Effect of dietary galacto-oligosaccharides on azoxymethane-induced aberrant crypt foci and colorectal cancer in Fischer 344 rats
M.V.W. Wijnands1,3,
H.C. Schoterman2,
J.P. Bruijntjes1,
V.M.H. Hollanders1 and
R.A. Woutersen1
1 TNO Nutrition and Food Research, Department of General Toxicology, Utrechtseweg 48, PO Box 360, 3700 AJ Zeist and
2 Borculo Domo Ingredients, PO Box 46, 7270 AA, Borculo, The Netherlands
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Abstract
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The aim of the present study was to investigate the effects of galacto-oligosaccharides (GOS, Elix'or) on the development of aberrant crypt foci (ACF) and colorectal tumours in rats treated with azoxymethane (AOM). Two groups of 102 male Fischer 344 rats were injected twice with AOM to induce colorectal tumours, and fed diets containing either a low [5% (w/w); LGOS] or a high [20% (w/w); HGOS] concentration of GOS. Four weeks after the last AOM injection, 18 animals from each group were killed and their colon was removed for scoring ACF. Half of the animals in the LGOS group were switched to an HGOS diet (L/HGOS) and half of those in the HGOS group to an LGOS diet (H/LGOS). Six weeks after the change in diet, nine animals per group were killed for scoring ACF. Ten months after the start of the study the remaining animals were killed for scoring colorectal tumours. The aberrant crypt multiplicity scored after 13 weeks and the colorectal tumour incidence in rats fed an HGOS diet were significantly lower than those in rats fed an LGOS diet. However, the induction of ACF by AOM, the proliferation rate and apoptotic index of the adenomas, and the size and multiplicity of colorectal tumours were not influenced by the amount of GOS in the diet. The aberrant crypt multiplicity, scored after 13 weeks, was predictive for the tumour outcome at the end of the study. It was concluded that an HGOS diet has a protective effect against the development of colorectal tumours in rats and that this protective effect is exerted during the promotion phase rather than the initiation phase of carcinogenesis.
Abbreviations: AC, aberrant crypt; ACF, aberrant crypt focus; AOM, azoxymethane; DMH, 1,2-dimethylhydrazine; GOS, galacto-oligosaccharides; LGOS, low GOS; HGOS, high GOS; SCFA, short-chain fatty acids.
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Introduction
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Over the last decades, dietary fibre has been recognized as an important food constituent. There is evidence from many epidemiological studies and animal models that it has protective properties against cancer, especially cancer of the alimentary tract, in humans and animals (18). Dietary fibre is, by definition, not digestible by the enzymes of the small intestines, but fermentable dietary fibres are degraded by microbial fermentation in the large intestines. Galacto-oligosaccharides (GOS) belong to the group of non-digestible carbohydrates that may be regarded as soluble dietary fibres, because they fit the generally accepted definition of dietary fibre including both biochemical and nutritional/physiological criteria (9). The main constituent of the GOS (Elix'or) syrup is a mixture of GOS produced by treatment of lactose with ß-galactosidase. The composition of the GOS is: (galactose)nglucose, where n is 17.
A previous experiment (10) showed that GOS were highly protective against the development of colorectal tumours in Wistar rats, treated with 10 weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH) at 50 mg/kg body weight, as was demonstrated by an inhibitory effect on tumour multiplicity (mean number of tumours per tumour-bearing animal), tumour incidence (percentage of tumour-bearing animals) and tumour size. The multiplicity of colorectal tumours in animals fed a high-GOS (HGOS) diet was statistically significantly lower than that in animals fed a low-GOS (LGOS) diet. Both the incidence and size of colorectal tumours were lower in animals fed a high-GOS diet than in those fed a low-GOS diet, although the differences were not statistically significant.
The aim of the present study was to investigate the effects of GOS on the development of aberrant crypt foci (ACF) and tumours in the colon and rectum of rats treated with azoxymethane (AOM). AOM, a metabolite of DMH, is a potent carcinogen. Two injections of AOM, 1 week apart, are sufficient to induce colorectal cancer in rats, resulting in a much shorter initiation phase than in the DMH model. This makes the AOM model more suitable for studying events taking place during either the initiation or promotion phase of carcinogenesis. It is generally accepted that carcinogenesis is a multihit/multistep process in which a tumour can develop after exposure of cells to an initiator, followed by exposure to a promoter. During the initiation phase a carcinogen causes changes in a target tissue, such that the altered cells become susceptible to the promotional effects of a promoter. During the promotion phase the initiated tissue may develop focal proliferative lesions, some of which may undergo further changes leading to the development of a malignant tumour (11).
ACF are considered putative preneoplastic lesions, which may develop into colorectal tumours. ACF do not occur in untreated rats, but appear in the colon and rectum within a few weeks (during the post-initiation phase) after treatment with carcinogens such as DMH or AOM.
In order to investigate whether the expected effect of GOS on tumorigenesis occurs during the initiation phase or the promotion phase, the LGOS diets were replaced by an HGOS diet, and vice versa, 7 weeks after the start of the study. Ten months after the start of the experiment the animals were killed and examined for the presence of colorectal tumours. The number, size and distance from the anus of all colorectal tumours were recorded. The growth rate of adenomas was studied by counting the relative number of proliferating and apoptotic adenoma cells after making the typical nuclear changes visible. The growth of a tumour is largely determined by the balance between increase in cell numbers (proliferation) and loss of cells by apoptosis. Modulation of either of these processes may play a role in enhancement or inhibition of carcinogenesis.
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Materials and methods
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Animals, diets and time schedule
Two hundred and four male specific-pathogen free Fischer 344 rats (Harlan Sprague Dawley, Indianapolis, IN, USA), 3 weeks old, were divided into two groups of 102 animals each. Rats were fed an AIN93-based diet containing a low [5% (w/w); LGOS] or high [20% (w/w); HGOS] concentration of GOS (Elix'or; Borculo Domo Ingredients, Borculo, The Netherlands). The diets contained approximately equal amounts of vitamins and minerals per unit of energy; they were prepared freshly every 2 months and stored at 20°C until use. GOS was a syrup containing 75% (w/w) dry substance. The composition of the dry substance was (by weight): 58.8% galacto-oligosaccharides, 21.3% lactose, 19.3% glucose and 1.1% galactose. The syrup was mixed with water to yield a GOS syrup containing 65% (w/w) dry substance. The GOS syrup was added to the diets in place of some of the wheat starch and water. The composition of the experimental diets is summarized in Table I
. Seven weeks after the start of the study (i.e. 4 weeks after the last AOM treatment), 18 animals from the LGOS and HGOS groups were killed and their colon removed for scoring ACF. Half of the animals in each group were then switched to the other diet. Thus, two additional experimental groups were formed: L/HGOS (which received the LGOS diet for the first 7 weeks of the study and the HGOS diet thereafter) and H/LGOS (which received the HGOS diet for the first 7 weeks of the study and the LGOS diet thereafter). Six weeks after the change in diet, nine animals per group were killed for scoring ACF again. Ten months after the start of the study the remaining animals were killed. The time schedule is summarized in Figure 1
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Fig. 1. Design of the study. AOM, treatment with azoxymethane; ACF, scoring aberrant crypt foci; LGOS and HGOS, low-and high galacto-oligosaccharides. L/HGOS, this group received the LGOS diet for the first 7 weeks of the study and the HGOS diet thereafter. H/LGOS, this group received the HGOS diet for the first 7 weeks of the study and the LGOS diet thereafter.
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Treatment and housing
All animals were treated with two subcutaneous injections with AOM (Sigma, Brussels, Belgium), 15 mg/kg body wt, in the second and third week after the start of the experiment, respectively. The animals were housed in macrolon cages with bedding (three animals per cage) from the start of the study up to 3 weeks after the treatment with carcinogen. Thereafter, the animals were housed in suspended stainless steel cages (three animals per cage) with wire mesh floor and front. Food and tap water were available ad libitum. The relative humidity was kept between 30% and 70%. The number of air changes was ~10 per hour. Artificial light was supplied from fluorescent tubes, in a 12 h light12 h dark cycle. The food intake, body weight and clinical signs of all animals were recorded regularly. Moribund animals were killed and autopsy was performed.
Aberrant crypt foci
The colons of the animals killed at the interim autopsies were removed, opened longitudinally, rinsed with saline, fixed flat between filtration paper and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. ACF were made visible by staining the colons with 0.1% methylene blue in saline for ~7 min. ACF can easily be identified using a light microscope at 40x magnification. Aberrant crypts have large, usually elongated openings. The lining epithelial cells are larger and more intensely stained with methylene blue than the surrounding normal epithelial cells. The results of previous experiments of other investigators have indicated that the development of larger ACF (four or more crypts per focus) might have particularly good predictive value for tumour yield at the end of the experiment (12,13). Therefore, the number of aberrant crypts (AC) in each focus was recorded to determine the AC multiplicity.
Autopsy, histology and histopathology
Ten months after the start of the experiment, all remaining animals were killed by exsanguination from the abdominal aorta under ether anesthesia. A thorough autopsy was performed. The colon was removed, cut open longitudinally, rinsed with saline and examined for the presence of neoplastic changes. The number, size and distance from the anus of all colorectal tumours were recorded. The remaining parts of the colon were collected as `Swiss rolls'. Collected tissues were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde, embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Serial sections were made when necessary to expose the stalk, if present, of a tumour. The collected tissues were examined microscopically and the type of the tumours (benign or malignant) was established and recorded. Microscopic classification of the tumours was done according to the criteria described by Whiteley et al (14). The tumour incidence (percentage of tumour-bearing animals) and multiplicity (mean number of tumours per tumour-bearing animal) were determined for adenomas, carcinomas and total tumours (adenomas and carcinomas). Data on properties of tumours in groups maintained on the same diet during the last phase of the study (from week 13 until autopsy at about 10 months) were combined.
Proliferation labelling index
Sections of all colorectal adenomas found were stained with a monoclonal antibody against Ki-67 and examined by light microscopy. The Ki-67 staining protocol included the following: after deparaffination, the slides were incubated for 10 min in 3% hydrogen peroxide in methanol to block endogenous peroxidase. Slides were rinsed in distilled water, immersed in 10 mM citric acid pH 6.0 and boiled for 15 min for antigen retrieval. After cooling at room temperature for ~30 min, the slides were rinsed in phosphate-buffered saline (PBS) and covered with 25% goat serum for 15 min. They were then incubated with primary antibody (Ki-67, Novocastra) for 1 h. Slides were rinsed in PBS and then incubated in secondary antibody (biotinylated rabbit anti-mouse; Dako, Denmark) for 30 min. After rinsing the slides in PBS, horseradish peroxidase-conjugated streptavidin (Dako) was applied for 30 min. Finally, peroxidase activity was developed for 10 min with 5 mg diaminobenzidine (Sigma) in 10 ml PBS and 5 µl hydrogen peroxidase. To increase the staining intensity, 100 µl CoCl2 was added to the staining solution. The slides were counterstained with nuclear Fast Red. In the adenomas, all labelled and unlabelled nuclei in 10 randomly selected areas with a total area of 0.046 mm2 were counted. The labelling index was expressed as the percentage of nuclei counted that stained brown.
Apoptosis
Apoptotic cells in colorectal adenomas were visualized using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) method. After deparaffination, the endogenous peroxidase was blocked by immersing the slides in 0.3% hydrogen peroxide in methanol for 30 min. After rinsing three times in PBS, the slides were incubated in proteinase K (15 µg/ml in PBS pH 7.4; Sigma) for 15 min at 37°C. Slides were rinsed twice in PBS and dried around the sample; TUNEL reaction mixture (Boehringer, Germany) was added. The sections were covered by a coverslip to avoid evaporation and incubated at 37°C for 60 min. Slides were rinsed twice in PBS for 10 min and then dried around the sample; a converter-POD was added and the sections were covered by a coverslip and incubated at 37°C for 30 min. Slides were rinsed twice in PBS for 10 min, diaminobenzidine was added for 10 min and then slides were rinsed in running water and counterstained with haematoxylin. In the adenomas all labelled and unlabelled nuclei in 10 randomly selected areas with a total area of 0.046 mm2 were counted. The apoptotic index was expressed as the percentage of nuclei that stained positively.
Statistical analysis
The multiplicity of AC and ACF, the multiplicity and size of the colorectal tumours, the Ki-67 labelling index and the apoptotic index were analysed using one-way analysis of variance (ANOVA). Levene's test was used to test whether variances among the groups were homogeneous. If Levene's test indicated homogeneous variances, the groups were compared by one-way ANOVA for equal variances, followed, if significant, by pooled variance t-tests. If Levene's test indicated heterogeneous variances, the groups were compared by one-way ANOVA for unequal variances, followed, if significant, by separate variance t-tests. Tumour incidences were analysed using Pearson's
2 test. A P value of <0.05 (two-tailed) was considered significant.
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Results
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Food consumption, energy intake and terminal body weight
The LGOS and HGOS diets had a similar caloric content (Table I
). The food consumption and (calculated) energy intake of animals fed the LGOS diet was slightly higher than that of animals fed the HGOS diet. This may explain the slightly higher final body weights of the animals maintained on the LGOS diet (Table II
). However, the differences were only marginal (
7%).
Aberrant crypt foci
ACF were present in all animals. After the first 7 weeks, the total number of ACF in animals fed an HGOS diet was lower than that in animals fed an LGOS diet (Table III
). The number of large ACF (four or more crypts) and the AC multiplicity were highest in the HGOS-fed animals (P < 0.05 for large ACF). From week 7 to week 13 (after the diet change), the number of total as well as large ACF increased in all animals as expected. However, the increase was less in rats that had been switched from an LGOS to an HGOS diet than in rats that had been switched from an HGOS to an LGOS diet. At 13 weeks, the AC multiplicity in the H/LGOS group was statistically significantly higher (P < 0.05) than that in all other groups. The number of total as well as large ACF was also highest in this group, but this was not statistically significant. To investigate the effect of the different diets during weeks 713 of the experiment, the results of the LGOS and H/LGOS groups were combined, as were those of the HGOS and L/HGOS groups. The number of total as well as large ACF was lower in the combined HGOS group than in the combined LGOS group (not statistically significant). The AC multiplicity was statistically significantly (P < 0.05) lower in the combined HGOS group.
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Table III. Mean number of ACF (±SD) in the colon and rectum of animals killed 7 or 13 weeks after the start of the study
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Properties of colorectal tumours
The overall mean incidence of colorectal tumours was 76.4%. The incidence of tumours in each diet group is presented in Table IV
. The incidence of adenomas is the percentage of adenoma-bearing animals with or without carcinoma. The incidence of carcinomas is the percentage of carcinoma-bearing animals with or without adenoma. The incidence of total tumours is the percentage of animals bearing adenoma or carcinoma or both. An HGOS diet resulted in a decreased incidence of colorectal adenomas and carcinomas. The decrease in incidence of all tumours combined (adenomas + carcinomas) was significant (P = 0.05).
The multiplicity of the tumours, expressed as the mean number of tumours per tumour-bearing animal, is presented in Table IV
. In general, the multiplicity was quite low in all animals. The experimental groups did not show statistically significant differences with respect to the multiplicity of adenomas, carcinomas or all tumour types combined.
The experimental groups did not show statistically significant differences with respect to the mean size of adenomas, carcinomas or all tumour types combined (Table IV
).
Most tumours were found in the distal two-thirds of the colon. Carcinomas were slightly more cranial (mean distance from the anus: 64 mm) than adenomas (mean distance from the anus: 44 mm) in all groups. In general, the location of the tumours was not influenced by the diet.
Labelling index and apoptotic index
To determine the labelling index in adenomas, using Ki-67 as a marker, a total number of 24205 nuclei was counted. The labelling index in the adenomas of LGOS-fed animals was 42.84 ± 7.22 and that in HGOS-fed animals was 40.72 ± 13.22. The difference was not statistically significant. To determine the apoptotic index in adenomas a total number of 14764 nuclei was counted. The apoptotic index in the adenomas of LGOS-fed animals was 0.76 ± 0.39 and that in HGOS-fed animals was 0.90 ± 0.64. The difference was not statistically significant.
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Discussion
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In the present study the incidence of colorectal tumours in rats fed an HGOS diet was lower than that in rats fed an LGOS diet. The results also demonstrate that an HGOS diet decreased the aberrant crypt multiplicity scored after 13 weeks but failed to affect the total number of ACF induced in rat colon by AOM. Although the effect of the different diets on the development of aberrant crypt foci was rather inconsistent, the aberrant crypt multiplicity scored after 13 weeks appeared to be predictive of the tumour outcome at the end of the study.
Several animal models are available for investigating the influence of dietary factors on the development of colorectal tumours. In the DMH model, rats are subjected to multiple (up to 10) high doses of carcinogen. This injection protocol leads to the development of a rather high number of colorectal tumours in almost all animals. The results of previously performed studies, conducted at our Institute, have shown that the incidence of colorectal tumours is hardly affected using this animal model. In most studies an effect is found on the multiplicity rather than on the incidence of colorectal tumours. An undesired effect of the DMH model is the frequent development of tumours at other sites, such as the duodenum and the Zymbal gland, causing health problems and death before the end of the experiment. AOM induces these unwanted effects at a much lower incidence than DMH. Another important advantage of the AOM model is the short injection protocol, which allows to study the effects of compounds during the initiation or the promotion phase of the carcinogenic process, separately.
In the present study, the effects of GOS during the initiation phase were determined by monitoring the development of putative preneoplastic ACF, induced in rats by two consecutive injections with AOM. A protective effect of an HGOS diet during the initiation phase of carcinogenesis should, theoretically, be reflected by a lower number and/or smaller size of ACF in animals fed an HGOS diet in comparison with animals fed an LGOS diet. The results of the present study, however, pointed to some inconsistent effects of GOS during the initiation phase, although the differences among the groups reached the level of statistical significance in one group only. Animals maintained on an HGOS diet for 7 weeks developed significantly more large (four or more crypts) ACF, but fewer total ACF, than animals maintained on an LGOS diet. After 13 weeks the aberrant crypt multiplicity (number of aberrant crypts per focus) was significantly higher in animals maintained on an HGOS diet during the first 7 weeks and on an LGOS diet from week 7 onwards. After combining the animals maintained on the same diets from week 7 to week 13 (LGOS + H/LGOS, and HGOS + L/HGOS), the number of total as well as large ACF was lower (not statistically significant) in the combined HGOS group than in the combined LGOS group. The AC multiplicity was statistically significantly (P < 0.05) lower in the combined HGOS group.
The results of studies into effects of different compounds on colon carcinogenesis in rats by other investigators have indicated that the predictive value of ACF for the tumour outcome is inconsistent. Some investigators have found a clear correlation between number and size of ACF, and the tumour outcome in studies with sodium phytate, a chemopreventive agent (12) or with ß-carotene and wheat bran fibre (15). In another study with wheat bran, Young et al. (16) found a correlation between the number but not the size of ACF and tumour outcome. Magnuson et al. (17) found just the opposite in rats fed cholic acid: not the number but the size of ACF correlated with tumour outcome. A plausible explanation was not always given. It has been demonstrated that ACF observed in the colon of humans and laboratory animals are histologically heterogeneous lesions (1820) and it has been speculated that only a selective group of ACF is susceptible to the modulating effects of chemicals and dietary components. Thorup et al. (21), who failed to find a correlation between ACF parameters and tumour outcome in animals fed a high fibre diet, even questioned the hypothesis that ACF are really preneoplastic lesions. These investigators have postulated that ACF and colorectal tumours could represent two parallel, independent consequences of cancer initiation. Nevertheless, the results from most previous studies indicate that the AC multiplicity is a more consistent predictor of tumour outcome than the number of ACF.
In the present study the ACF score and AC multiplicity after 13 weeks contrast with those after 7 weeks. Knowing the protective effect of GOS against the development of colorectal tumours, it can be concluded that, in this study, the AC multiplicity after 13 weeks but not that after 7 weeks has a predictive value for ultimate tumour outcome. This means that the beneficial effect of an HGOS diet is mainly exerted during the post-initiation phase. These data support the hypothesis that some ACF are transient lesions that are either eliminated or are remodelled to produce normal colonic crypts. Other ACF, the so-called persistent ACF, may develop into colorectal tumours. The occurrence of persistent nodules induced in the liver with various carcinogens is a comparable phenomenon (22). Based on the tumour size, proliferation index and apoptosis data found in the present study, it was concluded that the growth rate of colorectal adenomas was not significantly influenced by the different diets.
In a previous study (10) we showed that GOS have a significant inhibitory effect on the development of colorectal tumours. In that study, exposure of rats to the carcinogen DMH (10 weekly injections of 50 mg/kg) was very high, resulting in a high tumour incidence (almost 100%) and a high tumour multiplicity. In that study, GOS demonstrated a significant inhibitory effect on the multiplicity of colorectal tumours, but only a slight inhibitory effect on the incidence. Interestingly, the results of the present study with AOM showed a significant inhibitory effect of GOS on the incidence but not on the multiplicity of colorectal tumours. This may be due to the low number of tumours per animal, because an animal without a colorectal tumour will be omitted in the multiplicity score.
Although the present results are less pronounced than those observed in the DMH study, the results of both studies together strongly support the conclusion that a diet high in GOS inhibits the development of colorectal tumours in carcinogen-treated rats. This beneficial effect of an HGOS diet may be explained by the general properties of GOS. GOS escapes enzymatic digestion in the small intestines but is readily fermented in the caecum. The main fermentation products are short-chain fatty acids (SCFA), such as acetate, propionate and butyrate. We have analysed the stored caecum content of several animals from the DMH study for SCFA using gas chromatography. The results are presented in Table V
. The total amount of SCFA in the caecum of animals fed an HGOS diet was considerably higher than that in animals fed an LGOS diet. After correction for the differences in weight of the caecal content between the groups, the amounts of acetate, propionate and butyrate were significantly increased in the HGOS-fed animals. SCFA production decreases the pH in the large intestines (in the DMH study, the caecum pH in animals fed diets with cellulose as fibre source ranged from 6.4 to 6.6; the caecum pH in LGOS-fed animals was 6.2 and that in HGOS-fed animals was 5.8). At a low pH the formation of secondary bile acids, which are cytotoxic and are thought to enhance carcinogenesis (23,24), is inhibited and their solubility decreased (2527). Butyrate suppresses colorectal tumour formation (28) and both butyrate and propionate have been shown to inhibit proliferation and enhance differentiation of colon cancer cell lines (29,30). In the present study, however, the proliferation index was only marginally lower in HGOS-fed animals. Other investigators have reported that oligosaccharides may promote the growth of beneficial gut microflora, such as bifidobacteria and lactobacilli (31,32). This phenomenon may also have contributed to the protective effect of GOS on the development of colorectal tumours.
Taking into account the inhibitory effect of an HGOS diet on colorectal tumour multiplicity in rats, previously demonstrated in an experiment using the DMH model, it seems justifiable to conclude that an HGOS diet has a protective effect against the development of colorectal tumours in rats. The protective effect of GOS on colorectal carcinogenesis is exerted during the promotion phase rather than during the initiation phase of the carcinogenic process. SCFAs, the fermentation products of GOS, probably play a key role in the mechanism of protection.
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Notes
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3 To whom correspondence should be addressed 
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Acknowledgments
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We wish to thank Borculo Domo Ingredients, Borculo, The Netherlands, for financial support of this project.
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Received June 2, 2000;
revised September 7, 2000;
accepted September 8, 2000.