One-dimensional 13C NMR and HPLC-1H NMR techniques for observing carbon-13 and deuterium labelling in biosynthetic studies

For several decades isotope labelling techniques have been the indispensable tools used to unravel pathways of secondary product biosynthesis. NMR spectroscopy, together with mass spectrometry, is the most effective measuring technique used in the analysis of metabolites enriched with stable isotopes. ²H and ¹³C are the NMR-detectable nuclides which have been most frequently employed in plant secondary metabolite synthesis. Examples from the biosynthesis of phenylpropanoids, phenylphenalenones, and glucosinolates are used when discussing some aspects of one-dimensional NMR analysis of metabolites selectively labelled with ²H and ¹³C. Besides direct NMR detection of ¹³C-enriched metabolites, special emphasis is placed on indirect detection of ¹³C and ²H, especially by HPLC-¹H NMR coupling, to analyse the isotopomer pattern of compounds in low concentration. The examples discussed in this paper were obtained from studies with Anigozanthos preissii (root cultures) (Haemodoraceae) and Eruca sativa (Brassicaceae).     

NMR-based metabolomics reveals the metabolite profiles of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> under ferric iron stimulation

Vibrio parahaemolyticus is a halophilic bacterium endemic to coastal areas, and its pathogenicity has caused widespread seafood poisoning. In our previous research, the protein expression of V. parahaemolyticus in Fe³⁺ medium was determined using isobaric tags for relative and absolute quantitation (iTRAQ). Here, nuclear magnetic resonance (NMR) was used to detect changes in the V. parahaemolyticus metabolome. NMR spectra were obtained using methanol-water extracts of intracellular metabolites from V. parahaemolyticus under various culture conditions, and 62 metabolites were identified, including serine, arginine, alanine, ornithine, tryptophan, glutamine, malate, NAD⁺, NADP⁺, oxypurinol, xanthosine, dCTP, uracil, thymine, hypoxanthine, and betaine. Among these, 21 metabolites were up-regulated after the stimulation of the cells by ferric iron, and 9 metabolites were down-regulated. These metabolites are involved in amino acid and protein synthesis, energy metabolism, DNA and RNA synthesis and osmolality. Based on these results, we conclude that Fe³⁺ influences the metabolite profiles of V. parahaemolyticus.     

Nuclear Magnetic Resonance Studies of Poly(3-Hydroxybutyrate) and Polyphosphate Metabolism in Alcaligenes eutrophus

The metabolic pathways of poly(3-hydroxybutyrate) (PHB) and polyphosphate in the microorganism Alcaligenes eutrophus H16 were studied by ¹H, ¹³C, and ³¹P nuclear magnetic resonance (NMR) spectroscopy and by conventional analytical techniques. A. eutrophus cells accumulated two storage polymers of PHB and polyphosphate in the presence of carbon and phosphate sources under aerobic conditions after exhaustion of nitrogen sources. The solid-state cross-polarization/magic-angle spinning ¹³C NMR spectroscopy was used to study the biosynthetic pathways of PHB and other cellular biomass components from ¹³C-labeled acetate. The solid-state ¹³C NMR analysis of lyophilized intact cells grown on [1-¹³C]acetate indicated that the carbonyl carbon of acetate was selectively incorporated both into the carbonyl and methine carbons of PHB and into the carbonyl carbons of proteins. The ³¹P NMR analysis of A. eutrophus cells in suspension showed that the synthesis of intracellular polyphosphate was closely related to the synthesis of PHB. The roles of PHB and polyphosphate in the cells were studied under conditions of carbon, phosphorus, and nitrogen source starvation. Under both aerobic and anaerobic conditions PHB was degraded, whereas little polyphosphate was degraded. The rate of PHB degradation under anaerobic conditions was faster than that under aerobic conditions. Under anaerobic conditions, acetate and 3-hydroxybutyrate were produced as the major extracellular metabolites. The implications of this observation are discussed in connection with the regulation of PHB and polyphosphate metabolism in A. eutrophus.     

13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. ¹³C nuclear magnetic resonance (NMR) analysis of [1-¹³C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by ¹³C NMR by following the simultaneous metabolism of labeled [¹³C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH₂ of succinate and the CH₃ of acetate when the resting cells were metabolizing [1-¹³C]glucose and unlabeled glycogen were precisely quantified by using ¹³C-filtered spin-echo difference ¹H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.     

NMR analysis of plant nitrogen metabolism

The analysis of primary and secondary nitrogen metabolism in plants by nuclear magnetic resonance (NMR) spectroscopy is comprehensively reviewed. NMR is a versatile analytical tool, and the combined use of ¹H, ²H, ¹³C, ¹⁴N and ¹⁵N NMR allows detailed investigation of the acquisition, assimilation and metabolism of nitrogen. The analysis of tissue extracts can be complemented by the in vivo NMR analysis of functioning tissues and cell suspensions, and by the application of solid state NMR techniques. Moreover stable isotope labelling with ²H-, ¹³C- and ¹⁵N-labelled precursors provides direct insight into specific pathways, with the option of both time-course and steady state analysis increasing the potential value of the approach. The scope of the NMR method, and its contribution to studies of plant nitrogen metabolism, are illustrated with a wide range of examples. These include studies of the GS/GOGAT pathway of ammonium assimilation, investigations of the metabolism of glutamate, glycine and other amino acids, and applications to tropane alkaloid metabolism. The continuing development of the NMR technique, together with potential applications in the emerging fields of metabolomics and metabolic flux analysis, leads to the conclusion that NMR will play an increasingly valuable role in the analysis of plant nitrogen metabolism.     

Correlative and quantitative 1H NMR-based metabolomics reveals specific metabolic pathway disturbances in diabetic rats

Type 1 diabetes was induced in Sprague-Dawley rats using streptozotocin. Rat urine samples (8 diabetic and 10 control) were analyzed by ¹H nuclear magnetic resonance (NMR) spectroscopy. The derived metabolites using univariate and multivariate statistical analysis were subjected to correlative analysis. Plasma metabolites were measured by a series of bioassays. A total of 17 urinary metabolites were identified in the ¹H NMR spectra and the loadings plots after principal components analysis. Diabetic rats showed significantly increased levels of glucose (P < 0.00001), alanine (P < 0.0002), lactate (P < 0.05), ethanol (P < 0.05), acetate (P < 0.05), and fumarate (P < 0.05) compared with controls. Plasma assays showed higher amounts of glucose, urea, triglycerides, and thiobarbituric acid-reacting substances in diabetic rats. Striking differences in the Pearson’s correlation of the 17 NMR-detected metabolites were observed between control and diabetic rats. Detailed analysis of the altered metabolite levels and their correlations indicate a significant disturbance in the glucose metabolism and tricarboxylic acid (TCA) cycle and a contribution from gut microbial metabolism. Specific perturbed metabolic pathways include the glucose-alanine and Cori cycles, the acetate switch, and choline metabolism. Detection of the altered metabolic pathways and bacterial metabolites using this correlative and quantitative NMR-based metabolomics approach should help to further the understanding of diabetes-related mechanisms.     

Gender-Specific Metabolomic Profiling of Obesity in Leptin-Deficient ob/ob Mice by 1H NMR Spectroscopy

Despite the numerous metabolic studies on obesity, gender bias in obesity has rarely been investigated. Here, we report the metabolomic analysis of obesity by using leptin-deficient ob/ob mice based on the gender. Metabolomic analyses of urine and serum from ob/ob mice compared with those from C57BL/6J lean mice, based on the ¹H NMR spectroscopy in combination with multivariate statistical analysis, revealed clear metabolic differences between obese and lean mice. We also identified 48 urine and 22 serum metabolites that were statistically significantly altered in obese mice compared to lean controls. These metabolites are involved in amino acid metabolism (leucine, alanine, ariginine, lysine, and methionine), tricarbocylic acid cycle and glucose metabolism (pyruvate, citrate, glycolate, acetoacetate, and acetone), lipid metabolism (cholesterol and carnitine), creatine metabolism (creatine and creatinine), and gut-microbiome-derived metabolism (choline, TMAO, hippurate, p-cresol, isobutyrate, 2-hydroxyisobutyrate, methylamine, and trigonelline). Notably, our metabolomic studies showed distinct gender variations. The obese male mice metabolism was specifically associated with insulin signaling, whereas the obese female mice metabolism was associated with lipid metabolism. Taken together, our study identifies the biomarker signature for obesity in ob/ob mice and provides biochemical insights into the metabolic alteration in obesity based on gender.     

Isolation and Characterization of a Novel Atrazine Metabolite Produced by the Fungus Pleurotus pulmonarius, 2-Chloro-4-Ethylamino-6-(1-Hydroxyisopropyl)Amino-1,3,5-Triazine

The white rot fungus Pleurotus pulmonarius exhibited metabolism of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in liquid culture, producing the dealkylated metabolites desethylatrazine, desisopropylatrazine, and desethyl-desisopropylatrazine. A fourth, unknown metabolite was also produced. It was isolated and was identified as 2-chloro-4-ethylamino-6-(1-hydroxyisopropyl)amino-1,3,5-triazine by gas chromatography-mass spectrometry, Fourier transformed infrared spectroscopy, and ¹H nuclear magnetic resonance analysis. The structure of this metabolite was confirmed by chemical synthesis of the compound and comparison with the fungally produced metabolite.     

Catharinensine,an oxindole alkaloid from Peschiera catharinensis

The structure of catharinensine isolated from Peschiera catharinensis was deduced from a detailed analysis of its ¹H NMR, ¹³C NMR and mass spectra. The synthesis of 17-demethoxyrhynchophylline confirmed its structure, and led us to suggest the configuration allo A for catharinensine. Coronaridine, isovoacangine, heyneanine, 16-epiaffinine, decarbomethoxyvoacamine and conodurine were also isolated.     

Glucose and glycine metabolism in regenerating tobacco protoplasts: followed nondestructively by nuclear magnetic resonance spectroscopy

The metabolic states and the uptake and metabolism of [1-¹³C]glucose, [2-¹³C]glycine, and [¹⁵N]glycine in intact Nicotiana tabacum L. (cv Xanthi) mesophyll protoplasts were measured by ¹³C and ¹⁵N nuclear magnetic resonance spectroscopy. Changes in the concentration of metabolites during the first two days of culture in darkness were followed. Protoplasts isolated in 0.55 molar mannitol medium showed a drop in the concentration of all the intracellular metabolites during the first 28 hours of culture. Uptake of glucose and synthesis of glucose-derived metabolites were observed, indicating activity of glycolysis and the tricarboxylic acid cycle. Addition of glycine caused the accumulation of serine in dark cultured protoplasts, via the photorespiratory pathway. Glutamate dehydrogenase and glutamine synthetase activities in photorespiratory NH₄⁺ assimilation were observed. Glucose uptake and metabolism and cell division were inhibited by 3 millimolar glycine, suggesting that the accumulating serine or the release of ammonia during serine synthesis had toxic effects in this system.     

Interactions between Carbon and Nitrogen Metabolism in Fibrobacter succinogenes S85: a 1H and 13C Nuclear Magnetic Resonance and Enzymatic Study

The effect of the presence of ammonia on [1-¹³C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by ¹³C and ¹H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The ¹³C enrichment of succinate and acetate was precisely quantified by ¹³C-filtered spin-echo difference ¹H-NMR spectroscopy. The presence of ammonia did not modify the ¹³C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the ¹³C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by ¹³C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and ¹H NMR. When cells were incubated in vivo with [1-¹³C]glucose, only ¹³C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.     

Simultaneous tracers and a unified model of positional and mass isotopomers for quantification of metabolic flux in liver

Computational models based on the metabolism of stable isotope tracers can yield valuable insight into the metabolic basis of disease. The complexity of these models is limited by the number of tracers and the ability to characterize tracer labeling in downstream metabolites. NMR spectroscopy is ideal for multiple tracer experiments since it precisely detects the position of tracer nuclei in molecules, but it lacks sensitivity for detecting low-concentration metabolites. GC-MS detects stable isotope mass enrichment in low-concentration metabolites, but lacks nuclei and positional specificity. We performed liver perfusions and in vivo infusions of ²H and ¹³C tracers, yielding complex glucose isotopomers that were assigned by NMR and fit to a newly developed metabolic model. Fluxes regressed from ²H and ¹³C NMR positional isotopomer enrichments served to validate GC-MS-based flux estimates obtained from the same experimental samples. NMR-derived fluxes were largely recapitulated by modeling the mass isotopomer distributions of six glucose fragment ions measured by GC-MS. Modest differences related to limited fragmentation coverage of glucose C1–C3 were identified, but fluxes such as gluconeogenesis, glycogenolysis, cataplerosis and TCA cycle flux were tightly correlated between the methods. Most importantly, modeling of GC-MS data could assign fluxes in primary mouse hepatocytes, an experiment that is impractical by ²H or ¹³C NMR.     

13C and31P NMR spectroscopic studies on glucose metabolism inTribolium confusum

Nuclear magnetic resonance (NMR) technology was applied to study the glucose metabolism inTribolium confusum (Coleoptera).¹³C signals of D-(1-¹³C)glucose eaten by beetles were clearly detected in such metabolites of the glucose metabolism as glycogen, trehalose, triacylglycerol, alanine and proline by¹³C-NMR. After glucose feeding the³¹P-NMR spectra ofT. confusum showed the signal intensity increases in arginine-phosphate, sugar-phosphate and uridine diphosphoglucose. The results demonstrated the potential of NMR analysis for the study of glucose metabolism inT. confusum.     

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.¹ A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer ''epicuticular'' layer as well as the blend consisting of the cutin biopolymer and ''intracuticular'' waxes.² Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Schäffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.^3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) ¹³C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS ¹³C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH₂O) chemical moieties.Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state ¹³C NMR reveals differences in the relative numbers of CHO and CH₂O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-μm length scale.     

High-resolution magic angle spinning nuclear magnetic resonance analysis of metabolic changes in melanoma cells after induction of melanogenesis

Melanin synthesis affects melanoma behavior and tumor responsiveness to therapy; therefore, we investigated metabolic changes in melanoma cells after induction of melanogenesis. Amelanotic and melanotic melanoma cells were labeled with ¹³C precursors and changes in their metabolism was analyzed by high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). HRMAS NMR demonstrated clear differences in the pattern of metabolic intermediates between amelanotic and melanotic cells. Although the exact nature of the metabolites requires further investigations, our comparative studies clearly show that induction of melanogenesis is associated with changes of glucose and sodium acetate metabolism, demonstrating HRMAS NMR as a powerful and noninvasive technique to investigate such process.     

Schistosoma mansoni: utilization of exogenous metabolites by eggs in vitro

Schistosoma mansoni eggs were isolated from the livers of mice infected for 8 wk and were purified by a series of settling and sieving procedures. Aliquots of eggs were suspended in saline with added Eagle's minimal essential medium supplemented with NaHCO₃, glutamine, penicillin, and streptomycin to which a variety of radioisotope labelled metabolites were added. The uptake and utilization of tritiated thymidine demonstrated a high rate of DNA synthesis, particularly in the more immature eggs studied. RNA synthesis as shown with tritiated uridine was also significant. Large amounts of ¹⁴C-labelled isoleucine and arginine were incorporated into protein. Little glycolytic activity was demonstrated on prolonged incubation with ¹⁴C-labelled glucose. A high rate of catabolism of amino acids to CO₂ was observed, as was a very high rate of acetate metabolism. Degradation of radiolabelled glutamate after incubation of eggs with ¹⁴C acetate revealed labelling consistent with metabolism via the Krebs cycle. Thus, Schistosoma mansoni eggs utilize a wide variety of exogenous metabolites. They show active DNA and RNA metabolism, incorporation of amino acids into protein, and intermediary metabolism characterized by a low rate of glycolysis and an active Krebs cycle.     

Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways

Background

Metabolic phenotyping has become an important ‘bird''s-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems.

Methodology/Principal Findings

The analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 ¹H and ¹³C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each ¹³C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient ¹³C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts.

Conclusions/Significance

Overall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of ¹³C atoms of given metabolites on development-dependent changes in the 56 identified ¹³C-HSQC signals, we have determined the changes in metabolic networks that are associated with energy and nitrogen metabolism.     

3-Chlordivaricatsäure,ein neues depsid aus Thelomma-arten

The structure of 3-chlorodivaricatic acid, a new depside from the lichen Thelomma mammosum was elucidated by ¹H NMR, ¹³C NMR, mass and UV spectroscopy. The synthesis of 5-chlorodivaricatic acid is described.     

Neurochemical Changes in the Rat Occipital Cortex and Hippocampus after Repetitive and Profound Hypoglycemia During the Neonatal Period: An Ex Vivo 1H Magnetic Resonance Spectroscopy Study

The brain of a human neonate is more vulnerable to hypoglycemia than that of pediatric and adult patients. Repetitive and profound hypoglycemia during the neonatal period (RPHN) causes brain damage and leads to severe neurologic sequelae. Ex vivo high-resolution ¹H nuclear magnetic resonance (NMR) spectroscopy was carried out in the present study to detect metabolite alterations in newborn and adolescent rats and investigate the effects of RPHN on their occipital cortex and hippocampus. Results showed that RPHN induces significant changes in a number of cerebral metabolites, and such changes are region-specific. Among the 16 metabolites detected by ex vivo ¹H NMR, RPHN significantly increased the levels of creatine, glutamate, glutamine, γ-aminobutyric acid, and aspartate, as well as other metabolites, including succine, taurine, and myo-inositol, in the occipital cortex of neonatal rats compared with the control. By contrast, changes in these neurochemicals were not significant in the hippocampus of neonatal rats. When the rats had developed into adolescence, the changes above were maintained and the levels of other metabolites, including lactate, N-acetyl aspartate, alanine, choline, glycine, acetate, and ascorbate, increased in the occipital cortex. By contrast, most of these metabolites were reduced in the hippocampus. These metabolic changes suggest that complementary mechanisms exist between these two brain areas. RPHN appears to affect occipital cortex and hippocampal activities, neurotransmitter transition, energy metabolism, and other metabolic equilibria in newborn rats; these effects are further aggravated when the newborn rats develop into adolescence. Changes in the metabolism of neurotransmitter system may be an adaptive measure of the central nervous system in response to RPHN.