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1
*
Laboratory of Medicinal Chemistry and Radiopharmacy and
Laboratory of Biomedical Magnetic Resonance, Université Catholique de Louvain, B-1200 Brussels, Belgium
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • electron paramagnetic resonance • licorice • food • gut • mice • cachou
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Goda et al. 1996
), monitoring of pharmacokinetics of
paramagnetic spin labels (Gallez et al. 1996
), detection
of short-lived radicals by spin-trapping techniques
(Jiang et al. 1995
) and formation of free radicals from
therapeutic drugs (Mäder et al. 1995
). To date,
most EPR studies carried out with animals have involved the use of
spectrometers operating at 1 GHz, which constitutes a good compromise
between the sensitivity of the detection (signal-to-noise ratio) and
the depth of wave penetration into tissues (1 cm). Such studies could
be applied to larger samples or even to humans, thanks to the
development of EPR spectrometers operating at lower frequency,
typically in the MHz range with a higher wave penetration (several
centimeters) (Halpern and Bowman, 1991
).
Observing the fate of free radicals arising from food after oral
administration could be important in evaluating their reactivity in
vivo. However, no report exists to date in the literature that relies
on applications of in vivo EPR to detect the fate of free radicals
coming from food. The aim of this study was to demonstrate that it is
feasible to detect directly in vivo free radicals coming from food
using low frequency EPR spectroscopy. Because polyphenols are easily
oxidized into stable radicals, we assumed that these radicals could be
detected in food. We chose licorice, which contains several types of
polyphenols, compounds that are easily oxidized into radicals
(Hatano et al. 1997
). We first demonstrate that stable
free radicals were present in the licorice-flavored sweets. Then,
using low frequency EPR spectrometer, we were able to detect these free
radicals directly and noninvasively after oral administration to mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; they were analyzed using Bruker ER-200 tt and a EMX-320 X-Band
(9 GHz; Bruker, Ettlingen, Germany) EPR spectrometers for the
characterization of the radicals by their g-value and peak-to-peak
linewidth. The g-value is related to the line position through the
resonance condition h
= gßB, where h is Planck’s constant,
is the microwave frequency, ß is the Bohr magneton and B is the
applied magnetic field. The linewidth is the distance between the
maximum peak and the minimum peak of the first derivative spectrum.
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). Great sensitivity
of detection was obtained by laying the mouse stomach down on top of
the coil so that it was close to the area of reception. The mouse was
placed on the same spot during the course of the study. Because the
sensitivity of the measurement decreases dramatically when the distance
from the resonator increases, and because the stomach region was put
directly on the loop resonator, the measurements essentially reflect
the fate of these radicals in the stomach region. Animal protocols were
approved by the local ethics committee. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For a given spin concentration, narrow lines imply large
amplitudes and hence a higher detectability compared with broad lines.
Therefore, sample 11, which presented a relatively strong EPR signal,
was used further for in vivo experiments. Only for this sample was it
possible to record an EPR signal with a reasonable signal-to-noise
ratio using the 1.1-GHz EPR spectrometer. A typical EPR spectrum
recorded in vivo and the kinetic monitoring of the EPR signal
intensities are shown in Figure 1
. The spectrum (upper panel, A) is compared with
an EPR spectrum recorded from the stomach of a food-deprived mouse
(upper panel, B). No EPR signal was observed in
the latter case. After administration of sample 11, the signal
intensity (height of the first derivative peak) recorded from the
stomach region was rather stable because the signal decreased only
~20% over the time of observation (Fig. 1
, lower panel).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
These compounds, called chalcones, were able to oxidize into stable
radicals as demonstrated by EPR spectroscopy (Hatano et al. 1997
). Because polyphenols are easily oxidized into radicals,
we assumed that free radicals could be detected in preparations
containing licorice. A first aim of this study was to demonstrate the
presence of free radicals in licorice preparations, particularly in
sweets.
In the study of Hatano et al. (1997)
, which elucidated
the structure of the chalcones, the EPR spectrum (obtained after
air-oxidation of licorice phenolics in alkaline solution) presented
a hyperfine splitting pattern (coupling of the unpaired electron to
nuclei in the molecule). Using our preparations, neither the crude
preparations nor the air-oxidized alcaline solutions of sweets
presented a hyperfine splitting. The EPR spectrum obtained in our case
could be the sum of different oxidized phenolics leading to an
unresolved EPR spectrum with a broad line. One of the samples (sample
11) presented a narrow EPR line. The EPR spectrum is very intense and
sharp, probably due to intermolecular electron spin exchange. This
narrow EPR signal could be the result of modification of the
microenvironment of the paramagnetic centers during the preparation of
these sweets.
Direct evidence of the presence of radicals was obtained in vivo using
low frequency EPR spectrometers. Low frequency permits measurement in
live animals due to the increased microwave penetration. A great
sensitivity of detection was obtained by laying the mouse stomach down
on top of the coil so that the organ under study was close to the area
of reception. It was possible to record an EPR signal in vivo with a
reasonable signal-to-noise ratio. The signal intensity recorded from
the stomach region was rather stable because the signal decreased only
~20% over the time of observation (Fig. 1)
. That indicates a very
low reactivity of these radicals in the biological media. It is likely
that the decrease in this signal was due to the gastric emptying rather
than a reactivity of the radical toward biological components present
in the stomach.
In conclusion, this study is the first report demonstrating
noninvasively and in vivo the presence of free radicals coming from
food. We are further investigating the possibility of monitoring, using
low-frequency EPR, the fate of other polyphenols present in food
(e.g., fruits, vegetables or tea) and of free radicals coming from
irradiated food. This study emphasized that the noninvasive detection
of free radicals in the gut using low frequency EPR is not limited to
the use of spin labels (Gallez et al. 1996
).
Manuscript received December 13, 1999. Revision accepted February 21, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1. Gallez B., Mäder K., Swartz H. M. Non invasive measurement of the pH inside the gut using pH-sensitive nitroxides: an in vivo EPR study. Magn. Reson. Med. 1996;36:694-697[Medline]
2. Gallez B., Jordan B., Baudelet C., Misson P. D. Pharmacological modifications of the partial pressure of oxygen in tumors. Evaluation using in vivo EPR oximetry. Magn. Reson. Med. 1999;42:627-630[Medline]
3.
Goda F., Bacic G., O’Hara J. A., Gallez B., Swartz H. M., Dunn J. F. The relationship between pO2 and perfusion in two murine tumors after X-ray irradiation: a combined Gd-DTPA dynamic MRI and EPR oximetry study. Cancer Res 1996;56:3344-3349
4. Halpern H. J., Bowman M. K. Low-frequency EPR spectrometers: MHz range. Eaton G. R. Eaton S. Ohno K. eds. EPR Imaging and In Vivo EPR 1991:45-63 CRC Press Boca Raton, FL.
5. Hatano T., Takagi M., Ito H., Yoshida T. Phenolic constituents of liquorice. VII. A new chalcone with a potent radical scavenging activity and accompanying phenolics from liquorice. Chem. Pharm. Bull. 1997;45:1485-1492
6. Jiang J., Liu K. J., Shi X., Swartz H. M. Detection of short-lived free radicals by low-frequency electron paramagnetic resonance spin-trapping in whole live mice. Arch. Biochem. Biophys. 1995;313:248-252
7. Mäder K., Bacic G., Swartz H. M. In vivo detection of anthralin derived free radicals by low frequency electron paramagnetic resonance spectroscopy. J. Investig. Dermatol. 1995;104:514-517[Medline]
8. Nilges M. J., Walczak T., Swartz H. M. 1 GHz in vivo ESR spectrometer operating with a surface-probe. Phys. Med. 1989;2:195-201
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