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Human Nutrition and Metabolism
Research Communication

Use of an Oral/Intravenous Dual-Label Stable-Isotope Protocol to Determine Folic Acid Bioavailability from Fortified Cereal Grain Foods in Women¹

Nutrition & Consumer Science Division, Institute of Food Research, Norwich, UK and ^* School of Chemistry, University of Exeter, Exeter, UK

²To whom correspondence should be addressed. E-mail: paul.finglas{at}bbsrc.ac.uk.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Folic acid fortification, mandatory in the United States, is currently being considered by the UK. The hypothesis that the matrix of some cereal-product vehicles may result in low fortificant bioavailability was tested using a dual oral/intravenous (i.v.) isotopic-label approach, which was evaluated concurrently. Fifteen women received 225 µg oral folate (capsules, fortified white bread and fortified branflakes), mainly as folic acid labeled with ¹³C on 6 carbons of the benzoyl ring (¹³C₆-PteGlu), followed by i.v. injection of 100 µg folic acid labeled with ²H on 4 hydrogens of the glutamic acid group (²H₄-PteGlu). The urinary excretion ratio (UER) in intact folate of the percentage of labeled oral dose excreted divided by the percentage of i.v. dose excreted was used as the primary index of absorption. The geometric mean (95% confidence interval) UER for folic acid capsules was 3.68 (1.90, 7.14) at 24 h and 2.18 (1.24, 3.83) at 48 h. Because these were significantly in excess of 1.0, indicative of 100% absorption of the oral dose, it was concluded that oral and i.v. labeled folic acid are handled differently by the body and that "absolute" absorption cannot be calculated. Compared with the 48-h UER for folic acid capsules, the "relative" 48-h UER for white bread and branflakes was 0.71 and 0.37, respectively, indicating that some cereal-based vehicles may inhibit absorption of fortificant. However, even the validity of this "relative" approach is questioned.

KEY WORDS: • folic acid • bioavailability • absorption • stable isotope • cereal

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Folates are B-group vitamins that function in multiple coenzyme forms in acceptance, redox processing and transfer of 1-carbon units. They play a key role in the synthesis of DNA and the methylation of homocysteine to regenerate methionine (1 ). Folate deficiency causes impairment of these and other critical functions related to 1-carbon metabolism, and has a major effect on public health. Periconceptional supplementation of women with folic acid has been shown to significantly reduce the incidence and reoccurrence of neural tube defects (NTD)⁶ (2 ,3 ). Marginal folate deficiency is also associated with elevated plasma homocysteine, an emerging risk marker for vascular diseases, which is linked to certain cancers (4 ,5 ).

Effective January 1, 1998, manufacturers of enriched cereal-grain foods in the United States of America have been required by the Food and Drug Administration to add folic acid at a concentration of 140 µg/100 g product (6 ). In the UK, the Committee on Medical Aspects of Food and Nutrition Policy (COMA) has recently recommended that all flour be fortified with folic acid at 240 µg/100 g (7 ). This would give an increase of 200 µg/d in average intake of folic acid for women of childbearing age based on an estimate of the bioavailability of folic acid from fortified foods of 85%, compared with that for folic acid alone (8 ), although uncertainties in these estimates were acknowledged (9 ). Although humans absorb folic acid well in the absence of food, little is known about its absorption from fortified foods. Previous studies of the bioavailability of folic acid added to staple cereal-grain foods in South Africa found that the relative bioavailability of folic acid added to corn or rice was 50–60%, and that for fortified bread was 30–40% (10 ). This was based on a short-term change in plasma folate concentration relative to that for an aqueous folic acid control. Similar results were obtained with the chronic administration of folic acid supplements and fortified foods, when changes in erythrocyte folate were measured. This substantiates the acute-dose approach (10 –12 ). Although these studies indicate incomplete absorption, the concentration of added folic acid per serving (900–1000 µg) was high compared with amounts likely to be present in any single meal containing fortified foods and/or food naturally rich in folate. Fractional folate absorption may be reduced with increasing dose.

The development of stable isotope methods has permitted more sensitive and specific investigation of folate bioavailability in humans (13 ,14 ). The most developed methods are based on a single-dose dual-label approach in which two isotopically labeled forms of folic acid are administered, comprising an oral [¹³C] dose and an intravenous (i.v.) [²H] dose. Urinary excretion (24 h) of both isotopes, in isolated intact folate is measured; the percentage of the oral and i.v. doses excreted is calculated and expressed as the urinary excretion ratio (UER) (15 –17 ).

If stable isotope–labeled folates given orally and intravenously are subject to similar in vivo distribution and retention by the body, this single-dose, dual-label technique should allow estimation of the absolute fractional absorption of folic acid (PteGlu) from fortified food products. However, the phenomenon for some oral doses of PteGlu to exhibit a UER higher than the theoretical maximum of 1.0 has been reported (15 –17 ). On the first report of this phenomenon (15 ), it was suggested that the comparative excretions of folates from oral administration (derived by comparison of their UER) would be a valid way of at least obtaining relative absorptions. Interestingly, despite comments on this phenomenon, it is noteworthy that all authors reported that analysis of their data indicated that such UER were not statistically different from 1.0. It is important, therefore, to ascertain definitively whether UER can be used to estimate absolute fractional absorption of folate of oral origin. If the answer is "no," it then must be established whether UER can be used in a comparative manner to estimate relative absorptions.

The purpose of the current study was to make a critical evaluation of the dual-label method and, at the same time, determine potential matrix effects on the bioavailability of fortificant folic acid from cereal-based foods likely to provide the vehicles for fortification strategies.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isotopically labeled folates.

Folic acid was prepared in two isotopically labeled forms: ¹³C₆PteGlu and ²H₄-PteGlu (18 ). Similarly labeled folates had equivalent absorption after simultaneous oral administration (17 ).

Preparation of capsules and i.v. solutions.

Gelatin capsules containing ¹³C₆-PteGlu were manufactured by RP Scherer Ltd. (Swindon, UK). The contents of 20 capsules were assayed for PteGlu, using an HPLC procedure with UV detection (19 ), and found to contain 56.0 µg/capsule. Capsules were stored in the dark at +4°C. The folate content was checked every 6 mo and found to be stable for the period of study.

Solutions with a target concentration of 100 mg/L ²H₄-PteGlu for i.v. injection were prepared by the Pharmacy Manufacturing Unit, Ipswich NHS Hospital Trust (Ipswich, UK). The ²H₄-PteGlu preparation was added to sterile water. A small volume of 0.4 mol/L sodium bicarbonate (pharmaceutical grade) was then added to aid complete dissolution, to a maximum of 1% (v:v), taking care that the final pH of the solution was <pH 8. The resulting solution was thoroughly mixed, filter-sterilized (through 0.22 µm), and aliquots pipetted into glass ampoules, which were then flushed with nitrogen and heat-sealed; 10% of the ampoules were selected for virulence testing and the remainder stored frozen at -18°C. The mean concentration of PteGlu in five vials, as determined by HPLC (19 ), was 87.2 mg/L.

Preparation of fortified foods.

Two fortified foods containing ¹³C₆PteGlu were prepared, i.e., a breakfast cereal (bran flakes) and a white sliced bread. A batch of 4 kg of fortified breakfast cereal was produced by Kellogg’s (Manchester, UK). A solution of ¹³C₆-PteGlu was spray-dried onto unfortified bran flakes. Fortified and unfortified bran flakes were stored at +4°C in the dark. The mean concentration of folate in five samples of fortified and unfortified bran flakes, measured by microbiological assay (20 ), was 2480 and 330 µg/kg, respectively. Using commercial ingredients, Western Research Laboratories (Maidenhead, UK) used the Chorleywood Bread Process to produce standard white sliced bread (5 x 800-g loaves). ¹³C₆PteGlu (15 mg) was thoroughly blended, in small sequential steps, into 200 g flour to ensure that the folic acid was uniformly distributed. This was then blended into 2 kg flour and used in the bread mix for the preparation of dough, which was itself thoroughly mixed. Fortified and unfortified sliced loaves were stored frozen at -18°C. Two slices of bread were removed from each loaf and crumbled in a food processor to produce a single sample from each loaf. Each sample was analyzed for folate by microbiological assay (20 ). After losses during the baking process, the mean concentration of folate in fortified and unfortified white bread was 1910 and 210 µg/kg, respectively.

Study volunteers.

The study was approved by the Norwich and District Ethics Committee (Norfolk & Norwich Area Health Care Trust). Fifteen healthy women (aged 19–43 y) were recruited subject to the following criteria: neither pregnant nor planning a pregnancy; never having a NTD-diagnosed birth (or first-degree relative with one); not taking any medication including nutritional supplements, and not suffering from a chronic illness. An initial fasting blood sample (30 mL) was taken from all volunteers to check for normal blood biochemistry, serum vitamin B-12, plasma and erythrocyte folate and total homocysteine concentrations.

Experimental design.

The women attended three test days scheduled at least 3 wk apart to minimize the risk of isotope carry-over between doses. Volunteers were asked to avoid excessive consumption of folate-rich or fortified foods (a list was provided) for three days both before and after each of the test days. After an overnight fast, volunteers received an oral dose of folate as follows: 1) four gelatin capsules containing ¹³C₆-PteGlu (224 µg total); 2) 91 g fortified bran flakes containing a total of 225 µg folate (196 µg ¹³C₆-PteGlu and 29 µg folate of natural origin); or 3) 118 g white sliced bread containing a total of 225 µg folate (200 µg ¹³C₆-PteGlu and 25 µg folate of natural origin). All doses were observed by a member of the study team to be digested within 15 min, with the aid of water only, and an i.v. injection of 100 µg ²H₄-PteGlu in saline was administered 15 min after the start of consumption. The women were given a light, low folate lunch 4 h after the oral dose. Three 24-h urine samples were collected from each woman (24-h period before treatment, 0- to24-h period post-treatment and 24- to 48-h period post-treatment).

Habitual dietary intake data were obtained using written, weighed, records completed every sixth day for 7 wk (21 ) during the study period, excluding test days and the 3 d either side when the women were asked to avoid excessive consumption of folate-rich or fortified foods. Dietary records were coded (22 ) and mean daily nutrient intakes calculated.

Analysis of plasma and red-cell folate, serum vitamin B-12 and plasma total homocysteine concentrations.

Plasma folate was determined by microbiological assay (20 ). Red-cell folate and serum vitamin B-12 were measured by chemiluminescent immunoassay (Beckman Access, High Wycombe, UK) and enzyme immunoassay (Abbott Laboratories, Maidenhead, UK), respectively, at a local hospital, and total plasma L-homocysteine by fluorescence polarization immunoassy [IMx System, Abbott Laboratories; (23 ,24 )].

Isolation and gas chromatography-mass spectrometry (GC-MS) analysis of total labeled urinary folate.

HPLC analysis of urine samples revealed that the only folate present was 5-CH₃H₄PteGlu. A modification of a previously published GC-MS procedure was used to determine total labeled urinary folate (25 ,26 ). Urine samples were collected into 2.5-L polyethylene bottles containing 3 g ascorbic acid. DL-Dithiothreitol (0.045 g), was added to three subsamples of 60 mL and stored at -40°C. Samples were thawed and 55 mL mixed with 5.5 mL potassium phosphate buffer (0.5 mol/L; pH 7.0), followed by adjustment to 7.0 with NaOH (1 mol/L). After 30 min, solutions were filtered and intact labeled folate isolated on an affinity column using folate binding protein (FBP) bound to Affigel 10 gel. Elutes of labeled urinary 5CH₃H₄PteGlu were cleaved to para-aminobenzoylglutamate (PABG), centrifuged for 10 min at 2000 x g and injected (1000 µL injection loop) onto an HPLC system consisting of a RP-18 column (5 µm, 125 x 4 mm; Merck, Darmstadt, Germany), fitted with a LiChrosphere RP-18 guard column (5 µm; Merck) and mobile phase of formic acid (0.1 mol/L)/acetonitrile (95:5, v/v) at a flow rate of 0.7 mL/min. A fluorescence-detector was used and set at 290/356 nm (excitation/emission). The PABG peak was collected and, together with standards of unlabeled PABG, ²H₄-PABG and ¹³C₆-PABG (designated as M0, M4 and M6, respectively) prepared in methanol, lyophilized to dryness and stored at -18°C. Lyophilized samples or standards were transferred to vials having screw caps with teflon seal inserts, using 3 x 200 µL methanol and evaporated to dryness. Trifluoroacetic anhydride (0.2 mL; Sigma, Poole, UK) and trifluorethanol (0.1 mL; Sigma) were added, the vials tightly capped and heated at 80°C for 1 h. After cooling, solutions were evaporated to dryness, redissolved in toluene (25 µL for samples, 100 µL for standards), transferred into autosampler vials and stored at -18°C before MS analysis.

M0, M4 and M6 PABG from urine samples were quantified by GC-MS. The limit of detection was 5 µg/L for M0, and 10 µg/L for M4 and M6. The excretion of each isotopomer in intact urinary folate was calculated from the total daily urine volume, and UER calculated.

Statistics.

The mean and SEM for descriptive data [age, body mass index (BMI), plasma and RBC folate concentration, serum B-12 concentration and plasma total homocysteine concentration] were calculated on mathematically untransformed data, as was the mean and SEM for average calculated daily folate intake data. Women acted as their own controls. Data for percentage dose of each isotopomer excreted into the urine in intact folate and UER were mathematically transformed (log₁₀) to normalize distributions. Results were then examined within-subject using one-way ANOVA and Least Significant (P < 0.05) Difference tests for planned comparisons.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Subject characteristics.

The age and BMI of the women were 32.4 ± 1.9 y and 23.3 ± 0.8 kg/m², respectively. The calculated 7-d folate intake was 288 ± 26 (175–538) µg/d. The women had normal plasma folate (24.7 ± 3.6 nmol/L), erythrocyte folate (1074.1 ± 123.7 nmol/L), serum vitamin B-12 (342.6 ± 31.8 pmol/L) and plasma total homocysteine (7.5 ± 0.4 µmol/L) concentrations.

Urinary excretion of label in intact folate.

Cumulative urinary excretions of labeled folate, by 24 and 48 h postdose, are given in Table 1 , together with the UER. Of the total amount of ¹³C₆-labeled folate excreted into urine over the 48-h period of collection after oral consumption of folic acid capsules (control), 90% was excreted in the first 24 h. In contrast, of the total amount of ²H₄-labeled folate of i.v. origin excreted into urine over the 48-h period of collection after oral consumption of folic acid capsules, only 53% was excreted in the first 24 h, indicating a substantial difference in the pattern of clearance of the two isotopically labeled folates.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Provided that doses are not excessive, oral folic acid absorbed by enterocytes will be reduced and methylated before transfer into the body proper where it will appear in the hepatic portal vein as [¹³C₆] 5CH₃H₄PteGlu before entering the systemic blood supply. In contrast, the i.v. dose of folic acid is introduced directly into the systemic circulation, part of which is continuously routed through the intestines and back via the hepatic portal vein. The current findings are consistent with work reporting a greater hepatic uptake of nonreduced folic acid from portal blood relative to 5CH₃H₄PteGlu (27 ). Differences in experimental design may account for our observation of differential oral/i.v. isotope handling by the body. In the current study, the women fasted overnight and rigorously controlled their diet during and between studies by restricting excessive consumption of high folate foods 3 d before and after each trial. Urine was also collected in two parts, which allowed any initial differences in excretion of the labeled folates to be more easily detected.

Although the current study indicates that absolute absorption of an oral dose cannot be calculated using urinary excretion ratios, it has previously been suggested (15 ) that the excretion of labeled folates from oral administration compared with a control of folic acid (derived by comparison of their UER) would be a valid way of at least obtaining relative absorptions, especially because the use of UER could compensate for differences in overall folate excretion. The 24-h UER for white bread and bran flakes, when compared with that for folic acid capsules, resulted in geometric mean relative ratios of 0.58 and 0.27, respectively, which suggests strongly that the matrix of these cereal-foods has some inhibitory effect on intestinal absorption of folic acid added to the food as a fortificant. However, it is important to note that the relative ratios at 48 h, 0.71 and 0.37, respectively, were significantly higher. It is therefore arguable that for this approach to have validity, the postdose urine collection time required for relative ratios to reach a constant must be ascertained, and this may be significantly greater than the maximum collection period employed in this and any of the other studies examined. Great care, therefore, must be taken in the design of dual-label, stable-isotope protocols.

In spite of our conclusions, we believe that the dual oral/i.v. approach developed by Gregory and co-workers (13 –17 ) may still have much merit, but suggest that the only folate theoretically appropriate for i.v. use would be 5CH₃H₄PteGlu, the circulating form of plasma folate. However, because different i.v. clearance patterns exist between the two isomeric [6R-, 6S-] forms of this folate (28 ), only the naturally occurring [6S-] form should be used. The recent commercial availability of a stable-isotope labeled version of the [6S]-isomer of 5CH₃H₄PteGlu (which was not available at the time of the current study) should permit further evaluation of the dual oral/i.v. approach.

	ACKNOWLEDGMENTS

 
We thank all volunteers who took part in the study for their enthusiastic support, K. J. Scott for technical assistance, and Karel Jakobs and Kees de Meer (Amsterdam Free University Hospital) for homocysteine analyses.

	FOOTNOTES

 
¹ This work is a publication of the Institute of Food Research’s Nutrition & Consumer Science Division in collaboration with the Department of Chemistry, University of Exeter. The project was funded by the Ministry of Agriculture, Fisheries and Food. L.V. was supported under EU Marie Curie training grant no. ERB4001GT961823.

³ Present address: Department of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden.

⁴ Present address: Department of Applied Chemistry & Microbiology, University of Helsinki, Viiki Food Science, Helsinki, Finland.

⁵ Present address: Tripos Receptor Research Ltd., Bude Stratton Business Park, Bude, Cornwall, EX23 8LY, UK.

⁶ Abbreviations used: BMI, bodv mass index; 5-CH₃-H₄PteGlu, 5-methyltetrahydrofolic acid; ¹³C₆-PteGlu, folic acid labeled with ¹³C on 6 carbons of the benzoyl ring; COMA, UK Committee on Medical Aspects of Food and Nutrition Policy; FBP, folate binding protein; GC-MS, gas chromatography-mass spectrometry; ²H₄-PteGlu, folic acid labeled with ²H on 4 hydrogens of the glutamic acid group; i.v., intravenous; M0 PABG-unlabeled para-aminobenzoylglutamate: M4 PABG, ²H₄ labeled para-aminobenzoylglutamate; M6 PABG, ¹³C₆ labeled para-aminobenzoylglutamate; NTD, neural tube defects; PABG, para-aminobenzoylglutamate: PteGlu, folic acid; UER, urinary excretion ratio.

Manuscript received 24 July 2001. Initial review completed 4 September 2001. Revision accepted 14 January 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Shane, B. (1995) Folate chemistry and metabolism. Bailey, L. B. eds. Folate in Health and Disease 1995:1-22 Marcel Dekker New York, NY. .

2. MRC Vitamin Study Research Group (1991) Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet 338:131-137.[Medline]

3. Czeizel, A. E. & Duda, I. (1992) Prevention of the first occurrence of neural tube defects by periconceptional vitamin supplementation. N. Engl. J. Med. 327:32-35.

4. Boushey, C. J., Beresford, S.A.A., Omenn, G. S. & Motulsky, A. G. (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease—probable benefits of increasing folic acid intakes. J. Am. Med. Assoc. 274:1049-1057.[Abstract/Free Full Text]

5. Mason, J. B. (1995) Folate status: effects on carcinogenesis. Bailey, L. B. eds. Folate in Health and Disease 1995:361-378 Marcel Dekker New York, NY. .

6. Food and Drug Administration (1996) Food standards: amendment of standards of identity for enriched grain products to require addition of folic acid. Fed. Regist. 61:8781-8797.

7. Department of Health (2000) Report on Health and Social Subjects 50. Folic Acid and Prevention of Disease. Report of the Committee on Medical Aspects of Food and Nutrition Policy 2000 The Stationery Office London, UK. .

8. Bailey, L. B. (1988) Dietary reference intakes for folate: the debut of dietary folate equivalents. Nutr. Rev. 56:294-299.

9. Institute of Medicine, Panel on Folate, Other B Vitamins, and Choline (1998) Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B₆, Folate, Vitamin B₁₂, Pantothenic Acid, Biotin, and Choline 1998 National Academy Press Washington, DC. .

10. Colman, N., Green, R. & Metz, J. (1975) Prevention of folate deficiency by food fortification. II. Absorption of folic acid from fortified staple foods. Am. J. Clin. Nutr. 28:459-464.[Abstract/Free Full Text]

11. Colman, N., Larsen, J. V., Barker, M., Barker, E. A., Green, R. & Metz, J. (1975) Prevention of folate deficiency by food fortification. III. Effect in pregnant subjects of varying amounts of added folic acid. Am. J. Clin. Nutr. 28:465-470.[Abstract/Free Full Text]

12. Colman, N., Barker, E. A., Barker, M., Green, R. & Metz, J. (1975) Prevention of folate deficiency by food fortification. IV. Identification of target groups in addition to pregnant women in a rural population. Am. J. Clin. Nutr. 28:761-763.[Abstract/Free Full Text]

13. Gregory, J. F., Bailey, L. B., Toth, J. P. & Cerda, J. J. (1990) Stable isotope methods for assessment of folate bioavailability. Am. J. Clin. Nutr. 51:212-215.[Abstract/Free Full Text]

14. Gregory, J. F. (1997) Bioavailability of folate. Eur. J. Clin. Nutr. 51S:54-59.[Medline]

15. Gregory, J. F., Bhandari, S. D., Bailey, L. B., Toth, J. P., Baumgartner, T. G. & Cerda, J. J. (1992) Relative bioavailability of deuterium-labeled monoglutamyl tertrahydrofolates and folic acid in human subjects. Am. J. Clin. Nutr. 55:1147-1153.[Abstract/Free Full Text]

16. Pfeiffer, C. M., Rogers, L. M., Bailey, L. B. & Gregory, J. F. (1997) Absorption of folate from fortified cereal-grain products and of supplemental folate consumed with or without food determined by using a dual-label stable isotope protocol. Am. J. Clin. Nutr. 66:1388-1397.[Abstract/Free Full Text]

17. Rogers, L. M., Pfeiffer, C. M., Bailey, L. B. & Gregory, J. F. (1997) A dual-label stable isotopic protocol is suitable for determination of folate bioavailability in humans: evaluation of urinary excretion and plasma folate kinetics of intravenous and oral doses of [¹³C₅] and [²H₂] folic acid. J. Nutr. 127:2321-2327.[Abstract/Free Full Text]

18. Maunder, P., Finglas, P. M., Mallet, A. I., Mellon, F. A., Razzaque, M. A., Ridge, B., Vahteristo, L. & Witthöft, C. (1999) The synthesis of folic acid, multiply labelled with stable isotopes, for bioavailability studies in human nutrition. J. Chem. Soc. Perkin Trans. 1:1311-1323.

19. Witthöft, C. & Bitsch, I. (1993) HPLC methods to analyse folate patterns in food and human plasma as a precondition to evaluate availability of food folates by biokinetic methods. Schlemmer, U. eds. Bioavailability ’93 1993:436-439 Schriftenreihe der Bundesforschungsanstalt fur Ernahrung Karlsruhe, Germany. .

20. Southon, S., Wright, A.J.A., Finglas, P. M., Bailey, A. L., Loughridge, J. & Walker, A. D. (1994) Dietary intake and micronutrient status of adolescents: effect of vitamin and trace element supplementation on indices of status and performance in tests of verbal and non-verbal intelligence. Br. J. Nutr. 71:897-918.[Medline]

21. Wright, A.J.A., Southon, S., Bailey, A. L., Finglas, P. M., Maisey, S. & Fulcher, R. A. (1995) Nutrient intake and biochemical status of non-institutionalised elderly subjects in Norwich: comparison with younger adults and adolescents from the same general community. Br. J. Nutr. 74:453-475.[Medline]

22. Holland, B., Welch, A. A., Unwin, I. D., Buss, D. H., Paul, A. A. & Southgate, D.A.T. (1991) McCance & Widdowson’s The Composition of Foods 5th rev. ed. 1991 Royal Society of Chemistry/Ministry of Agriculture Fisheries and Food, Cambridge, UK. .

23. Fiore, M., Mitchell, J., Doan, T., Nelson, R., Winter, G., Grandone, C., Zeng, K., Haraden, R., Smith, J. & Harris, K. (1988) The Abbott IMx Automated Benchtop Immunochemistry Analyzer System. Clin. Chem. 34:1726-1732.[Abstract/Free Full Text]

24. Sundrehagen, E. (1990) Enzymatic assay for homocysteine and a kit therefor. EP 623174/US 56 31127 1990 Axis Biochemicals ASA Oslo, Norway. .

25. Toth, J. P. & Gregory, J. F. (1988) Analysis of folacin by GCMS: structure and mass spectra of fluorinated derivatives of para-aminobenzoyl glutamic acid. Biomed. Environ. Mass Spectrom. 17:73-79.

26. Gregory, J. F. & Toth, J. P. (1988) Chemical synthesis of deuterated folate monoglutamate and in vivo assessment of urinary excretion of deuterated folates in man. Anal. Biochem. 170:94-104.[Medline]

27. Steinberg, S. E. (1979) Kinetics of the normal folate enterohepatic cycle. J. Clin. Investig. 64:83-88.

28. Mader, R. M., Steger, G. G., Rizovski, B., Djavanmard, M. P., Scheithauer, W., Jakesz, R. & Rainer, H. (1995) Stereospecific pharmacokinetics of rac-5-methyletetrahydrofolic acid in patients with advanced colorectal cancer. Br. J. Clin. Pharmacol. 40:209-215.[Medline]

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