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,2
* Departments of Nutritional Physiology,
Food and Food Supplement Analysis and
** Biomolecular Sciences, TNO Nutrition and Food Research, 3700 AJ Zeist, The Netherlands;
Division of Human Nutrition, Wageningen University, 6700 EV Wageningen, The Netherlands,
Department of Food Science, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden
2To whom correspondence should be addressed. E-mail: Verwei{at}voeding.tno.nl.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • folate-binding protein • 5-methyltetrahydrofolate • folic acid • gastric passage • milk products
Prevention of neural tube defects (1,2) and reducing the risk for cardiovascular disease (3,4) and colon cancer (5,6) can be achieved by an adequate folate intake. This can be reached by a high consumption of folate-rich food products or by the intake of supplements or fortified food products (7–9). The dominant folate compound in natural food products is 5-methyltetrahydrofolate (5-CH3-H4folate), whereas in supplements and fortified products, primarily folic acid is used.
Although the natural folate concentration in milk is low compared with folate-rich food products such as vegetables and citrus fruit, milk is responsible for 10–15% of the daily folate intake in European countries with a high milk consumption, such as The Netherlands (10) and Sweden (11). Milk can be considered as a potential matrix for folate (folic acid or 5-CH3-H4folate) fortification because it is widely consumed and might enhance folate bioavailability from the diet (12); in addition, folic acid and 5-CH3-H4folate appear to be highly bioaccessible from the milk matrix (13).
In untreated milk, 5-CH3-H4folate occurs bound to folate-binding proteins (FBP) (14–16). The role of FBP in folate bioavailability is unclear. It has been suggested that FBP protects folate from bacterial uptake and degradation (17,18) or may play a role in sequestering folate from the blood plasma into the mammary glands, thereby supplying folate to the newborn (19). FBP could also affect mucosal folate transport, although both inhibition and enhancement have been reported (20–23). The influence of FBP on folate absorption might depend on its binding to folate after gastric passage. In a previous study, the effect of FBP on the absorption of folic acid was investigated in rats (22) that were administered free folic acid or folic acid bound to bovine milk FBP. Under acidic gastric conditions (pH < 4.5), folic acid was released from FBP and recombined in the small intestine (pH 6–7). In a study (24) with 6-d-old goat kids, who were bottle-fed FBP-bound folic acid in goat’s milk, it was shown that gastric acidity and gastrointestinal digestive enzymes had little effect on the binding characteristics of FBP for folic acid. In our previous studies (13,25) using an in vitro dynamic gastrointestinal model, it was found that FBP partly survives gastrointestinal passage and lowers the folate bioaccessibility; these effects appeared to be higher in folic acid–fortified milk products compared with 5-CH3-H4folate–fortified milk products. This suggests a different extent of binding to FBP for folic acid and 5-CH3-H4folate.
The present study was performed to investigate the binding characteristics of FBP for folic acid and 5-CH3-H4folate and to establish the effect of FBP stability on folate binding during gastric passage in a controlled in vitro model simulating human gastric conditions. The binding of folic acid and 5-CH3-H4folate to an equimolar amount of FBP during gastric passage was compared to examine the effect of FBP on the bioavailability of folic acid and 5-CH3-H4folate from milk products.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Folate binding to FBP during gastric passage under static experimental conditions.
First, the binding characteristics of FBP were studied under static experimental conditions, i.e., in test tubes for 1–2 h. Whey powder was used as the source of FBP. The FBP suspensions were made by mixing 50 mg whey powder (
1 nmol FBP) with 6 mL 0.1 mol/L phosphate buffer (pH 7.2). An equimolar FBP:folate suspension was made by adding [3H]-folic acid or [14C]-5-CH3-H4folate combined with unlabeled folic acid or 5-CH3-H4folate to reach 1 nmol folate per 6 mL suspension (167 nmol/L). The FBP and folate concentrations used in this study were comparable to the natural FBP and folate concentrations in milk, i.e., 110–220 nmol folate/L and 160–210 nmol FBP/L, respectively (27). The mixtures of folic acid, 5-CH3-H4folate or a combination of both folate compounds with the FBP suspensions were incubated for 1 h at pH 7.2 at 20°C to allow association. Various test conditions were simulated in the incubation experiments (n = 2) (Table 1). In addition to the incubation at pH 7, an additional incubation period of 1 h at pH 3 (gastric pH conditions) with or without pepsin was tested. The pH of all mixtures was adjusted to pH 7 before elution over the Sephadex column to study the binding profile of the whey proteins.
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In this model, we studied the fate of the FBP-folate complex during gastric passage and the possible recombination or stability of the FBP-folate complex in the duodenum. The folic acid-fortified and the 5-CH3-H4folate-fortified FBP suspensions were tested separately in duplicate experiments. Artificial oral and gastric juices with lipase and pepsin were gradually added into the gastric compartment. The pH was measured continuously and regulated by the addition of HCl to follow the preset pH curve. This curve corresponded to the in vivo pH drop in the stomach of adults after consumption of milk products. In the duodenal compartment, the pH was controlled at 6.5 by the addition of sodium bicarbonate.
The FBP suspension was made by dissolving 2.75 g whey powder in 330 mL of 0.01 mol/L phosphate buffer. This resulted in a final FBP content of 55 nmol. An equimolar folate:FBP mixture was obtained by adding 55 nmol folic acid or 5-CH3-H4folate, as a mixture of [3H]-folic acid or [14C]-5-CH3-H4folate with unlabeled folic acid and 5-CH3-H4folate, to the FBP suspension. After a 1-h incubation at 20°C to induce association between folate and FBP, a test portion of 300 g was put into the gastric compartment of the gastrointestinal model. The remaining 30 g of the FBP suspension was used for the determination of the folic acid and 5-CH3-H4folate binding to FBP before gastric passage. Intestinal material from the duodenal compartment was collected during 0–30, 30–60, 60–90 and 90–120 min after starting the experiments. All collected samples were stored at 2–8°C and analyzed by gel filtration within 5 d. After 120 min, the stomach was almost completely emptied, according to the preset curve for gastric emptying in humans, and the residual contents in the stomach and duodenum were collected for calculation of the mass balance of folate. The folate content of the collected samples was determined by radioactivity measurements with a scintillation counter (Wallac 1409, PerkinElmer, Boston, MA).
Characterization of FBP in bovine whey
Gel filtration. The distribution of [3H]-folic acid or [14C]-5-CH3-H4folate over the whey proteins was studied by gel filtration on a Sephadex G75-column (2.6 cm x 30 cm). The column was equilibrated with 0.1 mol/L phosphate buffer (pH 7.2). The [3H]-folic acid or [14C]-5-CH3-H4folate-fortified FBP suspensions (3 mL), whether digested or not, were eluted with the phosphate buffer at a flow rate of 25 mL/h. The eluent was measured spectrophotometrically with an UV detector at 280 nm, as indicator of protein content, and subsequently collected as fractions at 8-min intervals during a total run time of 800 min. Portions (200–500 µL) of these fractions were quantified for their [3H]-folic acid or [14C]-5-CH3-H4folate content by measuring radioactivity with a scintillation counter after the addition of 4 mL of scintillation suspension.
The column was calibrated with the elution volumes of proteins within a low- molecular-weight gel filtration calibration kit, including blue dextran 2000 (200 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (14 kDa). The exact elution volume of FBP was determined by quantification of the FBP content in the collected fractions over time.
ELISA.
The FBP content of the samples from the experiments with the gastrointestinal model and the gel filtration samples was analyzed by a two-site ELISA according to Høier-Madsen et al. (31) to determine the FBP stability after gastric passage and the elution volume of FBP, respectively. To 1 g of sample 0.09 g of Triton X-100 was added. The sample was put on a shaking device and incubated for 45 min at 20°C. After this extraction, the sample was applied to a microtiter plate followed by the ELISA procedure as described previously (32).
SDS-PAGE and immunoblotting. The whey suspensions, before and after incubation with pepsin at pH 3, were subjected to a 12% gradient Tris-Glycine gel and subsequently blotted onto a nitrocellulose membrane. Immunodetection was carried out by incubating the blots with polyclonal rabbit antibodies against bovine FBP followed by goat-anti-rabbit IgG alkaline phosphatase conjugate, tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (33,34).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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50 mL) corresponded to proteins larger than 60 kDa and the second peak (elution volume 75 mL) to proteins between 30 and 40 kDa. The ELISA analysis showed a maximum FBP content at an elution volume of 75 mL (Fig. 1B), corresponding to the elution volume of the second peak, i.e., proteins between 30 and 40 kDa (Fig. 1A). No FBP was detected at the elution volume of the first peak of the UV-chromatogram, indicating that the proteins larger than 60 kDa did not contain ELISA-detectable FBP.
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Folate binding to FBP during gastric passage under static experimental conditions. The extent of binding to FBP for folic acid and 5-CH3-H4folate was studied under static experimental conditions simulating gastric passage. Incubation of the FBP suspension with folic acid or 5-CH3-H4folate at pH 7 showed that the major part (79%) of both folate compounds was initially bound to FBP before the incubation period at pH 3 (Fig. 1C, Table 1). The amount of bound folic acid (78%) did not differ after incubation at pH 3 with or without pepsin (Table 1). Incubation at pH 3 without pepsin did not affect the extent of binding to FBP for 5-CH3-H4folate (79%). However, the FBP-bound fraction of 5-CH3-H4folate decreased from 79 to 27% after incubation at pH 3 with pepsin for 1 h. At the same time, the fraction of free 5-CH3-H4folate increased from 7 to 63%. This indicated that a major portion of 5-CH3-H4folate could occur freely in the duodenal lumen. The whey proteins were also incubated with a mixture of folic acid and 5-CH3-H4folate (both in a 1:1 mol/L ratio with FBP) and the FBP-bound fractions were compared with those after the incubation with the single folate compounds. In this mixture of folic acid and 5-CH3-H4folate, there was a small decrease in FBP-bound folic acid (from 79 to 65%) and a pronounced decrease in FBP-bound 5-CH3-H4folate (from 79 to 38%).
FBP in the whey suspension showed two clear bands between 30 and 40 kDa with SDS-PAGE combined with immunoblotting. After pepsin incubation at pH 3, the intensity of the bands was lowered.
Folate binding to and fate of FBP during gastric passage under dynamic experimental conditions. The extent of binding of folic acid and 5-CH3-H4folate to FBP was investigated in duplicate experiments in the gastrointestinal model. The mass balance of folate in these experiments was 102 ± 1% (n = 4). The gel filtration analyses of the whey suspension (gastric intake) and the samples of the duodenal lumen gave an analytical recovery of 98 ± 2% (n = 20). The gastric passage of folic acid and 5-CH3-H4folate over time as measured in the duodenal compartment showed that most of the folate entered the proximal part of the intestine within 30–90 min after the start of the experiment (Fig. 2). The distribution of folic acid and 5-CH3-H4folate over the protein fractions was determined in the gastric intake (0 min) and in the duodenal samples collected during 0–30, 30–60, 60–90 and 90–120 min (Fig. 3). At initial test conditions, the major part of folic acid was bound to FBP and this fraction (76–81%) remained constant in the five successive samples over time during gastric passage (Table 2). The binding of folic acid to proteins larger than 60 kDa and the free folic acid fraction also remained unchanged over time. A similar initial FBP-bound fraction (79%) was observed for 5-CH3-H4folate before digestion. However, during gastric passage, the FBP-bound 5-CH3-H4folate fraction declined from 79 to 5% in 2 h. Consequently, the fraction of free 5-CH3-H4folate increased from 7% in the initial whey suspension before gastric passage to 93% in the duodenal sample collected between 90 and 120 min. The data from the duplicate experiments show a very low variation, which allows evaluation of the difference in the binding of folic acid and 5-CH3-H4folate to FBP during gastric passage.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Exposing FBP in a whey suspension to an equimolar mixture of folic acid and 5-CH3-H4folate resulted in a low binding of 5-CH3-H4folate (38%) and a relatively high binding of folic acid (65%) to FBP (Table 1), indicating that FBP has a higher affinity for folic acid than for 5-CH3-H4folate. This agrees with the results from previous studies in which FBP binding characteristics were investigated in in vitro experiments at pH 5.0 and 7.4 (38,39). This difference in affinity for FBP between folic acid and 5-CH3-H4folate was found to vary within the pH range of 7.4 to 10.1 (40).
The present study also showed that incubation at pH 3 had no effect on the extent of binding of folic acid and 5-CH3-H4folate to FBP once the pH of the incubation medium was returned to 7, reflecting the actual pH changes occurring during gastric and duodenal passage. An explanation may be that at low pH, dissociation of folate takes place, followed by a reassociation of folate to FBP at neutral pH. This is in line with other studies (22,35,36) showing that the dissociation of folic acid from FBP is completely reversible, even after pepsin treatment (22). We also found that incubation of the folic acid-FBP suspension at pH 3 with pepsin had no effect on the binding of folic acid to FBP (remained 78%). However, the FBP binding characteristics for folic acid are apparently different from the binding to 5-CH3-H4folate because there was a marked decrease in FBP-bound fraction (27%) after pepsin incubation of the 5-CH3-H4folate-FBP suspension. This different effect of pepsin on binding of folic acid and 5-CH3-H4folate to FBP suggests a difference in FBP binding characteristics for the folate vitamers.
In addition to the experiments under static conditions, experiments in a gastrointestinal model were performed because this model simulates the kinetic digestion and passage of the whey suspension from the stomach into the duodenum. These studies show that the amount of FBP-bound folic acid remained constant during the gastric passage from 0 to 120 min, indicating no change in the extent of folic acid binding to FBP (Table 2, Fig. 3). In contrast, the FBP-bound 5-CH3-H4folate fraction gradually decreased during gastric passage from 79 to 5% within 120 min. The results obtained with these static and dynamic in vitro experiments simulating gastric conditions give the first evidence that the extent of binding to FBP is higher for folic acid than for 5-CH3-H4folate after gastric passage. It should be noted that these FBP binding characteristics for folic acid and 5-CH3-H4folate were established for FBP in whey powder. To draw conclusions about the FBP binding characteristics in milk products, results obtained in the present study should be extrapolated with caution. Direct extrapolation of the binding characteristics of FBP in whey protein concentrate to those in milk products might not be feasible because a previous study (41) showed different binding properties of FBP in raw milk, pasteurized milk and whey protein concentrate. Nevertheless, this difference between folic acid and 5-CH3-H4folate in extent of binding to FBP is also supported by our previous studies (13,25) in which the effect of FBP on the bioaccessibility of folic acid and 5-CH3-H4folate from fortified dairy products was investigated in the in vitro gastrointestinal model (bioaccessibility is, in these studies, defined as the free folate fractions that are available for absorption during gastrointestinal passage). The bioaccessibility of folic acid from folic acid-fortified milk and yogurt was lower (P < 0.05), i.e., 11–14 and 47%, respectively, after the addition of FBP to the fortified milk (13) and yogurt (25). However, FBP did not lower the bioaccessibility of 5-CH3-H4folate from fortified milk (13) and lowered the bioaccessibility of 5-CH3-H4folate from fortified yogurt by 26% (25). These findings indicate that FBP in whey powder, milk and yogurt have different binding characteristics for folic acid and 5-CH3-H4folate.
In this regard, one point to consider is the presence of endogenous folate in the whey protein concentrate (4 µg 5-CH3-H4folate/g whey powder). This endogenous folate could compete with the added (exogenous) folic acid and 5-CH3-H4folate and as a result influence the extent of binding to FBP. However, this does not alter our general conclusions on the extent of binding to FBP because we measured the relative binding of folic acid and 5-CH3-H4folate to FBP before and after gastric passage rather than focusing on the absolute quantification of the binding activity of FBP. Because both folate compounds could be used for the fortification of dairy products, already containing endogenous FBP and 5-CH3-H4folate, this study provides information about the extent of binding of folic acid and 5-CH3-H4folate to FBP in the duodenal lumen after consumption of fortified dairy products.
An in vivo study (24) with 6-d-old goat kids supports our in vitro studies because it showed that folic acid remained bound to FBP throughout the stomach and small intestine. Analysis of the goat’s jejunal and ileal contents with gel filtration showed that a major part of the labeled folic acid (85–90%) was eluted in fractions corresponding to a molecular weight of 39 kDa (i.e., FBP-bound folic acid). On the basis of these results, the authors suggested that goat’s milk FBP is resistant to digestion by gastric and intestinal enzymes. However, the fact that folic acid was bound to FBP in the goat’s intestine, does not necessarily mean that FBP was completely resistant to degradation. The stability of FBP can be investigated only by quantitative determination of FBP before and after exposure to gastric and/or intestinal enzymes. Therefore, we studied the extent of binding to FBP in parallel with the quantitative determination of FBP. In contrast to the observed difference in FBP’s binding characteristics for folic acid and 5-CH3-H4folate, FBP stability in the 5-CH3-H4folate/FBP and folic acid/FBP mixtures did not differ after gastric passage on the basis of the ELISA measurements. From both mixtures, 70% of the initial amount of FBP was recovered in the duodenum after gastric passage for 120 min. In our previous studies, in which folic acid- and 5-CH3-H4folate–fortified dairy products were tested in the gastrointestinal model (13,25), the FBP content was quantified by ELISA in the samples collected after passage through the stomach and small intestine. It appeared that bovine FBP in a dairy matrix was less stable in combination with 5-CH3-H4folate (0–17%) than with folic acid (13–34%). Thus, a major portion of FBP passed through the stomach intact and was largely digested by pancreatic enzymes along the passage through the small intestine. Apparently, this further digestion of FBP in the small intestine was dependent on the folate compound, folic acid or 5-CH3-H4folate, present in the dairy matrix.
We conclude that a major part of folic acid is still bound to FBP after gastric passage, whereas a large portion of 5-CH3-H4folate is released from FBP. This difference in extent of binding to FBP for the two folate compounds can influence the folate bioavailability (i.e., release from the food matrix and intestinal transport) from milk products. To examine this further, studies are underway in our laboratory concerning the effect of FBP on intestinal transport of folic acid and 5-CH3-H4folate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 30 July 2003. Initial review completed 15 August 2003. Revision accepted 21 October 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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