Alexis Gilbert

Accurate Quantitative Isotopic 13C NMR Spectroscopy for the Determination of the Intramolecular Distribution of 13C in Glucose at Natural Abundance

In order to understand (13)C isotope distributions in glucose and its metabolites, it is necessary to measure the internal (13)C distribution at natural abundance. These data, however, are not directly accessible, even by quantitative isotopic (13)C NMR spectrometry, due to anomerization at the C-1 position. A strategy has been developed that overcomes this difficulty by converting glucose via a three-step synthesis into 3,5,6-triacetyl-1,2-O-isopropylidene-alpha-D-glucofuranose (TAMAGF). This compound provides a satisfactory molecular probe to measure the site-specific (13)C/(12)C ratios in glucose by (13)C NMR. It is shown that the isotopic (13)C NMR signal gives sufficient precision (repeatability standard deviation < or = 0.8 per thousand) for routine use for the determination of the (13)C abundance of each carbon atom position in glucose. Thus, it can be seen that the internal (13)C distribution of glucose biosynthesized by the C3 and C4 metabolic pathways differs markedly. Furthermore, the method is suitable for determining the isotope ratio in the glucose moiety of sucrose and, possibly, in free fructose and the fructose moiety of sucrose.

Intramolecular 13C pattern in hexoses from autotrophic and heterotrophic C3 plant tissues

The stable carbon isotope 13C is used as a universal tracer in plant eco-physiology and studies of carbon exchange between vegetation and atmosphere. Photosynthesis fractionates against 13CO2 so that source sugars (photosynthates) are on average 13C depleted by 20‰ compared with atmospheric CO2. The carbon isotope distribution within sugars has been shown to be heterogeneous, with relatively 13C-enriched and 13C-depleted C-atom positions. The 13C pattern within sugars is the cornerstone of 13C distribution in plants, because all metabolites inherit the 13C abundance in their specific precursor C-atom positions. However, the intramolecular isotope pattern in source leaf glucose and the isotope fractionation associated with key enzymes involved in sugar interconversions are currently unknown. To gain insight into these, we have analyzed the intramolecular isotope composition in source leaf transient starch, grain storage starch, and root storage sucrose and measured the site-specific isotope fractionation associated with the invertase (EC 3.2.1.26) and glucose isomerase (EC 5.3.1.5) reactions. When these data are integrated into a simple steady-state model of plant isotopic fluxes, the enzyme-dependent fractionations satisfactorily predict the observed intramolecular patterns. These results demonstrate that glucose and sucrose metabolism is the primary determinant of the 13C abundance in source and sink tissue and is, therefore, of fundamental importance to the interpretation of plant isotopic signals.

The intramolecular 13C‐distribution in ethanol reveals the influence of the CO2‐fixation pathway and environmental conditions on the site‐specific 13C variation in glucose

Efforts to understand the cause of ¹²C versus ¹³C isotope fractionation in plants during photosynthesis and post-photosynthetic metabolism are frustrated by the lack of data on the intramolecular ¹³C-distribution in metabolites and its variation with environmental conditions. We have exploited isotopic carbon-13 nuclear magnetic resonance (¹³C NMR) spectrometry to measure the positional isotope composition (δ¹³C(i) , ‰) in ethanol samples from different origins: European wines, liquors and sugars from C₃, C₄ and crassulacean acid metabolism (CAM) plants. In C₃-ethanol samples, the methylene group was always ¹³C-enriched (∼2‰) relative to the methyl group. In wines, this pattern was correlated with both air temperature and δ(18)O of wine water, indicating that water vapour deficit may be a critical defining factor. Furthermore, in C₄-ethanol, the reverse relationship was observed (methylene-C relatively ¹³C-depleted), supporting the concept that photorespiration is the key metabolic process leading to the ¹³C distribution in C₃-ethanol. By contrast, in CAM-ethanol, the isotopic pattern was similar to but stronger than C₃-ethanol, with a relative ¹³C-enrichment in the methylene-C of up to 13‰. Plausible causes of this ¹³C-pattern are briefly discussed. As the intramolecular δ¹³C(i) -values in ethanol reflect that in source glucose, our data point out the crucial impact on the ratio of metabolic pathways sustaining glucose synthesis.

A 13C NMR spectrometric method for the determination of intramolecular δ13C values in fructose from plant sucrose samples

Recent developments in (13) C NMR spectrometry have allowed the determination of intramolecular (13) C/(12) C ratios with high precision. However, the analysis of carbohydrates requires their derivatization to constrain the anomeric carbon. Fructose has proved to be particularly problematic because of a byproduct occurring during derivatization and the complexity of the NMR spectrum of the derivative. Here, we describe a method to determine the intramolecular (13) C/(12) C ratios in fructose by (13) C NMR analysis of the acetyl-isopropylidene derivative. We have applied this method to measure the intramolecular (13) C/(12) C distribution in the fructosyl moiety of sucrose and have compared this with that in the glucosyl moiety. Three prominent features stand out. First, in sucrose from both C(3) and C(4) plants, the C-1 and C-2 positions of the glucosyl and fructosyl moieties are markedly different. Second, these positions in C(3) and C(4) plants show a similar profile. Third, the glucosyl and fructosyl moieties of sucrose from Crassulacean acid metabolism (CAM) metabolism have a different profile. These contrasting values can be interpreted as a result of the isotopic selectivity of enzymes that break or make covalent bonds in glucose metabolism, whereas the distinctive (13) C pattern in CAM sucrose probably indicates a substantial contribution of gluconeogenesis to glucose synthesis.

Improved Characterization of the Botanical Origin of Sugar by Carbon-13 SNIF-NMR Applied to Ethanol

Until now, no analytical method, not even isotopic ones, had been able to differentiate between sugars coming from C4-metabolism plants (cane, maize, etc.) and some crassulacean acid metabolism plants (e.g., pineapple, agave) because in both cases the isotope distributions of the overall carbon-13/carbon-12 and site-specific deuterium/hydrogen isotope ratios are very similar. Following recent advances in the field of quantitative isotopic carbon-13 NMR measurements, a procedure for the analysis of the positional carbon-13/carbon-12 isotope ratios of ethanol derived from the sugars of pineapples and agave using the site-specific natural isotopic fractionation-nuclear magnetic resonance (SNIF-NMR) method is presented. It is shown that reproducible results can be obtained when appropriate analytical conditions are used. When applied to pineapple juice, this new method demonstrates a unique ability to detect cane and maize sugar, which are major potential adulterants, with a detection limit in the order of 15% of the total sugars, which provides an efficient mean of controlling the authenticity of juices made from this specific fruit. When applied to tequila products, this new method demonstrates a unique ability to unambiguously differentiate authentic 100% agave tequila, as well as misto tequila (made from at least 51% agave), from products made from a larger proportion of cane or maize sugar and therefore not complying with the legal definition of tequila.

Site-specific 13C content by quantitative isotopic 13C nuclear magnetic resonance spectrometry: a pilot inter-laboratory study.

Isotopic (13)C NMR spectrometry, which is able to measure intra-molecular (13)C composition, is of emerging demand because of the new information provided by the (13)C site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic (13)C NMR as a routine tool. This paper describes the first collaborative study of intra-molecular (13)C composition by NMR. The main goals of the ring test were to establish intra- and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic (13)C NMR was then assessed on vanillin from three different origins associated with specific δ (13)Ci profiles. The standard deviation was, on average, between 0.9 and 1.2‰ for intra-variability. The highest standard deviation for inter-variability was 2.1‰. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of δ (13)Ci in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results.

Biochemical and physiological determinants of intramolecular isotope patterns in sucrose from C3, C4 and CAM plants accessed by isotopic 13C NMR spectrometry: a viewpoint

This paper discusses the biochemical and physiological factors underlying the site-specific, non-random distribution of ¹³C/¹²C isotope ratios within plant metabolites, which can be determined by isotopic ¹³C NMR spectrometry. It focuses on the key metabolite glucose and on enzyme activities and physiological processes that are responsible for the carbon isotope patterns in glucose from different biological origins. It further considers how intramolecular ¹³C/¹²C isotope ratios in glucose can be exploited to understand fundamental aspects of plant biological chemistry, how these are related to environmental parameters and how these influence metabolites beyond central sugar metabolism. It does not purport to be an extensive overview of intramolecular isotopic patterns. Rather, the aim is to show how a full understanding of ¹³C/¹²C fractionations occurring during plant metabolism can only be possible once the factors that define intramolecular isotope values are better delineated.

Comparison of IRMS and NMR spectrometry for the determination of intramolecular 13C isotope composition: Application to ethanol

Isotopic (13)C NMR is a relatively recent technique which allows the determination of intramolecular (13)C isotope composition at natural abundance. It has been used in various scientific fields such as authentication, counterfeiting or plant metabolism. Although its precision has already been evaluated, the determination of its trueness remains still challenging. To deal with that issue, a comparison with another normalized technique must be achieved. In this work, we compare the intramolecular (13)C isotope distribution of ethanol from different origins obtained using both Isotope Ratio Mass Spectrometry (IRMS) and Nuclear Magnetic Resonance (NMR) spectrometry techniques. The IRMS approach consists of the oxidation of ethanol to acetic acid followed by the degradation of the latter for the analysis of each fragments formed. We show here that the oxidation of ethanol to acetic acid does not bring any significant error on the determination of the site-specific δ(13)C (δ(13)C(i)) of ethanol using the IRMS approach. The difference between the data obtained for 16 samples from different origins using IRMS and NMR approaches is not statistically significant and remains below 0.3‰. These results are encouraging for the future studies using isotopic NMR, especially in combination with the IRMS approach.

Position-Specific Isotope Analysis of Xanthines: A 13C Nuclear Magnetic Resonance Method to Determine the 13C Intramolecular Composition at Natural Abundance

The natural xanthines caffeine, theobromine, and theophylline are of major commercial importance as flavor constituents in coffee, cocoa, tea, and a number of other beverages. However, their exploitation for authenticity, a requirement in these commodities that have a large origin-based price-range, by the standard method of isotope ratio monitoring by mass spectrometry (irm-MS) is limited. We have now developed a methodology that overcomes this deficit that exploits the power of isotopic quantitative (13)C nuclear magnetic resonance (NMR) spectrometry combined with chemical modification of the xanthines to enable the determination of positional intramolecular (13)C/(12)C ratios (δ(13)Ci) with high precision. However, only caffeine is amenable to analysis: theobromine and theophylline present substantial difficulties due to their poor solubility. However, their N-methylation to caffeine makes spectral acquisition feasible. The method is confirmed as robust, with good repeatability of the δ(13)Ci values in caffeine appropriate for isotope fractionation measurements at natural abundance. It is shown that there is negligible isotope fractionation during the chemical N-methylation procedure. Thus, the method preserves the original positional δ(13)Ci values. The method has been applied to measure the position-specific variation of the (13)C/(12)C distribution in caffeine. Not only is a clear difference between caffeine isolated from different sources observed, but theobromine from cocoa is found to show a (13)C pattern distinct from that of caffeine.

Accurate method for the determination of intramolecular 13C isotope composition of ethanol from aqueous solutions.

A new method, combining headspace solid phase microextraction (HS-SPME) with an online pyrolysis system coupled with isotope ratio mass spectrometry (IRMS), is developed for the determination of the intramolecular (13)C isotope composition of ethanol in aqueous solutions. The δ(13)C values of the pyrolytic fragments (CO, CH4, C2H4) are shown to be highly reproducible (sd <0.4‰). Furthermore, using 14 ethanol samples of known intramolecular isotope distribution, the CO and CH4 fragments are shown to arise solely from the methylene (CH2OH) and methyl (CH3) carbon atom positions of the original ethanol, respectively. Although the different steps (extraction and pyrolysis) fractionate between (12)C and (13)C, the isotopic fractionation is reproducible (sd <0.4‰), allowing correcting factors to be applied in order to back-calculate the original δ(13)CCH2OH and δ(13)CCH3 values of ethanol. The method thus allows the determination of the isotope composition of ethanol at the intramolecular and molecular levels, within a single run and a short experimental time (30 min), and with a very easy sample preparation. The method is then applied to alcoholic beverages to show its potential for authentication purposes.