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Human Nutrition and Food Science Department,
* Kinesiology and Health Promotion Department,
Biological Sciences Department, and
** Animal and Veterinary Science Department, California Polytechnic University, Pomona, CA 91768
2To whom correspondence should be addressed. E-mail: macaudill{at}csupomona.edu.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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60% relative to added folic acid. The objective of this long-term controlled feeding study was to assess the dietary folate equivalency of folic acid. After a 2-wk period of folate restriction, women (n = 42, 18–45 y old) consumed either 400 or 800 µg DFE/d derived from various combinations of food folate and folic acid for 12 wk. Folic acid was converted to DFE using the 1.7 multiplier from the DFE calculation and was consumed with a meal throughout the treatment period. Folate status response to the various treatments was assessed during wk 12–14. Serum folate, RBC folate, and plasma total homocysteine did not differ among the 400 µg DFE/d groups or among the 800 µg DFE/d groups. In contrast, consumption of 800 µg DFE/d led to higher (P 
0.05) serum and RBC folate than consumption of 400 µg DFE/d. These data support the validity of the 1.7 multiplier in the DFE equation and suggest that food folate bioavailability is 60% that of added folic acid when consumed as part of a mixed diet.
KEY WORDS: • folate • bioavailability • folic acid • dietary folate equivalent • DFE
In the United States and elsewhere, folic acid is added to enriched cereal grain products in an effort to increase folate consumption by women of childbearing age and reduce the incidence of neural tube defects (1). Enriched cereal grain products are delivering an estimated 200 µg/d folic acid (2,3) to the U.S. population, twice the intended amount (4). Folic acid is the most oxidized form of folate and exists as a monoglutamate. Most naturally occurring folates, referred to as food folate in this paper, exist in the reduced form as a mixture of mono- and polyglutamates (5).
The presence of folic acid in the U.S. food supply and the higher bioavailability of folic acid compared with food folate led to the expression of the 1998 folate recommended dietary allowance (RDA),3 400 µg/d, as dietary folate equivalents [DFE; (4)]. Dietary folate equivalents convert all forms of dietary folate, including folic acid in fortified products, to an amount that is equivalent to food folate and are defined as: DFE = µg natural food folate + 1.7 x µg synthetic folic acid (4,6). The 1.7 multiplier for converting folic acid to DFE was based on the assumptions that added folic acid (consumed with a meal as a supplement or fortificant) is 85% available (7) and food folate is 50% available (8); thus, the ratio, 85/50, yields the multiplier of 1.7 in the DFE calculation (6,9). The 85/50 ratio also infers that the bioavailability of food folate when consumed as part of a mixed diet is 60% (50/85 x 100) relative to added folic acid.
Although it is recognized that the bioavailability of food folate is less than that of folic acid when consumed as part of a mixed diet (4,9–11), only limited data exist from which to determine equivalency. Much of the uncertainty in the 85/50 ratio resides in the imprecision and variability of the denominator (9). The 50% estimate for food folate was based on a single study that was not designed to assess food folate bioavailability quantitatively (8). Brouwer et al. (12) reported that the bioavailability of food folate (relative to folic acid) as part of a mixed diet ranged from 60 to 98% depending upon the end-point used. Thus, the extent to which the bioavailabilities of added folic acid and food folate differ (13) and the validity of the 1.7 multiplier in the DFE calculation remain pressing issues (13,14).
The purpose of the present study was to examine the dietary folate equivalency of added folic acid. A long-term controlled folate feeding protocol was used in young women and provided either 400 or 800 µg DFE/d derived from various combinations of food folate and folic acid. Folic acid was converted to DFE utilizing the 1.7 multiplier from the DFE calculation and was consumed with a meal throughout the treatment period. Folate status response to the various treatments was assessed via several folate status indices including serum folate, RBC folate, urinary folate excretion, and plasma total homocysteine (tHcy).
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Healthy women, 18–45 y old, were recruited between January 2002 and April 2003 from the staff and student population at Cal Poly Pomona University as well as the surrounding community. The subjects were eligible for inclusion if the following criteria were met: no history of vascular, gastrointestinal, renal, or hepatic disease; normal blood glucose and lipid concentrations; normal blood chemistry; BMI between 18 and 32 kg/m2; methylenetetrahydrofolate reductase (MTHFR) 677 CC genotype; nonsmoker; not taking drugs known to interfere with folate metabolism; not a current user (within past 3 mo.) of supplemental vitamins/and or minerals; not pregnant or planning a pregnancy; and not lactating. The study was approved by the Institutional Review Board of California State Polytechnic University, Pomona and informed consent was obtained from each participant.
Experimental design
This was a 14-wk controlled folate feeding study. Subjects with the MTHFR 677 CC genotype (n = 42) consumed a low folate diet (135 µg/d food folate) for 2 wk followed by randomization to 1 of 6 treatment groups: 400 µg DFE/d total folate (T) derived from 1) 135 µg/d food folate (F) + 156 µg/d folic acid (n = 7; T400F135), 2) 230 µg/d food folate + 100 µg/d folic acid (n = 6;T400F230), and 3) 400 µg/d food folate (n = 7; T400F400); or 800 µg DFE/d derived from 4) 135 µg/d food folate + 392 µg/d folic acid (n = 6; T800F135), 5) 630 µg/d food folate + 100 µg/d folic acid (n = 8; T800F630), and 6) 800 µg/d food folate (n = 8; T800F800; Table 1). The folate restriction phase served to acclimate the participants to the experimental regimen and to assess the specificity and sensitivity of the response variables to changes in dietary folate intake. The 100 µg/d folic acid groups served to simulate the intended situation in the United States in which fortification of enriched cereal grain products would deliver an additional 100 µg/d folic acid. Two physiologically relevant folate intake levels were used, 400 and 800 µg DFE/d, in the event that the dietary folate equivalency of folic acid was dependent upon the amount of folate consumed. The 1.7 multiplier from the DFE equation was used to convert folic acid to DFE. Efforts were made to match the treatment groups on self-reported race/ethnicity because this factor was shown to influence folate requirements/status (15).
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The 5-d folate-restricted diet utilized during the first 2 wk was a slightly modified version of the menus previously described (16) and provided 133 ± 8 µg/d (mean ± SEM) food folate. The folate content of the 5-d treatment diets was 133 ± 8, 235 ± 22, 426 ± 20, 636 ± 23, or 835 ± 33 µg food folate and provided 2000–2300 kcal (8368–9623 kJ) with 54–66% from carbohydrate, 10–14% from protein, and 21–30% from fat (ESHA Food Processor Nutrient Data Base, version 7.81; ESHA Research). For the 133 µg/d treatment diet, certain foods were boiled 3 times to reduce folate content, and unfortified flour was used to make flour-containing food items (16).
Foods utilized to increase folate content of the menus included spinach, broccoli, asparagus, kidney and pinto beans, almonds, hazelnuts, sunflower seeds, wheat nuts, strawberries, boysenberries, whole wheat flour, and orange juice. All foodstuffs were weighed to the nearest gram. Subjects consumed morning and evening meals in the metabolic kitchen under the supervision of the investigators. Lunch, snacks, and an additional 14 meals of the subjects choosing were consumed off-site.
The nutrient content of each dietary treatment was estimated using ESHA Food Processor Nutrient Data Base (version 7.81; ESHA Research) and is shown in Table 2. Dietary intakes of choline and betaine were estimated using recently published data (17,18). Based on the nutrient content of the 135 µg food folate treatment diet, all subjects were given supplements to meet the dietary reference intakes [DRI; (4,19,20)] or 1989 RDA (21) for essential nutrients (except folate, biotin, pantothenic acid, vitamin D, chloride, and several trace minerals) not met by the diet. The dietary intakes of biotin, pantothenic acid, vitamin D, chloride, and several trace minerals were not analyzed due to incompleteness of the database. Iodized salt was utilized in the preparation of several food items to ensure adequate consumption of iodine and chloride. Year-round sun exposure minimized risks of vitamin D deficiency. The supplements were consumed at the morning meals throughout folate restriction and treatment under the supervision of the investigators. The diet and supplements provided a minimum of 85% of the recommended intakes for the nutrients analyzed not including folate (Table 2). In making these estimations, complete loss of water-soluble vitamins in food items boiled 3 times was assumed as well as a lack of thiamin, riboflavin, niacin, and iron in food items made with unfortified flour.
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Sample collection and processing
Baseline and weekly venous blood samples were collected from fasting subjects (9 h) in serum separator gel and clot-activator tubes (SST, Vacutainer; Becton Dickinson) and EDTA tubes (Vacutainer). Serum, whole blood, plasma, and peripheral leukocytes for serum folate, whole-blood folate, plasma tHcy, and MTHFR C677T genotype determinations, respectively, were processed and stored as previously described (16). The whole blood in EDTA tubes was also used for hematocrit determination. Baseline and weekly 24-h urine collections were also obtained, processed, and stored (16).
Analytical methods
Folate content of diet. The folate content of the diet was determined before starting the study and twice during the study. Each meal including beverage, was prepared, blenderized with 20 mL of 50 mmol/L Hepes:50 mmol/L Ches with 2% ascorbate and 0.2 mol/L of 2-mercaptoethanol buffer (pH 7.8), then dispensed into 50-mL conical tubes and stored at –20°C. Duplicates of the blenderized samples were thawed, homogenized, and subjected to trienzyme treatment (22) and double extraction (23). Total folate content was measured in triplicate microbiologically (24).
MTHFR C677T genotype. DNA for genotyping was extracted from leukocytes using a commercially available kit (DNeasy Tissue Kit; Qiagen) and determination of the MTHFR genotype was via PCR and Hinf1 restriction enzyme digestion as described by Frosst et al. (25) with minor modifications (26).
Plasma tHcy. An HPLC-method with fluorometric detection (27,28) was used to measure plasma tHcy concentrations in duplicate for wk 0, 2, and 12–14. The intra- and interassay CV for the internal control were 7 and 9%, respectively.
Blood and urinary folate. Microtiter plate adaptation with Lactobacillus casei as described by Tamura (24) was used to measure serum, erythrocyte, and urinary folate in triplicate for wk 0, 2, and 12–14. The intra- and interassay CV for the internal control were 10 and 12%, respectively.
Statistical analysis
All data summarization and analyses were performed using SPSS10.0 for Windows. Data are presented as means ± SEM. Differences were considered significant at P 0.05. To test for baseline differences (wk 0) in BMI, age, serum folate, RBC folate, urinary folate, and plasma tHcy among the various treatment groups, one-way ANOVA was performed and post hoc mean separations were accomplished using Tukey’s HSD procedure. The effects of folate restriction on serum folate, RBC folate, urinary folate, and plasma tHcy during the initial 2 wk of the experimental period were assessed using a paired t test.
Comparisons among the treatment groups consuming various combinations and levels of food folate and folic acid were made using the repeated-measures ANOVA facility of the GLM procedure with 1 within factor (wk 12–14) and 1 between factor (dietary treatment). Because substantial variation among individuals at baseline was observed, wk 0 was used as a covariate. Significant dietary treatment effects were examined by using simple contrasts in which the T400F400 and T800F800 treatments were used as reference groups.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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8% (range: 6–11%) of baseline weight. A total of 11 women reported using oral contraceptives during the study period (1 T400F135, 2 T400F230, 2 T400F400, 1 T800F135, 2 T800F630, 3 T800F800). The 6 dietary treatment groups were similar in ethnicity/race with 1–3 Mexican Americans, 1–2 African Americans, 0–2 Caucasians, and 1–3 Asians in each group. Serum folate, RBC folate, urinary folate, and plasma tHcy concentrations did not differ among the 6 different dietary treatment groups at baseline although serum folate tended to differ (P = 0.055; Table 3).
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0.05) from 31.3 ± 1.9 to 23.3 ± 1.5 nmol/L. Throughout the last 3 wk of the treatment phase (wk 12–14), the 400 or 800 µg DFE/d groups did not differ (Fig. 1A). Consumption of 800 µg DFE/d derived from any folate combination led to higher (P < 0.05) serum folate concentrations than 400 µg DFE/d derived from any folate combination (Fig 1A). No week effect or diet x week interaction was detected.
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0.05) from 1688 ± 61 to 1577 ± 54 nmol/L. Throughout the last 3 wk of the treatment phase (wk 12–14), the 400 or 800 µg DFE/d groups did not differ (Fig. 1B). Consumption of 800 µg DFE/d derived from any folate combination led to higher (P 0.05) RBC folate concentrations than 400 µg DFE/d derived from any folate combination (Fig. 1B). No week effect or diet x week interaction was detected.
Urinary folate excretion.
Folate restriction resulted in a 70% decline (P 0.05) from 76.4 ± 19.7 to 22.7 ± 1.7 nmol/d. Throughout the last 3 wk of the treatment phase (wk 12–14), the 400 µg DFE/d groups did not differ (Fig. 1C). Among the 800 µg DFE/d groups, the T800F135 group excreted more (P 0.05) urinary folate than the T800F630 and T800F800 groups (Fig. 1C). Consumption of 800 µg DFE/d derived predominately from folic acid (T800F135) led to higher (P 0.05) urinary folate excretion than 400 µg DFE/d derived from any folate combination (Fig. 1C). However, urinary folate excretion did not differ among groups consuming 800 µg DFE/d derived mostly from food folate (T800F630 and T800F800) and 400 µg DFE/d derived from any folate combination. No week effect or diet x week interaction was detected.
Plasma tHcy.
Folate restriction resulted in a 9% increase (P 0.05) from 5.4 ± 0.15 to 5.9 ± 0.19 µmol/L. Throughout the last 3 wk of the treatment phase (wk 12–14), the 400 or 800 µg DFE/d groups did not differ (Fig. 1D). Consumption of 800 µg DFE/d derived from any folate combination resulted in plasma tHcy concentrations similar to those after consumption of 400 µg DFE/d derived from any folate combination (Fig. 1D). No week effect or diet x week interaction was detected.
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.05) after folate restriction, and consumption of 800 µg DFE/d led to higher (P 0.05) serum and RBC folate concentrations than consumption of 400 µg DFE/d regardless of the folate source. The findings that serum and RBC folate during wk 12–14 did not differ among the groups consuming 400 µg DFE/d (T400F135, T400F230 and T400F400) or among the groups consuming 800 µg DFE/d (T800F135, T800F630 and T800F800) support the validity of the 1.7 multiplier in the DFE equation (4). In doing so, these data also suggest that the bioavailability of food folate as part of a mixed diet is 60% that of added folic acid.
Urinary folate excretion was a less reliable indicator of folate intake/status mainly due to the large variation in excretion values among individuals within the same treatment group. The T400F135, T400F230, T400F400, T800F630, and T800F800 groups did not differ during wk 12–14 despite the 100% higher folate intake in the last 2 groups. The lack of difference between the 2 treatment intake levels precluded the use of urinary folate excretion as a basis for estimating the dietary folate equivalency of folic acid. Of interest, however, was the higher (P 0.05) urinary folate excretion in subjects consuming 800 µg DFE/d derived primarily from folic acid (392 or 666 µg DFE/d) relative to the other treatment groups, which derived most of the 800 µg DFE/d as food folate. One explanation for this observation is that a substantial portion of the consumed folic acid escaped intestinal reduction (29), resulting in unmetabolized plasma folic acid that was excreted more readily in the urine than 5-methyltetrahydrofolate. A second explanation is an enhanced displacement of tissue 5-methyltetrahydrofolate in response to oral consumption of folic acid relative to naturally occurring food folates, primarily reduced formyl and methylated folates (30). Thus, when folic acid intake is high (i.e., 
500 µg/d), the dietary folate equivalency of folic acid is likely <1.7 due to excessive urinary folate excretion.
Plasma tHcy is a functional indicator of folate status and may be useful in assessing the bioequivalence of folic acid and food folate. However, 2 factors hindered the utility of plasma tHcy as a biomarker in the present study. The first was the nonlinear relation between dietary folate intake and plasma tHcy. Several investigators reported no further decreases in tHcy with folate intakes beyond 400 µg/d total folate or 200 µg/d folic acid (31,32). This is consistent with our finding that plasma tHcy concentrations did not differ between women consuming 800 or 400 µg DFE/d. The second factor was the low plasma tHcy concentrations at baseline and throughout the study. Further reductions in plasma tHcy were unlikely regardless of folate intake. Thus, the lack of difference among the 400 µg DFE/d groups may be because plasma tHcy reached a plateau or may be due to the 60% bioavailability of food folate relative to folic acid.
Only a few studies have evaluated the bioavailability of food folate (relative to folic acid) consumed as part of a mixed diet (8,12,33). For comparison purposes, the folate intakes administered in these studies were converted to DFE using the 1.7 multiplier from the DFE calculation (4). During the final phase of a metabolic study (d 71–92), Sauberlich et al. (8) provided 3 levels of folate intake including 156 µg DFE/d (20 µg/d food folate + 80 µg/d added folic acid), 200 µg DFE/d derived from food folate only, and 300 µg DFE/d derived from food folate only. At the end of the study, plasma folate concentrations did not differ between the 156 and 200 µg DFE/d groups. Subjects consuming 300 µg DFE/d had higher (P 0.05) plasma folate concentrations than either the 156 or 200 µg DFE/d groups. On the basis of these data, they concluded that food folate was no more than 50% available relative to folic acid consumed with a meal. Brouwer et al. (12) performed a 4-wk controlled intervention study involving a food folate group (560 µg DFE/d: 210 µg/d food folate from basal diet + 350 µg/d food folate from vegetables and citrus fruits), a folic acid group (635 µg DFE/d: 210 µg/d food folate from basal diet + 500 µg/d supplemental folic acid every other day), and a placebo group (210 µg DFE/d: 210 µg/d food folate). Estimates of food folate bioavailability from vegetables and citrus fruits relative to folic acid were 78% based on plasma folate, 98% based on RBC folate, and 60% based on plasma tHcy. Cuskelly et al. (33) conducted a 3-mo intervention trial in women assigned to a folic acid supplement (881 µg DFE/d: 201 µg/d food folate from basal diet + 400 µg/d folic acid), fortified foods (595 µg DFE/d: 138 µg/d food folate from basal diet + 269 µg/d folic acid), and extra dietary folate (410 µg DFE/d: 209 µg/d food folate from basal diet + 201 additional food folate). Based on RBC folate response, the calculated bioavailability estimates for food folate using the relative bioavailability equation described by Brouwer et al. (12) were 18% (relative to the folic acid fortified group) and 39% (relative to the folic acid supplement group).
The totality of evidence suggests that food folate is less bioavailable than folic acid when consumed as part of a mixed diet. The extent to which food folate and folic acid differ, however, is highly variable even within the same study. The results of the present study support the validity of the 1.7 multiplier but do not confirm it. The small sample size inherent in controlled feeding studies precluded the detection of the relatively small differences in blood folate levels observed among the 400 µg DFE/d treatment groups and among the 800 µg DFE/d treatment groups. To further assess the validity of the 1.7 multiplier, we calculated the bioavailability of food folate relative to folic acid using the equation described by Brouwer et al. (12) with modifications (see Appendix). In our first calculation, the T400F400 and T400F135 served as the reference groups and the T800F800 and T800F135 represented the dietary folate and folic acid groups, respectively. In our second calculation, the T400F230 and T400F135 served as the reference groups and the T800F630 and T800F135 represented the dietary folate and folic acid groups, respectively. Based on mean serum and RBC folate concentrations throughout weeks 12–14, the overall percentage of bioavailability of food folate (relative to folic acid) was 52% (range: 44–59%). Taken together, our data suggest that the dietary folate equivalency of folic acid ranges from 1.7 to 2.0 when consumed as part of a mixed diet.
Other factors that may affect results obtained from bioequivalency studies are the folate status of the host and the level of folate intake. The folate status of our study population throughout the treatment phase was similar to that in other studies conducted in women residing in the United States after folic acid fortification was initiated (34–37) but higher than in studies conducted in European countries (12,38) where folic acid fortification is not widespread. Similarly, the folate intake levels, 400 and 800 µg DFE/d, used in the present study are consistent with current folate consumption patterns in the United States (2,3,35–37) but are higher than folate consumption patterns in European countries (5,39). Nonetheless, the results of the present study are consistent with the study of Sauberlich et al. (8), which was conducted before folic acid fortification of U.S. cereal grain products and administered folate at levels < 400 µg DFE/d.
A unique aspect of this study was the use of food folate alone to provide the 400 or 800 µg DFE/d. A caveat of using food folate rather than folic acid as the main folate source is manipulation of other relevant nutrients. In the present study, the menu that was highest in food folate also contained more choline and betaine, important methyl donors that may influence one-carbon metabolic pathways (40). Despite higher intakes of these nutrients, plasma tHcy concentrations did not differ between women consuming 135 and 800 µg/d food folate. These data suggest that under conditions of folate sufficiency, increased dietary intake of choline and betaine will not lead to lower homocysteine concentrations in women with the MTHFR CC genotype.
In conclusion, data from this controlled folate feeding study in young women support the validity of the 1.7 multiplier in the U.S. DFE equation and suggest that food folate bioavailability is 60% of that of added folic acid when consumed as part of a mixed diet. These estimates are similar whether they are based on serum folate or RBC folate.
Calculations of food folate bioavailability (relative to folic acid) The equation defined by Brouwer et al. (12) was modified to accommodate the design of the present study. For the T800F800 and T800F135 comparisons, T400F400 and T400F135 served as the reference groups, respectively. For the T800F630 and T800F135 comparisons, T400F230 and T400F135 served as the reference groups, respectively. For each comparison, the additional folic acid and dietary folate provided was 236 and 400 µg, respectively. For each endpoint (change in concentration of serum folate and RBC folate), the relative bioavailability of the food folate to added folic was calculated as follows:
Relative bioavailability (%) equals:
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Actual Calculations
T800F800 versus T800F135 Comparisons
Serum folate (nmol/L): (35.8 – 22.4)/(41.0 – 23.1) x (236/400) x 100 = 44%
RBC folate (nmol/L): (1679 – 1319)/(1677 – 1267) x (236/400) x 100 = 52%
T800F630 versus T800F135 Comparisons
Serum folate (nmol/L): (36.0 – 19.9)/(41.0 – 23.1) x (236/400) x 100 = 53%
RBC folate (nmol/L): (1801 – 1389)/(1677 – 1267) x (236/400) x 100 = 59%
Overall Mean Bioavailability: 52%
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FOOTNOTES |
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3 Abbreviations used: DFE, dietary folate equivalents; DRI, dietary reference intakes; F, food folate in µg DFE/d; GLM, general linear model; HSD, honestly significant difference; MTHFR, methylenetetrahydrofolate reductase; RDA, recommended dietary allowance; T, total folate in µg DFE/d; tHcy, total homocysteine.
Manuscript received 16 November 2004. Initial review completed 17 December 2004. Revision accepted 18 February 2005.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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