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Circulating folic acid in plasma: relation to folic acid fortification^1,2,3

¹ From the Vitamin Metabolism and Aging Laboratory, Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA (RDK, SFC, PFJ, AMT, and JS), and the Framingham Heart Study, Boston University School of Medicine, Framingham, MA (RD)

² Supported by the US Department of Agriculture (agreement 1950-51520-008-00D; grant 2006-35200- 17198) and the Framingham Heart Study of the National Heart, Lung, and Blood Institute of the National Institutes of Health (contract N01-HC-25195).

³ Address reprint requests to J Selhub, Vitamin Metabolism and Aging Laboratory, Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111. E-mail: Jacob.selhub{at}tufts.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: The implementation of folic acid fortification in the United States has resulted in unprecedented amounts of this synthetic form of folate in the American diet. Folic acid in circulation may be a useful measure of physiologic exposure to synthetic folic acid, and there is a potential for elevated concentrations after fortification and the possibility of adverse effects.

Objective: We assessed the effect of folic acid fortification on circulating concentrations of folic acid and 5-methyltetrahydrofolate in the Framingham Offspring Cohort.

Design: This is a cross-sectional study that used plasma samples from fasting subjects before and after fortification. Samples were measured for folate distribution with the use of an affinity-HPLC method with electrochemical detection.

Results: Among nonsupplement users, the median concentration of folic acid in plasma increased from 0.25 to 0.50 nmol/L (P < 0.001) after fortification, and among supplement users the median increased from 0.54 to 0.68 nmol/L (P = 0.001). Among nonsupplement users, the prevalence of high circulating folic acid (85th percentile) increased from 9.4% to 19.1% (P = 0.002) after fortification. Among supplement users, the prevalence of high circulating folic acid increased from 15.9% to 24.3% (P = 0.02). Folic acid intake and total plasma folate were positively and significantly related to high circulating folic acid after adjustment for potential confounding factors (P for trend < 0.001).

Conclusions: Folic acid fortification has resulted in increased exposure to circulating folic acid. The biochemical and physiologic consequences of this are unknown, but these findings highlight the need to understand the effects of chronic exposure to circulating folic acid.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Since January 1998, folic acid fortification of flour and cereal grain has been mandatory in the United States and has also been implemented in Canada and other countries (1). The amount of fortification in the United States was set at 140 µg/100 g of cereal grain product, which was expected to increase the average folic acid intake attributable to fortification by 100 µg/d, although studies have indicated the increase to be closer to 200 µg/d (2, 3). Since the start of folic acid fortification, there has been a virtual elimination of folate deficiency and 50% decrease in the prevalence of hyperhomocysteinemia (4). These biochemical effects of fortification are temporally associated with a decrease in the incidence of neural tube defects (5, 6) and stroke associated mortality (7) but also with an increased incidence of colorectal cancer (8) and possible harm to large segments of the population with low vitamin B-12 status (9, 10). Considering that all Americans are now exposed to fortification and the uncertainty about its risks and benefits beyond those relating to the prevention of neural tube defects, it is imperative to clarify the long-term effects of fortification on unintended health outcomes (11).

Folic acid, the synthetic form of the vitamin folate, is not found in nature. Its conversion to the active form requires reduction to tetrahydrofolate, a reaction that is catalyzed by dihydrofolate reductase, which is expressed in the intestine and other peripheral tissues (12). Dihydrofolate reductase activity in the intestine converts absorbed folic acid to dihydrofolate, which is both reduced and methylated to 5-methyltetrahydrofolate (5MeTHF), the form which is predominantly found in blood. However, because the capacity of this conversion is limited, excessive intake of folic acid saturates this capacity and results in its appearance unaltered in circulation (13, 14). Thus, the presence of residual circulating folic acid under fasting conditions is indicative of saturating folic acid intake.

In the present study we used a highly sensitive HPLC method to analyze plasma samples from the sixth examination of the Framingham Offspring Cohort. Because these samples were taken before and after fortification was implemented, we could determine the prevalence of circulating folic acid associated with folic acid fortification.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study population
The Framingham Heart Study is an epidemiologic study of heart disease established in Framingham, MA, in 1948. The original Framingham cohort included 1644 husband-wife pairs and 378 persons whose parents had developed cardiovascular disease (15). The offspring of those subjects and the spouse of the offspring were invited to participate in the Framingham Offspring Study in 1971, and 5135 of the 6838 eligible persons participated in the first examination (16). The Offspring cohort undergoes repeat examination approximately every 3–4 y.

Between January 1995 and August 1998, members of the Offspring cohort (n = 3532) participated in the sixth study examination. Fortification of enriched cereal grain products was phased in over a 2-y period during this examination cycle (March 1996–January 1998). On the basis of discussions with food distributors in southern New England, we determined that there were few products fortified before September 1996 but that most products were fortified by July 1997. Exposure to fortification was determined on the basis of the examination date with the information noted above from the local food distributor. Participants whose sixth examination occurred before September 1996 are considered to be unexposed to fortification (before fortification). Participants who attended the examinations between September 1997 and August 1998 are considered as exposed to fortification (after fortification). Individuals who were examined between October 1996 and August 1997 (around fortification) were excluded from these analyses because of uncertainty of their folic acid intake. Consequently, the present cross-sectional analysis included participants whose examination date met our definitions of before fortification (n = 1103) or after fortification (n = 600). The Institutional Review Boards for Human Research at Boston University and Tufts-New England Medical Center approved the protocol.

Dietary data
Dietary intake was assessed by a semiquantitative food-frequency questionnaire (FFQ) (17). Subjects were asked to report on frequency of food consumption in the past year, vitamin and mineral supplements, and type of breakfast cereal most commonly consumed. To estimate intake of specific nutrients, the frequency of consumption was multiplied by the nutrient content of the specified portions. If 12 food items were left blank or if reported energy intakes were <600 kcal or >4000 kcal, the information was excluded from analysis.

To determine folic acid intake associated with fortification, folate intake from the FFQ was modified to reflect fortification as previously described (2). Briefly, foods enriched with folic acid after mandatory fortification was implemented were recalculated with the use of data from a study that measured the folate content of foods after fortification (18). The foods that were recalculated included bread, corn grits, rice, pasta, corn meal, muffins, pancakes, crackers, pizza, cookies, brownies, doughnuts, cakes, sweet rolls, and pie. Ready-to-eat cereals that were fortified before mandatory fortification were also modified if the measured folic acid content exceeded published database values. For all these enriched cereal grain products, the original total folate values used in the FFQ database were replaced by the more recently measured total folate values, and a new variable representing measured folic acid intake was created. Folic acid intake from supplements was assessed with the use of data from the FFQ that asks about supplement brand, type, and frequency of consumption. Analyses were separate for nonsupplement and supplement users.

After applying these modifications, total folate intake as micrograms of dietary folate equivalents (DFEs) was also calculated. This is done by adding the amount of folate consumed as natural folate (in µg) to the amount of folate consumed as folic acid (in µg) multiplied by 1.7 to account for the increased bioavailability of folic acid.

Biochemical methods
As part of the sixth examination, blood samples were taken from subjects who had fasted for 10 h. Folic acid and 5MeTHF concentrations were determined with the use of the affinity-HPLC method with electrochemical (coulometric) detection method (19) with the following modifications to increase throughput. Plasma samples (0.2 mL) were diluted with 1.2 mL of 50 mmol/L potassium tetraborate in 1% sodium ascorbate and vortexed. Twenty picomoles of ethyltetrahydrofolate was added as an internal standard. The mixture was boiled for 30 min and then cooled on ice and kept covered overnight at 4 °C. The mixture was centrifuged for 20 min at 4 °C at 36 000 x g and filtered. One milliliter of the supernatant fluid fraction was injected onto the affinity-HPLC system.

The revised affinity-HPLC system consisted of 2 affinity columns that were connected by a 10-port, 2-position switching valve to an analytic column (Betasil Phenyl 250 x 4.6 mm; Keystone Scientific Inc, Bellefonte, PA). This allows the first affinity column to be loaded with the sample and subsequently washed with a pH 7.0 phosphate buffer, while the second affinity column is eluted into the analytic column with an acetonitrile gradient at acid pH and into the electrochemical detector. At the end of the run, a signal is sent to switch the direction of the mobile phases to allow the elution of the loaded column for folate analysis and simultaneous loading of the second column with the new sample.

Folate activity was determined with the use of an ESA Four-Channel CoulArray Detector (Bioscience Inc, Chelmsford, MA). Quantification and identification of individual folates was done by comparison to external and internal folate standards of known concentration. The CV for folate detection by the described method ranged between 5.2% and 8.6% (intraassay) and 3.2% and 7.3% (interassay). The limit of quantification for folic acid was 0.19 nmol/L, and the limit of detection was 0.18 nmol/L. For these analyses, the limit of detectable folic acid (0.18 nmol/L) was used to describe detectable folic acid.

Statistical analysis
The statistical analyses were performed with SAS (version 9.1; SAS Institute, Cary, NC). Analyses assessing the effect of fortification on circulating folic acid, 5MeTHF, and total folate were calculated separately for users of B vitamin supplements and for users of non-B vitamin supplements. Data for total folate and 5MeTHF concentrations were positively skewed; therefore, these values were log transformed before analysis and reported as geometric means with 95% CIs. Folic acid intake values were positively skewed, and a square root transformation was used to normalize values. Circulating folic acid concentrations were skewed toward low values, and not all samples had detectable concentrations; thus, median concentrations and prevalence of detectable and high concentrations were reported. Detectable was defined as >0.18 nmol/L, the limit of detection for the method, and high was defined as 1.35 nmol/L, the 85th percentile for the cohort at the sixth examination cycle.

To determine the effect of folic acid fortification on circulating folic acid we compared median values in subjects both exposed and unexposed to fortification with the use of the Kruskal-Wallis test. SAS PROC GLM was used to assess the differences in prevalence of detectable and high circulating folic acid with adjustment for age and sex with the use of the Tukey's adjustment for multiple comparisons. We also used SAS PROC GLM to compare 5MeTHF and total plasma folate geometric means in subjects exposed and unexposed to folic acid fortification. To ensure values were related to fortification and not to differences in dietary patterns of folate- or folic acid-containing foods, we also adjusted for folate intake with the use of the food composition database not modified to reflect folic acid fortification.

To identify determinants of circulating folic acid 85% for this cohort, age- and sex-adjusted logistic regression analysis was used to calculate odds ratios for high circulating folic acid. We created categories for the continuous variables of age, body mass index (in kg/m²), number of cigarettes smoked per day, and alcohol intake and for the quartile categories of intakes of caffeine, natural folate, synthetic folic acid, vitamin B-6, and vitamin B-12 and the plasma concentrations of folate, vitamin B-6, and vitamin B-12. Relations between dietary vitamin intakes were also adjusted for total energy intake, and supplement users were excluded from these analyses. P values reported are for the comparison between the reference value and each of the categories. A test for a trend was performed by entering independent variables as continuous variables in the logistic regression model. The P for trend was the resulting P values for the associated logistic regression coefficient.

To describe graphically the relation between circulating folic acid and folate intake, we plotted predicted circulating folic acid concentrations and 95% CIs by folic acid intake with the use of SAS PROC LOESS with a smoothing value of 0.3. A test for a trend was performed by entering folic acid intake as a continuous variable in the model with the use of PROC GLM and adjusting for age, sex, and total energy.

We also plotted the prevalence and 95% CIs of high circulating folic acid by categories of DFE intake with the use of SAS PROC GLM. Within each DFE intake category, the average percentage of total DFE intake attributed to natural folate and synthetic folic acid was also calculated. Analyses were adjusted for age, sex, and total energy. P for trend was calculated by entering DFE intake as a continuous variable in the regression model relating high circulating folic acid to folic acid intake as a percentage of total folate intake.

	RESULTS

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Characteristics of the population
Circulating concentrations of folic acid, total plasma folate, and 5MeTHF were measured in 705 nonsupplement users and 398 supplement users not exposed to fortification and in 355 nonsupplement users and 245 supplement users exposed to fortification (Table 1). At the time of the sixth examination the range of ages was 29–86 y and the average in each group was between 57 and 60 y. The proportion of men within users of non-B vitamin supplements and of B vitamin supplements was similar for before and after fortification, but men were less likely to use B vitamin supplements than were women. The proportion of subjects who used supplements increased after fortification. Increased prevalence of supplement use during this time period was reported in other cohorts, particularly in older white populations (20), which describes a large proportion of our cohort. We are not able to assess why more people used supplements; it may be due to a perceived health benefit.

Among subjects who did use B vitamin supplements, the median circulating concentrations of folic acid increased after fortification compared with before fortification (0.54 before compared with 0.68 after fortification, P = 0.001) (Table 1). The prevalence of detectable circulating folic acid was 72.5% before fortification and 80.7% after fortification (P = 0.13). The prevalence of high circulating folic acid was 15.9% before fortification and 24.3% after fortification (P = 0.02). Compared with before fortification, total plasma folate concentrations were 29.9% higher after fortification (P < 0.001), and 5MeTHF concentrations were 28.7% higher (P < 0.001) after fortification.

Relation between circulating folic acid and folate intakes
Predicted circulating concentrations of folic acid by folic acid intake for the entire population is shown in Figure 1. A positive linear association was observed between folic acid intake and circulating folic acid concentrations (P for trend < 0.001).

View larger version (10K): [in this window] [in a new window]   FIGURE 1. Predicted concentrations of circulating folic acid (error bars are 95% CIs) by folic acid intake. The number of subjects in each group starting at 0 µg was 251, 193, 177, 100, 123, 119, 92, 47, 92, 110, 110, 49, 36, 28, 18, 15, 25, 18, 16, 10, and 29. A nonparametric loess smoother was fitted to the data, and 95% CIs were calculated with the use of PROC LOESS with a smooth value of 0.3. A test for a trend was performed by entering folic acid intake as a continuous variable in the model with the use of PROC GLM and adjustment for age, sex, and total energy. P for trend was < 0.001 for the relation between folic acid intake and circulating folic acid concentration.

Figure 2

View larger version (14K): [in this window] [in a new window]   FIGURE 2. The proportion of subjects (error bars are 95% CIs) with high circulating folic acid status (85%) according to folate intake. Folate intake is expressed as the µg/d of dietary folate equivalents (DFEs). Total DFE intake = (1.7 x µg of folic acid intake/d) + µg/d of natural folate intake. Within each DFE category, the average percentage of total DFE intake attributed to natural folate (white bars) and synthetic folic acid (black dots) was also calculated. The results were calculated with the use of PROC GLM adjusted for age, sex, and total energy. P for trend was < 0.001 for the relation between the percentage of folic acid intake as total folate intake in DFEs with prevalence of high circulating folic acid.

Table 2

View this table: [in this window] [in a new window]   TABLE 2 Determinants of circulating folic acid (FA) concentrations 85%1

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Folic acid in circulation is almost certainly derived from synthetic folic acid from fortified foods and supplements. Given the positive relation between folic acid intake and circulating folic acid, it is not surprising that participants taking supplements and exposed to fortification had the highest concentrations. Although even in this group, concentrations tended to be low. This is probably because plasma samples were taken from fasted participants, which will not represent total exposure to synthetic folic acid. Folic acid is eventually converted to 5MeTHF in the peripheral tissues, so the amount measured in fasted plasma represents the residual unmetabolized folic acid. With the use of nonfasted samples, Pfieffer et al (21) has reported that those with high total serum folate (>50 nmol/L) had a higher percentage of folic acid as total folate than did samples in which total serum folate was <50 nmol/L. Taken together, these results imply that higher folic acid intake will increase exposure to circulating folic acid.

Because circulating folic acid was never quantified in a population sample, the effect of fortification on folic acid concentrations was previously unknown. An earlier study by Sweeney et al (22) predicted the current fortification program in the United States, which was estimated to supply an average of 100 µg of folic acid, would be less likely to have folic acid in circulation than would a program that supplies 400 µg/d. However, our findings show that approximately three-quarters of persons using supplements, which typically provide 400 µg of folic acid, in the time before fortification and persons not using supplements in the time after fortification, have detectable circulating folic acid. The median circulating folic acid concentration and the prevalence of high folic acid concentrations was also comparable in these 2 groups.

One possible explanation for the higher than expected prevalence of detectable folic acid as a result of fortification is the way "detectable" folic acid is defined. Our assay for folic acid can detect folic acid at concentrations of 0.18 nmol/L, whereas the assay used in the Sweeney study can only detect concentrations 0.71 nmol/L (22). A second possible explanation for the difference in expected prevalence of detectable folic acid concentrations is that the estimated consumption of folic acid from fortification is higher than the 100 µg/d assumed by Sweeney et al (22) in their study. Finally, another important factor to consider is how long the population has been exposed to fortification. The exposure time in the present study was 1–2 y, compared with 14 wk in the study of Sweeney et al (22). Some evidence shows that repeated consumption of folic acid leads to accumulation in circulation (22, 23), which could mean even small intakes over a long period of time will result in more circulating folic acid. In the present study, 55% of persons before fortification and not taking supplements still had detectable circulating folic acid, despite a low mean intake of folic acid in these subjects. There is no evidence that folic acid in circulation could occur naturally, so detectable concentrations before fortification is probably from consumption of fortified breakfast cereals. Approximately 64% of nonsupplement users unexposed to fortification reported consuming fortified breakfast cereals in the FFQ.

It seems as if most of the population is now exposed to circulating folic acid, and evidence suggests that higher intakes will further increase concentrations. There has been much speculation about whether this poses a health risk (24). Because of a lack of information on what concentration is normal or may pose risk, we arbitrarily defined high as >85th percentile. In studies that have found an adverse effect of folate, it is in persons with the highest total folate status (9, 10) or the highest folic acid intakes (25-27). In the one study that did measure circulating folic acid, there was a trend toward lower natural killer cell cytotoxicity with increased concentrations in plasma in women >60 y (28). This association was independent of 5MeTHF and total folate concentrations.

In conclusion, this study indicated both supplement use and folic acid fortification are associated with increased exposure to circulating folic acid. It also raises the possibility that prolonged exposure and high intakes may further increase concentrations. Given the high prevalence of supplement use in the United States and increased folic acid intake attributable to fortification, ongoing monitoring of folic acid status is needed. To evaluate all the potential benefits and risks and optimal intakes of folic acid for all segments of the population, the physiologic and safety ramifications of lifetime exposure to circulating folic acid need to be elucidated.

	ACKNOWLEDGMENTS

 
The author's responsibilities were as follows—RDK, AMT, PFJ, and JS: contributed to study design, writing, and revising of the manuscript; RDK, SFC, and PFJ: contributed to data analysis; RDK, SFC, RD, and PFJ: contributed to data collection. None of the authors had a personal or financial conflict of interest.

	REFERENCES

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