An intracellular metabolic signature as a potential donor-independent marker of the osteogenic differentiation of adipose tissue mesenchymal stem cells

This paper describes an untargeted NMR metabolomics study to identify potential intracellular donor-dependent and donor-independent metabolic markers of proliferation and osteogenic differentiation of human adipose mesenchymal stem cells (hAMSCs). The hAMSCs of two donors with distinct proliferating/osteogenic characteristics were fully characterized regarding their polar endometabolome during proliferation and osteogenesis. An 18-metabolites signature (including changes in alanine, aspartate, proline, tyrosine, ATP, and ADP, among others) was suggested to be potentially descriptive of cell proliferation, independently of the donor. In addition, a set of 11 metabolites was proposed to compose a possible donor-independent signature of osteogenesis, mostly involving changes in taurine, glutathione, methylguanidine, adenosine, inosine, uridine, and creatine/phosphocreatine, choline/phosphocholine and ethanolamine/phosphocholine ratios. The proposed signatures were validated for a third donor, although they require further validation in a larger donor cohort. We believe that this proof of concept paves the way to exploit metabolic markers to monitor (and potentially predict) cell proliferation and the osteogenic ability of different donors.publishe

Metabolomic applications in stem cell research: a review

This review describes the use of metabolomics to study stem cell (SC) characteristics and function, excluding SCs in cancer research, suited to a fully dedicated text. The interest in employing metabolomics in SC research has consistently grown and emphasis is, here, given to developments reported in the past five years. This text informs on the existing methodologies and their complementarity regarding the information provided, comprising untargeted/targeted approaches, which couple mass spectrometry or nuclear magnetic resonance spectroscopy with multivariate analysis (and, in some cases, pathway analysis and integration with other omics), and more specific analytical approaches, namely isotope tracing to highlight particular metabolic pathways, or in tandem microscopic strategies to pinpoint characteristics within a single cell. The bulk of this review covers the existing applications in various aspects of mesenchymal SC behavior, followed by pluripotent and neural SCs, with a few reports addressing other SC types. Some of the central ideas investigated comprise the metabolic/biological impacts of different tissue/donor sources and differentiation conditions, including the importance of considering 3D culture environments, mechanical cues and/or media enrichment to guide differentiation into specific lineages. Metabolomic analysis has considered cell endometabolomes and exometabolomes (fingerprinting and footprinting, respectively), having measured both lipid species and polar metabolites involved in a variety of metabolic pathways. This review clearly demonstrates the current enticing promise of metabolomics in significantly contributing towards a deeper knowledge on SC behavior, and the discovery of new biomarkers of SC function with potential translation to in vivo clinical practice.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for co-funding the BIOIMPLANT project (PTDC/BTM-ORG/28835/2017) through the COMPETE2020 program and European Union fund FEDER (POCI-01–0145- FEDER-028835). CSHJ and KR are grateful to the same project for funding their contracts with the University of Aveiro. DSB acknowl- edges the Sociedade Portuguesa de Química and FCT for her PhD grant SFRH/BD/150655/2020. AMG acknowledges the CICECO-Aveiro Institute of Materials project, with references UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Part- nership Agreement. The NMR spectrometer used in this work is part of the National NMR Network (PTNMR) and, partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).publishe

Non-statistical isotope fractionation as a novel ``retro-biosynthetic'' approach to understanding alkaloid metabolic pathways

International Symposium on Chromatography of Natural Products (ISCNP), Lublin, POLAND, 2016During the biosynthesis of natural products, isotope fractionation occurs due to the selectivity of enzymes for the heavier or lighter isotopomers. As only some of the positions in the molecule are implicated in a given reaction mechanism, position-specific fractionation occurs. Thus, the position-specific C-13/C-12 ratios in these compounds can be related to their known precursors and to the known isotope effects of enzymes involved in their biosynthesis, or similar reaction mechanisms. This can be accessed by isotope ratio monitoring NMR spectrometry. In this short review, how isotope fractionation occurs and when it is manifest is described. Then, the way that C-13 NMR spectrometry has been applied to study certain aspects of the biosynthesis of the solanaceous alkaloids S-(-)-nicotine and tropine is outlined. Notably, it is shown how similar isotope fractionation is found in the steps of the pathway to the common intermediate, N-methyl-Delta(1)-pyrrolinium, but that in the moieties derived from different origins no such similarity is found, the isotopic composition of these atoms reflecting their specific metabolic ancestry. In a second example, tramadol, it is shown how this technique can be used in retro-biosynthesis to give direction as to what precursors and pathway intermediates are probable. It is shown how the observed fractionation in the site-specific C-13/C-12 ratios can be effectively explained by known metabolism and the properties of enzymes proposed for the pathway. Furthermore, it can give indications of possible mechanisms of those enzymes that are as yet to be described for a number of key steps. (C) 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved

Heat Waves Change Plant Carbon Allocation Among Primary and Secondary Metabolism Altering CO Assimilation, Respiration, and VOC Emissions

Processes controlling plant carbon allocation among primary and secondary metabolism, i.e., carbon assimilation, respiration, and VOC synthesis are still poorly constrained, particularly regarding their response to stress. To investigate these processes, we simulated a 10-day 38°C heat wave, analysing real-time carbon allocation into primary and secondary metabolism in the Mediterranean shrub Halimium halimifolium L. We traced position-specific 13 C-labeled pyruvate into daytime VOC and CO emissions and during light-dark transition. Net CO assimilation strongly declined under heat, due to three-fold higher respiration rates. Interestingly, day respiration also increased two-fold. Decarboxylation of the C1-atom of pyruvate was the main process driving daytime CO release, whereas the C2-moiety was not decarboxylated in the TCA cycle. Heat induced high emissions of methanol, methyl acetate, acetaldehyde as well as mono- and sesquiterpenes, particularly during the first two days. After 10-days of heat a substantial proportion of 13 C-labeled pyruvate was allocated into de novo synthesis of VOCs. Thus, during extreme heat waves high respiratory losses and reduced assimilation can shift plants into a negative carbon balance. Still, plants enhanced their investment into de novo VOC synthesis despite associated metabolic CO losses. We conclude that heat stress re-directed the proportional flux of key metabolites into pathways of VOC biosynthesis most likely at the expense of reactions of plant primary metabolism, which might highlight their importance for stress protection

Non-statistical C-13 Fractionation Distinguishes Co-incident and Divergent Steps in the Biosynthesis of the Alkaloids Nicotine and Tropine

During the biosynthesis of natural products, isotopic fractionation occurs due to the selectivity of enzymes for the heavier or lighter isotopomers. As only some of the positions in the molecule are implicated in a given reaction mechanism, position-specific fractionation occurs, leading to a non-statistical distribution of isotopes. This can be accessed by isotope ratio monitoring C-13 NMR spectrometry. The solanaceous alkaloids S-(-)-nicotine and hyoscyamine (atropine) are related in having a common intermediate, but downstream enzymatic steps diverge, providing a relevant test case to: (a) elucidate the isotopic affiliation between carbon atoms in the alkaloids and those in the precursors; (b) obtain information about the kinetic isotope effects of as yet undescribed enzymes, thus to make predictions as to their possible mechanism(s). We show that the position-specific C-13/C-12 ratios in the different moieties of these compounds can satisfactorily be related to their known precursors and to the known kinetic isotope effects of enzymes involved in their biosynthesis, or to similar reaction mechanisms. Thus, the pathway to the common intermediate, N-methyl-Delta(1)-pyrrolinium, is seen to introduce similar isotope distribution patterns in the two alkaloids independent of plant species, whereas the remaining atoms of each target compound, which are of different origins, reflect their specific metabolic ancestry. We further demonstrate that the measured 13C distribution pattern can be used to deduce aspects of the reaction mechanism of enzymes still to be identified

Insights into the role of methionine synthase in the universal C-13 depletion in O- and N-methyl groups of natural products

International audienceMany O-methyl and N-methyl groups in natural products are depleted in C-13 relative to the rest of the molecule. These methyl groups are derived from the C-1 tetrahydrofolate pool via L-methionine, the principle donor of methyl units. Depletion could occur at a number of steps in the pathway. We have tested the hypothesis that methionine biosynthesis is implicated in this depletion by using a combined experimental and theoretical approach. By using isotope ratio monitoring C-13 NMR spectrometry to measure the position-specific distribution of C-13 within L-methionine of natural origin, it is shown that the S-methyl group is depleted in C-13 by similar to 20%o relative to the other positions in the molecule. In parallel, we have conducted a basic theoretical analysis of the reaction pathway of methionine synthase to assess whether the enzyme cobalamin-independent L-methionine synthase (EC 2.1.1.14) that catalyzes the synthesis of L-methionine from 5-methyl-tetrahydrofolate and homocysteine plays a role in causing this depletion. Calculation predicts a strong normal C-13 kinetic isotope effect (1.087) associated with this enzyme. Hence, depletion in C-13 in the S-methyl of L-methionine during biosynthesis can be identified as an important factor contributing to the general depletion seen in many O methyl and N-methyl groups of natural products

Difficulties in Differentiating Natural from Synthetic Alkaloids by Isotope Ratio Monitoring using (13) C Nuclear Magnetic Resonance Spectrometry

International audienceWithin the food and pharmaceutical industries, there is an increasing legislative requirement for the accurate labeling of the products origin. A key feature of this is to indicate whether the product is of natural or synthetic origin. With reference to this context, we have investigated three alkaloids commonly exploited for human use: nicotine, atropine, and caffeine. We have measured by (13) C nuclear magnetic resonance spectrometry the position-specific distribution of (13) C at natural abundance within several samples of each of these target molecules. This technique is well suited to distinguishing between origins, as the distribution of the (13) C isotope reflects the primary source of the carbon atoms and the process by which the molecule was (bio)synthesized. Our findings indicate that labeling can be misleading, especially in relation to a supplied compound being labeled as synthetic even though its (13) C profile indicates a natural origin

Human baby hair amino acid natural abundance N-15-isotope values are not related to the N-15-isotope values of amino acids in mother's breast-milk protein (vol 45, pg 1365, 2017)

International audienc