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A Comparison of the Effect of Advice to Eat Either '5-A-Day'fruit And European Journal of Clinical Nutrition (2003) 57, 316–323 ß 2003 Nature Publishing Group All rights reserved 0954–3007/03 $25.00 www.nature.com/ejcn ORIGINAL COMMUNICATION A comparison of the effect of advice to eat either ‘5-a-day’ fruit and vegetables or folic acid-fortified foods on plasma folate and homocysteine PAL Ashfield-Watt1{, JM Whiting1, ZE Clark1, SJ Moat1, RG Newcombe2, ML Burr2 and IFW McDowell1* 1Cardiovascular Sciences Research Group, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff, Wales, UK; and 2Department of Epidemiology, Statistics and Public Health, University of Wales College of Medicine, Cardiff, Wales, UK Objective: To assess and compare the effects of natural folate (100 mg) with those of folic acid from fortified sources (100 mg=day) on plasma folate and homocysteine. Design: Randomized controlled trial (parallel groups). Setting: Men and women living in South Wales, UK. Subjects: A total of 135 healthy individuals recruited from the local workforce and blood donor sessions. All subjects possessed the ‘wild-type’ CC genotype for C677T polymorphism in methylenetetrahydrofolate reductase (MTHFR). Interventions: Subjects underwent one of the following dietary interventions for 4 months: (1) fortified diet — usual diet plus 100 mg=day folic acid from fortified foods; (2) natural folate diet — usual diet plus 100 mg=day folate from natural sources; (3) control — usual diet. Results: The fortified group increased reported intake of folic acid from fortified foods compared to other groups (P < 0.001) achieving an extra 98 mg=day (95% CI 88 – 108). The natural folate group increased reported intake of natural source folates compared with the other two groups (P < 0.001), but achieved a mean increase of only 50 mg=day (95% CI 34 – 66). Plasma folate increased (P < 0.01) by a similar amount in both intervention groups compared to controls (fortified group 2.97, 95% CI 0.8 – 5.1; natural group 2.76, 95% CI 0.6 – 4.9. Plasma homocysteine, vitamins B6 and B12 were not significantly changed. Conclusions: Subjects achieved increases in folate intake using fortified foods more easily than by folate-rich foods, however both sources increased plasma folate by a similar amount. These levels of intake were insufficient to reduce homocysteine concentrations in MTHFR CC homozygotes, but may be more effective in other genotypes. Sponsorship: This study was supported by the UK Ministry of Agriculture, Fisheries and Foods (latterly UK, Food Standards Agency, Project Reference N05002). The Kellogg Company of Great Britain provided funding for reimbursement of subjects for the cost of fortified foods. European Journal of Clinical Nutrition (2003) 57, 316 – 323. doi:10.1038=sj.ejcn.1601545 Keywords: natural folates, fortified foods, 5-a-day fruits and vegetables, plasma folate, plasma homocysteine Introduction Sub-optimal folate status is associated with increased risk of *Correspondence: Dr IFW McDowell, Department of Medical neural tube defects (NTD; Medical Research Council, 1991), Biochemistry, University of Wales College of Medicine, Cardiff, Wales CF14 4XN, UK. cardiovascular disease (CVD; Boushey et al, 1995) and cancer E-mail: [email protected] (Kim, 1999). Although a causal link has been proven only for { Current address: Strangeways Research Laboratory, Wort’s Causeway, NTD (Medical Research Council, 1991), possible health bene- The University of Cambridge CB1 8RN, UK. fits of folate at all stages of the life cycle have stimulated Guarantor: Dr IFW McDowell. Contributors: PALA-W, MLB, IFWMcD, RGN designed the study. PALA- much research into optimal folate nutrition. Plasma W and JMW collected the data presented here. ZEC and SJM were homocysteine is inversely related to blood measures of responsible for the genotyping and biochemical assessments. RGN folate and is therefore a responsive marker of folate status analysed the data. PALA-W wrote the first draft of the paper and the (Selhub et al, 1993). Epidemiological studies show an other authors commented on this and subsequent drafts. Received 18 June 2001; revised 27 May 2002; association between elevated homocysteine and both NTD accepted 28 May 2002 and CVD (Stone et al, 1999). ‘5-a-day’ fruit and vegetables vs folic acid-fortified foods PAL Ashfield-Watt et al 317 Dietary folates occur as natural folates in green leafy indicated mean intakes of 2.3 portions=day of fruit and vegetables, citrus fruits, liver and dairy products and as vegetables in this region of South Wales. An increase to added synthetic folic acid in fortified food products. five portions per day would raise natural folate intake by The major function of folates in vivo is the transfer of approximately 100 mg=day and may be more easily achieved single carbon units, particularly for DNA synthesis and at the population level than the higher folate doses homocysteine remethylation. Homocysteine is regulated by previously investigated. Alternatively, folate intake may be two pathways: transulfuration catalysed by cystathionine- increased using foods fortified with folic acid. We have b-synthase with co-factor pyridoxal phosphate (vitamin B6) previously demonstrated that a diet enhanced with fortified and remethylation catalysed by methionine synthase with foods (200 mg=day extra folate) significantly increases co-factor vitamin B12 and substrate 5-methyltetrahydrofolate plasma folate and reduces plasma homocysteine (Ashfield- (Finkelstein, 1998). Folate is the strongest nutritional pre- Watt et al, 2001). Low-dose natural and fortified folate dictor of homocysteine concentrations (Boushey et al, 1995). approaches have not been directly compared. This study Methylenetetrahydrofolate reductase (MTHFR) converts sought to investigate the relative efficacy of 100 mg=day 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate folate from natural sources (fruit and vegetables) compared which is required for remethylation of homocysteine. with 100 mg=day folic acid from fortified foods in terms of A common mutation in MTHFR (C677T) is present in the changes in folate status. homozygous form in approximately 12% of the Caucasian population (Clark et al, 1998) and is associated with raised Methodology homocysteine concentrations especially when folate intake Subjects were healthy men and women who were of the and plasma folate are low (Harmon et al, 1996). Homozy- ‘wild-type’ CC genotype for the MTHFR C677T polymorph- gotes for the ‘wild-type’ C allele (CC genotype) comprise ism. These were individuals who had previously been approximately half the general population. screened for, but not recruited to a study of the effect of In the UK and EU the reference nutrient intake for folate is C677T variant MTHFR genotype on response to folic acid 200 mg=day for adults (Department of Health, 1991; Commis- interventions (reported elsewhere, Pullin et al, 2001; sion of the European Community, 1993). It is recommended Ashfield-Watt et al, 2002). Buccal cell samples were collected that women intending pregnancy consume an additional at the screening sessions for determination of MTHFR geno- 400 mg=day to protect against NTD. Limited success of stra- type. The study was approved by Bro Taf Research Ethics tegies to increase folate intakes during pregnancy and the Committee. All subjects gave informed, written consent. possible relationship between folate and chronic disease Subjects were eligible to participate in the study if they have stimulated interest in population interventions to fulfilled the following criteria: (1) non-smoking; (2) no increase folate status. history of cardiovascular disease or epilepsy; (3) aged Cross-sectional studies have shown dietary folate intake between 18 and 65; (4) not taking supplements containing to be positively associated with plasma folate and inversely vitamins B6,B12 or folic acid; (5) not taking any drugs known with plasma homocysteine (Tucker et al, 1996). However, to interfere with folate metabolism, eg methotrexate, bile most successful dietary intervention studies have investi- acid sequestrants; and (6) women not pregnant nor planning gated synthetic folic acid either as a supplement or a food pregnancy during the timeframe of the study. fortificant (Brouwer et al, 1999a; Dierkes, 1995). Natural Subjects were allocated to treatment groups which were folate intervention studies have reported limited success balanced with respect to age and gender using a randomiza- due to lack of compliance with dietary advice, destruction tion scheme produced by the study statistician (RGN). A of natural food folates during processing and poor bioavail- total of 135 subjects were assessed at baseline (dietary assess- ability (Sauberlich et al, 1987; Cuskelly et al, 1996). Impor- ment and venepuncture) and randomly allocated to one of tantly, Cuskelly et al demonstrated that the additional three dietary interventions. Subjects were monitored regu- 400 mg=day folate intake required for pregnancy could not larly throughout the intervention period and were reassessed be achieved using natural source folates alone. Large at clinic after 4 months. The study profile is illustrated in increases in natural folate intake have been reported, but Figure 1. were achieved under conditions which could not be gene- rally applied to free-living individuals (Brouwer et al, 1999b). Dietary assessment and interventions Low-dose natural folate strategies would be easier to achieve Folate intake was assessed using a semi-quantitative and may produce important increases in folate
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