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Vol. 29, Issue 11, 1440-1445, November 2001
Laboratoire Synthèse Electrosynthèse et Etude de Systèmes à Intérêt Biologique, Unité Mixte Recherche 6504 du Centre National de la Recherche Scientifique, Université Blaise Pascal, Aubière, France (B.C., A.-M.D.); and Unité d'Ecologie et de Physiologie du Système Digestif, Institut National de la Recherche Agronomique, Jouy-en Josas, France (L.E., S.R.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion and Conclusion
References
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One- and two-dimensional 1H NMR spectroscopy were used to study the biotransformation of two dietary glucosinolates, sinigrin (SIN), and glucotropaeolin (GTL) by the human digestive microflora in vitro. The molecular structures of the new metabolites issued from the aglycone moiety of the glucosinolate were identified, and the modulation of carbon metabolism was studied by quantifying bacterial metabolites issued from the xenobiotic incubation in the presence or absence of a source of free glucose. Unambiguously and for the first time, it was shown that SIN and GTL were transformed quantitatively into allylamine and benzylamine, respectively. The comparison of the kinetics of transformation of SIN and GTL with and without glucose clearly showed that the presence of glucose did not modify either the nature of the metabolites or the rate of transformation of the glucosinolates (complete degradation within 30 h). The main end products of the glucose moiety of glucosinolates were characteristic of anaerobic carbon metabolism in the digestive tract (acetate, lactate, ethanol, propionate, formate, and butyrate) and similar to those released from free glucose. This work represents the first application of 1H NMR spectroscopy to the study of xenobiotic metabolism by the human digestive microflora, demonstrating allyl- and benzylamine production from glucosinolates. Whether these amines are produced in vivo from dietary glucosinolates remains to be established. This would reduce the availability of other glucosinolate metabolites, notably cancer-protective isothiocyanates.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion and Conclusion
References
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Glucosinolates are
sulfur-containing phytochemicals present in edible cruciferous plants,
such as Brussels sprouts, cabbage, radish, etc. Their common structure
comprises a 
-thioglucose group, a sulfonated oxime moiety, and a
variable side chain derived from methionine, tryptophan, or
phenylalanine. Upon disruption of plant tissues during food processing
or ingestion, glucosinolates are hydrolyzed by the endogenous enzyme
"myrosinase" (thioglucoside glucohydrolase EC 3.2.3.1.) to yield
isothiocyanates, nitriles, and other minor products (Fenwick et al.,
1983
). Numerous experimental studies performed in animal and cellular
models implicated isothiocyanates as the main bioactive agents
responsible for the anti-cancer properties of cruciferous vegetables
(Musk and Johnson, 1993; Nugon-Baudon and Rabot, 1994; Verhoeven et
al., 1997). In comparison, the fate of glucosinolates following
ingestion, in particular their breakdown site and rate, has received
little attention. When plant myrosinase is active, glucosinolates are
rapidly hydrolyzed in the food or in the proximal gut (De Vos and
Blijleven, 1988; Campbell et al., 1995). Should the enzyme be
deactivated by cooking (Jongen, 1996), glucosinolates reach the large
bowel, where they are broken down by the resident microflora (Rabot et
al., 1993; Michaelsen et al., 1994). A few recent experiments performed
in humans and gnotobiotic rats associated with a human fecal flora have
demonstrated that microbial breakdown of glucosinolates leads to the
formation of isothiocyanates (Shapiro et al., 1998; Elfoul et al.,
2001). The conversion is incomplete, however, suggesting that other
metabolites are produced; at present, nothing is known of the identity
of these latter products.
The aim of this study was to investigate the metabolism of
glucosinolates by the human colonic microflora in vitro, using 1H NMR spectroscopy. Indeed, NMR signals
are real fingerprints of molecules, and a wide range of metabolites can
be measured simultaneously and without a priori hypothesis. In
addition, despite a rather low sensitivity, this method is very
convenient since it can be performed directly on biological samples
without prior purification. Finally, quantitative data can be
collected. 1H NMR spectroscopy has proved to be a
powerful tool for analysis of the metabolite composition of biological
fluids (Nicholson and Wilson, 1989; Fan, 1996; Kalic et al., 2000) and
for the study of drug metabolism (Gartland et al., 1991; Holmes et al.,
1995; Bollard et al., 1996; Foxall et al., 1996). More recently, this technique has been applied to the study of microbial metabolism (Matheron et al., 1998; Brecker and Ribbons, 2000; Weber and Brecker, 2000) and, in particular, microbial degradation of xenobiotics (Gaines
et al., 1996; Besse et al., 1998; Combourieu et al., 1998a,b, 2000;
Poupin et al., 1998; Delort and Combourieu, 2000).
In this work, we have used 1D1 and 2D 1H NMR spectroscopy to elucidate the microbial biotransformation of two structurally different glucosinolates, sinigrin and glucotropaeolin. Sinigrin (SIN; Fig. 1) is a simple aliphatic glucosinolate prevalent in a wide range of cruciferous vegetables, whereas glucotropaeolin (GTL; Fig. 1) is an aromatic glucosinolate specifically present in garden cress and papaya fruit. In comparative experiments, SIN and GTL were added to the culture media, either alone or concurrently with free glucose. In this way, we sought to determine whether the availability of a simple source of energy and carbon, as is likely to be the case in the digestive tract, would hamper glucosinolate metabolism by digestive bacteria. In addition, data were collected from the NMR spectra to identify and quantify metabolites produced by the fermentation of the glucosinolate sugar moiety.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion and Conclusion
References
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Incubation Experiments. Freshly passed stools were collected from a healthy adult human subject who had not taken any antibiotics for at least 3 months preceding the study and who usually consumed a Western style diet.
Feces were transferred into an anaerobic glove box where they were thoroughly mixed with the incubation buffer, using an Ultra-turrax blender (Janke and Kunkel GmbH, Staufen, Germany). Incubation buffer was 0.1 M sterile potassium phosphate, pH 7.0, with added yeast extract (Difco) 2 g · l
1.
The suspension was adjusted at 1 g · 100 ml1 by further addition of incubation buffer
and divided into 20 ml fractions, transferred to amber glass vials,
either alone (control without substrate) or together with SIN
(Sigma-Aldrich, St. Louis, MO) and GTL (Merck, Darmstadt, Germany) (6 mM each) or SIN, GTL, and glucose (6 mM each). Sterile controls
containing SIN and GTL were prepared to check the stability of the
substrates in the absence of bacterial cells (controls without flora).
SIN, GTL, and glucose were added as freshly prepared aqueous stock
solutions sterilized by filtration (Millex-GS 0.22 µm; Millipore
Corporation, Bedford, MA). Vials were tightly closed with butyl-rubber
stoppers and sealed with aluminum caps to maintain anoxic conditions
and to avoid loss of volatile isothiocyanates. They were incubated at
37°C in a shaking bath (50 rpm). All incubations were performed in duplicate.
Samples were collected by a puncture through the stoppers at 0, 3, 6, 18, and 30 h. They were centrifuged to remove bacteria (8000g, 10 min, 4°C), and supernatants were stored at
20°C until analysis.
HPLC Analysis.
Supernatants were analyzed for residual intact SIN and GTL using HPLC
analysis of desulfoglucosinolates, as recommended by the
International Standardization Organization for glucosinolate analysis
in Brassica (Anonymous, 1990), except that no methanol extraction was required before the desulfation step.
NMR Spectroscopy.
Preparation of the samples for NMR and quantification of the
metabolites were performed as previously described (Combourieu et al.,
1998a). No purification was performed before analysis, and pH was
adjusted to 7 to avoid changes in chemical shifts. TSPd4 (10% v/v of an 8 mM solution in
D2O) constituted a reference for chemical shifts
(0 ppm) and quantification. All 1H NMR spectra
were recorded on a Bruker Avance 300 spectrometer (Bruker, Newark, DE)
at 300.13 MHz at 25°C with a 5-mm
1H-13C-15N
inverse probe equipped with z-gradients.
1D 1H NMR experiments.
Water was suppressed by presaturation or by the classical double-pulsed
field gradient echo sequence: WATERGATE (Price, 1999). In both cases,
64 scans were collected (relaxation delay, 5 s; acquisition time,
3.64 s; 32,000 data points). A 0.3-Hz line broadening was applied
before Fourier transformation, and a baseline correction was performed
on spectra before integration with Bruker software. The
1H NMR spectra obtained were compared with those
of the controls.
2D 1H NMR experiments. Gradients-correlation spectroscopy was used to identify all the end products of carbon metabolism. The data were acquired as 2048 × 256 point files, accumulating eight transients per t1 increment. Zero-filling in t1 and unshifted sinusoidal window function in both time domains were employed before Fourier transformation. 2D phase-sensitive total correlation spectroscopy (TOCSY) experiments with water resonance suppression by a WATERGATE sequence (put at the end of the sequence) were used to assign all members of a coupled spin network. Spectral widths were adjusted in both dimensions to encompass all 1H signals of interest. The "mixing period" (corresponding to several cycles of MLEV-17 spin-lock sequence) was 20 to 80 ms. The responses of eight scans for each of 512 t1 increments were acquired. Zero-filling in t1 and sine window function in both dimensions were applied before 2D Fourier transformation.
GC-MS Analysis.
Samples were extracted three times with CDCl3,
and the organic layers were dried over MgSO4. The
extracts were analyzed directly by GC-MS on an HP 5890 Series II Plus
GC equipped with an HP 5989 B mass spectrometer (Hewlett Packard, Palo
Alto, CA). Separation was achieved on a 30-m OPTIMA-5-MS capillary
column (0.25-mm i.d. and 0.25-µm film) using the following
temperature program: 55°C, 2-min hold, increased by 5°C · min1 to 80°C, 15-min hold. Helium was used as
a carrier gas at a linear velocity of 41 cm · s1. The temperatures of injector, interface,
and source were 250, 250, and 200°C, respectively. The ionization
mode was electronic impact at 70 eV, and the detection mode chosen was
single ion monitoring, which increases sensitivity by a factor of 10.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion and Conclusion
References
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Identification of Metabolites Issued from the Aglycone Moiety of SIN and GTL by 1D and 2D TOCSY 1H NMR Spectroscopy. Incubations were performed with a mixture of SIN and GTL in the presence or the absence of glucose. In both cases, the same signals were present in the NMR spectra.
An example of kinetics monitored in the absence of glucose is presented in Fig. 2; these 1D 1H NMR spectra contained simultaneously the resonances of SIN, GTL, and their derivatives and also the resonances of characteristic glucose metabolites.
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CH2 and CH, respectivelyof
SIN. The second methylene group of the SIN side chain cannot be
assigned in a 1D spectrum.
At 18 h, the signals of GTL (4.16 and 7.43 ppm) decreased, whereas
two new resonances at 4.20 and 7.49 ppm increased (named Y on Fig. 2).
Also, close to the signals of SIN resonating at 5.30 and 6.05 ppm, two
multiplets appeared at 5.40 and 5.95 ppm (named X on Fig. 2). At
30 h, a well resolved doublet of triplets was visible at 3.62 ppm.
This signal was probably present at 18 h but overlapped with the
glucose protons. At the end of the kinetics, the resonances of SIN and
GTL were no longer present, whereas the intensities of those of the new
metabolites were at maximum.
To assign the different signals present in the 1D
1H NMR spectra, homonuclear 2D
1H TOCSY experiments were performed on the
samples taken during the incubation of the fecal flora with SIN and
GTL. Water, which generates strong t1 noise
and a dramatic loss of sensitivity, was suppressed by the classical
double-pulsed field gradient echo WATERGATE sequence included at the
end of the pulse program. An 80-ms mixing time was used for the
experiments to give a total correlation of all the protons of a chain
with each other. Expanded regions of 2D spectra, recorded for the 18- and 30-h samples are presented in Fig. 3,
a and b, respectively. In Fig. 3a, the side chain protons of SIN are
clearly visible; correlations of the two vinyl protons (5.30 and 6.05 ppm) and correlations of these protons with
CH2(a) (3.56 ppm) can easily be seen. It was
noted that no correlation between this methylene group and the glucose moiety was observed since five bonds and a sulfur atom separate these
two types of spins. On this spectrum, additional signals resonating at
3.62, 5.40, and 5.95 ppm were visible. The proton resonating at 5.95 ppm correlated with the one resonating at 5.40 ppm, as shown from the
cross peaks, suggesting that they belong to the same molecule. These
resonances were the major ones in the spectrum collected at 30 h
(Fig. 3b). The presence of saccharide protons (corresponding to the
free glucose or glucose moiety of glucosinolates) at only residual
level in the 30-h 1D spectrum (at half the intensity of the three
peaks) indicated that this molecule is an aglycone product.
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), we had to consider a chemical mechanistic hypotheses to
identify the product molecules.
Tang et al. (1972) studied the bacterial degradation of benzyl
isothiocyanate and found that it was transformed into benzylamine. Consequently, the hypothesis of an amine formation was checked. In the
case of SIN, commercial allylamine (Fig. 1) was added to the samples,
and its signals overlapped perfectly with those present in Fig. 2. To
confirm this assignment definitely, liquid/liquid extraction of a 30-h
supernatant was carried out with deuterated chloroform as a solvent.
The residue was directly analyzed by 1H NMR and
GC-MS. Apart from residual CHCl3 and water, the
1H NMR spectrum exhibited only five well resolved
signals [at a concentration of less than 100 µM, as estimated by
comparison with satellites (1%) of residual non deuterated solvent
(0.1%)] at different chemical shifts from those observed in the
buffer (a typical solvent effect) but with the same multiplicity (data not shown). The following ions were detected in the mass spectrum: m/z 57 (M+), 56, 42, 41, 30, 29, 27, and 15, which correspond to 2-propen-1-amine (allylamine), as shown in the spectrum of the commercial compound.
Regarding the transformation of GTL, the TOCSY (80-ms mixing time)
experiment also indicated that the signal at 4.20 ppm was correlated
(by J-coupling) to the signal at 7.49 ppm, suggesting that the
corresponding protons belong to the same molecule (data not shown).
These signals overlapped perfectly with those of commercial benzylamine
(Fig. 1). The fragmentation obtained from GC-MS analyses of the 30-h
CDCl3 extract was identical to that of the
commercial product: m/z 107 ([M]+), 91 ([-CH2-C6H5]+),
79, 51, 30.
Kinetics of Glucosinolate Degradation.
Figure 4a shows the time course of GTL
degradation and benzylamine formation in the absence and presence of
glucose. The concentrations were calculated from the integrals of
1H NMR signals in 1D spectra of GTL and
benzylamine [CH2(a)] and TSPd4, as previously described (Combourieu et
al., 1998a). First, it should be noted that the transformation of GTL
into benzylamine was quantitative. Second, no differences were observed
due to the presence of glucose; the nature of the metabolite and the kinetics of its appearance were similar. GTL was degraded within 30 h. The concentration of allylamine could not be estimated
during the process since its signals overlapped with those of glucose and the vinyl protons of SIN; however, after 30 h, the methylene protons in the spectrum could be integrated. At that particular time,
the concentrations of allylamine were 3.9 and 3.5 mM in the presence or
the absence of glucose, respectively. In the first case, no residual
saccharide protons could be detected, whereas in the second case, a
small amount (<0.5 mM) of glycone moiety was observed. As the
integration of NMR signals was difficult in the case of SIN, HPLC was
used. The time course of SIN concentration in the presence or the
absence of glucose are presented in Fig. 4b. In both cases, SIN was
degraded almost completely within 30 h; the presence of free
glucose had no effect on the kinetics. Finally, to compare HPLC and NMR
as methods for quantification, GTL degradation was also monitored by
HPLC (Fig. 4c). The results obtained with HPLC are in very good
agreement with those obtained with NMR (Fig. 4a). The very small
differences in calculated concentrations could be due to the
"excitation hole" produced by water signal suppression in the range
of ±0.6 ppm from water resonance (4.7 ppm). In conclusion, these
experiments showed that SIN and GTL were degraded in a similar fashion
and that glucose had no effect on these biotransformations.
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Identification of Metabolites Issued from the Glucose Moiety of SIN
and GTL by 1D and 2D-gCOSY 1H NMR Spectroscopy.
Some major signals in which the intensity increased with time were
detected in 1D 1H NMR spectra (Fig. 2 and Fig.
5) in the incubation experiments without
glucose. A characteristic singlet, resonating at 1.92 ppm, corresponds
to CH3 of acetate (Fan, 1996; Matheron et al., 1998). In the downfield region, a singlet at 8.46 ppm increased until
6 h and disappeared at the end of the kinetics; this typical signal was assigned to formate (Fan, 1996; Matheron et al., 1997).
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Kinetics of Glucose Moiety Degradation. By measuring 1H NMR integrals in 1D spectra of the different metabolites (CH3 of acetate, CH3 of propionate, CH3 of lactate, CH of formate, and CH3 of ethanol), it was possible to compare the kinetics of formation of these metabolites when the human fecal flora was incubated with SIN and GTL in the presence or absence of glucose. It should be noted that the butyrate concentration could not be measured since its signals partially overlapped with other unknown signals and thus could not be integrated properly.
These data were compared with a control incubation containing
-D-glucose (6 mM); under these conditions, the same
metabolites as those produced in the presence of glucosinolates were
obtained, and glucose was completely exhausted after 3 h of
incubation (data not shown).
The time courses of the concentration of acetate, lactate, ethanol,
propionate, and formate are reported in Fig.
6, a-e, respectively. First, it should
be noted that the initial rates of formation of the different
metabolites were similar when glucose was alone or in combination with
glucosinolates. When glucosinolates were alone, the initial rate was
lower except in the case of propionate. This could reflect a limitation
due to the rate of glycone hydrolysis from glucosinolates. Second, end
products (acetate, propionate, and ethanol) accumulated and
intermediates (lactate and formate) disappeared differently according
to the incubation conditions.
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Discussion and Conclusion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion and Conclusion
References
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By using 1D and 2D 1H NMR spectroscopy, we
have shown unambiguously that SIN and GTL are transformed
quantitatively by the human fecal flora into allylamine and
benzylamine, respectively. These findings are in contrast with previous
studies showing that human digestive bacteria produced isothiocyanates
from glucosinolates (Shapiro et al., 1998; Getahun and Chung, 1999;
Elfoul et al., 2001). However, it is worth noting that in these
experiments low amounts of isothiocyanates were recovered accounting
for 10 to 20% of the initial amounts of glucosinolates. As amines were
not investigated, it is possible that up to 80 to 90% of the missing recovery was actually present in this form in the samples.
A question remains: do allylamine and benzylamine result from
transformation of the corresponding isothiocyanates? Although isothiocyanates were not detected, they could be present at a concentration less than to 0.03 mM (limit of NMR detection). These intermediates do not accumulate because they are likely to be transformed very rapidly in allylamine and benzylamine. Indeed, Tang et
al. (1972) demonstrated the conversion of benzyl isothiocyanate into
benzylamine upon incubation with a suspension of Enterobacter cloacae isolated from papaya pulp. In order to check that such a
transformation was possible under abiotic conditions, allyl isothiocyanate (Sigma-Aldrich) and benzyl isothiocyanate (Fluka, Buchs,
Switzerland) were incubated in the buffer in the absence of cells for
48 h at 37°C under the same anoxic conditions as those employed
with bacterial incubations. The supernatants were directly analyzed by
1D 1H NMR spectroscopy. Allylamine and
benzylamine were detected in the medium (data not shown), indicating
the high sensitivity of isothiocyanates to hydrolysis. One can imagine
that this chemical reaction could take place in vivo; alternatively,
this reaction could also be enzymatically catalyzed. The biological
significance of a possible release of allylamine and benzylamine from
SIN and GTL in vivo remains to be established. One can already conclude that the conversion of isothiocyanates to amines, whether of biotic or
abiotic origin, would reduce the delivery of biologically active isothiocyanates to the tissues and thus decrease their
cancer-protective potential.
The comparison of the kinetics of transformation of SIN and GTL with and without glucose clearly showed that the presence of glucose did not modify either the nature of the metabolites or the rate of transformation, suggesting that the availability of a simple source of energy is not an important factor in the degradation of these complex thioglucosides.
When glucosinolates are hydrolyzed, free glucose is released, which can
be further metabolized anaerobically by the bacteria of the human
flora. It was interesting to test whether this glucose moiety was
metabolized in the same way as free glucose. Regardless of the
experimental conditions, the main metabolites were similar, including
acetate, lactate, ethanol, propionate, formate, and butyrate. These
metabolites are characteristic of anaerobic carbon metabolism in the
digestive tract (Wolin et al., 1998, 1999). Acetate, propionate,
and ethanol, which are end products of bacterial fermentation,
accumulated in the incubation medium, whereas lactate and formate,
which are intermediates, decreased in the course of the incubations
(MacFarlane and Gibson, 1996). Their kinetics of formation or
degradation were different according to the experimental conditions;
this results from the limitation of available free glucose and proves
that metabolism of free glucose was quicker than hydrolysis of the
glycone moiety of glucosinolates.
In this work, 1D and 2D TOCSY and gCOSY 1H NMR
experiments were used both to elucidate the molecular structure of
glucosinolate derivatives and to quantify the concentrations of
metabolites. Different compounds of biochemical interest were analyzed
simultaneously in NMR spectra, including SIN, GTL, allylamine, and
benzylamine. Considering that the preparation of one sample takes 5 min
and that recording a 1D 1H NMR spectrum and a 2D
1H NMR spectrum takes about 10 min and 60 min,
respectively, NMR spectroscopy is a very powerful technique and can be
used routinely. Another advantage is that this technique is without
a priori hypothesis; consequently, unexpected metabolites, such
as the amine derivatives, can be detected. Although
1H NMR is used increasingly in pharmacology and
medicine to analyze biological fluids, this work represents the first
application to the study of the degradation of naturally occurring
xenobiotics from edible plants by the human digestive microflora. Wolin
et al. (1998, 1999) applied 13C NMR for studying
carbon metabolism by the human digestive microflora, using
[3-13C]glucose as a substrate. In regard to
xenobiotics, no 13C-enriched molecules are
commercially available, and thus, 1H NMR is the
most suited current methodology.
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Acknowledgments |
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We thank Dominique Harakat for excellent technical work in performing GC-MS spectra. We thank Professor D. Aitken for carefully reading the manuscript.
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Footnotes |
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Received March 19, 2001; accepted July 23, 2001.
This research was supported by the European Community under the program FAIR CT97 3029 entitled Effects of Food-Borne Glucosinolates on Human Health and by a fellowship awarded to one of the authors (L.E.) by the French Ministry of Education, Research, and Technology.
Anne-Marie Delort, Laboratoire Synthèse Electrosynthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504 du CNRS, Université Blaise Pascal, 63177 Aubière Cedex, France. E-mail: amdelort{at}chimtp.univ-bpclermont.fr
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Abbreviations |
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Abbreviations used are: 1D, one dimensional; 2D, two dimensional; SIN, sinigrin; GTL, glucotropaeolin; HPLC, high-pressure liquid chromatography; TSPd4, 3-(trimethyl-silyl)-1-propanesulfonic acid-tetra deuterated; TOCSY, total correlation spectroscopy; GC-MS, gas chromatography-mass spectrometry; gCOSY, gradients-correlation spectroscopy.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion and Conclusion
References
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, 
) and benzylamine (
, 
) measured
by 1H NMR (see Fig. 
), glucosinolates (X) and glucose (