Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

KLF5 governs sphingolipid metabolism and barrier function of the skin.

Stem cells are fundamental units of tissue remodeling whose functions are dictated by lineage-specific transcription factors. Home to epidermal stem cells and their upward-stratifying progenies, skin relies on its secretory functions to form the outermost protective barrier, of which a transcriptional orchestrator has been elusive. KLF5 is a Krüppel-like transcription factor broadly involved in development and regeneration whose lineage specificity, if any, remains unclear. Here we report KLF5 specifically marks the epidermis, and its deletion leads to skin barrier dysfunction in vivo. Lipid envelopes and secretory lamellar bodies are defective in KLF5-deficient skin, accompanied by preferential loss of complex sphingolipids. KLF5 binds to and transcriptionally regulates genes encoding rate-limiting sphingolipid metabolism enzymes. Remarkably, skin barrier defects elicited by KLF5 ablation can be rescued by dietary interventions. Finally, we found that KLF5 is widely suppressed in human diseases with disrupted epidermal secretion, and its regulation of sphingolipid metabolism is conserved in human skin. Altogether, we established KLF5 as a disease-relevant transcription factor governing sphingolipid metabolism and barrier function in the skin, likely representing a long-sought secretory lineage-defining factor across tissue types.

Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity.

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression.

N-Glycan Profile of Cerebrospinal Fluids from Alzheimer's Disease Patients Using Liquid Chromatography with Mass Spectrometry.

Glycosylation, an essential post-translational protein modification, is known to be altered in a variety of diseases, including neurodegenerative diseases such as Alzheimer's disease (AD), which is one of the most common neurodegenerative disorders that results in cognitive and memory impairments. To investigate the progression of such a condition, cerebrospinal fluid (CSF), a unique biofluid that may possess significant biochemical and neurochemical changes due to the disease, is utilized. However, due to the low concentration of proteins in CSF, a large volume of the biofluid is often required to comprehensively characterize the glycome in CSF. In this work, a glycomic study of CSF was performed using as little as 10 µL of CSF. This approach was executed with permethylation of released N-glycans with minimal sample cleanup, in conjunction with an online purification system attached to liquid chromatography and a high-resolution mass spectrometer. This technique was then applied to clinical samples. Preliminary data suggest that fucosylated and bisecting GlcNAc structures were higher in abundances in females with AD, while both females and males exhibited lower abundances of high-mannose structures. Although there seems to be statistically significant differences between disease state and disease-free CSF, due to the lack of number of samples, further validation study should be conducted.

NPC1L1-dependent intestinal cholesterol absorption requires ganglioside GM3 in membrane microdomains.

Intestinal cholesterol absorption is a key regulator of systemic cholesterol homeostasis. Excessive dietary cholesterol and its intestinal uptake lead to hypercholesterolemia, a major risk factor for cardiovascular disease. Intestinal cholesterol uptake is mediated by Niemann-Pick C1-like 1 (NPC1L1), a transmembrane protein localized in membrane microdomains (lipid rafts) enriched in gangliosides and cholesterol. The roles of gangliosides, such as monosialodihexosylganglioside (GM3) and its synthesizing enzyme GM3 synthase (GM3S), in NPC1L1-dependent cholesterol uptake have not been examined previously. Here, we examined NPC1L1-dependent cholesterol uptake in a cell model as well as in wild-type and apoE-deficient mice fed normal or high-cholesterol diets. We showed that NPC1L1-dependent cholesterol uptake was impaired in GM3S-deficient cells and that GM3S deficiency promoted resistance to hypercholesterolemia in both wild-type and apoE-deficient mice fed the high-cholesterol but not the normal diet. Our findings suggest that GM3 and related gangliosides are essential for NPC1L1-mediated intestinal cholesterol absorption and are potential targets for hypercholesterolemia therapy.

Altered expression of ganglioside GM3 molecular species and a potential regulatory role during myoblast differentiation.

Gangliosides (sialic acid-containing glycosphingolipids) help regulate many important biological processes, including cell proliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The structural diversity of gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecular species of a given ganglioside are suggested to have distinct biological properties and regulate specific and distinct biological events. Elucidation of the function of each molecular species is important and will provide new insights into ganglioside biology. Gangliosides are also suggested to be involved in skeletal muscle differentiation; however, the differential roles of ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of gangliosides (particularly GM3) during differentiation of mouse C2C12 myoblast cells and key roles played by distinct GM3 molecular species at each step of the process.

Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures.

Permethylation is a common derivatization method for MS-based glycomic analyses. Permethylation enhances glycan ionization efficiency in positive MS analysis and improves glycan structural stability. Recent biological glycomic studies have added to the growing body of knowledge and suggest the need for complete structural analysis of glycans. However, reverse phase LC analysis of permethylated glycans usually results in poor isomeric separation. To achieve isomeric separation of permethylated glycans, a porous graphitic carbon (PGC) column was used. PGC columns are well-known for their isomeric separation capability for hydrophilic analyses. In this study, we have optimized temperature conditions to overcome the issues encountered while separating permethylated glycans on a PGC column and found that the highest temperature examined, 75 °C, was optimal. Additionally, we utilized tandem MS to elucidate detailed structural information for the isomers separated. Glycan standards were also utilized to facilitate structural identifications through MS/MS spectra and retention time comparison. The result is an efficient and sensitive method capable of the isomeric separation of permethylated glycans. This method was successfully applied for the isomeric characterization of N-glycans released from the breast cancer cell lines MDA-MB-231 and MDA-MB-231BR (brain seeking). A total of 127 unique glycan structures were identified with 39 isobaric structures, represented as 106 isomers, with 21 nonisomeric glycans. Thirty seven structures exhibited significant differences in isomeric distribution (P < 0.05). Additionally, alterations in the distribution of isomeric sialylated glycans, structures known to be involved in cell attachment to the blood-brain barrier during brain metastasis, were observed.

Recent advances in mass spectrometric analysis of glycoproteins.

Glycosylation is one of the most common posttranslational modifications of proteins that plays essential roles in various biological processes, including protein folding, host-pathogen interaction, immune response, and inflammation and aberrant protein glycosylation is a well-known event in various disease states including cancer. As a result, it is critical to develop rapid and sensitive methods for the analysis of abnormal glycoproteins associated with diseases. Mass spectrometry (MS) in conjunction with different separation methods, such as capillary electrophoresis (CE), ion mobility (IM), and high performance liquid chromatography (HPLC) has become a popular tool for glycoprotein analysis, providing highly informative fragments for structural identification of glycoproteins. This review provides an overview of the developments and accomplishments in the field of glycomics and glycoproteomics reported between 2014 and 2016.

Characterization of isomeric glycan structures by LC-MS/MS.

The characterization of glycosylation is critical for obtaining a comprehensive view of the regulation and functions of glycoproteins of interest. Due to the complex nature of oligosaccharides, stemming from variable compositions and linkages, and ion suppression effects, the chromatographic separation of glycans, including isomeric structures, is necessary for exhaustive characterization by MS. This review introduces the fundamental principles underlying the techniques in LC utilized by modern day glycomics researchers. Recent advances in porous graphitized carbon, reverse phase, ion exchange, and hydrophilic interaction LC utilized in conjunction with MS, for the characterization of protein glycosylation, are described with an emphasis on methods capable of resolving isomeric glycan structures.

Direct comparison of derivatization strategies for LC-MS/MS analysis of N-glycans.

Protein glycosylation is a common post-translational modification that has significant impacts on protein folding, lifespan, conformation, distribution and function. N-Glycans, which are attached to asparagine residues of proteins, are studied most often due to their compatibility with enzymatic release. Despite the ease of N-glycan release, compositional and structural complexity coupled with poor ionization efficiency during liquid chromatography mass spectrometry (LC-MS) make quantitative glycomic studies a significant challenge. To overcome these challenges, glycans are almost always derivatized prior to LC-MS analyses to impart favorable characteristics, such as improved ionization efficiency, increased LC separation efficiency and the production of more informative fragments during tandem MS. There are a number of derivatization methods available for LC-MS analysis of glycans, each of which imparts different properties that affect both glycan retention on LC columns and MS analyses. To provide guidance for the proper selection of derivatizing reagents and LC columns, herein, we describe a comprehensive assessment of 2-aminobenzamide, procainamide, aminoxyTMT, RapiFluor-MS (RFMS) labeling, reduction and reduction with permethylation for N-glycan analysis. Of the derivatization strategies examined, RFMS provided the highest MS signal enhancement for neutral glycans, while permethylation significantly enhanced the MS intensity and structural stability of sialylated glycans.

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization.

The biosynthesis of glycans is a template-free process; hence compositionally identical glycans may contain highly heterogeneous structures. Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure. Structural elucidation of glycans is an essential component of glycobiology. Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans. Although mass spectrometry (MS)-based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization. Permethylation is an efficient derivatization method that improves glycan structural stability. In this report, permethylated glycans are isomerically separated; thus facilitating structural analysis of a mixture of glycans by LC-MS/MS. Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers. Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions, achieved by permethylation and PGC-LC at high temperatures, respectively. Assigning monosaccharide residues to specific glycan antennae was also achieved. Galactose linkages were also distinguished from each other by CID/HCD tandem MS. This was attainable because of the different bond energies associated with monosaccharide linkages. Graphical Abstract LC-MS and tandem MS of terminal galactose isomers.

Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT.

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.

Remodeling of marrow hematopoietic stem and progenitor cells by non-self ST6Gal-1 sialyltransferase.

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

Enzymatic basis for N-glycan sialylation: structure of rat α2,6-sialyltransferase (ST6GAL1) reveals conserved and unique features for glycan sialylation.

Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.

myo-Inositol and Phytate Are Toxic to Formosan Subterranean Termites (Isoptera: Rhinotermitidae).

Several rare and common monosaccharides were screened for toxic effects on the Formosan subterranean termite, Coptotermes formosanus Shiraki, with the aim of identifying environmentally friendly termiticides. myo-Inositol and phytic acid, which are nontoxic to mammals, were identified as potential termite control compounds. Feeding bioassays with termite workers, where both compounds were supplied on filter paper in concentrations from 160.2 to 1,281.7 µg/mm(3), showed concentration-dependent toxicity within 2 wk. Interestingly myo-inositol was nontoxic when administered to termites in agar (40 mg/ml) in the absence of a cellulosic food source, an unexplained phenomenon. In addition, decreased populations of termite hindgut protozoa were observed upon feeding on myo-inositol but not phytate-spiked filter paper. Radiotracer feeding studies using myo-inositol-[2-(3)H] with worker termites showed no metabolism after ingestion over a 2-d feeding period, ruling out metabolites responsible for the selective toxicity.

An IROA Workflow for correction and normalization of ion suppression in mass spectrometry-based metabolomic profiling data.

Ion suppression is a major problem in mass spectrometry (MS)-based metabolomics; it can dramatically decrease measurement accuracy, precision, and signal-to-noise sensitivity. Here we report a new method, the IROA TruQuant Workflow, that uses a stable isotope-labeled internal standard (IROA-IS) plus novel companion algorithms to 1) measure and correct for ion suppression, and 2) perform Dual MSTUS normalization of MS metabolomic data. We have evaluated the method across ion chromatography (IC), hydrophilic interaction liquid chromatography (HILIC), and reverse phase liquid chromatography (RPLC)-MS systems in both positive and negative ionization modes, with clean and unclean ion sources, and across different biological matrices. Across the broad range of conditions tested, all detected metabolites exhibited ion suppression ranging from 1% to 90+% and coefficient of variations ranging from 1% to 20%, but the Workflow and companion algorithms were highly effective at nulling out that suppression and error. Overall, the Workflow corrects ion suppression across diverse analytical conditions and produces robust normalization of non-targeted metabolomic data.

Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.

Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.

The bacterial microbiota regulates normal hematopoiesis via metabolite-induced type 1 interferon signaling.

Antibiotic therapy, especially when administered long term, is associated with adverse hematologic effects such as cytopenia. Signals from the intestinal microbiota are critical to maintain normal hematopoiesis, and antibiotics can cause bone marrow suppression through depletion of the microbiota. We reported previously that STAT1 signaling is necessary for microbiota-dependent hematopoiesis, but the precise mechanisms by which the gut microbiota signals to the host bone marrow to regulate hematopoiesis remain undefined. We sought to identify the cell type(s) through which STAT1 promotes microbiota-mediated hematopoiesis and to elucidate which upstream signaling pathways trigger STAT1 signaling. Using conditional knockout and chimeric mice, we found that the microbiota induced STAT1 signaling in non-myeloid hematopoietic cells to support hematopoiesis and that STAT1 signaling was specifically dependent on type I interferons (IFNs). Indeed, basal type I IFN signaling was reduced in hematopoietic progenitor cells with antibiotic treatment. In addition, we discovered that oral administration of a commensal-derived product, NOD1 ligand, rescues the hematopoietic defects induced by antibiotics in mice. Using metabolomics, we identified additional microbially produced candidates that can stimulate type I IFN signaling to potentially rescue the hematopoietic defects induced by antibiotics, including phosphatidylcholine and γ-glutamylalanine. Overall, our studies define a signaling pathway through which microbiota promotes normal hematopoiesis and identify microbial metabolites that may serve as therapeutic agents to ameliorate antibiotic-induced bone marrow suppression and cytopenia.

Adipose tissue-specific ablation of Ces1d causes metabolic dysregulation in mice.

Carboxylesterase 1d (Ces1d) is a crucial enzyme with a wide range of activities in multiple tissues. It has been reported to localize predominantly in ER. Here, we found that Ces1d levels are significantly increased in obese patients with type 2 diabetes. Intriguingly, a high level of Ces1d translocates onto lipid droplets where it digests the lipids to produce a unique set of fatty acids. We further revealed that adipose tissue-specific Ces1d knock-out (FKO) mice gained more body weight with increased fat mass during a high fat-diet challenge. The FKO mice exhibited impaired glucose and lipid metabolism and developed exacerbated liver steatosis. Mechanistically, deficiency of Ces1d induced abnormally large lipid droplet deposition in the adipocytes, causing ectopic accumulation of triglycerides in other peripheral tissues. Furthermore, loss of Ces1d diminished the circulating free fatty acids serving as signaling molecules to trigger the epigenetic regulations of energy metabolism via lipid-sensing transcriptional factors, such as HNF4α. The metabolic disorders induced an unhealthy microenvironment in the metabolically active tissues, ultimately leading to systemic insulin resistance.

Lipidomic Profiles of Plasma Exosomes Identify Candidate Biomarkers for Early Detection of Hepatocellular Carcinoma in Patients with Cirrhosis.

Novel biomarkers for HCC surveillance in cirrhotic patients are urgently needed. Exosomes and their lipid content in particular represent potentially valuable noninvasive diagnostic biomarkers. We isolated exosomes from plasma of 72 cirrhotic patients, including 31 with HCC. Exosomes and unfractionated plasma were processed for untargeted lipidomics using ultra-high-resolution mass spectrometry. A total of 2,864 lipid species, belonging to 52 classes, were identified. Both exosome fractionation and HCC diagnosis had significant impact on the lipid profiles. Ten lipid classes were enriched in HCC exosomes compared with non-HCC exosomes. Dilysocardiolipins were detected in 35% of the HCC exosomes but in none of the non-HCC exosomes (P < 0.001). Cardiolipins and sphingosines had the highest differential effects (fold change of 133.08, q = 0.001 and 38.57, q < 0.001, respectively). In logistic regression analysis, high abundances of exosomal sphingosines, dilysocardiolipins, lysophosphatidylserines, and (O-acyl)-1-hydroxy fatty acids were strongly associated with HCC [OR (95% confidence interval (CI)), 271.1 (14.0-5,251.9), P < 0.001; 46.5 (2.3-939.9), P = 0.012; 14.9 (4.3-51.2), P < 0.001; 10.3 (3.2-33.1), P < 0.001]. Four lipid classes were depleted in HCC exosomes compared with non-HCC exosomes. In logistic regression analysis, lack of detection of sulfatides and acylGlcSitosterol esters was strongly associated with HCC [OR (95% CI): 215.5 (11.5-4,035.9), P < 0.001; 26.7 (1.4-528.4), P = 0.031]. These HCC-associated changes in lipid composition of exosomes reflected alterations in glycerophospholipid metabolism, retrograde endocannabinoid signaling, and ferroptosis. In conclusion, this study identified candidate biomarkers for early detection of HCC as well as altered pathways in exosomes that may contribute to tumor development and progression. PREVENTION RELEVANCE: This study identifies lipids in circulating exosomes, that could serve as biomarkers for the early detection of hepatocellular carcinoma as well as altered pathways in exosomes that may contribute to tumor development and progression.