Introduction

Sub-optimal folate status is associated with increased risk of neural tube defects (NTD; Medical Research Council, 1991), cardiovascular disease (CVD; Boushey et al, 1995) and cancer (Kim, 1999). Although a causal link has been proven only for NTD (Medical Research Council, 1991), possible health benefits of folate at all stages of the life cycle have stimulated much research into optimal folate nutrition. Plasma homocysteine is inversely related to blood measures of folate and is therefore a responsive marker of folate status (Selhub et al, 1993). Epidemiological studies show an association between elevated homocysteine and both NTD and CVD (Stone et al, 1999).

Dietary folates occur as natural folates in green leafy vegetables, citrus fruits, liver and dairy products and as added synthetic folic acid in fortified food products. The major function of folates in vivo is the transfer of single carbon units, particularly for DNA synthesis and homocysteine remethylation. Homocysteine is regulated by two pathways: transulfuration catalysed by cystathionine--synthase with co-factor pyridoxal phosphate (vitamin B₆) and remethylation catalysed by methionine synthase with co-factor vitamin B₁₂ and substrate 5-methyltetrahydrofolate (Finkelstein, 1998). Folate is the strongest nutritional predictor of homocysteine concentrations (Boushey et al, 1995).

Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate which is required for remethylation of homocysteine. A common mutation in MTHFR (C677T) is present in the homozygous form in approximately 12% of the Caucasian population (Clark et al, 1998) and is associated with raised homocysteine concentrations especially when folate intake and plasma folate are low (Harmon et al, 1996). Homozygotes for the 'wild-type' C allele (CC genotype) comprise approximately half the general population.

In the UK and EU the reference nutrient intake for folate is 200 g/day for adults (Department of Health, 1991; Commission of the European Community, 1993). It is recommended that women intending pregnancy consume an additional 400 g/day to protect against NTD. Limited success of strategies to increase folate intakes during pregnancy and the possible relationship between folate and chronic disease have stimulated interest in population interventions to increase folate status.

Cross-sectional studies have shown dietary folate intake to be positively associated with plasma folate and inversely with plasma homocysteine (Tucker et al, 1996). However, most successful dietary intervention studies have investigated synthetic folic acid either as a supplement or a food fortificant (Brouwer et al, 1999a; Dierkes, 1995). Natural folate intervention studies have reported limited success due to lack of compliance with dietary advice, destruction of natural food folates during processing and poor bioavailability (Sauberlich et al, 1987; Cuskelly et al, 1996). Importantly, Cuskelly et al demonstrated that the additional 400 g/day folate intake required for pregnancy could not be achieved using natural source folates alone. Large increases in natural folate intake have been reported, but were achieved under conditions which could not be generally applied to free-living individuals (Brouwer et al, 1999b). Low-dose natural folate strategies would be easier to achieve and may produce important increases in folate status in the general population.

Folate intake may be enhanced through other widely publicised health messages such as the 5-a-day fruit and vegetable recommendation of the World Health Organization (WHO, 1990). In the UK, the average daily intake of fruits and vegetables falls short of this target (Department of Health, 2000a). Our pilot data from 85 healthy volunteers indicated mean intakes of 2.3 portions/day of fruit and vegetables in this region of South Wales. An increase to five portions per day would raise natural folate intake by approximately 100 g/day and may be more easily achieved at the population level than the higher folate doses previously investigated. Alternatively, folate intake may be increased using foods fortified with folic acid. We have previously demonstrated that a diet enhanced with fortified foods (200 g/day extra folate) significantly increases plasma folate and reduces plasma homocysteine (Ashfield-Watt et al, 2001). Low-dose natural and fortified folate approaches have not been directly compared. This study sought to investigate the relative efficacy of 100 g/day folate from natural sources (fruit and vegetables) compared with 100 g/day folic acid from fortified foods in terms of changes in folate status.

Methodology

Subjects were healthy men and women who were of the 'wild-type' CC genotype for the MTHFR C677T polymorphism. These were individuals who had previously been screened for, but not recruited to a study of the effect of C677T variant MTHFR genotype on response to folic acid interventions (reported elsewhere, Pullin et al, 2001; Ashfield-Watt et al, 2002). Buccal cell samples were collected at the screening sessions for determination of MTHFR genotype. The study was approved by Bro Taf Research Ethics Committee. All subjects gave informed, written consent.

Subjects were eligible to participate in the study if they fulfilled the following criteria: (1) non-smoking; (2) no history of cardiovascular disease or epilepsy; (3) aged between 18 and 65; (4) not taking supplements containing vitamins B₆, B₁₂ or folic acid; (5) not taking any drugs known to interfere with folate metabolism, eg methotrexate, bile acid sequestrants; and (6) women not pregnant nor planning pregnancy during the timeframe of the study.

Subjects were allocated to treatment groups which were balanced with respect to age and gender using a randomization scheme produced by the study statistician (RGN). A total of 135 subjects were assessed at baseline (dietary assessment and venepuncture) and randomly allocated to one of three dietary interventions. Subjects were monitored regularly throughout the intervention period and were reassessed at clinic after 4 months. The study profile is illustrated in Figure 1.

Figure 1.

(a) Folic acid diary. (b) Guidelines given to subjects. This example applies to the natural folate diet.

Full figure and legend (31K)

Dietary assessment and interventions

Folate intake was assessed using a semi-quantitative food frequency questionnaire. This questionnaire was developed from the questionnaire of Yarnell et al (1983) which had been validated in this area of South Wales. This questionnaire was modified to include questions on specific folate rich foods such as spinach, Brussels sprouts and other green vegetables, on folate-rich fruit and on vegetable extract spreads, eg Marmite. In total 32 foods were relevant to estimating total folate intakes. Subjects were asked to report how often they ate each of these foods (no. of days per week, fortnightly, monthly/rarely). Frequency of intake of folate-rich plus frequently consumed folate poor foods (eg dairy products, meats, unfortified breads) was multiplied by standard average portion sizes (Crawley, 1993) and then by the folate content derived from UK nutrient composition tables to provide folate intake data (Holland et al, 1991).

Data on fortified foods were obtained directly from manufacturers and supermarkets and updated regularly. In the UK an overage (additional amount of fortificant added during manufacture to maintain vitamin activity at the level stated on the packet during processing and storage) is added to baked products such as breads. No overage is added to cereal products. We have found in previous studies that fortified cereals are more widely available and therefore the more popular means of increasing folic acid intake. This means that our assessment of folic acid from fortified sources is unlikely to underestimate the amount of folic acid consumed by subjects in this trial.

Subjects underwent one of three possible dietary interventions for a 4 month period: (1) control diet—subjects were advised to eat their normal diet throughout the study period and asked to pay particular attention to maintaining their usual intake of folic acid-fortified products; (2) fortified diet—subjects were advised to eat an extra 100 g/day folic acid from fortified food products including fortified cereals and breads and not to exceed this amount; (3) natural folate diet—subjects were advised to eat an extra 100 g/day folate from natural sources, particularly folate-rich fruit and vegetables and to maintain their usual intake of fortified products.

Due to the amount of variation in folic acid fortification levels both within and between brands, care was taken to assess accurately the baseline folate intake from fortified sources of all subjects. For this reason, a detailed history of cereal intake over the previous month was taken in order to calculate the average baseline folate intake from this source. Subsequently, subjects randomized to control or natural intervention were required to maintain baseline intakes of folic acid-fortified foods throughout the study. Subjects in the fortified group were advised to increase folic acid intake by 100 g/day (10 points) and not to exceed this amount. All subjects were provided with a comprehensive list of the folate scores derived from information from cereal and bread manufacturers. This enabled subjects to vary their intake of cereal and bread types whilst following the dietary advice given at baseline. The scores were derived by calculating the amount of folate/folic acid per average portion (g/day), rounding to the nearest 10 g and then dividing by 10. Each active intervention therefore required an increase of 10 points (100 g) per day.

Monitoring compliance

To help understand the dietary changes required, all subjects were given a 2 week folate diary at the baseline visit which they completed and returned in a pre-paid envelope. To monitor compliance throughout the study, further food diaries were sent to subjects after 2 months and then 2 weeks before their final visit. Subjects who did not return charts or whose charts indicated poor compliance were contacted by the study nutritionist and encouragement given. The food diary consisted of a chart on which subjects recorded the number of folate points scored from the various food groups over a 2 week period. The folate scores from a wide range of folate-rich foods and frequently eaten moderate folate sources were printed on the reverse of the chart (Figure 2).

Figure 2.

(a) Folic acid diary. (b) Guidelines given to subjects. This example applies to the natural folate diet.

Full figure and legend (101K)

All subjects were asked to complete charts during the first 2 weeks of the study to familiarize subjects with the required dietary changes. However, subjects undertaking the natural folate intervention recorded their usual folate intake for the first week of the first chart in order to gauge their baseline folate intake. During the second and subsequent weeks they aimed to achieve on average 10 points more per day than during week l. The changes in fortified foods were easier for subjects to quantify, therefore subjects were not required to maintain baseline intakes during the first week of the study.

Sample collection

Following an overnight fast, venous blood was collected into EDTA vacutainers for determination of plasma folate and plasma total homocysteine (Hcy). Samples for measurement of pyridoxal phosphate and vitamin B₁₂ were collected into lithium heparin vacutainers. Blood samples were centrifuged within 10 min and the plasma stored at -70°C until assayed.

Samples were thawed and analysed in batches to ensure that pre- and post-intervention samples for subjects were analysed together to reduce assay variation. Plasma homocysteine was measured by enzymatic immunoassay and plasma folate and vitamin B₁₂ by competitive protein binding methods using an Abbot IMX instrument (between batch CVs 5.3, 9.3 and 4.0%). Plasma folate results were converted from g/l to nmol/l using a conversion factor of 2.265, based on the formula weight of folic acid.

Plasma pyridoxal phosphate (PLP) was measured as previously described (Bailey et al, 1999), with the following modifications: 100 l of the cyanide derivatives were injected onto the HPLC column (APEX ODS 3 m (25 cm4 mm). PLP was eluted isocratically at a flow rate of 1 ml/minute using a 2 M acetate buffer, containing 1 mM heptane–sulphonic acid, adjusted to pH 3.75 with potassium hydroxide, and detected fluorometrically at an excitation and emission wavelengths of 325 and 418 nm (between batch CV 5.1%).

Genotyping

Heteroduplex analysis involving DNA extraction from buccal cells, polymerase chain reaction (PCR) and electrophoresis was used for MTHFR genotyping (Clark et al, 1998). The buccal cells were obtained from the inner lining of the cheek using a 'cyto-brush'.

Statistical methods

The primary statistical test was analysis of covariance (ANCOVA) comparing treatment levels between the three groups using the corresponding pre-treatment value as covariate. Skewed variables which were log–normal (vitamins B₆ and B₁₂) were log-transformed before applying parametric tests. Bonferroni correction was applied to multiple comparisons as appropriate. The relation between variables at baseline was assessed using Pearson's correlation co-efficient for normally distributed and log–normal variables. Stepwise regression analysis was used to describe the relationship between homocysteine and B group vitamins. The effect of interventions is reported in the text relative to changes in the control group. Changes reported in the table are the absolute increments calculated in each group as the difference between pre- and post-treatment values.

Results

Baseline characteristics

The three dietary groups were balanced with regard to primary variables (see Table 1). Each group comprised 16 males and 29 females. The mean (s.d.) ages for the three groups were: control 41.2 (11.2), fortified 41.1 (11.6) and natural 41.8 (11.3). The estimated mean (s.d.) total folate intake was 242 g/day (95% CI 230–255). Eighteen percent of total folate intake was from fortified sources (45 g/day 95% CI 38–51). Dietary folate intake was moderately correlated with plasma folate at baseline (r=0.32, P<0.001, n=133). Plasma homocysteine was significantly and inversely correlated with plasma folate (r=-0.45, P<0.001, n=128) and log vitamin B₁₂ (r=-0.27, P=0.002, n=128). There was no significant correlation with log pyridoxal phosphate (r=0.04, P=0.69, n=128).

Table 1 - Baseline and follow-up dietary and biochemical parameters.

Full table (51K)

A regression model was fitted to baseline plasma homocysteine which included plasma folate, log B₁₂, log pyridoxal phosphate and pyridoxic acid as predictor variables. Of these, only plasma folate and B₁₂ significantly predicted homocysteine concentrations. The regression co-efficients for predictor variables were: plasma folate -0.449, P<0.001; log B₁₂ -4.060, P=0.01, log pyridoxal phosphate 0.92, P=0.547, pyridoxic acid 0.08, P=0.073. Plasma folate was the strongest predictor, explaining 19.8% of the variability of homocysteine. Adding vitamin B₁₂ to the model increased the predictive power to 22.9%.

Changes in dietary intake following intervention

Dietary and biochemical data are presented in Table 1. Subjects in the fortified group reported greater increases in folic acid intakes from fortified foods compared to either control (difference 93 g/day, CI 80–106) or the natural folate group (difference 87 g/day, CI 73–100), P<0.001 for both. There was a small, non-significant increase (6.7 g/day) in folate intake from fortified sources in the natural folate group compared to controls.

Subjects in the natural folate group reported significantly greater natural folate intakes compared with either the fortified group (difference 45 g/day, 95% CI 24–65) or controls (difference 62 g/day, 95% CI 42–83), P<0.001.

Total dietary folate intake (the sum of fortified and natural folates) increased significantly in both intervention groups compared with controls (P<0.001 for both), but the increase in total folate in the fortified group was significantly greater than in the natural folate group (difference 43 g/day, 95% CI 16–70, P<0.01).

Both interventions significantly raised plasma folate concentrations compared with controls (P=0.01). The change in plasma folate was similar in both intervention groups (fortified group 2.97 nmol/l, 95% CI 0.8–5.1, natural folate group 2.76 nmol/l, 95% CI 0.6–4.9).

There was no statistically significant effect of intervention on plasma homocysteine (P=0.65). Compared with controls, the fortified and natural folate interventions reduced homocysteine by 0.47 and 0.61 mol/l, respectively. There were no significant differences in effects on plasma B₆ or B₁₂ concentrations between groups at follow-up.

Discussion

Plasma concentrations of homocysteine, folic acid and the other B group vitamins measured were similar to those reported elsewhere for healthy subjects in Britain (Chambers et al, 2000) and Europe (Graham et al, 1997) with plasma folate being the strongest predictor of homocysteine concentrations. Vitamin B₁₂, but not B₆, was weakly predictive of homocysteine concentrations. This is in keeping with other studies which have indicated a stronger relationship between B₆ and post-methionine load homocysteine concentrations than with fasting homocysteine concentrations (Boushey et al, 1995).

Subjects had dietary folate intakes which were similar to reported national average values, 242 vs 238 g/day (Department of Health, 2000a). Although less than half (47%) of the subjects ate cereals fortified with folic acid at baseline, this intervention group complied well with advice to increase folate intake from fortified sources and reported no difficulties. The target of 100 g extra folic acid daily was generally achieved with a mean increase in the fortified group of 98 (s.d. 32) g/day.

Subjects in the natural folate group reported more difficulty in attaining the target level of intake. Only 13% achieved the target 100 g/day extra natural food folates. Several of those unable to reach the target already had high intakes of fruit and vegetables at baseline. However, some subjects may have achieved greater folate intakes than indicated by the dietary folate estimate as nutrient composition data are not available on the wide range of exotic fruits now available. Also, changes to portion sizes are difficult to detect using the structured format of the food frequency questionnaire. Diet charts indicated greater compliance in some subjects than did food frequency questionnaire estimates. The difficulty experienced by subjects in this study, however, reflects the small response to government policies to increase fruit and vegetable intakes (Cox et al, 1996) and limited success of natural dietary interventions intended to achieve larger increases in dietary folate intake (>100 g) in free-living, 'unsupervised' subjects (Cuskelly et al, 1996).

We observed a significant increase in natural folate intake, albeit short of the target, which was associated with a significant increase in plasma folate. This finding supports the promotion of fruit and vegetables as valuable sources of natural folate. The similar change in plasma folate in both intervention groups was unexpected given the widely reported poorer bioavailability of natural source folates compared with folic acid. The bioavailability of folate has been more widely studied than that of many other micronutrients, but remains contentious. Sauberlich et al estimated that natural folates have only approximately 50% of the bioavailability of folic acid, although more recently, Brouwer et al estimated that the relative bioavailability of nautral folates compared to synthetic folic acid is much higher (98%; Brouwer et al, 1999b). These authors suggest that their higher estimate may be due to the source of natural folate in their study (citrus fruit and vegetables) compared with the wider range of foods used in the former study. The almost exclusive use of fruit and vegetables to increase folate intake in the current study may have produced an increase in the more bioavailable folates.

Both interventions increased plasma folate by approximately 3 nmol/l (95% CI approximately 1–5) compared with controls, a change which we consider to be real given the high degree of subject matching at baseline. Two other studies which have investigated the effect of similar levels of folic acid fortified foods on plasma folate and homocysteine have reported conflicting results. Malinow et al (1998) reported a 3.8 nmol/l increase following intervention with fortified cereal providing 127 g/day folic acid which produced a non-significant 0.5 mol/l reduction in homocysteine concentrations in patients with coronary artery disease. These results are very similar to the effects which we observed in healthy subjects in the current study. However, Jacques et al reported a much greater increase in plasma folate (12 nmol/l) and significant reduction in homocysteine (0.7 nmol/l) in a group of subjects assessed following early implementation of mandatory fortification regulations in the USA compared with a group of subjects assessed before mandatory fortification was implemented (Jacques et al, 1999). Fortification in the USA is estimated to increase folic acid intakes by approximately 100 g/day (Institute of Medicine, 1998).

Methodological differences between these studies complicates comparison. The cross-over study of Malinow et al used a 5 week intervention period which may have been too short to attain new steady state folate concentrations. Schorah et al (1998) reported significant changes in plasma folate after 4 weeks of folic acid supplementation and in homocysteine after 8 weeks. The current study had a much longer intervention period (4 months) and therefore should have been adequate for equilibration of cellular folate stores. The larger effect observed in the study of Jacques et al may indicate that the average increase in dietary folate intakes in the USA as a result of mandatory fortification is greater than the estimated 100 g. This has been suggested by other authors (Rader et al, 2000; Lewis et al, 1999).

It has been suggested that folate and homocysteine responses to folate interventions depend on baseline concentrations (Graham et al, 1997). Ward et al (1997) suggest 'a threshold of plasma homocysteine in terms of ability to respond to folic acid'. They observed that there was no response to folate interventions in those with the highest baseline plasma folate and lowest homocysteine concentrations. The results reported here are in keeping with a concentration-dependent effect.

A further important factor which is likely to have influenced folate and homocysteine responses in the current study was the genetic composition of the group. Subjects for this study were CC 'wild-type' homozygotes for the C677T MTHFR polymorphism. It is widely accepted that subjects with the CC genotype have the highest plasma folate and lowest homocysteine concentrations of the three MTHFR genotypes. TT homozygotes occupy the extreme opposite positions while CT heterozygotes have intermediate concentrations (Harmon et al, 1996; Jacques et al, 1996). We have recently shown that, in subjects drawn from the same population as the current study, MTHFR genotype modulates the homocysteine lowering response to folate enhancing interventions with TT homozygotes responding most (Ashfield-Watt et al, 2002). This study, which comprised equal numbers of subjects with each MTHFR C677T genotype (n=42 of each), supports previous observations made in cohorts where only a small number of TT homozygotes were studied (Malinow et al, 1997; Nelen et al, 1998). Fohr et al (2002) have recently reported that subjects with the MTHFR TT genotype have a greater response to synthetic folates than other genotypes, but did not include a dietary intervention in their study. The gene frequency of C677T variant MTHFR in South Wales is 0.32, giving genotype frequencies of 12% TT, 40% CT and 48% CC (in keeping with other Caucasian populations; Clark et al, 1998). This is in contrast to the current study where the frequency of the CC genotype was 100%. Therefore, a greater reduction in homocysteine can be expected in the general population because of the greater number of TT homozygotes.

The level of fortification of staple foodstuffs recommended in the UK by the Committee on Medical Aspects of food (COMA) for health reasons is 240 g/100 g flour product which is estimated to produce an increase of 201 g/day folic acid intakes generally (Department of Health, 2000b). While it is unlikely that this level of extra folate intake could be achieved by increasing natural dietary folate, it is encouraging that achievable increases from natural folate sources can significantly increase folate status. Such measures are therefore complementary to the current situation of voluntary fortification of cereal products and promote health by providing a wide range of other nutrients.

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