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.

Heat Waves Change Plant Carbon Allocation Among Primary and Secondary Metabolism Altering CO2 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 13C-labeled pyruvate into daytime VOC and CO2 emissions and during light-dark transition. Net CO2 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 CO2 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 13C-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 CO2 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.

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

Within the food and pharmaceutical industries, there is an increasing legislative requirement for the accurate labeling of the product's 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 13C nuclear magnetic resonance spectrometry the position-specific distribution of 13C 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 13C 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 13C profile indicates a natural origin.

Correction to: Human baby hair amino acid natural abundance 15N-isotope values are not related to the 15N-isotope values of amino acids in mother's breast-milk protein.

In the article (Romek et al. 2013) we reported the values of δ15N (‰) and δ13C (‰) obtained by.

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

Many O-methyl and N-methyl groups in natural products are depleted in 13C 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 13C NMR spectrometry to measure the position-specific distribution of 13C within l-methionine of natural origin, it is shown that the S-methyl group is depleted in 13C by ∼20‰ 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 13C kinetic isotope effect (1.087) associated with this enzyme. Hence, depletion in 13C 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.

Non-statistical 13C 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 (13)C 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 (13)C/(12)C 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-Δ(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 (13)C distribution pattern can be used to deduce aspects of the reaction mechanism of enzymes still to be identified.

A retro-biosynthetic approach to the prediction of biosynthetic pathways from position-specific isotope analysis as shown for tramadol.

Tramadol, previously only known as a synthetic analgesic, has now been found in the bark and wood of roots of the African medicinal tree Nauclea latifolia. At present, no direct evidence is available as to the biosynthetic pathway of its unusual skeleton. To provide guidance as to possible biosynthetic precursors, we have adopted a novel approach of retro-biosynthesis based on the position-specific distribution of isotopes in the extracted compound. Relatively recent developments in isotope ratio monitoring by (13)C NMR spectrometry make possible the measurement of the nonstatistical position-specific natural abundance distribution of (13)C (δ(13)Ci) within the molecule with better than 1‰ precision. Very substantial variation in the (13)C positional distribution is found: between δ(13)Ci = -11 and -53‰. Distribution is not random and it is argued that the pattern observed can substantially be interpreted in relation to known causes of isotope fractionation in natural products. Thus, a plausible biosynthetic scheme based on sound biosynthetic principals of precursor-substrate relationships can be proposed. In addition, data obtained from the (18)O/(16)O ratios in the oxygen atoms of the compound add support to the deductions made from the carbon isotope analysis. This paper shows how the use of (13)C NMR at natural abundance can help with proposing a biosynthetic route to compounds newly found in nature or those difficult to tackle by conventional means.

Human baby hair amino acid natural abundance 15N-isotope values are not related to the 15N-isotope values of amino acids in mother's breast milk protein.

Since exclusively breast-suckled infants obtain their nutrient only from their mother's milk, it might be anticipated that a correlation will exist between the (15)N/(14)N isotope ratios of amino acids of protein of young infants and those supplied by their mother. The work presented here aimed to determine whether amino nitrogen transfer from human milk to infant hair protein synthesized within the first month of life conserves the maternal isotopic signature or whether post-ingestion fractionation dominates the nitrogen isotope spectrum. The study was conducted at 1 month post-birth on 100 mother-infant pairs. Isotope ratios (15)N/(14)N and (13)C/(12)C were measured using isotope ratio measurement by Mass Spectrometry (irm-MS) for whole maternal milk, and infant hair and (15)N/(14)N ratios were also measured by GC-irm-MS for the N-pivaloyl-O-isopropyl esters of amino acids obtained from the hydrolysis of milk and hair proteins. The δ(15)N and δ(13)C (‰) were found to be significantly higher in infant hair than in breast milk (δ(15)N, P < 0.001; δ(13)C, P < 0.001). Furthermore, the δ(15)N (‰) of individual amino acids in infant hair was also significantly higher than that in maternal milk (P < 0.001). By calculation, the observed shift in isotope ratio was shown not to be accounted for by the amino acid composition of hair and milk proteins, indicating that it is not simply due to differences in the composition in the proteins present. Rather, it would appear that each pool-mother and infant-turns over independently, and that fractionation in infant N-metabolism even in the first month of life dominates over the nutrient N-content.