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The Journal of Nutrition Vol. 128 No. 2 February 1998,
pp. 234-238
CSIRO (Australia) Division of Human Nutrition, Adelaide 5000, Australia
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Wheats used for feeding poultry differ considerably in the ratio of soluble to insoluble non-starch polysaccharides (NSP) and apparent metabolizable energy (AME). We have examined effects of whole and white flour from a wheat of low (12.02 MJ/kg of dry matter) and high (14.52 MJ/kg of dry matter) AME in rats fed a cholesterol-free diet. NSP concentrations were higher in whole flour from the low AME wheat but similar in both white flours. In contrast to chickens, food intake and body weight gain of rats were unaffected by diet. Plasma cholesterol concentrations were lower in rats fed whole wheat and low AME wheat. Cecal bile acids and neutral sterol pools were larger in rats fed whole wheat but were unaffected by wheat type. Negative correlations were found between digesta steroid pools and plasma cholesterol, consistent with greater fecal steroid excretion. Cecal digesta was greater in rats fed whole wheat and low AME wheat. Digesta pH was lower in rats fed whole wheat, but there was a significant interaction between wheat and flour type with lower pH in rats fed low AME white flour. Total volatile fatty acids (VFA) and acetate and butyrate pools were larger in rats fed whole wheat than in those fed white flour. Total and individual VFA pools were larger in rats fed low AME flours than in those fed high AME flours, apparently due to greater cecal starch fermentation in the former. Factors affecting wheat AME in chickens affect important metabolic variables in rats and may have similar actions in other species including humans.
KEY WORDS: rats · wheat flour · plasma lipids · steroid metabolism · volatile fatty acids
The quantity and quality of dietary non-starch polysaccharides (NSP,4 major components of dietary fiber) in cereal products have a substantial influence on the nutritional properties of those products. Numerous studies have shown that oats and barley, which are relatively high in soluble NSP, are effective in lowering plasma cholesterol in humans (Anderson et al. 1991 Most of the studies in dietary NSP and carbohydrates have focussed on differences among cereal types and have not taken into account the fact that there can be substantial variability within species due to the influence of environment and cultivar (Bolton and Blair 1974 Clearly, a variation of over 150% in AME of wheat when assayed in one animal species has the potential to have a substantial but, as yet, unquantified effect in others. Two areas with implications for human health that are affected by NSP are steroid metabolism and large bowel VFA production. Consequently, we have examined the effects of diets formulated with two wheats of different AME in rats. One wheat was of low AME (12.02 MJ/kg dry matter) and the other of high AME (14.52 MJ/kg dry matter). These wheats were incorporated either as whole milled flour or as white milled flour.
Ethical considerations.
All procedures used (diet formulation, anesthesia and sampling) were under the supervision of the Animal Care and Ethics and Experimental Animals Committees of the Division of Human Nutrition and conformed to published guidelines (National Health and Medical Research Council, CSIRO and Australian Agricultural Council 1985).
Preparation of wheat flours.
The AME of the two wheats used in this experiment was assayed previously in poultry (Choct et al. 1995 Care of animals.
Four groups of eight rats (150-160 g of body weight) of the Hooded Wistar strain were used. They were bred in the Experimental Animal Colony of the Division of Human Nutrition and housed in stainless steel cages with wire-mesh bottoms under conditions of controlled heating and lighting as described previously (Illman et al. 1993 Diets.
The diets were based on the AIN93 formulation (Reeves et al. 1993 Sampling.
After 18 d of dietary treatment, rats were anesthetized with halothane and blood drawn from the abdominal aorta after a midline laparotomy. The blood was collected into ice-cold tubes containing EDTA as anticoagulant. Then the rats were killed by rupture of the diaphragm; the cecum was excised and the contents were extruded, weighed and diluted with water for determination of VFA, pH, and bile acids and neutral sterols.
Analytical procedures.
Plasma was prepared by centrifugation of blood at 2500 × g for 10 min; its total cholesterol concentration was determined (after saponification) by gas-liquid chromatography on a 0.32 mm × 25 m capillary column of AT 210 (Alltech Associates, Sydney, Australia) using epi-coprostanol (Steraloids, Wilton, New Hampshire) as internal standard. Concentrations of VFA were measured in diluted cecal contents by a modification of our method for plasma as described previously (Illman et al. 1993 Statistical methods.
Data are shown as the means for eight observations per group (rats) with a pooled SEM. The data were analyzed as a 2 × 2 factorial analysis examining the effect of wheat type (low and high ME) and flour type (whole wheat or white) and the interactions between the two. Simple correlations between variables were calculated by linear regression analysis using individual values. A value of P < 0.05 was taken as the criterion of significance. Statistical calculations were performed with a commercial package (Statgraphics, STSC, Rockville, MD) using a desk top computer (Digital Venturis 466, DEC, Sydney, Australia).
Flour composition.
The starch and protein concentrations of the two wheats were similar (Table 1). The NSP content of the low AME whole wheat flour was 8.9 g/kg dry matter more than that of the high AME whole wheat flour. Most of this difference was in soluble NSP. As would be expected, the total NSP concentration of the white flours was lower than that of the whole wheat flours. Although the total NSP concentration of the low AME white flour was 11.4 g/kg higher than that of the high ME white flour, there was little difference in soluble NSP.
Feed intake and body weight gain.
The rats found all of the experimental diets palatable and food intake did not differ among groups (data not shown). Weight gain also was unaffected by diet; the final body weights were 305, 302, 306 and 306 g (pooled SEM 7, n = 8) for rats fed the low AME whole wheat flour, high AME wheat flour, low AME white flour and high AME white flour diets, respectively.
Plasma cholesterol.
Plasma cholesterol concentrations were significantly (P < 0.001) lower in rats fed the white flours than in those fed whole wheat flours, regardless of wheat type (2.21 and 2.74 mmol/L, pooled SEM 0.04, n = 16). There was a significant (P < 0.001, pooled SEM 0.06) interaction between wheat type and flour fraction. Thus, plasma cholesterol was 2.04 mmol/L in rats fed the low AME white flour compared with 2.39, 2.62 and 2.86 mmol/L in rats fed the high AME white flour, high AME whole wheat and low AME whole wheat diets, respectively.
Weight and pH of cecal contents.
Cecal digesta weights were affected significantly by both the type of wheat and flour (Table 2). Digesta was lowest in rats fed the high AME white flour and was equally higher in those fed the low AME white flour and the high AME whole flour. The greatest digesta weight was found in rats fed the low AME whole wheat flour.
Cecal VFA.
Pools (i.e., concentrations × cecal water volume) of total VFA were significantly larger in animals fed whole wheat flour than in those fed white flour (Table 3). However, there was also a significant effect of wheat type with larger pools in rats fed flours from the low AME wheat. Acetate was the acid present in greatest quantity, and pools were significantly greater in rats fed whole wheat flour and low AME wheat. In contrast, there was no effect of flour type on the propionate pool, which was affected significantly by the type of wheat fed. The propionate pool was of the same size in rats fed the low and high AME whole wheat diets and the low AME white flour but was smaller in those fed the high AME white flour. In animals fed whole wheat, butyrate was the second most abundant VFA, but in those fed either white flour, butyrate ranked third. The butyrate pool was highest in rats fed the low AME whole wheat diet. There was a significant effect of AME and flour type, with the smallest effect found in rats fed the high AME white flour.
Cecal steroids.
The pool of total bile acids was influenced significantly by the type of flour but not by the AME of the wheat, with larger pools in rats fed both white flours (Table 4). In keeping with the differences in total bile acids, the pools of lithocholate, deoxycholate, chenodeoxycholate and cholate (but not hyodeoxycholate) were significantly higher in rats fed the white wheat flour than in those fed the whole wheat flour. For chenodeoxycholate and cholate, there was a significant interaction between AME and flour type pools, which were significantly smaller in rats fed the high AME white flour than in those fed the corresponding low AME flour. The pool of cholate, the only one to be affected by flour type, was significantly smaller in rats fed the high AME white flour than in those fed the low AME white flour. Neither
We believe that this is the first time that wheat flours made from samples selected on the basis of substantial differences in their AME have been examined in rats. The two wheats used in this study were chosen from a previous experiment in poultry in which those AME values were established (Choct et al. 1995 We wish to thank R. Trimble and L. McGrath for valuable technical assistance.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Newman et al. 1989, Whyte et al. 1992) and animals (De Shrijver et al. 1992, Illman and Topping 1985). In humans, such a reduction would diminish the risk of early-onset coronary disease. Wheat products (e.g., wheat bran) that are high in insoluble NSP appear to have little or no cholesterol-lowering activity (Anderson et al. 1991, Illman et al. 1993) but are good laxative agents, acting in part through the promotion of fecal bulk (Topping and Wong 1994). Wheat bran also promotes large bowel health by raising colonic volatile fatty acids (VFA), which are produced by the bacterial fermentation of NSP and complex carbohydrates. In contrast to wheat bran, wheat flour, which is relatively low in insoluble NSP and high in soluble NSP, lowers plasma cholesterol in humans (Anderson et al. 1991) and rats (Illman et al. 1993).
). This variability is afforded much more attention in the nutrition of domesticated species where the apparent metabolizable energy (AME) of the feed is a major consideration for efficient production. Formerly, a value of 14.5 MJ/kg of dry matter was accepted widely for the AME of wheat for broiler chickens (Janssen et al. 1979). However, it has become clear that the AME of wheat varies widely and can range from as low as 10.35 MJ/kg dry matter to 15.9 MJ/kg dry matter (Mollah et al. 1983, Rogel et al. 1987). This great variability seems to reside in the soluble NSP content of the grain, which is related inversely to its AME for broiler chickens (Annison 1991). Supplementation of low AME wheats with NSP-degrading enzymes lowers the content of soluble NSP and improves growth of broiler chickens (Choct et al. 1995).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). The white flours were extracted by a commercial mill (W. Thomas and Co., Port Adelaide, SA). The wholemeal flours were prepared by milling the whole grain used to prepare the white flours in a Wiley knife mill (Thomas-Wiley, Philadelphia) to a particle size sufficient to pass through a 2-mm screen.
).
) and contained 75% milled whole wheat or the flour derived from that quantity of grain. This formulation was similar to that used in a previous study (Illman et al. 1993); the diets differed in NSP but were balanced for other nutrients. Protein was kept at 20% by the addition of casein; in the white flour diets, the NSP removed by milling was replaced with sucrose. All diets were supplemented with vitamins, minerals, choline bitartrate and methionine (Illman et al. 1993).
). Bile acids and neutral sterols were extracted from cecal contents with methanol according to the method of Glatz et al. (1985). 23-nor-Deoxycholate and 5
-cholan-24-ol were used as internal standards for sterols and bile acids that were converted to the methyltrifluoroacetate and trifluoroacetate derivatives, respectively, for measurement by gas-liquid chromatography by using the same column used for plasma cholesterol determination (Illman et al. 1993). Cholestane was used as an internal standard for neutral sterols and nor-deoxycholate for bile acids. All of the reference materials were obtained from Steraloids.
and Theander and Westerlund (1993). Starch in wheat was determined by a total starch assay procedure (Amyloglucosidase/
-amylase method; Megazyme, Sydney, Australia). Protein was calculated as nitrogen (measured by using a Kjeldahl automatic analyzer, Foss Electric, Copenhagen, Denmark) multiplied by 5.7 (American Association of Cereal Chemists 1983).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Non-starch polysaccharide (NSP), starch and protein concentrations of low and high apparent metabolizable energy (AME) wheat milling fractions
View this table:
Table 2.
Weight and pH of cecal digests of rats fed whole and white flours of wheats of low and high apparent metabolizable energy (AME)1
View this table:
Table 3.
Pools of individual and total volatile fatty acids (VFA) in cecal digesta of rats fed whole and white flours of wheats of low and high apparent metabolizable energy (AME)1
- nor -muricholate was detected in any of the samples.
View this table:
Table 4.
Pools of individual and total bile acids in cecal digesta of rats fed whole and white flours of wheats of low and high apparent metabolizable energy (AME)1
View this table:
Table 5.
Pools of individual and total neutral sterols in cecal digesta of rats fed whole and white flours of wheats of low and high apparent metabolizable energy (AME)1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The present data show quite clearly that these wheats and their milling fractions had a substantial effect on a number of important metabolic variables in a non-avian species. Of the parameters investigated, two that were not affected were food intake and body weight gain. The lack of effect on weight gain is quite surprising in view of the well-documented role of AME in chicken production but may reflect the relatively high energy demands of the latter species and the fact that the rats were fed for only 3 wk. However, these data mean that effects on other variables were not related to differences in food consumption or the rate of growth.
) and rats (Illman et al. 1993) that plasma cholesterol concentrations were significantly higher when the diet contained whole wheat rather than white wheat flour. These earlier differences were explained in terms of the relative enrichment of the white flour with soluble NSP compared with whole wheat flour because these polysaccharides lower plasma cholesterol both as whole foods and isolates. The same NSP fraction appears to be responsible for differences among wheats in their nutritional performance in chickens (Choct 1992) where they appear to reduce digestive efficiency and impair growth (Annison 1991). Degradation of NSP by enzymes leads to a lowering of viscosity and improved AME (Choct et al. 1995). These studies extend those previous findings in rats and chickens and show that a relatively small difference in soluble NSP content of the two white flours produced a substantial difference in nutritional effect. However, it appears that these polysaccharides may not have been the sole determinant of differences between wheat and flour types. For example, plasma cholesterol concentrations were higher in rats fed whole flour from wheat of low AME compared with rats fed white flour from the same source. This was despite the fact that soluble NSP were higher in the whole flour. It follows that either soluble NSP are not the sole determinant of the nutritional properties of white flour or that other factors may come into play in the milled product.
), the concentrations required are high and it appears unlikely that the last mechanism operates in vivo (Illman et al. 1988). This conclusion is supported by this experiment in which there was a positive correlation (r = +0.565, P < 0.01) between cecal butyrate and plasma cholesterol but no relationship between plasma cholesterol and cecal propionate (r = 
0.046, P > 0.05). Rather similar data were obtained in a previous study in rats in which it was concluded that large bowel propionate and butyrate vary with the concentrations of total steroids and the products of bacterial metabolism of cholesterol and bile acid (Illman et al. 1993). One difference between this experiment and the previous report is the lack of any apparent relationship between cecal propionate and plasma cholesterol. This may be due to the fact that in contrast to this study, the wheat fractions tested in the earlier study all came from the same batch.
; Ikegami et al. 1990). In rats, reduction of small intestinal cholesterol absorption has been noted in the presence of viscous polysaccharides (Gee et al. 1983). Gallaher et al. (1993a and 1993b) have shown that this process is extremely sensitive to increases in digesta viscosity so that relatively small changes could affect plasma cholesterol substantially. In these animals, which were fed a diet not supplemented with cholesterol and cholic acid, the differences in plasma cholesterol reflect changes in endogenous steroid metabolism. By a process of elimination, it appears that the most likely mechanism for cholesterol lowering is enhanced steroid excretion. Both cecal bile acid (r = 0.461, P < 0.01) and neutral sterol (r = 0.518, P < 0.01) pools correlated negatively with plasma cholesterol concentrations. The former two are indices of fecal excretion (De Schrijver et al. 1992, Illman and Topping 1985) so that these significant relationships support increased loss of cholesterol from the body with lower plasma cholesterol concentrations. Despite the differences in plasma cholesterol between the two wheats and their fractions, there was no significant effect of wheat type on fecal bile acids or neutral sterols. As discussed above, it appears that NSP content alone cannot explain the effects of diet on plasma cholesterol. However, it must be noted that we did not measure the viscosity of either the flours or the gut contents of the animals so that we cannot exclude differences in a physiological variable that may not be related closely to NSP as measured analytically. It is equally possible that some other wheat component (e.g., starch) may have been responsible. It is known that a fraction of starch can escape small intestinal digestion and enter the large bowel of humans, rats and other species (Annison and Topping 1994). This starch, called resistant starch (RS), is fermented by the colonic bacteria as are NSP and contributes to the effective "fiber" intake of animals and humans. A number of factors influence amylolysis in vivo and the presence of NSP in foods is known to lower the small intestinal digestion of starch. If small intestinal starch digestion was lower in the low AME flours, it could account for the differences in plasma cholesterol because RS appears to be an effective agent for lowering plasma cholesterol in rats (Yunes et al. 1995) but not in humans (Heijnen et al. 1996).
) and also in large bowel VFA in this study. As expected from the NSP content of the diets, large bowel digesta mass was greater in rats fed the whole wheat flours. Nevertheless, there was also an effect of cultivar with significantly more digesta in rats fed the low AME flours. Greater entry of starch into the cecum could explain this effect as well as the larger pools of total VFA, acetate, propionate and butyrate in rats fed the low AME flours. All of these variables were higher in animals fed whole wheat flour except for propionate. The effect of wheat type on butyrate is especially interesting because it is believed that RS favors production of this acid by the large bowel microflora (Weaver et al. 1992). Butyrate is thought to play an important role in the maintenance of a normal cell phenotype in the human colon and in reducing the risk of colonic cancer (Kruh et al. 1994). Thus it would appear that either whole wheat or white wheat flours from low AME cultivars might be of benefit in that regard. pH may be important in human colon cancer risk, with a low pH being protective (Malhotra 1982). Negative correlations were found between total VFA (r = 0.794, P < 0.001)), acetate (r = 0.707, P < 0.001), propionate (r = 0.455, P < 0.01) and butyrate (r = 0.850, P < 0.001) and cecal pH. Given that VFA are acids (albeit relatively weak ones), these correlations are to be expected and their greater pools in rats fed whole wheat flour and also in those fed the low AME products explain the lower pH in those animals.
ACKNOWLEDGMENTS
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FOOTNOTES |
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Manuscript received 11 June 1997. Initial reviews completed 5 August 1997. Revision accepted 10 September 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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