A multi-tissue metabolome atlas of primate pregnancy.

Pregnancy induces dramatic metabolic changes in females; yet, the intricacies of this metabolic reprogramming remain poorly understood, especially in primates. Using cynomolgus monkeys, we constructed a comprehensive multi-tissue metabolome atlas, analyzing 273 samples from 23 maternal tissues during pregnancy. We discovered a decline in metabolic coupling between tissues as pregnancy progressed. Core metabolic pathways that were rewired during primate pregnancy included steroidogenesis, fatty acid metabolism, and arachidonic acid metabolism. Our atlas revealed 91 pregnancy-adaptive metabolites changing consistently across 23 tissues, whose roles we verified in human cell models and patient samples. Corticosterone and palmitoyl-carnitine regulated placental maturation and maternal tissue progenitors, respectively, with implications for maternal preeclampsia, diabetes, cardiac hypertrophy, and muscle and liver regeneration. Moreover, we found that corticosterone deficiency induced preeclampsia-like inflammation, indicating the atlas's potential clinical value. Overall, our multi-tissue metabolome atlas serves as a framework for elucidating the role of metabolic regulation in female health during pregnancy.

Dissecting the relationship between plasma and tissue metabolome in a cohort of women with obesity: Analysis of subcutaneous and visceral adipose, muscle, and liver.

Untargeted metabolomics of blood samples has become widely applied to study metabolic alterations underpinning disease and to identify biomarkers. However, understanding the relevance of a blood metabolite marker can be challenging if it is unknown whether it reflects the concentration in relevant tissues. To explore this field, metabolomic and lipidomic profiles of plasma, four sites of adipose tissues (ATs) from peripheral or central depot, two sites of muscle tissue, and liver tissue from a group of nondiabetic women with obesity who were scheduled to undergo bariatric surgery (n = 21) or other upper GI surgery (n = 5), were measured by liquid chromatography coupled with mass spectrometry. Relationships between plasma and tissue profiles were examined using Pearson correlation analysis subject to Benjamini-Hochberg correction. Plasma metabolites and lipids showed the highest number of significantly positive correlations with their corresponding concentrations in liver tissue, including lipid species of ceramide, mono- and di-hexosylceramide, sphingomyelin, phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine, dimethyl phosphatidylethanolamine, ether-linked PC, ether-linked PE, free fatty acid, cholesteryl ester, diacylglycerol and triacylglycerol, and polar metabolites linked to several metabolic functions and gut microbial metabolism. Plasma also showed significantly positive correlations with muscle for several phospholipid species and polar metabolites linked to metabolic functions and gut microbial metabolism, and with AT for several triacylglycerol species. In conclusion, plasma metabolomic and lipidomic profiles were reflective more of the liver profile than any of the muscle or AT sites examined in the present study. Our findings highlighted the importance of taking into consideration the metabolomic relationship of various tissues with plasma when postulating plasma metabolites marker to underlying mechanisms occurring in a specific tissue.

Nuclear magnetic resonance-based tissue metabolomic analysis clarifies molecular mechanisms of gastric carcinogenesis.

Gastric cancer (GC) is one of the deadliest cancers worldwide, and the progression of gastric carcinogenesis (GCG) covers multiple complicated pathological stages. Molecular mechanisms of GCG are still unclear. Here, we undertook NMR-based metabolomic analysis of aqueous metabolites extracted from gastric tissues in an established rat model of GCG. We showed that the metabolic profiles were clearly distinguished among 5 histologically classified groups: control, gastritis, low-grade gastric dysplasia, high-grade gastric dysplasia (HGD), and GC. Furthermore, we carried out metabolic pathway analysis based on identified significant metabolites and revealed significantly disturbed metabolic pathways closely associated with the 4 pathological stages, including oxidation stress, choline phosphorylation, amino acid metabolism, Krebs cycle, and glycolysis. Three metabolic pathways were continually disturbed during the progression of GCG, including taurine and hypotaurine metabolism, glutamine and glutamate metabolism, alanine, aspartate, and glutamate metabolism. Both the Krebs cycle and glycine, serine, and threonine metabolism were profoundly impaired in both the HGD and GC stages, potentially due to abnormal energy supply for tumor cell proliferation and growth. Furthermore, valine, leucine, and isoleucine biosynthesis and glycolysis were significantly disturbed in the GC stage for higher energy requirement of the rapid growth of tumor cells. Additionally, we identified potential gastric tissue biomarkers for metabolically discriminating the 4 pathological stages, which also showed good discriminant capabilities for their serum counterparts. This work sheds light on the molecular mechanisms of GCG and is of benefit to the exploration of potential biomarkers for clinically diagnosing and monitoring the progression of GCG.

Comprehensive Metabolomic and Lipidomic Profiling of Human Kidney Tissue: A Platform Comparison.

Metabolite profiling of tissue samples is a promising approach for the characterization of cancer pathways and tumor classification based on metabolic features. Here, we present an analytical method for nontargeted metabolomics of kidney tissue. Capitalizing on different chemical properties of metabolites allowed us to extract a broad range of molecules covering small polar molecules and less polar lipid classes that were analyzed by LC-QTOF-MS after HILIC and RP chromatographic separation, respectively. More than 1000 features could be reproducibly extracted and analyzed (CV < 30%) in porcine and human kidney tissue, which were used as surrogate matrices for method development. To further assess assay performance, cross-validation of the nontargeted metabolomics platform to a targeted metabolomics approach was carried out. Strikingly, from 102 metabolites that could be detected on both platforms, the majority (>90%) revealed Spearman's correlation coefficients ≥0.3, indicating that quantitative results from the nontargeted assay are largely comparable to data derived from classical targeted assays. Finally, as proof of concept, the method was applied to human kidney tissue where a clear differentiation between kidney cancer and nontumorous material could be demonstrated on the basis of unsupervised statistical analysis.

Cooperative STAT3-NFkB signaling modulates mitochondrial dysfunction and metabolic profiling in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) continues to pose a significant health challenge and is often diagnosed at advanced stages. Metabolic reprogramming is a hallmark of many cancer types, including HCC and it involves alterations in various metabolic or nutrient-sensing pathways within liver cells to facilitate the rapid growth and progression of tumours. However, the role of STAT3-NFκB in metabolic reprogramming is still not clear. APPROACH AND RESULTS: Diethylnitrosamine (DEN) administered animals showed decreased body weight and elevated level of serum enzymes. Also, Transmission electron microscopy (TEM) analysis revealed ultrastructural alterations. Increased phosphorylated signal transducer and activator of transcription-3 (p-STAT3), phosphorylated nuclear factor kappa B (p-NFκß), dynamin related protein 1 (Drp-1) and alpha-fetoprotein (AFP) expression enhance the carcinogenicity as revealed in immunohistochemistry (IHC). The enzyme-linked immunosorbent assay (ELISA) concentration of IL-6 was found to be elevated in time dependent manner both in blood serum and liver tissue. Moreover, immunoblot analysis showed increased level of p-STAT3, p-NFκß and IL-6 stimulated the upregulation of mitophagy proteins such as Drp-1, Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK-1). Meanwhile, downregulation of Poly [ADP-ribose] polymerase 1 (PARP-1) and cleaved caspase 3 suppresses apoptosis and enhanced expression of AFP supports tumorigenesis. The mRNA level of STAT3 and Drp-1 was also found to be significantly increased. Furthermore, we performed high-field 800 MHz Nuclear Magnetic Resonance (NMR) based tissue and serum metabolomics analysis to identify metabolic signatures associated with the progression of liver cancer. The metabolomics findings revealed aberrant metabolic alterations in liver tissue and serum of 75th and 105th days of intervention groups in comparison to control, 15th and 45th days of intervention groups. Tissue metabolomics analysis revealed the accumulation of succinate in the liver tissue samples, whereas, serum metabolomics analysis revealed significantly decreased circulatory levels of ketone bodies (such as 3-hydroxybutyrate, acetate, acetone, etc.) and membrane metabolites suggesting activated ketolysis in advanced stages of liver cancer. CONCLUSION: STAT3-NFκß signaling axis has a significant role in mitochondrial dysfunction and metabolic alterations in the development of HCC.

Comprehensive brain tissue metabolomics and biological network technology to decipher the mechanism of hydrogen-rich water on Radiation-induced cognitive impairment in rats.

BACKGROUND: Hydrogen-rich water (HRW) has been shown to prevent cognitive impairment caused by ionizing radiation. This study aimed to investigate the pharmacological effects and mechanisms of HRW on ionizing radiation by coupling the brain metabolomics and biological target network methods. METHODS AND RESULTS: HRW significantly improves the cognitive impairment in rats exposed to ionizing radiation. Based on metabolomics and biological network results, we identified 54 differential metabolites and 93 target genes. The KEGG pathway indicates that glutathione metabolism, ascorbic acid and aldehyde acid metabolism, pentose and glucuronic acid interconversion, and glycerophospholipid metabolism play important roles in ionizing radiation therapy. CONCLUSION: Our study has systematically elucidated the molecular mechanism of HRW against ionizing radiation, which can be mediated by modulating targets, pathways and metabolite levels. This provides a new perspective for identifying the underlying pharmacological mechanism of HRW.

Metabolomics analyses of cancer tissue from patients with colorectal cancer.

The alteration of metabolism is essential for the initiation and progression of numerous types of cancer, including colorectal cancer (CRC). Metabolomics has been used to study CRC. At present, the reprogramming of the metabolism in CRC remains to be fully elucidated. In the present study, comprehensive untargeted metabolomics analysis was performed on the paired CRC tissues and adjacent normal tissues from patients with CRC (n=35) using ultra­high­performance liquid chromatography­mass spectrometry. Subsequently, bioinformatic analysis was performed on the differentially expressed metabolites. The changes in these differential metabolites were compared among groups of patients based on sex, anatomical tumor location, grade of tumor differentiation and stage of disease. A total of 927 metabolites were detected in the tissue samples, and 24 metabolites in the CRC tissue were significantly different compared with the adjacent normal tissue. The present study revealed that the levels of three amino acid metabolites were increased in the CRC tissue, specifically, N­α­acetyl­Îµ­(2­propenal)­Lys, cyclo(Glu­Glu) and cyclo(Phe­Glu). The metabolites with decreased levels in the CRC tissue included quinaldic acid (also referred to as quinoline­2­carboxilic acid), 17α­ and 17ß­estradiol, which are associated with tumor suppression activities, as well as other metabolites such as, anhydro­ß­glucose, Asp­Arg, lysophosphatidylcholine, lysophosphatidylethanolamine (lysoPE), lysophosphatidylinositol, carnitine, 5'­deoxy­5'­(methylthio) adenosine, 2'­deoxyinosine­5'­monophosphate and thiamine monophosphate. There was no difference in the levels of the differential metabolites between male and female patients. The differentiation of CRC also showed no impact on the levels of the differential metabolites. The levels of lysoPE were increased in the right side of the colon compared with the left side of the colon and rectum. Analysis of the different tumor stages indicated that 2­aminobenzenesulfonic acid, P­sulfanilic acid and quinoline­4­carboxylic acid were decreased in stage I CRC tissue compared with stage II, III and IV CRC tissue. The levels of N­α­acetyl­Îµ­(2­propenal)­Lys, methylcysteine and 5'­deoxy­5'­(methylthio) adenosine varied at different stages of tumorigenesis. These differential metabolites were implicated in multiple metabolism pathways, including carbohydrate, amino acid, lipid, nucleotide and hormone. In conclusion, the present study demonstrated that CRC tumors had altered metabolites compared with normal tissue. The data from the metabolic profile of CRC tissues in the present study provided supportive evidence to understand tumorigenesis.

LC-QToF-Based Metabolomics Identifies Aberrant Tissue Metabolites Associated with a Higher-Fat Diet and Their 'Reversion to Healthy' with Dietary Probiotic Supplementation.

Over 33% of Americans are labeled as obese, leading the World Health Organization to designate obesity as a major public health problem. One consequence of obesity is the development of metabolic syndrome, a condition which has been correlated to an increased risk for developing cardiovascular disease and Type 2 diabetes. Prolonged ingestion of a higher-fat diet, one cause of obesity, results in alterations to the gut microbiome. These alterations are implicated to have a profound role in the evolution and progression of obesity-linked diseases. Probiotics are associated with positive health effects such as limiting pathogen colonization, aiding in digestion, and vitamin synthesis. Using Ossabaw pigs as a model for obesity, and in conjunction with our previous research, we performed an in-depth, nontargeted, metabolomic analysis on select organs to elucidate the effects of dietary supplementation with the probiotic Lacticaseibacillus paracasei. We focused our analysis on the effects of probiotic supplementation on a higher-fat (obesogenic) diet and a nutritionally balanced diet. Notably, our findings reveal that the brain cortex is highly sensitive to dietary influencers, and with probiotic supplementation, several aberrant metabolites associated with a higher-fat diet revert to healthy levels, thus demonstrating the potential for a probiotic intervention for obesity-linked disease.

Ceramide metabolism associated with chronic dietary nutrient surplus and diminished insulin sensitivity in the liver, muscle, and adipose tissue of cattle.

High dietary energy and protein supply is common practice in livestock nutrition, aiming to maximize growth and production performance. However, a chronic nutritional surplus induces obesity, promotes insulin insensitivity, and triggers low-grade inflammation. Thirty Holstein bulls were randomly assigned to two groups, low energy and protein (LEP), and high energy and protein (HEP) intake, provided from the 13th to the 20th month of life. Body weight, carcass composition, laminitis score, and circulating insulin and glucose concentrations were assessed. The expression and extent of phosphorylation of insulin signaling proteins were measured in the liver, muscle, and adipose tissue. The sphingolipid metabolome was quantified by a targeted liquid chromatography-mass spectrometry based metabolomics approach. The HEP bulls were obese, had hyperinsulinemia with euglycemia, and expressed clinical signs of chronic laminitis. In the liver, protein kinase B (PKB) phosphorylation was decreased and this was associated with a higher tissue concentration of ceramide 16:0, a sphingolipid that diminishes insulin action by dephosphorylating PKB. In the adipose tissue, insulin receptor expression was lower in HEP bulls, associated with higher concentration of hexosylceramide, which reduces the abundance of functional insulin receptors. Our findings confirm that diet-induced metabolic inflammation triggers ceramide accumulation and disturbs insulin signaling. As insulin insensitivity exacerbates metabolic inflammation, this self-reinforcing cycle could explain the deterioration of metabolic health apparent as chronic laminitis. By demonstrating molecular relationships between insulin signaling and sphingolipid metabolism in three major tissues, our data extend our mechanistic understanding of the role of ceramides in diet-induced metabolic inflammation.

Detection of the Disorders of Glycerophospholipids and Amino Acids Metabolism in Lung Tissue From Male COPD Patients.

Background: At present, few studies have reported the metabolic profiles of lung tissue in patients with COPD. Our study attempted to analyze the lung metabolome in male COPD patients and to screen the overlapping biomarkers of the lung and plasma metabolomes. Methods: We performed untargeted metabolomic analysis of normal lung tissue from two independent sets (the discovery set: 20 male COPD patients and 20 controls and the replication set: 47 male COPD patients and 27 controls) and of plasma samples from 80 male subjects containing 40 COPD patients and 40 controls. Results: We found glycerophospholipids (GPs) and Amino acids were the primary classes of differential metabolites between male COPD patients and controls. The disorders of GPs metabolism and the valine, leucine and isoleucine biosynthesis metabolism pathways were identified in lung discovery set and then also validated in the lung replication set. Combining lung tissue and plasma metabolome, Phytosphingosine and l-tryptophan were two overlapping metabolites biomarkers. Binary logistic regression suggested that phytosphingosine together with l-tryptophan was closely associated with male COPD and showed strong diagnostic power with an AUC of 0.911 (95% CI: 0.8460-0.9765). Conclusion: Our study revealed the metabolic perturbations of lung tissues from male COPD patients. The detected disorders of GPs and amino acids may provide an insight into the pathological mechanism of COPD. Phytosphingosine and l-tryptophan were two novel metabolic biomarkers for differentiating COPD patients and controls.

Assessment of solid phase microextraction as a sample preparation tool for untargeted analysis of brain tissue using liquid chromatography-mass spectrometry.

This work presents an evaluation of solid-phase microextraction (SPME) SPME in combination with liquid chromatography-high resolution mass spectrometry (LC-HRMS) as an analytical approach for untargeted brain analysis. The study included a characterization of the metabolite coverage provided by C18, mixed-mode (MM, with benzene sulfonic acid and C18 functionalities), and hydrophilic lipophilic balanced (HLB) particles as sorbents in SPME coatings after extraction from cow brain homogenate at static conditions. The effects of desorption solvent, extraction time, and chromatographic modes on the metabolite features detected were investigated. Method precision and absolute matrix effects were also assessed. Among the main findings of this work, it was observed that all three tested coating chemistries were able to provide comparable brain tissue information. HLB provided higher responses for polar metabolites; however, as these fibers were prepared in-house, higher inter-fiber relative standard deviations were also observed. C18 and HLB coatings offered similar responses with respect to lipid-related features, whereas MM and C18 provided the best results in terms of method precision. Our results also showed that the use of methanol is essential for effective desorption of non-polar metabolites. Using a reversed-phase chromatographic method, an average of 800 and 1200 brain metabolite features detected in positive and negative modes, respectively, met inter-fibre RSD values below 30% (n=4) after removal of fibre and solvent artefacts from the associated datasets. For features detected using a lipidomics method, a total of 900 and 1800 features detected using C18 fibers in positive and negative mode, respectively, met the same criteria. In terms of absolute matrix effects, the majority of the model metabolites tested showed values between 80 and 120%, which are within the acceptable range. Overall, the findings of this work lay the foundation for further optimization of parameters for SPME-LC-HRMS methods suitable for in vivo and ex vivo brain (and other tissue) untargeted studies, and support the applicability of this approach for non-destructive tissue metabolomics.

Metabolomics based predictive classifier for early detection of pancreatic ductal adenocarcinoma.

The availability of robust classification algorithms for the identification of high risk individuals with resectable disease is critical to improving early detection strategies and ultimately increasing survival rates in PC. We leveraged high quality biospecimens with extensive clinical annotations from patients that received treatment at the Medstar-Georgetown University hospital. We used a high resolution mass spectrometry based global tissue profiling approach in conjunction with multivariate analysis for developing a classification algorithm that would predict early stage PC with high accuracy. The candidate biomarkers were annotated using tandem mass spectrometry. We delineated a six metabolite panel that could discriminate early stage PDAC from benign pancreatic disease with >95% accuracy of classification (Specificity = 0.85, Sensitivity = 0.9). Subsequently, we used multiple reaction monitoring mass spectrometry for evaluation of this panel in plasma samples obtained from the same patients. The pattern of expression of these metabolites in plasma was found to be discordant as compared to that in tissue. Taken together, our results show the value of using a metabolomics approach for developing highly predictive panels for classification of early stage PDAC. Future investigations will likely lead to the development of validated biomarker panels with potential for clinical translation in conjunction with CA-19-9 and/or other biomarkers.

Mass Spectrometry Imaging of Metabolites.

Mass spectrometry imaging (MSI) is a technique which is gaining increasing interest in biomedical research due to its capacity to visualize molecules in tissues. First applied to the field of clinical proteomics, its potential for metabolite imaging in biomedical studies is now being recognized. Here we describe how to set up experiments for mass spectrometry imaging of metabolites in clinical tissues and how to tackle most of the obstacles in the subsequent analysis of the data.

Metabolic risk factors associated with sudden cardiac death (SCD) during acute myocardial ischemia.

Sudden cardiac death (SCD) is the leading cause of death worldwide. Myocardial ischemia (MI) is the most common underlying causal disorder for SCD. Metabolic risks leading to SCD during acute MI are still not fully understood. Here, using tissue metabolomics, we aimed to investigate myocardial metabolic alterations relevant to SCD events in an acute MI rat model induced by coronary artery ligation (CAL). Thirty-four rats were successfully performed CAL, of which 13 developed lethal ventricular tachyarrhythmia (LVTA)-SCD and 7 developed severe atrioventricular block (AB)-SCD. Fourteen rats that survived within 70 min after the ligation were served as peer controls. The partial least squares-discriminant analysis plots demonstrated clear separations between the SCD rats and controls, indicating obvious differences in myocardial metabolome between these rats. The levels of isoleucine, lactate, glutamate choline, phosphorylcholine, taurine and asparagine in ischemic myocardia were positively associated with LVTA-SCD events; in contrast, the levels of alanine, urea, phenylalanine, linoleic acid, elaidic acid and stearic acid were inversely correlated with LVTA-SCD events. The levels of glutamate and urea were positively and negatively relevant to AB-SCD events, respectively. The dangerous metabolites indicated that lower levels of energy substrates, severe hypoxia, the inhibition of transamination and hyper sympathetic excitement and reactive oxygen species in myocardia were vulnerable to SCD during acute MI. The results suggest fatal metabolic alterations correlated with SCD events during acute MI, which could offer novel clues for the prevention or treatment of acute MI-related SCD.

Betaine supplementation causes increase in carnitine metabolites in the muscle and liver of mice fed a high-fat diet as studied by nontargeted LC-MS metabolomics approach.

SCOPE: Betaine (BET) reduces diet-induced liver lipid accumulation, and may relieve obesity-related metabolic disturbances. The aim of our study was to analyze metabolite alterations after supplementation of BET, polydextrose (PDX, a soluble dietary fiber), or their combination (BET PDX) via drinking water to C57BL/6J mice fed a high-fat (HF) diet. METHODS AND RESULTS: BET supplementation increased BET levels in plasma, muscle, and liver (p < 0.05), and the nontargeted LC-MS metabolite profiling revealed an increase in several metabolites in the carnitine biosynthesis pathway after BET supplementation both in liver and muscle. These included carnitine and acetylcarnitine (1.4-fold, p < 0.05), propionylcarnitine and γ-butyrobetaine (1.5-fold, p < 0.05), and several other short-chain acylcarnitines (p < 0.05) in muscle. These changes were slightly higher in the BET PDX group. Furthermore, BET reduced the HF diet induced accumulation of triglycerides in liver (p < 0.05). The supplementations did not attenuate the HF diet induced increase in body weight gain or the increase in adipose tissue mass. Instead, the combination of BET and PDX tended to increase adiposity. CONCLUSION: Our results suggest that increased availability of BET in different tissues, especially in muscle, after BET supplementation has an impact on carnitine metabolism, and this could further explain the link between BET and lipid metabolism.