Appareil Digestif, Environnement et Nutrition, Institut Fédératif de Recherche Multidisciplinaire sur les Peptides, and Centre d'Investigation Clinique-Institut National de la Santé et de la Recherche Médicale-Centre Hospitalier Universitaire, Rouen, France
Submitted 6 September 2002 ; accepted in final form 1 April 2003
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ABSTRACT |
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intestine; protein metabolism; nutrients
Additionally, enteral infusion of a high glutamine load in volunteers
altered whole body leucine fluxes in a manner indicating a reduced protein
oxidation and an increased protein synthesis
(16). The beneficial effects
of glutamine on gut mucosa could be partly due to a stimulation of protein
synthesis as shown in animal studies, in vitro
(17) and in vivo
(41). In previous studies, we
have shown that glutamine was well absorbed in human intestine
(13) and stimulated 40%
duodenal protein synthesis in hypercatabolic subjects, although this effect
only approached statistical significance
(4). Furthermore, an
improvement of protein balance in the gut could result from an inhibition of
proteolysis. In main organs such as the liver and the muscle, the degradation
of proteins results from the activity of the three major systems, the
lysosomal (cathepsins), the Ca2+-activated (calpains),
and the ATP-ubiquitin-dependent proteolytic pathways. These pathways have been
reported in rat intestinal mucosa
(35), but data in human mucosa
are still limited. The aim of this study was to investigate the effects of
enteral glutamine on gut mucosal protein synthesis and on the mRNA levels for
the main proteolytic systems in healthy volunteers. The effects of glutamine
on whole body protein turnover were also assessed.
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MATERIALS AND METHODS |
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Twenty healthy volunteers, male and female, participated in the study. They were 22 (median, range 2025) yr old and had a mean body mass index of 21.7 kg/m2 (19.523.9). All subjects were in good general health with no hepatic, renal, or cardiac dysfunction or any medical or surgical digestive past history. The study was approved by the local ethics committee, and the subjects gave their written informed consent. The subjects were divided into two groups of 10 subjects (group 1 and group 2), and each subject was studied on two occasions in random order. During 3 days, all subjects consumed a controlled diet providing 30 kcal and 1.2 g protein·kg body wt-1·day-1. The meals were prepared by the hospital dietary unit. The study was started at 8:00 AM on day 4 after a 12-h overnight fast. A nasogastric feeding tube was placed in the stomach. Subjects from group 1 received during 6 h either glutamine (0.8 mmol·kg body wt-1·h-1 in saline) or the same infusion rate of normal saline (4.5 ml·kg body wt-1·h-1), in a random order with a 15-day interval among studies. Subjects from group 2 received either glutamine (0.8 mmol·kg body wt-1·h-1) or an isonitrogenous and isoosmolar mixture of amino acids (Gly/Ala/Ser/Pro/Asp/Asn/His, 2/2/4/4/7/10/10). We conceived this amino acid solution instead of a balanced solution with all 20 amino acids because we wanted this control solution to be both isonitrogenous and isoosmolar to glutamine to limit any interference due to fluid movements and variations of intestinal cell volume. Thus enterally infused solutions were isoosmolar (saline: 308 mosM; glutamine: 311 mosM; nonessential amino acids: 310 mosM), and rate of fluid infusion was similar (4.5 ml·kg body wt-1·h-1). After baseline blood and air samples were obtained, a bolus infusion of tracers was performed providing 6 µmol/kg body wt of [L-ring-2H5]phenylalanine [90% mol percent enrichment (MPE); Mass Trace, Woburn, MA], 6 µmol/kg body wt of [L-1-13C]leucine (99% MPE; Mass Trace) and 6 µmol/kg body wt of [L-2H4]tyrosine (99% MPE; Mass Trace). After the bolus, 6 µmol·kg body wt-1·h-1 of [L-ring-2H5]phenylalanine and 6 (group 1) or 4 (group 2) µmol·kg body wt-1·h-1 of [L-1-13C]leucine were intravenously infused during 6 h. In addition, subjects from group 1 received 6 µmol·kg body wt-1·h-1 of [L-2H3]leucine (99% MPE; Mass Trace) via the nasogastric tube during 6 h. Tracers were tested for sterility and pyrogenicity and were diluted in saline in the hospital pharmacy on the evening before study and kept at 4°C until use.
Arterialized blood samples were taken from a vein in the contralateral hand of the intravenous infusions, the hand being placed in a heating box, before the infusion (baseline sample) and at regular intervals during the last 2 h of infusion. Before and during isotope infusion, carbon dioxide production was repeatedly measured over 20-min periods by means of indirect calorimetry (Deltatrac II; Datex, Helsinki, Finland); the variation coefficient of VCO2 measurement was <10%. Breath samples were collected in bags for analysis of 13CO2 enrichment by infrared spectrometry (IRIS; Wagner).
The upper endoscopy (Olympus XQ10) was performed, for practical and safety reasons, 30 min after the end of the intravenous tracer and enteral amino acid infusions. At endoscopy, intragastric residual volumes ranged from 20 to 150 ml, which were aspirated before biopsy sampling in the duodenum. Eight mucosal biopsies were taken from the distal duodenum: two biopsies were fixed in formalin for histological assessment and two series of three biopsies were immediately frozen in liquid nitrogen, either as such for isotope analysis or sunk in guanidium isothiocyanate for mRNA analysis, and stored at -80°C.
Sample Analysis
Mucosal tissue samples were processed as previously described (4). Briefly, mucosal tissue samples were quickly rinsed in ice-cold 0.9% NaCl and immediately ground. Proteins were precipitated with 10% TCA, and free amino acids were then separated from the supernatant. The supernatant was prepared as plasma to measure free amino acid enrichment. The protein pellet was dissolved in 1 M NaOH and then hydrolyzed in 6 M HCl at 110°C for 18 h to allow analysis of the enrichment of amino acid released from protein hydrolysis.
The enrichments of [2H5]phenylalanine, [13C]leucine, [13C]ketoisocaproate ([13C]KIC), and [2H3]leucine were determined in the plasma, the mucosal intracellular free amino acid pools, and the mucosal proteins by gas chromatography-mass spectrometry (model MSD 5972; Hewlett Packard, Palo Alto, CA) by using tert-butyldimethylsilyl derivatives as previously described (4, 8). Appropriate standard curves were run simultaneously for determination of the enrichments.
Calculations
The fractional synthesis rate (FSR) of duodenal mucosal protein was calculated as follows: FSR (%/day) = (Et - Eo)/Ep x 1/t x 24 x 100, where Et is the enrichment in tissue protein at time t (%); Eo is the natural abundance of the labeled amino acid in intestinal mucosal protein (%); Ep is the enrichment of the precursor pool at plateau (%); and t is the duration of the tracer infusion (h). The baseline isotopic enrichment was determined in normal duodenal biopsies from ambulatory patients undergoing endoscopy for medical reasons. The precursor pool used was the intracellular free amino acid pool. Whole body leucine, KIC, and phenylalanine fluxes were calculated according to standard isotopic dilution equations as previously described (8), by using steady-state plasma enrichment level during the last 2 h of continuous tracer infusion. Although [2H4]tyrosine enrichment was measured, tyrosine kinetics were estimated from phenylalanine kinetics as previously described (40).
Plasma amino acid concentrations. Plasma amino acid concentrations were measured by using an amino acid analyzer (Biotronik LC3000; Eppendorf).
Plasma hormone concentrations. Plasma concentrations of insulin and IGF-I were analyzed by radioimmunoassay.
RT-PCR. Mucosal RNA were extracted from biopsies by a modified
extraction method as previously described
(9). The quality and quantity
of total RNA were determined by spectrophotometry by using the absorbance at
A260/A280 nm. The integrity was also controlled by
visualization of 18S and 28S ribosomal bands. RNA expression of ubiquitin,
cathepsin D, and m-calpain was studied by RT-PCR. The RT reaction was
performed as previously described
(9). RT products were amplified
by PCR by using sense and antisense primers specific for proteolytic systems
and GAPDH as an internal standard: ubiquitin,
5'-GTTGAGCCCAGTGACACCAT-3' and
5'-CTCTGAGACGGAGCACCAG-3'; cathepsin D,
5'-CATTGTGGACACAGGCACTT-3' and
5'-GTAGTAGCGGCCGATGAAGA-3'; m-calpain,
5'-TTGAACAAGTCGCTGGTGAG-3' and
5'-GTAGACATGGTGCCGGACTT-3'; GAPDH,
5'-GTCATCCATGACAACTTTGG-3' and
5'-GAGCTTGACAAAGTGGTCGT-3'. The PCR reaction mixture (25 µl)
consisted of sense and antisense primers (50 pmol each), 1 unit of Thermoprime
Plus ADN Polymerase (ABgene), 200 µM of each of the four dNTPs, 1 x
PCR buffer (Promega) supplemented with 2.5 mM MgCl2 (Promega), 0.5
µCi of -33P]ATP (Amersham), and 5 µl of RT samples.
Simultaneous amplification was performed by 21 (ubiquitin, cathepsin D, and
GAPDH) or 28 cycles (m-calpain) consisting of denaturation for 30 s at
95°C, primer annealing for 40 s at 60°C, and primer extension for 40 s
at 72°C by using a thermal cycler (model PTC 200; MJ Research).
PCR products were electrophoresed on a 6% polyacrylamide gel. After 2 days of exposure the autoradiogram bands were analyzed by an image analyzer (Biocom; Lecphor). The levels of amplified product were normalized to constant amounts of GAPDH mRNA. The number of PCR cycles in each system was chosen within linear phase to use this assay as a relative measure of gene expression. For each RNA, RT-PCR was performed three times.
Statistical Analysis
Results are expressed as median (range). To evaluate the effects of glutamine, amino acids, or saline, statistical analyses were assessed by using a nonparametric paired (Wilcoxon) or unpaired test (Mann Whitney U-test).
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RESULTS |
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Plasma, intracellular, and protein enrichments are displayed in Table 1. In group 1, FSR was increased after glutamine infusion compared with saline infusion whatever tracer was used (Fig. 1): intravenous tracers (P < 0.05 for [2H5]phenylalanine and P = 0.06 for [13C]leucine) or enteral tracer (P < 0.05 for [2H3]leucine). The plateau of plasma enrichments was obtained after 3 h of infusion for the different tracers (data not shown). After glutamine infusion, both leucine endogenous rate of appearance (Ra) and nonoxidative disposal (Table 2) decreased by 10% (P < 0.05) compared with saline infusion. Phenylalanine fluxes were not affected by glutamine infusion.
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The mRNA level for ubiquitin was significantly decreased (P < 0.05, Table 3) after glutamine infusion compared with saline infusion, whereas cathepsin D and m-calpain mRNA levels remained unchanged.
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Enteral glutamine infusion increased plasma glutamine concentration compared with baseline (P < 0.05, Table 4). Plasma essential amino acid concentrations decreased after both saline and glutamine infusions compared with baseline (P < 0.05); plasma essential amino acid concentrations were significantly lower after glutamine compared with saline infusion (P < 0.05).
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Enteral glutamine infusion increased plasma insulin concentration at 6 h compared with saline (P < 0.05) but had no influence on IGF-I concentration (Table 5).
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Group 2: Glutamine vs. Amino Acids Infusion
In group 2, whatever the tracer used, FSR was increased to the same extent by glutamine and amino acid infusion compared with saline infusion (P < 0.05, unpaired test); there was no significant difference for FSR after glutamine or amino acid infusion (Table 1). Phenylalanine and leucine fluxes were not different between glutamine and amino acid infusion, except for a slight yet significant (P < 0.05) increase of leucine oxidation after glutamine infusion (Table 2).
The ubiquitin mRNA level was significantly decreased after glutamine compared with amino acid infusion (P < 0.05, Table 3). The intraindividual comparison of mRNA expression (Fig. 2) showed that the mRNA level decreased in 9 of 10 subjects for ubiquitin and in 6 of 10 subjects for cathepsin D. However, cathepsin D and m-calpain mRNA expression was not statistically different after glutamine or amino acid infusion.
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Plasma nonessential amino acids increased after both amino acids and glutamine infusions compared with baseline (P < 0.05, Table 4). Plasma glutamine concentration was also increased after both amino acid and glutamine infusions compared with baseline (P < 0.05). Essential amino acid concentration was decreased only after glutamine infusion compared with baseline (P < 0.05).
Plasma insulin concentration at 6 h was increased to the same extent by glutamine and amino acids compared with saline (P < 0.05, Table 5). IGF-I concentration was not significantly influenced.
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DISCUSSION |
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Available studies (3,
29) on human gut mucosal
protein synthesis are still limited and have measured the FSR of gut mucosal
protein mainly in the postabsorptive state in healthy humans. In these
studies, duodenal protein FSR approached 3050% per day, a value much
higher than that of other tissues such as liver or muscle
(32). Surprisingly, feeding
had no effect on gut FSR (3).
In celiac patients, gut FSR was still higher than in healthy subjects,
probably due to the high proliferation rate of the mucosa
(28). In contrast, jejunal FSR
was reduced in short-bowel patients treated with a somatostatin analog
(31). In a previous study from
our group (4), a moderate
enteral glutamine supply increased gut FSR 40% in hypercatabolic humans,
an effect however, that only approached statistical significance (P =
0.069) in this small group of subjects. In controlled clinical studies with
glutamine in patients with short-bowel syndrome, only modest effects on gut
absorptive functions were reported; however, gut protein synthesis was not
assessed in these studies (5,
36). In the present study, by
using both intravenous and enteral tracers (group 1), a high load of
enteral glutamine increased markedly (almost twofold) mucosal protein FSR
compared with saline. Gut mucosal FSR measured with the enteral leucine tracer
was still higher than with both intravenous tracers, which is in accordance
with previous reports (44)
showing that gut mucosa preferentially incorporates luminal amino acids for
its protein synthesis. In group 2, FSR was increased to a similar
extent by infusion of an isonitrogenous mixture of amino acids, indicating
that a high load of glutamine or nonessential amino acids is able to stimulate
gut FSR. In the present study, intravenous infusion of tracers was stopped 30
min before sampling of biopsies. The calculation of FSR relies on the
enrichment in the amino acid intracellular pool and in the mucosal protein at
the time of sampling of the biopsy. Although it cannot easily be measured
repeatedly in the duodenum, it can be assumed that intracellular enrichment
increases over time in parallel to plasma enrichment; achievement of a steady
state of duodenal intracellular free amino acid enrichment after 2 to 3 h as
for plasma enrichment (3,
4) looks likely. In our present
study, although the enrichment may begin to decline both in the intracellular
and mucosal protein pool over 30 min after the end of tracer infusion, this
seems unlikely to induce a major bias in the calculation of FSR and especially
hamper its comparison among groups studied with the same protocol. Indeed,
both leucine and phenylalanine intravenous tracers, although having different
behavior in the splanchnic bed, led to similar estimates of FSR and showed its
increase in response to glutamine. In addition, we have compared this study
with previous ones by means of tissue/plasma enrichment ratios. Indeed, by
using the phenylalanine enrichments, which are the most accurate ones, the
ratio of protein to plasma enrichment in the present study averaged 0.06 and
0.07 in the saline and glutamine groups, respectively, which is quite similar
to 0.05 and 0.05 in the control and glutamine groups, respectively, from our
previous study (4). Similarly,
the ratio of intracellular to plasma enrichment in the present study averaged
0.31 and 0.26 in the saline and glutamine groups, respectively, which is quite
similar to 0.34 and 0.24 in the control and glutamine groups, respectively,
from our previous study (4).
Thus it seems likely that the FSR results in our present study are good
estimates and should not differ markedly from that measured during ongoing
tracer infusion. In addition, in the present study, subjects from group
1 also received an enteral infusion of tracer,
[2H3]leucine, together with intravenous
[2H5]phenylalanine and [13C]leucine. This
enteral tracer infusion was stopped at the same time as the enteral glutamine
or saline infusion, i.e., 30 min before biopsies were taken. Yet, at the
beginning of the endoscopy, intragastric residual volumes ranged from 20 to
150 ml. So [2H3]leucine was still present in the
gastrointestinal lumen at the time of biopsy sampling. With the use of
[2H3]leucine enrichment in intracellular pool and
mucosal protein pool, calculated FSR confirms the results obtained with
intravenous tracers.
In the present study, glutamine intake is about four times that used in our previous study (4), and the total nitrogen intake as glutamine or amino acid mixture is in the same range (0.020.03 g N·kg body wt-1·h-1)as that given by means of full enteral nutrition in previous studies (3, 4). However, in our control group, the nitrogen intake is provided only as a mixture of nonessential amino acids, whereas the fed group in the study by Bouteloup-Demange et al. (3) received a mixed protein meal. These authors observed no significant effect of feeding on mucosal FSR. It is difficult, however, to make a direct comparison between the present study and that from Bouteloup-Demange et al. (3), because in the latter, subjects received a complete energy and protein diet. Among other differences with our amino acid mixture, this diet included branched-chain amino acids, which may have favored protein synthesis in the muscle, or in the liver, rather than in the gut (26). We also observed that our control nonessential amino acid mixture did stimulate gut FSR as potently as glutamine. This could be due either to the anabolic effects of one or several of the amino acids contained in the mixture or to the stimulation of glutamine de novo synthesis from these amino acids in the gut. Indeed, glutamine synthetase is very active in human stomach and also present in human duodenal mucosa (21). It may thus be hypothesized that the nonessential amino acids could have stimulated glutamine synthesis in gastrointestinal mucosa and thus mimic the effect of glutamine. Alternatively, the effects of the nonessential amino acids may have been mediated, in part, by systemic glutamine, because an increase of plasma glutamine concentration after amino acid mixture infusion was observed (Table 4). Our evaluation of whole body protein turnover from leucine and phenylalanine fluxes failed to detect a relevant effect of glutamine on whole body protein turnover. Fluxes calculated from KIC enrichments gave estimates of protein turnover similar to that of leucine (data not shown). This is at variance with a previous study indicating a 1215% significant increase of whole body protein synthesis and reduction of protein oxidation estimated from leucine fluxes (16). However, in this latter study, glutamine was compared with a high glycine load. In the present study, glutamine infusion, but not amino acid mixture infusion, induced a decrease of plasma essential amino acids. This was likely due to the increase in insulin secretion and its suppressive effect on whole body proteolysis as suggested from the leucine Ra data (Table 2).
Protein metabolism in the gut results from the balance between synthesis and degradation. However, the regulation of proteolysis in human gut has been poorly documented until now, and we are not aware of studies reporting the mRNA levels for components of the proteolytic pathways in human gut. The influence of nutrients on gut proteolysis has been studied only in animals (1, 30). Several pathways for the degradation of protein have been described in major organs such as the liver and the muscle (39), including the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways. This last pathway is believed to be the most important one (39). In the gastrointestinal tract, little information is available, although all three processes have been identified in the small intestine of the rat (19, 35). The expression of the components of proteolytic pathways was increased during fasting in rats (35) but not during short-term underfeeding in ewes (30). Increased gut proteolysis may contribute to mucosal atrophy in the absence of adequate nutritional supply, in addition to a reduction of mucosal protein synthesis (43).
Cathepsin D is the major aspartic proteinase of the lysosomal compartment, contributing to the degradation of long-lived cytoplasmic proteins. It has been reported that cathepsin D may contribute to intestinal damage and to the activation of systemic inflammatory response after hemorrhagic shock in rat (14). During total parenteral nutrition in rats, intestinal cathepsin activity increases and could be implicated in gut barrier alteration (24). In the present study, the mRNA level for cathepsin D in duodenum was not modified by glutamine infusion.
The Ca2+-activated calpain system is ubiquitous in cells but has rarely been documented in intestine. The mRNA level for m-calpain in intestine increases during fasting in rats (35). In addition, m-calpain has been implicated in the differentiation of enterocytes (19). The use of a calpain inhibitor in a model of experimental colitis in rat reduced the degree of colitis (11). In piglets, a mixture of luminal amino acids did not influence intestinal mRNA level for m-calpain in isolated jejunal segments (1). In the present study, the mRNA level for m-calpain was not influenced by enteral infusion of glutamine compared with saline (group 1) and was not different after glutamine or amino acid infusion (group 2), suggesting that the Ca2+-activated proteolytic pathway is not influenced by nitrogen intake in human duodenum.
The ATP-ubiquitin-dependent proteolytic pathway associated to proteasome is
known to degrade short-lived or abnormal proteins. This system could be
important for the turnover of gut mucosal proteins, which are very short lived
(7). In rat duodenum, ubiquitin
expression is modulated by feeding
(18). In an in vitro model of
piglet jejunal segment, luminal amino acids markedly decreased the intestinal
mRNA level for ubiquitin (1).
In the present study, glutamine infusion significantly decreased ubiquitin
mRNA level in human duodenal biopsies compared both with saline and with an
isonitrogenous mixture of amino acids. Therefore, glutamine may limit mucosal
protein degradation through the ATP-ubiquitin-dependent proteolytic pathway,
an effect that seems to be specific for glutamine and was not documented
previously in any tissue. The ATP-ubiquitin-dependent proteolytic pathway also
participates in the regulation of the inflammatory response. Indeed, IB
ubiquitinylation allows NF-
B translocation in nucleus and thus
transcription of proinflammaory genes
(22), including IL-8
(34). In contrast, an
inhibitor of proteasome reduced NF-
B activation
(23). In an experimental model
of colitis in rats, pretreatment with glutamine decreased intestinal mucosal
lesions and intestinal IL-8 production
(2). We also reported that
enteral glutamine reduced IL-8 production by cultured duodenal mucosa from
healthy subjects (9). Decreased
ubiquitin mRNA level, as observed in the present study, could explain the
previously observed influence of glutamine on IL-8 production
(2,
9,
12). Indeed, glutamine may
decrease I
B ubiquitinylation and consequently limit NF-
B
translocation and, finally, IL-8 gene transcription. Accordingly, glutamine
prevented LPS-induced activation of NF-
B in human intestinal epithelial
cells (27). Thus nutritional
modulation of the ubiquitin pathway may be critical to limit intestinal
inflammation and mucosal damage.
The mechanism by which glutamine and amino acids modulate mucosal protein metabolism deserves further investigation. Insulin may contribute, in part, to glutamine and amino acid effects on FSR, because a moderate increase of plasma insulin concentration was observed after both glutamine and amino acid infusion. Indeed, recent data indicate that insulin can support intestinal protein synthesis (6). On the other hand, insulin reduces muscular ATP-ubiquitin-dependent proteolytic pathway activity (10); however, in the present study, the inhibiting effect of glutamine on intestinal ubiquitin mRNA is unlikely mediated by insulin, because insulin was not differentially affected by glutamine compared with amino acid mixture infusion. An indirect effect of glutamine through IGF-I secretion seems also unlikely, because IGF-I secretion was not altered.
In summary, enteral infusion of glutamine in humans stimulates protein synthesis in a nonspecific way but may limit mucosal proteolysis through a specific inhibition of the ubiquitin pathway. Glutamine may thus be beneficial to gut protein balance in humans. Whether the antiproteolytic effect of glutamine may also contribute to the regulation of the inflammatory response in intestinal mucosa deserves further investigation.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* M. Coëffier and S. Claeyssens contributed equally to this work.
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REFERENCES |
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