstract Aims Lipid metabolism might be compromised in type 1 diabetes, and the understanding of lipid physiology is critically important. This study aimed to compare the change in plasma lipid concentrations during carbohydrate dietary changes in individuals with type 1 diabetes and identify links to early‐stage dyslipidaemia. We hypothesized that (1) the lipidomic profiles after ingesting low or high carbohydrate diet for 12 weeks would be different; and (2) specific annotated lipid species could have significant associations with metabolic outcomes. Methods Ten adults with type 1 diabetes (mean ± SD: age 43.6 ± 13.8 years, diabetes duration 24.5 ± 13.4 years, BMI 24.9 ± 2.1 kg/m2, HbA1c 57.6 ± 2.6 mmol/mol) using insulin pumps participated in a randomized 2‐period crossover study with a 12‐week intervention period of low carbohydrate diet (< 100 g carbohydrates/day) or high carbohydrate diet (> 250 g carbohydrates/day), respectively, separated by a 12‐week washout period. A large‐scale non‐targeted lipidomics was performed with mass spectrometry in fasting plasma samples obtained before and after each diet intervention. Longitudinal lipid levels were analysed using linear mixed‐effects models. Results In total, 289 lipid species were identified from 14 major lipid classes. Comparing the two diets, 11 lipid species belonging to sphingomyelins, phosphatidylcholines and LPC(O‐16:0) were changed. All the 11 lipid species were significantly elevated during low carbohydrate diet. Two lipid species were most differentiated between diets, namely SM(d36:1) (β ± SE: 1.44 ± 0.28, FDR = 0.010) and PC(P‐36:4)/PC(O‐36:5) (β ± SE: 1.34 ± 0.25, FDR = 0.009) species. Polyunsaturated PC(35:4) was inversely associated with BMI and positively associated with HDL cholesterol (p

[This corrects the article DOI: 10.1371/journal.pmed.1003012.].

BackgroundThere is growing evidence that Alzheimer disease (AD) is a pervasive metabolic disorder with dysregulation in multiple biochemical pathways underlying its pathogenesis. Understanding how perturbations in metabolism are related to AD is critical to identifying novel targets for disease-modifying therapies. In this study, we test whether AD pathogenesis is associated with dysregulation in brain transmethylation and polyamine pathways.Methods and findingsWe first performed targeted and quantitative metabolomics assays using capillary electrophoresis-mass spectrometry (CE-MS) on brain samples from three groups in the Baltimore Longitudinal Study of Aging (BLSA) (AD: n = 17; Asymptomatic AD [ASY]: n = 13; Control [CN]: n = 13) (overall 37.2% female; mean age at death 86.118 ± 9.842 years) in regions both vulnerable and resistant to AD pathology. Using linear mixed-effects models within two primary brain regions (inferior temporal gyrus [ITG] and middle frontal gyrus [MFG]), we tested associations between brain tissue concentrations of 26 metabolites and the following primary outcomes: group differences, Consortium to Establish a Registry for Alzheimer's Disease (CERAD) (neuritic plaque burden), and Braak (neurofibrillary pathology) scores. We found significant alterations in concentrations of metabolites in AD relative to CN samples, as well as associations with severity of both CERAD and Braak, mainly in the ITG. These metabolites represented biochemical reactions in the (1) methionine cycle (choline: lower in AD, p = 0.003; S-adenosyl methionine: higher in AD, p = 0.005); (2) transsulfuration and glutathione synthesis (cysteine: higher in AD, p < 0.001; reduced glutathione [GSH]: higher in AD, p < 0.001); (3) polyamine synthesis/catabolism (spermidine: higher in AD, p = 0.004); (4) urea cycle (N-acetyl glutamate: lower in AD, p < 0.001); (5) glutamate-aspartate metabolism (N-acetyl aspartate: lower in AD, p = 0.002); and (6) neurotransmitter metabolism (gamma-amino-butyric acid: lower in AD, p < 0.001). Utilizing three Gene Expression Omnibus (GEO) datasets, we then examined mRNA expression levels of 71 genes encoding enzymes regulating key reactions within these pathways in the entorhinal cortex (ERC; AD: n = 25; CN: n = 52) and hippocampus (AD: n = 29; CN: n = 56). Complementing our metabolomics results, our transcriptomics analyses also revealed significant alterations in gene expression levels of key enzymatic regulators of biochemical reactions linked to transmethylation and polyamine metabolism. Our study has limitations: our metabolomics assays measured only a small proportion of all metabolites participating in the pathways we examined. Our study is also cross-sectional, limiting our ability to directly test how AD progression may impact changes in metabolite concentrations or differential-gene expression. Additionally, the relatively small number of brain tissue samples may have limited our power to detect alterations in all pathway-specific metabolites and their genetic regulators.ConclusionsIn this study, we observed broad dysregulation of transmethylation and polyamine synthesis/catabolism, including abnormalities in neurotransmitter signaling, urea cycle, aspartate-glutamate metabolism, and glutathione synthesis. Our results implicate alterations in cellular methylation potential and increased flux in the transmethylation pathways, increased demand on antioxidant defense mechanisms, perturbations in intermediate metabolism in the urea cycle and aspartate-glutamate pathways disrupting mitochondrial bioenergetics, increased polyamine biosynthesis and breakdown, as well as abnormalities in neurotransmitter metabolism that are related to AD.

Background and aimsAdipose tissue plays a pivotal role in storing excess fat and its composition reflects the history of person's lifestyle and metabolic health. Broad profiling of lipids with mass spectrometry has potential for uncovering new knowledge on the pathology of obesity, metabolic syndrome, diabetes and other related conditions. Here, we developed a lipidomic method for analyzing human subcutaneous adipose biopsies. We applied the method to four body areas to understand the differences in lipid composition between these areas.Materials and methodsAdipose tissue biopsies from 10 participants were analyzed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The sample preparation optimization included the optimization of the lipid extraction, the sample amount and the sample dilution factor to detect lipids in an appropriate concentration range. Lipidomic analyses were performed for adipose tissue collected from the abdomen, breast, thigh and lower back. Differences in lipid levels between tissues were visualized with heatmaps.ResultsLipidomic analysis on human adipose biopsies lead to the identification of 186lipids in 2 mg of sample. Technical variation of the lipid-class specific internal standards were below 5%, thus indicating acceptable repeatability. Triacylglycerols were highly represented in the adipose tissue samples, and lipids from 13 lipid classes were identified. Long polyunsaturated triacylglycerols in higher levels in thigh (qConclusionThe method presented here is suitable for the analysis of lipid profiles in 2 mg of adipose tissue. The amount of fat across the body is important for health but we argue that also the distribution and the particular profile of the lipidome may be relevant for metabolic outcomes. We suggest that the method presented in this paper could be useful for detecting such aberrations.

Feces are the product of our diets and have been linked to diseases of the gut, including Chron’s disease and metabolic diseases such as diabetes. For screening metabolites in heterogeneous samples such as feces, it is necessary to use fast and reproducible analytical methods that maximize metabolite detection. As sample preparation is crucial to obtain high quality data in MS-based clinical metabolomics, we developed a novel, efficient and robust method for preparing fecal samples for analysis with a focus in reducing aliquoting and detecting both polar and non-polar metabolites. Fecal samples (n = 475) from patients with alcohol-related liver disease and healthy controls were prepared according to the proposed method and analyzed in an UHPLC-QQQ targeted platform in order to obtain a quantitative profile of compounds that impact liver-gut axis metabolism. MS analyses of the prepared fecal samples have shown reproducibility and coverage of n = 28 metabolites, mostly comprising bile acids and amino acids. We report metabolite-wise relative standard deviation (RSD) in quality control samples, inter-day repeatability, LOD (limit of detection), LOQ (limit of quantification), range of linearity and method recovery. The average concentrations for 135 healthy participants are reported here for clinical applications. Our high-throughput method provides a novel tool for investigating gut-liver axis metabolism in liver-related diseases using a noninvasive collected sample.

Abstract Background With the shift of research focus towards the pre-dementia stage of Alzheimer’s disease (AD), there is an urgent need for reliable, non-invasive biomarkers to predict amyloid pathology. The aim of this study was to assess whether easily obtainable measures from structural MRI, combined with demographic data, cognitive data and apolipoprotein E (APOE) ε4 genotype, can be used to predict amyloid pathology using machine-learning classification. Methods We examined 810 subjects with structural MRI data and amyloid markers from the European Medical Information Framework for Alzheimer’s Disease Multimodal Biomarker Discovery study, including subjects with normal cognition (CN, n = 337, age 66.5 ± 7.2, 50% female, 27% amyloid positive), mild cognitive impairment (MCI, n = 375, age 69.1 ± 7.5, 53% female, 63% amyloid positive) and AD dementia (n = 98, age 67.0 ± 7.7, 48% female, 97% amyloid positive). Structural MRI scans were visually assessed and Freesurfer was used to obtain subcortical volumes, cortical thickness and surface area measures. We first assessed univariate associations between MRI measures and amyloid pathology using mixed models. Next, we developed and tested an automated classifier using demographic, cognitive, MRI and APOE ε4 information to predict amyloid pathology. A support vector machine (SVM) with nested 10-fold cross-validation was applied to identify a set of markers best discriminating between amyloid positive and amyloid negative subjects. Results In univariate associations, amyloid pathology was associated with lower subcortical volumes and thinner cortex in AD-signature regions in CN and MCI. The multi-variable SVM classifier provided an area under the curve (AUC) of 0.81 ± 0.07 in MCI and an AUC of 0.74 ± 0.08 in CN. In CN, selected features for the classifier included APOE ε4, age, memory scores and several MRI measures such as hippocampus, amygdala and accumbens volumes and cortical thickness in temporal and parahippocampal regions. In MCI, the classifier including demographic and APOE ε4 information did not improve after additionally adding imaging measures. Conclusions Amyloid pathology is associated with changes in structural MRI measures in CN and MCI. An automated classifier based on clinical, imaging and APOE ε4 data can identify the presence of amyloid pathology with a moderate level of accuracy. These results could be used in clinical trials to pre-screen subjects for anti-amyloid therapies.

Abstract Background There is an urgent need for novel, noninvasive biomarkers to diagnose Alzheimer’s disease (AD) in the predementia stages and to predict the rate of decline. Therefore, we set up the European Medical Information Framework for Alzheimer’s Disease Multimodal Biomarker Discovery (EMIF-AD MBD) study. In this report we describe the design of the study, the methods used and the characteristics of the participants. Methods Participants were selected from existing prospective multicenter and single-center European studies. Inclusion criteria were having normal cognition (NC) or a diagnosis of mild cognitive impairment (MCI) or AD-type dementia at baseline, age above 50 years, known amyloid-beta (Aβ) status, availability of cognitive test results and at least two of the following materials: plasma, DNA, magnetic resonance imaging (MRI) or cerebrospinal fluid (CSF). Targeted and untargeted metabolomic and proteomic analyses were performed in plasma, and targeted and untargeted proteomics were performed in CSF. Genome-wide SNP genotyping, next-generation sequencing and methylation profiling were conducted in DNA. Visual rating and volumetric measures were assessed on MRI. Baseline characteristics were analyzed using ANOVA or chi-square, rate of decline analyzed by linear mixed modeling. Results We included 1221 individuals (NC n = 492, MCI n = 527, AD-type dementia n = 202) with a mean age of 67.9 (SD 8.3) years. The percentage Aβ+ was 26% in the NC, 58% in the MCI, and 87% in the AD-type dementia groups. Plasma samples were available for 1189 (97%) subjects, DNA samples for 929 (76%) subjects, MRI scans for 862 (71%) subjects and CSF samples for 767 (63%) subjects. For 759 (62%) individuals, clinical follow-up data were available. In each diagnostic group, the APOE ε4 allele was more frequent amongst Aβ+ individuals (p

Background: physiological differences between males and females could contribute to the development of Alzheimer’s Disease (AD). Here, we examined metabolic pathways that may lead to precision medicine initiatives. Methods: We explored whether sex modifies the association of 540 plasma metabolites with AD endophenotypes including diagnosis, cerebrospinal fluid (CSF) biomarkers, brain imaging, and cognition using regression analyses for 695 participants (377 females), followed by sex-specific pathway overrepresentation analyses, APOE ε4 stratification and assessment of metabolites’ discriminatory performance in AD. Results: In females with AD, vanillylmandelate (tyrosine pathway) was increased and tryptophan betaine (tryptophan pathway) was decreased. The inclusion of these two metabolites (area under curve (AUC) = 0.83, standard error (SE) = 0.029) to a baseline model (covariates + CSF biomarkers, AUC = 0.92, SE = 0.019) resulted in a significantly higher AUC of 0.96 (SE = 0.012). Kynurenate was decreased in males with AD (AUC = 0.679, SE = 0.046). Conclusions: metabolic sex-specific differences were reported, covering neurotransmission and inflammation pathways with AD endophenotypes. Two metabolites, in pathways related to dopamine and serotonin, were associated to females, paving the way to personalised treatment.

Abstract Transient receptor potential canonical 5 (TRPC5), a calcium-permeable, non-selective cation channel is expressed in the periphery, but there is limited knowledge of its regulatory roles in vivo. Endogenous modulators of TRPC5 include a range of phospholipids that have an established role in liver disease, including lysophosphatidylcholine (LPC). Cholestasis is characterized by impairment of excretion of bile acids, leading to elevation of hepatic bile acids. We investigated the contribution of TRPC5 in a murine model of cholestasis. Wild-type (WT) and TRPC5 knock-out (KO) mice were fed a diet supplemented with 0.5% cholic acid (CA) for 21 days. CA-diet supplementation resulted in enlargement of the liver in WT mice, which was ameliorated in TRPC5 KO mice. Hepatic bile acid and lipid content was elevated in WT mice, with a reduction observed in TRPC5 KO mice. Consistently, liver enzymes were significantly increased in cholestatic WT mice and significantly blunted in TRPC5 KO mice. Localized dyslipidaemia, secondary to cholestasis, was investigated utilizing a selected lipid analysis. This revealed significant perturbations in the lipid profile following CA-diet feeding, with increased cholesterol, triglycerides and phospholipids, in WT, but not TRPC5 KO mice. Our results suggest that activation of TRPC5 contributes to the development of cholestasis and associated dyslipidemia. Modulation of TRPC5 activity may present as a novel therapeutic target for liver disease.

Background: Improved understanding of the pathophysiology causing diabetic kidney disease (DKD) is imperative. The aim of this study was to uncover associations between serum metabolites and renal outcomes.Methods: Non-targeted serum metabolomics analyses were performed in samples from 637 persons with type 1 diabetes using two-dimensional gas chromatography coupled to time-of-flight mass-spectrometry. Longitudinal data at follow-up (median 5.5 years) on renal events were obtained from national Danish health registries. A composite renal endpoint (n = 123) consisted of estimated glomerular filtration rate (eGFR) decline from baseline (≥30%), progression to end-stage renal disease and all-cause mortality. Metabolites with significant associations (p < 0.05) in any of the cross-sectional analyses with eGFR and albuminuria were analyzed for specific and composite endpoints. Adjustments included traditional cardiovascular risk factors and correction for multiple testing.Results: A data-driven partial correlation analysis revealed a dense fabric of co-regulated metabolites and clinical variables dominated by eGFR. Ribonic acid and myo-inositol were inversely associated with eGFR, positively associated with macroalbuminuria (p < 0.02) and longitudinally associated with higher risk of eGFR decline ≥30% (HR 2.2–2.7, CI [1.3–4.3], p < 0.001). Ribonic acid was associated with a combined renal endpoint (HR 1.8, CI [1.3–2.3], p = 0.001). The hydroxy butyrate 3,4-dihydroxybutanoic acid was cross-sectionally associated with micro- and macroalbuminuria, urinary albumin excretion rate and inversely associated with eGFR (p < 0.04) while branched chain amino acids were associated with eGFR and lower risk of the combined renal endpoint (p < 0.02).Conclusions: Alterations in serum metabolites, particularly polyols and amino acids, were associated with renal endpoints in type 1 diabetes highlighting molecular pathways associated with progression of kidney disease. External validation is needed to further assess their roles and potentials as future therapeutic targets.

BACKGROUND:The metabolic basis of Alzheimer disease (AD) is poorly understood, and the relationships between systemic abnormalities in metabolism and AD pathogenesis are unclear. Understanding how global perturbations in metabolism are related to severity of AD neuropathology and the eventual expression of AD symptoms in at-risk individuals is critical to developing effective disease-modifying treatments. In this study, we undertook parallel metabolomics analyses in both the brain and blood to identify systemic correlates of neuropathology and their associations with prodromal and preclinical measures of AD progression. METHODS AND FINDINGS:Quantitative and targeted metabolomics (Biocrates AbsoluteIDQ [identification and quantification] p180) assays were performed on brain tissue samples from the autopsy cohort of the Baltimore Longitudinal Study of Aging (BLSA) (N = 44, mean age = 81.33, % female = 36.36) from AD (N = 15), control (CN; N = 14), and "asymptomatic Alzheimer's disease" (ASYMAD, i.e., individuals with significant AD pathology but no cognitive impairment during life; N = 15) participants. Using machine-learning methods, we identified a panel of 26 metabolites from two main classes-sphingolipids and glycerophospholipids-that discriminated AD and CN samples with accuracy, sensitivity, and specificity of 83.33%, 86.67%, and 80%, respectively. We then assayed these 26 metabolites in serum samples from two well-characterized longitudinal cohorts representing prodromal (Alzheimer's Disease Neuroimaging Initiative [ADNI], N = 767, mean age = 75.19, % female = 42.63) and preclinical (BLSA) (N = 207, mean age = 78.68, % female = 42.63) AD, in which we tested their associations with magnetic resonance imaging (MRI) measures of AD-related brain atrophy, cerebrospinal fluid (CSF) biomarkers of AD pathology, risk of conversion to incident AD, and trajectories of cognitive performance. We developed an integrated blood and brain endophenotype score that summarized the relative importance of each metabolite to severity of AD pathology and disease progression (Endophenotype Association Score in Early Alzheimer's Disease [EASE-AD]). Finally, we mapped the main metabolite classes emerging from our analyses to key biological pathways implicated in AD pathogenesis. We found that distinct sphingolipid species including sphingomyelin (SM) with acyl residue sums C16:0, C18:1, and C16:1 (SM C16:0, SM C18:1, SM C16:1) and hydroxysphingomyelin with acyl residue sum C14:1 (SM (OH) C14:1) were consistently associated with severity of AD pathology at autopsy and AD progression across prodromal and preclinical stages. Higher log-transformed blood concentrations of all four sphingolipids in cognitively normal individuals were significantly associated with increased risk of future conversion to incident AD: SM C16:0 (hazard ratio [HR] = 4.430, 95% confidence interval [CI] = 1.703-11.520, p = 0.002), SM C16:1 (HR = 3.455, 95% CI = 1.516-7.873, p = 0.003), SM (OH) C14:1 (HR = 3.539, 95% CI = 1.373-9.122, p = 0.009), and SM C18:1 (HR = 2.255, 95% CI = 1.047-4.855, p = 0.038). The sphingolipid species identified map to several biologically relevant pathways implicated in AD, including tau phosphorylation, amyloid-β (Aβ) metabolism, calcium homeostasis, acetylcholine biosynthesis, and apoptosis. Our study has limitations: the relatively small number of brain tissue samples may have limited our power to detect significant associations, control for heterogeneity between groups, and replicate our findings in independent, autopsy-derived brain samples. CONCLUSIONS:We present a novel framework to identify biologically relevant brain and blood metabolites associated with disease pathology and progression during the prodromal and preclinical stages of AD. Our results show that perturbations in sphingolipid metabolism are consistently associated with endophenotypes across preclinical and prodromal AD, as well as with AD pathology at autopsy. Sphingolipids may be biologically relevant biomarkers for the early detection of AD, and correcting perturbations in sphingolipid metabolism may be a plausible and novel therapeutic strategy in AD.

BACKGROUND:The metabolic basis of Alzheimer disease (AD) pathology and expression of AD symptoms is poorly understood. Omega-3 and -6 fatty acids have previously been linked to both protective and pathogenic effects in AD. However, to date little is known about how the abundance of these species is affected by differing levels of disease pathology in the brain. METHODS AND FINDINGS:We performed metabolic profiling on brain tissue samples from 43 individuals ranging in age from 57 to 95 y old who were stratified into three groups: AD (N = 14), controls (N = 14) and "asymptomatic Alzheimer's disease" (ASYMAD), i.e., individuals with significant AD neuropathology at death but without evidence for cognitive impairment during life (N = 15) from the autopsy sample of the Baltimore Longitudinal Study of Aging (BLSA). We measured 4,897 metabolite features in regions both vulnerable in the middle frontal and inferior temporal gyri (MFG and ITG) and resistant (cerebellum) to classical AD pathology. The levels of six unsaturated fatty acids (UFAs) in whole brain were compared in controls versus AD, and the differences were as follows: linoleic acid (p = 8.8 x 10-8, FC = 0.52, q = 1.03 x 10-6), linolenic acid (p = 2.5 x 10-4, FC = 0.84, q = 4.03 x 10-4), docosahexaenoic acid (p = 1.7 x 10-7, FC = 1.45, q = 1.24 x 10-6), eicosapentaenoic acid (p = 4.4 x 10-4, FC = 0.16, q = 6.48 x 10-4), oleic acid (p = 3.3 x 10-7, FC = 0.34, q = 1.46 x 10-6), and arachidonic acid (p = 2.98 x 10-5, FC = 0.75, q = 7.95 x 10-5). These fatty acids were strongly associated with AD when comparing the groups in the MFG and ITG, respectively: linoleic acid (p < 0.0001, p = 0.0006), linolenic acid (p < 0.0001, p = 0.002), docosahexaenoic acid (p < 0.0001, p = 0.0024), eicosapentaenoic acid (p = 0.0002, p = 0.0008), oleic acid (p < 0.0001, p = 0.0003), and arachidonic acid (p = 0.0001, p = 0.001). Significant associations were also observed between the abundance of these UFAs with neuritic plaque and neurofibrillary tangle burden as well as domain-specific cognitive performance assessed during life. Based on the regional pattern of differences in brain tissue levels of these metabolites, we propose that alterations in UFA metabolism represent both global metabolic perturbations in AD as well as those related to specific features of AD pathology. Within the middle frontal gyrus, decrements in linoleic acid, linolenic acid, and arachidonic acid (control>ASYMAD>AD) and increases in docosahexanoic acid (AD>ASYMAD>control) may represent regionally specific threshold levels of these metabolites beyond which the accumulation of AD pathology triggers the expression of clinical symptoms. The main limitation of this study is the relatively small sample size. There are few cohorts with extensive longitudinal cognitive assessments during life and detailed neuropathological assessments at death, such as the BLSA. CONCLUSIONS:The findings of this study suggest that unsaturated fatty acid metabolism is significantly dysregulated in the brains of patients with varying degrees of Alzheimer pathology.

Urinary tract infection is one of the most common bacterial infections worldwide. Current diagnosis of urinary tract infection chiefly relies on its clinical presentation, urine dipstick tests and urine culture. Small molecules found in bio-fluids related with both infection and recovery would facilitate diagnosis and management of UTI. Mass spectrometry-based fingerprinting of plasma and urine at 3 time points, pre-infection (t = -24h), infection (t = 24h) and post 3-day treatment (t = 112h), were acquired in the following four groups: mice which were healthy, infected but not treated, infected and treated with ciprofloxacin, and infected and treated with Relinqing® granules (n = 6 per group). A metabolomics workflow including multivariate analysis and ROC regression was employed to select metabolic features that correlated with UTI and its treatment. Circa 4,000 molecular features were acquired for each sample. The small acid 3-hydroxybutyrate in plasma was found to be differentiated for urinary tract infection, with an area under the curve = 0.97 (95% confidence interval: 0.93-1.00, accuracy = 0.91, sensitivity = 0.92 and specificity = 0.91). The level of 3-hydroxybutyrate in plasma was depleted after infection with a fold change of -22 (q < 0.0001). Correlation between plasma 3-hydroxybutyrate and urine bacterial number in all groups and time points was r = -0.753 (p < 0.0001). The findings show that 3-hydroxybutyrate is depleted in blood and strongly associated with UTI at both infection and post-treatment stage in a UTI mouse model. Further work is envisaged to assess the clinical potential of blood tests to assist with UTI management.

Introduction: Parkinson’s disease (PD) is the second most common neurodegenerative disorder, without any widely available curative therapy. Metabolomics is a powerful tool which can be used to identify unexpected pathway-related disease progression and pathophysiological mechanisms. In this study, metabolomics in brain, plasma and liver was investigated in an experimental PD model, to discover small molecules that are associated with dopaminergic cell loss. Methods: Sprague Dawley (SD) rats were injected unilaterally with 6-hydroxydopamine (6-OHDA) or saline for the vehicle control group into the medial forebrain bundle (MFB) to induce loss of dopaminergic neurons in the substantia nigra pars compacta. Plasma, midbrain and liver samples were collected for metabolic profiling. Multivariate and univariate analyses revealed metabolites that were altered in the PD group. Results: In plasma, palmitic acid (q = 3.72 × 10−2, FC = 1.81) and stearic acid (q = 3.84 × 10−2, FC = 2.15), were found to be increased in the PD group. Palmitic acid (q = 3.5 × 10−2) and stearic acid (q = 2.7 × 10−2) correlated with test scores indicative of motor dysfunction. Monopalmitin (q = 4.8 × 10−2, FC = −11.7), monostearin (q = 3.72 × 10−2, FC = −15.1) and myo-inositol (q = 3.81 × 10−2, FC = −3.32), were reduced in the midbrain. The liver did not have altered levels of these molecules. Conclusion: Our results show that saturated free fatty acids, their monoglycerides and myo-inositol metabolism in the midbrain and enteric circulation are associated with 6-OHDA-induced PD pathology.

BACKGROUND AND AIMS:The shortage of organs for transplantation has led to increased use of organs procured from donors after cardiac death (DCD). The effects of cardiac death on the liver remain poorly understood, however. Using livers obtained from DCD versus donors after brain death (DBD), we aimed to understand how ischemia/reperfusion (I/R) injury alters expression of pro-inflammatory markers ceramides and influences graft leukocyte infiltration. METHODS:Hepatocyte inflammation, as assessed by ceramide expression, was evaluated in DCD (n = 13) and DBD (n = 10) livers. Allograft expression of inflammatory and cell death markers, and allograft leukocyte infiltration were evaluated from a contemporaneous independent cohort of DCD (n = 22) and DBD (n = 13) livers. RESULTS:When examining the differences between transplant stages in each group, C18, C20, C24 ceramides showed significant difference in DBD (p

Abstract Symbiotic gut microorganisms (microbiome) interact closely with the mammalian host's metabolism and are important determinants of human health. Here, we decipher the complex metabolic effects of microbial manipulation, by comparing germfree mice colonized by a human baby flora (HBF) or a normal flora to conventional mice. We perform parallel microbiological profiling, metabolic profiling by 1H nuclear magnetic resonance of liver, plasma, urine and ileal flushes, and targeted profiling of bile acids by ultra performance liquid chromatography–mass spectrometry and short‐chain fatty acids in cecum by GC‐FID. Top‐down multivariate analysis of metabolic profiles reveals a significant association of specific metabotypes with the resident microbiome. We derive a transgenomic graph model showing that HBF flora has a remarkably simple microbiome/metabolome correlation network, impacting directly on the host's ability to metabolize lipids: HBF mice present higher ileal concentrations of tauro‐conjugated bile acids, reduced plasma levels of lipoproteins but higher hepatic triglyceride content associated with depletion of glutathione. These data indicate that the microbiome modulates absorption, storage and the energy harvest from the diet at the systems level.

Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.

Abstract We identify biomarkers for disease progression in three type 2 diabetes cohorts encompassing 2,973 individuals across three molecular classes, metabolites, lipids and proteins. Homocitrulline, isoleucine and 2-aminoadipic acid, eight triacylglycerol species, and lowered sphingomyelin 42:2;2 levels are predictive of faster progression towards insulin requirement. Of ~1,300 proteins examined in two cohorts, levels of GDF15/MIC-1, IL-18Ra, CRELD1, NogoR, FAS, and ENPP7 are associated with faster progression, whilst SMAC/DIABLO, SPOCK1 and HEMK2 predict lower progression rates. In an external replication, proteins and lipids are associated with diabetes incidence and prevalence. NogoR/RTN4R injection improved glucose tolerance in high fat-fed male mice but impaired it in male db/db mice. High NogoR levels led to islet cell apoptosis, and IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. This comprehensive, multi-disciplinary approach thus identifies biomarkers with potential prognostic utility, provides evidence for possible disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

Summary: Background: Type 1 diabetes is a complex heterogenous autoimmune disease without therapeutic interventions available to prevent or reverse the disease. This study aimed to identify transcriptional changes associated with the disease progression in patients with recent-onset type 1 diabetes. Methods: Whole-blood samples were collected as part of the INNODIA study at baseline and 12 months after diagnosis of type 1 diabetes. We used linear mixed-effects modelling on RNA-seq data to identify genes associated with age, sex, or disease progression. Cell-type proportions were estimated from the RNA-seq data using computational deconvolution. Associations to clinical variables were estimated using Pearson's or point-biserial correlation for continuous and dichotomous variables, respectively, using only complete pairs of observations. Findings: We found that genes and pathways related to innate immunity were downregulated during the first year after diagnosis. Significant associations of the gene expression changes were found with ZnT8A autoantibody positivity. Rate of change in the expression of 16 genes between baseline and 12 months was found to predict the decline in C-peptide at 24 months. Interestingly and consistent with earlier reports, increased B cell levels and decreased neutrophil levels were associated with the rapid progression. Interpretation: There is considerable individual variation in the rate of progression from appearance of type 1 diabetes-specific autoantibodies to clinical disease. Patient stratification and prediction of disease progression can help in developing more personalised therapeutic strategies for different disease endotypes. Funding: A full list of funding bodies can be found under Acknowledgments.