Quantitative ¹³C Traces of Glucose Fate in Hepatitis B Virus-Infected Hepatocytes

Quantitative characterization of 13C-labeled metabolites is an important part of the stable isotope tracing method widely used in metabolic flux analysis. Given the long relaxation time and low sensitivity of 13C nuclei, direct measurement of 13C-labeled metabolites using one-dimensional 13C NMR often fails to meet the demand of metabolomics studies, especially with large numbers of samples and metabolites having low abundance. Although HSQC-based 2D NMR methods have improved sensitivity with inversion detection, they are time-consuming and thus unsuitable for high-throughput absolute quantification of 13C-labeled metabolites. In this study, we developed a method for absolute quantification of 13C-labeled metabolites using naturally abundant TSP as a reference with the first increment of the HMQC pulse sequence, taking polarization transfer efficiencies into consideration. We validated this method using a mixture of 13C-labeled alanine, methionine, glucose, and formic acid together with a mixture of alanine, lactate, glycine, uridine, cytosine, and hypoxanthine, which have natural 13C abundance with known concentrations. We subsequently applied this method to analyze the flux of glucose in HepG2 cells infected with hepatitis B virus (HBV). The results showed that HBV infection increased the cellular uptake of glucose, stimulated glycolysis, and enhanced the pentose phosphate and hexosamine pathways for biosynthesis of RNA and DNA and nucleotide sugars to facilitate HBV replication. This method saves experimental time and provides a possibility for absolute quantitative tracking of the 13C-labeled metabolites for high-throughput studies

Solid-State NMR Analyses Reveal the Structure Dependence of the Molecular Dynamics for ω-Amino Acids

The molecular dynamics of metabolites is structure dependent and vitally important for the interactive functions in their potential applications as natural materials. To understand the relationship between molecular structure and dynamics, the molecular motions of four structurally related ω-amino acids (β-alanine, γ-aminobutyric acid, 5-aminovaleric acid, and 6-aminocaproic acid) were investigated by measuring their proton spin–lattice relaxation times (<i>T</i>₁, <i>T</i>_1ρ) as a function of temperature (180–440 K). ¹³C CPMAS NMR and DSC analyses were performed to obtain complementary information. All of these ω-amino acids showed no phase transition in the temperature range studied but had outstandingly long proton <i>T</i>₁ at 300 MHz and even at 20 MHz for the deuterated forms. The molecular dynamics of all these ω-amino acids were dominated by the reorientation motions of amino groups and backbone motions except in β-alanine. The activation energies for amino group reorientations were positively correlated with the strength of hydrogen bonds involving these groups in the crystals and the carbon-chain lengths, whereas such energies for the backbone motions were inversely correlated with the carbon-chain lengths. These findings provided essential information for the molecular dynamics of ω-amino acids and demonstrated the combined solid-state NMR methods as a useful approach for understanding the structural dependence of molecular dynamics

Combined NMR and LC-MS Analysis Reveals the Metabonomic Changes in <i>Salvia miltiorrhiza</i> Bunge Induced by Water Depletion

Plant metabonomic analysis is essential for understanding plant systems responses to osmotic stresses. To understand the comprehensive metabolic responses of Salvia miltiorrhiza Bunge (SMB) to continuous and exhaustive water depletion, we characterized the SMB metabonomic variations induced by three different drying processes using the combined NMR and LC-DAD-MS method. NMR results showed that SMB extracts were dominated by 29 primary metabolites such as sugars, carboxylic acids and amino acids, which were comprehensively reported for the first time, and 8 secondary metabolites including polyphenolic acids and diterpenoids. LC-DAD-MS methods detected 44 secondary metabolites, among which 5 polyphenolic acids together with genipin, umbelliferone and tormentic acid were found for the first time in this plant. We found that aqueous methanol was efficient in extracting both primary metabolites and polyphenolic acids, whereas chloroform−methanol was effective in selectively extracting diterpenoids. We further found that air- and sun-drying markedly affected both primary and secondary metabolisms of SMB by enhancing tanshinone and glutamate-mediated proline biosynthesis and altering carbohydrate and amino acid metabolisms. The shikimate-mediated biosynthesis of polyphenolic acids was promoted by air-drying but suppressed by sun-drying. These findings fill the gap of our understandings to the metabolic responses of S. miltiorrhiza Bunge to water depletion and demonstrated effectiveness of the combined NMR and LC-DAD-MS methods in plant metabonomic analysis

Metabonomic Analysis Reveals the CCl₄-Induced Systems Alterations for Multiple Rat Organs

CCl₄-induced metabonomic changes have been extensively studied for mammalian liver, and such changes have not been reported for other organs. To investigate the CCl₄ effects on other organs, we analyzed the CCl₄-induced metabonomic changes in rat kidney, lung, and spleen using ¹H NMR-based metabonomics approaches with complementary information on serum clinical chemistry and histopathology. We found that acute CCl₄ exposure caused significant level elevation for creatine and decline for glucose, taurine, trimethylamine, uridine, and adenosine in rat kidney. CCl₄-treatment also induced elevation of amino acids (isoleucine, leucine, valine, threonine, alanine, lysine, ornithine, methionine, tyrosine, phenylalanine, and histidine), creatine, and betaine in rat lung together with depletion of glycogen, glucose, taurine, glycine, and hypoxanthine. Furthermore, CCl₄ caused elevation of lactate, alanine, betaine, and uracil in rat spleen accompanied with decline for glucose, choline, and hypoxanthine. These observations indicated that CCl₄ caused oxidative stresses to multiple rat organs and alterations of their functions including renal osmotic regulations, accelerated glycolysis, and protein and nucleotide catabolism. These findings provide essential information on CCl₄ toxicity to multiple rat organs and suggest that systems toxicological views are required for metabonomic studies of toxins by taking many other organs into consideration apart from so-called targeted ones

Age-Related Topographical Metabolic Signatures for the Rat Gastrointestinal Contents

Symbiotic gut microbiota is essential for mammalian physiology and analyzing the metabolite compositions of gastrointestinal contents is vital for understanding the microbiome–host interactions. To understand the developmental dependence of the topographical metabolic signatures for the rat gastrointestinal contents, we systematically characterized the metabolite compositional variations of the contents in rat jejunum, ileum, cecum, and colon for two age-groups using ¹H NMR spectroscopy and multivariate analysis. Significant topographical metabolic variations were present for the jejunal, ileal, cecal, colonic contents, and feces, reflecting the absorption functions for each intestinal region and the gut microbiota therein. The concentrations of amino acids, lactate, creatine, choline, bile acids, uracil and urocanate decreased drastically from jejunal to ileal contents followed with steady decreases from cecal content to feces. Short-chain fatty acids (SCFAs) and arabinoxylan-related carbohydrates had highest levels in cecal content and feces, respectively. Such topographical metabolic signatures for the intestinal contents varied with animal age highlighted by the level changes for lactate, choline, taurine, amino acids, carbohydrates, keto-acids, and SCFAs. These findings provided essential information for the topographical metabolic variations in the gastrointestinal tract and demonstrated metabolic profiling as a useful approach for understanding host–microbiome interactions and functional status of the gastrointestinal regions

Gallic Acid Intake Induces Alterations to Systems Metabolism in Rats

Gallic acid (GA) and its metabolites are polyphenolic compounds present in daily diets and herbal medicines. To understand the GA effects on the endogenous metabolism of mammals, we systematically analyzed the metabonomic responses of rat plasma, liver, urine, and feces to a single GA dosage of 120 and 600 mg/kg, which were below the no-obvious-adverse-effect-level of 1 g/kg for rats. Clinical chemistry and histopathological assessments were conducted to provide complementary information. Our results showed that GA intake induced significant metabonomic changes in multiple rat biological matrices. Such changes were more outstanding in liver than in the other matrices and clearly showed dose- and time-dependence. The results suggested GA-induced promotion of oxidative stress as the major effect. High-dose GA caused significant metabolic changes involving glycogenolysis, glycolysis, TCA cycle, and metabolism of amino acids, purines, and pyrimidines, together with gut microbiota functions. Low-dose GA only caused some urinary metabonomic changes and to a much less degree. The GA-induced liver metabonomic changes were not completely recoverable within a week, although such recovery completed in plasma, urine, and feces within 80 h. These findings provided new essential information on the effects of dietary polyphenols and demonstrated the great potential of this nutrimetabonomics approach

Dynamic Metabonomic Responses of Tobacco <i>(Nicotiana tabacum)</i> Plants to Salt Stress

Metabolic responses are important for plant adaptation to osmotic stresses. To understand the dosage and duration dependence of salinity effects on plant metabolisms, we analyzed the metabonome of tobacco plants and its dynamic responses to salt treatments using NMR spectroscopy in combination with multivariate data analysis. Our results showed that the tobacco metabonome was dominated by 40 metabolites including organic acids/bases, amino acids, carbohydrates and choline, pyrimidine, and purine metabolites. A dynamic trajectory was clearly observable for the tobacco metabonomic responses to the dosage of salinity. Short-term low-dose salt stress (50 mM NaCl, 1 day) caused metabolic shifts toward gluconeogenesis with depletion of pyrimidine and purine metabolites. Prolonged salinity with high-dose salt (500 mM NaCl) induced progressive accumulation of osmolytes, such as proline and myo-inositol, and changes in GABA shunt. Such treatments also promoted the shikimate-mediated secondary metabolisms with enhanced biosynthesis of aromatic amino acids. Therefore, salinity caused systems alterations in widespread metabolic networks involving transamination, TCA cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis, shikimate-mediated secondary metabolisms, and the metabolisms of choline, pyrimidine, and purine. These findings provided new insights for the tobacco metabolic adaptation to salinity and demonstrated the NMR-based metabonomics as a powerful approach for understanding the osmotic effects on plant biochemistry

Revealing the Metabonomic Variation of Rosemary Extracts Using ¹H NMR Spectroscopy and Multivariate Data Analysis

The molecular compositions of rosemary (Rosmarinus officinalis L.) extracts and their dependence on extraction solvents, seasons, and drying processes were systematically characterized using NMR spectroscopy and multivariate data analysis. The results showed that the rosemary metabonome was dominated by 33 metabolites including sugars, amino acids, organic acids, polyphenolic acids, and diterpenes, among which quinate, cis-4-glucosyloxycinnamic acid, and 3,4,5-trimethoxyphenylmethanol were found in rosemary for the first time. Compared with water extracts, the 50% aqueous methanol extracts contained higher levels of sucrose, succinate, fumarate, malonate, shikimate, and phenolic acids, but lower levels of fructose, glucose, citrate, and quinate. Chloroform/methanol was an excellent solvent for selective extraction of diterpenes. From February to August, the levels of rosmarinate and quinate increased, whereas the sucrose level decreased. The sun-dried samples contained higher concentrations of rosmarinate, sucrose, and some amino acids but lower concentrations of glucose, fructose, malate, succinate, lactate, and quinate than freeze-dried ones. These findings will fill the gap in the understanding of rosemary composition and its variations

Combined NMR and LC−DAD-MS Analysis Reveals Comprehensive Metabonomic Variations for Three Phenotypic Cultivars of <i>Salvia Miltiorrhiza</i> 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

Understanding the Molecular Dynamics Associated with Polymorphic Transitions of dl-Norvaline with Solid-State NMR Methods

dl-Norvaline (NVA) is an important pharmaceutical intermediate and undergoes two polymorphic transitions between 140 and 300 K. To understand molecular dynamics of NVA accompanied with these transitions, we conducted a comprehensive study on its molecular motions at multiple time scales (10−9-1 s) with various solid-state NMR methods. 13C CPMAS NMR spectra revealed four sets of clearly resolved signals for NVA carbons corresponding to at least three crystal modifications with two polymorphic transitions. Proton and 13C relaxation results showed that, apart from the reorientations of methyl and amino groups, NVA had another relaxation process following the second transition with the activation energy of 16−21 kJ/mol corresponding to the side-chain motions. This was further confirmed with the data from dipolar and chemical shift experiments. No motions were detected at CODEX time scale (ms-s). These results provide essential information for understanding the mechanistic aspects of the polymorphic transitions in aliphatic α-amino acids with linear side-chains