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Nutrition Research Division, Health Products and Food Directorate, Health Canada, PL2203C Banting Research Centre, Ottawa, ON K1A 0L2, Canada and * Institute of Biochemistry and Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6 Canada
2To whom correspondence should be addressed. E-mail: steve brooks{at}hc-sc.gc.ca.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • folate bioavailability • dietary fiber • inulin • polydextrose • fermentation • rats
Folate is a generic term that refers to compounds based on the simplest form of folic acid [pteroylglutamic acid; (1 )]. Although humans can synthesize all components of folate, they lack the conjugase enzyme that condenses them. Folate thus remains an essential human dietary component (2 ). Both deficiency and excess folate intake are physiologically important. Folate deficiency is associated with anemia and neural tube defects, which constitute important public health problems. To address this problem, folate fortification of cereal-based products is mandatory in Canada (3 ). Excess folate intake has been associated with both detrimental and beneficial effects. On the one hand, it may confound diagnosis of vitamin B-12 deficiency, a common problem in the elderly (4 ), whereas on the other hand, it may attenuate hyperhomocysteinemia, an independent risk factor for cardiovascular disease (5 ). If dietary fiber (DF) does enhance production of bioavailable folates by human gut bacteria, fortification requirements could be decreased, thus limiting potential long-term increased exposure to folates by groups such as the elderly.
Work with rats suggests that dietary folate is not the only source of biologically available folate and that folate synthesized by cecal bacteria may also be absorbed and stored in the liver (6 –9 ). This suggests that diets containing DF and undigested and fermented dietary material (UFDM) provide more folate than diets low in DF because of the increased bacterial fermentation associated with DF and UDFM. A demonstration of this effect was provided by experiments with folate-depleted/replenished rats fed diets high in xylan or California small white beans (6 ) as well as in rats fed wheat germ or Brewers yeast (7 ). The contribution of bacterially derived folate to liver stores has also been inferred from increased cecal and fecal folate after feeding rats human milk, which is partly fermented by these animals (8 ). This increase was not observed when rats were fed milk plus succinylsulfathiazole in an attempt to inhibit cecal bacteria growth (9 ). In humans, higher urinary excretion of specific folacin isomers was observed after feeding diets high in California small white beans (10 ). Direct absorption of bacterially synthesized folate has been confirmed by radioactively labeling the bacterial folate pool (11 ) although an estimation of the absolute absorption suggested that this effect was small. The argument that colonic bacteria constitute an important source of folate is supported by a positive association between DF intake and serum folate concentrations in humans (12 ).
These results imply that foods high in DF should increase folate bioavailability, but this has not been observed consistently. For example, diets containing soft white wheat bran, hard red wheat bran, pectin, lower concentrations of xylan and cabbage did not increase rat liver folate stores above that expected from the dietary folate content (6 ,13 ,14 ) even though the DF from these food components is readily fermented by colonic bacteria. In addition, the antibacterial agent most commonly used to inhibit colonic growth and, presumably, reduce folate production in the colon (succinylsulfathiazole) is an analog of folic acid and may interfere with folate absorption and metabolism. This could confound the conclusions of these studies.
The present study directly assessed the contribution of bacterially derived folate to liver folate stores in a more systematic fashion using the depleted/replenished rat model. The animal-based folate assay has been validated by many laboratories (7 ,8 ,10 ,11 ,15 –17 ) and has proven useful in assessing the bioavailability of the many different chemical forms of folate. It has been shown that tissue folate concentrations are highest in liver (18 ) and that a dose response relationship occurs between dietary and liver folate (6 ,7 ). For this reason, liver folate was measured and used as an indicator of folate bioavailability in these studies. The assay requires the depletion of liver folate stores before feeding a test diet. During the refeeding phase, liver stores are replenished, and the extent of repletion is a function of the folate content in the diet and its bioavailability. The liver response to added folate in depleted rats, as for growth (4 ), is much more dramatic than that in nondepleted rats. Relative bioavailability is determined by comparison with liver folate in replenished rats fed standard diets containing known amounts of completely available folic acid.
In the present study, several different DF sources (wheat bran, oat bran, ground corn and wheat germ) as well as UFDM (inulin and polydextrose) were added to a basal diet to determine the contribution of DF and UFDM to liver folate stores in the presence and absence of an antibiotic, succinylsulfathiazole, to better define the relationship between fermentation and folate production. The role of the antibiotic was to inhibit bacterial folate synthesis, whereas the role of DF and UFDM was to stimulate fermentation and, consequently, increase bacterial folate synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The study was approved by the Health Canada Animal Care Committee and the rats were housed and cared for in accordance with the guidelines of the Canadian Council on Animal Care. Weanling male Sprague-Dawley rats (Charles River Canada, St. Constant, QC, Canada; 45.3 ± 2.8 g, mean ± SD) were individually housed in randomly assigned mesh-bottomed stainless steel cages. The room temperature was maintained at 23 ± 2°C and lights were on a 12-h light/dark cycle.
In the depletion phase, the rats were fed a low folate diet based on the AIN 93G diet (19 ) containing folate-free vitamin mix and vitamin-free casein (Harland Teklad, Madison, WI). The folate concentration of the depletion diet was 0.37 ± 0.02 mg/kg diet. Rats had free access to tap water. On d 28 of the depletion period, liver folate concentration was 5.2 ± 0.9 nmol/g wet liver and the rats weighed 247 ± 22 g (mean ± SD) at this point (n = 7).
At this time, the 126 folate-depleted rats were randomly divided into 9 diet groups that were formulated to be isoenergetic and isonitrogenous (Table 1). One half of the rats in each diet group also received succinylsulfathiazole (Sigma Chemical, St. Louis, MO) at a concentration of 5 g/kg diet to inhibit intestinal bacterial folate synthesis. The standard diets were supplemented with folate to give 
0.5 (low), 0.6, (medium) and 0.9 (high) mg folate/kg diet. Some of the test diets containing DF and UFDM were supplemented with folic acid (Sigma Chemical) to increase their total folate content so that it fell within the range of the low, medium and high standard diets. The folate content of the individual diets is listed in Tables 2and 3. The energy content of the diets was calculated from composition data using the factors of 16.6 kJ/g protein or carbohydrate, 37.4 kJ/g fat and 8.3 kJ/g DF or UFDM. Food consumption and body weights were recorded weekly (not shown) and changes in physical appearance were recorded if observed. Fecal samples were collected daily during the repletion phase and stored at -20°C until used for analysis.
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Analyses.
Liver, feces and dietary samples were analyzed for total folate in triplicate on different days; the analysis was based on a method of the American Association of Cereal Chemists with minor modification (20 ). Short-chain fatty acids (SCFA) were analyzed by the method of Weaver et al. (21 ) using a Hewlett-Packard 5880A Series Gas chromatograph with a Nukol fused silica capillary column (60 m x 0.25 mm x 0.25 µm film thickness coated with nitroterephthalic acid–modified polyethylene glycol polymer; Supelco, Bellefonte, PA). Total DF in diets and fecal samples was determined by a combination of enzymatic and gravimetric methods, using the rapid Health Protection Branch method (22 ). Inulin was determined according to McCleary et al. (23 ) and polydextrose was analyzed as described by Craig et al. (24 ). Total nitrogen in rat feces was determined by the Kjeldahl method using a Kjeltec analytical system (Tecator, Hoganas, Sweden). The concentration of 2–6-diaminopimelic acid (DAPA) in rat feces was determined based on a modification of published methods (25 ,26 ). Muramic acid (MA) was determined as described by Hadzija (27 ). Complete blood counts were determined by the hematology laboratory, Animal Resource Division, Health Canada, using a Coulter S Plus IV (Coulter Electronics, Hialeah, FA). Blood films were stained using Accustain Wright Stain (Sigma Diagnostics, Sigma Chemical) and differential enumeration was performed manually (28 ).
Statistical analyses.
Data were analyzed by a two-way ANOVA (diet x drug) unless otherwise indicated. Data were checked for a potential correlation between means and SD before ANOVA. When a correlation was observed, the data were transformed using the Box-Cox formula: T(Y) = (Y
- 1)/ where Y is the response variable and is the transformation parameter (29
). Values of were chosen to minimize the mean-square error. Post-hoc analyses, when warranted, were performed by Tukey’s Honestly Significant Difference test for unequal sample sizes (Sigma Stat version 5, 97 edition, Jandel Scientific, San Rafael, CA). A value of P < 0.05 was taken as the criterion of significant difference. Values in the text are means ± SD
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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No physical signs of folate deficiency such as a tendency for the eyelids to stick together, thinning of the hair on the head or loss of weight were observed after the 28-d depletion period. Hematologic variables measured at the end of the depletion phase, however, were indicative of anemia. Depleted rats had lower hemoglobin concentrations (129 ± 3.8 vs. 143 ± 5 g/L for replenished rats), lower hematocrits (0.36 ± 0.01 vs. 0.40 ± 0.02 for replenished rats), higher mean cell hemoglobin 20.7 ± 1.0 vs. 19.7 ± 0.8 for replenished rats) and lower erythrocyte counts (6.2 ± 0.3 vs. 7.3 ± 0.4 x 1012 cells/L for replenished rats). No differences in leukocyte concentrations, RBC distribution width, platelet volume or mean cell hemoglobin concentration were observed. Food consumption over the course of the depletion period was 17.0 ± 3.8 g/d and the body weight at the end of the depletion period was 248 ± 8 g (n = 126). One rat died during the repletion phase (wheat bran diet plus succinylsulfathiazole), but this was unrelated to the experimental treatment. All other rats remained in good health until the end of the experiment.
During the repletion phase, rats were fed one of nine different diets containing DF or UFDM with or without addition of an antibiotic, succinylsulfathiazole (Table 1). The presence of the antibiotic had no apparent effect on appetite or growth as shown by the lack of a significant effect of diet or drug on weekly food intake or body weight (Tables 2and 3). The test diets contained differing amounts of both endogenous and added folate so that the total intake was within the range of folate consumption of the rats fed the three standard diets. Folate intakes were calculated by multiplying the measured folate content of the diet (Table 1) by food intake (Tables 2and 3).
Fermentation.
The diets contained differing amounts of alphacel plus DF or UFDM to maintain approximately constant total amounts of DF plus UFDM. Alphacel was added to maintain energy density, whereas DF and UFDM were added to stimulate fermentation. Differences in diet fermentability were mirrored by differences in fiber excretion. Rats consuming alphacel excreted 0.98 ± 0.08 g fiber/g dietary alphacel. Fermentation of nonalphacel DF was estimated by subtracting the contribution of alphacel from the diets and feces. Wheat bran fiber was the most poorly fermented DF in the absence of antibiotic (Fig. 1 ). All of the other DF and UFDM were fermented to approximately the same extent. The antibiotic significantly reduced the fermentability of the oat bran, ground corn and wheat germ fibers but had no effect on wheat bran fiber, inulin or polydextrose fermentability.
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Rats fed diets with succinylsulfathiazole excreted less folate than those fed diets without the antibiotic (P < 0.000001; compare Tables 2and 3). Succinylsulfathiazole also had a diet-dependent influence on folate excretion. For example, in rats fed standard diets, dietary folate had no effect on folate excretion in the absence of antibiotic (Table 2) but was associated with dietary folate in the presence of succinylsulfathiazole (Table 3). In the absence of the antibiotic, folate excretion was higher in rats fed wheat bran, oat bran, wheat germ and inulin (Table 2) compared with rats fed the standard diets, whereas in the presence of the antibiotic, folate excretion was higher in rats fed wheat bran, oat bran and wheat germ (Table 3).
Bacterial growth.
Bacterial growth and excretion were measured indirectly as fecal DAPA, MA and nitrogen (Fig. 3 ). ANOVA revealed significant effects of diet, antibiotic, and a diet x antibiotic interaction. The effect of diet was similar for all three compounds but different in magnitude so that differences were not always significant. For example, rats fed wheat bran, oat bran and wheat germ excreted more DAPA, MA and nitrogen than rats fed the standard diets. However, rats fed inulin excreted more DAPA and MA, but not nitrogen, whereas rats fed ground corn and polydextrose excreted more MA only.
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Relationship between liver folate and dietary folate.
Rat liver folate concentration is an indicator of the total amount of bioavailable folate, which could potentially include folate produced through cecal bacterial fermentation. If bacterially derived folate were bioavailable, liver folate concentrations should exceed those predicted by the standard curve (obtained by adding folate alone to the diet). This was tested by plotting liver folate concentration at the end of the repletion phase as a function of total folate intake over the repletion phase (Fig. 4 ). A linear relationship between dietary folate intake (µg/d) and liver folate stores (µmol/g wet tissue) was apparent for rats fed the standard diets in the absence as well as in the presence of succinylsulfathiazole. In these graphs, points lying above the standard diet regression line indicate a higher liver folate concentration than can be explained by dietary folate intake alone. On the other hand, points below the line indicate a lower liver folate concentration than expected. Data were analyzed by calculating the liver folate/dietary folate ratio for each rat followed by a two-way ANOVA (diet x antibiotic). Data from rats fed low, medium and high standard diets were pooled because the three groups did not differ. Comparing liver folate concentrations of rats fed standard diets to those fed diets with added DF or UFDM showed no effect of added DF or UFDM.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The lack of effect of fermentation and antibiotic in this study differs from a previously published study (16 ) that measured liver folate concentrations in Fisher-344 male rats fed diets containing xylan or wheat bran. In that study, the authors found no effect of phthalylsulfacetamide on folate absorption when fed at 5 g/100 g diet, a level identical to that of the present study. They did, however, observe increased liver folate concentrations when xylan or wheat bran was added to the diet. This led those authors to attribute the effect of wheat bran and xylan to folate bioavailability from cecal fermentation. The results and conclusions of that study are the exact opposite of those in the present study.
One of the reasons for the lack of an effect of fermentation on liver folate concentrations may have been a low rate of folate production in the cecum. It is possible to estimate the dietary equivalent of the fecal folate by comparing the excretion rate with the rate of folate intake. In rats fed the standard diets with added folate (but without added succinylsulfathiazole), the excretion rate was constant at 3.5 µg/d. Using this baseline value, it is possible to estimate that the intestinal microflora utilizing DF or UFDM produced 5.7 µg/d (wheat bran), 3.8 µg/d (oat bran), 1.8 µg/d (ground corn), 5.7 µg/d (wheat germ), 3.4 µg/d (inulin) and 0 µg/d (polydextrose) additional folate. This represented 45% (wheat bran), 32% (oat bran), 20% (wheat germ), 30% (ground corn), 29% (inulin) and 0% (polydextrose) of the total ingested folate. On the basis of these values, we expected to see a significantly greater liver folate/dietary folate ratio than was observed in rats fed the standard diets, which are not fermented.
Another potential reason for the lack of an effect of fermentation is a low absorption of cecal folate in Sprague-Dawley rats. Rong et al. (11
) investigated folate absorption directly by injecting a radioactive folate precursor into the cecum of male Sprague-Dawley rats. They showed that de novo bacterially synthesized folate was absorbed by the large intestine and could be recovered in the liver after 72 h. Although they did not quantify the amount of folate contributed by the intestinal bacteria, they estimated that 1 nmol of radioactive folate appeared in the liver after 3 d. Extrapolating this to a 28-d repletion period gives 9 nmol folate. In our experiments, rats fed a high amount of dietary folate (wheat germ) accumulated 130 nmol of liver folate during the repletion phase. This means that bacterially derived folate would represent only 7% of the total accumulation. Rats fed highly fermentable diets low in folate (oat bran) accumulated only slightly less liver folate (127 nmol) so that the total potential bacterial contribution is still only 7%. This low contribution is within the statistical variability of the present experiments, meaning that any effect of bacterially derived folate could have been missed. The total liver folate accumulation in Sprague-Dawley rats (130 nmol folate) is approximately double that observed in Fisher-344 rats, where an accumulation of 77 nmol of liver folate was observed for rats fed a high wheat bran diet for 27 d (16
). About half of this accumulation was apparently due to fermentation (41 nmol liver folate), a value much higher than the estimate of 9 nmol folate/28 d obtainable from fermentation in Sprague-Dawley rats (see above). Thus, although DF and UFDM may enhance bacterial folate synthesis in Sprague-Dawley rats, this folate does not appear to be absorbed to any great extent by these rats and, hence, does not contribute to their folate status. Fisher-344 rats, on the other hand, can apparently absorb 4.6 times more folate from their cecum than can Sprague-Dawley rats. These calculations, therefore, suggest that part of the difference may lie in a superior ability of Sprague-Dawley rats to absorb dietary folate.
Succinylsulfathiazole was added to provide an indirect assessment of the contribution from fermentation to total folate intake. Although the addition of succinylsulfathiazole decreased bacterial folate excretion, it also lowered liver folate concentrations equally in control and test rats. A simple explanation for this is a succinylsulfathiazole-associated inhibition of folate absorption in the small intestine. The fact that succinylsulfathiazole belongs to a group of sulfonamides that all have the same structure as p-aminobenzoic acid (part of the folic acid molecule) may make it an effective competitive inhibitor of the absorption process although no data exist concerning the effect of succinylsulfathiazole on folate absorption. The reduction in folate excretion was likely due to succinylsulfathiazole-associated: 1) inhibition of folic acid synthesis in bacteria [(30
); Tables 2and 3]; 2) reduction in the number of intestinal bacteria suggested by lower DAPA and MA excretion [(30
,31
); Fig. 3
]; and 3) reduction in fermentation suggested by lower cecal acetic and propionic acids (Fig. 2)
. However, this interpretation is overly simplistic because there are 350 species of bacteria in the rat intestinal tract and many will not respond to the addition of a single antibiotic. In addition, not all bacteria produce folate in concentrations large enough to be measured in the feces. Previous experiments have shown the complicated nature of the intestinal bacteria population. Gant et al. (32
) observed an initial decline in the number of fecal Escherichia coli after 3–4 d followed by a gradual return to normal 20–25 d after the addition of 5 g succinylsulfathiazole/100 g diet. This rebound was interpreted as the growth of succinylsulfathiazole-resistant organisms. E. coli are important because they are thought to be the major folate-producing species in the rat intestine (33
–35
). Our results showed reduced folate excretion in rats fed succinylsulfathiazole for 28 d (Tables 2and 3) which, according to Gant et al. (32
), should be enough time for the recolonization of E. coli in the cecum. The results may be explained either by a loss of folate-synthesizing capacity in succinylsulfathiazole-resistant E. coli or by their replacement with nonfolate-producing bacteria. The growth of drug-resistant species is a more likely scenario because Miller (30
) observed no change in the total number of excreted bacteria even though the number of intestinal facultative anaerobes was reduced after antibiotic treatment. Our results, therefore, show that caution is warranted when using succinylsulfathiazole as a means of demonstrating the contribution of bacterial fermentation to folate absorption (9
).
In summary, we observed no effect of DF or UFDM on liver folate stores in depleted and replenished male Sprague-Dawley rats. If the human response is similar to that observed in Sprague-Dawley rats, these results would suggest that the current levels of folic acid fortification in cereal-based products do not have to be readdressed with respect to the influence of DF and UFDM on overall folate intake.
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FOOTNOTES |
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3 Abbreviations used: DAPA, diaminopimelic acid; DF, dietary fiber; MA, muramic acid; UFDM, undigested and fermented dietary material.
Manuscript received 1 October 2002. Initial review completed 15 November 2002. Revision accepted 28 January 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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