The Journal of Nutrition Vol. 128 No. 11 November 1998, pp. 1907-1912

Urinary Excretion of [²H₄]Folate by Nonpregnant Women Following a Single Oral Dose of [²H₄]Folic Acid Is a Functional Index of Folate Nutritional Status^1,2

Jesse F. Gregory III³, Jerry Williamson, Lynn B. Bailey, and John P. Toth

Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0370

	ABSTRACT

Abstract Introduction Methods Results Discussion References

In a 10-wk study with nonpregnant women (21-27 y, n = 5-6 per group), subjects were fed a diet containing ~68 nmol/d (30 µg/d) folate from food that was supplemented with folic acid in apple juice to yield a constant intake of 454, 680 or 907 nmol/d (200, 300 or 400 µg/d) to evaluate folate status and long-term in vivo kinetics. Reported here is an additional phase of this protocol conducted to determine the relationship between short-term urinary excretion after a single isotopically labeled dose and various measures of folate nutritional status. It was hypothesized that urinary excretion from a single [glutamate-²H₄]folic acid ([²H₄]folic acid) dose would increase in proportion to folate nutritional status due to saturable cellular uptake and retention processes along with saturation of renal reabsorption. Each subject was given 1.13 µmol (500 µg) of [²H₄]folic acid orally on the morning of d 70 of the study, followed by a complete 24-h urine collection. Urine was analyzed to determine the isotopic enrichment of urinary folate by gas chromatography-mass spectrometry and the concentration of urinary folate by HPLC. Urinary excretion of [²H₄]folate was greatest at the 907 nmol/d intake and was positively correlated with serum folate concentration but was not correlated with erythrocyte folate. Excretion of [²H₄]folate tended to be greatest when plasma homocysteine concentrations were low (<8 µmol/L), although this relation was not significant. These results suggest that 24-h urinary excretion after a single oral dose of isotopically labeled folate is a functional indicator of folate nutritional status that complements other measures of folate nutriture.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Evaluation of nutritional status for folate is complicated by the variety of metabolic functions of this vitamin along with its complex pattern of interorgan transport and turnover (Steinberg 1984). Assessments based on plasma folate concentration are convenient and reflect long-term status but are most sensitive to recent intake of folate (Lindenbaum and Allen 1995). Measurement of erythrocyte folate concentration serves as an indirect indicator of long-term status because uptake of folate occurs mainly during erythropoiesis (Lindenbaum and Allen 1995). Plasma homocysteine has gained widespread use as a functional indicator of one-carbon metabolic status (Lindenbaum and Allen 1995). Although elevated plasma homocysteine concentration occurs during even marginally suboptimal folate status, (O'Keefe et al. 1995), this indicator also is influenced by vitamin B-6 and B-12 nutriture and age (Selhub et al. 1993). Other procedures such as the deoxyuridine suppression test and measurement of uracil misincorporation into genomic DNA also provide functional information regarding folate status (Blount et al. 1997, Lindenbaum and Allen 1995). The deoxyuridine suppression test yields a specific evaluation of cellular folate status; however, this procedure is no more informative diagnostically than measurement of erythrocyte folate concentration (Tamura et al. 1990). Uracil content of DNA isolated from blood samples has been shown to be sensitive to folate depletion and repletion in human subjects (Blount et al. 1997), but the sensitivity of this procedure as an indicator of marginal folate status has not been evaluated. Moderate folate deficiency also has been shown to reduce the methylation of lymphocyte DNA in postmenopausal women (Jacob et al. 1998), which suggests the possible application of DNA hypomethylation as a functional indicator of folate deficiency. Urinary excretion of formiminoglutamate either before or after a histidine load provides additional metabolic evidence of folate and/or vitamin B-12 deficiency (Sauberlich et al. 1974), although this method has had little use recently due to problems of analytical specificity (Lindenbaum and Allen 1995). Urinary folate excretion is not viewed as a reliable indicator of folate status because it is only weakly related to plasma folate concentration at low levels of folate intake (Sauberlich et al. 1974).

This study was conducted to determine the relationship between controlled folate status and the extent of urinary excretion of deuterium-labeled folate derived from a bolus dose of deuterated folic acid. Results of several studies have suggested that information regarding folate status may be derived from measurements of the urinary excretion of folate resulting from a bolus dose of isotopically labeled folic acid. Early studies conducted using bolus doses of nonlabeled folate showed that plasma clearance of the administered folate was much faster in folate deficiency than in folate-adequate individuals. This effect has been attributed to enhanced folate uptake or retention by tissues during deficiency (Chanarin et al. 1958, Herbert and Zalusky 1962, Metz et al. 1961). Decreased urinary excretion of [³H]folate from a bolus dose of [³H]folic acid has been reported in folate-deficient rats relative to adequately nourished controls (Eisenga et al. 1992). Analysis of tissue content of [³H]folate derived from the tracer dose indicated that folate deficiency caused increased tissue uptake of [³H]folate in nonhepatic tissues, whereas uptake of [³H]folate by the liver was greater in folate-adequate rats. Urinary folate excretion by humans after various intravenous doses of nonlabeled folic acid has been found to be substantially increased at doses above ~10 µg/kg body weight (Cooperman et al. 1970). Various folates labeled with either radioisotopes or stable isotopes have been used in protocols intended to evaluate intestinal absorption; these studies generally have no bearing on postabsorptive retention because of the use of "flushing doses" or "saturation" with nonlabeled folate to enhance excretion (Gregory 1995).

On the basis of such previous studies, it was hypothesized that short-term (i.e., 24-h) excretion of deuterium-labeled folate after a bolus oral dose would increase in proportion to folate status. We report here a test of this hypothesis that was conducted immediately after a 10-wk period of precisely controlled dietary folate intake and detailed evaluation of folate status (O'Keefe et al. 1995). In spite of the fact that this long-term protocol involved chronic administration of [3',5'-²H₂]folic acid ([²H₂]folic acid)⁴ to permit evaluation of whole-body folate kinetics (Gregory et al. 1998), the use of a separate deuterated tracer, [glutamate-²H₄]folic acid ([²H₄]folic acid), allowed the short-term excretion study to be conducted without interference from the [²H₂]folate tracer because of the high specificity of the gas chromatography-mass spectrometry (GCMS) analysis.

	SUBJECTS AND METHODS

Abstract Introduction Methods Results Discussion References

Protocol.  The details of the protocol and diet composition have been reported previously (Gregory et al. 1998, O'Keefe et al. 1995). All subjects were nonpregnant females (n = 18, age 21-27 y, weight 47-67 kg) who had normal blood chemistry and were in good health as reflected by a medical history and examination by a physician. This study was approved by the University of Florida Institutional Review Board. Informed consent was obtained from each subject. Subjects were randomly assigned to three treatment groups (n = 6 per group), each group having different constant folate intake throughout the 10-wk period. One subject withdrew for personal reasons midway through the study. The protocol was conducted on an out-patient basis at the University of Florida Clinical Research Center. Adequacy of vitamin and mineral intake was ensured by administration of folate-free supplements (O'Keefe et al. 1995). The dietary intake of energy, protein, and fat and of supplemental vitamins and minerals was reported previously (O'Keefe et al. 1995). Dietary folate from food sources was ~680 nmol/d (30 µg/d) for all groups. Additional folate was provided as synthetic folic acid added to commercial pasteurized apple juice consumed at breakfast and evening meals (nonlabeled folic acid, wk 1-2; [²H₂]folic acid, wk 3-10) to yield intakes of total folate of 454, 680 or 907 nmol/d (200, 300 or 400 µg/d). The quantities of dietary folate, labeled and nonlabeled folic acid administered throughout the 10-wk protocol were described in detail previously (Gregory et al. 1998).

On the morning of d 70 of the controlled dietary study, all subjects were given their usual light breakfast (O'Keefe et al. 1995) and apple juice containing the [²H₂]folic acid dose, to which 1.13 µmol (500 µg) [²H₄]folic acid was also added. Subjects collected all urine for the next 24 h into acid-washed brown plastic 2-L bottles containing 5 g of dry sodium ascorbate; the bottles were kept refrigerated (2-4°C) during the collection period. Immediately after the 24-h collection was completed, the total volume was measured, and 50-mL portions were transferred to polyethylene vials, saturated with nitrogen gas, then stored at 30°C until analyzed.

Folic acid sources administered.  Nonlabeled folic acid was obtained commercially (Sigma Chemical, St. Louis, MO). [²H₄]Folic acid was synthesized in this laboratory and was analyzed by HPLC, proton nuclear magnetic resonance and GCMS to verify purity and identity (Gregory and Toth 1988). The concentration of [²H₄]folic acid in the stock solution added to apple juice was determined spectrophotometrically using the pH 7.0 molar absorptivity coefficient of 27,600 L/(mol · cm) (Blakley 1969).

Analytical methods.  Documentation of folate nutritional status. As described previously, serum and erythrocyte folate concentrations were determined by microbiological assay with Lactobacillus casei (Tamura 1990). Total plasma homocysteine concentrations were determined by a fluorometric HPLC procedure (Vester and Rasmussen 1991).

Determination of urinary folate by HPLC and preparation of urinary folate for GCMS analysis. Urinary folate concentration was determined by HPLC after affinity chromatography (Gregory and Toth 1988, Stites et al. 1997). A portion of the urinary folate eluted from the affinity chromatography column was intentionally cleaved at the 9C-10N bond, and the resulting para-aminobenzoylglutamate (pABG) isolated and derivatized to form the N-trifluoroacetyl-p-aminobenzoylglutamate lactam -trifluoroethyl ester (Gregory and Toth 1988).

GCMS analysis of derivatized pABG (derived from urinary folate) was performed as previously described in electron-capture negative ionization mode with selected-ion monitoring at mass-to-charge ratios (m/z) 426, 428 and 430 (Gregory and Toth 1988, Wei et al. 1996), corresponding to nonlabeled (¹H), ²H₂ and ²H₄ species. All analyses were performed using a Hewlett-Packard Model 5989 GCMS system (Palo Alto, CA) with methane as reagent gas. Ratios of observed peak areas from the selected-ion monitoring GCMS analysis were converted by simultaneous equations to molar ratios of these isotopomers (i.e., ²H₂/¹H and ²H₄/¹H). In this computation, all values were corrected for the natural abundance of stable isotopes. Known mixtures of pABG derivatives (prepared from known mixtures of [²H₄], [²H₂] and [¹H]folic acid) were employed to prepare calibration standards that were used to verify the accuracy of this calculation. All standard mixtures and samples were analyzed in duplicate or triplicate.

Statistical analysis.  All data are presented as means ± SEM. Absolute excretion of [²H₄]folate was calculated from 24-h urine volume, concentration of total urinary folate, and molar ratios of isotopomers and expressed as a percentage of the administered dose. Differences among dietary groups with respect to urinary excretion were evaluated using one-way ANOVA, with multiple comparisons using the Student-Newman-Keuls procedure. Variability of [²H₄]folate excretion, relative to the group mean, was substantially greater at the 907 nmol/d level of folate intake, which necessitated logarithmic transformation to normalize distributions and variances in ANOVA. The relationship between [²H₄]folate excretion and serum folate, erythrocyte folate, and plasma homocysteine concentration was evaluated by one-way ANOVA for log-transformed data partitioned approximately as tertiles of each value (e.g., [homocysteine] <8, 8-12, and >12 µmol/L). Finally, the strength of relationships among all indicators of folate nutritional status was evaluated using the Pearson product-moment correlation procedure. All analyses were performed as described by Glantz (1992) using SigmaStat Version 1.0 software (Jandel, San Rafael, CA). Differences with P < 0.05 were considered to be significant.

	RESULTS

Abstract Introduction Methods Results Discussion References

As previously reported, the 10-wk dietary regimen of this study yielded clear differences in the folate nutritional status of the subjects (Gregory et al. 1998, O'Keefe et al. 1995). The group receiving 454 nmol total folate/d (200 µg/d) was in marginally inadequate and declining folate status as judged by serum and erythrocyte folate and plasma homocysteine concentration. Groups receiving 680 and 907 nmol/d were in adequate status according to these criteria.

Mean values for total folate excretion and [²H₄]folate enrichment of urinary folate are presented in Table 1. Total urinary folate excretion increased markedly at the 907 nmol/d intake. There was no significant difference in ²H₄ isotopic enrichment of urinary folate, although the expected downward trend with increasing folate intake was observed.

Under the conditions of this protocol, the 24-h excretion of urinary [²H₄]folate was low, with all mean values <1%/24 h (Fig. 1). The observed levels of [²H₄]folate excretion were readily measurable by the procedures used in this study. Differences in mean excretion and within-group variability are illustrated in the data of Figure 1, presented as mean (± SEM) values determined before transformation. ANOVA of log-transformed data indicated that the urinary excretion of [²H₄]folate was significantly affected by folate intake (P = 0.0157), with [²H₄]folate excretion at the 907 nmol/d intake significantly greater than at the lower two intake levels.

View larger version (50K): [in this window] [in a new window]   Fig 1. Urinary excretion of [2H4]folate over 24 h after an oral dose of [2H4]folic acid by nonpregnant women consuming various levels of total folate. Values are means ± SEM; n = 5 (454 nmol/d), n = 6 (others). Bars designated by a different small letter are significantly different after logarithmic transformation of data, P < 0.05.

The relationship between urinary [²H₄]folate excretion and other indicators of folate status was also examined. In view of the relatively small number of subjects in this study, values for each indicator (serum and erythrocyte folate and plasma homocysteine concentration) were classified as tertiles, analogous to the approach used by others to facilitate examination of such relationships (Selhub et al. 1993, Tucker et al. 1996). Again, ANOVA was performed after logarithmic transformation. In this analysis, a significant relationship was found between serum folate concentration and [²H₄]folate excretion (P = 0.0380), with [²H₄]folate excretion significantly greater for subjects with serum folate concentration >10 nmol/L than for those with concentrations <6 nmol/L (Fig. 2A). There was no significant relationship between [²H₄]folate excretion and erythrocyte folate concentration (Fig. 2B). Also, no significant differences in [²H₄]folate excretion were observed as a function of plasma homocysteine concentration (Fig. 2C). As shown in Figure 2C, variation in [²H₄]folate excretion increased with decreasing homocysteine concentration, which made detection of a significant relationship difficult with the small number of subjects employed.

View larger version (23K): [in this window] [in a new window]   View larger version (58K): [in this window] [in a new window]   Fig 2. Relationship between various indicators of folate status (classed as tertiles) and urinary excretion of [2H4]folate over 24 h after an oral dose of [2H4]folic acid by nonpregnant women consuming various levels of total folate; (A) serum folate; (B) erythrocyte folate; and (C) plasma homocysteine. Values are means ± SEM; n = 5 (454 nmol/d), n = 6 (others). Bars designated by a different small letter are significantly different after logarithmic transformation of data, P < 0.05.

The strength of the overall linear relationships among [²H₄]folate excretion and all other criteria, as evaluated by the Pearson product-moment correlation procedure, yielded the results shown in Table 2. Significant correlations were observed between folate intake and all outcome variables examined. The linear correlation of [²H₄]folate excretion and other outcome variables was significant only in the case of serum folate. Serum folate was significantly correlated with erythrocyte folate concentration, whereas the relationship between erythrocyte folate and plasma homocysteine concentration was not significant.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This study was conducted to determine whether short-term excretion of labeled (i.e., ²H₄) folate from a bolus oral dose would be reflective of folate nutritional status. The 10-wk study previously described (O'Keefe et al. 1995) provided an excellent opportunity to test this hypothesis. Folate intake was primarily in the form of synthetic folic acid, which exhibits high and consistent bioavailability in the context of this protocol (Pfeiffer et al. 1997). In view of the relationships among [²H₄]folate excretion, folate intake and serum folate, we conclude that [²H₄]folate excretion does reflect folate status.

Indicators of folate status that reflect the functional adequacy of various folate-dependent metabolic processes are required. We believe that measurement of short-term isotopic excretion in the present protocol does provide a functional measure of one aspect of the in vivo physiologic handling of folate, i.e., cellular uptake and retention. The urinary excretion of folate is influenced by many factors. Intestinal absorption is a key factor. Malabsorption of the oral [²H₄]folic acid dose would yield low percentage excretion and would be potentially associated with inadequate folate status. Thus, the protocol described here involved the use of an oral, rather than an injected dose. It should also be noted that a recent study has shown that deuterated forms of folic acid exhibit quite different short-term kinetics in vivo when administered orally vs. intravenously presumably because of the hepatic first-pass effect experienced after intestinal absorption (Rogers et al. 1997). Modeling studies of long-term folate metabolism from this 10-wk study (based on the [²H₂]folic acid tracer) have indicated that the fraction of ingested folate that appears in urine increases in proportion to folate intake (Gregory et al. 1998), which is consistent with observations of this study regarding folate intake and [²H₄]folate excretion. This finding is also consistent with results in rats showing that reduced urinary excretion of labeled folate in folate deficiency is related, in part, to the enhanced uptake and retention of labeled folate molecules by nonhepatic tissues (Eisenga et al. 1992).

Urinary excretion of folate with increasing intake is likely also related to saturation of the renal tubular reabsorption system as plasma folate concentration increases (Birn et al. 1993), although nonspecific pathways also appear to play a role in renal folate reabsorption (Muldoon et al. 1996). The relationship between folate intake and urinary total folate excretion is due in part to this effect. Additionally, the in vivo retention of folate is a function of saturable processes. Specific binding to several intracellular folate-binding proteins is one aspect (Wagner 1995). Additionally, metabolic trapping occurs by polyglutamylation of folates in tissues catalyzed by folylpolyglutamate synthetase. Product inhibition of this enzyme by the long-chain polyglutamyl folates that predominate in tissues (Cichowicz and Shane 1987) would reduce cellular retention of newly absorbed monoglutamyl folate molecules after cellular uptake when dietary intakes and tissue folate stores are high. Thus, we conclude that the 24-h urinary excretion of [²H₄]folate is a functional indicator of folate status that is related to the concentration dependence of multiple folate transport/retention processes.

In summary, this isotopic procedure adds to the growing list of functional indicators of folate status. Disadvantages include the needs for complete urine collection and two analytical methods (i.e., measurement of urinary folate concentration by HPLC and isotopic enrichment by GCMS). It is likely that the quantitative HPLC step could be eliminated through the use of a stable isotopic internal standard in GCMS to allow calculation of total urinary folate in a manner analogous to that reported by Santhosh-Kumar et al. (1995). The HPLC and GCMS methods are not inordinately difficult, although they are fairly labor intensive. The complete urine collection required reduces the applicability of this method in surveys of nutritional status but not in controlled research studies. For future application of this method, we recommend, for reasons of economy and ease of synthesis, that the labeled folate dose be provided as [3',5'-²H₂]folic acid (Gregory 1990), rather than the [²H₄]folic acid preparation used in this study or the [¹³C₅]folic acid preparation recently developed (Pfeiffer et al. 1997). The current lack of commercial availability of [²H₂]folic acid may also limit application of the method; however, a single synthesis typically yielding 0.5-1.0 g of product would suffice for many trials at the dosage level described here.

	FOOTNOTES

Manuscript received 30 March 1998. Initial reviews completed 13 May 1998. Revision accepted 24 June 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Birn H., Selhub J., Christensen E. I. Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule. Am. J. Physiol. 1993; 264:C302-C310[Abstract/Free Full Text]
• Blakley, R. L. (1969) The Biochemistry of Folic Acid and Related Pteridines. Wiley, New York, NY.
• Blount B. C., Mack M. M., Wehr C. M., MacGregor J. T., Hiatt R. A., Wang G., Wickramasinghe S. N., Everson R. B., Ames B. N. Folate deficiency causes uracil misincorporation into human DNA and chromosomal breakage: implications for cancer and neuronal damage. Proc. Natl. Acad. Sci. U.S.A. 1997; 94:3290-3295[Abstract/Free Full Text]
• Chanarin I., Mollin D. L., Anderson B. B. The clearance from the plasma of folic acid injected intravenously in normal subjects and patients with megaloblastic anemia. Br. J. Haematol. 1958; 4:435-446[Medline]
• Cichowicz D. J., Shane B. Mammalian folylpoly--glutamate synthetase. 2. Substrate specificity and kinetic properties. Biochemistry 1987; 26:513-521[Medline]
• Cooperman J. M., Pesci-Bourel A., Luhby A. L. Urinary excretion of folic acid activity in man. Clin. Chem. 1970; 16:375-381
• Eisenga B. H., Collins T. D., McMartin K. E. Incorporation of ³H-label from folic acid is tissue-dependent in folate-deficient rats. J. Nutr. 1992; 122:977-985
• Glantz, S. A. (1992) Primer of Biostatistics, 3rd ed. McGraw-Hill, New York, NY.
• Gregory J. F. . Improved synthesis of [3`,5`-²H₂] folic acid: extent and specificity of deuterium labeling. J. Agric. Food Chem. 1990; 38:1073-1076
• Gregory, J. F. (1995). The bioavailability of folate. In: Folate: Nutritional and Clinical Perspectives (Bailey,L., ed.), pp. 195-235. M. Dekker, New York, NY.
• Gregory J. F., Toth J. P. Chemical synthesis of deuterated monoglutamate and in vivo assessment of urinary excretion of deuterated folates in man. Anal. Biochem. 1988; 170:94-104[Medline]
• Gregory J. F., Williamson J., Liao J.-F., Bailey L. B., Toth J. P. Kinetic model of folate metabolism in nonpregnant women consuming [²H₂]folic acid: isotopic labeling of urinary folate and the catabolite para-acetamidobenzoylglutamate indicates slow, intake-dependent, turnover of folate pools. J. Nutr. 1998; 128:1896-1906[Abstract/Free Full Text]
• Herbert V., Zalusky R. Interrelations of vitamin B12 and folic acid metabolism: folic acid clearance studies. J. Clin. Investig. 1962; 41:1263-1276
• Jacob R. A., Gretz D. M., Taylor P. C., James S. J., Pogribny I. P., Miller B. J., Henning S. M., Swendseid M. E. Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women. J. Nutr. 1998; 128:1204-1212[Abstract/Free Full Text]
• Lindenbaum, J. & Allen, R. H. (1995). Clinical spectrum and diagnosis of folate deficiency. In: Folate: Nutritional and Clinical Perspectives (Bailey, L., ed.), pp. 43-73. M. Dekker, New York, NY.
• Metz J., Stevens K., Krawitz S., Brandt V. The plasma clearance of injected doses of folic acid as an index of folic acid deficiency. J. Clin. Pathol. 1961; 14:622-625
• Muldoon R. T., Ross D. M., McMartin K. E. Folate transport pathways regulate urinary excretion of 5-methyltetrahydrofolate in isolated perfused rat kidney. J. Nutr. 1996; 126:242-250
• O'Keefe, C. A., Bailey L. B., Thomas E. A., Hofler S. A., Davis B. A., Cerda J. J., Gregory J. F. Controlled dietary folate affects folate status in nonpregnant women. J. Nutr. 1995; 125:2717-2725
• Pfeiffer C. M., Rogers L. M., Bailey L. B., Gregory J. F. Absorption of folate from fortified cereal-grain products and of supplemental folate consumed with or without food determined using a dual-label stable-isotope protocol. Am. J. Clin. Nutr. 1997; 66:1388-1397[Abstract/Free Full Text]
• Rogers L. M., Pfeiffer C. M., Bailey L. B., Gregory J. F. A dual-label stable-isotopic protocol is suitable for determination of folate bioavailability: evaluation of urinary excretion and plasma folate kinetics of intravenous and oral doses of [¹³C₅] and [²H₂]folic acid. J. Nutr. 1997; 127:2321-2327[Abstract/Free Full Text]
• Santhosh-Kumar C. R., Deutsch J. C., Hassell K. L., Kolhouse N. M., Kolhouse J. F. Quantitation of red blood cell folates by stable isotope dilution gas chromatography-mass spectrometry utilizing a folate internal standard. Anal. Biochem. 1995; 225:1-9[Medline]
• Sauberlich, H. E., Dowdy, R. P. & Skala, J. H. (1974) Laboratory Tests for the Assessment of Nutritional Status, pp. 49-60. CRC Press, Cleveland, OH.
• Selhub J., Jacques P. F., Wilson P. W., Rush D., Rosenberg I. H. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. J. Am. Med. Assoc. 1993; 270:2693-2698[Abstract/Free Full Text]
• Steinberg S. E. Mechanisms of folate homeostasis. Am. J. Physiol. 1984; 246:G319-G324[Abstract/Free Full Text]
• Stites T. E., Bailey L. B., Scott K. C., Toth J. P., Fisher W. P., Gregory J. F. Kinetic modeling of folate metabolism using chronic administration of deuterium-labeled folic acid in adult men. Am. J. Clin. Nutr. 1997; 65:53-60[Abstract/Free Full Text]
• Tamura, T. (1990) Microbiological assay of folates. In: Folic Acid Metabolism in Health and Disease (Picciano, M. F., Stokstad, E.L.R., Gregory, J. F., eds.), pp. 121-137. John Wiley & Sons, New York, NY.
• Tamura T., Soong S. J., Sauberlich H. E., Hatch K. D., Cole P., Butterworth C. E. Evaluation of the deoxyuridine suppression test by using whole blood samples from folic acid-supplemented subjects. Am. J. Clin. Nutr. 1990; 51:80-86[Abstract/Free Full Text]
• Tucker K. L., Selhub J., Wilson P. W. F., Rosenberg I. H. Dietary intake pattern relates to plasma folate and homocysteine concentrations in the Framingham Heart Study. J. Nutr. 1996; 126:3025-3031
• Vester B., Rasmussen K. High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum. Eur. J. Clin. Chem. Clin. Biochem. 1991; 29:549-554[Medline]
• Wagner, C. (1995) Biochemical role of folate in cellular metabolism. In: Folate in Health and Disease (Bailey, L. B., ed.), pp. 23-42. M. Dekker, New York, NY.
• Wei M.-M., Bailey L. B., Toth J. P., Gregory J. F. . Bioavailability for humans of deuterium-labeled monoglutamyl and polyglutamyl folates is affected by selected foods. J. Nutr. 1996; 126:3100-3108

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