Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry

The Aconitum alkaloids aconitine, mesaconitine, and hypaconitine are the main toxic components in a commonly used traditional Chinese herbal medicine Fu Zi. To provide guidelines for the safe use of this medicine, metabolic changes in Wistar rats caused by these compounds were investigated by means of integrated analysis of two metabonomic approaches: ¹H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF–MS). Rats were given a single dose of aconitine, mesaconitine, hypaconitine, or vehicle. The largest metabolic changes were observed 6 h after treatment. Every group receiving a dose had higher urine concentrations of glucose, acetate, dimethylglycine, succinate, and alanine and had lower concentrations of creatinine, citrate, 2-oxoglutarate, N-acetylated metabolites, and trimethylamine-N-oxide (TMAO) than did the control group. These results may reflect the perturbation of renal tubular function within the first 24 h after treatment. The results also revealed a larger perturbation of metabolic profiles in the aconitine group than in the mesaconitine and hypaconitine groups, illustrating how these alkaloids exhibit different toxicities. An analysis of plasma samples collected 7 days postdose showed that there were higher levels of lactate, alanine, and lipids along with lower levels of glucose, β-hydroxybutyrate, and creatine in the plasma of the aconitine and mesaconitine groups than there were in the control and hypaconitine groups. The GC/TOF–MS data from the plasma samples showed that the number of metabolites, with significant changes or with a tendency to change, in the aconitine and mesaconitine groups were dissimilar, suggesting a possible difference in the acute toxicity mechanisms of these alkaloids.     

Identification and measurement of methylamines in elasmobranch tissues using proton nuclear magnetic resonance (1H-NMR) spectroscopy

Methylamines are frequently present in high concentrations in biological samples, but their separation and quantification are difficult. Data presented show that methylamines commonly occurring in biological material can be uniquely identified and quantified by proton nuclear magnetic resonance spectroscopy by recording spectra at both neutral and acid pH. Use of a high sensitivity probe permits this analysis even in the presence of high water concentrations, allowing accurate quantification with minimum preparative technique. The method was tested on tissues of the dogfish. Trimethylamine oxide was found in amounts ranging from 42 mmol kg⁻¹ fresh weight in liver, up to 115 mmol kg⁻¹ fresh weight in heart. Betaine was found to range from 10 mmol kg⁻¹ fresh weight in liver to 49 mmol kg⁻¹ fresh weight in brain. Creatine was not found in heart or liver, but was present in body wall muscle and in brain. Further analysis using high-performance liquid chromatography allowed determination of urea/methylamine ratios, which ranged from 1.9 in liver to 3.7 in body wall muscle. Accepted: 7 October 1997     

Measurement of fluxes through the pentose phosphate pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy

Erythrocytes from individuals with sickle cell anemia have previously been shown to have increased levels of intracellular oxidants and increased oxidative damage. Oxidative damage has been implicated in the events leading to the painful crises and hemolytic anemia found in sickle cell anemia. Since the pentose phosphate pathway (PPP) is an important source of reducing capacity in erythrocytes, we have investigated the fluxes through the PPP in normal and sickle cell erythrocytes using [2-¹³C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that sickle cell erythrocytes have a flux through the PPP of 0.13±0.02 μmol/h per ml erythrocytes that is comparable to that in normal erythrocytes, 0.21±0.02 μmol/h per ml erythrocytes. However, when stimulated with methylene blue, sickle cell erythrocytes show a decreased response, 0.59±0.10 μmol/h per ml erythrocytes, compared to normal erythrocytes, 1.64±0.10 μmol/h per ml erythrocytes. When homogeneous populations of sickle cell erythrocytes are isolated by density gradient centrifugation, the rate of flux through the PPP in methylene blue-stimulated sickle cell erythrocytes, 1.16±0.16 μmol/h per ml erythrocytes, approaches that in methylene blue-stimulated normal erythrocytes. In addition, by analyzing the dose response to methylene blue, we have found that the decreased stimulation of the PPP by methylene blue in heterogeneous populations of sickle cell erythrocytes is a failure of methylene blue to simulate the PPP rather than a deficiency in the PPP in sickle cell erythrocytes.     

Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry

The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Multinuclear magnetic resonance, electrospray ionization-mass spectroscopy, and parametric method 5 studies of a new derivative of gossypol with 2-thiophenecarbohydrazide as well as its complexes with LI+, Na+, K+, RB+, and Cs+ cations

A new derivative of racemic gossypol with 2-thiophenecarbohydrazide (GHHT) and its complexes with monovalent cations have been synthesized and studied by electrospray ionization-mass spectroscopy (ESI-MS), multinuclear nuclear magnetic resonance (NMR), as well as by the Parametric Method 5 (PM5) methods. It is demonstrated that GHHT forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by three types of intramolecular hydrogen bonds. The spectroscopic methods have provided clear evidence that GHHT and its complexes exist in the DMSO-d6 solution in the N-imine-N-imine tautomeric forms. The structures of the GHHT and its complexes with Li+, Na+, K+, Rb+, and Cs+ cations are visualized and discussed in detail.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress

Breast cancer cells that have undergone partial epithelial–mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H₂S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.