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Keywords:

  • 13C isotope distribution;
  • carbohydrate;
  • fructose;
  • glucose;
  • isomerization;
  • isotope fractionation;
  • isotopic 13C NMR;
  • sucrose

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • Recent developments in 13C NMR spectrometry have allowed the determination of intramolecular 13C/12C 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 13C/12C ratios in fructose by 13C NMR analysis of the acetyl-isopropylidene derivative.
  • We have applied this method to measure the intramolecular 13C/12C 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 C3 and C4 plants, the C-1 and C-2 positions of the glucosyl and fructosyl moieties are markedly different. Second, these positions in C3 and C4 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 13C pattern in CAM sucrose probably indicates a substantial contribution of gluconeogenesis to glucose synthesis.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Photosynthetic CO2 fixation discriminates against 13C, so that organic matter formed by plants is naturally 13C-depleted compared with atmospheric CO213C = −8‰) (Troughton et al., 1974; O’Leary et al., 1992). However, the isotope fractionation associated with net CO2 assimilation differs between CO2 assimilation pathways: C3 plants have typical δ13C values between −22‰ and −30‰, C4 plants between −9‰ and −13‰, and Crassulacean acid metabolism (CAM) plants between −10‰ and −20‰ (for a review, see Farquhar et al., 1989). The photosynthetic 13C/12C isotope fractionation in plants is mainly caused by diffusional and enzymatic effects, and models based on photosynthetic CO2 fixation have been developed to explain the isotopic composition in primary plant organic matter (Brugnoli & Farquhar, 2000). However, further fractionations may occur after carbon fixation (so-called post-photosynthetic fractionations), thereby changing the isotopic signal of net fixed carbon and causing differences in the isotope composition between plant organs and/or plant organic compounds (Cernusak et al., 2009).

For instance, leaves of C3 plants are systematically 13C-depleted compared with sink organs such as roots or stems (Badeck et al., 2005). This difference between organs is not yet clearly understood, although several reasons have been discussed recently (Badeck et al., 2005; Cernusak et al., 2009). Furthermore, within a single plant organ, quite large differences in δ13C values occur between metabolites. Typically, lipids and lignin are 13C-depleted by up to 7‰ compared with carbohydrates (sucrose, starch) (Park & Epstein, 1961; Winkler et al., 1978). Such differences in δ13C are caused by enzymatic isotope effects (for a review, see Schmidt & Gleixner, 1998) in which the following are well recognized: the oxidation of pyruvate to acetyl-CoA by pyruvate dehydrogenase fractionates against 13C, leading to 13C-depleted acetyl-CoA, which is, in turn, consumed to synthesize fatty acids (Melzer & Schmidt, 1987); the condensation of triose phosphates to fructose-1,6-bisphosphate favours 13C (Gleixner & Schmidt, 1997), thereby explaining the 13C enrichment in the C-3 and C-4 positions of glucose (Rossmann et al., 1991; Gilbert et al., 2009). This isotope effect pervades metabolism, and probably explains the 13C enrichment in leaf-evolved CO2 and in transitory starch (Tcherkez et al., 2004). Positional intramolecular isotope effects of enzymes are thus critical for the 13C distribution within plants and the isotopic composition of CO2 evolved by plant respiration. In other words, enzymatic isotope effects and the intramolecular δ13C values in metabolites will dictate the isotopic CO2 exchange fluxes between plants and the atmosphere (Yakir & Wang, 1996; Barbour & Hanson, 2009).

It has long been recognized that, in order to understand isotope fractionation in plant metabolism, an analysis of isotope redistributions at the intramolecular level is required. This has, however, proved challenging, and relatively few compound-specific δ13C values in metabolites (Collister et al., 1994; Schmidt et al., 2004) and enzymatic isotope effects (Tcherkez & Farquhar, 2005; Mauve et al., 2009) have been investigated. Mostly, only data for whole-molecule δ13C values (δ13Cg, see Table 1 for definitions) have been obtained by isotope ratio mass spectrometry (IRMS), a technique that does not give direct access to the positional δ13C values (δ13Ci). These can only be accessed by IRMS via complex and tedious chemical degradations. The most complex example of this approach is that of Rossmann et al. (1991), who used IRMS following a series of chemical and biochemical degradations to measure the intramolecular δ13C values in glucose of C3 (obtained by the hydrolysis of sucrose in beet syrup) and C4 (from starch of maize flour) origins. Although this work clearly showed the nonuniform 13C distribution in natural glucose with a good precision, the use of this technique is impractical for large sample sets.

Table 1.   Symbols used in this article
SymbolMeaning
δ13CCarbon isotope composition: carbon isotopic ratio of the molecule relative to the international standard (Vienna Pee Dee Belemnite, V-PDB)
δ13Cg13C mean isotopic composition of a whole molecule measured by isotope ratio mass spectrometry (IRMS)
δ13Ci13C isotopic composition of the carbon position i measured by 13C NMR
fiMolar fraction for a carbon site i measured by 13C NMR = area of the peak corresponding to the carbon position i divided by the sum of all the carbon sites of the molecule (inline image)
FiStatistical molar fraction for a carbon site i: molar fraction for the carbon site i in the case of a homogeneous 13C distribution within the molecule (Fi = 1/6 for glucose and fructose derivatives)
AiIsotopic abundance for a carbon site i
AgIsotopic abundance of a whole molecule

Quantitative NMR, however, offers the possibility to determine intramolecular δ13Ci values at natural abundance. It analyses directly the target molecule without the need for prior chemical degradation. Site-specific natural isotopic fractionation studied by NMR (SNIF-NMR) was developed for δ2H determinations in the 1980s and is now routinely used for metabolic and climatic analyses and as a tool in authentication (Martin et al., 1986; Remaud et al., 1997; Augusti et al., 2008). However, the use of NMR for the study of intramolecular 13C distributions presents more of a challenge because the range of isotopic variation in natural compounds is c. 10-fold less for 13C than for 2H (c. 50‰ and 500‰, respectively, on the δ scale). Hence, isotopic 13C NMR requires 10 times higher precision. Furthermore, for the effective use of quantitative 13C NMR at natural abundance, a protocol for efficient proton decoupling of 13C–1H interactions is required, as was a means to reduce the extended analytical duration. These obstacles have been overcome by the use of adiabatic decoupling sequences (Tenailleau & Akoka, 2007) and relaxation agents (Caytan et al., 2007b), respectively. Using these protocols, the intramolecular δ13C distributions in vanillin (Caytan et al., 2007a), paracetamol (Silvestre et al., 2009) and natural glucose (Gilbert et al., 2009) have been established recently.

However, the analysis of carbohydrates by 13C NMR is still impeded by isomerization equilibria and inadequate separation of the isomers. In both glucose and fructose, two configurational changes may occur in solution (Fig. 1): mutarotation (α/β forms) and C6 vs C5 cyclization (pyranose/furanose forms) (Flood et al., 1996; Yamabe & Ishikawa, 1999). The isomers have different chemical shifts that generate complex spectra, such that quantitative 13C NMR cannot be carried out reliably with isomeric mixtures. We have previously developed a methodology based on the use of acetyl-isopropylidene derivatives to prevent isomeric interconversions, and this has been applied to glucose analysis (Gilbert et al., 2009). However, the same methodology is insufficient to determine the intramolecular δ13C in fructose. First, fructose forms two products when reacting with iodine in acetone (first step of the derivatization): DAFP (2,3;4,5-di-O-isopropylidene-β-d-fructopyranose) and DAFP-1,2 (1,2;4,5-di-O-isopropylidene-β-d-fructopyranose) (Fig. 2) (Verhart et al., 1992; Kang et al., 1995). The former, the major product, is that desired for 13C NMR analyses, but the formation of the byproduct (DAFP-1,2) may be associated with isotope fractionation and so potentially might distort the δ13C value of DAFP. Second, the product of the second step of the derivatization (1-O-acetyl-2,3;4,5-di-O-isopropylidene-β-d-fructopyranose, MADAFP) has several carbon atoms with similar chemical groups (C-3, C-4 and C-5), giving inadequate resolution in the 13C NMR spectra.

image

Figure 1. Mutarotational equilibrium of d-fructose involving the linear form, α/β and pyranose/furanose configurations. Numbers in parentheses represent the proportion of each form for d-fructose in aqueous solution at room temperature.

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image

Figure 2. Procedure for the derivatization of fructose and glucose into 1-O-acetyl-2,3;4,5-di-O-isopropylidene-β-d-fructopyranose (MADAFP) and 3,5,6-triacetyl-1,2-Ο-isopropylidene-α-d-glucofuranose (TAMAGF) for analysis by isotopic 13C NMR. In the first step, an isomer of 2,3;4,5-di-O-isopropylidene-β-d-fructopyranose (DAFP) is obtained (1,2;4,5-di-O-isopropylidene-β-d-fructopyranose, DAFP-1,2), which can be separated from the product DAFP by a selective hydrolysis using 80% aqueous acetic acid (step 2). See text for other abbreviations.

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Here, we describe a method for the determination of δ13Ci in fructose and apply it to obtain the intramolecular 13C patterns in both the fructosyl and glucosyl moieties obtained from sucrose.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals

Silica (63–200 μm mesh) thin layer chromatography (TLC) plates (aluminium sheets, silica gel 60 F254), sulfuric acid (98%), acetone (99.9%), acetic acid (99%), acetic anhydride (99%), pyridine (99%), ethanol (99%), sodium chloride, magnesium sulfate, sodium thiosulfate, sodium carbonate and solvents (> 99% purity) dichloromethane, petroleum ether, diethylether, cyclohexane, ethylacetate, ethanol and sulfuric acid (98%) were purchased from VWR (Fontenay-sous-Bois, France). Solvents used for the purification steps (recrystallization and column chromatography) were redistilled before use. Molecular iodine was from Sigma Aldrich (Lyon, France). Hexadeuterated acetone (acetone-d6) was purchased from Eurisotop (Saint-Aubin, France) and tris(2,4-pentadionato)chromium-iii [Cr(Acac)3] (97%) from Acros Organics (Courtaboeuf, France). Cane (Saccharum officinarum) and beet (Beta vulgaris) sucrose and fructose samples were from VWR. Pineapple juice (Ananas comosus) was from a commercial source (local supermarket). Cation exchange (50WX8-200 mesh, H+ form) and anion exchange (1X8200-400 mesh, Cl form) resins were from Sigma-Aldrich.

Extraction of sugars from pineapple juice

The procedure used for the separation of sugars from the other constituents in pineapple juice has been described by Gonzalez et al. (1999). Briefly, pure juice was passed through cationic and anionic resin columns to eliminate amino acids and organic acids, respectively. The resulting crude solution was evaporated to yield a viscous syrup which was used for the derivatization without further purification.

Chemical synthesis of the fructose derivative MADAFP

The syntheses of 3,5,6-triacetyl-1,2-Ο-isopropylidene-α-d-glucofuranose (TAMAGF) and MADAFP were carried out as follows (the derivatization sequence is depicted in Fig. 2).

Step 1: synthesis of DAFP and 1,2;5,6-di-O-isopropylidene-α-d-glucofuranose (DAGF)  This step is similar to that used by Gilbert et al. (2009) for glucose. The sugar sample was ground and then dried with P2O5 under vacuum for one night; 2.5 g sucrose was introduced into a 250-mL round-bottomed flask containing 125 mL acetone. Molecular iodine (93 mg, 0.05 eq.) was added at 50°C and the reaction mixture was refluxed at 80°C for 6–8 h. After completion of the reaction (monitored by TLC with petroleum ether/diethylether, 4 : 1), the reaction mixture was cooled to room temperature and aqueous Na2S2O3 (0.1 M) was added until discoloration of the solution was complete. After evaporation, a white solid was obtained to which aqueous Na2S2O3 (0.1 M, 75 mL) and dichloromethane (25 mL) were added. After separation of the organic layer, the aqueous phase was extracted twice with dichloromethane (35 mL). The organic layers were pooled, washed with 100 mL aqueous NaCl (10% w/v), dried over MgSO4, filtered and evaporated to give a mixture of DAGF and DAFP as a yellowish solid. This was used in step 2 without further purification. For the derivatization of pineapple extract, H2SO4 98% (1 mL g−1 of sugar) was used as a catalyst instead of iodine. At the end of the reaction, the excess sulfuric acid was neutralized with a saturated NaHCO3 solution and the process was continued as already detailed.

Step 2: synthesis of 1,2-O-isopropylidene-α-d-glucofuranose (MAGF)  The solid obtained in step 1 was treated with 80 mL aqueous acetic acid (80% v/v). The reaction mixture was stirred for 15–18 h and the reaction was monitored by TLC (petroleum ether : ethyl ether, 4 : 1). After completion, ethanol (50 mL) was added to the reaction mixture, which was then evaporated to give a white solid. To this, H2O (100 mL) was added and the aqueous layer was extracted twice with dichloromethane (50 mL). The remaining aqueous layer containing MAGF was evaporated with ethanol in order to obtain a dry white solid. The organic layers containing DAFP were pooled, washed twice with 100 mL aqueous Na2CO3 (15% w/v), dried over MgSO4, filtered and evaporated to give a yellowish oil which crystallized spontaneously.

Step 3: acetylation of glucose and fructose derivatives  MAGF and DAFP from step 2 were treated with acetone (1.5 eq./OH) in pyridine (10 mL g−1 of product). The reaction mixture was stirred overnight and MAGF/DAFP disappearance was monitored by TLC (cyclohexane : ethylacetate, 2 : 1). After completion (15–17 h), the reaction mixture was evaporated and then co-evaporated with toluene, and the viscous yellow liquid obtained was dissolved in dichloromethane. The organic layer was washed with aqueous NaHCO3 (15% w/v) and distilled water. The organic layers were pooled and dried over MgSO4, filtered and evaporated under vacuum. The acetylated compounds obtained (TAMAGF and MADAFP) were then purified.

Purification procedures  The resulting crude products (yellow solids) were purified on a silica chromatography column (63–200 μm) with a mixture of cyclohexane : ethylacetate (4 : 1) and further recrystallized from hot light petroleum ether (boiling point, 40–65°C). During the chemical derivatization of sucrose (or of a mixture of glucose and fructose), a byproduct coming from fructose was obtained, which is an isomer of DAFP: DAFP-1,2 (see Fig. 2). This compound was eventually hydrolysed into 1,2-O-isopropylidene-β-d-fructopyranose during step 2 (Fig. 2), which was, in turn, acetylated during step 3. This compound could have contaminated the glucose derivative (TAMAGF) as it was eluted in the same chromatographic fraction. To avoid this, the byproduct was eliminated by the recrystallization of pure TAMAGF in hot light petroleum ether (boiling point, 40–65°C).

NMR spectrometry

Quantitative 13C NMR spectra were recorded using a Bruker 400 Avance I spectrometer fitted with a 5-mm-i.d. dual+ probe 13C/1H carefully tuned at the recording frequency of 100.64 MHz.

Sample preparation  TAMAGF (280 mg) was dissolved in deuterated acetone (acetone-d6, 500 μL), submitted to ultrasonication for 15 min (to eliminate any oxygen trapped during dissolution) and filtered into a 5-mm-o.d. tube. MADAFP (300 mg) was dissolved in acetone-d6 (500 μL) and 20 μL of Cr(Acac)3 solution (0.1 M in acetone) was added. The solution was then ultrasonicated for 15 min and then filtered into a 5-mm-o.d. tube.

Spectral acquisition conditions  The temperature of the probe was set at 303 K. The offsets for both 13C and 1H were set at the middle of the frequency range for each molecule. Inverse-gated decoupling was applied in order to avoid any nuclear Overhauser effect. The repetition delay (RD) between each 90° pulse was set at 10 × T1max of the molecule under investigation. The longest T113C values (T1max) measured using the sample preparation described above were 1.2 s and 7.5 s for TAMAGF and MADAFP, respectively. The decoupling sequence used a cosine adiabatic pulse with appropriate phase cycles, as described by Tenailleau & Akoka (2007). For TAMAGF, the acquisition conditions were as follows: acquisition time, 0.8 s; 480 scans with RD = 15 s, leading to a signal-to-noise ratio of c. 650. For MADAFP, the conditions were as follows: acquisition time, 1.0 s; 460 scans with RD = 75 s (T1max = 7.5 s), leading to a signal-to-noise ratio of c. 750. Each measurement made was the average of three to five independent NMR records.

NMR data processing  Free induction decay was submitted to an exponential multiplication inducing a line broadening of 1.6 Hz for TAMAGF and 1.4 Hz for MADAFP. Curve fitting was carried out in accordance with a Lorentzian mathematical model using Perch Software (Perch NMR Software™; University of Kuopio, Kuopio, Finland).

Isotopic data  Isotope 13C/12C ratios were calculated from processed spectra using the method of Silvestre et al. (2009). Briefly, the positional isotopic distribution in a molecule was obtained from 13C mole fractions fi (where i stands for the C-atom position considered) as follows: fi = Si /Stot, where Si is the 13C signal (i.e. the area under the peak associated with the C atom in position i) and Stot is the sum of the areas of all 13C signals of the molecule. Each Si had to be corrected to compensate for the slight loss in intensity caused by satellites (13C–13C interactions) by multiplying by (1 + × 0.011), where n is the number of carbon atoms directly attached to the C-atom position i and 1.1% (= 0.011) is the average natural 13C abundance [see Tenailleau et al. (2004) and Silvestre et al. (2009) for a detailed explanation]. If Fi denotes the statistical mole fraction (homogeneous 13C distribution) at any C-atom position i, the site-specific relative deviation in the 13C abundance is di = fi /Fi – 1. The di values were converted to δ13C values using the isotope composition of the whole molecule obtained by IRMS. This link is established through the relationship Ai = fi /Fi × Ag, where Ag is the isotopic abundance of the whole molecule (see Table 1 for notations). A numerical application of the calculations on a fructose sample (MADAFP) is shown in Supporting Information Table S1.

Elemental analyser (EA)-IRMS

The 13C abundance of the whole molecule, designated as δ13Cg, was determined by IRMS. A sample (c. 0.8 mg) was sealed in a tin capsule and introduced into an EA Flash HT (ThermoFinnigan, Courtaboeuf, France) equipped with a Porapack Q column to separate CO2 and N2. The dry CO2 was swept on-line into a Delta-V Advantage spectrometer (ThermoFinnigan) and the δ13C was determined by reference to a working standard of glutamic acid standardized against calibrated international reference material (NBS-22, IAEA-CH-6, IAEA-CH-7). Thus:

  • image(Eqn 1)

where R is the 13C/12C isotope ratio of the sample and Rstd is the 13C/12C isotope ratio of Vienna Pee Dee reference standard (V-PDB) (Rstd = 0.0112372).

Results and Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Selection of the derivatization procedure

As explained in the Introduction, the sugars (fructose and glucose) need to be constrained so as to adopt a single configuration before isotopic 13C NMR analysis. In contrast with glucose, fructose can adopt several isomeric forms, all of which are present at a significant concentration (Fig. 1). Furthermore, although there are several configuration-selective derivatization methods for glucose (Pearson & Spessard, 1975; Pham-Huu et al., 2002), this is not the case for fructose, so that fructose derivatization may lead to a mixture of products. The acetyl-isopropylidene derivatization used here fixes the configuration of the anomeric C-1 carbon, allows the separation of the glucose and fructose moieties when a mixture of glucose and fructose and/or sucrose is used, and avoids any 2H vs 1H exchange between the hydroxyl group(s) of the molecule and the residual free deuterium atoms in the deuterated NMR solvent used for the frequency lock (Gilbert et al., 2010). For fructose, the derivatization selectively attacks β-d-fructopyranose (Fig. 1). Therefore, the consumption of β-d-fructopyranose by the reaction displaces the isomerization equilibrium and all other fructose configurations are eventually converted to β-d-fructopyranose. The yield of the derivatization obtained from either glucose or fructose is above 90% for each step of the procedure. Similarly, 90% of substrate fructose is consumed by derivatization. However, the first step (I2 in acetone) produced 90% DAFP (that is, 80% of substrate fructose) and 10% of byproduct DAFP-1,2 (that is, 9% of substrate fructose) (Fig. 2). The final product of DAFP-1,2 (MAFP-1,2) was eventually eliminated by solvent recrystallization. The occurrence of this byproduct did not influence the δ13C analysis in fructose (see later).

Quality and acquisition of NMR spectra

The spectra obtained with glucose and fructose derivatives (TAMAGF and MADAFP) are shown in Fig. 3. In order to obtain spectra of sufficient quality to measure the 13C : 12C ratios in the six carbon positions of these sugars, a number of parameters must be carefully controlled. The choice of the best solvent can substantially improve the resolution of the spectrum. In MADAFP, three carbon atoms (C-3, C-4 and C-5 of fructose) have very close chemical shift values. Of the solvents tested, deuterated acetone (acetone-d6) clearly gave the most effective peak separation (Table S2). As MADAFP does not contain any free hydroxyl residues, the risk of 2H–1H exchange with 2H of residual water in the solvent does not arise (Gilbert et al., 2010). An additional problem is the presence of a quaternary carbon (C-2) in MADAFP, which has a very long relaxation time (c. 19 s). This problem was substantially overcome by the addition of the relaxation agent Cr(Acac)3. This reduced the relaxation time to c. 7.5 s (whilst only weakly affecting the relaxation time of other C atoms); thus, the experimental acquisition time was significantly reduced to 30 h for each set of three MADAFP spectra.

image

Figure 3. Proton-decoupled 13C NMR spectra of 3,5,6-triacetyl-1,2-Ο-isopropylidene-α-d-glucofuranose (TAMAGF) (a) and 1-O-acetyl-2,3;4,5-di-O-isopropylidene-β-d-fructopyranose (MADAFP) (b) obtained in the conditions described in the Materials and Methods section. The numbering used is the conventional numbering for fructose and glucose.

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NMR repeatability and reliability

An assessment of the analytical repeatability on the glucose derivative TAMAGF gave a standard deviation of 0.9‰ (= 10) on the δ scale. The fructose derivative MADAFP, obtained from a commercial fructose, was analysed 10 times in the same conditions and the results are reported in Table 2. The maximum standard deviation observed for positional δ13Ci values was 0.9‰.

Table 2.   Mean isotopic composition δ13Ci and standard deviation determined from 10 independent measurements of the fructose derivative 1-O-acetyl-2,3;4,5-di-O-isopropylidene-β-d-fructopyranose (MADAFP) obtained from commercial fructose with the protocol described in Fig. 2, or obtained under conditions wherein the proportion of byproduct (1,2;4,5-di-O-isopropylidene-β-d-fructopyranose, DAFP-1,2) was c. 50%
 C-1C-2C-3C-4C-5C-6
δ13Ci mean (‰)−27.1−19.9−24.3−20.5−32.0−31.8
SD (‰)0.60.70.90.80.80.9
δ13Cic. 50% byproduct−25.2−20.6−29.5−20.7−29.0−30.7

The intramolecular δ13C values in TAMAGF derived from beet sucrose are shown in Fig. 4a. With the exception of the C-1 position, there is very good agreement between the values obtained here (dashed line) and those obtained with (bio)chemical degradation and IRMS (Rossmann et al., 1991) (dotted line). This clearly shows that the NMR-based method described here gives satisfactory results for 13C natural abundance. Nevertheless, there remains a difference of c. 3‰ in the C-1 atom position. Although the α/β mutarotation of glucose enriches the α-conformer in 13C with an equilibrium isotope effect of c. 4‰ (Mauve et al., 2009), and the substrate of the derivatization is α-glucofuranose (Fig. 2), this cannot be the cause of this fractionation, as the yield of the derivatization was c. 90%; thus, mutarotation could cause an isotopic shift of no more than 0.4‰. Furthermore, when derivatization was stopped with Na2S2O3 at a glucose conversion yield of only 50%, the δ13C value in C-1 (and in other positions as well) remained similar (data not shown; see also Gilbert et al., 2009), indicating that the discrepancy cannot be caused by any isotope effect associated with derivatization.

image

Figure 4. Isotope composition (δ13C) for each carbon site in glucosyl (dashed line) and fructosyl (solid line) moieties of natural sucrose. (a) Beet (C3). (b) Cane (C4). (c) Pineapple (Crassulacean acid metabolism, CAM). Error bar is ± 1SD obtained from 10 repetitions of fructose derivative measurement and 10 repetitions of glucose derivative measurement (0.9‰ in both cases). Results obtained by Rossmann et al. (1991) for a beet glucose are also given in (a) (dotted line).

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There are no special trends for the NMR features of C-1 which could explain this discrepancy. In particular, the longitudinal relaxation time of the C-1 position of TAMAGF is not significantly different from that of the other tertiary carbons of the molecule (Table 3), thus implying no supplementary error for the determination of the δ value for this site. One other potential source of error is the use of the relaxation agent Cr(Acac)3. Even though its utilization has been shown to bring no bias in the δ values of vanillin (Caytan et al., 2007b), we investigated the effect of Cr(Acac)3 on the δ values measured on the glucose derivative (Table 3). The δ values obtained with a preparation with and without Cr(Acac)3 showed no significant differences, supporting the results of Caytan et al. (2007b). Furthermore, the δ values obtained for a longer RD were the same as those obtained with the delay used routinely (RD > 10 × T113C). Thus, it can be concluded that the RD used routinely is not a source of error in the determination of the δ values in glucose. Therefore, the NMR analysis per se can also be dismissed as the origin of the 13C enrichment in C-1. Hence, it can be concluded that the isotopic enrichment observed here in C-1 is not an artefact of the methodology, but reflects a difference between our sucrose sample and that analysed by Rossmann et al. (1991).

Table 3.   Values of δ13C (‰) for each carbon position in 3,5,6-triacetyl-1,2-Ο-isopropylidene-α-d-glucofuranose (TAMAGF)
 C-1C-2C-3C-4C-5C-6
  1. NMR analysis was carried out with or without a relaxation agent [Cr(Acac)3, tris(2,4-pentadionato)chromium-iii] and with different values of the relaxation delay (RD). In all cases, SD was < 0.9‰.

RD = 10 s with Cr(Acac)3−21.3−22.4−23.7−25.5−28.4−28.0
RD = 12 s with Cr(Acac)3−21.7−22.0−23.9−25.5−29.0−27.3
T113C (s) with Cr(Acac)30.90.91.01.01.00.6
RD = 15 s without Cr(Acac)3−21.1−22.6−23.4−25.7−29.0−27.7
T113C (s) without Cr(Acac)31.01.21.21.21.20.7

Is there a 12C/13C isotope fractionation associated with fructose derivatization?

The chemical derivatization of fructose might similarly be associated with an isotope fractionation that would influence the measurement of the 13C abundance. However, even though the β-fructopyranose and β-fructofuranose conformers (Fig. 1) might have different isotope compositions as a result of an equilibrium isotope fractionation, this would be eliminated as the derivatization method selectively attacks the β-fructopyranose. As the yield is large (c. 90%), this would displace all the β-fructofuranose to β-fructopyranose, and negate any isotopic differences. In addition, there was little change in the isotopic β-factors of the carbon atoms (Galimov, 1985, 2006), implying that fractionation in any event is small. Nonetheless, the production of the byproduct DAFP-1,2 could alter the measured 13C abundance if such a byproduct had a different isotope composition from that of the main product DAFP. Experiments carried out at low temperature (40°C), specifically to increase the ratio of DAFP : DAFP-1,2 to c. 1 : 1 caused site-dependent changes in the δ13C of MADAFP, with the highest being 5‰ for C-3 (Table 2). Hence, it can be deduced that, with the typical DAFP proportion of 10%, the isotopic shift in MADAFP caused by the formation of DAFP-1,2 would be, at most, 1‰ in C-3 and between 0‰ and 0.4‰ in all the other C-atom positions. This level is within the error of repeatability for MADAFP (Table 2). We can therefore conclude that any variation in δ13Ci caused by derivatization-associated isotope effects is insignificant compared with that caused by natural δ13C variation between C-atom positions.

Intramolecular δ13C in glucosyl and fructosyl moieties of plant sucrose

The method validated here can be used to investigate in parallel the intramolecular 13C distribution in the glucosyl and fructosyl moieties of sucrose. The isotopic patterns are shown in Fig. 4 for samples from C3 (beet sugar), C4 (cane sugar) and CAM (pineapple juice extract) plants. Independently of the pathway of photosynthetic CO2 assimilation, the C-3, C-4, C-5 and C-6 atoms have very similar relative δ13C values in fructose and glucose. It is worth noting that, for the C3 plant (beet), these positions show strong similarity to those found by Rossmann et al. (1991) for glucose. Three prominent features stand out from Fig. 4. First, in sucrose from both C3 and C4 plants, the C-1 and C-2 positions of the glucosyl and fructosyl moieties are markedly different, especially so when compared with the other four carbons. Second, the C-1 and C-2 positions in C3 and C4 plants show a similar profile. Third, the glucosyl and fructosyl moieties of sucrose from CAM metabolism have a profile quite distinct from that found for C3 and C4 plants.

For the C-1 and C-2 positions in C3- and C4-sucrose, the fructosyl C-1 is up to 10‰ depleted compared with C-2, whereas, in the glucosyl moiety, the difference is in the opposite sense, with C-1 enriched by up to 5‰ (Fig. 4a,b). Based on the arguments given above, we can be confident that there is no analytical flaw that might have caused this level of 13C enrichment in the C-2 atom of fructose. Rather, these contrasting δ13C values can be explained as a result of the isotopic selectivity of enzymes that specifically break or make covalent bonds in these positions. The two most pertinent reactions are glucose-6-phosphate isomerase (EC 5.3.1.9) that isomerizes glucose-6-phosphate to fructose-6-phosphate, and invertase (EC 3.2.1.26) that cleaves sucrose to glucose and fructose.

For the isomerization of glucose to fructose, the predicted fractionations calculated using β-factors (Galimov, 1985, 2006) are 1.021 and 0.979 for C-1 and C-2, respectively. At equilibrium, this translates into the C-2 atom position in fructose being 13C-enriched by 21‰ and C-1 being 13C-depleted by 21‰ compared with the same position in glucose. Thus, theoretical considerations give values in the same sense and, in the case of C-2, of the same size as those obtained experimentally.

The isotopic fractionation associated with invertase has been studied by Mauve et al. (2009) during the hydrolysis of sucrose by the yeast (Saccharomyces cerevisiae) invertase in vitro. These authors concluded that the invertase-associated fractionation should produce free fructose 13C-depleted in the C-2 position by c. 12‰. The impact of this fractionation occurring during sucrose hydrolysis by invertase in plants would be to enrich residual sucrose in the C-2 position of the fructose moiety, exactly as observed here.

In fructose and glucose from CAM sucrose, the C-6 and C-1 positions are strongly 13C-depleted (up to 25‰) compared with the C-5 and C-2 positions (Fig. 4c). This very distinctive 13C pattern in CAM sucrose can be interpreted as indicating a substantial contribution of gluconeogenesis to glucose synthesis, a process shown to be significant in CAM plants (Holtum & Osmond, 1981). That is, the recovery of pyruvate produced by malic enzyme to phosphoenolpyruvate and glucose is likely to be associated with the isotope effects that are responsible for the 13C depletion in C-1 and C-6. Although no data on isotope effects are yet available for the plant malic enzyme, the chicken enzyme assayed in vitro has been shown to fractionate by 21‰ in the C-3 position of malate, thereby depleting 13C in the C-3 position in phosphoenolpyruvate (Edens et al., 1997). This is the position that gives rise to both C-1 and C-6 positions in glucose through gluconeogenesis.

From a physiological point of view, such a marked difference between glucosyl and fructosyl moieties of sucrose may be critical for the isotopic mass balance of plant organs. For example, this could influence the δ13C composition of CO2 evolved by plant metabolism, because the pentose phosphate pathway decarboxylates the C-1 position in glucose, and so the natural 13C enrichment in C-1 may compensate for the isotope effect of 6-phosphogluconate decarboxylase.

Conclusions

Our results clearly show that it is now possible to access the site-specific isotope fractionation in some plant hexoses by exploiting 13C NMR to determine directly the intramolecular δ13Ci values. Based on an initial study of the post-photosynthetic fractionations associated with sucrose and glucose metabolism, it is clear that considerable variation is present and that this can be linked to metabolic processes. Of special interest is that the data obtained can be interpreted directly in terms of isotope effects associated with specific enzymes, in the present example those of glucose-6-phosphate isomerase and invertase. Further examination of the critical role of these enzymes in defining specific isotope fractionation will be developed in a subsequent study.

More generally, the methodology reported herein will find application in studies aiming to better define the allocation of sugars in plants, notably the relative distributions of fructose and glucose to different pathways. For example, it offers a means potentially to quantify the allocation of fructose-1,6-bisphosphate and glucose-6-phosphate between glycolysis and the pentose phosphate pathway, or to evaluate the contributions to the glucose pool in CAM plants of photosynthetic and gluconeogenic origins for glucose. It may also find application in the analysis of the remobilization of polymers, such as starch and fructans, known to be isotopically distinct (Lehmeier et al., 2010) Although it is recognized that, overall, glucose is sequestered to a greater degree than fructose – notably in the formation of polymers, such as cellulose and inositol, and through glycosylation reactions – the consequent metabolism of fructose could potentially be better defined in exploiting the isotopic differences identified herein. Nevertheless, it must be accepted that measurements of the intramolecular 13C values of hexoses are highly specialized and that this analytical protocol will be limited to those cases in which a relatively small number of measurements can define the isotopic redistribution patterns. Notwithstanding, we have recently shown that the 13C/12C ratio measured by 13C NMR in ethanol derived from glucose can act as a sensitive probe of the isotopic values at the C1 + C6 and C2 + C5 positions (Gilbert et al., 2011). Such a correlation should hold for the fermentation of any hexose to ethanol by a defined pathway. Ethanol is recovered by a simple distillation of fermentation medium and the NMR measurement is rapid (2 h) compared with that for glucose or fructose (12–25 h). This option may help to give access to intramolecular data in a wider number of applications.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Alexis Gilbert thanks the Scientific Council of the Pays de la Loire Region (France) and the CNRS for a co-funded doctoral bursary.

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  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Table S1 Example of calculation of δ13Ci for each carbon position of fructose derivative from the area under the relevant peak in the 13C NMR spectrum

Table S2 Differences in Hertz between nuclear shielding of the carbon atom signals in the 13C NMR spectrum of 1-O-acetyl-2,3;4,5-di-O-isopropylidene-β-D-fructopyranose (MADAFP) corresponding to C-3, C-4 and C-5 of fructose

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