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Department of Biochemistry and Molecular Biology, The University of Queensland, St Lucia, Queensland 4072, Australia and * Centre for Food Technology, Department of Primary Industries, Hamilton, Queensland 4007, Australia
3To whom correspondence should be addressed. E-mail: p.nixon{at}mailbox.uq.edu.au.
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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-formyltetrahydrofolate • 5-methyltetrahydrofolate • bioavailability • dietary interactions
Adequate daily intake of folate vitamins is essential to prevent disorders such as megaloblastic anemia (1
), neural tube defects in the developing fetus (2
) and some cancers (3
), and to decrease the risk of vascular disease (4
). Because folate is required for the synthesis of DNA and some amino acids, it is in highest demand during periods of rapid cell growth, such as in newborns. Often, a newborn’s only nutrition source is its mother’s milk, and although milk contains a relatively low concentration of folate, the newborn obtains adequate folate nutrition from milk.
The folate in milk is entirely bound by an excess of folate-binding protein (FBP).4 The function of FBP in milk is unclear, but it may ensure the folate content of milk by sequestering folate from the blood plasma into the mammary gland (5
). It is also very effective in stabilizing the very labile tetrahydrofolate (H4folate) and moderately labile 5-methyltetrahydrofolate (5-CH3H4folate) in vitro (6
). FBP is resistant to gastric digestion, and although it releases folate at the pH of the stomach, the two recombine in the more alkaline environment of the small intestine (7
,8
). FBP binds folates in a 1:1 molar stoichiometry (9
), and folate bound to FBP is less available to microorganisms that inhabit the intestinal tract (10
), making more folate available for absorption.
A few studies have investigated whether FBP has a direct effect on folate absorption. Several investigators have observed that although free folic acid is rapidly absorbed from the jejunum, absorption of folic acid, either bound to purified FBP or in the presence of crude milk, occurs primarily in the ileum (8
,11
,12
,13
). Whether overall absorption is affected by FBP is unclear; some investigators observed an increase (12
,14
), some observed no difference (8
) and some observed less overall absorption (11
,15
) of folate when bound to FBP. Tani and Iwai (16
) observed lower excretion of folate into the urine of rats when folic acid was administered with bovine FBP, and attributed this result to a more gradual absorption of folic acid, thereby decreasing the blood folate peak and reducing urinary folate loss. One study, using crude milk rather than purified FBP, examined the period after absorption and found that kidney and plasma folate levels were higher after 4 wk in rats fed a milk-containing diet than in rats fed a milk-free diet, despite the diets having equal folic acid content (17
).
Most of these studies utilized folic acid, with only two investigating the effect of FBP on absorption of the predominant dietary folate, 5-CH3H4folate (14
,15
); none have extended studies to include other tetrahydrofolates. Naturally occurring folates are all derivatives of H4folate with 5-CH3H4folate as the predominant in most tissues and foods. Because both H4folate and 5-CH3H4folate are stabilized greatly by complexation with FBP (6
), such complexation may decrease losses of those labile dietary folates during food storage, preparation and perhaps during digestion and absorption. It is necessary, therefore, to investigate the effect of FBP on folate nutrition when the source of dietary folate is 5-CH3H4folate and even H4folate, although the labile nature of the latter raises methodological challenges.
We examined the absorption of single doses of 5-CH3H4folate by rats, and also tissue folate levels after 4 wk of daily feeding various tetrahydrofolate derivatives complexed with FBP. Results differed with the use of crude or purified FBP; therefore, other whey proteins and casein were tested in combination with purified FBP. The finding that casein affects the outcome is important because dairy casein is the conventional source of protein in semipurified laboratory rodent diets (18
,19
).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Folates, FBP, whey fractions and casein.
The calcium salt of 5-CH3H4folate and H4folate trihydrochloride were purchased from Schircks Laboratories (Jona, Switzerland), weighed in an argon atmosphere into small portions, then stored dry over liquid nitrogen. The calcium salt of 5-formyltetrahydrofolate (5-HCOH4folate) was from Lederle Laboratories (St Leonards, Australia). Folate solutions were prepared immediately before use. Tritiated folic acid (740–1332 MBq/mmol) was purchased from Amersham (Sydney, Australia) and tritiated 5-CH3H4folate (814–1110MBq/mmol; 5-CH3H4[3H]folate) was purchased from Moravek Laboratories, Brea, CA. All folates were monoglutamates. FBP was purified by affinity chromatography of whey protein concentrate (WPC; Bonlac Foods, Melbourne, Australia) by the method of Salter (20
) as applied in this laboratory (21
), concentrated by ultrafiltration, then lyophilized by freeze drying. The affinity chromatography product is 
90% pure. The folate-binding capacity of purified FBP was determined by quenching of tryptophan fluorescence upon binding of folate (22
). WPC depleted of FBP (D-WPC) was produced by lyophilizing material not binding to the affinity column during loading of WPC. WPC fractions enriched in whey lipids (L-WPC), in ß-lactoglobulin (LG-WPC) or in 
-lactalbumin (LA-WPC) were a kind gift from Bonlac Foods, Melbourne, Australia. LG-WPC and LA-WPC were prepared by precipitation of -lactalbumin at pH 4.2 and 65°C (23
). Electrophoretic analysis of these fractions showed that WPC, LG-WPC and LA-WPC all contained the same major proteins, but LG-WPC and LA-WPC had a greater proportion of ß-lactoglobulin and -lactalbumin, respectively. L-WPC had a lower protein content and contained no ß-lactoglobulin. The folate-binding capacity of whey fractions was determined by the enhancement of the fluorescence of N-pteroyl-N-(fluorescein thiocarbamoyl)-L-lysine upon binding to FBP (24
). Acid-precipitated, vitamin-free Alacid-714 casein from Kiwi Co-operative Dairies (Hawera, New Zealand) was supplied by Dyets (Bethlehem, PA) and used either as supplied, or after solubilization by the addition of KOH until the pH was stable at 8, then heating to 50°C, and then neutralizing to pH 6 with HCl. The folate content of the casein was 1.4 pmol/mg.
Single-dose bioavailability studies
After overnight food deprivation with provision of unlimited water, rats were administered a solution containing 126 kBq and 0.23 nmol of 5-CH3H4[3H]folate (molar equivalent of 100 ng folic acid) by gavage. For test groups, preparations of WPC, casein (acid-precipitated or solubilized), FBP, D-WPC, L-WPC, LG-WPC or of LA-WPC were added to the folate solution to test the effects of these fractions. A nonpurified diet was provided 3 h after the dose was administered and 72 h later, rats were killed by an intraperitoneal injection of 0.5 mL Nembutal. Liver and kidneys were weighed and extracted by homogenization in 0.42 mol/L perchloric acid, then centrifugation at 8000 x g for 5min. The supernatant was neutralized with KOH and the resulting potassium perchlorate precipitate separated by a second centrifugation. The final tissue supernatants were counted for radioactivity in Beckman Ready Protein scintillation fluid, on a Beckman LS3801 scintillation counter.
One-month nutritional studies
Diet composition.
To facilitate daily addition of folate formulations to the diet, rats were fed a liquid diet. A number of diets were tested to ensure that components, including insoluble casein, remained in an even suspension for 24 h. A liquid AIN-93M diet was discarded due to settling. The control Lieber-DeCarli diet (25
) satisfied this test and was adopted. The suppliers (Dyets) confirmed that their experience was identical to ours. The Lieber-DeCarli control diet was modified such that p-aminobenzoic acid and folic acid were omitted, 10 g/kg succinyl sulfathalazole was added, and the vitamin-free casein portion was packaged separately. This modified diet was obtained from Dyets (Catalog #717767). Diet was prepared fresh each day and folate, FBP and whey fractions were added as indicated in the individual experiments. Vitamin-free casein was added, either untreated or after first being solubilized, to bring the total protein amount to 3.1 g/(rat · d). The liquid diet (75 mL/rat) was provided at 1700 h, and rats immediately began eating. All diet bottles were empty by morning, ensuring that all rats in a group received the same dose of folate. At the commencement of each experiment, a few rats would take some time to become accustomed to the liquid diet, but any rats not consuming the entire overnight dose on the third night were replaced.
Control folate deprivation. Preliminary experiments were performed to determine the lowest daily folate dose above which there would be no increase in tissue folate after 26 d of feeding. This was to ensure that the folate dose would not cause apparent disease, yet be limiting so that any enhancement of folate bioavailability by FBP would be readily apparent. For 26 d, each rat was fed daily the liquid diet with acid-precipitated casein, containing either 15, 30, 45 or 60 nmol/d of folic acid. Blood was collected at d 0 from the tail vein and after 26 d by cardiopuncture; at 26 d, the liver, kidney and intestinal mucosa were extracted. Student’s t test was used to compare results in a tissue for a particular dosage with the results for the next smaller dose.
FBP experiments.
For 28 d, rats were fed the liquid diet prepared daily with various additions. Folate (5-CH3H4folate, H4folate or 5-HCOH4folate) was added at 45 nmol/d for each rat, which is 50% of the AIN-76 and AIN-93 recommended level for rats (18
,19
). Also added to the diet of some groups were the indicated amounts of FBP, WPC and LG-WPC.
Rats (8 per group) were weighed weekly. All groups gained weight during the 28 d and the groups did not differ. At 0 and at 14 d, blood samples were collected into heparinized tubes from the tail vein with the rat anesthetized under halothane gas. After 28 d, rats were killed with an intraperitoneal injection of 0.5 mL Nembutal. Blood was collected by cardiopuncture and the liver, kidneys and intestinal mucosal cells were extracted.
Folate measurement.
Blood samples were collected into heparinized tubes, a portion of whole blood retained, and the remainder centrifuged at 10,000 x g for 10 min to separate plasma. The hematocrit was measured. Whole blood was lysed in 10 g/L ascorbate and incubated at 37°C for 30 min, which is sufficient time to allow endogenous plasma conjugase to remove any polyglutamate peptide from red cell folate (26
). Plasma and whole blood lysates were stored at -20°C until analysis. Tissue samples, stored at -70°C until analysis, were homogenized in 50 mmol/L sodium acetate, pH 3.9, and then incubated at 37°C for 30 min with conjugase purified from bovine liver (27
). Plasma, whole blood lysates and tissue homogenate supernatants were diluted in 5 g/L ascorbate and then assayed for folate by the Lactobacillus casei microbiological growth assay (28
).
Statistical methods. A one-way ANOVA was conducted for each experiment to test for overall significance of differences, with the significance level set at P < 0.05. A Tukey-Kramer post-test was then performed. In experiments containing only two groups, comparisons were made using a Student’s t test. All statistical calculations were carried out using GraphPad Prism, Version 3.02 (San Diego, CA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Single-dose studies were initiated after the first 28-d feeding study (see below) to guide further 28-d studies. In single-dose studies, retention of radioactivity in the liver and kidney of rats administered free 5-CH3H4[3H]folate was not different from the retention in rats administered the same folate bound to purified FBP. However, when the folate was administered in the presence of LG-WPC, sufficient to provide equimolar FBP activity, retention in liver tended to be greater (P < 0.2; Fig. 1
). Using only four rats in each group has reduced the statistical power to detect differences. LA-WPC behaved similarly to LG-WPC (data not shown).
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). Fortifying the depleted WPC with purified FBP tended to increase retention in the liver (P = 0.2) and kidney (P = 0.2). Lipid-enriched WPC administered as the FBP source decreased the retention of tritium (P < 0.05) but the addition of purified FBP to the L-WPC tended to normalize the retention of tritium in liver (P = 0.2) and kidney (P = 0.2; Fig. 3
).
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). Compared with folate administered with no FBP, folate administered with FBP and LG-WPC tended to be retained more in the presence of insoluble casein (P < 0.1) and was retained in the presence of solubilized casein (P < 0.001) (Fig. 4)
. FBP with LG-WPC increased folate retention more than did formulations without folate-binding activity, in the presence of both insoluble (P < 0.01) and solubilized (P < 0.05) casein (Fig. 4)
.
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). Weight gains did not differ among groups and the hematocrits did not fall over the 26 d nor vary among groups, indicating no apparent disease. For subsequent 28-d studies, 45 nmol/d was chosen as the daily folate provision per rat, half the recommendation of the AIN76 and AIN93 diets (18
,19
). In the experiments reported below, blood plasma folate determinations at 0, 14 and 28 d confirmed that the blood plasma folate concentration fell progressively over 28 d, whereas the hematocrit did not vary with time or among groups.
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upper panel). However, when rats were fed a liquid diet containing 5-CH3H4folate and solubilized casein (Fig. 6
), the presence of FBP increased the kidney folate concentration (P < 0.05) and tended to increase (P = 0.2) the liver folate concentration compared with rats administered no FBP. If the FBP was accompanied either by unfractionated WPC or by WPC enriched in ß-lactoglobulin, then the effect was even greater in both the liver (P < 0.05) and kidney (P < 0.001). Intestinal mucosa folate levels were not affected by the various treatments (Figs. 5
, 6)
.
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After 4 wk of administering 5-HCOH4folate in a diet containing acid-precipitated casein in the presence of a threefold molar excess of purified FBP, the kidney folate concentration was decreased (P < 0.005), compared with the kidney of rats fed free 5-HCOH4folate (Fig. 5
lower panel). Liver and intestinal mucosa were unaffected by FBP. In diets containing 5-HCOH4folate and solubilized casein, FBP (either purified or with WPC or LG-WPC) did not affect folate concentrations in any of the tested tissues (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and binds very tightly with a dissociation constant in the low nanomolar range (29
). Therefore, if the ratio of FBP to folate is >1, as it is in milk, essentially all folate would be bound. FBP may be expected to affect the uptake and subsequent bioavailability of dietary folate, but in these experiments its effect depended greatly on interactions with other components of the diet. Initial studies (Fig. 5)
suggested that pure FBP might behave differently from FBP in whole WPC. To explore such possible effects directly, single-dose studies were conducted using 5-CH3H4[3H]folate. The results of single-dose studies then guided the design of the 1-mo studies conducted later.
Single-dose studies suggested that crude WPC might increase tissue folate concentration more than purified FBP (Fig. 1)
. Other whey fractions were investigated to determine which components had the greatest effect on the activity of FBP. Studies with WPC depleted of FBP activity confirmed that any retention effect of WPC is dependent on the FBP content. Retention was decreased in the presence of WPC enriched in whey lipids, but could be increased in the presence of WPC enriched in either -lactalbumin or ß-lactoglobulin. Thus, FBP appears to increase folate bioavailability more in a hydrophilic environment than in a hydrophobic environment.
In part, FBP appears to be highly hydrophobic. Milk FBP is soluble but is related to membrane-associated FBP (M-FBP) because they are both translated from the same gene (30
). M-FBP associates with cell membranes via a glycosylphosphatidyl inositol anchor (31
,32
); the soluble form may result from proteolytic cleavage (33
) or by removal of the anchor by a lipase (34
). M-FBP has been shown to be involved in transport of folate into the cell via caveolin-mediated potocytosis (35
), requiring clustering of the FBP-bound folate into the caveolae and interaction with cholesterol (36
), thereby sequestering folate into a lipid-rich environment. FBP has a high content of aromatic amino acids (37
), and we propose that some of these are exposed on the surface, allowing FBP to associate with cholesterol or self-associate by hydrophobic interactions. Thus, it is possible that FBP associates with the lipids present in L-WPC. Such an association could sequester folate and partially prevent its binding to the intestinal transport mechanism, thereby decreasing the bioavailability of the folate.
Laboratory rodent purified diets following AIN76 or AIN93 (18
,19
) recommendations are based on acid-precipitated, insoluble casein as the sole source of protein. Resolubilization of the casein requires alkali. It has been well established that acid-precipitated casein provides sufficient essential amino acids (18
,19
), but here we document a nutrient interaction in which the state of the casein appears to affect the behavior of FBP in either enhancing or diminishing the bioavailability of folates bound to FBP. Below we attempt to explore the mechanisms that could mediate such an interaction. Such interactions are subtle, and their detection requires comparison of results from the use of both purified and less purified diets. Similar effects are likely to be observed in future studies of the effects of other vitamin-binding proteins, such as riboflavin-binding protein (RBP), which has structural similarities to FBP (see below).
The effect of FBP on the bioavailability of 5-CH3H4folate and of 5-HCOH4folate was dependent on the state of the accompanying casein. These results suggest that the structural form of casein interacts in some way with FBP or with the free folate or both. At the normal pH of milk (6.5), the casein is present in micelle structures that have a net negative charge due to the polar 
-casein on the surface. This charge keeps the micelles dispersed and practically soluble (38
). During acid precipitation, -casein loses its negative charge and the dried product has a very low level of hydration. Resolubilization requires the addition of alkali (39
). The hydrophobic nature of FBP may cause it to associate with the acid-precipitated casein, thus removing folate from the intestinal transport mechanisms and decreasing folate bioavailability. Resolubilized casein is polar and therefore less likely to associate so readily with FBP.
During the 28-d feeding studies, FBP had differing effects on the bioavailability of different folate derivatives. 5-CH3H4folateis moderately unstable, H4folate is extremely labile and 5-HCOH4folate is very stable. The pH of the diet is 5.3, and pure H4folate and 5-CH3H4folate in acetate buffer at this pH have half-lives of 25 min and 10 h, respectively, but binding to FBP has been shown to greatly enhance the stability of these two derivatives, extending the half-lives to >22 d and 800 d, respectively (6
). For H4folate, there is likely to be considerable loss in the absence of FBP due to degradation in the hours between diet preparation and consumption, and for 5-CH3H4folate, there may also be some loss. In rats fed labile folates, perhaps a major effect of FBP is to limit degradation of the vitamin. However, 5-HCOH4folate would not benefit from a stabilizing effect of FBP and in the presence of acid-precipitated casein, the complex might be sequestered into micelle aggregates, explaining in part the decrease (P < 0.005) in kidney folate concentration under these conditions.
In 28-d experiments in which the casein had been solubilized before being added to the diet, FBP, and to a greater extent WPC and LG-WPC, increased the bioavailability of 5-CH3H4folate, but had no effect on the bioavailability of H4folate or 5-HCOH4folate. The different results for H4folate and 5-HCOH4folate may reflect differences in ligand affinity for FBP, a characteristic that has been well studied for folic acid (29
), but poorly for other folates. The affinity of FBP for folic acid is greater than that for the reduced folates, and the rank order of affinity for the reduced folates is 5-CH3H4folate > H4folate > 5-HCOH4folate (40
). Knowledge of the structure of FBP and the amino acids lining its binding site should provide some insight into the reasons for the differences among folate derivatives in affinity for FBP.
The three-dimensional structure of FBP has not been published, but it has high sequence homology with RBP (41
) whose structure has been published without deposition of its coordinates (42
). In RBP, the ligand-binding site is bound on one side by a tyrosine residue and on the other by a tryptophan residue. Another six tryptophan residues are in close proximity to the binding site. The isoalloxazine ring of riboflavin is stacked into the binding site between the tyrosine and tryptophan rings. All of the tryptophan residues of RBP, the tyrosine residue in the binding site and 16 of the cysteine residues, are conserved in FBP, suggesting great similarity of the structures of FBP and RBP. It follows that folate might bind to FBP by a similar stacking of its pteridine ring between the conserved tyrosine and tryptophan residues that are homologous with those in the active site of RBP. Folate receptors from human and murine sources have two different isoforms (FR- and FR-ß) which have opposite stereospecificities for the (6S)- and (6R)-diasteroisomers of 5-CH3H4folate and 5-HCOH4folate, respectively (43
,44
). The residues that confer specificity toward the different folate diastereoisomers have been identified (44
,45
), and all are hydrophobic with the exception of one glutamate residue in FR-. By using a homology model for the structure of FBP based on the known structure of RBP, these residues were positioned within the binding cleft (46
). Thus, the folate-binding site in FBP is likely to be highly hydrophobic. Therefore, it would follow that the hydrophobic methyl group of 5-CH3H4folate should fit more easily into such a pocket than the hydrophilic formyl group of 5-HCOH4folate, consistent with the affinity of the former being greater than that of the latter.
The folates of unfortified foods are derivatives of tetrahydrofolylpolyglutamate, and the polyglutamate adduct further affects the interactions of natural folates with FBP. Folylpolyglutamates do bind to FBP (47
), but there has been no analysis of the relative affinity, by glutamate peptide chain length, of folates for FBP. However, as the chain length of folylpolyglutamates increases, their affinity for FBP likely also increases, by analogy with the increased affinity of methotrexate polyglutamates for the FBP from cultured KB cells (48
).
The physical properties of FBP appear to be affected also by heat treatment. Raw cow’s milk FBP binds folic acid with positive cooperativity, whereas that property is lost under selected conditions after pasteurization (49
) without greatly affecting binding capacity or binding affinity. Whatever properties are altered by pasteurization might be associated with the observation (12
) that pasteurized bovine and goat milk did not affect the uptake of folic acid into isolated intestinal cells, whereas uptake was enhanced by unheated human and goat milk. Our study was designed to assess the effects of FBP from commercial WPC, which was spray dried and thus briefly heated beyond pasteurization. Because the FBP and WPC fractions were not exposed to heat beyond that of the WPC, and the casein was similarly spray dried, we do not believe that the folate-binding properties of these fractions were differentially affected by their preparation. However, it is possible that different results would be obtained if FBP was prepared from raw milk.
It is not clear how other dietary components modify the effects of FBP on folate absorption. Our experiments measured retention of folate by tissues, particularly the liver and kidneys. The effects of other dietary components on the behavior of FBP may be mediated after folate absorption; the particular composition of dietary amino acids, for example, could affect folate metabolism. We argue that this hypothesis is untenable because the effect of casein depended on its physical state rather than its chemical composition. Hence, we conclude that the effects reported here must be mediated within the lumen of the gastrointestinal tract. Whatever the mechanism, the behavior of FBP in the pure state appears to differ from that when other proteins surround it, an important point to recognize when the affinity purification of FBP is relatively facile.
The presence of milk in feed given to weanling rats appears to facilitate folate absorption (17
). This must be studied in milk that is both enriched in and depleted of FBP before ascribing the observation to the FBP content. It is possible that FBP in milk fed to newborns might have a much larger effect on folate nutrition than in weanling rats (17
) or here in young adult rats.
Our results suggest that FBP-rich foods could be combined with folate-rich foods to enhance the bioavailability of natural folates in human diets. However, these results also indicate that the effects of FBP depend upon other dietary components in complex interactions, making it impossible to extrapolate to full diets and other species with any confidence. Although the full investigation of dietary interactions in humans would likely be unaffordable, perhaps a limited study that focused on the joint effects of FBP concentrate and WPC on folate bioavailability in humans is warranted.
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FOOTNOTES |
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2 The support of the Dairy Research and Development Corporation, Australia, is gratefully acknowledged.
4 Abbreviations used: 5-CH3H4folate, 5-methyltetrahydrofolate; 5-CH3H4[3H]folate, tritiated 5-methyltetrahydrofolate; D-WPC, whey protein concentrate depleted of folate-binding activity; FBP, folate-binding protein; FR-, folate receptor ; FR-ß, folate receptor ß; 5-HCOH4folate, 5-formyltetrahydrofolate; H4folate, tetrahydrofolate; L-WPC, lipid-enriched whey protein concentrate; LA-WPC, -lactalbumin-enriched whey protein concentrate; LG-WPC, ß-lactoglobulin-enriched whey protein concentrate; M-FBP, membrane-associated FBP; RBP, riboflavin-binding protein; WPC, whey protein concentrate.
Manuscript received 7 July 2002. Initial review completed 7 August 2002. Revision accepted 17 October 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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