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© 2007 American Society for Nutrition J. Nutr. 137:923-929, April 2007

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Whole-Grain and Refined Wheat Flours Show Distinct Metabolic Profiles in Rats as Assessed by a 1H NMR-Based Metabonomic Approach1

Anthony Fardet2,*, Cécile Canlet3, Gaëlle Gottardi3, Bernard Lyan2, Rafaël Llorach2, Christian Rémésy2, André Mazur2, Alain Paris3 and Augustin Scalbert2

2 UMR 1019-Unité de Nutrition Humaine, Institute National de la Recherche Agronomique, Centre de Recherche de Clermont-Ferrand/Theix, F-63122 St-Genès-Champanelle, France and 3 UMR 1089-Xénobiotiques, Institute National de la Recherche Agronomique-Ecole National Vétérinaire de Toulouse, BP 3, 31931 Toulouse Cedex 9, France

* To whom correspondence should be addressed. E-mail: afardet{at}clermont.inra.fr.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The protection against diabetes and cardiovascular disease provided by whole-grain cereal consumption has been attributed to the fiber and micronutrients present in the bran. But exactly how this occurs remains unclear due to both diversity of bran constituents and the complexity of the metabolic responses to each of these constituents. We investigated the metabolic responses of 2 groups of rats (n = 10/group) fed 2 diets, for 2 wk each, in a crossover design. One diet contained 60 g/100 g whole-grain wheat flour (WGF) and the other contained 60 g/100 g refined wheat flour (RF). Markers of oxidative stress [urinary isoprostanes and malondialdehydes (MDA), plasma ferric-reducing ability of plasma, MDA, and vitamins E and C] and lipid status (liver and plasma triglycerides and cholesterol) were measured. Urine samples collected during the feeding periods and plasma and liver samples collected at the end of experiment were analyzed by 1H NMR spectroscopy. Metabonomic analyses showed that each group reached a new metabolic balance within 48 h of changing the diet. Urinary excretion of some tricarboxylic acid cycle intermediates, aromatic amino acids, and hippurate was significantly greater in rats fed the WGF diet. Although the diets did not affect conventional lipid and oxidative stress markers, there were decreases in some liver lipids and increases in liver reduced glutathione and betaine as shown by metabonomic analyses. These suggest that the WGF diet improved the redox and lipid status.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Epidemiological studies suggest that whole-grain cereal consumption protects against metabolic diseases such as atherosclerosis, diabetes, and cancer (1). Whole-grain products differ from refined products by their bran content, which is rich in fiber, micronutrients, and other phytochemicals. These compounds are thought to improve bowel physiology and lipid and antioxidant status, and help suppress tumor growth (2,3). The hypocholesterolemic effects of some cereal products are attributed to fiber, particularly soluble fiber (4). Some authors have also suggested that certain antioxidants in whole-grain cereals improve redox status and decrease the risk of associated disorders (2,5,6). However, apart from some studies on colored rice varieties that are particularly rich in antioxidants (7), these effects have not been established in in vivo studies.

Exactly how whole-grain cereals protect against metabolic diseases remains largely unknown, partly because of the diversity of the active constituents in whole-grain products and partly because of the complexity of the metabolic responses to each of them.

1H NMR spectroscopy or MS can be used in a metabonomic study to analyze simultaneously several hundreds or thousands of metabolites in biological fluids such as urine, plasma, or tissues (8). Multivariate statistical analysis is then used to identify markers influenced by an intervention (9). This approach is capable of describing the effects of diet on metabolism, because it can assay many metabolites in a single sample; traditional approaches focused on specific biomarkers often fail to detect metabolic changes. Metabonomic studies on the influence of complex foods or diets on metabolic profiles have been used only rarely to date. The regular consumption of chamomile tea was shown to influence urinary metabolic profiles in humans (10). These effects were persistent and still observed 14 d after ending chamomile consumption, possibly because of some modifications of the gut microbiota. Ingesting soy isoflavone affects human endogenous metabolism, including osmolyte fluctuation and energy metabolism (11). A metabolomic study also revealed some effects of epicatechin consumption on the endogenous metabolism of rats (12).

We have postulated that there are detectable, unequivocal differences in the metabolism of animals consuming whole-grain cereals and those consuming refined grain. We fed rats diets containing 60% whole-grain wheat flour (WGF)4 or refined wheat flour (RF) and monitored their general metabolism using a 1H-NMR-based metabonomic approach and more conventional metabolic biomarkers of fiber fermentation, lipid status, and oxidative stress. The changes in some of these markers suggest that whole-grain cereals have previously unrecognized metabolic effects that may explain their health benefits.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Animals and diets. We used male Wistar rats weighing 378 g that were fed the RF diet for 2 wk. They were assigned to 1 of 2 groups (n = 10); 1 group was fed a semipurified diet containing 60% RF for 2 wk (d 0– 14) followed by a diet containing 60% WGF for 2 wk (d 15–28) (RF-WGF). The other group (WGF-RF) was fed the 2 diets in the reverse order. The diets were distributed as moistened powders. WGF and RF were purchased from Celnat. The WGF diet contained whole wheat germ (2.5–3% of the whole grain). WGF contained 1.78 g ash/100 g, 621 g flour/kg, 103 g casein/kg, 89 g/kg sucrose, 97 g/kg starch, and 50 g/kg soybean oil; the RF diet contained 0.62 g ash/100 g, 572 g/kg flour, 125 g/kg casein, 95 g/kg sucrose, 104 g/kg starch, and 61 g/kg soybean oil. The diets thus had equal proportions of carbohydrate, protein, and lipid (70/22/8). The diets also contained vitamin (10 g/kg diet) (AIN-93Vx, UPAE, INRA Jouy-en-Josas, France) and mineral (35 g/kg diet) (AIN-93G, MP Biomedical) mixtures (13). Rats were allowed free access to 25 g fresh food per day and tap water. The rats were first housed 2 per cage, then 1 per cage during the 2nd feeding period and were kept in a temperature-controlled room (22°C) with a dark period from 2000 to 0800. They were handled according to the recommendations of the Institutional Ethics Committee (Clermont-Ferrand University). The body weights of rats were recorded each week. Food intake was recorded on d 13, 21, and 27. Urine volume was recorded 2x/d: postprandially (PP) at 0900 and in the postabsorptive (PA) period at 1600 on d 13, 14, 15, 16, 17, 19, 21, 23, 25, 27, and 28. Feces were collected during the final 3 d.

    Sampling procedures. Rats were anaesthetized at the end of the dark period with sodium pentobarbital (40 mg/kg) and maintained at 37°C during sample collection. An abdominal incision was made and blood was withdrawn from the abdominal aorta into heparinized tubes. It was centrifuged at 12,000 x g for 2 min and the supernatant (plasma) was collected and stored at –80°C for lipid analysis (triglycerides and cholesterol), antioxidant capacity [ferric-reducing ability of plasma (FRAP) and malondialdehydes (MDA)], antioxidant quantification (vitamins E and C) and 1H NMR analyses. The cecum (wall with contents) was then removed and weighed. Cecal contents were collected into 2-mL microfuge tubes, frozen, and stored at –20°C for assays of SCFA. The liver was weighed and freeze-clamped into liquid nitrogen and stored at –80°C for the measurement of triglycerides, cholesterol, and betaine concentrations, and 1H NMR analyses. The urine collected twice per day (see above) was stored at –20°C for analysis of MDA and isoprostane [8-epi-prostaglandin F2{alpha} (8-epi-PGF2{alpha})] concentrations and 1H NMR analyses.

    Analytical procedures. SCFA concentrations were measured by GLC on the supernatants (8000 x g; 5 min at 4°C) of cecal contents (18). Plasma triglycerides and total cholesterol were determined enzymatically using kits purchased from BioMérieux (Triglycerides PAP 150 and Cholesterol RTU). Liver triglycerides and cholesterol were extracted and analyzed as described by Mazur et al. (14). The FRAP was determined as described previously (15). The TBARS in plasma and urine samples were measured by the modified procedure of Lee et al. (16) by reading absorbance at 532 nm. TBARS were measured in urine collected during the PP period (from 1600 to 0900, i.e. 17 h) on d 27 and expressed as nmol equivalents of MDA excreted during 17 h. Plasma vitamin E ({alpha}-tocopherol) was analyzed by HPLC-UV according to Lyan et al. (17). Plasma ascorbic acid was determined according to Tessier et al. (18). Isoprostane (8-epi-PGF2{alpha}) in urine collected during the PP period was determined with a commercial kit (Oxford Biomedical Research). 8-epi-PGF2{alpha} was measured on d 27 and expressed in ng of 8-epi-PGF2{alpha} excreted in 17 h.

    Sample preparation for 1H NMR spectroscopy. Urine samples were prepared for NMR spectroscopy by mixing 500 µL urine with 200 µL phosphate buffer in D2O (pH 7.4) containing 10 mmol/L deuterated trimethylsilylpropionate as chemical shift reference ({delta} 0.0 ppm). The buffered urine samples were then centrifuged at 4600 x g for 10 min to remove any precipitates and aliquots of the resulting supernatant (600 µL) were placed in 5-mm NMR tubes. Plasma samples were prepared for NMR spectroscopy by mixing 400 µL plasma and 200 µL D2O. 1H chemical shifts were referenced internally to the lactate resonance at 1.33 ppm, measured relative to the chemical shift of trimethylsilyl [2,2,3,3-2H4] propionate used as a reference. The solutions were transferred to 5-mm (o.d.) NMR tubes. Samples of liver tissue (~100 mg) were homogenized with a Polytron PT 2100 in 2 mL acetonitrile/H2O (50:50, v:v) containing 0.1% BHT in an ice-water bath. The homogenates were centrifuged at 5000 x g for 10 min at 4°C and the supernatants were removed and lyophilized. The resulting powders were reconstituted in 1 mL D2O and extracted with 2 mL chloroform/methanol (75/25, v:v) and finally centrifuged (5000 x g for 15 min at 4°C). The precipitates are referred to as the water-soluble liver extracts. The lipophilic supernatants were dried under a stream of nitrogen and reconstituted in 600 µL CDCl3. These are referred to as the chloroform/methanol liver extracts. The reconstituted solutions were transferred to NMR tubes.

    NMR spectroscopic analyses. All 1H NMR spectra were obtained on a Bruker DRX-600 Avance NMR spectrometer operating at 600.13 MHz for 1H resonance frequency using an inverse detection 5 mm 1H-13C-15N cryoprobe attached to a CryoPlatform (the preamplifier cooling unit). 1H experiments on urine samples were recorded using the Improved Watergate sequence (19) to suppress water resonance, accumulating 128 free induction decays into 32-K data points on a 12-ppm spectral width. The 1H NMR spectra of plasma samples were acquired at 300 K using the 1 dimensional Carr-Purcell-Meiboom-Gills spin echo pulse sequence (20), accumulating 256 free induction decays. The 1H NMR spectra of liver extracts were acquired at 300 K using a standard 1 dimensional single pulse (21). All NMR spectra were data-reduced using AMIX (version 3.1, Bruker Analytik) to integrate 0.01-ppm-wide regions for urine samples and 0.04-ppm-wide regions for plasma and liver samples corresponding to the region {delta} 10.0–0.5 ppm. The regions {delta} 6.5–4.5 ppm in the urine spectra, {delta} 5.1–4.5 ppm in the plasma spectra, and {delta} 5.0–4.7 ppm in the aqueous extracts were set to 0 integral value to remove the variability of water resonance suppression and cross-relaxation effects on the urea signal. The regions {delta} 7.5–6.8 ppm (chloroform) and {delta} 3.6–3.3 ppm (methanol) were removed from all chloroform/methanol liver extract spectra.

    Measurement of liver betaine concentration by liquid chromatography-MS/MS. The betaine concentration in the aqueous liver extract was measured essentially as described by Holm et al. (22); water-soluble liver extracts were injected into a reverse-phased 150- x 2-mm C12 column (Jupiter Proteo 90A, Phenomenex) packed with 4-µm diameter particles; the mobile phase consisted of an isocratic eluent (98% water + 1% formic acid and 2% acetonitrile + 1% formic acid); the column effluent flow rate into the mass spectrometer was 200 µL/min and the injection interval (run time) was 5 min; detection was with an API 2000 Triple-quadrupole tandem mass spectrometer (Applied Biosystems) in the positive-ion mode equipped with electrospray interface, the collision energy was 37 eV, the declustering potential was 21 V, the capillary voltage was set at 5000 V, the ion source temperature was 550°C, and betaine was detected with the m/z 118–58 transition. Data were processed using Sciex software (v1.4.1, Applied Biosystems).

    Data analysis. Values of energy and food intake, weight gain, feces weight, urine volume, relative cecum, liver and heart weights, fermentation, lipid and antioxidant status, and liver betaine concentration are all given as means ± SEM, and significance between the 2 groups at the end of the experiment (P < 0.05) was determined by Student's t test (STATVIEW software; version 5.0; SAS Institute). 1H NMR spectral data were analyzed by ANOVA and linear discriminant analyses (LDA) Dumas et al. (23). Briefly, ANOVA was used to select significant variables from the set of initial urinary variables as candidates for LDA. This factorial procedure is equivalent to a principal component analysis performed on the means (barycenters) of the groups for each date. The factorial map from this multidimensional analysis gives a hierarchy between the factors that may explain the total variance, in particular whether there are 1) significant trajectories revealed in urine fingerprints by changing diet, and 2) significant differences between urine fingerprints collected during PP and PA periods. The statistical calculations were made using S-Plus 2000 software (v2.0 Insightful Corporation) including MASS and Multidim libraries and SAS (v8.01, SAS Institute).


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Food intake, weight gain, cecal fermentation, lipid, and antioxidant status. Energy intakes did not differ between the groups during the last feeding period, but the WGF-RF rats had a significantly higher daily mean weight gain and food intake than did the RF-WGF rats (Table 1). The feces produced by the RF-WGF rats were over twice the weight of those produced by the WGF-RF rats, but the WGF-RF rats excreted significantly more urine. Total cecum, liver and heart weights, body weight, cecal pH, or total SCFA pool in the cecum did not differ between the 2 groups at the end of the experiment. The SCFA ratios were also quite similar, except for a significant higher concentration of butyric acid in RF-WGF group. Triglyceride and cholesterol concentrations in plasma and liver did not differ between groups and all the tested biomarkers of oxidative stress were similar in plasma (FRAP, MDA, vitamins E and C) and urine (MDA and 8-epi-PGF2{alpha}) in the 2 groups (Table 1).


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  		TABLE 1 Fermentation, lipid, and antioxidant status of rats after 2 wk of consuming WGF and RF diets

 
    Influence of the WGF and RF diets on urinary metabolic profiles. Chemometric analyses were performed on all urine NMR spectra (Supplemental Fig. 1 available on line) of samples collected in the morning (PP) and afternoon (PA) between d 13 and 28 for all rats consuming either the RF or WGF diets.

First, an ANOVA was performed on selected variables that significantly distinguished WGF and RF diets. Two hundred spectral regions of 0.01 ppm among the 750 of the entire spectra were selected and used for the LDA. The urinary profiles of rats fed WGF or RF were quite distinct (according to the first component LD1, which represents 30.2% of the between-group variance), as were the 2 urine sampling times (according to LD2, which represents 13.8% of the between-group variance) (Fig. 1). Changing from WGF to RF (d 14–15) or conversely produced a new metabolic balance within 48 h (Fig. 1). The urine samples collected in the morning and afternoon showed similar metabolic trajectories.


Figure 1
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  		Figure 1  LDA score plot of the 1H NMR urinary spectra highlighting the separation before, between, and after the diet change (d 14–15) and between the urine sampling times (PP and PA). RF-WGF group, dotted lines; WGF-RF group, solid lines. Each polygon represents the limits of the metabolic profiles obtained for the 10 rats of a given group at a given day and urine sampling time. Urine samples were collected from d 13 to 28.

 
The most discriminating variables were selected on the basis of canonical correlations higher than 0.70. The loading plot showed that tyrosine, tryptophan, hippurate, creatine, citrate, phenylalanine, fumarate, and 6 unidentified signals all contributed significantly to the first axis and increased when the RF diet was replaced by the WGF diet (fold changes >1; Table 2). Similarly, pyruvate, TMAO/taurine, and 2 unidentified compounds significantly decreased after WGF consumption (fold changes <1; Table 2). The same variables were found for urine samples collected in the morning and in the afternoon. The metabolites contributing most to the second axis (explaining mostly the difference between PA and PP states) were lactate, citrate, 2-oxoglutarate (2-OG), creatinine and 6 unidentified compounds. They were higher in the PP period (fold changes >1; Table 3), whereas dimethylamine (DMA) and tryptophan were lower. The changes in the relative intensities of some of these compounds from d 13 to d 28 are shown in Figure 2A–F. Tryptophan and hippurate in both PP and PA urine samples clearly increased in rats changed from the RF diet to the WGF diet, and remained higher for the following 2 wk (Fig. 2B,C). The TMAO/taurine pattern was opposite; it decreased when the rats changed from the RF diet to the WGF diet (Fig. 2D). The citrate and DMA concentrations were not affected by changing the diet (Fig. 2E,F).


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  		TABLE 2 Urinary metabolites significantly affected in rats after 2 wk consuming WGF or RF diets1

 

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  		TABLE 3 Urinary metabolites significantly affected by the sampling time1

 

Figure 2
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  		Figure 2  Normalized intensities of specific 1H NMR urinary spectral regions on d 13–28 (see Fig. 1 for day and group numbering): A, unassigned compound; B, tryptophan; C, hippurate; D, TMAO/taurine; E, citrate; F, DMA. Data are means ± SEM, n = 10.

 
    Influence of the whole-grain and refined grain diets on plasma and liver metabolic profiles. LDA were used to identify the metabolite variables (see spectra and metabolites identification on the Supplemental Fig. 2 available on line) that contributed most significantly to the effects of the WGF and RF diets on the plasma and liver (Table 4). Two unidentified compounds, lysine and lipid function CH2-CO, significantly increased in rats fed the WGF diet. Glucose and some unidentified sugars, TMAO/betaine, and reduced glutathione (GSH) in liver aqueous extracts all increased when rats were fed the WGF diet, whereas inosine and 3 unidentified compounds increased in rats fed the RF diet. There were lower concentrations of the lipid functions CH2-OCOR, CH2-CO, and CH2-CH2-CO in the chloroform/methanol liver extracts of rats on the WGF diet. The betaine concentration in the liver was measured by liquid chromatography-MS/MS because the TMAO and betaine signals overlapped. The betaine concentration (P < 0.05) was higher in the livers of rats fed the WGF diet (1.35 ± 0.16 µmol/g liver) than in those on the RF diet (0.86 ± 0.12 µmol/g liver).


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  		TABLE 4 Main metabolites in the plasma, and water soluble/chloroform-methanol liver extracts that significantly discriminate between RF-WGF rats and WGF-RF rats1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
    Effect of WGF consumption on markers of oxidative stress. Neither WGF nor RF had any significant influence on the commonly used metabolic marker measured, apart from expected effects on butyrate production in the cecum (24). The absence of effects of WGF consumption on oxidative stress markers is somewhat surprising, because there are antioxidant compounds like ferulic acid, lignins, phytic acid, zinc, copper, selenium, and manganese in the grain envelope and vitamin E in the germ (6). To our knowledge, the antioxidant effects of whole-grain cereals have never been confirmed in humans, especially for wheat, the most widely consumed cereal in Western countries, and studies in animals are scarce. The lack of effect on plasma MDA was reported in studies on women who consumed dietary supplements of wheat bran (25) and on pigs fed wheat bran (74 g/d) for 14 d (26). Only cereal varieties particularly rich in anthocyanins, such as red or black rice, were shown to affect some oxidative stress biomarkers (7). The lack of antioxidant effect of WGF in this study may be due to the low antioxidant concentrations or to poor bioavailability (27). The antioxidant effects of whole-grain cereals may be more readily observed in animals kept under prooxidative conditions, as in pigs fed readily oxidized linseed oil (26).

    Diurnal metabolic changes. Our metabonomic approach allowed us to compare urine collected from rats that were in PP (high activity, meal consumption during the night) and PA states (day). The excretions of citrate, 2-OG, lactate, and creatinine were increased in the PP state, whereas the excretion of DMA and tryptophan was higher in the PA period. Similar patterns of citrate, 2-OG, and creatinine urinary excretions have been described (28,29). These metabolites, together with lactate, reflect the greater activity of the rats during the night. DMA is a breakdown product of dietary choline, formed in the gut by the microbiota (30). Its higher excretion in the PA period may be explained by the time taken by the microbiota to metabolize it.

    Metabolic changes in rats fed RF and WGF diets. The metabolic profiles of rats fed WGF and RF diets differed, as reflected in their urine samples collected during PP and PA periods. The markers differentiating the 2 diets were similar for both types of urine samples. A new metabolic balance was reached within 48 h when switching from 1 diet to the other (Fig. 1). The consumption of the WGF diet resulted in increased urinary excretion of citrate, fumarate, tyrosine, tryptophan, phenylalanine, hippurate, and creatine. The high excretion of citrate could be caused by the higher intake of magnesium by rats on the WGF diet. A low magnesium status has been associated with hypocitraturia (31), and magnesium is ~4 times more abundant in WGF than in RF (32). Hippurate is synthesized from benzoate and glycine in the kidney and liver and benzoate is also a breakdown product of several dietary polyphenols (33). Rats on the WGF diet may have excreted more hippurate in their urine because of the phenolic compounds in wheat bran (34).

Changes in metabolite concentrations were less pronounced in the plasma and liver than in the urine due to homeostatic control. The plasma of rats fed the WGF diet had a higher concentration of lysine, a difference likely explained by its higher concentration (x1.5) in WGF compared with RF (32). Some lipid signals were less intense (CH2-CO, CH2-OCOR, and CH2-CH2-CO) in the livers of rats fed the WGF diet, whereas total hepatic tryglycerides were unaffected as estimated by a conventional method.

The livers of rats fed the WGF also had more GSH, glucose, and betaine than the livers of RF-fed rats. The relatively high concentration of betaine in the liver might be due to the high betaine content of WGF. Wheat bran is a major dietary source of betaine (1.3 g/100 g) (35) and the concentration in white flour is, in comparison, very low (1.6 mg/100 g) (36). As bran accounts for 15–17% of whole-grain wheat, there would be ~140 times more betaine in WGF than in RF. A higher betaine concentration in plasma and urine was previously observed in pigs fed a whole-grain rye diet (37). Betaine participates in the conversion of homocysteine to methionine. A dietary supplement of betaine stimulates the conversion of homocysteine to methionine in the liver of ethanol-treated rats (38), and methionine added to freshly isolated hepatocytes increases intracellular GSH (39,40). Betaine treatment also improves the antioxidant status of guinea pigs (41) and rats (42). The significantly higher betaine and GSH concentrations in the livers of these rat therefore suggests that WGF consumption has an antioxidant effect.

We conclude that our metabonomic approach shows that the consumption of WGF produces metabolic changes, some of which may protect the organism against oxidative stress. This high throughput approach may unravel subtle metabolic changes that measurements of classic markers of lipid and oxidative stress may fail to reveal. The metabolic effects of WGF help to explain the beneficial effects of whole-grain cereals on health. A particular attention should be paid to the high betaine concentration in whole-grain cereals which has been so far largely neglected when exploring the health benefit of whole-grain cereals.


   ACKNOWLEDGMENTS
 
We thank P. Lamby, C. Lab, S. Mercier, and C. Demange for technical assistance, and C. Besson for animal handling.


   FOOTNOTES
 
1 Supplemental Figures 1 and 2 are available with the online posting of this paper at jn.nutrition.org. Back

4 Abbreviations used: DMA, dimethylamine; 8-epi-PGF2{alpha}, 8-epi-prostaglandin F2{alpha}; FRAP, ferric-reducing ability of plasma; GSH, reduced glutathione; LDA, linear discriminant analysis; MDA, malondialdehydes; PA, postabsorptive; PP, postprandial; 2-OG, 2-oxoglutarate; RF, refined wheat flour; RF-WGF group, group of rats consuming the refined wheat flour-based diet first and then the whole-grain wheat flour-based diet; WGF, whole-grain wheat flour; WGF-RF group, group of rats consuming the whole-grain wheat flour-based diet first and then the refined wheat flour-based diet. Back

Manuscript received 7 August 2006. Initial review completed 29 August 2006. Revision accepted 3 January 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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