Absorption, Transport, and Storage

Food folates (polyglutamate derivatives) are hydrolyzed to monoglutamate forms in the gut before absorption across the intestinal mucosa. This cleavage is accomplished by a γ-glutamylhydrolase, more commonly called folate conjugase. The monoglutamate form of folate is actively transported across the proximal small intestine by a saturable pH-dependent process. When pharmacological doses of the monoglutamate form of folate are consumed, it is also absorbed by a nonsaturable mechanism involving passive diffusion.

Monoglutamates, mainly 5-methyl-tetrahydrofolate, are present in the portal circulation. Much of this folate can be taken up by the liver, where it is metabolized to polyglutamate derivatives and retained or released into the blood or bile. Approximately two-thirds of the folate in plasma is protein bound. A variable proportion of plasma folate is bound to low-affinity protein binders, primarily albumin, which accounts for about 50 percent of bound folate. Low levels of high-affinity folate binders are also present in plasma.

Cellular transport of folate is mediated by a number of different folate transport systems, which can be characterized as either membrane carriers or folate-binding protein-mediated systems. These transport systems are not saturated by folate under physiological conditions, and folate influx into tissues would be expected after any elevation in plasma folate after supplementation.

Folate concentrations in liver of 4.5 to 10 μg/g were reported after liver biopsies (Whitehead, 1973). Because the adult male liver weighs approximately 1,400 g, the total quantity of folate in the liver would be approximately 6 to 14 mg. If the liver is assumed to contain 50 percent of the body stores of folate, the estimated total body folate store would be 12 to 28 mg. Using the same assumption, Hoppner and Lampi (1980) determined average liver folate concentrations to be approximately 8 μg/g (range 3.6 to 14.8 μg/g) after autopsy; the liver folate content would be approximately 11 mg and total body folate 22 mg.

Metabolism and Excretion

Before being stored in tissue or used as a coenzyme, folate monoglutamate is converted to the polyglutamate form by the enzyme folylpolyglutamate synthetase. When released from tissues into circulation, folate polyglutamates are reconverted to the monoglutamate form by γ-glutamylhydrolase. Folates must be reduced enzymatically and resynthesized to the polyglutamate form to function in single-carbon transfer reactions.

The metabolic interrelationship between folate and vitamin B₁₂ may explain why a single deficiency of either vitamin leads to the same hematological changes. Both folate and vitamin B₁₂ are required for the formation of 5,10-methylenetetrahydrofolate and involved in thymidylate synthesis by way of a vitamin B₁₂-containing enzyme. The formation of 5,10-methylene tetrahydrofolate depends on the regeneration of the parent compound (tetrahydrofolate) in the homocysteine-to-methionine conversion. This reaction involves the removal of a methyl group from methyl folate and the delivery of this group to homocysteine for the synthesis of methionine. Folate is involved as a substrate (5-methyl-tetrahydrofolate) and vitamin B₁₂ as a coenzyme. The 5,10-methylenetetrahydrofolate delivers its methyl group to deoxyuridylate to convert it to thymidylate for incorporation into DNA. In either a folate or vitamin B₁₂ deficiency, the megaloblastic changes occurring in the bone marrow and other replicating cells result from lack of adequate 5,10-methylenetetrahydrofolate.

The major route of whole-body folate turnover appears to be via catabolism to cleavage products. The initial step in folate catabolism involves the cleavage of intracellular folylpolyglutmate at the C9-N10 bond, and the resulting p-aminobenzoylpolyglutamates are hydrolyzed to the monoglutamate, which is N-acetylated before excretion.

Folate freely enters the glomerulus and is reabsorbed in the proximal renal tubule. The net effect is that most of the secreted folate is reabsorbed. The bulk of the excretion products in humans are folate cleavage products. Intact urinary folate represents only a very small percentage of dietary folate. Biliary excretion of folate has been estimated to be as high as 100 μg/day (Herbert and Das, 1993; Whitehead, 1986); however, much of this is reabsorbed by the small intestine (Weir et al., 1985). Fecal folate losses occur, but it is difficult to distinguish actual losses from losses of folate synthesized by the intestinal microflora (Krumdieck et al., 1978).

Bioavailability of Folic Acid

When consumed under fasting conditions, supplements of folic acid are nearly 100 percent bioavailable (Gregory, 1997). Daly and coworkers (1997) reported incremental increases in erythrocyte folate in response to graded doses of folic acid, which provides evidence for the high bioavailability of supplemental folate. Additional work may be necessary to improve the precision of the estimate of bioavailability (Pfeiffer et al., 1997a).

No published information was found regarding the effect of food on the bioavailability of folate supplements. Pfeiffer and coworkers (1997a) recently examined the bioavailability of C¹³-labeled folic acid (administered in apple juice) when given with or without a serving of food; they found a slight (about 15 percent) but insignificant reduction when folic acid was consumed with a portion of food. From these experimental data the bioavailability of folic acid consumed with food is estimated to be 85 percent. Studies have not been conducted to define the bioavailability of folic acid consumed with entire meals. It is assumed that the bioavailability would be somewhat lower than that observed with folic acid alone or with a small portion of food.

Bioavailability of Folate Added to Foods

The recently approved U.S. fortification of breads and grains with folate has raised interest in the bioavailability of folate provided in the form of folic acid. On the basis of erythrocyte folate response over a 3-month study, it was concluded that the folate in a supplement and in fortified bread and breakfast cereal consumed in the context of normal diet was equally bioavailable (Cuskelly et al., 1996). Pfeiffer and colleagues (1997a) evaluated the bioavailability of folate from cereal-grain foods fortified experimentally with C¹³-labeled folic acid. In a series of single-dose trials with human subjects, there was a slight but insignificant difference between the control (water with folic acid) and any of the tested foods (white and whole-wheat bread, pasta, and rice). This finding indicates high bioavailability of the folate in the form of added folic acid.

Overall, the very different studies of Cuskelly et al. (1996) and Pfeiffer et al. (1997a) complement each other and strongly indicate that folate added to cereal-grain foods is highly available and efficacious. These two studies contradict previous reports of low (30 to 60 percent) bioavailability of folate in experimentally fortified cereal-grain foods in South Africa (summarized by Colman [1982]). In the South African studies of folate-deficient pregnant women, the response criterion used to estimate bioavailability was either 2-hour changes in serum folate or changes in erythrocyte folate over time. The quantity of folate consumed in the fortified foods was not directly measured in these studies. If the amount was overestimated, that would explain the lower reported bioavailability (33 to 60 percent) compared with the recent estimates (85 to 100 percent) by Pfeiffer et al. (1997a) and Cuskelly et al. (1996). The experimental fortification of these South African foods in the 1970s may have little relevance to the current fortification process in the United States and Canada.

The value used in this report—85 percent bioavailability of folic acid consumed with a meal—is probably an underestimate, the effect of which may be an underestimation of the folate requirement.

Bioavailability of Food Folate

Perhaps the best data on which to base an estimate of the bioavailability of food folate are provided by Sauberlich and coworkers (1987). On the basis of changes in blood folate values, the authors concluded that the bioavailability of food folate was no more than 50 percent that of folic acid. Although this study was not designed as a quantitative study of food folate bioavailability, the results provide strong evidence in that regard. Similarly, the data of Cuskelly and colleagues (1996) suggest that food folate is less bioavailable than the synthetic form, as evidenced by a smaller increase in erythrocyte folate in the group that received an increased level of folate from food rather than from the synthetic form. The percentage bioavailability of folate could not be determined from this study because food consumption was not controlled.

A stable isotope investigation of the relative bioavailability of monoglutamyl and polyglutamyl folates consumed in water (control) or added to lima beans or tomatoes found that the relative bioavailability of deuterated polyglutamyl folates was equivalent to that of the monoglutamyl tracer (Wei et al., 1996). However, the bioavailability of polyglutamyl folate added to orange juice was approximately 33 percent lower (p < 0.05) than that of the monoglutamyl folate tracer. The authors concluded that naturally occurring polyglutamyl folates in orange juice are approximately 67 percent available—slightly more available than the food folate bioavailability estimate of Sauberlich. Related issues have been discussed in several reviews on this subject (Gregory, 1989, 1995, 1997).

Bioavailability Estimates and Assumptions

Many controlled studies on folate requirements have used a defined diet (food folate) supplemented with folic acid. Because folic acid taken with food is 85 percent bioavailable but food folate is only about 50 percent bioavailable, folic acid taken with food is 85/50 (i.e., 1.7) times more available. Thus, if a mixture of folic acid plus food folate has been fed, dietary folate equivalents (DFEs) are calculated as follows to determine the Estimated Average Requirement (EAR):

Expressed differently, to be comparable with food folate, only half as much folic acid is needed if taken on an empty stomach, or

When food folate was the sole source of folate in studies used to determine requirements, no corrections were applied to convert to DFEs. Adjustments made for DFEs are indicated, if applicable, where folic acid was a source of folate. Adjustments cannot be made for epidemiological studies if data are lacking on the folate sources. If future research indicates that food folate is more than 50 percent bioavailable, this could lower the estimated requirements that appear later in the chapter.

Fiber

Experimental data do not support the hypothesis that dietary fiber per se reduces folate bioavailability (Bailey, 1988; Gregory, 1989). Human studies (Russell et al., 1976) confirmed the negative findings of both rat and chick bioassays regarding the identification of an inhibitory action of various dietary fiber sources. Certain forms of fiber (e.g., wheat bran) may decrease the bioavailability of certain forms of folate under some conditions (Bailey et al., 1988; Keagy et al., 1988), but many forms of fiber appear to have no adverse effects (Gregory, 1997).

Experimental evidence in rats indicates that synthesis of folate by intestinal bacteria influences folate status (Keagy and Oace, 1989; Krause et al., 1996). Rong and colleagues (1991) reported that bacterially synthesized folate in the rat large intestine is incorporated into host tissue polyglutamates. The applicability of these data to humans is unknown. Suggestive evidence of a positive association between dietary fiber intake and folate status in humans was reported by Houghton and coworkers (1997). Zimmerman (1990) provided evidence that the monoglutamate form can be transported into the mucosa of the human colon by facilitated diffusion, allowing for the possibility of subsequent absorption of folate synthesized in the large intestine.

Alcohol

Data from surveys of chronic alcoholics suggest that inadequate intake is a major cause of the folate deficiency that has often been observed in chronic alcohol users (Eichner and Hillman, 1971; Herbert et al., 1963). Ethanol intake may aggravate folate deficiency by impairing intestinal folate absorption and hepatobiliary metabolism (Halsted et al., 1967, 1971, 1973) and by increasing renal folate excretion (McMartin et al., 1986; Russell et al., 1983).

Nonsteroidal Anti-inflammatory Drugs

When taken in very large therapeutic doses (e.g., 3,900 mg/day), nonsteroidal anti-inflammatory drugs, including aspirin, ibuprofen, and acetaminophen, may exert antifolate activity (Baggott et al., 1992; Eichner et al., 1979; Lawrence et al., 1984; Willard et al., 1992). However, routine use of low doses of these drugs has not been reported to impair folate status.

Anticonvulsants

Numerous studies have cited evidence of impaired folate status associated with chronic use of the anticonvulsants diphenylhydantoin (phenytoin and Dilantin^®) and phenobarbital (Collins et al., 1988; Klipstein, 1964; Malpas et al., 1966; Reynolds et al., 1966). Diphenylhydantoin is known to inhibit the intestinal absorption of folate (Elsborg, 1974; Young and Ghadirian, 1989). Few studies, however, have controlled for potential differences in dietary folate intake between groups of anticonvulsant users and nonusers (Collins et al., 1988). Thus, definitive conclusions cannot be drawn relative to adverse effects of these drugs on folate status.

Methotrexate

Methotrexate is a folate antagonist that has been used frequently and successfully in the treatment of nonneoplastic diseases such as rheumatoid arthritis, psoriasis, asthma, primary biliary cirrhosis, and inflammatory bowel disease (Morgan and Baggott, 1995). Methotrexate has been especially effective in the treatment of rheumatoid arthritis (Felson et al., 1990), with efficacy established in numerous trials (Morgan et al., 1994). Patients with rheumatoid arthritis are frequently reported to be folate deficient, and folate stores are decreased in patients with rheumatoid arthritis who take methotrexate (Morgan et al., 1987, 1994; Omer and Mowat, 1968). Some of the side effects of methotrexate administration, such as gastrointestinal intolerance, mimic severe folate deficiency (Jackson, 1984). When patients are also given high-folate diets or supplemental folate, there is a significant reduction in toxic side effects with no reduction in drug efficacy. It has been recommended that patients undergoing chronic methotrexate therapy for rheumatoid arthritis increase folate consumption (Morgan et al., 1994) or consider folate supplements (1 mg/day) (Morgan et al., 1997).

Other Drugs with Antifolate Activity

The following diseases have been treated with drugs having antifolate activity: malaria with pyrimethamine, bacterial infections with trimethoprim, hypertension with triamterene, Pneumocystis carinii infections with trimetrexate (Morgan and Baggott, 1995), and chronic ulcerative colitis with sulfasalazine (Mason, 1995).

Oral Contraceptives

A number of early studies of oral contraceptive agents containing high levels of estrogens suggested an adverse effect on folate status (Grace et al., 1982; Shojania et al., 1968, 1971; Smith et al., 1975). However, oral contraceptive use has not been reported to influence folate status in large-scale population surveys (LSRO/FASEB, 1984) or in metabolic studies in which dietary intake was controlled (Rhode et al., 1983).

Method Used to Set the Adequate Intake

The AI reflects the observed mean folate intake of infants consuming exclusively human milk. Hematological and growth rate changes that have been measured in controlled studies of infants are not considered to be specifically attributable to the adequacy of dietary folate intake.

Serum and erythrocyte folate values of newborn infants are significantly higher than maternal blood concentrations, possibly reflecting an active transport process in utero (Ek, 1980; Landon and Oxley, 1971). These high blood folate values decline during the first 6 months in concert with the decline in the rate of cell division (Landon and Oxley, 1971).

The AI is the quantity of dietary folate that maintains blood folate concentrations comparable with those of the infant exclusively fed human milk. When human milk is consumed exclusively, the infant's serum or plasma folate concentration has been reported to range from 35 to over 60 nmol/L (16 to 30 ng/mL) whereas erythrocyte values averaged from 650 to over 930 nmol/L (300 to 430 ng/mL) (Ek and Magnus, 1979; Smith et al., 1985; Tamura et al., 1980). These values reported in infants are much higher than adult values (Smith et al., 1985), which makes the use of adult norms inappropriate for infants. Additionally there are no reports of full-term infants who are exclusively and freely fed human milk manifesting any signs of folate deficiency.

The folate concentration of human milk remains relatively constant regardless of maternal dietary folate intake unless there is a severe maternal deficiency (Metz, 1970). The reported concentration of folate in human milk varies with the methods used, and these have changed substantially over the past decade. However, recent data from the laboratories of Picciano and colleagues (Lim et al., 1997) are consistent with the data of Brown and colleagues (1986) and O'Connor and colleagues (1991), all of whom reported average human milk folate concentrations to be 85 μg/L. The human milk folate concentration used to estimate AIs for infants thus is 85 μg/L.

Ages 0 through 6 Months. The AI for infants 0 through 6 months of age, derived by using the average volume of milk of 0.78 L/day (see Chapter 2) for this age group and the average folate concentration in human milk after 1 month of lactation (85 μg/L), is 66 μg/day, which is rounded to 65 μg. This equals approximately 9.4 μg/kg of reference body weight. Because this is food folate, the amount is the same in dietary folate equivalents (DFEs).

Ages 7 through 12 Months. If the reference body weight ratio method described in Chapter 2 to extrapolate from the AI for folate for infants ages 0 through 6 months is used, the AI for folate for the older infants would be 80 μg/day after rounding. The second method (see Chapter 2), extrapolating from the Estimated Average Requirement (EAR) for adults and adjusting for the expected variance to estimate a recommended intake, gives a comparable AI of approximately 80 μg/day.

The five studies summarized in Table 8-1 illustrate the data from controlled studies that measured folate intake and assessed the infants' status. They include studies in which infants were fed either human milk or formula. Asfour and colleagues (1977) concluded that although the observed serum and erythrocyte concentrations in three groups of infants fed formula were borderline, the folate values were sufficient to maintain growth, hematopoiesis, and clinical well-being. However, the criteria of growth, hematopoiesis, and clinical well-being are too nonspecific for evaluating folate status. Therefore, these data suggest that none of the folate levels (3.6, 4.3, or 5.0 μg/kg) maintained folate adequacy in all the infants tested based on serum or erythrocyte folate concentrations. Ek and Magnus (1982) provided data that infant formula containing folate at 78 μg/L supported blood folate concentrations comparable with those of infants fed human milk. Smith and coworkers (1983) reported that serum folate values of infants fed human milk were approximately 45 nmol/L (20 ng/mL) at age 6 weeks and 65 nmol/L (30 ng/mL) at age 12 weeks, whereas erythrocyte folate concentrations were approximately 1,000 nmol/L (460 ng/mL) at age 6 weeks and 940 nmol/L (430 ng/mL) at age 12 weeks. Smith and coworkers (1985) reported that throughout the first 6 months, serum folate concentrations were significantly higher in infants fed formula than in those fed human milk; erythrocyte folate concentrations of approximately 2,200 nmol/L (1,000 ng/mL) at age 4 months clearly show that 158 μg/L of formula is in excess of what is needed. Salmenpera and colleagues (1986) reported that infants fed exclusively human milk all maintained adequate plasma folate concentrations with values twofold to more than threefold higher than maternal concentrations throughout the study.

TABLE 8-1

Folate Intake and Status of Infants by Study.

Folate AI Summary, Ages 0 through 12 Months

Data from the research studies included in Table 8-1 supports the AI of 65 μg/day of folate for young infants and of 80 μg/day for older infants.

The extent to which the AIs for folate could be lowered and still meet the physiological needs for infants fed human milk is unknown.

Special Considerations

No data were found to support the need to adjust dietary intake of folate on the basis of the type of infant formula compared with human milk to achieve the same folate status other than that inherent in DFE equivalency.

Method Used to Estimate the Average Requirement

No data were found on which to base an EAR for children. In the absence of additional information, EARs and RDAs for these ages have been estimated by using the method described in Chapter 2, which extrapolates from adult values. The resulting EARs are 120 and 160 μg/day of DFEs for children ages 1 through 3 and 4 through 8 years, respectively.

Folate EAR and RDA Summary, Ages 1 through 8 Years

The RDA for folate is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for folate; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for folate the RDA is 120 percent of the EAR).

Method Used to Estimate the Average Requirement

As for younger children, EARs and RDAs for these ages have been extrapolated from adult values by using the method described in Chapter 2. Although body size varies because of gender in these age groups, no conclusive data indicating a difference in requirements for adults were determined, thus no difference based on gender is proposed for these age groups.

Folate EAR and RDA Summary, Ages 9 through 18 Years

The RDA for folate is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for folate; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for folate the RDA is 120 percent of the EAR).

Evidence Considered in Estimating the Average Requirement

No single indicator was judged a sufficient basis for deriving an EAR for adults. That is, it was not deemed appropriate to base the EAR on an examination limited to studies that provided data only on erythrocyte folate, plasma homocysteine, or any other single laboratory value. The main approach to determining the EAR for adults uses a combination of erythrocyte folate, plasma homocysteine, and plasma or serum folate. The focus used was on the adequacy of specific quantities of folate consumed under controlled metabolic conditions to maintain normal blood concentrations of these indicators. Cutoff points for the normal range were based on the occurrence of documented biochemical abnormalities.

The types of studies considered were primarily those in which maintenance or restoration of folate status was evaluated in controlled metabolic conditions. In these studies folate was provided either as food or as food plus folic acid. Intakes related to these status indicators were computed by calculating DFEs, which gives higher intakes when folic acid is used as part of the protocol than what the authors describe when reporting their work (see “Bioavailability”).

In addition to data on maintenance or restoration of folate status, several other types of experimental data were critiqued and compared. These included kinetic estimates of body pool size and daily turnover (Gailani et al., 1970; Herbert, 1962b, 1968; Krumdieck et al., 1978; Russell et al., 1983; Stites et al., 1997; Von der Porten et al., 1992), quantitation of urinary folate catabolites as an index of folate turnover (McPartlin et al., 1993), and repletion of severe clinical folate deficiency (Hansen and Weinfeld 1962; Herbert, 1962a, 1968; Marshall and Jandl, 1960; Zalusky and Herbert, 1961). Analyses of relationships of dietary folate intake and biochemical indices of folate status from the Third National Health and Nutrition Examination Survey are in progress and were thus unavailable for use in this report.

Metabolic Studies

Two principal studies of healthy human subjects were critiqued and compared; the amounts of folate ranged from 100 to 489 μg/day of DFEs (O'Keefe et al., 1995; Sauberlich et al., 1987). Two additional studies (Jacob et al., 1994; Milne et al., 1983) were also considered but were given less weight because of the study design. These four studies are summarized in Table 8-2.

TABLE 8-2

Key Controlled Metabolic Studies Providing Evidence Used to Derive the Estimated Average Requirement (EAR).

100 to 150μg/day of DFEs. Sauberlich and colleagues (1987) conducted a controlled depletion-repletion metabolic study (28 days of depletion followed by 64 days of graded repletion phases, each phase lasting 21 days) with nonpregnant women. Plasma and erythrocyte folate concentrations continued to fall in response to repletion with 100 μg of food folate (equal to 100 μg DFEs) for 21 days. This continued depletion led to the conclusion that 100 μg of dietary folate is below the average requirement.

Jacob and coworkers (1994) conducted a controlled depletion-repletion metabolic study (30 days depletion at 25 μg/day of folate [25 μg DFEs] followed by 15 days repletion at 151 μg/day of DFEs) with adult males. Although 150 μg of DFEs was insufficient to decrease the elevated plasma homocysteine concentration below 16 μmol in 4 of the 10 subjects or to return plasma folate to predepletion concentrations, the repletion period was too short to allow appropriate evaluation of the primary response variables. Thus, no conclusion about the adequacy of 150 μg/day of DFEs can be reached from this study.

Approximately 200μg/day of DFEs. Sauberlich and colleagues (1987) evaluated the repletion response of two subjects and reported that erythrocyte folate continued to fall in response to 200 μg of food folate (200 μg DFEs) for 21 days. Data are not sufficient for estimating the erythrocyte folate response to a longer repletion phase.

Milne and coworkers (1983) used serum and erythrocyte folate to evaluate maintenance of folate status in 40 men consuming 200 μg/day of food folate (200 μg DFEs) for periods of 2 to 8 months. Both serum and erythrocyte folate decreased significantly over time regardless of initial status but not below the cutoff values of 7 nmol/L (3 ng/mL) and 305 nmol/L (140 ng/ml), respectively. This study was designed primarily for a different purpose, however, and had several limitations for the estimation of average requirements: the diet was changed during the study, subjects were included for different periods of time, and some of the subjects resumed their normal diet (for 10 days to 2 months) during the study. Thus, the findings from this study were judged equivocal.

Approximately 320μg/day of DFEs. O'Keefe and colleagues (1995) conducted a controlled metabolic study in which five women were fed a diet that provided 319 μg/day of DFEs (30 μg from food sources and 170 μg from folic acid). Three of the five had erythrocyte folate concentrations less than 305 nmol/L (140 ng/mL) and serum folate concentrations less than 7 nmol/L (3 ng/mL). Two of the subjects had elevated homocysteine concentrations (greater than 16 μmol/L) and a third subject had a homocysteine concentration greater than 14 μmol/L. (These data were obtained directly from the investigators of the published study.) These findings suggest that approximately half would have had normal erythrocyte folate and plasma homocysteine concentrations if 320 μg/day of DFEs had been consumed.

Approximately 500μg/day of DFEs. O'Keefe and coworkers (1995) fed subjects 270 μg as folic acid with 30 μg of food folate, corresponding to 489 μg of DFEs. This level of intake maintained normal plasma homocysteine, erythrocyte folate, and serum folate values with no significant increase or decrease throughout the 70-day maintenance study. Therefore, 489 μg/day of DFEs could be considered to be above the average requirement.

Summary. Of the controlled metabolic studies reviewed above, greatest weight was given to the study by O'Keefe for five reasons: (1) it was designed as a maintenance study for the purpose of estimating the folate requirement; (2) although it included only five subjects, this sample size exceeds that in the Sauberlich study, which was also rigorously controlled; (3) it evaluated the metabolic response of homocysteine in addition to erythrocyte and serum folate; (4) the diet was fed for 70 days in contrast to very short repletion phases in other metabolic studies (i.e., 15 days [Jacob et al., 1994], and 21 days [Sauberlich et al., 1987]); and (5) it provided folate largely in the form of folic acid, thus minimizing the possibility that folate intake was underestimated. Moreover, considering the evidence that problems with methods have led to underestimates of the folate content of food, it is likely that the subjects in the Sauberlich et al. (1987) and Milne et al. (1983) studies received more folate than reported.

Other Evidence Considered

Epidemiological data support an Estimated Average Requirement (EAR) of approximately 320 μg/day of DFEs. A primary example is the study by Selhub and colleagues (1993). In this study the prevalence of a homocysteine value greater than 14 μmol/L was significantly greater among individuals in the lowest four deciles of folate intake (less than 280 μg/day) as determined from a food frequency questionnaire. Reported intakes in this study were obtained prior to folate fortification and do not include supplements, but they include synthetic folic acid from ready-to-eat or cooked cereals (which frequently contained added folate) and thus would be higher if expressed in DFEs.

The amount of folate utilized daily has been estimated by measuring the catabolic products excreted in the urine and then expressing the sum as folate equivalents by multiplying the value by two (because the molecular weight of folate is approximately two times that of catabolites) (McPartlin et al., 1993). This approach may underestimate folate requirements because folate coenzymes may be recycled and not catabolized when utilized and because measurement of urinary catabolites does not account for endogenous folate lost from the body as a mixture of catabolites and intact folates in the feces (Caudill et al., 1998).

Results of other studies were considered (Table 8-3). Several (Gailani et al., 1970; Herbert, 1962a, b; Zalusky and Herbert, 1961) were found less useful than the previously cited metabolic studies for estimating the folate requirement because the diets were deficient in more than one nutrient. Ancillary information is provided by the studies using stable isotope methods to estimate in vivo folate pool size and the rate of daily utilization. With use of the estimate of the total body pool folate of 20 mg as extrapolated from liver folate measurements (Hoppner and Lampi, 1980; Whitehead, 1973), and the assumption of a 1 percent daily turnover rate of folate (Krumdieck et al., 1978; Stites et al., 1997; Von der Porten et al., 1992), the daily quantity of folate utilized is calculated to be approximately 200 μg. When 200 μg/day is corrected for the 50 percent bioavailability of food folate, the DFE is 400 μg/day.

TABLE 8-3

Additional Studies of the Folate Status of Adults.

Folate EAR and RDA Summary, Ages 19 through 50 Years

With greatest weight given to the metabolic maintenance study by O'Keefe along with data considered from the other studies reviewed above, it was concluded that the data support an EAR of approximately 320 μg/day of DFEs for the age group 19 through 50 years. A special recommendation is made for women capable of becoming pregnant (see “Recommendations for Neural Tube Defects Risk Reduction”).

The RDA for folate is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for folate; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for folate the RDA is 120 percent of the EAR).

Evidence Considered in Estimating the Average Requirement

The EAR for men and women ages 51 years and older is based on evaluation of three types of studies: metabolic (Jacob et al., 1998), observational folate status assessment of population subgroups (Bates et al., 1980; Garry et al., 1982, 1984; Jägerstad, 1977; Jägerstad and Westesson, 1979; Koehler et al., 1996; Ortega et al., 1993; Rosenberg, 1992; Sahyoun et al., 1988), and epidemiological (Selhub et al., 1993).

Jacob and colleagues (1998) conducted a depletion-repletion metabolic study in eight post-menopausal women aged 49 to 63 years. A folate depletion diet (56 μg/day [56 μg/day of DFEs]) was fed for 35 days, followed by three repletion periods in which graded amounts of folate were added to the diet. After being converted to DFEs, the three repletion amounts were 150, 450, and 850 μg/day for 28, 13, and 8 days, respectively. Plasma homocysteine concentrations remained elevated (greater than 12 μmol/L) in five of the eight women in response to either 150 or 450 μg/day of DFEs. Plasma folate remained low (less than 7 nmol/L [3 ng/mL]) in five of the eight subjects in response to 150 μg/day of DFEs but returned to normal in all subjects in response to 450 μg/day. The short repletion periods limit conclusions regarding the adequacy of these intake levels. From the plasma folate changes, which do respond quickly, it appears that 450 μg/day was adequate for all subjects and 150 μg/day was inadequate for a large percentage of the group. Extrapolating from these data, approximately 300 μg/day would result in normal folate status in approximately 50 percent of the group and would therefore be consistent with an EAR of 320 μg/day.

The observational folate status assessment studies that provide data on both folate intake and biochemical measures of folate status (Table 8-4) provide evidence that tends to support an EAR for older adults that is equivalent to that for younger adults: 320 μg/day of DFEs. Data from Selhub and colleagues (1993) (Figure 8-1) show that the mean homocysteine concentration begins to stabilize when folate intakes are approximately 300 μg/day. Figure 8-2 presents data showing the relationship of plasma homocysteine to plasma folate concentrations (Lewis et al., 1992).

TABLE 8-4

Observational Status Assessment Studies Considered in Setting the Estimated Average Requirement (EAR) for Folate in the Elderly.

FIGURE 8-1

Mean plasma homocysteine (Hcy) concentrations (and 95% confidence intervals) by deciles of intake of folate. Means are adjusted for age, gender, and other vitamin intakes. Asterisk indicates significantly different from mean in the highest decile (p < (more...)

FIGURE 8-2

Relationship of plasma homocysteine concentrations to plasma folate concentrations in 209 adult males. A indicates lower limit of normal plasma folate as used by the Second National Health and Nutrition Examination Survey (6.8 nmol/L). B indicates lower (more...)

Folate EAR and RDA Summary, Ages 51 Years and Older

Data from metabolic folate status assessment and epidemiological studies support an EAR for adults ages 51 years and older of 320 μg/day of DFEs. The EAR for this age group is expected to be the same as that for younger age groups because the aging process does not appear to impair folate absorption or utilization nor do studies separate those over age 70 from those 51 to 70 years.

The RDA for folate is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for folate; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for folate the RDA is 120 percent of the EAR).

Evidence Considered in Estimating the Average Requirement

For pregnant women the maintenance of erythrocyte folate, which reflects tissue stores, was selected as the primary indicator of adequacy. When this indicator was not measured, serum folate was evaluated with the recognition that hemodilution contributes to a normal reduction in serum folate concentration during gestation. Homocysteine concentrations have not been shown to reflect folate status during pregnancy, possibly because of hormonal changes, hemodilution, or other unknown factors associated with pregnancy (Andersson et al., 1992; Bonnette et al., 1998).

Population-Based Studies. A number of population-based studies confirm that folic acid consumed in conjunction with diet prevents folate deficiency in pregnant women as assessed by maintenance of normal folate concentration in erythrocytes, serum, or both. The folate has been provided either by supplements (Chanarin et al., 1968; Dawson, 1966; Hansen and Rybo, 1967; Lowenstein et al., 1966; Qvist et al., 1986; Willoughby, 1967; Willoughby and Jewel, 1966) or fortified food (Colman et al., 1975) (see Table 8-5).

TABLE 8-5

Supplementation Studies in Pregnancy.

Willoughby and Jewel (1966, 1968) conducted a series of studies involving approximately 3,500 pregnant women beginning at 12 weeks of gestation who were assigned to different levels of folate supplementation (0, 100, 350, or 450 μg/day). Their dietary folate was estimated to be less than 100 μg/day. A supplementation level of 100 μg/day in conjunction with the low-folate diet was insufficient to prevent deficient (less than 7 nmol/L [3 ng/mL]) serum concentrations in 33 percent of the group (Willoughby and Jewel, 1966) or to prevent megaloblastic anemia in 5 percent of the group (Willoughby, 1967). In contrast, 300 μg/day of supplemental folate was sufficient to maintain a mean serum folate concentration that was comparable with the mean in healthy nonpregnant control subjects (Willoughby and Jewel, 1966) and to prevent megaloblastic anemia (Willoughby, 1967). These data agree with those of Dawson (1966), who found that taking 150 μg/day of folate supplements (beginning at 28 weeks) in addition to diet resulted in low serum folate concentrations (less than 7 nmol/L [3 ng/mL]) in 30 percent of the group at delivery. Also confirming these findings, Hansen and Rybo (1967) reported that 100 μg of folic acid plus diet was not sufficient to prevent serum folate reduction (defined as less than 4 nmol/L [2 ng/mL]) in 15 percent of the group whereas a folate supplement of 500 μg/day resulted in a mean serum folate concentration of 13 nmol/L(6 ng/mL) at 36 to 38 weeks of gestation.

Lowenstein and colleagues (1966) compared serum and erythrocyte folate and bone marrow morphology of women taking 500 μg/day of supplemental folate with those of women taking a placebo. In the folate-supplemented women, mean serum and erythrocyte folate levels were approximately 21 and 870 nmol/L (10 and 400 ng/mL), respectively, at 36 and 38 weeks of gestation and postpartum. Bone marrow aspirates at 38 weeks were essentially normal. In contrast, a large percentage of the placebo-treated subjects had serum and erythrocyte folate concentrations that were less than normal.

Chanarin and colleagues (1968) compared erythrocyte folate concentrations in 103 pregnant women supplemented with 100 μg of folate from 25 weeks of gestation until delivery with those of 103 unsupplemented pregnant control subjects. Dietary intake was analyzed in 111, 24-hour duplicate diets and reported to be 676 μg/day. Supplementation of the usual diet with 100 μg/day resulted in maintenance of erythrocyte folate concentration throughout pregnancy whereas a significant reduction in erythrocyte folate was observed in the unsupplemented subjects.

Colman et al. (1975) evaluated the efficacy of folate-fortified maize to maintain erythrocyte folate concentrations in 70 pregnant women. Erythrocyte folate response was compared between women receiving maize fortified to provide 300, 500, or 1,000 μg of folic acid and a control group. (Additional groups consumed folic acid in tablet form to assess the relative bioavailability of the fortified maize.) Maize containing 300 μg of folic acid in addition to dietary folate, the lowest level tested, was effective in preventing the progression of folate depletion in the eighth month of pregnancy.

Controlled Metabolic Study. Caudill and colleagues (1997) conducted a metabolic study in which either of two levels of folate was consumed for 12 weeks by pregnant women during the second trimester (14 weeks to 25 weeks of gestation). Their folate status was compared with that of nonpregnant control subjects. Folate was provided as a combination of dietary folate (120 μg/day) and folic acid (either 330 or 730 μg/day) consumed with the diet. After correcting for bioavailability, the intakes of the groups were approximately 60 μg/day of DFEs and more than 1,300 μg/day of DFEs, respectively. Folate status was normal (serum folate greater than 7 nmol/L [3 ng/mL] and erythrocyte folate values greater than 305 nmol/L [140 ng/mL]) in all subjects consuming the diet with 680 μg/day of DFEs and was not different from that of the nonpregnant control subjects with the same folate intake.

The data provided by the only diet-controlled metabolic study that has been conducted in pregnant women (Caudill et al., 1997) agree with the findings from the population studies and confirm that a combination of approximately 300 μg of folate from supplements, fortified food, or both plus dietary folate (assumed to be approximately 100 μg/day before folate fortification) has been shown to be sufficient to maintain normal folate status during pregnancy. When expressed as DFEs, the consistent finding across the numerous population studies and the controlled metabolic study is that 600 μg/day of DFEs is adequate to maintain normal folate status.

Three of the four studies provided data that 100 to 150 μg/day of supplemental folate plus a low-folate diet was inadequate to maintain normal serum and hematological indices, which were the only outcomes measured in all of the subjects. The accuracy of the dietary estimates could not be ascertained, but they were lower than the one analyzed intake estimate (676 μg/day) reported by Chanarin and coworkers (1968).

Other Evidence Considered. McPartlin and colleagues (1993) quantitated the urinary excretion of the major folate catabolites in six pregnant women and six nonpregnant control subjects. These investigators converted the quantity of urinary catabolites to urinary folate equivalents and estimated that the recommended folate intake for second-trimester pregnant women would be 660 μg/day.

Folate EAR and RDA Summary, Pregnancy

From these data, low dietary folate intake plus 100 μg of supplemental folate (equivalent to approximately 200 μg/day of DFEs) is inadequate to maintain normal folate status in a significant percentage of population groups assessed. The EAR therefore was derived by adding this quantity in DFEs (200 μg/day) to the EAR for non-pregnant women (320 μg/day) to provide an EAR of 520 μg/day of DFEs.

The RDA for folate is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for folate; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for folate the RDA is 120 percent of the EAR). Data from the controlled metabolic study support an RDA of 600 μg/day of DFEs based on maintenance of normal erythrocyte folate concentrations and agree with the findings from the series of population studies that 600 μg/day of DFEs is adequate to maintain normal folate status in groups of pregnant women.

Method Used to Estimate the Average Requirement

The EAR for the lactating woman is estimated as the folate intake necessary to replace the folate secreted daily in human milk plus the amount required by the nonlactating woman to maintain folate status. The average daily amount of folate secreted in human milk is estimated to be 85 μg/L, as described in the previous section “Infants Ages 0 through 12 Months” (see “Human Milk”). The dietary intake needed to provide this amount must account for the estimated 50 percent bioavailability of food folate (see “Bioavailability”).

Other Evidence Considered

There are no metabolic studies in which lactating women consumed controlled amounts of dietary folate. It is unclear whether the reduction in maternal folate concentration observed in lactating women (Keizer et al., 1995; Qvist et al., 1986; Smith et al., 1983) is related to the discontinuation of use of prenatal folate supplements, loss of maternal body folate stores, or other factors. For example, in a recent study of lactating adolescents (Keizer et al., 1995), both breastfeeding mothers and mothers of formula-fed infants showed a decline in erythrocyte folate between 4 and 12 weeks postpartum, suggesting that the postpartum decline in folate status may not be related to lactation. The decrease was prevented by supplemental folate (300 μg/day).

In a recent study in which folate status was compared in supplemented and nonsupplemented lactating women (Mackey et al., 1997), dietary folate intake was estimated to be 400 μg/day. In the unsupplemented lactating women, plasma homocysteine concentrations increased significantly but remained well within the normal range (6 to 7 μmol/L); this increase, therefore, does not appear to be of nutritional significance.

Folate EAR and RDA Summary, Lactation

The calculation used to obtain the extra amount of folate needed to cover lactation is

When this quantity is added to the EAR for the nonlactating non-pregnant woman (320 μg/day), the result is rounded down, giving an EAR of 450 μg/day of DFEs. Women who are only partially breastfeeding would need less.

The RDA for folate is set by assuming a coefficient of variation (CV) of 10 percent (see Chapter 1) because information is not available on the standard deviation of the requirement for folate; the RDA is defined as equal to the EAR plus twice the CV to cover the needs of 97 to 98 percent of the individuals in the group (therefore, for folate the RDA is 120 percent of the EAR).

Classification, Anatomy, and Embryology

NTDs are the most common major congenital malformations of the central nervous system. They arise as a result of a disturbance of the embryonic process of neurulation and are midline defects that affect neural tissues, their coverings anywhere along the neuraxis, or both. They are heterogeneous malformations, and the terms used to define them here (see Box 8-1) are based on clinical descriptions and the presumed embryological defect (Lindseth, 1996; Volpe, 1995). The terminology in the literature may vary.

BOX 8-1

Forms of Neural Tube Defects. Anencephaly: a fatal form characterized by partial absence of brain tissue, presumably caused by failure of closure of the anterior neuropore. Meningomyelocele: a midline defect of the spinal cord in which the neural tissue (more...)

NTDs are not to be confused with spina bifida occulta, a common radiographic finding that does not involve neural elements, or encephalocele, a protrusion of meninges and brain tissue outside the cranium, most frequently in the occipital region.

In the less-severe forms of NTD a child may otherwise be normal and, with appropriate surgical and medical care, can lead a productive life, including parenthood. Less than 20 percent of NTDs show associations with malformations in nonneural tissues, chromosomal defects, or specific genetic syndromes (Khoury et al., 1982). Techniques have been established for prenatal screening for NTDs by measuring maternal serum and amniotic fluid α-fetoprotein and by fetal ultrasound (Hobbins, 1991).

Neurulation is the first organogenetic process to be initiated and completed. It begins in the human at approximately 21 days post-fertilization and is complete by 28 days. Thus, neurulation is ongoing at the time that a woman may first recognize her pregnancy by a missed menstrual period. Closure of the neural tube begins separately and consecutively in at least three sites: the cervical-hindbrain boundary, the forebrain-midbrain boundary, and the rostral extremity of the forebrain. Closure spreads to the intervening regions with completion of neural tube formation at neuropores in the forebrain (anterior neuropore), the hindbrain, and the lumbosacral region (posterior neuropore). NTDs appear to arise from failure or inadequacy of this closure process. Different forms of NTD could arise at different times in neurulation, possibly from distinct mechanisms. Although many specific molecules are involved in the successful completion of neurulation, none have been implicated in the mechanisms underlying the common human NTDs.

Prevalence of NTDs

United States and Canada. National birth-defect registry data are not available, but a decrease in the prevalence of NTDs at birth has been observed during the past 30 years. This is not entirely explained by increased widespread prenatal screening and diagnostic techniques (De Wals et al., 1999; Yen et al., 1992). Although the comparison of results of studies using different methods for case identification should be made cautiously, regional variation in the risk of NTDs is likely (Table 8-6). It is not known whether the especially low rate observed in Hawaii is caused by genetic or by environmental factors (Cragan et al., 1995). The populations studied included women who had taken vitamin supplements at the time of conception, but the frequency of folate supplementation was estimated only in California (Velie and Shaw, 1996).

TABLE 8-6

Total Prevalence Rates of Neural Tube Defect in Selected Areas of North America, from Birth Defect Registry Data, 1985–1994.

Other Countries. The incidence of the common forms of NTD varies worldwide from less than 1 to approximately 9 per 1,000 total births, with the highest incidence reported in Great Britain and Ireland (Copp and Bernfield, 1994). Other populations with high incidence include northern Chinese and Australian Aborigines (Bower et al., 1984; Moore et al., 1997). Although Sikhs have a high NTD incidence, the defects are often thoracic and associated with minimal deficit, suggesting a distinct etiology (Baird, 1983). The decrease in NTDs among Irish immigrants to the United States could be explained by genetic dilution through interethnic marriages. However, some studies of migrant populations in which NTD incidence decreases with changes in locale suggest a nutritional etiology (Borman et al., 1986; Carter, 1974).

Etiology of NTDs

The causes of these abnormalities have been the subject of intensive research over many decades. Differences in the pathogenesis and the epidemiology of different categories of NTD have led to the idea that NTDs are highly heterogeneous in etiology (Dolk et al., 1991). Substantial familial aggregation indicates that anencephaly, myelomeningocele, and craniorachischisis are related pathogenetically and genetically. Evidence from epidemiological studies of NTDs indicates that heredity is a major contributor. Indeed, the recurrence risk in a sibling birth is 3 to 5 percent (Laurence, 1990). For the most cases the inheritance is believed to be polygenic, potentially involving multiple genes. Such polygenic traits are influenced by environmental factors, thus the etiology appears to be multifactorial (Laurence, 1990). Recently, attention has turned to assessing the genetic basis of NTD and to evaluating the role of vitamins, specifically folate, in the prevention of NTDs. Coverage of other risk factors for NTDs is beyond the scope of this report.

Genetic Evidence. An assessment of heritability for common forms of NTDs has been put at 60 percent (Emery, 1986). Data from demographic, family, and mouse model studies have prompted a search for candidate genes that predispose individuals to an NTD. A defect in enzymes involved in homocysteine metabolism is suggested by altered folate, vitamin B₁₂, homocysteine, and methylmalonate values in mothers of infants with NTDs (Mills et al., 1995; Steegers-Theunissen et al., 1994); the prevention of some human NTDs by folate administration; and the prevention of NTDs in some rodent models by methionine (Essien, 1992; Vanaerts et al., 1994). These enzymes are 5,10-methylenetetrahydrofolate reductase (MTHFR), cystathionine β-synthase, and methionine synthase (Figure 8-3). Interestingly, families with homocystinuria caused by severe mutations in genes for each of these enzymes do not exhibit NTDs (Haworth et al., 1993; Kang et al., 1991b). Moreover, deletion of cystathionine β-synthase in the mouse yields a phenocopy of human homocystinuria but not an NTD (Watanabe et al., 1995). Subsequent work on other genetic markers of risk has not provided conclusive results (van der Put et al., 1997a, b).

FIGURE 8-3

Major pathways depicting involvement of vitamin B₁₂ and folate in homocysteine metabolism.

Two studies have shown a statistically significant association between mothers of children with NTDs and a common variation within the gene for MTHFR (van der Put et al., 1995; Whitehead et al., 1995). This gene codes for a thermolabile variant of the enzyme that shows about 50 percent enzymatic activity and is associated with elevated serum homocysteine concentrations (Kang et al., 1991a). This association would account for approximately 15 percent of NTD cases (van der Put et al., 1995). The polymorphism was recently associated with low erythrocyte folate values (Molloy et al., 1997), which suggests that these values by themselves could account for the increased NTD risk. However, similar studies using linkage assessments are needed in suitable (genetically homogeneous) NTD populations with adequate numbers and types of controls.

No correlation has been found between two common mutations in cystathionine β-synthase and NTD prevalence in an Irish population (Ramsbottom et al., 1997). The gene for methionine synthase has only recently been cloned in mammals (Chen et al., 1997; Li et al., 1996). There are no reports of an association between mutations of this gene and NTDs.

The genetic mouse models of NTD suggest a variety of other candidate genes for human NTDs (Baldwin et al., 1992; Tassabehji et al., 1993). However, no reports assess whether any of the causative genes for mouse models show linkage with the common forms of human NTD. Because the likely heterogeneity of human NTDs may make it impossible to demonstrate linkage for any one candidate gene, candidate genes will need to be assessed in individuals with NTDs by sequence analysis.

A summary of evidence from animal studies on the etiology of NTD appears in Appendix M. Animal models of NTD have been examined and manipulated to elucidate the mechanisms of abnormal neurulation and to test etiologic hypotheses suggested by human epidemiological data.

Nutrition Evidence. Studies of migrant populations suggest a nutritional etiology for NTD (Borman et al., 1986; Carter, 1974). Lower socioeconomic class also correlates with NTD incidence (Elwood and Elwood, 1980; Laurence, 1990). Differences in diet and in supplement use could contribute to the inverse relationship of socioeconomic status with incidence of NTD, but this has not been analyzed in recent years.

Nutritional markers, particularly maternal serum vitamin B₁₂ and serum and erythrocyte folate, as well as their metabolic indicators of adequacy, have been assessed in relation to the risk of NTD. The results have been inconsistent, some showing no association with NTD prevalence (Wald, 1994). Others have demonstrated low or low normal levels of both vitamin B₁₂ and erythrocyte folate and suggested that both vitamins represent independent risk factors for NTD (Kirke et al., 1993). Methylmalonic acid is elevated in maternal serum of midterm NTD pregnancies (Adams et al., 1995). Some of the women who gave birth to infants with NTDs had elevated homocysteine values (Mills et al., 1995; Steegers-Theunissen et al., 1994). These studies support the proposition that NTD is associated with altered status of vitamin B₁₂, folate, or both during pregnancy.

Teratology Studies. Drugs identified as causes of NTD in humans include folate antagonists (specifically aminopterin, previously used as an antitumor agent) (Thiersch, 1952); carbamazepine (Rosa, 1991) and valproate (commonly used antiepileptic drugs) (Blaw and Woody, 1983; Gomez, 1981; Stanley and Chambers, 1982); and retinoids, including isotretinoin (used to treat acne) (Dai et al., 1989; Hill, 1984) and etretinate (used to treat psoriasis) (Happle et al., 1984). Clomiphene (an oocyte maturation agent) is also suspected as a teratological cause of human NTDs (Wilson, 1973). These agents account for less than 0.1 percent of all NTDs. In general, the induced malformations are not restricted to NTD, and the precise mechanisms of these teratological effects are not clear. Indeed, as with other teratogens, the pharmacological and teratological mechanisms may differ because the embryo, especially at neurulation stages or earlier, is a qualitatively different organism from other developmental stages and the adult.

Risk of NTD According to Maternal Intake of Folate

The possibility that folate might be involved in NTD was first raised by Hibbard (1964). This was followed by observational studies of the effect of both dietary folate and folate supplements on NTD (Table 8-7), nonrandomized intervention studies of folate supplementation (Table 8-8), and randomized prevention studies, most of which were conducted with women who had prior NTD pregnancies (Table 8-8). The best evidence comes from the four randomized prevention trials (Czeizel and Dudas, 1992; Kirke et al., 1992; Laurence et al., 1981; Wald et al., 1991), but the observational evidence (Table 8-7) strongly supports the intervention studies and provides the only evidence concerning dietary folate.

TABLE 8-7

Observational Studies of Folate and Risk of Neural Tube Defect.

TABLE 8-8

Controlled Trials Relating Folate Supplementation and Risk of Neural Tube Defect in the Periconceptual Period.

NTD Risk Associated with Different Levels of Dietary Folate. Data from two observational studies (Shaw et al., 1995c; Werler et al., 1993) indicate a statistically significant decreasing risk of NTD with increasing dietary folate in unsupplemented women. In both studies, the median dietary folate in the control group was approximately 300 μg/day. In the hospital-based case-control study carried out from 1988 to 1991 (Werler et al., 1993) (Tables 8-7 and 8-9), mothers were interviewed by telephone within 6 months after delivery. The interview included detailed questions about use of vitamin supplements and a semiquantitative food frequency questionnaire. In the population-based study from 1989 to 1991 (Shaw et al., 1995c) (Tables 8-7 and 8-9), folate supplement use and dietary folate intake during the periconceptional period were retrospectively assessed by using a face-to-face interview and a semiquantitative food frequency questionnaire with mothers of children with NTDs and randomly selected controls. Interviews were completed an average of 5 months after delivery. The proportion of women reporting no use of a folate supplement before conception or in the first trimester was 39 percent (207/526) for cases and 29 percent (149/523) for controls (Velie and Shaw, l996). From these data, the average risk of NTD in the fraction of the population taking no supplement can be estimated to be 1.3 per 1,000.

TABLE 8-9

Relative Risk of Neural Tube Defect Based on Reported Folate Intake During the Periconception Period.

In a case-control study in Australia, a negative association was found between NTD occurrence and free and total folate intake in early pregnancy (Bower and Stanley, 1989). However, the published results contain only the combined data on supplement use and dietary intake.

The results from the studies of Werler et al. (1993) and Shaw et al. (1995c) can be used to draw a tentative risk curve. Point estimates in the two studies are remarkably concordant (Figure 8-4). There is a quasilinear decreasing NTD risk for dietary folate values between 100 and 400 μg/day but no further decrease is observed for higher intake values. A possible explanation for the risk observed at higher intakes could be overreporting of consumption of folate-rich foods by some women. Also, imprecision in risk estimates because of the small sample numbers cannot be excluded.

FIGURE 8-4

Risk of neural tube defect (NTD) according to folate intake based on two retrospective studies. Intake values appear next to each point, in micrograms. Midpoint values for each reference intake category have been used for defining folate intake, and relative (more...)

NTD Risk Associated with Periconceptional Folate Supplement Use. The only randomized trial on the effect of periconceptional vitamin supplementation (800 μg/day of folic acid) on the risk of a first occurrence of NTD was prematurely terminated after 4,753 women had been enrolled (Czeizel and Dudas, 1992) (Table 8-8). No case of NTD was observed in the group taking multivitamins containing 800 μg of folate daily compared with six cases in the group receiving a trace element supplement (p = 0.029). The effect of supplemental folate alone was not assessed. Although no protective effect was observed in one case-control study (Mills et al., 1989), a significant reduction in risk associated with supplementation was seen in one cohort (Milunsky et al., 1989) and four other case-control studies (Bower and Stanley, 1992b; Mulinare et al., 1988; Shaw et al., 1995c; Werler et al., 1993) (Table 8-7). The optimal timing for supplementation seems to be during the 4 weeks before and after conception (Mulinare et al., 1988).

In the United States the risk reduction achieved with a daily supplement of 400 μg of folate, the most usual dose in multivitamins, was 70 percent (relative risk 0.3; 95 percent confidence interval [CI], 0.2 to 0.6) in New England (Werler et al., 1993) and 35 percent (relative risk 0.65; 95 percent CI, 0.45 to 0.94) in California (Shaw et al., 1995c) in unselected populations with average daily dietary folate intake of about 300 μg. The intake values assume a high level of compliance with supplementation, and the points represent values adjusted for bioavailability because they are given in dietary folate equivalents. It is not clear whether supplements at doses lower than 400 μg/day of folic acid provide the same level of protection as 400 μg/day or whether higher doses are associated with increased risk reduction.

The Medical Research Council Trial (Wald et al., 1991) (Table 8-8), which addressed reduction of the recurrence of NTD, used a factorial design to investigate folate in a dose of 4.0 mg/day and a mixture of other vitamins (A, thiamin, riboflavin, B₆, C, D, and nicotinamide). This study found a 71 percent decreased NTD incidence in offspring of women taking the folate supplement relative to those on no supplements but no reduction with the other vitamins. In a nonrandomized trial on the risk of NTD recurrence conducted in the United Kingdom, a daily supplement of 360 μg in addition to normal diet was apparently protective (Smithells et al., 1981).

Because NTDs represent a heterogeneous group of congenital malformations both etiologically and pathogenetically, it is probable that there are cases not preventable even by large doses of folate, as was the case in the Medical Research Council Vitamin Study (Wald et al., 1991). More studies are needed to evaluate whether fortification of foods is similarly associated with reduced risk or with a valid proxy for NTD risk.

NTD Risk According to Maternal Folate Status. The erythrocyte folate concentration is a marker of long-term folate status. Studies looking for an association of erythrocyte folate with NTD risk based on estimating erythrocyte folate levels in blood samples taken early in pregnancy are preferred because maternal folate status is likely to change during pregnancy and postpartum. In four studies with blood specimens taken during pregnancy, erythrocyte folate values were higher in women with a normal pregnancy than in women carrying a fetus with an NTD (Kirke et al., 1993; Laurence et al., 1981; Smithells et al., 1976) or with a fetus having another type of malformation (Bunduki et al., 1995). This difference was not found in one study with only eight NTD cases (Economides et al., 1992).

In a case-control study in three maternity hospitals in Dublin, Ireland, from 1986 to 1990, erythrocyte folate values were measured in frozen samples taken at a median gestational age of 15 weeks (Daly et al., 1995; Kirke et al., 1993). The percentage of women using folate supplements was 5 percent. A negative apparently nonlinear association was observed between NTD risk and erythrocyte folate concentration (Table 8-10). It is not known whether the risk would continue to decrease as erythrocyte folate values increased to higher than 1,241 nmol/L (570 ng/mL), which was the mean erythrocyte concentration of the controls who had concentrations in the highest category in Table 8-10. However, the population studied had a relatively high incidence of NTD, around 2 per 1,000 births. Extrapolation of results should be made with great care because the NTD risk in the U.S. population could be lower at every level of erythrocyte folate.

TABLE 8-10

Distribution of Cases and Controls and Risks of Neural Tube Defect (NTD) by Erythrocyte Folate Concentration.

Determinants of Erythrocyte Folate. In a recent study in women aged 22 to 35 years in the Minneapolis-St. Paul area, folate supplements and folate-fortified cereals were found to be independent predictors of erythrocyte folate levels (Brown et al., 1997). An apparently nonlinear correlation was observed between folate intake from various sources and erythrocyte folate. These results are concordant with those of a controlled experiment in women who were randomly assigned to receive 0, 100, 200, or 400 μg/day of supplemental folate (Daly et al., 1997). In this randomized placebo trial, Daly and coworkers estimated the quantity of additional folate associated with an erythrocyte folate concentration of greater than 870 nmol/L (400 ng/mL), which is the amount previously shown to be associated with a significant reduction in NTD risk. The initial erythrocyte folate concentrations of the women were in the normal range (327 to 870 nmol/L [150 to 400 ng/mL], median 707 nmol/L [325 ng/mL]). The median incremental changes in erythrocyte folate concentration in the 100-, 200-, and 400-μg/day groups were + 146 nmol/L (67 ng/mL), + 283 nmol/L (130 ng/mL), and + 435 nmol/L (200 ng/mL), respectively.

The relative effectiveness of different interventions in increasing erythrocyte folate concentrations was evaluated in a 3-month randomized trial in 62 healthy women aged 17 to 40 years in Northern Ireland (Cuskelly et al., 1996). Erythrocyte folate concentrations improved significantly only in the groups taking folate supplements or food fortified with folate; there was no increase in the group provided extra food folate or dietary advice. Because food intake was not controlled, further studies are needed to evaluate more precisely the relative efficacy of different supplementation regimens in reducing NTD risk.

Mechanism. The mechanism by which folate could reduce NTD risk is not known. Increasing folate intake and thus the concentrations of folate derivatives in tissues might overcome a metabolic deficiency in the production of proteins or in DNA synthesis at the time of neural tube closure (Mills et al., 1995). Another hypothesis is that folate does not prevent the occurrence of NTD but selectively increases the abortion rate of affected fetuses (Hook and Czeizel, 1997). Certainly, more research is needed to understand the effect of folate on embryonic and fetal development.

Recommendations for NTD Risk Reduction

To summarize the data, a reduced risk of NTD has been observed for women who took a folate supplement of 360 to 800 μg/day in addition to a dietary folate intake of 200 to 300 μg/day. Folate intake is positively associated with erythrocyte folate concentration (Bower and Stanley, 1989; Brown et al., 1997; Cuskelly et al., 1996; Daly et al., 1997), and NTD risk is inversely associated with both folate intake (Bower and Stanley, 1989; Shaw et al., 1995c; Werler et al., 1993) and erythrocyte folate concentration (Daly et al., 1995).

Although it is recognized that there are still uncertainties about the relationships among folate intake, erythrocyte folate, and NTD risk and the extent to which there are differences in the absorption of folate from food compared with supplements, the evidence is still judged sufficient to support a recommendation to reduce the risk of NTD. The recommendation made here for women capable of becoming pregnant is for intake that exceeds the Recommended Dietary Allowance (RDA) for folate. In particular, it is recommended that women capable of becoming pregnant consume 400 μg of folate daily from supplements, fortified foods, or both in addition to consuming food folate from a varied diet. At this time the evidence for a protective effect from folate supplements is much stronger than that for food folate. It is certainly conceivable that, if taken in adequate quantity, food folate will be shown to be as effective as folic acid, but it remains to be demonstrated. When more data are available, this recommendation will be revised.

An even larger dose of folate has been recommended to prevent recurrence in women with a previous NTD-affected pregnancy (CDC, 1991). However, some NTDs are not prevented by increasing folate intake.

To date there is no conclusive evidence to support any population screening for genetic markers of NTD risk. In the event that the correlation between the 5,10-MTHFR T⁶⁷⁷ allele and NTD is confirmed, screening women for the gene that codes for the thermolabile variant would identify only about 15 percent of those at risk for NTD. Thus, recommending consumption of 400 μg folate daily from supplementation or fortified foods for all women capable of becoming pregnant would be a more effective prevention measure than screening for the variant (Mills and Conley, 1996).

Dysplasia and Metaplasia

Dysplastic and metaplastic changes have been reported to reverse in response to high-dose supplemental folate. Butterworth and coworkers (1982) conducted a prospective, controlled clinical intervention trial giving supplements of 10 mg/day of folate to 47 women with dysplastic changes in the epithelium of the uterine cervix. They observed a significant attenuation of dysplasia, but the alteration in cytology may have been an attenuation of dysplasia or simply a reduction in megaloblastic cellular changes. A subsequent intervention trial by the same research group (Butterworth et al., 1992b) was unable to reproduce the results. However, the subjects in this second intervention study initially had the lowest grade of dysplasia, which has a greater than 60 percent spontaneous rate of reversion to normal. Heimburger and colleagues (1988) observed a significant reduction in metaplastic change in bronchial epithelial tissue in response to 10 mg of folate with 500 μg of vitamin B₁₂ given daily for 4 months to 36 subjects compared with changes in 37 subjects given a placebo. These findings may be questioned because of spontaneous variation in bronchial cytology, small sample size, short duration of trial, and the very high doses of folate and vitamin B₁₂ used.

It has been hypothesized that poor folate status by itself is not carcinogenic but may enhance an underlying predisposition to cancer (Heimburger et al., 1987; Mason and Levesque, 1996). Support for this theory includes data from a case-control intervention trial of patients with cervical dysplasia who also were at significantly higher risk for cervical cancer because of cervical infection with human papilloma virus-16 (HPV-16) (Butterworth et al., 1992a). Subjects with the HPV-16 infection had a fivefold greater risk of having dysplasia if they also had diminished erythrocyte folate values (660 nmol/L [303 ng/mL]) (Butterworth et al., 1992a). On the basis of these data and other data from study of the colorectum (Lashner, 1993), Mason and Levesque (1996) suggested that even a minor decrease in folate status may promote carcinogenesis.

Potential mechanisms for folate-related enhancement of carcinogenesis include the induction of DNA hypomethylation (Kim et al., 1997), increased chromosomal fragility or diminished DNA repair (Kim et al., 1997), secondary choline deficiency, diminution in natural killer cell surveillance, misincorporation of uridylate for thymidylate in DNA synthesis, and facilitation of tumorigenic virus metabolism (Mason and Levesque, 1996).

Cervical Neoplasia

Although several studies suggest that increased consumption of folate reduces the relative risk of cervical neoplasia (Brock et al., 1988; Potischman et al., 1991; Verreault et al., 1989; Ziegler et al., 1990, 1991), statistical significance was not attained in these studies after adjustments were made for confounding variables. These studies had several limitations: folate intake was assessed with a food frequency instrument that had not been validated for folate intake (Mason and Levesque, 1996); because subjects were not stratified for HPV infections as was done by Butterworth and colleagues (1992a), the inverse association between folate intake and cervical neoplasia in high-risk subjects was not examined; and the subjects had either carcinoma in situ or invasive cancer—advanced stages of neoplasia that may be unresponsive to folate (Heimburger et al., 1987; Mason and Levesque, 1996). Therefore, the effect of folate status on carcinogenesis in the cervix remains uncertain.

Colorectal Cancer

Data supporting the modulation of carcinogenesis by folate status are the strongest for the colorectum. Patients with chronic ulcerative colitis are at increased risk for colonic cancer and also coexisting folate deficiency. Sulfasalazine, a drug taken chronically by these patients, inhibits folate absorption (Halsted et al., 1981) and metabolism (Selhub et al., 1978). Lashner and coworkers (1989) observed that the rate of colonic neoplasia was 62 percent lower in folate-supplemented patients with chronic ulcerative colitis than in unsupplemented patients and that sulfasalazine therapy was associated with an increase in the risk of dysplasia. These observations were not statistically significant but pointed to an important area of investigation. Lashner (1993) subsequently compared prospectively the erythrocyte folate concentrations in patients with neoplastic changes in the colorectum with those for disease-matched control patients without neoplasia. The mean erythrocyte concentration was significantly lower in the individuals with neoplasia (988 nmol/L [454 ng/mL]) than in the control patients (1,132 nmol/L [520 ng/mL]) but was still well within the normal range, which is in line with observations of erythrocyte folate concentrations and dysplasia in the uterine cervix (Butterworth et al., 1992a). Meenan and colleagues (1996) described the lack of association between erythrocyte folate levels and colonic biopsy specimens in healthy individuals, indicating the potential difficulty in predicting localized folate deficiency. In a subsequent report (Meenan et al., 1997), epithelial cell folate depletion occurred in neoplastic but not adjacent normal colonic mucosa.

In general, epidemiological studies support an inverse relationship between folate status and the rate of colorectal neoplasia (Mason and Levesque, 1996). Two large, well-controlled prospective studies support the inverse association between folate intake and incidence of colorectal adenomatous polyps (Giovannucci et al., 1993) and colorectal cancers (Giovannucci et al., 1995). In these two studies, moderate-to-high alcohol intake greatly increased the neoplastic risk of a low-folate diet. There was a significant 35 percent lower risk of adenoma in those in the highest quintile of folate intake (approximately 800 μg/day) relative to those in the lowest quintile (approximately 200 μg/day, relative risk approximately 0.65). The adverse effect of high alcohol intake coupled with a low-folate diet was confirmed by Glynn and colleagues (1996), who observed a significant fourfold increase in risk of colorectal cancer. Physicians' Health Study participants with the MTHFR polymorphism had reduced risk of colon cancer, but low folate intake or high alcohol consumption appeared to negate some of the protective effect (Ma et al., 1997) (see Appendix L for further discussion of MTHFR polymorphism).

More evidence for or against a causal relationship between folate status and colorectal cancer will be provided by data from prospective controlled intervention trials that are currently under way.

Lung, Esophageal, and Stomach Cancer

As reviewed by Mason and Levesque (1996), data are not sufficient for making conclusions regarding the possible role of folate in reducing the risk of cancer of the lung, esophagus, or stomach.

Reducing the Risk of NTD

Uncertainties still exist about the relationships among folate intake, erythrocyte folate, and NTD risk and about the extent to which the effect of food folate should be distinguished from the effect of folate from supplements or fortified foods, but the evidence is judged sufficient to support a specific recommendation to reduce the risk of NTD.

Reducing the Risk of Cardiovascular Disease, Cancer, and Psychiatric and Mental Disorders

The evidence that folate may reduce the risk of cardiovascular disease, certain types of cancer, and psychiatric and mental disorders is provocative and promising. However, it is not yet sufficiently substantiated and is somewhat conflicting. It is premature to consider reduction of any of these risks as a basis for setting an EAR or Adequate Intake (AI).

Adverse Effects

No adverse effects have been associated with the consumption of the amounts of folate normally found in fortified foods (Butterworth and Tamura, 1989). Therefore, this review is limited to evidence concerning intake of supplemented folate. The experimental data in animal studies and in vitro tissue and cell culture studies were considered briefly to determine whether they supported the limited human data.

Neurological Effects. The risk of neurological effects described in this section applies to individuals with vitamin B₁₂ deficiency. Vitamin B₁₂ deficiency is often undiagnosed but may affect a substantial percentage of the population, especially older adults (see Chapter 9). Three types of evidence suggest that excess supplemental folate intake may precipitate or exacerbate the neurological damage of vitamin B₁₂ deficiency. First, numerous human case reports show onset or progression of neurological complications in vitamin B₁₂-deficient individuals receiving supplemental folate (Table 8-12). Second, studies in monkeys (Agamanolis et al., 1976) and fruit bats (van der Westhuyzen and Metz, 1983; van der Westhuyzen et al., 1982) show that vitamin B₁₂-deficient animals receiving supplemental folate develop signs of neuropathology earlier than do controls. The monkey studies used dietary methods to induce vitamin B₁₂ deficiency whereas the fruit bat studies used a well-described method involving nitrous oxide (Metz and van der Westhuyzen, 1987). Third, a metabolic interaction between folate and vitamin B₁₂ is well documented (Chanarin et al., 1989). Although the association between folate treatment and neurological damage observed in human case reports does not provide proof of causality, the hazard associated with excess supplemental folate cannot be ruled out. The hazard remains plausible given the findings from animal studies and the demonstrated biochemical interaction of the two nutrients. The resulting neurological damage may be serious, irreversible, and crippling.

TABLE 8-12

Dose and Duration of Oral Folate Administration and the Occurrence of Neurological Manifestations in Patients with Pernicious Anemia.

For many years, it has been recognized that excessive intake of folate supplements may obscure or mask and potentially delay the diagnosis of vitamin B₁₂ deficiency. Delayed diagnosis can result in an increased risk of progressive, unrecognized neurological damage.

Evidence from animal as well as in vitro tissue and cell culture studies (Baxter et al., 1973; Hommes and Obbens, 1972; Kehl et al., 1984; Loots et al., 1982; Olney et al., 1981; Spector, 1972; Weller et al., 1994) suggests that folate in the form of folic acid is neurotoxic and epileptogenic in animals; however, there is no clear evidence of folate-induced neurotoxicity in humans. Concerns have been raised about the possibility of decreased effectiveness of treatment if individuals treated with anticonvulsant drugs take high doses of folate. However, the UL does not apply to drug-drug interactions or to high doses taken under medical supervision (see “Anticonvulsants” and “Methotrexate”).

General Toxicity. In one nonblinded uncontrolled trial, oral doses of 15 mg/day of folate for 1 month were associated with mental changes, sleep disturbances, and gastrointestinal effects (Hunter et al., 1970). However, studies using comparable or higher doses, longer durations, or both failed to confirm these findings (Gibberd et al., 1970; Hellstrom, 1971; Richens, 1971; Sheehy, 1973; Suarez et al., 1947).

Reproductive and Developmental Effects. Many studies have evaluated the periconceptional use of supplemental folate (in doses of approximately 0.4 to 5.0 mg) to prevent neural tube defects (Table 8-13). No adverse effects have been demonstrated, but the studies were not specifically designed to assess adverse effects. No reports were found of adverse effects attributable to folate in long-term folate supplement users or in infants born each year to mothers who take supplements, but this has not been investigated systematically. Because it is possible that subtle effects might have been missed, investigations designed to detect adverse effects are needed.

TABLE 8-13

Assessing Adverse Reproductive Effects from Studies Involving Supplemental Folate.

Carcinogenicity. In a large epidemiological study, positive associations were found between supplemental folate intake and the incidence of cancer of the oropharynx and hypopharynx and of total cancer (Selby et al., 1989). However, the authors of this study suggest that these associations might have been related to unmeasured confounding variables such as alcohol and smoking. Additionally, other studies suggest that folate might be anticarcinogenic (see “Cancer”) (Campbell, 1996).

Hypersensitivity. Individual cases of hypersensitivity reactions to oral and parenteral folate administration were reported (Gotz and Lauper, 1980; Mathur, 1966; Mitchell et al., 1949; Sesin and Kirschenbaum, 1979; Sparling and Abela, 1985). Such hypersensitivity is rare, but reactions have occurred at supplemental folate doses as low as 1 mg/day (Sesin and Kirschenbaum, 1979).

Intestinal Zinc Absorption. Although there has been some controversy regarding whether supplemental folate intake adversely affects intestinal zinc absorption (Butterworth and Tamura, 1989), a comprehensive review of the literature reveals that folate supplementation has either no effect on zinc nutriture or an extremely subtle one (Arnaud et al., 1992; Butterworth et al., 1988; Hambidge et al., 1993; Keating et al., 1987; Milne et al., 1984; Tamura, 1995; Tamura et al., 1992). In a study of prenatal folate supplementation, Mukherjee et al. (1984) noted a significant association between the occurrence of fetomaternal complications and the combination of low maternal plasma zinc and high maternal plasma folate concentrations. However, this study may have failed to control for potential confounding factors. Furthermore, these findings are not supported by Tamura and colleagues (1992), who found high serum folate concentrations to be associated with favorable pregnancy outcomes including higher birth weight and Apgar scores of newborns, reduced prevalence of fetal growth retardation, and lower incidence of maternal infection close to the time of delivery.

Summary

The weight of the limited but suggestive evidence that excessive folate intake may precipitate or exacerbate neuropathy in vitamin B₁₂-deficient individuals justifies the selection of this endpoint as the critical endpoint for the development of a UL for folate.

Adults

Data Selection. To evaluate a dose-response relationship and derive a UL for folate, case reports were used that involved oral administration of folate in patients with vitamin B₁₂ deficiency who showed development or progression of neurological complications. Because a number of apparently healthy individuals are vitamin B₁₂ deficient (see Chapter 9), these individuals are considered part of the general population in setting a UL.

Identification of a No-Observed-Adverse-Effect Level (NOAEL) and a Lowest-Observed-Adverse-Effect Level (LOAEL). The literature was reviewed to find cases in which vitamin B₁₂-deficient patients who were receiving oral doses of folate experienced progression of neurological disorders. Data were not available on which to set a NOAEL. A LOAEL of 5 mg of folate is based on the data presented in Table 8-12:

• at doses of folate of 5 mg/day and above, there were more than 100 reported cases of neurological progression;
• at doses of less than 5 mg/day of folate (0.33 to 2.5 mg/day), there are only eight well-documented cases;
• in most cases throughout the dose range, folate supplementation maintained the patients in hematological remission over a considerable time span; and
• the background intake of folate from food was not specified, but all except for three cases (those reported by Allen and co-workers [1990]) occurred before the fortification of breakfast cereal with added folate.

Uncertainty Assessment. An uncertainty factor (UF) of 5 was selected. Compared with the UFs used to date for other nutrients for which there was also a lack of controlled, dose-response data, a UF of 5 is large. The selection of a relatively large UF is based primarily on the severity of the neurological complications observed but also on the use of a LOAEL rather than a NOAEL to derive the UL. The UF is not larger than 5 on the basis of the uncontrolled observation that millions of people have been exposed to self-treatment with about one-tenth of the LOAEL (i.e., 400 μg in vitamin pills) without reported harm.

Derivation of a UL. The LOAEL of 5 mg/day of folate was divided by a UF of 5 to obtain the UL for adults of 1 mg/day or 1,000 μg/day of folate from supplements for fortified food. A UL of 1,000 μg/day is set for all adults rather than just for the elderly because of (1) the devastating and irreversible nature of the neurological consequences, (2) data suggesting that pernicious anemia may develop at a younger age in some racial or ethnic groups (Carmel and Johnson, 1978), and (3) uncertainty about the occurrence of vitamin B₁₂ deficiency in younger age groups. In general, the prevalence of vitamin B₁₂ deficiency in females in the childbearing years is very low and the consumption of supplemental folate at or above the UL in this subgroup is unlikely to produce adverse effects.

Folate UL Summary, Adults

Other Life Stage Groups

There are no data on other life stage groups that can be used to identify a NOAEL or LOAEL and derive a UL. For infants the UL was judged not determinable because of a lack of data on adverse effects in this age group and concern about the infant's ability to handle excess amounts. To prevent high levels of intake, the only source of intake for infants should be from food. No data were found to suggest that other life stage groups have increased susceptibility to adverse effects of high supplemental folate intake. Therefore, the UL of 1,000 μg/day is also set for adult pregnant and lactating women. The UL of 1,000 μg/day for adults was adjusted for children and adolescents on the basis of relative body weight as described in Chapter 3. Values have been rounded down.

Special Considerations

Individuals who are at risk of vitamin B₁₂ deficiency (e.g., those who eat no animal foods [vegans] and other individuals identified in Table 9-4) may be at increased risk of the precipitation of neurological disorders if they consume excess folate.