Abstract

Elevated total plasma homocysteine (tHcy) concentrations are considered a risk factor for neural tube defects (NTD) and cardiovascular disease. Supplementation with folic acid decreases the risk of women having children with NTD. In both sexes, it decreases tHcy levels. We investigated the efficacy of natural dietary folate in improving folate and homocysteine status. We performed a 4-wk dietary controlled, parallel design intervention trial with 66 healthy subjects (18–45 y) divided into 3 treatment groups: the dietary folate group, the folic acid group and the placebo group. Each day each group was fed a different diet. The dietary folate group received a diet high in vegetables and citrus fruit (total folate content ∼560 μg) plus a placebo tablet. The folic acid group received a diet naturally low in folate (∼210 μg) plus 500 μg folic acid and placebo tablet on alternate days, i.e., 250 μg folic acid/d. And the placebo group received the same low-folate diet as the folic acid group plus a placebo tablet. After 4 wk of intervention, folate status improved, and tHcy concentrations decreased in both the dietary folate and the folic acid groups. From the amount of additional folate (350 μg/d) and folic acid (250 μg/d) consumed, the relative bioavailability of dietary folate compared to folic acid was calculated to be 60–98%, depending on the endpoint used. In conclusion, increasing the consumption of vegetables and citrus fruit, both good sources of folate, will improve folate status and decrease tHcy concentrations. This may contribute to the prevention of cardiovascular disease and NTD in the general population

Increased intake of folate can have a high impact on public health. Folic acid supplementation around conception decreases the risk of women having offspring with neural tube defects (NTD)4 (Czeizel & Dudás 1992, Medical Research Council Vitamin Study Research Group 1991). In both men and women it improves the folate status and decreases elevated total plasma homocysteine (tHcy) concentration, which is considered to be a risk factor for cardiovascular disease (Boushey et al. 1995) and NTD (Mills et al. 1995, Steegers-Theunissen et al. 1994).

In several countries, women planning to become pregnant are advised to take 400–500 μg of folate per day (Expert Advisory Group 1992, US Public Health Service 1992). It is generally thought that this intake can only be reached by using folic acid, the synthetic form of folate. Daily doses of 200–300 μg of synthetic folic acid not only improve the folate status, but also decrease tHcy levels (Brouwer et al. 1999, Jacob et al. 1994, O'Keefe et al. 1995, Ward et al. 1997). The effectiveness of these low doses of folic acid suggests that a nutritional intervention with foods rich in folate could also be feasible and successful. We, therefore, tested the hypothesis that increased intake of dietary folate from vegetables and citrus fruits improves the folate status and decreases tHcy concentrations.

SUBJECTS AND METHODS

Subjects.

Healthy men and women aged 18–45 y were recruited. A total of 66 women and 28 men applied for enrollment in the study. Exclusion criteria were smoking; known gastrointestinal disorders; screening tHcy concentrations of >20 μmol/L; or use of vitamins, minerals, yeast or seaweed, malaria prophylactics, or anti-convulsants in the 4 mo prior to the experiment. Food products fortified with folic acid are not available in the Netherlands. Therefore, the habitual diet of the subjects contained no folic acid. None of the women were pregnant or planning to become pregnant within the first 6 mo after the study. Based on the selection criteria, 62 women and 25 men were eligible for participation. We included 52 of the women and 25 men in the trial. Ten of these subjects received treatment as part of a study on carotene bioavailability that will be published separately. Because only 77 subjects were required, all 25 men and 52 of the women entered the trial: 2 women decided not to participate for personal reasons and 8 were chosen at random for exclusion. Thus, the present study included 67 subjects. The study was approved by the Medical Ethical Committee of the Division of Human Nutrition and Epidemiology of Wageningen Agricultural University, and all participants gave their written informed consent.

Design.

The trial had a parallel design with three treatments. Group sizes were selected to be able to detect, with a power of 80%, an 11% decrease in tHcy concentrations after 4 wk of intervention with 500 μg of folic acid every-other-day (Brouwer et al. 1999). The treatments for each group were as follows: dietary folate group, a diet high in natural folate plus a placebo tablet (n = 23); folic acid group, a diet low in folate plus supplemental folic acid (n = 22); and placebo group, the same low-folate diet as the folic acid group plus a placebo tablet (n = 22).

All subjects were assigned to groups on the basis of initial tHcy concentration, energy intake, vegetarianism, and gender.

Diets.

Before the trial started, trained dietitians, using a validated food frequency questionnaire (Feunekes et al. 1993), estimated the habitual energy intake of the subjects. This method was validated for estimating fat and energy intake; however, it is not suitable for estimating folate intake. Therefore, we did not estimate habitual folate intake. The energy content of the diets was calculated using the Dutch Nutrient Data Base (Brants & Hulshof 1995, Stichting NEVO 1993).

Each day, all subjects received a basal diet with a similar nutrient composition (Table 1). The quantity of the basal diet was based on the habitual energy intake for each subject. Body weights, without shoes, jackets, or heavy clothing, were recorded twice weekly. Energy intake was adjusted when necessary to compensate for any weight change. If subjects complained about having either too much or not enough food, we weighed them more often. In exceptional cases, for example when the subjects were hungry or underwent more than usual physical activity, we provided energy buns, which had a similar macronutrient composition as the rest of the diet and a known folate content. The meals contained conventional foods and beverages. Six different menus were provided over the 4-wk intervention period.

TABLE 1

Daily intake of nutrients and energy during dietary intervention period1

Energy/NutrientIntervention
Dietary folate groupFolic acid group2Placebo group
Folate, μg/d3    
Calculated 594 ± 27 226 ± 9 226 ± 9 
Analyzed 560 ± 184 210 ± 49 210 ± 49 
Folic acid, μg/d  0 2502  0 
Protein, energy% 14.1 13.7 13.6 
Fat, energy% 31.7 30.7 30.5 
Carbohydrates, energy% 53.0 55.6 55.9 
Alcohol, energy%  1.3  1.5  0.5 
Dietary fiber, g/MJ  4.8  4.2  4.1 
Energy, MJ/d 9.89 ± 2.53 9.61 ± 2.57 9.85 ± 2.47 
Energy, kcal/d 2364 ± 605 2297 ± 614 2354 ± 590 
Energy/NutrientIntervention
Dietary folate groupFolic acid group2Placebo group
Folate, μg/d3    
Calculated 594 ± 27 226 ± 9 226 ± 9 
Analyzed 560 ± 184 210 ± 49 210 ± 49 
Folic acid, μg/d  0 2502  0 
Protein, energy% 14.1 13.7 13.6 
Fat, energy% 31.7 30.7 30.5 
Carbohydrates, energy% 53.0 55.6 55.9 
Alcohol, energy%  1.3  1.5  0.5 
Dietary fiber, g/MJ  4.8  4.2  4.1 
Energy, MJ/d 9.89 ± 2.53 9.61 ± 2.57 9.85 ± 2.47 
Energy, kcal/d 2364 ± 605 2297 ± 614 2354 ± 590 
1

Values are based on the analysis of six complete duplicate diets (one for each day of the menu cycle) plus its calculated contribution from the free-choice items (see Methods section).

2

The folic acid group received one 500 μg folic acid tablet and one placebo tablet on alternate days.

3

The folate content represents the daily amount for a subject receiving 11 MJ/d. Differences in folate content were similar for all energy levels.

TABLE 1

Daily intake of nutrients and energy during dietary intervention period1

Energy/NutrientIntervention
Dietary folate groupFolic acid group2Placebo group
Folate, μg/d3    
Calculated 594 ± 27 226 ± 9 226 ± 9 
Analyzed 560 ± 184 210 ± 49 210 ± 49 
Folic acid, μg/d  0 2502  0 
Protein, energy% 14.1 13.7 13.6 
Fat, energy% 31.7 30.7 30.5 
Carbohydrates, energy% 53.0 55.6 55.9 
Alcohol, energy%  1.3  1.5  0.5 
Dietary fiber, g/MJ  4.8  4.2  4.1 
Energy, MJ/d 9.89 ± 2.53 9.61 ± 2.57 9.85 ± 2.47 
Energy, kcal/d 2364 ± 605 2297 ± 614 2354 ± 590 
Energy/NutrientIntervention
Dietary folate groupFolic acid group2Placebo group
Folate, μg/d3    
Calculated 594 ± 27 226 ± 9 226 ± 9 
Analyzed 560 ± 184 210 ± 49 210 ± 49 
Folic acid, μg/d  0 2502  0 
Protein, energy% 14.1 13.7 13.6 
Fat, energy% 31.7 30.7 30.5 
Carbohydrates, energy% 53.0 55.6 55.9 
Alcohol, energy%  1.3  1.5  0.5 
Dietary fiber, g/MJ  4.8  4.2  4.1 
Energy, MJ/d 9.89 ± 2.53 9.61 ± 2.57 9.85 ± 2.47 
Energy, kcal/d 2364 ± 605 2297 ± 614 2354 ± 590 
1

Values are based on the analysis of six complete duplicate diets (one for each day of the menu cycle) plus its calculated contribution from the free-choice items (see Methods section).

2

The folic acid group received one 500 μg folic acid tablet and one placebo tablet on alternate days.

3

The folate content represents the daily amount for a subject receiving 11 MJ/d. Differences in folate content were similar for all energy levels.

In addition to the basal diet (containing ∼200 μg folate/d, by calculation), we supplied subjects in the placebo and folic acid groups with foods low in folate (containing ∼25 μg folate/d, by calculation), while the dietary folate group received foods rich in folate (containing ∼395 μg folate/d, by calculation) (Table 1). The main sources of dietary folate were vegetables (spinach, green peas, broccoli, Brussels sprouts, green beans, or a mixed vegetable dish, ∼320 μg folate/d) and citrus fruit (1 orange or 2 tangerines and orange juice, ∼75 μg folate/d). Soup or ragout with fewer vegetables, rice or pasta salad, and non-citrus fruit (apples/pears) instead of citrus fruit were provided to the folic acid and placebo groups. The energy content of the foods supplied in addition to the basal diet was similar for all subjects in all three intervention groups (1380 kJ/d).

All foodstuffs were weighed for each subject. On weekdays at noon, hot meals were prepared and served under supervision at the Division of Human Nutrition and Epidemiology. If subjects could not finish their meal, dessert was packed and the subject took the remaining food home. At lunch time, food for the rest of the day was provided and subjects took the food home. On Friday, food for the weekend was provided for the subjects to eat at home. Subjects checked whether their own package of food was complete before leaving the Division. If the participant later discovered that something was missing, the missing item(s) was delivered to their house. If subjects could not come to the Division to eat their hot meal, food was either collected or delivered to their house. In addition to the food supplied, subjects were allowed a limited number of free-choice items low in folate and fat chosen from a list. The free-choice items were mainly non-alcoholic soft-drinks, alcoholic drinks [subjects were allowed to have no more than one beer (200 mL) each day], candy, and sweetbread fillings. All participants kept a diary in which they recorded illness, medication used, consumption of free-choice items, and any deviations from their diet. At the end of the trial, subjects were asked to complete an anonymous questionnaire regarding problems and noncompliance during the study.

Duplicate portions of the low and high folate diet were collected on each of the 29 trial days for a fictitious participant with a daily energy intake of 11 MJ. The folate content was analyzed in a sub-sample (one from each menu; 12 samples total) by microbiological assay with Lactobacillus casei as described by Horne & Patterson (1988) and modified by Tamura et al. (1990). The assay had an intra-assay CV of <10%. Before extraction, 20 g sodium ascorbate/L was added to the sample. This was followed by suspending the sample in 10 volumes of a buffer containing 5 mmol 2-mercaptoethanol/L and 50 mmol potassium tetraborate/L (Seyoum and Selhub 1993). A chicken pancreas conjugase preparation was used to convert polyglutamates into monoglutamates. Macronutrients were analyzed in pooled samples. The energy and nutrient content of the free-choice items were estimated from data in food consumption tables (Stichting NEVO 1993).

Tablets.

In addition to the food, the folic acid group received a tablet containing 500 μg folic acid every-other-day and a placebo tablet every-other-day, while the other two groups received one placebo tablet daily. Subjects were unaware whether they received folic acid or placebo tablets. Because of the varying amounts of vegetables, participants were aware of their diet category. All subjects noted on a tablet calendar whether they took the tablet. Supplementation compliance was monitored by counting the remaining tablets and inspection of the tablet calendars.

Blood sampling and analysis.

Venous blood samples were collected after an overnight fast at the start (d 0 and 1), after 2 wk (d 15), and at the end of the intervention period (d 29 and 30). tHcy and plasma folate concentrations were assessed in all samples. Red blood cell folate concentrations were determined on d 1 and 30.

Blood samples were drawn into EDTA vacutainer tubes (Venoject II, Terumo, Madrid, Spain). For the determination of tHcy and plasma folate concentration, samples were immediately placed on ice and centrifuged within 60 min at 3000 x g for 10 min. Plasma was separated and stored at -35°C for folate determination and at -80°C for total homocysteine determination. For the determination of folate concentrations in red blood cells, whole blood was diluted fourfold with an ascorbic acid solution (10 g/L) and stored at -35°C. Before measurement, the hemolysates were further diluted with IMx Folate RBC Lysis Reagent (Abbott Laboratories, North Chicago, IL). To be able to express the folate concentration in red blood cells, hematocrits were also measured. Corrections were made for the concentration of folate in plasma. tHcy was measured by HPLC with fluorimetric detection (Araki & Sako 1987). The intra- and inter-assay CV were <8%. Folate concentrations in blood were determined with the Abbott IMx Folic Acid assay, which is based on ion-capture technology for the IMx automated immunoassay system (Abbott Laboratories). The intra-assay CV was <6%, and the inter-assay CV was <10%. All samples from each subject were analyzed concurrently.

Calculations of bioavailability of folic acid.

We calculated the bioavailability of dietary folate from vegetables and citrus fruits relative to the bioavailability of folic acid. This calculation assumes a linear response between 0 μg added folic acid and the amount administered and between 0 μg added dietary folate and the amount administered. For each endpoint (change in concentration of plasma folate, red blood cell folate, and tHcy), it was calculated as follows:

\[Relative\ bioavailability\ (\%)\ {=}\ \frac{\begin{array}{l}Change\ in\ endpoint\ in\ dietary\ folate\ group\ during\\intervention\ minus\ change\ in\ placebo\ group\end{array}}{\begin{array}{l}Change\ in\ endpoint\ in\ folic\ acid\ group\ during\\intervention\ minus\ change\ in\ placebo\ group\end{array}}{\times}\ \frac{Additional\ folic\ acid\ provided\ (250\ {\mu}g)}{Additional\ dietary\ folate\ provided\ ({\mu}g)}{\times}100\]

For each subject, average values for tHcy and plasma folate concentration were taken for d 0 and d 1 (wk 0) and for d 29 and d 30 (wk 4). Response to the various treatments was calculated for each subject as the change in each endpoint, between the start (wk 0) and the end (wk 4) of the intervention period.

Statistics.

One-way ANOVA on log-transformed data was used to analyze differences in baseline levels of tHcy, plasma folate, and red blood cell folate concentrations among the three groups. Changes in folate and tHcy concentrations were normally distributed as checked by visual inspection of the normal probability plots (univariate procedure; SAS Institute, Cary, NC). To analyze differences in endpoint response between the intervention groups and the placebo group, Student's t-tests were used with a significance level of P < 0.025 to maintain an overall significance level of P < 0.05. Values in the text are means ± sd.

RESULTS

Diets and adherence.

The nutrient and energy intakes of the subjects are shown in Table 1. The energy intake from the free-choice items was fixed for each level of daily energy intake and was 11.2% ± 1.3 of total energy.

Neither the questionnaires, nor the diaries, revealed any deviations from the protocol that might have affected the results. Two subjects missed three supervised hot meals, six subjects two supervised meals, and 16 subjects missed one supervised meal. In all these cases, the meals were eaten outside the Division. The remaining 42 subjects attended all supervised hot meals. One man withdrew from the study on the second day because he could not adhere to the diet.

Counting the remaining tablets and checking the tablet calendars revealed that seven subjects in the folic acid group failed to take one tablet during the intervention period. None of the subjects failed to take more than one folic acid tablet during the study.

Stratification was successful because tHcy, energy intake, vegetarianism, and gender were not different among the groups. The mean age was 23.0 ± 7.5 y, and the body mass index was 22.4 ± 2.0 kg/m2. Over the 29 d of the trial, subjects' body weights decreased by 0.6 ± 0.9 kg.

Folate status.

Baseline plasma folate and red blood cell folate concentrations were not significantly different among the three groups. After 4 wk of intervention, the plasma folate concentrations increased in both the dietary folate and the folic acid group (Table 2). The most distinct increase in plasma folate concentrations occurred during the first 2 wk. Red blood cell folate concentrations also increased in both the dietary folate group and the folic acid group (Table 2).

TABLE 2

Effect of dietary folate and folic acid on plasma folate, red blood cell folate, and total plasma homocysteine concentrations in humans fed natural-food folate, supplemental folic acid, or placebo1

Dietary folate group (additional folate: 350 μg/d) (n = 23)2Folic acid group (additional folic acid: 500 μg/2d) (n = 22)Placebo group (no additional folate or folic acid) (n = 22)
Plasma folate, nmol/L    
Week 0 13.8 ± 3.0 14.6 ± 4.7 13.2 ± 3.4 
Week 2 20.1 ± 4.0 19.0 ± 4.5 12.9 ± 3.6 
Week 4 20.4 ± 3.5 20.4 ± 4.1 12.7 ± 2.9 
Change from baseline 6.5 ± 3.0** 5.8 ± 3.1** −0.6 ± 1.7 
Red blood cell folate, nmol/L    
Week 0 338 ± 81 339 ± 78 347 ± 79 
Week 4 400.1 ± 114 382 ± 70 345 ± 69 
Change from baseline 59.3 ± 55.5** 42.9 ± 50.5* −1.2 ± 38.6 
Plasma homocysteine, μmol/L    
Week 0 11.0 ± 4.6 10.8 ± 3.6 10.2 ± 2.5 
Week 2 10.1 ± 4.1 9.3 ± 2.5 9.8 ± 2.3 
Week 4 9.5 ± 3.1 9.0 ± 2.6 10.7 ± 2.8 
Change from baseline −1.5 ± 1.7** −1.8 ± 1.8** 0.6 ± 1.5 
Dietary folate group (additional folate: 350 μg/d) (n = 23)2Folic acid group (additional folic acid: 500 μg/2d) (n = 22)Placebo group (no additional folate or folic acid) (n = 22)
Plasma folate, nmol/L    
Week 0 13.8 ± 3.0 14.6 ± 4.7 13.2 ± 3.4 
Week 2 20.1 ± 4.0 19.0 ± 4.5 12.9 ± 3.6 
Week 4 20.4 ± 3.5 20.4 ± 4.1 12.7 ± 2.9 
Change from baseline 6.5 ± 3.0** 5.8 ± 3.1** −0.6 ± 1.7 
Red blood cell folate, nmol/L    
Week 0 338 ± 81 339 ± 78 347 ± 79 
Week 4 400.1 ± 114 382 ± 70 345 ± 69 
Change from baseline 59.3 ± 55.5** 42.9 ± 50.5* −1.2 ± 38.6 
Plasma homocysteine, μmol/L    
Week 0 11.0 ± 4.6 10.8 ± 3.6 10.2 ± 2.5 
Week 2 10.1 ± 4.1 9.3 ± 2.5 9.8 ± 2.3 
Week 4 9.5 ± 3.1 9.0 ± 2.6 10.7 ± 2.8 
Change from baseline −1.5 ± 1.7** −1.8 ± 1.8** 0.6 ± 1.5 
1

Values are means ± sd. The absolute values were not normally distributed, but the changes from baseline were. Significant difference in change from baseline in intervention group versus placebo group, Student's t-test *P <0.01, **P < 0.001.

2

One of the subjects withdrew from the study after 2 d of intervention.

TABLE 2

Effect of dietary folate and folic acid on plasma folate, red blood cell folate, and total plasma homocysteine concentrations in humans fed natural-food folate, supplemental folic acid, or placebo1

Dietary folate group (additional folate: 350 μg/d) (n = 23)2Folic acid group (additional folic acid: 500 μg/2d) (n = 22)Placebo group (no additional folate or folic acid) (n = 22)
Plasma folate, nmol/L    
Week 0 13.8 ± 3.0 14.6 ± 4.7 13.2 ± 3.4 
Week 2 20.1 ± 4.0 19.0 ± 4.5 12.9 ± 3.6 
Week 4 20.4 ± 3.5 20.4 ± 4.1 12.7 ± 2.9 
Change from baseline 6.5 ± 3.0** 5.8 ± 3.1** −0.6 ± 1.7 
Red blood cell folate, nmol/L    
Week 0 338 ± 81 339 ± 78 347 ± 79 
Week 4 400.1 ± 114 382 ± 70 345 ± 69 
Change from baseline 59.3 ± 55.5** 42.9 ± 50.5* −1.2 ± 38.6 
Plasma homocysteine, μmol/L    
Week 0 11.0 ± 4.6 10.8 ± 3.6 10.2 ± 2.5 
Week 2 10.1 ± 4.1 9.3 ± 2.5 9.8 ± 2.3 
Week 4 9.5 ± 3.1 9.0 ± 2.6 10.7 ± 2.8 
Change from baseline −1.5 ± 1.7** −1.8 ± 1.8** 0.6 ± 1.5 
Dietary folate group (additional folate: 350 μg/d) (n = 23)2Folic acid group (additional folic acid: 500 μg/2d) (n = 22)Placebo group (no additional folate or folic acid) (n = 22)
Plasma folate, nmol/L    
Week 0 13.8 ± 3.0 14.6 ± 4.7 13.2 ± 3.4 
Week 2 20.1 ± 4.0 19.0 ± 4.5 12.9 ± 3.6 
Week 4 20.4 ± 3.5 20.4 ± 4.1 12.7 ± 2.9 
Change from baseline 6.5 ± 3.0** 5.8 ± 3.1** −0.6 ± 1.7 
Red blood cell folate, nmol/L    
Week 0 338 ± 81 339 ± 78 347 ± 79 
Week 4 400.1 ± 114 382 ± 70 345 ± 69 
Change from baseline 59.3 ± 55.5** 42.9 ± 50.5* −1.2 ± 38.6 
Plasma homocysteine, μmol/L    
Week 0 11.0 ± 4.6 10.8 ± 3.6 10.2 ± 2.5 
Week 2 10.1 ± 4.1 9.3 ± 2.5 9.8 ± 2.3 
Week 4 9.5 ± 3.1 9.0 ± 2.6 10.7 ± 2.8 
Change from baseline −1.5 ± 1.7** −1.8 ± 1.8** 0.6 ± 1.5 
1

Values are means ± sd. The absolute values were not normally distributed, but the changes from baseline were. Significant difference in change from baseline in intervention group versus placebo group, Student's t-test *P <0.01, **P < 0.001.

2

One of the subjects withdrew from the study after 2 d of intervention.

Homocysteine.

At baseline, the median (25%, 75%) of the tHcy concentration was 10.1 (8.0, 11.6) μmol/L. Baseline tHcy concentrations were not significantly different among the groups. Four weeks of intervention decreased the tHcy concentrations in both the dietary folate and the folic acid group (Table 2). After correction for changes in the placebo group, the mean decrease was 2.0 μmol/L (95% CI, 1.0–3.0) in the dietary folate group and 2.4 μmol/L (1.4–3.4) in the folic acid group. Concentrations of tHcy decreased gradually during the entire 4-wk intervention period (Table 2).

Bioavailability.

The bioavailability of folate from vegetables and citrus fruits relative to folic acid was, for the different endpoints, 60% based on tHcy concentration, 78% based on plasma folate concentration, and 98% based on red blood cell folate concentration.

DISCUSSION

This controlled dietary intervention study demonstrated that a diet rich in vegetables and citrus fruits favorably affects plasma folate, red blood cell folate, and tHcy concentrations in young, healthy volunteers. Many studies have shown that the synthetic monoglutamate folic acid decreases tHcy concentrations and improves folate status (Brouwer et al. 1999, Cuskelly et al. 1996, Jacob et al. 1994, O'Keefe et al. 1995, Ward et al. 1997). However, studies investigating the effects of natural food folate on tHcy and folate status are scarce. A cross-sectional study showed that intake of dietary folate was inversely correlated with tHcy concentrations and positively correlated with the plasma folate concentration (Tucker et al. 1996). Cuskelly et al. (1996) found a nonsignificant increase of 11% (95% CI: -6, 29) in red blood cell folate concentration in young, healthy women after 3 mo of intervention with natural-food folate. Our study shows a significant 17% (CI: 8–26) increase in red blood cell folate concentration after 4 wk of intervention (Table 2). This discrepancy can be explained by differences in the study hypotheses and designs. Cuskelly et al. (1996) investigated whether fortified foods, supplements, and consumption of natural-food folate would have equal effects on folate status in a free living situation in which the subjects selected their own foods. They included no more than 10 women in each group, and the average intake of additional dietary folate was calculated to be 200 μg/d (Cuskelly et al. 1996). In contrast, our study was a controlled dietary intervention study with 22 subjects in each group. We supplied the subjects in the dietary folate group with vegetables and fruits containing 350 μg of additional dietary folate per day, a relatively high amount. Nevertheless, because their study lasted three times longer than ours, a higher response would have been expected in the study of Cuskelly et al. (1996).

The 350 μg of additional dietary folate consumed daily in the dietary folate group was 1.4 times the 250 μg of folic acid provided to subjects in the folic acid group. Depending on the endpoint chosen, the bioavailability of dietary folate from vegetables and citrus fruit relative to folic acid was calculated to be between 60 and 98%. This estimate is higher than the ≤50% suggested by the study of Sauberlich et al. (1987) who determined the bioavailability of folate from a mixed diet by using responses in plasma folate. Based on the response in red blood cell folate concentration, our estimate of relative folate bioavailability of 98% is high compared to the 39% estimated from the study of Cuskelly et al. (1996). The difference in bioavailability between the studies might be explained by the fact that we used only vegetables and citrus fruits as a source of folate, whereas the other two studies fed a wider range of food products (Cuskelly et al. 1996, Sauberlich et al. 1987). However, the difference might also be attributable to the higher degree of compliance in fruit and vegetables consumption in our study.

In our study, the relative bioavailability was considerably lower when calculated from the change in tHcy (60%) than from the change in folate concentrations in plasma (78%) and red blood cells (98%). These differences cannot be explained by differences in intestinal absorption between synthetic folic acid and folate from foods, but would appear to be related to the chemical form of the vitamin ingested. Tablets contain the fully oxidized form of pteroylglutamic acid, whereas in food the vitamin exists with two or four additional hydrogen atoms and is conjugated to glutamic acid. Food folate appears to be less effective in reducing tHcy concentrations than folic acid. On the other hand, it tends to accumulate more in red blood cells than in plasma.

For practical reasons we provided 500 μg of folic acid on alternate days instead of 250 μg each day. It is likely that the estimate of bioavailability of the dietary folate based on changes in tHcy (60%) would have been slightly lower if we had provided 250 μg of folic acid each day. We assumed linearity in response between 0 added folic acid and the 500 μg/2d. However, Kelly et al. (1997) showed that an intake of folic acid in addition to that in the diet of >266 μg/d results in significant amounts of unmetabolized folic acid in the blood. This suggests that not all folic acid supplied is available for the remethylation of homocysteine to methionine. The homocysteine-lowering response is thus not linear over the whole range, which implies an overestimation of the relative bioavailability for dietary folate.

Folate is the nutrient most likely to be responsible for the reduction in tHcy concentrations because it was present in large quantities in the diet of the dietary folate group and because folate concentrations in plasma and red blood cells increased significantly during the intervention period. In addition to folate, vitamin B-12 is also involved as a cofactor in the remethylation process. However, vitamin B-12 is only present in animal products and, therefore, could not have caused the observed reduction in tHcy concentrations in our study. Apart from the remethylation to methionine, homocysteine can also be converted to cysteine by transsulfuration by the vitamin B-6-dependent enzyme, cystathionine synthase. Even though vitamin B-6 is present in vegetables and fruit, and even though an intake of vitamin B-6 above the recommended daily allowances might protect women against coronary heart disease (Rimm et al. 1998), it is not likely to have caused the observed effect on tHcy in fasting subjects. Vitamin B-6 administration in tablet form was shown to have a marked effect on tHcy concentrations after methionine loading, whereas the effect of vitamin B-6 administration on fasting tHcy concentrations is regarded as negligible (Ubbink et al. 1994).

This study was designed to determine the efficacy of dietary folate from vegetables and citrus fruits to improve the folate status and to decrease tHcy concentrations. Therefore, we chose the strategy of supplying the subjects with a high amount of folate in a controlled setting rather than to examine the effects that can be observed under field conditions. As a consequence, the intake of 350 g of vegetables and one piece of citrus fruit and 200 mL of citrus fruit juice given in our study in addition to the basal diet (total folate content in the dietary folate group: 560 μg/d) was higher than what can be expected to be eaten by the general population.

In summary, we have shown that the intake of folate-dense vegetables and citrus fruits significantly enhances the folate status and decreases tHcy concentrations in healthy volunteers.

The authors thank the volunteers for their participation and E. Siebelink, C. Schuurman, J. Dijkstra, D. Boonstra, M. Jimmink, and N. de Bock for their assistance during the intervention study. We are grateful to J. Selhub, M. Nadeau, and H. Elzerman (Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA) for measuring folate in the duplicate portions. We thank R. Passas, Abbott Diagnostics, Maidenhaid, UK, for his support concerning the IMx diagnostic testing kits. We also thank E. Haddeman, Unilever Research Vlaardingen, for his support. Finally, we thank the laboratory staff of the Division of Human Nutrition and Epidemiology of the Wageningen Agricultural University, the Department of Chemical Endocrinology of the University Hospital Nijmegen St. Radboud, and the Laboratory of Metabolic Diseases of the Wilhelmina Children's Hospital in Utrecht for their support and expert technical assistance.

LITERATURE CITED

Araki
,
A.
&
Sako
,
Y.
(
1987
)
Determination of free and total homocysteine in human plasma by high performance liquid chromatography with fluorescence detection
.
J. Chromatogr.
422
:
43
52
.

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 cardiovascular disease
.
JAMA
274
:
1049
1057
.

Brants
,
H.A.M
&
Hulshof
,
K.F.A.M.
(
1995
)
A quantitative assessment of plasma homocysteine as a risk factor for cardiovascular disease
.
De ontwikkeling van een voedingsmiddelentabel met foliumzuurgehalten
TNO Nutrition
Zeist, the Netherlands
.

Brouwer
,
I. A.
,
van Dusseldorp
,
M.
,
Thomas
,
C.M.G.
,
Duran
,
M.
,
Hautvast
,
J.G.A.J.
,
Eskes
,
T.K.A.B.
&
Steegers-Theunissen
,
R.P.M.
(
1999
)
Low-dose folic acid supplementation decreases plasma homocysteine: A randomized trial
.
Am. J. Clin. Nutr.
69
:
99
104
.

Cuskelly
,
G. J.
,
McNulty
,
H.
&
Scott
,
J. M.
(
1996
)
Effect of increasing dietary folate on red-cell folate: Implications for prevention of neural tube defects
.
Lancet
347
:
657
659
.

Czeizel
,
A. E.
&
Dudás
,
I.
(
1992
)
Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation
.
N. Engl. J. Med.
327
:
1832
1835
.

Expert Advisory Group
(
1992
)
Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation
.
Folic acid and the prevention of neural tube defects
Department of Health
London, UK
.

Feunekes
,
G.I.J
,
van Staveren
,
W. A.
,
de Vries
,
J.H.M
,
Burema
,
J.
&
Hautvast
,
J. G.A.J.
(
1993
)
Relative and biomarker-based validity of a food-frequency questionnaire estimating intake of fats and cholesterol
.
Am. J. Clin. Nutr.
58
:
489
496
.

Horne
,
D. W.
&
Patterson
,
D.
(
1988
)
Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates
.
Clin. Chem.
34
:
2357
2359
.

Jacob
,
R. A.
,
Wu
,
M.-M.
,
Henning
,
S. M.
&
Swenseid
,
M. E.
(
1994
)
Homocysteine increases as folate decreases in plasma of healthy men during short-term dietary folate and methyl group restriction
.
J. Nutr.
124
:
1072
1080
.

Kelly
,
P.
,
McPartlin
,
J.
,
Goggins
,
M.
,
Weir
,
D. W.
&
Scott
,
H. M.
(
1997
)
Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements
.
Am. J. Clin. Nutr.
65
:
1790
1795
.

Medical Research Council Vitamin Study Research Group
(
1991
)
Prevention of neural tube defects: Results of the Medical Research Council Vitamin Study
.
Lancet
338
:
131
137
.

Mills
,
J. L.
,
McPartlin
,
J. M.
,
Kirke
,
P. N.
,
Lee
,
Y. L.
,
Conley
,
M. R.
,
Weir
,
D. G.
&
Scott
,
J. M.
(
1995
)
Homocysteine metabolism in pregnancies complicated by neural-tube defects
.
Lancet
345
:
149
151
.

O'Keefe
,
C. A.
,
Bailey
,
L. B.
,
Thomas
,
E. A.
,
Hofler
,
S. A.
,
Davis
,
B. A.
,
Cerda
,
J. J.
&
Gregory
,
J. F.
, III
(
1995
)
Controlled dietary folate affects folate status in nonpregnant women
.
J. Nutr.
125
:
2717
2725
.

Rimm
,
E. B.
,
Willett
,
W. C.
,
Hu
,
F. B.
,
Sampson
,
L.
,
Colditz
,
G. A.
,
Manson
,
J. E.
,
Hennekes
,
C.
&
Stampfer
,
M. J.
(
1998
)
Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women
.
JAMA
279
:
359
364
.

Sauberlich
,
H. E.
,
Kretsch
,
M. J.
,
Skala
,
J. H.
,
Johnson
,
H. L.
&
Taylor
,
P. C.
(
1987
)
Folate requirement and metabolism in nonpregnant women
.
Am. J. Clin. Nutr.
46
:
1016
1028
.

Seyoum
,
E.
&
Selhub
,
J.
(
1993
)
Combined affinity and ion pair column chromatographies for the analysis of food folate
.
J Nutr. Biochem.
4
:
488
494
.

Steegers-Theunissen
,
R.P.M
,
Boers
,
G.H.J
,
Trijbels
,
J.M.F
,
Finkelstein
,
J. D.
,
Blom
,
H. J.
,
Thomas
,
C.M.G.
,
Borm
,
G. F.
,
Wouters
,
M.G.A.J.
&
Eskes
,
T.K.A.B.
(
1994
)
Maternal hyperhomocysteinemia: A risk factor for neural-tube defects?
.
Metabolism
43
:
1475
1480
.

Stichting NEVO
(
1993
)
Dutch Nutrient Data Base 1993.
Voorlichtingsbureau voor de Voeding
,
Den Haag
.

Tamura
,
T.
,
Freeberg
,
L. E.
&
Cornwell
,
P. E.
(
1990
)
Inhibition of EDTA of growth of Lactobacillus casei in the folate microbiological assay and its reversal by added manganese or iron
.
Clin. Chem.
36
:
1993
.

Tucker
,
K. L.
,
Selhub
,
J.
,
Wilson
,
P.W.F
&
Rosenberg
,
I. H.
(
1996
)
Dietary intake pattern relates to plasma folate and homocysteine concentrations in the Framingham Heart Study
.
J. Nutr.
126
:
3025
3031
.

Ubbink
,
J. B.
,
Vermaak
,
W.J.H
,
van der Merwe
,
A.
,
Becker
,
P. J.
,
Delport
,
R.
&
Potgieter
,
H. C.
(
1994
)
Vitamin requirements for the treatment of hyperhomocysteinemia in humans
.
J. Nutr.
124
:
1927
1933
.

US Public Health Service
(
1992
)
Recommendation for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects
.
Morbid. Mortal. Weekly Rep.
41
:
1
7
.

Ward
,
M.
,
McNulty
,
H.
,
McPartlin
,
J.
,
Strain
,
J. J.
,
Weir
,
D. G.
&
Scott
,
J. M.
(
1997
)
Plasma homocysteine, a risk factor for cardiovascular disease, is lowered by physiological doses of folic acid
.
Q. J. Med.
90
:
519
524
.

Abbreviations

     
  • NTD

    neural tube defect

  •  
  • tHcy

    total plasma homocysteine

Footnotes

2

This work was supported by the Zorg Onderzoek Nederland/Dutch Prevention Fund (28–2559), The Hague, and Unilever Research Vlaardingen. Tablets were supplied by Pharmachemie BV, Haarlem, the Netherlands. Abbott Diagnostics, Maidenhead, UK provided the IMx diagnostic kits.

Author notes

1

Presented in part at Bioavailability meeting 1997, Wageningen, the Netherlands and at the Homocysteine Metabolism Conference, April 1998, Nijmegen, the Netherlands [Brouwer, I. A., van Dusseldorp, M., West, C. E., Meyboom, S., Thomas, C.M.G., Duran, M., van het Hof, K. H., Eskes, T.K.A.B., Hautvast, J.G.A.J., Steegers-Theunissen, R.P.M. (1998) High daily intake of dietary folate decreases homocysteine and improves folate status: A dietary controlled trial in young healthy volunteers. Netherlands J. Med. 52: S41 (abstract)].