Recommended daily allowances (RDAs) are based on the nutrient level that is ade- quate for 97.5% of healthy individuals, and are calculated by adding two standard deviations to the estimated average requirement (EAR), which meets the needs of 50% of individuals. If data are insufficient to establish an EAR, an adequate intake (AI) is given. The tolerable upper intake level (UL) represents the maximum amount that can be consumed per day with no increased risk of adverse effects [17]. The recommended intake levels and upper limits for folate, vitamin B6, vitamin B12, and choline during pregnancy are shown in Table 1.
Table 1 Dietary requirement of one-carbon nutrients Dietary requirements during pregnancy
Age
Folate (μg/day) Vitamin B6 (mg/day) Vitamin B12 (μg/day) Choline (mg/day)
RDA UL RDA UL RDA UL AI UL
<19 600 800 1.9 80 2.6 ND 450 3000
>19 600 1000 1.9 100 2.6 ND 450 3500
RDA recommended daily allowance, UL upper limit, ND no data, AI adequate intake Adapted from Dietary Reference Intakes, Institute of Medicine
Folate
The RDA for folate is expressed as dietary folate equivalents (DFE) and was established using data from human feeding studies in which folate intake was highly controlled [17]. Folate status is typically assessed by measuring serum folate; red blood cell (RBC) folate can provide an indication of long-term status.
Deficiency is usually considered to be <3 ng/mL in serum or <140 ng/mL in RBCs [17].
Pregnancy substantially increases the requirement for folate, due to accelera- tions in cell division and tissue expansion of the placenta and fetus [25]. Studies in pregnant women indicated a need for 200 or more additional DFE per day as compared to the non-pregnant state. Although the protective effects of folic acid on neural tube defects (NTD) are well-established, this was not considered in setting the RDA, because the critical window for neural tube closure occurs before most women recognize they are pregnant [17]. Rather, a separate recom- mendation intended for women planning to become pregnant suggests an intake of 400 μg per day of synthetic folic acid in addition to folate from the diet, as this intake level of folic acid maximized NTD risk reduction among most women.
The major symptom of folate deficiency is megaloblastic anemia resulting from impaired DNA synthesis in red blood cells [26]. Folate deficiency can arise from dietary inadequacy, vitamin B12 deficiency (due to the trapping of folate as 5-methyl-THF), and gene–nutrient interactions [17, 27]. Additional symptoms of folate deficiency may include fatigue, weight loss, oral sores, heart palpitations, and appetite loss [17]. Folate deficiency is now rare in the United States due to folic acid fortification of the food supply. More than 85% of adults achieve the estimated average requirement [28], and pregnant women consuming supplemen- tal folic acid have circulating serum folate concentrations far exceeding the defi- ciency cut-off [29]. Nonetheless, among pregnant and non-pregnant women of reproductive age not consuming supplements, the median intake is less than half of the recommended 400 μg of synthetic folic acid per day [30], suggesting that women who are pregnant, or of reproductive age, remain a subgroup of concern.
In addition, women carrying multiple fetuses are at greater risk of folate defi- ciency anemia and may have a higher requirement; 1000 μg/day has been sug- gested as a recommended intake [31].
Toxicity from food folate (including fortified foods) has not been reported [17].
However, excessively high doses of folic acid supplements may “mask” a vitamin B12 deficiency; therefore, an upper limit was set at 1000 μg folic acid/day for preg- nant women >19 years. More recently, high folic acid consumption was shown to impair MTHFR activity in mice, suggesting there may be clinical implications for individuals consuming high-dose folic acid supplements, particularly among those who are MTHFR deficient [32].
Folate Vs Folic Acid
Although both folates and folic acid can fulfill the dietary requirement, there are several key differences in their absorption [33], which result in a higher bioavail- ability of folic acid. Because of the difference in bioavailability between folate and folic acid, the unit DFE, or dietary folate equivalent, is used. Since food folate is approximately 60% as bioavailable as folic acid, either 1 μg of naturally occurring food folate or 0.6 μg of folic acid is equivalent to 1 μg DFE [17]. Thus, the amount of DFEs in a meal is equivalent to μg of food folate + (1.7× μg of folic acid). To maximize protection from neural tube defects, the National Academy of Medicine, the Centers for Disease Control and Prevention, and the US Public Health Service recommend that pregnant women consume at least 400 μg of their daily intake as synthetic folic acid, either from supplements or fortified foods [34].
Vitamin B12
The EAR for vitamin B12 was established based on data from individuals with pernicious anemia, vegetarians/vegans, and the body’s ability to absorb and store vitamin B12. For pregnant women, 0.2 μg/day was added to account for fetal accu- mulation of the vitamin [17].
Deficiency can be assessed by one of several biomarkers; a combination of two is more definitive [35]. Plasma or serum vitamin B12 is commonly used but may fail to detect early deficiency, as it remains normal even after tissue levels begin to decline. More specific are methylmalonic acid, which accumulates when methylmalonyl- CoA mutase is unable to function properly without its vitamin B12 cofactor, and holotranscobalamin, which reflects the fraction of vitamin B12 cur- rently bound to transcobalamin. Homocysteine, although not an ideal biomarker since it is affected by the status of several vitamins, may reflect vitamin B12 status, particularly in populations where folate intake levels are high [36].
Like folic acid, long-term vitamin B12 deficiency results in macrocytic anemia, and can lead to potentially permanent neurological symptoms of unknown etiology;
therefore, detecting a vitamin B12 deficiency in the early stages is crucial. Excessive intakes of folic acid can “mask” detection of a vitamin B12 deficiency because metabolism of folic acid bypasses the methionine synthase reaction (see Fig. 1) which requires vitamin B12 as a cofactor and is needed to regenerate the forms of folate that function in DNA biosynthesis. In contrast, naturally occurring food folates are typically converted to 5-methyl-THF during absorption and therefore are unable to bypass the methyl-trap phenomenon [9]. Although the average intake of vitamin B12 in pregnant women is well above the EAR [17], a recent controlled feeding study in third-trimester pregnant women that provided ~3x the RDA for
12 weeks (an average of 8.6 μg per day) did not result in elevated serum vitamin B12 levels, but rather maintained them within normal range, with no change from baseline. This finding suggests that intakes higher than the current RDA (2.6 μg per day) may be necessary to maintain vitamin B12 status throughout pregnancy [37].
Because animal products are the only source of vitamin B12, vegetarians and vegans are at greater risk of deficiency and must consume supplements and/or fortified foods [38]. In addition, women chronically exposed to nitrous oxide, which is commonly used in dental procedures, may be at risk of deficiency since methionine synthase can act on nitrous oxide to form a radical which destroys the enzyme and the B12 cofactor [5].
Toxicity has not been shown (likely because absorption is limited to ~2 μg at a time) and there is no upper limit [17, 39].
Vitamin B6
Vitamin B6 recommendations for adult men and non-pregnant women were estab- lished based on optimal plasma concentrations of PLP (>20 nmol/L). Plasma PLP is the standard marker for vitamin B6 status; it is responsive to intake levels and reflects liver stores [40] Although PLP levels decline to approximately 10 nmol/L during pregnancy, this is considered a normal physiological occurrence, and the RDA for pregnant women does not attempt to correct this [17]. Rather, 0.5 mg/day was added to the EAR to account for placental and fetal accumulation, based on animal studies, as well as changes in maternal weight and metabolism. Symptoms of deficiency have been reported to include microcytic anemia, depression, seizures, inflammation, and sores of the mouth [41]. Deficiency in vitamin B6 can also lead to hyperhomocysteinemia, due to its role in the transsulfuration pathway. Because vitamin B6 is widely distributed in commonly consumed foods, most Americans are not at risk of deficiency. In addition, symptoms are rarely seen above intakes of 0.5 mg/day [17]. The upper intake level has been set at 100 mg based on possible risk of sensory neuropathy, but risk of toxicity is very low, as this level of intake is rarely seen and can only be achieved through pharmacological doses [17].
Oral contraceptives are associated with decreased levels of PLP, with one study finding that 78% of women currently taking birth control and 40% of past users had PLP levels below 20 nmol/L compared to 20% of women who had never used hor- mones [42]. However, it is uncertain whether this reflects a true B6 deficiency or simply a redistribution of body stores.
Choline
Very little data were available to the IOM committee for derivatization of an EAR, and thus establishment of an RDA, for choline. As a result, an AI was developed based on the choline intake level needed to maintain normal liver function in men [17]. The
current AI for non-pregnant women is 425 mg choline/day, and was adjusted to 450 mg choline/day for pregnant women [17], based on the observation that pregnant rodents easily become choline deficient due to large amounts of choline being trans- ported to the placenta and the fetus, both of which have poor ability to synthesize choline [43, 44]. Recent data from a 12-week feeding study in third- trimester pregnant women indicate that this recommendation may not be adequate, because pregnant women consuming the AI level have lower plasma concentrations of choline-derived methyl donors and display altered partitioning between the two choline pathways used to synthesize PC and betaine. Notably, both of these can be improved by increas- ing maternal choline intake [45, 46]. A higher maternal choline intake also decreases the placental transcript level of soluble fms-like tyrosine kinase (sFLT1), which is elevated in women with preeclampsia [47], and corticotropin- releasing hormone, which lowers cortisol concentration in the cord blood [48]. Collectively, these data support a dietary recommendation exceeding the current AI for achieving optimal pregnancy outcome and improving the long-term well-being of the child.
Excessive choline consumption can lead to a fishy body odor, which is caused by increased production and excretion of the choline metabolite, trimethylamine.
Furthermore, a high dose of choline can result in hypotension (low blood pressure), increased sweating, vomiting, and salivation secondary to excess production of the neurotransmitter, acetylcholine. To prevent these adverse effects, an upper limit was established at 3500 mg/day for adults [17].