Food and Nutrition Bulletin

This paper provides a general review on folate and vitamin B₁₂ nutrition and metabolism and the metabolic interrelationship between these vitamins. The effects of some common polymorphisms in folate and vitamin B₁₂ genes and the influence of vitamin B₆ and riboflavin status on folate and vitamin B₁₂ metabolism are also discussed.

This review describes current knowledge of the main causes of vitamin B₁₂ and folate deficiency. The most common explanations for poor vitamin B₁₂ status are a low dietary intake of the vitamin (i.e., a low intake of animal-source foods) and malabsorption. Although it has long been known that strict vegetarians (vegans) are at risk for vitamin B₁₂ deficiency, evidence now indicates that low intakes of animal-source foods, such as occur in some lacto-ovo vegetarians and many less-industrialized countries, cause vitamin B₁₂ depletion. Malabsorption of the vitamin is most commonly observed as food-bound cobalamin malabsorption due to gastric atrophy in the elderly, and probably as a result of Helicobacter pylori infection. There is growing evidence that gene polymorphisms in transcobalamins affect plasma vitamin B₁₂ concentrations. The primary cause of folate deficiency is low intake of sources rich in the vitamin, such as legumes and green leafy vegetables, and the consumption of these foods may explain why folate status can be adequate in relatively poor populations. Other situations in which the risk of folate deficiency increases include lactation and alcoholism.

Human deficiencies of folate and vitamin B₁₂ result in adverse effects which may be of public health significance, but the magnitude of these deficiencies is unknown. Therefore, we examine the prevalence data currently available, assess global coverage of surveys, determine the frequency with which vitamin status assessment methods are used, and identify patterns of status related to geographical distribution and human development. Surveys were identified through PubMed and the Vitamin and Mineral Nutrition Information System at the World Health Organization (WHO). Since different thresholds were frequently used to define deficiency, measures of central tendency were used to compare blood vitamin concentrations among countries. The percentage of countries with at least one survey is highest in the WHO Regions of South-East Asia and Europe. Folate and vitamin B₁₂ status were most frequently assessed in women of reproductive age (34 countries), and in all adults (27 countries), respectively. Folate status assessment surveys assessed plasma or serum concentrations (55%), erythrocyte folate concentrations (21%), or both (23%). Homocysteine was assessed in one-third of the surveys of folate and vitamin B₁₂ status (31% and 34% respectively), while methylmalonic acid was assessed in fewer surveys of vitamin B₁₂ status (13%). No relationship between vitamin concentrations and geographical distribution, level of development, or population groups could be identified, but nationally representative data were few. More representative data and more consistent use of thresholds to define deficiency are needed in order to assess whether folate and vitamin B₁₂ deficiencies are a public health problem.

Deficiencies of folate or of vitamin B₁₂ are widespread and constitute a major global burden of morbidity affecting all age groups. Detecting or confirming the presence of folate or vitamin B₁₂ deficiency and distinguishing one from the other depends, ultimately, on laboratory testing. Tests to determine the presence of folate or vitamin B₁₂ deficiency are used singly or in combination to establish the nutritional status and prevalence of deficiencies of the vitamins in various populations. The efficacy of interventions through the use of fortification or supplements is monitored using the same laboratory tests. Tests currently in use have limitations that can be either technical or have a biological basis. Consequently, each single test cannot attain perfect sensitivity, specificity, or predictive value. Laboratory indicators of vitamin B₁₂ or folate status involve measurement of either the total or a physiologically relevant fraction of the vitamin in a compartment such as the blood. Thus, assays to measure vitamin B₁₂ or folate in plasma or serum as well as folate in red blood cells are in widespread use, and more recently, methods to measure vitamin B₁₂ associated with the plasma binding protein transcobalamin (holotranscobalamin) have been developed. Alternatively, levels of surrogate biochemical markers that reflect the metabolic function of the vitamin can be used. Surrogates most commonly used are plasma homocysteine, for detection of either vitamin B₁₂ or folate deficiency and methylmalonic acid for detection of vitamin B₁₂ deficiency. The general methods as well as their uses, indications, and limitations are presented.

In recent years there has been growing interest in the vitamins folic acid and vitamin B₁₂ because of the realization that the status of these vitamins in populations is less than adequate, and that such inadequacy may be linked to adverse public health outcomes. This concern has prompted the United States, Canada, and other countries to fortify grain products with folic acid, while additional countries are considering doing so in the near future. This presentation provides a new approach which relies on the combination of the concentrations in blood of vitamins and their respective functional indicators to establish cutoff points for assessing folate and vitamin B₁₂ status in populations. The premise is based on the fact that the relationship between plasma vitamin concentrations and their respective functional indicators is inverse and biphasic, with a steep slope at low concentrations of the vitamin and a more moderate slope at higher plasma vitamin concentrations. We propose that the intersection of these two slopes be used as a guideline for assessing the status of these vitamins and the adequacy of fortification programs. The cutoff would be 10 nmol/L for serum folate and 340 nmol/L for red blood cell folate, based on lowest plasma homocysteine. For serum vitamin B₁₂, the cutoff would be 150 pmol/L based on lowest methylmalonic acid and 300 pmol/L based on lowest homocysteine.

Based on biochemical evidence, a high prevalence of biochemical evidence of vitamin B₁₂ or folate deficiency has been reported in a number of areas in the world. The evidence that these biochemical abnormalities lead to a comparable prevalence of anemia is reviewed. The overall contribution of vitamin B₁₂ deficiency to the global burden of anemia is probably not significant, except perhaps in women and their infants and children in vegetarian communities. In developed countries, folate-deficiency anemia is uncommon. In some developing countries, this anemia is still seen, but there are no comprehensive data on the relative prevalence compared with anemia due to malaria, iron-deficiency, hemoglobinopathy, and HIV disease. It seems unlikely that folate deficiency makes a major contribution to the burden of anemia in developing countries. Iron-deficiency anemia may coexist with vitamin B₁₂ and especially folate deficiency, and may confound the hematological features of the vitamin deficiencies whose prevalence would then be underestimated. Supplementation of the diet of pregnant women with folic acid can virtually eliminate folate-deficiency anemia in these women. There are very few data on the hematological effect of vitamin B₁₂ supplementation or fortification at the population level. The addition of vitamin B₁₂ to the supplementation of the diet of pregnant women with iron and folic acid does not produce an increased hematological response, at least in nonvegetarian populations. There are numerous reports of the effect of folic acid fortification of food on tests of folate status, but only a single published report on the hematological response was found.

The role that nutritional factors play in mammalian development has received renewed attention over the past two decades as the scientific literature has exploded with reports that folic acid supplementation in the periconceptional period can protect embryos from a number of highly significant malformations. As is often the case, the relationship between B vitamin supplementation and improved pregnancy outcomes is more complicated than initially perceived, as the interaction between nutritional factors and selected genes must be considered. In this review, we attempt to summarize the complex clinical and experimental literature on nutritional factors, their biological transport mechanisms, and interactions with genetic polymorphisms that impact early embryogenesis. While not exhaustive, our goal was to provide an overview of important gene–nutrient interactions, focusing on folic acid and vitamin B₁₂, to serve as a framework for understanding the multiple roles they play in early embryogenesis.

The importance of folate in reproduction can be appreciated by considering that the existence of the vitamin was first suspected from efforts to explain a potentially fatal megaloblastic anemia in young pregnant women in India. Today, low maternal folate status during pregnancy and lactation remains a significant cause of maternal morbidity in some communities. The folate status of the neonate tends to be protected at the expense of maternal stores; nevertheless, there is mounting evidence that inadequate maternal folate status during pregnancy may lead to low infant birthweight, thereby conferring risk of developmental and long-term adverse health outcomes. Moreover, folate-related anemia during childhood and adolescence might predispose children to further infections and disease. The role of folic acid in prevention of neural tube defects (NTD) is now established, and several studies suggest that this protection may extend to some other birth defects. In terms of maternal health, clinical vitamin B₁₂ deficiency may be a cause of infertility or recurrent spontaneous abortion. Starting pregnancy with an inadequate vitamin B₁₂ status may increase risk of birth defects such as NTD, and may contribute to preterm delivery, although this needs further evaluation. Furthermore, inadequate vitamin B₁₂ status in the mother may lead to frank deficiency in the infant if sufficient fetal stores of vitamin B₁₂ are not laid down during pregnancy or are not available in breastmilk. However, the implications of starting pregnancy and lactation with low vitamin B₁₂ status have not been sufficiently researched.

The essentiality of folate and vitamin B₁₂ for the synthesis of DNA may interfere with a successful pregnancy outcome when the mother is deficient in these micronutrients. The objective of this paper is to assess the effects of folate and vitamin B₁₂ deficiencies on pregnancy outcomes, other than neural tube defects (NTD), and the effects of these deficiencies on infant and child development.

Supplementation studies identified by two Cochran Reviews were selected to assess the impact of folate deficiency on pregnancy outcomes, and a systematic review of the literature was used to assess their effects on infant and child developments.

Folate supplementation consistently resulted in improvement of hematological and folate status indicators. Seven supplemental studies consistently found no differences in the risk of total fetal loss, early or late miscarriage, stillbirth, preeclampsia, perinatal death, neonatal death, preterm birth, small-for-gestational age, or infant death in supplemented women compared with their controls. Two of those studies found greater placental weights (difference 96 g; 95% CI, 30.7 to 161.2 g), birthweights (difference 312 g; 95% CI, 108.5 to 515.4 g), and a lower risk for newborns weighing < 2,500 g in supplemented women (RR = 0.94; 95% CI, 0.90 to 0.99). Abnormal vitamin B₁₂ and homocysteine serum concentrations were more readily associated with poor pregnancy outcomes. The very few studies addressing the effects of folate and vitamin B₁₂ deficiencies on infant and child development were inconclusive.

Early supplementation with folate to pregnant women improves hematological and folate status indicators, but has little or no effect on pregnancy outcomes, other than on NTD. Vitamin B₁₂ deficiencies and low homocysteine are more readily associated with poor pregnancy outcomes.

Homocysteine is a sulfur amino acid whose metabolism stands at the intersection of two pathways: remethylation, which requires folic acid and vitamin B₁₂ coenzymes; and transsulfuration, which requires pyridoxal-5′-phosphate, the vitamin B₆ coenzyme. Data from a number of laboratories suggest that mild elevations of homocysteine in plasma are a risk factor for occlusive vascular disease. In the Framingham studies, we have shown that plasma homocysteine concentration is inversely related to the intake and plasma levels of folate and vitamin B₆ as well as vitamin B₁₂ plasma levels. Almost two-thirds of the prevalence of high homocysteine is attributable to low vitamin status or intake. Elevated homocysteine concentrations in plasma are a risk factor for prevalence of extracranial carotid-artery stenosis ≥ 25% in both men and women. Prospectively elevated plasma homocysteine is associated with increased total and cardiovascular mortality, increased incidence of stroke, increased incidence of dementia and Alzheimer's disease, increased incidence of bone fracture, and higher prevalence of chronic heart failure. It was also shown that elevated plasma homocysteine is a risk factor for preeclampsia and maybe neural tube defects (NTD). This multitude of relationships between elevated plasma homocysteine and diseases that afflict the elderly, pregnant women, and the embryo points to the existence of a common denominator which may be responsible for these diseases. Whether this denominator is homocysteine itself, or homocysteine is merely a marker, remains to be determined.

Folate deficiency in the periconceptional period contributes to neural tube defects; deficits in vitamin B₁₂ (cobalamin) have negative consequences on the developing brain during infancy; and deficits of both vitamins are associated with a greater risk of depression during adulthood. This review examines two mechanisms linking folate and vitamin B₁₂ deficiency to abnormal behavior and development in infants: disruptions to myelination and inflammatory processes. Future investigations should focus on the relationship between the timing of deficient and marginal vitamin B₁₂ status and outcomes such as infant growth, cognition, social development, and depressive symptoms, along with prevention of folate and vitamin B₁₂ deficiency.

Dementia has reached epidemic proportions, with an estimated 4.6 million new cases worldwide each year. With an aging world population, the prevalence of dementia will increase dramatically in the next few decades. Of the predicted 114 million who will have dementia in 2050, about three-quarters will live in less developed regions. Although strongly age-related, dementia is not an inevitable part of aging but is a true disease, caused by exposure to several genetic and nongenetic risk factors. Prevention will be possible when the nongenetic risk factors have been identified. Apart from age, more than 20 nongenetic risk factors have been postulated, but very few have been established by randomized intervention studies. Elevated blood concentrations of total homocysteine and low-normal concentrations of B vitamins (folate, vitamin B₁₂, and vitamin B₆) are candidate risk factors for both Alzheimer's disease and vascular dementia. Seventy-seven cross-sectional studies on more than 34,000 subjects and 33 prospective studies on more than 12,000 subjects have shown associations between cognitive deficit or dementia and homocysteine and/or B vitamins. Biologically plausible mechanisms have been proposed to account for these associations, including atrophy of the cerebral cortex, but a definite causal pathway has yet to be shown. Raised plasma total homocysteine is a strong prognostic marker of future cognitive decline, and is common in world populations. Low-normal concentrations of the B vitamins, the main determinant of homocysteine concentrations, are also common and occur in particularly vulnerable sections of the population, such as infants and elderly. Large-scale randomized trials of homocysteine-lowering vitamins are needed to see if a proportion of dementia in the world can be prevented.

Long known as an uncommon but serious medical disorder requiring medical management, vitamin B₁₂ deficiency is now seen to be common worldwide, but it is in a quite different form than traditionally envisioned. Most of the newly recognized deficiency is subclinical in nature, its health impact and natural history are uncertain, and its prevalence has been greatly inflated by also including persons with “low-normal” vitamin B₁₂ levels, few of whom are deficient. The spread of folic acid fortification has also introduced concerns about folate's potentially adverse neurologic consequences in persons with undetected vitamin B₁₂ deficiency. Fortification with vitamin B₁₂ may prove more complicated than fortification with folic acid, however, because the bioavailability of vitamin B₁₂ is limited. Bioavailability for those who need the vitamin B₁₂ the most is especially poor, because they often have malabsorption affecting either classical intrinsic factor-mediated absorption or food-vitamin B₁₂ absorption. Moreover, new evidence shows that many elderly persons respond poorly to daily oral doses under 500 μg (1 μg = 0.74 nmol), even if they do not have classical malabsorption, which suggests that proposed fortification with 1 to 10 μg may be ineffective. Those least in need of vitamin B₁₂ usually have normal absorption and are thus at greatest risk for whatever unknown adverse effects of high-dose fortification might emerge, such as the effects of excess accumulation of cyanocobalamin. Studies are needed to define the still unproven health benefits of vitamin B₁₂ fortification, the optimal levels of fortification, the stability of such fortification, interactions with other nutrients, and any possible adverse effects on healthy persons. The answers will permit formulation of appropriately informed decisions about mandatory fortification or (because fortification may prove a poor choice) about targeted supplementation in subpopulations with special needs for additional vitamin B₁₂, such as vegetarians, nursing mothers, and the elderly.

Folate and vitamin B₁₂ deficiencies represent important and evolving global health challenges that contribute to the global burden of anemia, neurologic conditions, neurodevelopmental disorders, and birth defects. We present a review of population-based programs designed to increase consumption of folates and vitamin B₁₂.

A folic acid supplementation program targeting couples prior to marriage in China has led to optimal consumption of supplements containing folic acid and a significant reduction of neural tube defects (NTD). Supplementation programs that use mass community education show some promise, but have not been shown to be as effective as targeted education. The success of supplementation programs hinges on a strong and persistent educational component and access to the supplements.

Fortification with folic acid has been shown to reduce the prevalence of NTD in the countries where it has been implemented. Challenges to fortification programs include identifying the appropriate delivery vehicles, setting the optimal fortification level, sustaining the quality assurance of the fortification level, and addressing regulatory challenges and trade barriers of commercially fortified flours.

Supplementation and fortification are cost-effective and viable approaches to reducing the burden of NTD, anemia, and other conditions resulting from folate deficiency. The experience with interventions involving folic acid could provide a model for the subsequent development of supplementation and fortification programs involving vitamin B₁₂.

Neural tube defects (NTD) are among the most common and devastating birth defects. Annually, in China, between 80,000 and 100,000 pregnancies result in children born with NTD. Northern China has the highest known rate of NTD in the world. Birth defects are becoming the leading cause of infant mortality in the urban and developed areas in China. The results of studies conducted in the early 1990s and early 2000s showed significant geographic and seasonal variations of folate status among Chinese women of childbearing age, with lower serum and red blood cell folate levels in northern China. In the north, 32% to 35% of women had low plasma folate and low red blood cell folate, and folate levels were significantly lower in spring than in fall. Since 1993, Peking University Health Science Center (formerly Beijing Medical University), collaborating with the US Centers for Disease Control and Prevention (CDC), has conducted a large-scale study to evaluate a public health campaign in China among women preparing for marriage in order to determine the effectiveness of daily supplementation of 400 μg of folic acid alone in preventing NTD in both the north and the south of China. The results showed that among the fetuses or infants of the women who took periconceptional folic acid, the reduction in risk of NTD was 85% in the northern region and 40% in the southern region. Daily intake of 400 μg of folic acid may also reduce the risk of nonsyndromic orofacial clefts. We found no evidence that daily consumption of folic acid before and during early pregnancy influenced the risk of miscarriage or twinning. In 2001, the Chinese Ministry of Health and the Chinese Disabled Person Federation released a National Action Plan for Reducing Birth Defects and Disabilities in China for 2002–2010. The Action Plan aims to improve birth outcomes, to reduce infant mortality by reducing the risk of birth defects and disabilities, and to ensure that every baby is born healthy.

Evidence from controlled trials suggests that ingestion of 0.4 mg of folic acid per day in the periconceptional period is effective in preventing neural tube defects (NTD). For this reason, most countries recommend that women planning pregnancy take folic acid supplements in the periconceptional period, and some countries even fortify stable foods with folic acid. Denmark exemplifies a country with a relatively conservative attitude with respect to taking action in these matters. In 1999, a national information campaign was launched that recommended women planning pregnancy take 0.4 mg of folic acid periconceptionally, but with the moderation that women who eat a healthy diet do not need to take folic acid supplement. The campaign was repeated during 2001. The results of the latter campaign were evaluated by using data from a national survey among pregnant women conducted simultaneously with the campaign by the Danish National Birth Cohort. An increase in the proportion of folic acid users took place concomitantly with the launching of the information events, but the increase was limited. Among women who did not plan their pregnancy, a small proportion had taken folic acid supplements periconceptionally, and this proportion did not change concomitantly with the campaign. Young age and low education were factors associated with low likelihood of taking folic acid. It seems that different and more efficient actions are needed if a more substantial proportion of Danish women and their fetuses are going to benefit from the knowledge that folic acid supplementation in the periconceptional period can prevent NTD.

In November of 1993, the Dutch government recommended daily folic acid supplementation of 0.4 or 0.5 mg for all women planning pregnancy, starting 4 weeks before conception until 8 weeks after. In 1995, a one-time mass media campaign was conducted, and due to this campaign, the use of folic acid in this recommended period increased from 4.8% in 1995 to 21% in 1996. Subsequently, no structural strategies were undertaken until 2003, and at that time, 22% of lower-educated women used folic acid in the recommended period. The prevalence of neural tube defects (NTD) decreased from 11.44/10,000 before the official recommendations to 6.52/10,000 thereafter. Currently, community pharmacies proactively approach women before their first pregnancy on the subject of folic acid supplementation. This is done by means of a sticker placed on packages of oral contraceptives with the text “Child wish?*”. Ask for information about folic acid in your pharmacy,” and a brochure (leaflet) containing detailed information about folic acid. (*“Child wish” refers to women who plan to become pregnant.) Data from an explorative, comparative study strongly suggest that this intervention is effective. Sustainable education programs through which women are informed repeatedly to take folic acid supplements during the periconceptional period are urgently needed.

Determining the micronutrient contents in fortified foods depends not only on the health goal (additional intake to complement the diet), but also on ensuring that fortification does not raise micronutrient intakes beyond the Tolerable Upper Intake Level (UL), i.e., the safe limit. Technological incompatibility and cost may also restrict the fortification contents. For folic acid, the limiting factor is safety, while for vitamin B₁₂, it is cost. However, adequate fortification contents that are both safe and efficacious can be estimated for both nutrients. In order to obtain the maximum benefit from the fortification programs, three different formulas responding to three categories of consumption, as specified by the median and 95th percentile of consumption, are proposed.

The model presented is based on the estimation of a Feasible Fortification Level (FFL), which then is used to determine the average, minimum, and maximum contents of the nutrients during production, taking into consideration the acceptable variation of the fortification process. Finally, the regulatory parameters, which support standards and enforcement, are calculated by reducing the proportion of the nutrient that is degraded during the usual marketing process of the fortified food. It is expected that this model will establish a common standard for food fortification, and improve the reliability and enforcement procedures of these programs.

The model was applied to flours as vehicles for folic acid in the United States, Guatemala, and Chile. Analysis of the data revealed that, with the exception of Chile, where wheat flour consumption is very high and probably within a narrow range, supplementation with folic acid is still needed to cover individuals at the low end of consumption. This is especially true when the difference in flour consumption is too wide, as in the case of Guatemala, where the proportional difference between consumption at the 95th percentile of the nonpoor group is as high as 100 times the consumption at the 5th percentile of the extremely poor group. Adoption of fortification content for staple foods near the safe limit brings together the need of restricting the voluntary addition of the specific nutrient to other foods and to dietary supplements.

Canada mandated that cereal grains, especially white flour, be fortified with folic acid by mid-1998. Between 0.1 and 0.2 mg of additional synthetic folic acid per day has been provided through this initiative. The current paper describes how this took place, and the epidemiological effects that occurred in the Canadian population, including higher blood folate levels and a decline in the prevalence of neural tube defects.

The Chilean Ministry of Health legislated to add folic acid (2.2 mg/100 g) to wheat flour to reduce the risk of neural tube defects (NTD), beginning in January 2000. This policy resulted in a significant increase in serum and red blood cell folate in women of childbearing age 1 year after fortification. The frequency of NTD was studied in all births, both live and stillbirths, in a prospective hospital-based design including 25% of national births during 1999–2000 (prefortification period) and 2001–2002 (postfortification period). During the prefortification period, there was a total of 120,566 newborns, and the NTD rate was 17.1/10,000 births. During the postfortification period (2001–2002) there was a total of 117,704 newborns, and the NTD rate was significantly reduced by 43% to 9.7/10,000 births (RR = 0.57; 95% CI, 0.45 to 0.71). This implies a reduction of 43% in the rate of NTD. The costs per NTD case and infant death averted were 1,200 international dollars (I$) and I$11,000, respectively. The cost per disability-adjusted life year (DALY) averted was I$91, or 0.8% of the country's per capita GDP. On the overall, fortification resulted in net cost savings of I$1.8 million.

Fortification of wheat flour with folic acid has proven to be an effective and cost saving strategy for the primary prevention of NTD in a middle-income country in a postepidemiological transition, and in a dramatically short period of time.