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Department of Biochemistry and Molecular Biology, The University of Queensland, St. Lucia, Queensland 4072, 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 INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • folate-binding protein • tetrahydrofolate • 5-methyltetrahydrofolate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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), prevents some neural tube defects in newborns (2
), decreases the risk of selected cancers (3
) and decreases the blood plasma homocysteine concentration (4
) in some populations, and hence their risk of vascular disease. Given that the supply of folates in the human diet is limited, it is important to recognize factors that affect the stability of folates, some of which are quite labile when isolated.
Although folic acid is quite stable, its natural, reduced derivatives have varying stability. The predominant dietary folate is 5-methyltetrahydrofolate (5-CH3H4folate),4
which is moderately stable. Tetrahydrofolate (H4folate) is also present in foods and is very labile, especially in an oxygen atmosphere and when heated. One method for measuring folates is the microbiological growth assay (5
) requiring the vitamin to remain intact over an incubation of at least 18 h at 37°C to allow growth of Lactobacillus casei, but this assay is too slow to detect labile H4folate with full recovery. Faster analysis methods such as HPLC minimize time- and temperature-dependent degradation, and do measure intact tetrahydrofolates. Nevertheless, some reported levels of H4folate may be underestimates because of the difficulty in keeping the vitamin intact during extraction and analysis.
Whatever the analytical method, folate from biological samples such as food or animal tissues requires extraction before analysis. Extraction usually involves boiling or autoclaving of samples to free the folate from endogenous binding proteins. Although folate degradation during extraction and analysis can be slowed by the addition of a reducing agent such as ascorbate, ß-mercaptoethanol (6
), dithiothreitol (7
) or dithioerythritol (DTE) (8
), some loss of activity during analysis can be expected. Folate-binding protein (FBP), present in milk (9
) and on most mammalian cell surfaces as a folate receptor (10
), binds folates with high affinity and 1:1 molar stoichiometry (11
). It has no known enzyme activity, but may have a role in sequestering folate from the blood plasma into the mammary gland and then delivering it to the newborn (12
). All folate present in milk is bound to FBP because the latter is present in a molar concentration exceeding that of the former.
Selhub and colleagues (13
) developed a method that allows detection of low levels of folate by affinity concentration and purification of the vitamin from extracts and subsequent analysis by HPLC. The affinity chromatography uses isolated bovine milk FBP immobilized onto a solid support as the capture reagent (14
,15
). Folates eluted from the affinity column are analyzed immediately by HPLC. The method is very sensitive and is suitable for both tissue (16
) and food samples (17
). Reducing agents are included during the extraction procedure but are washed away after loading the sample onto the affinity column. Despite the highly labile nature of H4folate, it can be recovered from samples by using this method even if left bound to the affinity matrix for several weeks in the absence of reducing agents, suggesting that the stability of H4folate is increased by its binding to the FBP.
This paper reports the first definitive measurements of the ability of FBP to stabilize 5-CH3H4folate and H4folate over long periods against degradation. Whether soluble or immobilized, FBP enhanced folate stability many fold.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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5-CH3H4folate and H4folate were purchased dry from Schircks Laboratories (Jona, Switzerland), weighed into small portions in an argon atmosphere and stored dry in cryovials over liquid nitrogen until ready to use. The folates were dissolved in the indicated buffers and a zero-time sample analyzed immediately by HPLC. The samples were then incubated at 4, 22, 37 or 72°C for timed periods before sampling and analysis by HPLC.
Degradation of folate bound to soluble FBP.
FBP was purified from dried whey protein concentrate (WPC) (Bonlac, Melbourne, Australia) by affinity chromatography on a folic acid-agarose column (18
) as applied in this laboratory (19
). WPC was dissolved in water to 20 g/L then loaded onto the column, which was subsequently washed with water, then 50 g/L NaCl and then water again. FBP was eluted at pH 2.5, concentrated by ultrafiltration, lyophilized by freeze-drying and was >90% pure when analyzed by polyacrylamide gel electrophoresis under denaturing conditions.
5-CH3H4folate and H4folate (50 µmol/L) were dissolved with 100 µmol/L FBP in the indicated buffers. Solutions were incubated at 4, 22, 37 and 72°C, with samples taken at zero time and then at appropriately timed intervals. Before injection onto the column, the FBP-folate complex was dissociated and the FBP removed. Dissociation was achieved by one of two methods. The initial method was used for samples incubated at neutral pH and proved suitable for analysis of 5-CH3H4folate only; H4folate samples proved too unstable during this process. The 5-CH3H4folate:FBP complex was acidified by HCl to dissociate the complex, and then the FBP was removed by filtration through a Microcon-30 filter (Millipore, Sydney, Australia) by centrifugation for 5 min at room temperature and 10,000 x g. The filtrate containing free folate was analyzed. The second method was used for separation of 5-CH3H4folate and H4folate from their complexes with FBP. The complex was dissociated by the addition of perchloric acid to a final concentration of 15 g/L, causing the FBP to precipitate and allowing removal of FBP by centrifugation within 1 min. The supernatant was analyzed. The faster analysis time for this method made it more suitable for H4folate analysis than the first method, but results of 5-CH3H4folate analysis did not differ between the two methods.
Degradation of folate bound to immobilized FBP.
FBP was immobilized onto Sepharose 4B (Amersham Biosciences, Sydney, Australia) by the method of Selhub et al. (14
). Several 1-mL columns were prepared in glass Pasteur pipettes plugged with polyester wool, and equilibrated with 1 mol/L potassium phosphate, pH 7. Folate (250 nmol of 5-CH3H4folate or of H4folate) was applied to each of 10 columns at each incubation temperature. The columns were then washed with 2 mL of 1 mol/L potassium phosphate buffer, 0.2 g/L NaN3, pH 7, and incubated at 4, 22 or 37°C as indicated. At zero time and after appropriate incubation periods, a column was washed with 2 mL water and the folate was eluted by 2 x 1-mL aliquots of 20 mmol/L trifluoroacetic acid. The eluate was collected into tubes containing 10 µmol of piperazine and 0.5 µmol of DTE (Sigma, Sydney, Australia). Folate was entirely eluted in the second fraction, and 100 µL of this fraction was analyzed by HPLC.
HPLC separations.
Initially, folates and degradation products were separated by ion-pair, reverse-phase HPLC (8
) using an Alltech C18 Econosphere 150 x 4.6mm column (Sydney, Australia). Tetrabutyl-ammonium phosphate (TBAP) (PIC-A reagent, Waters, Sydney, Australia) was the ion-pair reagent and separation of folates was achieved at pH 6.8 by an acetonitrile gradient. DTE at 0.5 mmol/L was included in all solvents, which were deoxygenated by an in-line ERMA ERC-3511 vacuum degasser. However, this system is designed to separate many forms of polyglutamylated folates and was unnecessarily complex for the demands of experiments restricted to pteroylmonoglutamates. Hence, an isocratic method was developed with separation using a Waters XTerra C18 150 x 4.6 mm column and degassed solvent containing 20 mmol/L sodium acetate, 50 mL/L acetonitrile and 0.5 mmol/L DTE in water adjusted to pH 5.0. Thus, initial experiments used the gradient elution system, but subsequent experiments utilized the isocratic elution system; the method used for each experiment is indicated with the results. Eluted folates and breakdown products were detected by their absorbance at 280 nm, and the area under the elution peaks was compared with the areas of standards to determine the concentration of each species present. The rates of degradation of 5-CH3H4folate and of H4folate were measured by constructing semilog plots of the remaining folate as a percentage of the initial concentration against time. A least-squares linear regression line was fitted to the data, by use of GraphPad Prism (San Diego, CA) version 3.02, and the degradation rate was described by the slope. The half-life of degradation was calculated (Ln 2/rate), and the standard error represents how well the data fit the regression line. Student’s t test was used to compare the linear regressions describing the degradation rates of free folates with those for bound folates (either soluble or immobilized), under each set of specified conditions.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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) was well achieved by both separation methods (e.g., Fig. 1
). Degradation of free folates was accompanied by an equivalent increase in the appearance of pABG. An additional product whose peak height increased as the folate decreased was observed when 5-CH3H4folate was incubated in acetate buffer at pH 5 or when H4folate was in TBAP at pH 6.8. This peak was not identified but may be a pteridine derivative, the anticipated second product of folate cleavage. Under all other conditions, no putative pteridine was detected eluting from the column. The expected immediate product of oxidation of 5-CH3H4folate is 5-methyldihydrofolate (5-CH3H2folate) (21
), but our analytical system could not detect this product because the presence of DTE in the HPLC solvent reduced any 5-CH3H2folate to 5-CH3H4folate (22
). Therefore, we measured degradation to pABG of the combined pool of 5-CH3H4folate and 5-CH3H2folate. However, for FBP-bound 5-CH3H4folate, the samples were acidified to dissociate the folate from FBP, and acidification would have cleaved any 5-CH3H2folate present to yield pABG (23
,24
).
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Semilog plots of remaining folate, as a percentage of the initial folate concentration, against time (Fig. 2
) indicated that the degradation was first order because the majority of data sets were well described by a least-squares linear regression. Table 1
summarizes the half-lives of folate degradation free in solution, or bound to either soluble or immobilized FBP, under various conditions. For very long half-lives, the precision of measurement was limited by the observation periods, although some exceeded 5 or even 14 mo. Longer periods of observation likely would have improved the precision of the estimated half-life, but were impractical.
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) also observed faster degradation of 5-CH3H4folate in phosphate buffer, perhaps because of the presence of contaminating trace heavy metals, which is typical of phosphate salts. Lucock et al. (7
) measured a half-life of 20.7 h for 5-CH3H4folate degradation at 25°C in Tris buffer at pH 7.2, which is comparable with the half-life that we measured at 22°C in TBAP (16.2 h). Free H4folate was much less stable than free 5-CH3H4folate with half-lives measured in minutes rather than hours. H4folate at neutral pH was less stable than at pH 5. Additional measurements at pH 3.2 yielded an even longer half-life, >36 h. This increase in stability of free H4folate at acid pH is consistent with the observations of Paine-Wilson and Chen (25
).
Reducing agents helped to stabilize free H4folate; 0.5mmol/L DTE increased the half-life in TBAP by 150% at 4°C, but 5 g/L (28.5 mmol/L) ascorbate was even more protective. Measurements of the half-lives of H4folate degradation in the presence of acetate buffer, pH 5, with 5 g/L ascorbate were 3.5 d, 14.6 h, 1.3 h and 37.4 min at 4, 22, 37 and 72°C, respectively, giving a 53-fold increase in stability of H4folate at 4°C. Ascorbate has been shown to produce formaldehyde when heated in solution at 100°C (26
), and formaldehyde converts H4folate to the more stable 5,10-methylenetetrahydrofolate with a dissociation constant of 77 µmol/L (27
). However, for 99% of the H4folate in the current studies to be converted to 5,10-methylenetetrahydrofolate, 27% of the available ascorbate would require prior conversion to formaldehyde. This is unlikely to have occurred in stock ascorbic acid stored dry at ambient temperature, or during the course of our experiments, which were undertaken at lower than boiling temperatures. Possibly some formaldehyde would be generated at 72°C, but that would be inconsistent with the short half-life observed at this temperature. We conclude that the stabilization of free H4folate solutions by ascorbate, in our experiments, was due principally to its removal of oxygen.
By far, the most effective agent to protect these folates against degradation proved to be FBP, with degradation rates in the presence of FBP measured in days, rather than minutes or hours (P < 0.0001 for most comparisons, Table 1
). In many cases, no significant degradation was detected over time periods exceeding 1 y. The half-life of the moderately stable 5-CH3H4folate had a modest twofold increase by binding to FBP in TBAP at 4°C and pH 6.8, but it increased 
1000-fold in some samples such as phosphate at pH 7 and 4°C, where essentially no degradation of bound folate was detected after > 400 d. When bound to soluble FBP at neutral pH, even the extremely labile H4folate could be recovered 100% intact after > 100 d incubation at 22°C. When either folate was bound, stability was greater in phosphate buffer at pH 7 than in acetate buffer at pH 5, which is in direct contrast to free folate. At pH 5, dissociation of the complex is likely to be very slight but much greater than at higher pH, and perhaps sufficient to allow some generation of free folate over long periods of time and hence greater rates of degradation.
FBP increased the stability of the studied folates under all conditions tested at temperatures up to 37°C. However, when incubated at 72°C, 5-CH3H4folate degraded very rapidly despite the presence of FBP. It is possible that the affinity of FBP for 5-CH3H4folate is weakened at elevated temperature, displacing the equilibrium between bound and free folate in favor of more free and therefore resulting in less protection of the ligand. Additionally, any 5-CH3H2folate produced by oxidation of 5-CH3H4folate (21
) would convert to pABG during acidification of the sample before analysis (23
,24
), resulting in an apparently faster degradation of bound 5-CH3H4folate than of free 5-CH3H4folate (Table 1)
.
Analysis of folate vitamins from natural sources will detect H4folate only if the analysis is very rapid or if the extracts are stabilized. The use of FBP affinity chromatography in the extraction methods of Selhub and colleagues (13
–17
) minimizes degradation of natural folates before HPLC analysis. These extraction methods could be combined with the current methods to examine the stability of folates in tissues, or in milk and other foods.
FBP also stabilizes H4folate in vivo. Pig blood plasma contains H4folate as the principal folate (28
), whereas only trace levels of H4folate are detected in the plasma of many other mammalian species. This observation is consistent with an unusually high level of FBP in pig plasma (29
) and the ability of pig plasma, but not plasma ultrafiltrate or albumin, to stabilize H4folate (30
).
In the current study, the stabilization of labile tetrahydrofolates by FBP was demonstrated directly over a range of temperature and pH conditions. It could be hypothesized that other folate-binders, including the enzymes of one-carbon metabolism, might also stabilize tetrahydrofolates. This hypothesis is consistent with the prolonged half-life of 19 d, measured by von der Porten et al. (31
), for the turnover of total body folates (predominantly 5-CH3H4folate) in humans. This value is much closer to the half-life of FBP-bound 5-CH3H4folate than to the half-life of free 5-CH3H4folate measured here. Additionally, most folate in foods and tissues is intracellular, and during extraction and analysis, it is possible that the folate has been freed, by boiling, from a complex with some protein. This protein is unlikely to be FBP because FBP is a receptor on cell surfaces (10
) and does not accumulate intracellularly. Thus, other folate-binders are likely to stabilize food folates and might also contribute to the bioavailability and nutritional value of food folates.
Natural food folates are all derivatives of H4folate and hence of varying stability during harvest, distribution, storage and food preparation. Although any 5-formyltetrahydrofolate should be quite stable, free 5-CH3H4folate has limited stability. Free H4folate and 10-formyltetrahydrofolate are quite unstable. FBP is easy to purify and could be used as an additive in foods to enhance the stability of natural food folates. However, such an approach is unlikely to be economical, given that fortification of foods with folic acid is both affordable and effective.
The predominant folate in milk is 5-CH3H4folate, and its concentration is much lower than that in intracellular sources, i.e., solid foods. Despite its low folate concentration, maternal milk is the sole source of folate for newborns during the period of maximal growth and hence of greatest folate utilization. Milk folate is entirely bound by an excess concentration of FBP; therefore, it may be assumed that milk folate is extremely stable. Thus, FBP may not only sequester folate into milk, but may also protect the folate from degradation during processing and storage, thereby ensuring maximum bioavailability. Consequently, dairy milk appears to have substantial folate nutritional value despite the low concentration.
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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-CH3H2folate, 5-methyldihydrofolate; 5-CH3H4folate, 5-methyltetrahydrofolate; DTE, dithioerythritol; FBP, folate-binding protein; H4folate, tetrahydrofolate; pABG, p-aminobenzoylglutamate; TBAP, tetrabutyl-ammonium phosphate; WPC, whey protein concentrate.
Manuscript received 24 April 2002. Initial review completed 21 May 2002. Revision accepted 10 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1. Herbert, V. (1987) Recommended dietary intakes (RDI) of folate in humans. Am. J. Clin. Nutr. 45:661-670.
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4. Landgren, F., Israelsson, B., Lindgren, A., Hultberg, B., Andersson, A. & Brattstrom, L. (1995) Plasma homocysteine in acute myocardial infarction: homocysteine-lowering effect of folic acid. J. Intern. Med. 237:381-388.[Medline]
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14. Selhub, J., Ahmad, O. & Rosenberg, I. H. (1980) Preparation and use of affinity columns with bovine milk folate-binding protein (FBP) covalently linked to Sepharose 4B. Methods Enzymol. 66:686-690.[Medline]
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19. Treloar, T., Grieve, P. A. & Nixon, P. F. (2000) One-step affinity purification of folate-binding protein, a minor whey protein. Aust. J. Dairy Technol. 55:96.
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22. Donaldson, K. O. & Keresztesy, J. C. (1962) Naturally occurring forms of folic acid. III. Characterization and properties of 5-methyldihydrofolate, an oxidation product of 5-methyltetrahydrofolate. J. Biol. Chem. 237:3815-3819.
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28. Natsuhori, M., Shimoda, M., Kokue, E., Hayama, T. & Takahashi, Y. (1991) Tetrahydrofolic acid as the principal congener of plasma folates in pigs. Am. J. Physiol. 261:R82-R86.
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31. von der Porten, A. E., Gregory, J. F., Toth, J. P., Cerda, J. J., Curry, S. H. & Bailey, L. B. (1992) In vivo folate kinetics during chronic supplementation of human subjects with deuterium-labeled folic acid. J. Nutr. 122:1293-1299.
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