Fuhua Hao

Metabonomics in Ulcerative Colitis: Diagnostics, Biomarker Identification, And Insight into the Pathophysiology

Nuclear magnetic resonance (NMR) spectroscopy and appropriate multivariate statistical analyses have been employed on mucosal colonic biopsies, colonocytes, lymphocytes, and urine from patients with ulcerative colitis (UC) and controls in order to explore the diagnostic possibilities, define new potential biomarkers, and generate a better understanding of the pathophysiology. Samples were collected from patients with active UC (n = 41), quiescent UC (n = 33), and from controls (n = 25) and analyzed by NMR spectroscopy. Data analysis was carried out by principal component analysis and orthogonal-projection to latent structure-discriminant analysis using the SIMCA P+11 software package (Umetrics, Umea, Sweden) and Matlab environment. Significant differences between controls and active UC were discovered in the metabolic profiles of biopsies and colonocytes. In the biopsies from patients with active UC higher levels of antioxidants and of a range of amino acids, but lower levels of lipid, glycerophosphocholine (GPC), myo-inositol, and betaine were found, whereas the colonocytes only displayed low levels of GPC, myo-inositol and choline. Interestingly, 20% of inactive UC patients had similar profiles to those who were in an active state. This study demonstrates the possibilities of metabonomics as a diagnostic tool in active and quiescent UC and provides new insight into pathophysiologic mechanisms.

Human Serum Metabonomic Analysis Reveals Progression Axes for Glucose Intolerance and Insulin Resistance Statuses

Understanding the metabolic basis of glucose intolerances and insulin resistance is essential to facilitate early diagnosis, satisfactory therapies and personalized treatments of type 2 diabetes (T2DM). Here, we analyzed the serum metabolic variations from 231 human participants with normal glucose tolerance (NGT, n = 80, M/F = 34/46, mean age 53 +/- 10 years), impaired glucose regulation (IGR, n = 77, M/F = 33/44, mean age 51 +/- 10 years) and T2DM (n = 74, M/F = 32/42, mean age 51 +/- 9 years) to establish the relationship between the serum metabolite compositions and the development of diabetes. By using the proton nuclear magnetic resonance spectroscopy in conjunction with the multivariate data analysis, we found that the development of both glucose intolerances and insulin resistances are closely correlated with the progressive changes of human serum metabonome. Compared with NGT subjects, the IGR and T2DM participants showed clear dysfunctions of choline metabolism, glucose metabolism, lipid and amino acid metabolisms, and disruptions of TCA cycle. The insulin resistance statuses were closely associated with the serum metabonomic changes in terms of glucose, fatty acid and protein/amino acid metabolisms. We also found greater metabonomic heterogeneity among the populations with T2DM and high insulin resistance status. These findings provide useful information to bridge the gaps in our understandings to the metabolic alterations associated with the progression of glucose intolerances and insulin resistance status.

Combined NMR and LC-DAD-MS analysis reveals comprehensive metabonomic variations for three phenotypic cultivars of Salvia Miltiorrhiza Bunge.

Metabonomic analysis is an important molecular phenotyping method for understanding plant ecotypic variations and gene functions. Here, we systematically characterized the metabonomic variations associated with three Salvia miltiorrhiza Bunge (SMB) cultivars using the combined NMR and LC-DAD-MS detections in conjunction with multivariate data analysis. Our results indicated that NMR methods were effective to quantitatively detect the abundant plant metabolites including both the primary and secondary metabolites whereas the LC-DAD-MS methods were excellent for selectively detecting the secondary metabolites. We found that the SMB metabonome was dominated by 28 primary metabolites including sugars, amino acids, and carboxylic acids and 4 polyphenolic secondary metabolites, among which N-acetylglutamate, asparate, fumurate, and yunnaneic acid D were reported for the first time in this plant. We also found that three SMB cultivars growing at the same location had significant metabonomic differences in terms of metabolisms of carbohydrates, amino acids, and choline, TCA cycle, and the shikimate-mediated secondary metabolisms. We further found that the same SMB cultivar growing at different locations differed in their metabonome. These results provided important information on the ecotypic dependence of SMB metabonome on the growing environment and demonstrated that the combination of NMR and LC-MS methods was effective for plant metabonomic phenotype analysis.

Comparison of serum metabolite compositions between obese and lean growing pigs using an NMR-based metabonomic approach

Childhood obesity has become a prevalent risk to health of children and teenagers. To develop biomarkers in serum for altered lipid metabolism, genetically obese (Ningxiang strain) and lean (Duroc×Landrace×Large Yorkshire strain) growing pigs were used as models to identify potential differences in the serum metabonome between the two strains of pigs after consuming the same diet for 46 days. At the end of the study, pigs were euthanized for analysis of the serum metabonome and determination of body composition. Obese pigs had higher fat mass (42.3±8.8% vs. 21.9±4.5%) and lower muscle mass (35.4±4.5% vs. 58.9±2.5%) than lean pigs (P<.01). Serum concentrations of insulin and glucagon were higher (P<.02) in obese than in lean pigs. With the use of an NMR-based metabonomic technology, orthogonal projection to latent structure with discriminant analysis showed that serum HDL, VLDL, lipids, unsaturated lipids, glycoprotein, myo-inositol, pyruvate, threonine, tyrosine and creatine were higher in obese than in lean pigs (P<.05), while serum glucose and urea were lower in obese pigs (P<.05). In addition, changes in gut microbiota-related metabolites, including trimethylamine-N-oxide and choline, were observed in sera of obese pigs relatively to lean pigs (P<.05). These novel findings indicate that obese pigs have distinct metabolism, including lipogenesis, lipid oxidation, energy utilization and partition, protein and amino acid metabolism, and fermentation of gastrointestinal microbes, compared with lean pigs. The obese Ningxiang pig may be a useful model for childhood obesity research.

High-Fat Diet Induces Dynamic Metabolic Alterations in Multiple Biological Matrices of Rats

Obesity is a condition resulting from the interactions of individual biology and environmental factors causing multiple complications. To understand the systems metabolic changes associated with the obesity development and progression, we systematically analyzed the dynamic metabonomic changes induced by a high-fat diet (HFD) in multiple biological matrices of rats using NMR and GC-FID/MS techniques. Clinical chemistry and histopathological data were obtained as complementary information. We found that HFD intakes caused systematic metabolic changes in blood plasma, liver, and urine samples involving multiple metabolic pathways including glycolysis, TCA cycle, and gut microbiota functions together with the metabolisms of fatty acids, amino acids, choline, B-vitamins, purines, and pyrimidines. The HFD-induced metabolic variations were detectable in rat urine a week after HFD intake and showed clear dependence on the intake duration. B-vitamins and gut microbiota played important roles in the obesity development and progression together with changes in TCA cycle intermediates (citrate, α-ketoglutarate, succinate, and fumarate). 83-day HFD intakes caused significant metabolic alterations in rat liver highlighted with the enhancements in lipogenesis, lipid accumulation and lipid oxidation, suppression of glycolysis, up-regulation of gluconeogenesis and glycogenesis together with altered metabolisms of choline, amino acids and nucleotides. HFD intakes reduced the PUFA-to-MUFA ratio in both plasma and liver, indicating the HFD-induced oxidative stress. These findings provided essential biochemistry information about the dynamic metabolic responses to the development and progression of HFD-induced obesity. This study also demonstrated the combined metabonomic analysis of multiple biological matrices as a powerful approach for understanding the molecular basis of pathogenesis and disease progression.

Systems Biological Responses to Chronic Perfluorododecanoic Acid Exposure by Integrated Metabonomic and Transcriptomic Studies

Perfluorocarboxylic acids (PFCAs) have been widely used in consumer and industrial products, such as food packaging, and found in the blood of both humans and wildlife. Although studies showed a high tendency toward biological accumulation and a variety of toxic effects for PFCAs, the mechanistic aspects of their toxicity remain unknown. In present study, we investigated the dosage-dependent metabonomic and transcriptomic responses of male rats to the exposure to perfluorododecanoic acid (PFDoA) over 110 days. Our NMR-based metabonomics results for both liver tissues and serum demonstrated that PFDoA exposure led to hepatic lipidosis, which was characterized by a severe elevation in hepatic triglycerides and a decline in serum lipoprotein levels. The results from transcriptomic changes induced by PFDoA corroborated these results with changes in gene transcript levels associated with fatty acid homeostasis. These results demonstrate that PFDoA induces hepatic steatosis via perturbations to fatty acid uptake, lipogenesis, and fatty acid oxidation. Several serum metabolites exhibited dose-dependences, providing thorough descriptions of changes induced by PFDoA exposure. These observations yielded novel insights regarding the toxicological mechanism of PFCAs at the systems level.

Revealing Different Systems Responses to Brown Planthopper Infestation for Pest Susceptible and Resistant Rice Plants with the Combined Metabonomic and Gene-Expression Analysis

Brown planthopper (BPH) is a notorious pest of rice plants attacking leaf sheaths and seriously affecting global rice production. However, how rice plants respond against BPH remains to be fully understood. To understand systems metabolic responses of rice plants to BPH infestation, we analyzed BPH-induced metabolic changes in leaf sheaths of both BPH-susceptible and resistant rice varieties using NMR-based metabonomics and measured expression changes of 10 relevant genes using quantitative real-time PCR. Our results showed that rice metabonome was dominated by more than 30 metabolites including sugars, organic acids, amino acids, and choline metabolites. BPH infestation caused profound metabolic changes for both BPH-susceptible and resistant rice plants involving transamination, GABA shunt, TCA cycle, gluconeogenesis/glycolysis, pentose phosphate pathway, and secondary metabolisms. BPH infestation caused more drastic overall metabolic changes for BPH-susceptible variety and more marked up-regulations for key genes regulating GABA shunt and biosynthesis of secondary metabolites for BPH-resistant variety. Such observations indicated that activation of GABA shunt and shikimate-mediated secondary metabolisms was vital for rice plants to resist BPH infestation. These findings filled the gap of our understandings in the mechanistic aspects of BPH resistance for rice plants and demonstrated the combined metabonomic and qRT-PCR analysis as an effective approach for understanding plant-herbivore interactions.

Systemic responses of mice to dextran sulfate sodium-induced acute ulcerative colitis using 1H NMR spectroscopy.

The interplay between genetic mutation and environmental factors is believed to contribute to the etiology of inflammatory bowel disease (IBD). While focused attention has been paid to the aforementioned research, time-specific and organ-specific metabolic changes associated with IBD are still lacking. Here, we induced acute ulcerative colitis in mice by providing water containing 3% dextran sulfate sodium (DSS) for 7 days and investigated the metabolic changes of plasma, urine, and a range of biological tissues by employing a (1)H nuclear magnetic resonance (NMR)-based metabonomics approach with complementary information on serum clinical chemistry and histopathology. We found that DSS-induced acute ulcerative colitis leads to significant elevations in the levels of amino acids in plasma and decreased levels in the membrane-related metabolites and a range of nucleotides, nucleobases, and nucleosides in the colon. In addition, acute-colitis-induced elevations in the levels of nucleotides in the liver were observed, accompanied by reduced levels of glucose. DSS-induced acute colitis also resulted in increased levels of oxidized glutathione and attenuated levels of taurine in the spleen. Furthermore, acute colitis resulted in depletion in the levels of gut microbial cometabolites in urine along with an increase in citric acid cycle intermediates. These findings suggest that DSS-induced acute colitis causes a disturbance of lipid and energy metabolism, damage to the colon and liver, a promoted antioxidative and anti-inflammatory response, and perturbed gut microbiotal communities. The information obtained here provided details of the time-dependent and holistic metabolic changes in the development of the DSS-induced acute ulcerative colitis, which could be useful in discovery of novel therapeutic targets for management of IBD.

Global Metabolomic Responses of Escherichia coli to Heat Stress

Microbial metabolomic analysis is essential for understanding responses of microorganisms to heat stress. To understand the comprehensive metabolic responses of Escherichia coli to continuous heat stress, we characterized the metabolomic variations induced by heat stress using NMR spectroscopy in combination with multivariate data analysis. We detected 15 amino acids, 10 nucleotides, 9 aliphatic organic acids, 7 amines, glucose and its derivative glucosylglyceric acid, and methanol in the E. coli extracts. Glucosylglyceric acid was reported for the first time in E. coli. We found that heat stress was an important factor influencing the metabolic state and growth process, mainly via suppressing energy associated metabolism, reducing nucleotide biosynthesis, altering amino acid metabolism and promoting osmotic regulation. Moreover, metabolic perturbation was aggravated during heat stress. However, a sign of recovery to control levels was observed after the removal of heat stress. These findings enhanced our understanding of the metabolic responses of E. coli to heat stress and demonstrated the effectiveness of the NMR-based metabolomics approach to study such a complex system.

Streptozotocin-Induced Dynamic Metabonomic Changes in Rat Biofluids

Diabetes mellitus is a complex polygenic disease caused by gene-environment interactions with multiple complications, and metabonomic analysis is crucial for pathogenesis, early diagnosis, and timely interventions. Here, we comprehensively analyzed the dynamic metabolic changes in rat urine and plasma, which were induced by the well-known diabetogenic chemical streptozotocin (STZ), using (1)H NMR spectroscopy in conjunction with multivariate data analysis. The results showed that a single intraperitoneal injection of STZ with a moderate dosage (55 mg/kg) induced significant urinary metabonomic changes within 24 h. These changes showed time-dependence and heterogeneity among the treated animals with an animal recovered within 11 days. STZ-induced metabonomic alterations were related to suppression of glycolysis and TCA cycle, promotion of gluconeogenesis and oxidation of amino acids, alterations in metabolisms of basic amino acids associated with diabetic complications, and disruption of lipid metabolism and gut microbiota functions. With diffusion-edited NMR spectral data, we further observed the STZ-induced significant elevation of monounsaturated fatty acids and total unsaturated fatty acids together with reductions in PUFA-to-MUFA ratio in the blood plasma. These findings provided details of the time-dependent metabonomic changes in the progressive development of the STZ-induced diabetes mellitus and showed the possibility of detecting the biochemical changes in the early stage of type 1 diabetic genesis.