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Department of Nutritional Sciences, University of Toronto and The Hospital for Sick Children, Toronto, Canada M5G 1X8 and * Department of Nutritional Sciences, University of Toronto and St. Michael’s Hospital, Toronto, Canada M5B 1W8
2To whom correspondence should be addressed. E-mail: deborah_l.o'connor{at}sickkids.ca.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • folate • folate bioavailability • humans • infants • pigs
Suboptimal intakes of folate in humans have been associated with a number of important health outcomes, most convincingly anemia during pregnancy and neural tube defects (1–3). Due to the weight of the evidence in the case of neural tube defects, elevated folic acid fortification of flour (140–150 µg/100 g) and enriched cereal-based products became mandatory in the United States and Canada in 1998 (4,5). Consequently, recent evidence indicates that blood levels of folate have risen, and circulating total homocysteine concentrations have fallen substantially in the North American population (6–10). Some remain concerned, however, that excess folic acid in the food supply may confound the diagnosis of vitamin B-12 deficiency, a common problem in the elderly (3,11–13). Further, most countries have not adopted an elevated folic acid fortification program. The long-term goal of our research program is to determine whether manipulation of the microbial milieu of humans might result in a complementary source of bioavailable folate. If this could be accomplished, dietary folate requirements and the level of folic acid fortification could be decreased.
Unlike humans, many species of bacteria, including those in the large intestine, can synthesize folate (14–18). Preliminary data exist to suggest that these folates, or a fraction of them, may be absorbed across the large intestine (19–23). Consistent with much earlier work (24–26), we demonstrated that rats fed diets containing human milk solids, known to be bifidogenic, have higher concentrations of folate in the cecum and plasma but not liver, compared with rats fed cow’s or goat’s milk solids (27–31). Similarly, Keagy and Oace (32) demonstrated an increased liver folate concentration among rats fed diets containing xylan, a partially soluble and fermentable hemicellulose, but not wheat bran. Thoma et al. (33) very recently reported that rats fed a diet containing citrus pectin had higher plasma, erythrocyte, and colonic tissue folate concentrations and lower total homocysteine concentrations, but not altered liver folate concentrations compared with rats fed diets containing cellulose. Sepehr et al. (34) found no difference in liver folate content among rats fed different sources of dietary fiber (wheat bran, oat bran, ground corn, wheat germ) or undigested and fermented dietary material (polydextrose, inulin).
An obvious limitation of extrapolating data from rats to the human condition is that rats are coprophagic; hence folate synthesized by microorganisms in the large intestine and excreted in feces can be absorbed in the small intestine. Early studies with humans reported that 300–500 µg/d of folate may be excreted in adult feces compared with dietary intakes < 100 µg/d (35,36). Although frequently cited, the total number of samples measured in these studies was small (n = 10), folate extraction procedures were likely incomplete, and the microorganism used in these analyses (Streptococcus faecalis) is not responsive to all forms of folate (37). Therefore, the purpose of the 2 experiments reported herein was to estimate the pool of folate in the feces of human infants and piglets to ascertain whether the quantity and form of folate in the large intestine are sufficient to potentially affect the folate status of the host organism if absorbed. Analysis of the quantity and form of folate in the feces of piglets will provide preliminary data to ascertain whether the piglet can be used as a model for future studies in this area.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Subjects.
Infants and their mothers (n = 22) were recruited into the study between June, 2001 and January, 2003 via word-of-mouth and publicity in local newspapers and in the postpartum units at Sunnybrook and Women’s College Health Sciences Centre, Women’s College Campus, and at St. Michael’s Hospital, Toronto, Canada. Infant/mother dyads were screened for confirmation of eligibility and informed written consent was obtained. Healthy term infants (
37 wk gestation) between 1 and 6 mo of age and fed exclusively human milk or a cow’s milk–based infant formula were eligible to participate. Infants were excluded from the study if a combination of human milk and formula was fed or if solid foods were introduced. Similarly, infants and nursing mothers were excluded if they had a medical condition or were taking drugs (e.g., azidothymidine, antibiotics) known to interfere with either folate metabolism or intestinal bacteria growth. Infants and mothers taking vitamin and mineral supplements were not excluded. Demographic data were collected by means of a self-administered questionnaire at the time of biological sample collection. None of the infants had chronic diarrhea or constipation at the time of the study. The study protocol was approved by the Research Ethics Boards at The Hospital for Sick Children, Sunnybrook and Women’s College Health Sciences Centre and St. Michael’s Hospital.
Sample collection. For all infants, stools were collected into lined diapers (Kushies Baby Products) for 3 consecutive d. The stools and diaper liners were transferred to aluminum cans (3.8 L) with a secure fitting lid and stored at –20°C in the infant’s home until the end of the 3-d collection period. They were then transferred on ice back to the laboratory and stored at –80°C until analysis.
Mothers of exclusively breast-fed infants were asked to express all milk from a single breast either manually or by using an electric breast pump, between 1300 and 1500 h, for 3 consecutive d (38). Milk was divided each day into 5-mL aliquots and stored in tubes with cryoseal screw tops, containing 10 g/L sodium ascorbate to protect labile folates. Aliquots of infant formula were also collected from mothers of exclusively formula-fed infants in tubes containing 10 g/L sodium ascorbate. Milk samples were stored at –20°C in the infant’s home until the end of the 3-d collection period, at which time they were transferred back to the laboratory on ice and stored at –80°C until analysis.
Microbiological analysis of milk and stools for folate. Stools and diaper liners were homogenized in 10 volumes of Hepes/Ches buffer (50 mmol/L Hepes, 50 mmol/L Ches, pH 7.85), containing 20 g/L sodium ascorbate and 10 mmol/L 2-mercaptoethanol (39,40) and then centrifuged (40,000 x g, 30 min, 4°C). The supernatant fraction was flushed with nitrogen gas and stored at –80°C, until further analysis. As evident from work on a subset of these samples (n = 7), boiling before centrifugation (vs. after) to destroy cellular matrices and liberate folates from binding proteins did not yield an increase in analyzed folate content.
Human milk, infant formula, and supernatant fractions of stools underwent several steps before microbiological analysis (41). They were first heated (5 min, 100°C), then treated with 
-amylase (EC 3.2.1.1) and protease (EC 3.4.24.31) (Sigma Chemical) to free folates from carbohydrate and protein matrices, respectively. They were then treated either with or without rat serum folate conjugase (Harlan Bioproducts for Science) to estimate short-chain (
3 glutamate residues) and long-chain (4 glutamate residues) pteroylpolyglutamates. Irrespective of the carbon substitution, the test microorganism, Lactobacillus casei (ATCC 7469) fully responds to both oxidized and reduced folates with 3 or fewer glutamate residues. Further, utilization of tetra-, penta-, hexa- and heptaglutamate forms of folate by L. casei is reported to be 65.5, 9.9%, 3.5, and 2.4%, respectively (42). For all samples, folate concentrations were determined by the microbiological microtiter plate assay, as described by Molloy and Scott with minor modification (43). The interassay CV for the microbiological assay was 11.2% based on repeated measurements of a pooled plasma control.
HPLC analysis of stools for the proportion of 5-methyltetrahydrofolate (5-methylTHF) that was monoglutamylated. The proportion of 5-methylTHF in stool samples that was monoglutamylated was determined on supernatant fractions treated with amylase and protease, either with or without conjugase treatment, as described above. Folates were purified from these enzyme-treated supernatants by affinity chromatography using immobilized bovine milk folate binding proteins (FBP). Purified folates were then separated and identified using ion-pair HPLC with electrochemical detection. Detailed descriptions and validation of these chromatographic procedures were published by Bagley and Selhub and Belz and Nau (44–46).
Briefly, an FBP affinity column was prepared by suspending 50 g of dried whey (ADM Nutraceutical) in 500 mL of water and adjusting the pH to 9 with 5 mol/L NaOH. This solution was stored overnight (4°C) and then centrifuged (10,000 x g, 30 min, 4°C). To prepare the affinity column matrix, the supernatant fraction of the whey suspension was allowed to react with AffiPrep 10 Support (Bio-Rad Laboratories) by stirring overnight at 4°C. The FBP-AffiPrep 10 matrix was washed sequentially with 20 mmol/L trifluoroacetic acid, 1 mol/L potassium phosphate, and water. The FBP-AffiPrep 10 matrix slurry (1 mL) was then transferred to a glass Pasteur pipette packed with glass wool. A fresh column was prepared each week.
Enzyme-treated samples were loaded onto the affinity column and washed sequentially with water and then the mobile phase, which contained equal portions of A (112 mmol/L potassium phosphate, 240 mmol/L phosphoric acid) and B (800 mL/L acetonitrile), and water. The recovery from the affinity column, as measured with tritiated folic acid (Amersham Pharmacia Biotech), was 85.5 ± 0.7% (mean ± SD). The total folate binding capacity exceeded 500 µmol/L solid phase.
The eluate from the affinity column was then injected into the HPLC. The HPLC system consisted of a P580 pump with ASI-100 autosampler, a 250 x 4.6 mm Betasil Phenyl analytical column installed within an oven set at 30°C, and an ED50 electrochemical detector with set-up shift and Ag/AgCl reference potential, managed by Chromeleon version 6.2 software. All parts were purchased from Dionex Corporation, except for the phenyl analytical column, which was purchased from Keystone Scientific. The mobile phase was delivered at a flow rate of 0.75 mL/min and for the first 10 min and maintained at 25% A, 7% B, and 68% water. From 10 to 40 min, the concentration of B was raised linearly to 20%, providing the gradient. The various folate derivatives were identified on the basis of retention time and comparison of the electrochemical response of each peak with that of the corresponding folate standard (gift from Merck KGaA).
Piglet study
Animal care. Male Yorkshire piglets (n = 10, 5 d old) were purchased locally and housed individually in cages bedded with wood chips, with a controlled temperature (23–24°C) and a 12-h light:dark cycle. The piglets were acclimated for 5 d before study initiation. Commencing on arrival, piglets were fed via feeders (4 times daily) a freshly reconstituted milk-based formula. Tap water was consumed ad libitum. The antibiotic-free diet (Research Diets) was formulated after one that has been used extensively by others and modified to meet the nutrient requirements of the NRC for piglets (Table 1) (47). It contained 1.386 µg folic acid/g as analyzed by microbiological assay in our laboratory. The amount of formula consumed by piglets and their weight was determined daily (±10 g) with a MBS 2010 Digital Baby Scale (My Weigh). The Animal Care Committee at The Hospital for Sick Children approved all aspects of the experimental protocol.
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Folate analyses. To assess the quantity and form of folates most easily available for absorption (the fluid fraction), fecal fluid and solids were separated by centrifugation (40,000 x g, 60 min, 4°C). The supernatant fecal fluid fraction was flushed with nitrogen gas and stored in 10 g/L sodium ascorbate at –80°C, until further analysis. The pellet was homogenized in 10 volumes of Hepes/Ches buffer, then heated (100°C, 15 min) and centrifuged (40,000 x g, 30 min, 4°C) (39). The supernatant of this fecal solid fraction was flushed with nitrogen gas and stored at –80°C as fecal solid, until further analysis. Procedures used to analyze piglet fecal fluid and fecal solids for folate were identical to those used in the human infant study as described above.
Statistical analysis. All data were analyzed using the Statistical Analysis System for Windows version 8.01 (1999–2000). All data were checked to ensure they were normally distributed (Proc Univariate procedure). The folate contents of human feces were not normally distributed and were log transformed. Statistical comparisons of milk and fecal folate content between human milk–fed and formula-fed infants and between folate content of fecal solids and fecal fluids of piglets were conducted by t test and differences were considered significant at the 5% level. Values in the text are means ± SD.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Subject characteristics. After screening of 14 breast-feeding mother/infant dyads and 15 formula-fed infants, 26 pairs were eligible to participate in the study. Three infants were ineligible because they were fed a combination of formula and human milk (n = 1), a soy-based formula (n = 1), or were too young by the time the study had closed (n = 1). Of the 26 enrolled infants, 2 families each did not complete the sample collection and 2 dropped from the study because they developed conditions making them ineligible to participate (one mother developed mastitis and an infant developed an ear infection requiring antibiotics). Therefore, 12 exclusively human milk–fed infants and 10 exclusively formula-fed infants completed the study with a mean age of 2.9 ± 1.1 mo. No differences existed between the 2 groups with respect to either age (maternal or infant) or gender distribution. Seven of 12 human milk–fed infants were taking oral vitamin D supplements but none were provided with supplemental folic acid. No formula-fed infants were provided with vitamin supplements. The mean age of lactating mothers was 31.3 ± 3.7 y; 9 of the 12 lactating mothers consumed vitamin supplements, all containing 400-1000 µg of folic acid. None were smokers and 8 women consumed alcohol at least once each week.
Fecal folate. The fecal folate content of all infants (n = 22) was 93.2 ± 92.8 nmol/d (41.1 ± 41.0 µg/d), of which 66% was short-chain folate (Table 2). The folate content of feces collected from formula-fed infants was more than 2-fold greater than that of human milk–fed infants (P < 0.05). The percentage of fecal short-chain folate did not differ between the 2 groups. The predominant folate species was 5-methylTHF, of which 52.5 ± 30.1% was monoglutamylated 5-methylTHF. The percentage of monoglutamylated 5-methylTHF in fecal solids did not differ between human milk–fed and formula-fed infants. Folic acid was detected in the feces of 2 human milk–fed infants and 6 formula-fed infants. Folic acid may have been undetected in other samples because they fell below the minimum detection limit of the HPLC (0.1 nmol/L).
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Piglet characteristics. The body weight of piglets on d 10 of life was 3.2 ± 0.5 kg. The formula and folate intakes of piglets at d 10 were 1.61 ± 0.37 L/d and 833.1 ± 191.8 nmol/d, respectively. The fecal solid and fluid weights of piglets at d 10 were 14.2 ± 7.6 and 8.3 ± 10.5 g/d, respectively, with a total fecal weight of 22.8 ± 14.1 g/d.
Fecal folate. The folate content of feces collected from piglets over a 24-h period was 301.3 ± 145.7 nmol/d (133.0 ± 64.3 µg/d), of which 269.8 ± 129.2 nmol/d (119.1 ± 57.0 µg/d) and 31.4 ± 27.4 nmol/d (13.9 ± 12.1 µg/d) of folate were found in fecal solids and fluids, respectively (Table 4). The folate contents and concentrations of fecal solids and fluids differed significantly (P < 0.05). The percentage of short-chain folate in fecal solids and fluids did not differ. The predominant form of folate found in the fecal solids was 5-methylTHF, of which the monoglutamylated fraction amounted to 29.3 ± 33.2%. Folic acid was detected in 4 of the 10 piglet fecal solid samples. Monoglutamylated folates of any kind, including 5-methylTHF in fecal fluids were below the minimum detection limit of our HPLC.
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Results from the present study suggest that a significant depot of folate exists in the large intestine of nursing human infants and piglets. Further, many of these folates are monoglutamylated, a form that can be readily absorbed across the small intestine. The amount of folate measured in the fecal solids of infants was 93.2 ± 92.8 nmol/d (41.1 ± 41.0 µg/d). This quantity represents, on average, 
63% of the adequate intake level for infants < 5 mo of age (65 µg/d). The total amount of folate excreted in the stool solids each day by infants in this study was 50% of that consumed, assuming an average daily milk intake of 0.78 L/d. If, as we and others hypothesize, absorption of folate occurs across the large intestine, the folate measured in stools in the present study is what remained after absorption. Therefore the folate content of stool solids presented herein is likely an underestimate of the true amount of folate available for absorption across the large intestine.
Approximately two-thirds of the folate found in the stool solids of human infants was short-chain folate. Further, approximately half of the 5-methylTHF was monoglutamylated. Although the mechanism by which folate is absorbed across the large intestine is not clear, several studies have confirmed that absorption can occur and that the process may be similar to what occurs in the small intestine (20–23). In the small intestine of adult humans and pigs, polyglutamylated forms of folate must be hydrolyzed into monoglutamates before transport into the enterocyte via the protein transporter, reduced folate carrier (48,49). The enzyme responsible for this hydrolysis is glutamate carboxypeptidase II, also known as folylpoly-
-glutamate carboxypeptidase or folate conjugase, found in the intestinal brush border of humans. To our knowledge, no studies to date have examined either the presence of folate conjugase or its activity in human feces. Nonetheless, the data reported herein suggest a significant proportion of fecal folate is in a form (monoglutamylated) that can be absorbed.
The mean folate content of feces of formula-fed infants was approximately 2-fold greater than that of human milk–fed infants although the opposite result might have been expected from findings of previous studies in rats (27–29). In the present study, the higher folate content of infant formula compared with human milk and hence the differential contribution of unabsorbed folates between formula-fed and human milk–fed infants likely account for some of this difference. Differences in the profile of the colonic microflora, specifically the relative proportion of folate synthesizing vs. consuming microorganisms, between humans and rats may also be a contributory explanation. In addition, the lower folate content of feces of human milk–fed infants may have been due, at least in part, to incomplete stool collections. Exclusively human milk–fed infants produce a softer and more liquid stool of greater volume compared with formula-fed infants (50,51). Consequently, the loss of fecal fluid may have resulted in a disproportionately lower fecal folate content in the human milk–fed compared to the formula-fed group.
The long-term goal of our research program is to determine whether manipulation of the microbial milieu of humans might result in a complementary source of bioavailable folate. As such, it is of interest to estimate what proportion of total fecal folate is of bacterial origin. Data presented herein allow us to make a theoretical, although admittedly preliminary, estimate about what proportion of total fecal folate is of bacterial origin. Endogenous losses by way of enterohepatic circulation and sloughing of intestinal cells likely account for a portion of the fecal folate measured. At least 1 study with rats suggested that it is unlikely that losses from enterohepatic circulation account for a significant amount of fecal folate because most biliary folate is reabsorbed in the small intestine, even after pharmacologic doses of the vitamin (52,53). In contrast, Krumdieck et al. (54) reported a similar rate of fecal and urinary excretion of 14C after administration of [14C]folate to a single human subject, suggesting that a measurable quantity of biliary folates and/or catabolic products is not reabsorbed. A fecal isotope excretion study in a monkey model suggested that 10% of fecal folate is of endogenous origin (55).
Nonetheless, 2 likely sources of folate in feces are folates from the diet and bacterial biosynthesis. Taking into account the human milk and formula folate concentrations reported herein and assuming infants consumed 0.78 L milk/d with bioavailability of folates in the range of 75–85%, we estimate that 13.7–22.8 and 47.9–79.9 nmol of fecal folate from exclusively human milk–fed and formula-fed infants, respectively, could be of dietary origin (3,56). Because the mean fecal folate content of human milk–fed and formula-fed infants was 47.1 ± 36.9 and 148.5 ± 109.3 nmol/d, respectively, folates of bacterial origin could have accounted for up to 24.3–33.4 and 68.6–100.6 nmol/d, respectively. Thus, a much smaller proportion of fecal folate appears to be of bacterial origin in human milk–fed infants compared with that of formula-fed infants.
It is interesting to note that 2 of the 12 exclusively human milk–fed infants had supplemental folic acid in their feces. Because a folic acid supplement was not provided to infants and these babies were fed human milk exclusively, we presume that the folic acid came from their mother’s milk. Selhub (57) previously reported the presence of supplemental folic acid in human milk collected from a single lactating mother before enhanced fortification of the food supply in North America. Whether this subject consumed a folic acid–containing supplement was not reported.
Piglet study
Much of the earlier work investigating the effect of microbial folate biosynthesis on the folate status of the host was done using the rat as a model (20,28,29,32,34). However, it is difficult to extrapolate these results to humans because rats consume their feces and recognized differences exist between rats and humans in the mechanism of folate absorption. As described previously, the mechanism of folate absorption in the large intestine is thought to resemble that of the small intestine (20–23). Both brush border and intracellular folate conjugases exist in the small intestine of humans, whereas brush border folate conjugase is lacking in rats (58,59). In contrast, both the brush border and intracellular folate conjugases were described in pigs with characteristics very similar to those in the human intestine (60). Therefore, the pig was recommended as the most suitable animal model for studies of folate absorption in humans (61).
In accordance with this recommendation, we analyzed the quantity and form of folate in the feces of piglets. The folate content of feces collected from piglets was 301.3 + 145.7 nmol/d, which represents 166% of the NRC folate requirement for 3- to 5-kg piglets and 36% of their actual intake (43). Applying the same logic used in the human infant study and assuming 75–85% bioavailability, we find that the amount of bacterially synthesized folate may be up to 93–176.3 nmol/d.
Unlike in the human infant study, we were able to perform complete fecal collections in the piglet study. Approximately one tenth of the total folate content of piglet feces was found in fecal fluid. As was the case with the human infant study, about two-thirds of piglet fecal folates were short chain. The principal form of folate found in the piglet fecal solids was 5-methylTHF, of which the monoglutamylated form amounted to 29.3 ± 33.2%. Monoglutamylated 5-methylTHF was undetectable by our HPLC in the fecal fluids due to its low concentration. Because monoglutamylated folates in the fluid fraction are not trapped inside of cellular matrices, they are theoretically more available for absorption across the large intestine compared with folate in the solid fraction. Perhaps the lower folate concentration in the fecal fluid represents increased absorption from this fraction.
In conclusion, the findings from this study suggest that the quantity of microbially synthesized folate in the large intestine of human infants is sufficiently large to potentially affect the folate status of the host. The results of the analysis of human infant feces for the predominant forms of folate suggest that a significant fraction of folate is in a form (monoglutamylated) that can be readily absorbed. Analysis of piglet feces produced results comparable to that of human infants, indicating the suitability of the piglet as a model for future studies in this area.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 29 December 2003. Initial review completed 13 January 2004. Revision accepted 23 February 2004.
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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