Prevention of Neural Tube Defects in India

1) NTDs develop by the end of the very first month of conception: Scope of the problem in India.

NTDs are life-threatening birth defects of the brain and/or spinal cord.¹ A systematic review on the birth prevalence of NTDs in India, predominantly using data on the number of cases of NTDs referred to large tertiary referral centers in cities across India, reported a birth prevalence of NTDs as 4.1 cases per 1000 total births (95% CI 3.1 to 5.4 per 1000-total births).³ Given 25- to 26-million births annually, with most pregnant women having these nutritional deficits, this translates to over 100,000 births of babies with NTDs each year in India (i.e., over one million more babies with NTDs born every decade). Another systematic review of observational studies (19 city hospital-based studies; one community-based study) revealed an overall pooled birth prevalence (random effect) of NTDs in India of 4.5 per 1000 total births (95% CI 4.2 to 4.9),⁴ which is still much too high given that NTDs are a largely preventable condition. Parenthetically, it should be noted that studies on the baseline prevalence of NTDs in India using hospital records have not changed significantly over half a century. This is not surprising since the dietary basis for the ongoing high prevalence of combined low-folate and low-vitamin B12 status (discussed below) has not significantly changed in India.

Of added significance, the only population-based door-to-door study¹⁹ from Balrampur District, in Uttar Pradesh (U.P.—India’s largest State; population >200 million), indicated the incidence of NTDs was 6·57 to 8.21 cases of NTDs per 1000 livebirths (95% CI, 4·2 to 15·6).¹⁹ When compared to all other hospital based studies across India,⁴ this community-based study,¹⁹ was the only one which fully met all six “quality assessments” based on STROBE criteria [strengthening the reporting of observational studies in epidemiology]²⁰ published 2 years later. These criteria⁴ included a clear description of the study setting and population, details on how the diagnosis of a NTD was made, whether informed consent was obtained, whether consecutive births were examined, and whether results of study were generalizable. Indeed, although this population-based was conducted in the least developed region of India [Figure 4; Map],² it was one among 168 other districts with similar low (health, social, and economic) composite index values across North India.¹⁹

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Figure 4:

A map of the Indian Government’s National Commission on Population Report (Government of India). Source: https://doi.org/10.4103/jiaps.jiaps_211_21. The original source of this Map was found in http://populationcommission.nic.in:80/mapbased.htm (accessed August 4, 2004, but it was subsequently taken down by the Government of India, Ministry of Health). This comprehensive Map highlights the composite index of major health, social, and economic indices, which paints a detailed portrait of the general state of the health of various communities within the many States of India. The composite index employed 12 variables including population growth, births, deaths, safe water and sanitation, female literacy rates, child marriage, fertility, family planning, sex ratio, immunization, access to skilled obstetric care and connection to other villages via paved roads. (See the Map for Color Codes that signify the composite index score of each region studied throughout India). Balrampur district (located just above the alphabets ‘SH’ in UTTAR PRADESH), in the northeastern side of the most populous North Indian State of Uttar Pradesh of over 200 million, borders Nepal and shaded in Red, is at the very bottom of the list of the Government of India’s (GOI) health–social–economic–composite index. This district, which had a composite index <46, had the highest incidence of NTDs (up to 8.2 per 1000 live births). There are an additional 168 districts, primarily in North India, with comparable composite index values as Balrampur district, consistent with widespread and serious deficits in health, social and economic development throughout North India

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Furthermore, the only other independent community-based study in India carried out 10 years later along similar lines as the Balrampur study¹⁹ but in another region of U.P. likewise identified that NTDs were still largely unchanged at 7.48 cases of NTDs per 1000 births.²¹ This validates the extrapolation of community-based data¹⁹ to a wider area across North India. Therefore, the data from the only two population-based studies from U.P. as well as several other older hospital-based data from major cities across India cannot possibly be summarily dismissed as not being relevant to India.

There has not yet been any comparable evaluation of NTDs with associated blood tests for folate and vitamin B12 in India. However, a recent population-based biomarker survey in women of childbearing age from South India,¹² which is higher on the GOI list of health–social–economic–composite index [Figure 4],² identified the supermajority [nearly 80%] of women at risk for NTDs.¹² Therefore, despite an apparent superior standard of living than found across rural U.P., these South Indian women also exhibited serious deficits in their folate and/or vitamin B12 status, thereby placing them at risk for having babies with NTDs.

2) Most NTDs may be prevented in India by rapid replenishment of depleted folate and vitamin B12 stores early in the periconceptional period.

Definition of the periconceptional period: The periconceptional period comprises a pre-conceptional phase, fertilization and conception, followed by a post-conceptional phase.²² The pre-conceptional phase, which begins ∼14 weeks before conception, coincides with the most active phase of ovarian follicular development. Following fertilization, the post-conceptional phase, which involves growth and development of the conceptus, lasts up to 10-weeks. Therefore, well before the periconceptional period, is when the prospective mother’s serum folate (and vitamin B12) status must be optimized to support progressive growth and development of the conceptus throughout embryonic and fetal life; this can prevent the supermajority of folate-responsive NTDs. This is the basis for recommending that robust serum folate- and vitamin B12- concentrations must be maintained throughout the reproductive years of Indian women. Such a goal can only be achieved by folate and vitamin B12 fortification of a candidate food vehicle that is consumed by most Indian women every day at full therapeutic doses that can rapidly optimize their folate and vitamin B12 status.

Evidence that folic acid reduces NTD: As early as 1952, when the orally administered anti-folate drug (aminopterin) was used for medical termination of pregnancy, some fetuses exposed early in pregnancy [but had not been spontaneously aborted] were found to have an NTD at birth.²³ (Parenthetically, this was the earliest report that drugs used in pregnancy can be dangerous for the fetus; only a decade later we learned of the profound danger of thalidomide use in pregnancy in inducing phocomelia, i.e., severe foreshortening or absence of limbs). Studies over subsequent decades hinted to a potential role of folates in preventing NTDs.²⁴ It was also known that a woman who gives birth to a baby with NTD has up to 10-fold increased risk of having a subsequent baby with NTDs.¹⁰ Moreover, because the decision on whether a baby develops an NTD occurs by the end of the very first month of conception, any prevention of NTDs with folate would need to encompass the periconceptional period. In a randomized controlled trial (RCT), when women who had given birth to a baby with NTD were treated with 4 mg folic acid daily before conception and throughout pregnancy, they showed nearly 75% protection against the delivery of a subsequent baby with NTD;²⁵ this assured prevention of the recurrence of NTDs. A year later (1992), a subsequent RCT from Hungary²⁶ then demonstrated that the first occurrence of NTDs could be prevented if prospective mothers consumed a little less than 1 mg of folic acid daily to ensure they had sufficient folate in their body when they conceived.^25-29

The extent of protection of the first occurrence of NTD (up to 81%)²⁸ by periconceptional supplementation with only 0.4 mg folic acid, was established in 1999 by a very large community interventional trial in China. Berry et al.²⁸ (1999) documented that in areas with a high prevalence of NTDs, there were many more instances of a protective effect by the administration of folate; conversely, where the prevalence of NTDs had been low, the extent of protection had also been lower. However, in contrast to China, NTD-prevention in India is likely to be unique since over 75% of Indian women consume a diet low in both folate and vitamin B12, which predisposes them to dual deficiencies.^10,11 It is estimated that close to 90% prevention of folic acid-responsive NTDs could be prevented if India had a robust program to optimize the folate status [which includes optimized vitamin B12 status] of women. This is projected to result in the prevention of 115,000 Indian babies born with NTDs each year.¹⁷

Thus, the evidence that folic acid reduces NTDs is supported by several types of studies that are still considered of great relevance in modern epidemiology.³⁰ There are studies to support the conclusion that folic acid fortification of food can improve the folate status of women of childbearing age.^31,32. Moreover, the use of folic acid reduces the prevalence of NTDs, with greater reduction wherever mandatory fortification has been instituted.³³ For examples, in Canada [where data included live births, stillbirths, and termination of pregnancy for fetal anomalies], the prevalence of NTDs decreased from 1.58 per 1000 births before folic acid food fortification to 0.86 per 1000 births during the post full-fortification period; a 46% reduction (95% confidence interval, 40 to 51).³⁴ As first noted in China,²⁸ the magnitude of the decrease was proportional to the pre-fortification baseline rate in each province³⁴ and geographical differences almost disappeared after fortification began.

In clinical medicine, the highest quality of evidence is obtained using a RCT format to determine if a medication(s), diagnostic test/procedure, or device provides unambiguous evidence of efficacy or effectiveness in benefiting patients. In this context, there is significant RCT-related evidence that folic acid increases serum folate concentrations, of the effectiveness of folic acid in reducing the first occurrence of NTD,²⁶ and in preventing recurrence of NTDs.^25,35 Additional evidence demonstrating a drop in NTDs in USA/Canada after the institution of folate fortification of food supports the significant value of this practice.³⁴ Other observational studies from resource-limited countries (Cameroon,³⁶ Chile,³⁷ and Tanzania³⁸) and a systematic review of programs from Chile, Argentina, Brazil, Canada, Costa Rica, Iran, Jordan, South Africa, and the USA³¹ have shown correlated improvements in blood folate levels with reductions in NTDs.

See Annexure 1(a). “How does folate get into the brain and what happens when its entry is perturbed?”

Therefore, it would be unethical to carry out additional RCTs using a control/placebo group where folic acid is withheld from women with a prior birth of a baby with NTD, while an experimental group receives folate. Parenthetically, an apparently misguided study sponsored by the Indian Council of Medical Research on this very topic has been discussed elsewhere in this White Paper³⁹ in Annexure 1(e) under “Do we need more evidence to justify widespread vitamin -folate fortification of food to prevent NTDs in India?”

Moreover, there is extensive basic science evidence that sufficient intracellular vitamin B12 is essential to enable intracellular folate to participate in the very complex interrelated pathways that are collectively referred to as One-Carbon Metabolism.⁴⁰ Importantly, these functions of vitamin B12 are apart from its essential role in maintaining the integrity of the central and peripheral nervous system in children and adults. Therefore, whenever vitamin B12 is either borderline low-normal or frankly deficient, it must be rapidly replenished to ensure optimal function of both folate and vitamin B12 in human physiology. For this reason, it would be unethical to withhold vitamin B12 from women of childbearing age in any RCT to show its role in NTD-protection.

See Annexure 1(b). for a discussion of “How does insufficient folate lead to midline defects?”

The serum folate concentration required to breach the “NTD-threshold” is much higher than that required to prevent megaloblastic anemia: A population-based threshold for “optimal” maternal red blood cell (RBC) folate concentration, as a biomarker for the prevention of NTDs, has been defined at ∼1000 nmol/L;⁴¹ this corresponds to a population plasma/serum folate concentration threshold for optimal NTD prevention of 11.3-ng/mL (using a microbiological assay and provided there is no associated evidence of vitamin B12 deficiency).¹⁸ At this concentration, the maternal hematopoietic system does not show morphological evidence of megaloblastic changes in the peripheral blood or bone marrow.¹⁰ This implies that the blood folate concentration in women of childbearing age needs to be much higher to prevent NTD-affected pregnancies than to prevent megaloblastic anemia.⁴² See Annexure 1(c) for added discussion on “A serum folate level required to overcome the risk of NTD—the NTD-threshold” and Annexure 1(d) for a discussion on “Embryonic and fetal tissues are much more sensitive to small reductions in maternal folate and vitamin B12 than hematopoietic cells.”

During early development, embryonic cells receive signals to initiate proliferation at precisely ordained times. At such times, various cohorts of high proliferative potential cells that are pre-destined to form the embryonic neural tube must have significantly more folate and vitamin B12 to support doubling of deoxyribonucleic acid (DNA) during short bursts of rapid cell division, when compared to other embryonic cells.⁴³ Therefore, if maternal borderline low-to-mid range concentrations of folate/vitamin B12 transferred to the embryo are insufficient to support these bursts of cell division, there can be a delay and/or arrest at either S or G2/M stage of the cell cycle; when such a megaloblastic arrest remains unrelieved, this can eventually result in apoptosis of these cells. Therefore, depending on which specific cohorts of neural tube precursors lose their capacity to proliferate and migrate to eventually fuse at the midline [in either the cranial and/or caudal region] during embryological development can explain the spectrum of NTDs observed at birth.^1,43

Is the available evidence sufficient to justify major health policy changes that are designed to prevent NTDs in India? Despite such a plurality of robust data (as described above), a common objection and related concerns posed by policy leaders in several countries have been to insist that “traditional gold standards of evidence (RCT’s and systematic reviews of these RCTs), which have been used to make decisions in clinical medicine, should likewise be used to inform public health policies.”

What has not been sufficiently emphasized in response [to such a position] is that many leading epidemiologists in recent years have published analytical reviews pointing towards a more comprehensive understanding of the traditional and modern approach to define “evidence” in assessments that can help decision making in the realm of public health. Indeed, these more nuanced assessments related to public health are not the same as what has been deemed essential to document advances in the practice of clinical medicine.

Since the deployment of food fortification with folate and vitamin B12 can potentially help save the lives of an estimated 1 million Indian babies that are otherwise lost with each decade of delay,² we will highlight the details why leading modern epidemiologists, who analyzed the pitfalls and drawbacks of traditional RCTs, have also identified alternative methods to generate ‘evidence’ in large populations like India. The key reasons are elaborated in Annexure 1(e), “Do we need more evidence to justify widespread vitamin B12 and folate fortification of food to prevent NTDs in India?”

How did the USA respond to the news of successful prevention of NTDs with folic acid? Several advanced nations have carefully evaluated the scientific evidence favoring the important role of folic acid (folate) in preventing NTDs. The subsequent amassing of political will to pursue a scientifically established approach adopted by many nations was not developed without careful evaluation and deliberation. As one example, the US Centers for Disease Control and Prevention (CDC) in conjunction the US Institute of Medicine (IOM), the US National Academy of Medicine (NAM), independently concluded that food fortification of a widely consumed food vehicle (wheat flour) with folate was a safe, cost-effective, large scale public health initiative that could effectively reduce the less than 5000 cases of NTDs each year in USA. Finally, just over 25 years ago, following expert testimony and discussion, the US Congress voted into law a mandate favoring fortification of wheat flour with folate, so that the entire US population would consume vitamin-fortified wheat flour and its products. Following successful implementation, careful public health follow-up by the CDC documented significant reduction in the incidence of NTDs, a reduction in anemia, and a near complete elimination of folate deficiency in the USA. Equally important, no adverse side effects have been identified from folate fortification in the US over the past 25 years. Therefore, the GOI can be assured that a very careful and cautious process of vetting of the data on effectiveness and safety has already been completed in the USA. This White Paper summarizes the extant data in India pointing to what is uniquely required for the Indian situation for the prevention of NTDs.

The complexity of dietary folate and vitamin-B12 insufficiency in India leads to greater challenges than those in the West. The situation in India today (population over 1.4 billion) is far more complex and serious than any other country worldwide. The baseline prevalence of NTDs in India using hospital records has not changed significantly, a fact coincident with the average Indian diet, which has likewise remained the same for most Indians since the Green Revolution (over 50 years ago). This is evidenced by similar documentation of folate and vitamin B12 insufficiency in the blood of women in South India in 1973⁴⁴ and in 2021;¹² in North India from 2007⁴⁵ and in 2019;⁴⁶ and in Western India in 2001⁴⁷ and 2021.⁷ Parenthetically, the caveat of underestimation of folate deficiency in the presence of a deficiency of vitamin B12⁶ is also applicable in many of these studies.

Thus, there is widespread persistence of combined low-folate and low-vitamin B12 status among most Indian women over 20 years of age in studies from North, South, West, and Northeast India.^7,12,46,48 These micronutrient deficiencies stem from an inadequacy in both vitamins in a vegetarian diet, as well as a ‘near-vegetarian’ diet that is consumed by most Indians.^5,14 This necessitates dual fortification with both of these vitamins. The implications of the close interrelationship and interdependence between vitamin B12 and folate are discussed in Annexure 1(f) under “Key basic science related interrelationships between folate and vitamin B12.”

3) NTDs in India are largely due to dietary insufficiency of folate and vitamin B12

(a). Dietary insufficiency of folate and vitamin B12 adversely affects the health and well-being of all Indians: Although the effectiveness of folate fortification of food to prevent the births of babies with NTD³⁰ has been demonstrated in many advanced countries, there are distinctly different problems faced by a much larger country like India, where the population of her largest state (U.P.) alone could contend as the fifth largest country in the world. The key issues expanded in this White Paper are as follows:

i). The clinical consequences of insufficient folate and vitamin B12 in humans: Indian women of childbearing age and their children have a high prevalence of dietary insufficiency of both folate and vitamin B12. This contributes to a proportionately high prevalence of women with low folate and vitamin B12 status (see next), predisposes to megaloblastic anemia, and interferes with maternal-to-embryonic/fetal nutrition, which places the conceptus at risk for NTDs and childhood-onset neuro-psychological-psychiatric defects. Moreover, apart from the important role of vitamin B12 being essential to enable proper functioning of folate in the body, vitamin B12 is also independently needed to maintain the integrity of the human nervous system throughout life. When left uncorrected, vitamin B12 deficiency can progress to severe demyelination with subacute combined degeneration of the spinal cord and brain.¹⁰ Because Indian girls and women of childbearing age need abundant stores of both vitamins to prepare them for the ordeal of pregnancy, they are at high risk for developing clinical sequelae from insufficiency of both vitamins if they are left unprotected. See Annexure 1(g) for a discussion on the “Distinction between folate- or vitamin B12- ‘deficiency’ versus ‘insufficiency’.”

ii). Perpetuation of Inter-Generational folate- and vitamin B12- insufficiency in India: Over many years of consuming a diet low in both these vitamins, a progressive reduction [insufficiency] of body stores of folate and vitamin B12 culminates in clinical manifestations of either frank folate and vitamin B12 deficiency or borderline low-normal values that portend an incipient deficiency of both vitamins; this is referred to in this White Paper as low-folate and low-vitamin B12 status; this is the root cause of vertical transmission of nutritional folate- and vitamin B12-insufficiency down from one generation of women to the next, and so on, ad infinitum.

In this construct, during pregnancy, an insufficient quota of maternal folate and vitamin B12 is ‘gifted’ across the placenta to the developing fetus, and then to the newborn girl from her mother’s milk. After weaning, this young girl grows up on the same nutritionally insufficient dietary content of folate and vitamin B12 as her mother, and over time, the adolescent will also grow up with a persistent low-folate and -vitamin B12 status. When this young woman becomes pregnant, she too will vertically pass down an insufficient ‘gift’ of low folate and vitamin B12 to her progeny [from the paucity of her low-folate and -vitamin B12 stores]. And so, her newborn will, just like her grandmother and mother, also pass down her own gift of insufficient vitamins vertically down to her own baby, and so on. Such a vertical family tree composed of unhealthy women who are perpetually at risk for NTDs and other potentially severe adverse effects of folate and vitamin B12 deficiency can embody Tolstoy’s observation on unhappy families: “All happy families are alike; each unhappy family is unhappy in its own way.” Fortunately, this unhealthy [and unhappy] state related to a vertical transmission of a low-folate and -vitamin B12 status from one generation to another ad infinitum can be reversed in a single generation by pharmacologic therapeutic dosing of both vitamins well before pregnancy to ensure that the maternal-to-child ‘gift’ of vitamins is optimized.

(b). Is vitamin B12 deficiency common in India? Although measurement of the serum vitamin B12 level is not perfect,¹⁰ these tests are clinically useful to help make the diagnosis of vitamin B12 deficiency when there is a serum vitamin B12 cut-off value of 200 pg/mL or less accompanied by clinical manifestations of either anemia or a spectrum of [often subtle] neurological findings. Some skeptics have long maintained that vitamin B12 deficiency is not common in India. This was apparently reinforced by a recent comprehensive national nutrition survey (CNNS) report,⁴⁹ the first nationally representative nutrition survey of children and adolescents in India, which noted that among adolescents aged 15–19 years, the prevalence of vitamin B12 deficiency was ‘only’ 34%. However, a recent meta-analysis showed that 53% of the population in India is vitamin B12 deficient.⁵⁰ These two studies did not provide a more comprehensive understanding of those individuals with borderline low-normal values, i.e., between 200-300 pg/mL, which has clinically been referred to as having vitamin B12 insufficiency. The significance of this group is that up to 15% of cases with serum vitamin B12 levels between 200-300 pg/mL,⁵¹ have clear-cut metabolic evidence of true vitamin B12 deficiency using more sensitive metabolite tests.⁵² Indeed, the addition of Indian women with borderline low-normal values (vitamin B12 insufficiency) to those who are frankly vitamin B12 deficient provides a larger percentage of Indians with ‘low vitamin B12 status,’ which provides a clearer picture of the overall percentage of the population in India whose stores need to be repleted with vitamin B12. This is illustrated in several recent examples across India that are uniformly at variance with data from women who are only a few years older than the girls and young women described in the CNNS report.⁴⁹

• i).

  In Northern India (Haryana),⁴⁶ among 866 women, 58% were vitamin B12-deficient and an additional 23% had marginal/insufficient serum vitamin B12 levels, thereby pointing to 81% with low vitamin B12 status.

• ii).

  In South India (Andhra Pradesh),¹² among 978 women, 48% had vitamin B12 deficiency, but an additional 26% had borderline low-normal vitamin B12 levels, so 74% had low vitamin B12 status.

• iii).

  In Western India (Maharashtra),⁷ 23% women had vitamin B12 deficiency, but an additional 40% had borderline low-normal vitamin B12 levels, so 63% of women had low-vitamin B12 status.

• iv).

  In Northeast India (Assam),⁴⁸ 51% had vitamin B12 deficiency, and an additional 21% had borderline low-normal vitamin B12 levels, so 72% of women had low-vitamin B12 status.

Thus, the number of Indian women of childbearing age with low-vitamin B12 status in India, from the North, South, West, and Northeast was 81%, 74%, 63%, and 72%, respectively.^7,12,46,48 which is much higher than the composite number derived from a meta-analysis pointing to 53% with vitamin B12 deficiency.⁵⁰ Because these data indicate a high percentage of women with low-vitamin B12 status, the existing Indian daily requirement for vitamin B12 needs reassessment. Moreover, such data immediately questions the scientific basis supporting reliance on any ‘natural and sustainable option’ of food-based intervention. Indeed, none of the so-called ‘natural’ vitamin B12 source foods have been shown to be of value in consistently improving low-vitamin B12 status in formal clinical trials.

Other elements of public health decision-making, including the quantification of benefits and the harms from administration of vitamin B12, as well as the harm from not administering vitamin B12 to those with low-vitamin B12 status, and the overall costs are touched upon in this White Paper [Main text and in Annexure 2].

(c). Presence of combined folate and vitamin B12 insufficiency warrants replenishment of both vitamins to interrupt the inter-generational vertical transmission of both vitamins from one generation to the next. Although the successful implementation of food fortification with folate in the USA provides a general blueprint for replication, the combined insufficiency of folate and vitamin B12 that likely affects many hundreds of millions of Indian women and children, as well as the late-middle-aged and elderly, is a far more complex challenge. Therefore, complete replenishment of low-folate and -vitamin B12 stores, ideally via fortification of a widely distributed food vehicle that can reach all Indians, will be essential to significantly improve the folate and vitamin B12 status of all Indians of both genders at all life stages.

India will, however need to independently blaze her own path in two distinct arenas:

The challenge relates to the urgent need to rapidly replenish Indian women with preexisting combined ‘low-folate and -vitamin B12 status’ with full therapeutic [pharmacological] doses of both folate and vitamin B12, and then switching to the use of smaller doses of both vitamins to supplement the intrinsically folate and vitamin B12 insufficient diet. This challenge necessitates identification of one or more food vehicles that can be centrally fortified and distributed to reach all Indians, including those who live in very remote areas.

Successful implementation of these two distinct challenges using scientifically sound clinical trials can dramatically reduce the incidence of NTDs and other adverse effects of low-folate and -vitamin B12 status throughout India. Here too, India must forge on ahead on its own, since this has never been attempted at such a large scale in any other resource-limited country. However, solving the issue of ensuring optimum folate and vitamin B12 status in India would pave the way to ensure that 90% of the world’s women (in other resource-limited countries who cannot procure sufficient folic acid and vitamin B12⁵³) are also aided.

(d). A low vitamin B12 level is not a benign finding in Indians at all life stages.

Vitamin B12 in women of childbearing age and in pregnancy: In women with low vitamin B12 status, there are additional risks related to pregnancy complications and pregnancy outcomes. Because the mother provides her fetus with vitamin B12 throughout gestation, the longer a mother has been on a low vitamin B12 diet, there is a greater likelihood she will have low -serum and -breast milk vitamin B12 concentrations that closely correlate with a low vitamin B12 concentration in the infant.⁵⁴ The risk of a baby girl growing up on the same inadequate vitamin B12 diet as her mother has added powerful implications for Indian babies.

Vitamin B12 in infancy and childhood: Longitudinal studies on infants fed a macrobiotic diet [which is very low in vitamin B12] in The Netherlands have had profound and long-lasting damage to brain function. At only 15-months, infants had markedly low vitamin B12 status as well as impaired psychomotor functioning,⁵⁵ and despite switching to a diet with marginally increased vitamin B12 content by their 6^th birthday, 20% of these children continued to have low vitamin B12 status associated with persistent cognitive dysfunction and tests of fluid intelligence up to a decade later, with potential negative consequences lasting well into adulthood.⁵⁶ This is yet another reason to ensure sufficient vitamin B12 in infants and children, and also to rapidly fill stores with full doses of vitamin B12 when they are found to be have insufficient levels in their blood.

An RCT among 6-30-month-old North Indian children demonstrated that compared to placebo, children who received both vitamin B12 and folic acid had 0.45 (95% CI 0.19, 0.73) and 0.28 (95% CI 0.02, 0.54) higher SD-units in the domains of gross motor and problem-solving functioning, respectively.⁵⁷ Thus, supplementation of both vitamins had beneficial effects on neurodevelopment in North Indian children. So, the ongoing danger for infants and toddlers and young Indian children is eminently clear from these reports; such studies cannot be reproduced because of serious ethical concerns. Therefore, the Indian Ministry of Health should spare no efforts to protect Indian mothers against dietary vitamin B12 deficiency: The risk of doing nothing can lead to a whole generation of vitamin B12-deficient children (growing into adulthood) who are incapable of making good decisions because of their lifelong neurologic deficits induced by vitamin B12 deficiency since birth.

Effect of vitamin B12 deficiency in young adults, and middle-aged and elderly: The topic of low serum vitamin B12 concentrations found in apparently healthy vegetarians compared to those on a non-vegetarian diet was noted in medical students from Bombay in 1956 (within a year after a report from England). Similar results were confirmed across India in children and adults and even more recently. Importantly, one-half or more of subjects with subclinical vitamin B12 deficiency— (i.e., those with lower-than-normal vitamin B12 concentrations but with no clinical findings on history and conventional physical examination)— have abnormal tests of brain function using highly specialized neurocognitive studies. Of added significance, changes in electro-encephalography, evoked potentials, and P300 event-related potentials, which are electrophysiologic markers of cognitive ability,⁵⁸ were reversed with vitamin B12 therapy, pointing to a causal relationship.

In 2003, a review of vitamin B12 deficiency in India pointed out that this was present at all life stages.⁵ A year later, another comprehensive review noted that vitamin B12 deficiency was a worldwide problem.⁵⁹ As noted above, a recent meta-analysis documented that 53% of the population in India is vitamin B12 deficient.⁵⁰ Ominously, persistence of hyperhomocysteinemia, either reflecting metabolic evidence of vitamin B12 [or folate] deficiency, is now also causally related to several additional occlusive vascular diseases and strokes, cerebral atrophy, premature dementia, and even Alzheimer’s disease.^1,10 Therefore, what was previously considered solely a serious risk to women of childbearing age and their newborn babies with NTDs has now moved to center stage by potentially affecting all strata of society, including those aging policy leaders who hold the keys to reversing the dietary insufficiency of these vitamins across India. Moreover, whereas inaction in optimizing folate and vitamin B12 status used to take an emotional and economic toll primarily on families of children with NTDs, the consequence of continued inaction can now impact all Indian family members at every life stage.

Parenthetically, even among 1092 pregnant women composed of Chinese, Malay, and Indian ethnic groups living in Singapore, a highly-developed country,⁶⁰ a total of 15·6 % of mothers were found to be vitamin B12 deficient (<148 pmol/L) and 41·8% of mothers were vitamin B12 insufficient (148 to 220.9 pmol/L). Those who were vitamin B12 deficient were more likely to belong to the Indian ethnic group, were vegetarians, tended to have higher concentrations of homocysteine, and were more likely to have insufficient concentrations of folate.⁶⁰. Importantly, the infants of mothers with vitamin B12 deficiency had 0·42 (95 % CI −0·70, −0·14) SD lower cognitive scores, compared with infants of mothers with sufficient vitamin B12. This study among the Indian diaspora, which documents a similar problem of low vitamin B12 status when they continue to consume a traditional Indian diet in Singapore, reiterates the need for adequate vitamin B12 and folate during pregnancy to avoid adverse consequences to the development of the newborn even in resource-rich countries.

Finally, even in the USA, among 20-30% of the elderly, there is a problem with ‘gastric food-vitamin B12 malabsorption.’ Other common causes include the use of metformin in Type 2 diabetes and long-term use of medications to reduce gastric hyperacidity, which can also reduce food-vitamin B12 absorption.¹⁰ These are universal problems that are likely also common in India.

(e). What is the evidence to give vitamin B12 with folic acid to reduce NTDs in India. The proposal for widespread administration of vitamin B12 with folate to prevent NTDs in India is primarily based on the following tenets underlying the normal physiology of the interrelationship of these two vitamins:

i). Supplemental folate prevents a significant number of NTDs in direct proportion to the prevalence of folate deficiency in the population.⁶¹ Because of the high prevalence of low-folate status in India, up to 90% of babies with NTDs can be prevented from developing NTDs and instead be born normally.³⁰

ii). Vitamin B12 is essential for the normal function of folate to help perpetuate One-Carbon Metabolism;⁴⁰ so, with a deficiency of vitamin B12, there is an inability of folate to carry out its critical functions within cells.¹⁰

iii). The evidence that vitamin B12-deficiency alone can lead to NTDs is not yet established; however, based on the close inter-relationship between vitamin B12 and folate, whenever the serum vitamin B12 is borderline low-normal or low in women of childbearing age, merely providing folate without vitamin B12 will fail to optimize folate function within cells. Review of several observational [low-grade evidence] studies suggests that low maternal vitamin B12 status increases the NTD-risk to the developing embryo.⁶²

iv). Several contemporary publications attest to a high prevalence of a low-vitamin B12 status in women above 20 years of age across India.^{7,12,46,48,50} Therefore, solely relying on the CNNS report,⁴⁹ which only studied girls and young women up to 19 years, and concluding that young Indian women need not be given vitamin B12, must be questioned. Therefore, it is imperative from a basic science and clinical medicine standpoint that vitamin B12 must be replaced together with folate to ensure an optimum folate and vitamin B12 status throughout India.

(f). The need for a radical approach to reversing longstanding dietary-inadequacy induced low-folate and -vitamin B12 status. In the absence of any attempt at radically improving the folate and vitamin B12 status among Indian women, there is no possibility of effectively interrupting the vertical transmission of this dietary insufficiency from one generation of Indian women to the next. Therefore, the challenge is to impartially weigh the clinical consequences arising from a longstanding [preventable] dietary insufficiency of both vitamins on the health and well-being of Indian women and children. Addressing this issue can have a profound impact on women and children before conception, during the first month of pregnancy, and throughout in utero development. For example, without intervention, following birth of the baby of a mother with insufficiency of both vitamins, there is a proportionate reduction of the content of both vitamins in breast milk that is passed on to the breastfed child; and following weaning, this folate and vitamin B12 insufficiency is further perpetuated as the child shares in her mother’s diet, which is intrinsically low in folate and vitamin B12 content. Since the average Indian diet remains relatively unchanged from one generation to the next, the passage of a chronic insufficiency of both vitamins from one generation of mothers to their children, grandchildren, and great-grandchildren [ad infinitum] needs a radical reversal from the existing status quo. This situation in India is distinctively different from that encountered in the West, where low-vitamin B12 status is not such a clinically significant issue.

(g). Do males have a low-folate and -vitamin B12 status too? Encouraging girls and women of childbearing age to consume folate- and vitamin B12-fortified food immediately raises the question of why boys and men are excluded from receiving such special foods. Since boys and men tend to consume the same diet as their mothers and wives, respectively, they too are likely to have similar low-folate and -vitamin B12 status, which renders them prone to deficiency of folate and vitamin B12. Therefore, it can be reasonably deduced that the high prevalence of Indian women with combined low-folate and vitamin B12 status will also be found in boys and men.

Even with finding a low serum folate and vitamin B12 concentration in the absence of anemia, there can be subtle cognitive or other neurological deficits attributed to low-vitamin B12 status (that are not commonly elicited in an outpatient clinic setting). However, several subtle neurocognitive deficits can be documented using sophisticated neurological tests such as P300 testing (which is not widely available across India). Since it is impractical to assess serum folate and vitamin B12 levels among hundreds of millions of Indians, the most expeditious approach is to rapidly replenish both folate and vitamin B12 in all men, women, and adolescents with full therapeutic doses of folate and vitamin B12.

This is particularly important since there are long-term effects of untreated hyperhomocysteinemia that can have devastating effects on vascular integrity (small vessel strokes) as well as premature brain atrophy, premature dementia, and even Alzheimer’s disease.^1,10 Moreover, no Indian who consumes a regular Indian diet is immune to such problems, including those Indians living outside of India in both the UK⁶³ and USA.⁵⁹ Thus, as with the entire population in the USA, all ages can eventually be encouraged to participate in consuming folate and vitamin B12 fortified food; even small children can be given lower dose [tasteless] tablets mixed into sweetened water.

(h). What is the rationale for rapid replenishment of folate and vitamin B12 in full therapeutic doses, especially in Indian women? Ensuring that women of childbearing age in India have sufficient stores of folate and vitamin B12 (and iron) before pregnancy has important implications in preventing the perpetuation of a population-wide, vertical inter-generational transmission of micronutrient insufficiency from mother-to-child down several generations—(as described above).

The critical importance of rapid restoration and optimization of stores of any deficient micronutrient is an axiom in Clinical Hematology.^1,10 Thus, all women with either deficiency or insufficiency in any one or both or all three key micronutrients [contributing to anemia] must be immediately replaced at the outset with full therapeutic [i.e., pharmacologically appropriate] doses of vitamin B12 and folate (and iron).^1,10 Only after this first step is completed would it be clinically appropriate to use a lower supplemental ‘maintenance dose’— that is primarily designed to supplement the ongoing daily dietary insufficiency, which contributed to the micronutrient deficiency in the first place.^1,10 See Annexure 1(h) for a discussion on “What is the basis for use of full therapeutic doses of folate and vitamin B12 to ensure rapid replenishment of folate and vitamin B12 among Indian women?”

Without this critically important two-step intervention in replacement therapy,¹ young woman of childbearing age can continue to limp through life without ever filling up their stores of these micronutrients. With a meager ‘bank-balance’ of micronutrients, these women cannot provide their developing progeny during in utero life and after birth with a healthy quota of micronutrients. Indeed, a deficiency of either iron, folate, or vitamin B12 in a pregnant woman can adversely affect the in utero and postnatal neurodevelopment of her baby, which can manifest in postnatal neurologic, psychologic, or psychiatric consequences in childhood.¹ This unhealthy state can be rapidly interrupted by full therapeutic dosing with folate and vitamin B12 (and iron) replacement.

By contrast, use of any non-clinically proven/approved (i.e., unorthodox) approach, such as replenishing deficient vitamin stores using either physiological doses or subtherapeutic dietary supplemental doses of both vitamins via inadequately fortified food, is to be discouraged. This is because during slow reversal of the low-vitamin status among a large population of Indian women over several months [approaching a year]⁶⁴ allows them to suffer the consequence of micronutrient deficiencies for themselves as well as their babies. For example, each month’s delay in optimization of folate and vitamin B12 status among Indian women places millions of them at an ongoing risk for [vitamin-responsive] anemias; the birth of potentially thousands of children who are conceived while their mothers are still not fully replenished enough to overcome the NTD-threshold and are therefore also at risk for postnatal neuro-psychologic and psychiatric dysfunction.¹ Given the evidence from the literature,¹ any such unorthodox practice among Indian women could be judged ethically unacceptable in a court of law.

Therefore, the challenge facing policy leaders is that only after initial complete replenishment of these vitamins using a pharmacological dose of folate and vitamin B12 over a short period of approximately 4 months, would it be reasonable to supplement the daily diet with a ‘lower-strength’ folate- and vitamin B12-fortified food that compensates for ongoing dietary insufficiency of these vitamins among the supermajority of Indians.

(i). The importance of treating a borderline low-normal serum folate- and vitamin B12- level. From a clinical perspective, an individual can have a combined low serum folate and/or low serum vitamin B12 concentration that reflects low-folate and -vitamin B12 status (and stores) of these vitamins in the body. This state can easily become transformed into a frank clinical deficiency with the superimposition of a physiological stress like pregnancy or lactation, or a pathological stress such as a dietary inadequacy or malabsorptive state, or hemolysis. Therefore, it is deemed prudent to treat all such scenarios with full therapeutic doses of 1- to 2-mg folate and 2-mg oral vitamin B12 daily for 4 months to rapidly replace depleted stores.^10,65 This approach of rapid replacement using full therapeutic [pharmacological] doses avoids the possibility of a slower response that allows for the superimposition of adverse risk to Indian women of childbearing age (who may become pregnant before their folate and vitamin B12 stores are abundantly replenished to support a pregnancy).

(j). Is vitamin B12 a risk factor for NTDs? In Ireland, which had a high prevalence of NTDs and no folic acid fortification, as was observed with folate supplements,⁶⁶ there appears to be a graded relationship between lower levels of circulating vitamin B12 and increased risk of an NTD-affected pregnancy.⁶⁷ These investigators used cut-offs for vitamin B12 to analyze risk by maternal vitamin B12 status: These cutoffs for vitamin B12 represented deficient (0–149-ng/L), borderline-deficient (150–199-ng/L), low-adequate (200–299-ng/L), adequate-good (300–399-ng/L) and good (≥400-ng/L) status.⁶⁷ This is the basis for our own clinical trial assessment of serum vitamin B12 levels being ‘adequate-to-good’ with values of ∼300–400-pg/mL, and ‘good’ being ∼400–500-pg/mL or above, among Indian women treated with vitamin-fortified-Tea. 

4) Rapid replenishment of folate and vitamin B12 with full therapeutic (pharmacologic) doses of tablets or vitamin-fortified foods (as guided by Clinical Trials).

(a). Rationale for initial rapid optimization of folate and vitamin B12 status in most Indian women before instituting low-dose maintenance supplementation of the chronic dietary insufficiency. An important concept often missed in nutrition policy discourse is that the amount of folate and vitamin B12 supplemented in any fortified food is designed to gradually make up for an existing shortfall in the diet—i.e., to ‘fill in the gap’ slowly over several months. By the same token, traditional fortification of food has never been intended to rapidly treat a serious existing clinical deficiency of folate and/or vitamin B12 in the population; that belongs to the realm of pharmacology that necessitates full-therapeutic dosing regimens for rapid restoration of normal physiology. Moreover, because an untreated deficiency of folate and vitamin B12 in women of childbearing age poses a potential grave risk for a conceptus developing an NTD, the speed of vitamin replenishment required to optimize the vitamin status takes on paramount importance. Thus, with a clinically significant low-folate and -vitamin B12 status [as is found in most Indian women], full pharmacological therapeutic dosing of folic acid and vitamin B12 is critically required to rapidly replete body stores of both vitamins, and thereby reverse their low-folate and -vitamin B12 status to effectively reduce the imminent risk to the conceptus of developing a NTD early in pregnancy.

A caveat that warrants emphasis is that among Indian women with long-standing low-vitamin B12 status who were given a low physiological daily dose of oral vitamin B12, it took nearly a full year to normalize their vitamin B12 levels.⁶⁴ This unnecessary delay in normalization of vitamin status for all Indian women with low vitamin B12 status must be avoided by the use of full therapeutic replacement doses of vitamins, the standard treatment for several decades.^1,10,11,68 Moreover, as noted above, recent population-based studies identified a threshold level for serum folate levels of >11.3 ng/mL beyond which NTD risk is reduced.¹⁸ However, this applies only to a minority of Indian women because over two-thirds have combined low-folate and -vitamin B12 status for which the NTD-threshold is invalid. Therefore, an estimated ‘generally safe’ range of serum folate well over 11.3 ng/mL (i.e., closer to 15–20 ng/mL) is not unreasonable.^1,18 Likewise, a general estimate to ensure replenishment of vitamin B12 in a “safe” range would be a serum vitamin B12 of 400-500 pg/mL.⁶⁷ See Annexure 1(i) for a discussion on “Why is the NTD-threshold not valid for the majority of Indian women?” Thus, as discussed above, since >70% of Indian women have a low-folate and -vitamin B12 status, the only solution for rapid optimization of folate and vitamin B12 status is to help them vault over this threshold by full therapeutic administration of up to 2-mg of folic acid and 2-mg of vitamin B12 daily for ∼4 months;^1,2,7,10,68 this will ensure prevention of NTDs within the shortest possible time. This will be especially valuable among young adolescent girls and women of childbearing age to help terminate the inter-generational cycle of nutritional folate and vitamin B12 deficiency.^1,2

Parenthetically, it cannot be overemphasized that restoring a clinically serious deficit in folate and vitamin B12 in the body is applicable to Indians of all life stages and gender. Restoring normal physiology prevents neurological deficits from vitamin B12 deficiency, megaloblastic anemias induced by either vitamin, and the dangers of long-term hyperhomocysteinemia on vascular occlusive disease as well as brain dysfunction.

Only after the first step using full therapeutic [pharmacological] doses of both vitamins for ∼4 months will smaller near-physiological ‘maintenance’ doses of both vitamins help to supplement the existing daily vitamin-deficit in the diet and thereby maintain long-term optimal folate and vitamin B12 status—see below).

(b). Will tablets of folic acid and vitamin B12 be sufficient for all Indians over the short and long term? What doses of both vitamins should be used over the long term? As discussed above, there will be a need for two distinct doses of folate and vitamin B12: The first to rapidly replenish a major deficit in body stores, followed by a lower dose of both vitamins administered long-term to compensate for daily dietary insufficiency. The first step can be accomplished by either use of a single tablet containing 2-mg each of folic acid and vitamin B12 for 4-months or by a daily portion of vitamin-fortified food that contains a similar amount of folic acid and vitamin B12 that is consumed for 4-months; these should suffice to replenish the underlying existing vitamin deficit.

Folate and vitamin B12 are relatively inexpensive vitamins.¹ The cost of adding 1 mg folic acid and 1 mg vitamin B12 as a daily dose (in either tablet form or a daily portion of vitamin-fortified food) to ensure a rapid full therapeutic replacement dose of low-vitamin status is only Rs. 0.2 per day.¹ Therefore, the GOI can ensure that fortification of any food vehicle should not significantly raise the basic price of each day’s portion of vitamin-fortified food.

Once low-folate and -vitamin B12 status has been reversed with rapid replenishment with full therapeutic [pharmacological] doses of folate and vitamin B12, it will be necessary to supplement what is generally missing in the Indian diet. This entails the use of smaller near-physiological ‘maintenance’ doses of both vitamins to help supplement the existing daily vitamin-deficit in the diet and ensure long-term optimal folate and vitamin B12 status.

An important question is: What do young educated Indian women prefer—combination folate and vitamin B12 tablets or vitamin-fortified food? Many Task Force members have delivered educational talks on the prevention of NTDs over two decades in the following States in India: Northwest (Punjab, New Delhi); Northeast (West Bengal, Assam, Manipur, Meghalaya, Nagaland); West (Maharashtra); North (Uttar Pradesh and Bihar); Central (Madhya Pradesh); and South India (Karnataka, Telangana, Kerala and Tamil Nadu). Informal polling of these young, educated school and college-aged women across India has highlighted their uniform distaste for long-term consumption of daily tablets during their reproductive lifetime. Instead, there is an enthusiastic preference for a long-term food-based approach for replenishment of folate and vitamin B12.⁷ This sentiment is consonant with views of young women in many other countries.

When given the option of consuming either a daily combination tablet containing folate and vitamin B12 every day for 4 months versus a specially formulated vitamin-fortified food for 4 months, most women again opted for the latter. However, a few thoughtfully observed that a combination vitamin tablet could provide more assurance of receiving a full daily dose of vitamins than their erratic consumption of a vitamin-fortified food (like wheat or rice) every day for 4 months. Therefore, this minority of Indian women could be amenable to taking a daily tablet for 4 months to ensure their folate and vitamin B12 stores are completely replenished. (But, as noted above, long-term compliance with consumption of a lower supplemental ‘maintenance’ dose to compensate for daily dietary inadequacy of both vitamins clearly warrants development of a vitamin-fortified food).

An all-purpose vitamin-fortified food for initial fortification with pharmacologic doses of folate and vitamin B12 (4 months), as well as over the long term using small supplemental doses to the diet, would be ideal.

The rationale for pharmacological dosing for therapeutic replacement of a dual deficiency/insufficiency of folate and folate and vitamin B12 over 4 months has been discussed in the previous section. However, the choice of the dose for long-term maintenance following rapid replenishment with folate and vitamin B12 is less clear.

The body stores of folate (before mandatory folate-fortified foods were available) can last only 4 months.¹⁰ Therefore, there is a need to prevent fully folate-replenished subjects from gradually declining into a state of folate insufficiency/deficiency over time when they continue to consume a diet that is inadequate in providing the daily requirement of folate. Therefore, one can argue that the ideal dose of folate for long-term fortification should be close to the recommended dietary allowance (RDA), which is derived from the nutrient requirement for folate; the RDA can ensure that intake levels meet the needs of nearly all (97%) of the Indian population. This was the approach employed for folate fortification of food in the USA/Canada. By contrast, use of the Estimated Average Requirement (EAR) for long-term [daily] maintenance of an optimum folate and vitamin B12 status, which is smaller than the RDA, is estimated to meet the nutrient requirement of only one-half of the healthy individuals in a life stage and gender group. Therefore, there appears to be a lack of clarity in justifying the use of a lower dose of folate (derived from the EAR over RDA) for food fortification in long-term maintenance. Additional debate on this issue can be left to informed, unbiased members within the Food Safety and Standards Authority of India (FSSAI).

With regards to daily vitamin B12 requirements: Although the previous RDA of vitamin B12 has been 2.4 µg/day, most Western experts feel that ∼6 to 7 µg/day of vitamin B12 is optimal.⁶⁹ Because Indian diets provide less than 0.5 µg/day of vitamin B12, a slightly higher-than-Western recommended dose may be suitable. In this context, we can leave it to the policymakers to decide if 5-micrograms or less of vitamin B12 daily⁵ appears to be too prodigal for most Indians over the long term.

On the other hand, once fully replenished with pharmacologic dosing over 4 months, vitamin B12 remains in the body for between 5 and 10 years. Therefore, the addition of the lower EAR dose for long-term maintenance of optimal vitamin B12 status in a fortified food would be reasonable, since not all subjects are likely to become vitamin B12 deficient at the same time over the longer duration of 5-10 years.

Finally, if there is post-implementation surveillance evidence that many Indian women are prone to having early deficits in folate, they can easily fall back on taking another short course (of 4-months or less) of a combination of either full-doses of vitamin tablets or full-doses of folate and vitamin B12 added to fortified food to restore optimum vitamin status. Any excess folate or vitamin B12 will easily be excreted in the urine.

Thus, choosing an acceptable food-vehicle for fortification with folate and vitamin B12 is complex for such a large country as India with distinct dietary habits in and even within her very large States. This is the basis for additional discussion in this White Paper on the safety of folate and vitamin B12, as well as a discussion of the variety of food vehicle options for fortification, the means to ensure successful implementation, and subsequent monitoring of the effectiveness of this campaign.

5) Safety of Folic acid and vitamin B12

Folic acid and vitamin B12 have been given in full therapeutic doses (1-to-2 mg each of folic acid and vitamin B12) to those with a deficiency of these vitamins since the 1960s with no major problems. This fact alone provides significant assurance on concerns about the danger of long-term administration of these vitamins.

(a). Safety of folic acid: There has also been 25 years of experience following the institution of a mandatory program of folic acid fortification in the USA and Canada, where there is an additional safeguard of a careful surveillance system to pick up early evidence of adverse effects arising from this practice. There have been no documented adverse effects in either the US or other countries using the WHO-recommended amounts of folate added to wheat flour. Indeed, the safety of folic acid fortification is now confirmed through multiple studies.^31,33

Less than a decade ago, an expert Committee⁷⁰ reviewed major national reports on the safety of folic acid fortification from the U.S. National Toxicology Program (in 2015), the UK Scientific Advisory Committee on Nutrition (in 2016) and other authoritative reports up to 2018. The conclusion from a scholarly review of the literature was that there was an overwhelming benefit arising from folic acid fortification in the prevention of NTD-births. Importantly, there was no evidence pointing to adverse consequences from imposing a mandatory folic acid-fortification program in many countries. As a consequence, there was a uniform recommendation in favor of folic acid fortification to improve the folate status of the entire population.⁷⁰

(Professor Patrick Stover, a member of the US National Academy of Science, Engineering, and Medicine concluded following a comprehensive review of the safety of folic acid used in fortification in the USA¹⁶): “Overall, the totality of the evidence of the literature fully supports the benefits of mandatory folic acid fortification in NTD prevention. Furthermore, there are no established risks for adverse consequences resulting from existing mandatory folic acid fortification programs that have been implemented in many countries.” Thus, there is strong evidence of the safety of providing folic acid through the fortification of staple foods.¹⁶ In 2019, another NIH Workshop⁷¹ concluded: “Observations indicating adverse effects from excess folic acid intake, elevated folate status, and unmetabolized folic acid (UMFA) remain inconclusive; the data do not provide the evidence needed to affect public health recommendations. Moreover, strong biological and mechanistic premises connecting elevated folic acid intake, UMFA, and/or high folate status to adverse health outcomes are lacking.”

The USA Food and Nutrition Board established an upper limit (UL) for synthetic forms of folate available in dietary supplements and fortified foods at 1,000-micrograms (µg); this is equivalent to 1,667 µg dietary folate equivalents (DFE) because 0.6 µg folic acid = 1-mcg DFE. However, and of particular significance to the issue of therapeutic [pharmacological] dosing of 2-mg folic acid daily for those individuals with low-folate status, these ULs do not apply to individuals taking a short course of high doses of supplemental folate under medical supervision. Thus, our recommendation to first rapidly replenish Indian women with 2-mg folic acid daily for an average of 4 months is a sufficient duration to fill up previously depleted stores of folate in most humans.

(b). Safety of vitamin B12: Of particular significance to NTD-prevention in India, the expert Committee⁷⁰ also recommended that “in countries with a high prevalence of B12 deficiency, consideration of B12 inclusion with folic acid should be a priority in NTD-prevention efforts” and “considering the coverage limitations of fortifiable cereal grains in many countries, it is important to invest in further research of potential new vehicles to enhance scale-up of the delivery of folic acid.”⁷⁰ Finally, “the overarching conclusion [of the expert Committee] is that large scale food-fortification should be a component of most national public health strategies, particularly where there is a fortifiable food vehicle consumed regularly by the majority of the population and produced by a reasonably small number of production facilities.”

The USA Institute of Medicine Food and Nutrition Board of the National Academy of Medicine has not established a tolerable upper intake level (UL) for vitamin B12.⁷² Parenthetically, this UL is the highest level of long-term daily intake of vitamin B12 that is likely to pose no risk for adverse health effects to the population. This reflects the negligibly low potential for toxicity even at large doses because the body does not store excess amounts of water-soluble vitamin B12, which is exceedingly well excreted in the urine.⁷²

(c). Added discussion of health risks and safety of food fortification with folic acid and vitamin B12: It must be reiterated that the intake of folic acid below the established tolerable UL of 1000 µg/day for the general population has also not been associated with adverse health outcomes in the USA over the past 2 decades of mandatory folate fortification in USA.¹⁶ In addition, there is no upper limit of toxicity for the intake of vitamin B12. The treatment of pernicious anemia related vitamin B12 deficiency with 1-2 mg tablets of vitamin B12 (cyanocobalamin) daily is standard practice in Hematology¹⁰ and Internal Medicine⁷³ and Nutrition^11,14 for several decades; this allows for up to 20-to-25-µg of vitamin B12 to be [passively] absorbed daily over the long term in patients with pernicious anemia and gastric food-vitamin B12 malabsorption.

Collectively, there is already a significant body of evidence that long term use of both folic acid (used in hemolytic states like sickle cells anemia) and vitamin B12 (in pernicious anemia) for over 50 years are safe; ^10,11 moreover, there is a large body of evidence in the literature to demonstrate effectiveness of folate fortification of food in improving maternal folic acid intake and in preventing NTDs. Furthermore, the U.S. National Toxicology Program report has emphatically asserted there was no conclusive evidence for adverse effects from folic acid.¹⁶ See Annexure 2 for ‘Frequently Asked Questions’ related to various off-target effects of folic acid in humans.

6) Status report of three key elements needed for prevention of NTDs in India

Meeting the challenge of a 90% reduction in the annual incidence of NTDs by raising the folate and vitamin B12 status of women throughout India is critically important and eminently feasible. This will require the development of three closely integrated [but independent] projects that are simultaneously rolled out across India. Use of the blueprint employed by the successful Pulse Polio Immunization (PPI) campaign that reached all of India is an excellent starting point: This includes the delivery of a well formulated message via a multimedia approach designed to create national awareness and pointing to the method to prevent the problem (polio) by immunization, and then implementation of the plan. The parallels to prevention of NTDs are obvious: For example, there is a great need to develop a national campaign explaining the current problem of NTDs and first pointing adolescent girls and Indian women of childbearing age to the way to substantially reduce their risk of having a baby with NTDs. This is accomplished by initial consumption of either [high-dose]-folate-and-vitamin B12 tablets or [high-dose]-folate-and-vitamin B12-fortified food for at least 4 months. Next is the use of a [low-dose]-folate-and-vitamin B12-fortified food over the long term. Others across India (children, adolescent boys and men) can then follow the lead of Indian women. Finally, the development of a robust network of Birth Defect Surveillance Registries in each State can capture the effectiveness of these interventions in the reduction of NTDs after a lag time of ∼2 years. So, the incidence of baseline birth defects across India can be captured during this lag time, whereas the effectiveness of the intervention can be documented later. In the meantime, studies can be carried out to document the relative efficacy of combined vitamin tablets versus combined vitamin-fortified foods in optimizing the folate and vitamin B12 status of women. However, we must first consider the current status of these 3 key elements in India.

(a). Status report: Key element 1. Current awareness about NTDs: Awareness of the potential for prevention of NTDs with folic acid [or need for vitamin B12] is almost non-existent across India, irrespective of their educational level or socio-economic status. Almost all women of childbearing age in India are unaware of the serious problem that having a child with NTD poses to them. Thus, this lack of awareness is universal in cities, towns, and villages across India. Over the years, many Task Force members have educated tens of thousands of Indian women students in high schools and colleges across several States in India on the importance of improving their nutrition. We delivered lectures across Northwest (Punjab, New Delhi), Northeast (West Bengal, Assam, Manipur, Meghalaya, Nagaland), West (Maharashtra), North (Uttar Pradesh and Bihar), Central (Madhya Pradesh), and South India (Karnataka, Telangana, Kerala and Tamil Nadu). However, none of us have encountered a single woman with any practical knowledge of the importance of ensuring her own optimum folate and vitamin B12 nutrition to reduce her own risk of having a baby with an NTD in the future.

Over the years, sporadic attempts by various state governments or voluntary agencies have undertaken small-scale programs and ‘awareness’ campaigns via ‘health-improvement camps’ and related programs; provision of tablets of folic acid for women; supply of folic acid/vitamin B12-fortified wheat flour, rice, and salt.^74,75 However, because these efforts were limited in scope and necessarily short-term, they have not achieved meaningful results.

With the hope of improving the iron and folate status of women of childbearing age and reducing the high incidence of anemia in Indian women, the GOI has had a program for weekly distribution of supplements containing a combination of iron-folic acid tablets (composed of 100 mg elemental iron and 500-mcg folic acid). However, in practice, both adolescent and young women we have encountered have uniformly admitted to poor compliance (adherence) in taking these tablets regularly; this is invariably triggered by the well-known side effects of oral iron on the gastrointestinal system. In Dibrugarh, Assam, where such data was published, revealed that only 15% of women completed a course of iron-folate tablets provided.⁷⁶ A more recent national survey conducted across all 28 states, 8 union territories and 707 districts between 2019-2021⁷⁷ revealed that although 88% had access to iron and folic acid tablets during pregnancy, less than half of pregnant women (44%) used these tablets for 100 days.⁷⁷ Apart from the failure of the iron-folic acid program, this intervention is invariably instituted well past the time when the decision for neural tube closure is made. There is also no education on the critical requirement of vitamin B12, which is inadequate in their diet. Moreover, women need education so that when side effects arise, they are solely due to the side effects of iron. So, when women discontinue these iron-folate tablets, they lose the ability to optimize their folate status, leaving them vulnerable to the risk of having a baby with an NTD. Once again, this highlights the need for a program to allow for the administration of full therapeutic doses of both folate and vitamin B12 several months before contemplating pregnancy.

When awareness campaigns have been instituted on the topic of prevention of NTDs, these have been hampered by a lack of appropriate messaging, and other pitfalls that develop from a lack of sustained Government leadership, focus, and funding. This has led to narrow pockets of targeted women (most often urban women) and waning of interest when there is insufficient financial support. Indeed, reaching Indian women will be particularly difficult since they are busy, often working hard in double shifts (in handling their affairs at home as well as working outside the home to earn a wage). Therefore, all such awareness programs that target women need to be instituted at a national level, ideally along the lines of the successful PPI campaign.

(b). Status report: Key element 2. Availability of vitamin-fortified foods/tablets and the challenge to identify an acceptable indian food for fortification with folate and vitamin B12. Ideally, a diversified balanced diet containing a regular intake of carbohydrates, proteins, fats, and micronutrients can be accessed by anyone who consumes cereal grains or rice, dairy products and other animal-source foods, vegetables, lentils, nuts, and fruits.¹⁴ While such a diet can provide the necessary micronutrients for optimum health, such diets are not affordable by a large segment of the Indian population. This is evidenced by the finding of a high percentage of young Indian women with low folate and vitamin B12 status. Therefore, fortification of a universally consumed food in India is the next-best option to replenish folate and vitamin B12 [Figure 5].

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Figure 5:

A collage to highlight the fact that NTDs that can arise from a low folate and vitamin B12 status in Indian women, which can be largely prevented by one or more candidate foods for fortification with therapeutic doses of folate and vitamin B12. The small round photographs include (from top left, clockwise): a newborn with spina bifida; another child with hydrocephalus associated with a NTD; use of a daily cup of Indian black tea as a food vehicle for fortification; an Indian woman who was fully repleted with vitamins in preparation for pregnancy and the availability of other candidate foods like wheat/rice/salt for fortification with both vitamins. (Courtesy of Dr. RM Vora and Bhavin K Paleja, Sangli)

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India is not among the 80+ countries that have adopted the use of folic acid-fortified wheat flour to reduce the risk of NTDs.^17,78 In such countries, large-scale conversion of wheat to flour is carried out centrally in large roller mills; fortification is then achieved by ensuring the addition of ∼140 µg of folic acid per 100 g of wheat flour. This folate-fortified wheat flour is then widely distributed throughout the population, which must also necessarily rely on the provision of such ready-made flour for purchase in easily accessible shops. Therefore, the success of the distribution of any such fortified food-vehicles for a vast country like India will be highly dependent on (and restricted by) the extent to which each of these fortified foods are efficiently distributed in stores, purchased at an easily affordable price, and consumed by women living in the smallest villages and hamlets of India. In this context, the choice of a food-vehicle for large-scale fortification and its widespread distribution to easily reach every Indian on the subcontinent is critically important to achieving ultimate success. This warrants consideration of several uniquely Indian issues that must be accommodated: An overarching obstacle is that India poses several unique challenges which stem from the fact that ∼70% of the population lives in over 650,000 villages, which poses several inherent logistical problems related to several factors: included among these are issues of equitable distribution, acceptability of choice of fortified food vehicle, and affordability. Another practical issue is that there are several types of whole grains grown across India that are dependent on local geographic and climatic conditions that permit the growth of one or more of such grains. These grains include wheat (gehu), rice (chawal), pearl millet (bajra), finger millet (ragi), sorghum (jowar), corn/maize (makka), barley (jau), and less commonly, amaranth (rajgira). Even in a single large State in India, different grains may be consumed in different seasons by a single family. This can potentially complicate plans for choosing a single grain for fortification, because no such contextually appropriate single grain exists for fortification with folate and vitamin B12.

Thus, the challenge in identifying a common food-based vehicle that is acceptable to all Indian women of every geographic, social, economic, cultural, and ethnic distinction (whose diet is dictated by these differences) is a formidable one. Nevertheless, in general, poverty [or any crisis] can force an entire population to accept any type of easily available, subsidized, staple food. In such a situation, most rigidly held food preferences fall away and become less selective, thereby allowing for greater flexibility in acceptance of any less commonly consumed staple fortified food. The GOI’s vast Public Distribution System that is responsible for distributing both food and non-food essentials at subsidized prices through ‘Ration Shops’ can potentially reach all remote villages of India with a message that the subsidized staple food provided contains health-promoting vitamins; thus, a previously unfamiliar staple food can potentially become transformed into an acceptable vehicle for fortification with folic acid and vitamin B12. Interviewing focus groups, composed of women of childbearing age and their village leaders from several States across India, can help the GOI in understanding, planning, and acting on whether such an adaptation to consumption of a previously unfamiliar staple food is both plausible and actionable.

Currently, there are no tablets containing 2 mg folate and 2 mg vitamin B12 either singly or in combination to rapidly replenish Indians with a baseline low-folate and -vitamin B12 status. This must be rectified. See Annexure 1(j) for a detailed discussion on: “The track record of the Indian Government’s studies on vitamin-fortification of food: Arguments in favor and against fortification of wheat, rice, salt, and tea.”

(c). Status report: Key element 3. Birth defect surveillance registries across India: Although there are small birth defect registries in some cities, India does not have a nationwide network of Birth Defects Surveillance Registries in each State. This is needed to accurately assess the baseline incidence of birth defects like NTDs and evaluate the effectiveness of any intervention containing folate and vitamin B12 over time. These registries can also confirm whether an intervention is working even in the most remote parts of India. This will be critically important after the rollout of the planned intervention to reduce NTDs. Therefore, the development of a robust interconnected network of such birth defect registries in every State of India forms the 3^rd key element.

The benefits of continuous surveillance and vigilance of birth defects through such a birth defect network of registries in the USA show the power of how a similar registry can help India. Indeed, how the USA responded to reducing the annual births of fewer than 5000 babies with NTDs each year is an instructive example.⁷⁹ So, the moment the solution to preventing the first occurrence of NTDs with folic acid was published in 1992, within the same year, the U.S. Public Health Service recommended that all women capable of becoming pregnant should consume 400-micrograms (µg) of folic acid daily to prevent NTDs. Moreover, a mere 6 years later, after realizing that folic acid tablets led to poor compliance among US women of childbearing age, the US Food and Drug Administration (FDA) mandated that sufficient folic acid must be mixed into wheat flour to ensure that American women are protected from the risk of giving birth to babies with NTDs.^10,11,80 Even after folate fortification of flour, important follow-up studies in the USA then identified that Hispanic women were not sufficiently being protected from NTDs because their staple food was corn flour-derived [and not wheat flour-derived]. This led to a corrective action to mandate folic acid fortification of corn flour to resolve this oversight.

Such a focused sequence of actions is an excellent blueprint for successful control of NTDs in India using a similar robust National Birth Defect Surveillance Registry across India. See Annexure 1(k) for a discussion of “Potential for folate fortification to unmask other midline congenital defects.”

7) Prevention of NTDs by a closely coordinated tripartite program

This initiative for successful reversal of low-folate and -vitamin B12 status in India must necessarily rest on three pillars: Judicious planning and roll-out of a National Educational Awareness Campaign coincident with the availability and [subsidized] provision of pharmacologic replacement of folate and vitamin B12 tablets (2-mg of each for 4-months) for young adolescents and women who choose this route. Since folate- and vitamin B12-fortification of a widely consumed food vehicle is the most efficient way for delivery of both water-soluble vitamins to a large population, this will necessitate identification of one or more centrally processed food-vehicle(s) fortified with pharmacological daily doses of folate and vitamin B12 for 4 months. This vehicle should meet criteria of being easily affordable, widely available, acceptable and efficacious (after completing Clinical Trials) in optimizing the low-folate and -vitamin B12 status of all Indians. The various options for vitamin-fortification of candidate foods are discussed in detail in Annexure 1(j), The track record of the Indian Government’s studies on vitamin-fortification of food: Arguments in favor and against fortification of wheat, rice, salt, and tea. Eventually, the effectiveness of the vitamin-fortified foods chosen can be evaluated by assessing their capacity for optimization of folate and vitamin B12 status and reduction of NTDs using the National Birth Defects Surveillance Registry, the third pillar. However, each of these three pillars has intrinsic challenges:

(a). The first pillar—A national educational awareness campaign.

i). Educating hundreds of millions of adolescents and women of childbearing age in India: The major challenge will be in explaining to Indian women of the problem NTDs, how they can reduce their risk of having a baby with NTD, how they can do this by consumption of either a combination tablet of folate and vitamin B12 or a vitamin-fortified food containing full doses of these vitamins for a minimum of 4 months; and finally by consumption of a daily vitamin-fortified food containing a smaller dose over the long term [Figure 6].

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Figure 6:

Elements of a comprehensive national awareness campaign include key agencies and educators who can target various adolescents and women of childbearing age. The agencies should include: the Government of India, Ministry of Health and Family Welfare (MOHFW), the Spina Bifida Foundation, Educational Institutions, and Marriage Registry Offices. Other agencies that have already had experience in facilitating major initiatives [such as the Pulse Polio Immunization Campaign] can also be recruited. All health care institutions in large cities and smaller towns can also reach large numbers of the population of women, as can family physicians and specialists working with both women’s health issues and with children. This will necessarily need to involve educators, such as teachers and student counsellors, health care personnel including physicians, nurses and ASHA (Accredited Social and Healthcare Activists) workers, and recruitment of social media and networks (radio, television, newspapers) to reach target groups of pre-adolescents and adolescents, and women of childbearing age who are contemplating pregnancy. (Courtesy of Dr. RM Vora and Bhavin K Paleja, Sangli)

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This type of education poses serious obstacles that stem from the fact that nearly 70% of the Indian population lives in rural areas where their primary food source is locally grown, processed, and consumed. This will thwart any attempt from a logistical standpoint for them to consume a centrally fortified food [sourced from outside the region] even if it is designed to optimize their folate and vitamin B12 status. Because Indians also tend to consume a variety of grains in their diet, asking them to consume a single vitamin-fortified food that is distinct from their preferred grain may foster resistance. There will also be several questions related to the safety of the food, the side effects, if any, and the cost. Many of these questions are included in the Annexures (especially in Annexure 2 - Frequently Asked Questions). Therefore, much will be gained by a well-planned and well-choreographed All-India National Education Awareness Program designed to create awareness across India about NTDs and their prevention by an adequate intake of folic acid and vitamin B12.

This initiative must reach all adolescent girls and Indian women of childbearing age who reside in small towns, villages, and hamlets, as well as those who reside in large and small cities. Later, there will be a trickle-down effect to include all other Indians as a function of time.

Educational activities should be undertaken at all high schools and colleges to highlight the significant benefits conferred by optimizing nutrition among women.

This educational initiative to educate women across India about NTDs and their prevention can use the recent PPI campaign as a blueprint for success (Box 1).

Finally, a clear message should also include information for the prevention of recurrence of NTDs (i.e., a second or more babies born among those women who previously had a baby with NTDs). ¹⁰ This is accomplished using 4-mg folic acid throughout pregnancy plus a 2-mg vitamin B12 tablet daily for at least 4 months to fill up their low vitamin B12 stores.

ii). Education of all health care personnel across India: A related challenge is to educate all obstetricians, traditional health care workers, non-governmental organisations working on rendering health in remote areas, and primary health centres to emphasize the need for all women of childbearing age to increase their folate and vitamin B12 status by consuming available folate and vitamin B12 fortified food or tablets well before pregnancy. Moreover, emphasis on education that full therapeutic doses of folate and vitamin B12 need to be administered, and not just smaller supplemental dosage, is of paramount importance.

(b). The second pillar—Ensuring widespread availability of subsidized folate and vitamin B12 -tablets and vitamin-fortified food

i). Vitamin tablets versus vitamin-fortified foods. The conventional food items that have been fortified with folate have been wheat [or corn] flour, which is consumed by many, but not by all Indians. Whereas rice, salt, and tea are other candidates, the efficacy of most of these food vehicles using pharmacological doses of both folate and vitamin B12 is still to be demonstrated by randomized clinical trials (RCTs) in India. Eventually, one or more of these foods that can be centrally processed and fortified with both vitamins can be widely distributed and consumed by all Indians; an alternative is to harness the Public Distribution Scheme for delivery of food to reach large numbers of Indians. However, here too, additional education and incentivization will be required to improve the appeal of such a subsidized distributed food. Eventually, two doses of vitamin fortified foods: (i) in “full replacement doses” to rapidly reverse the existing low-folate and -vitamin B12 status over 4-months, followed by (ii) both vitamins in “small maintenance doses” for supplementation of ongoing dietary insufficiency that is intrinsic to an Indian diet.

In the highly successful recent PPI campaign, there was outstanding coordination between the content of the message (i.e., the national awareness campaign for prevention of polio), which immediately preceded the widespread availability and distribution of the method for prevention of infection (by the vaccine). Finally, there was practical information on implementation (i.e., how and when the entire process of vaccination) would be implemented. Coordination between the message, the method planned, and the actual implementation of the plan was well-choreographed by the GOI. Therefore, while a National Educational Awareness Campaign prevention of NTDs is being developed, efforts should be made to prepare and make widely available, affordable, combined folic acid and vitamin B12 tablets and one or more folate and vitamin B12-fortified foods that have completed RCTs that demonstrate efficacy in improving both serum folate and vitamin B12 concentrations. Such RCTs must pass the rigorous vetting by reviewers of a peer-reviewed journal.

ii). Interim need for affordable combination tablets of folic acid and vitamin B12. Given the widespread problem of a low-folate and -vitamin B12 status among Indian women of childbearing age, any initiation of a national educational awareness campaign will necessarily raise the practical question: “Where can I get full-strength folic acid and vitamin B12 tablets to protect me from having a baby with a NTD?” This will necessarily require the GOI to ensure the availability of combined high-dose folate- and vitamin B12-tablets and -vitamin-fortified foods. Assuming there is no clear timeline when an approved, efficacious, vitamin-fortified food will be approved and ready for roll-out for widespread distribution, an alternative and immediate option is for women to consume a daily tablet containing 2 mg folic acid and 2 mg vitamin B12 for 4 months. However, the cost for purchasing tablets containing a combination of 1 to 2 mg of folic acid and 1 to 2 mg vitamin B12 (cyanocobalamin) to rapidly improve folate and vitamin B12 status is currently prohibitive and out of reach of most women. (The same is true with a lower-dose combination of folate and vitamin B12 tablets for supplementation of the Indian diet over the long term). Therefore, the GOI must proactively ensure widespread availability of tablets containing 2 mg of both vitamins. Vitamin tablets are not a long-term practical solution for prevention of NTDs in India; however, they are an excellent option for a minority of women who prefer to take full-dose vitamin tablets regularly for 4 months in hopes of rapidly optimizing their folate and vitamin B12 (in anticipation of conception after 6 months).

Alternatively, a suitable vitamin-fortified food, e.g., a daily cup of tea containing 2 mg folic acid plus 2 mg vitamin B12 (which is undergoing Clinical Trials presently), or yet another food-fortified containing a full therapeutic daily dose of both vitamins [that has undergone RCTs and is approved] can be deployed.

Once the folate- and vitamin B12-status of all Indian women are optimized within 4 months (to an estimated ‘safe’ serum folate concentration ∼15-20 ng/mL and serum vitamin B12 concentration of ∼400-500 pg/mL), they can be switched to a low-dose vitamin-fortified food that has completed RCTs, demonstrated efficacy, and is approved for wide spread use by the FSSAI. For this purpose, a vitamin-fortified food needs to contain a smaller daily dose of folic acid (0.4 mg corresponding to the RDA) and vitamin B12 (less than 5 µg, perhaps corresponding to the EAR or the RDA) for long term consumption to compensate for a chronic insufficiency of both these vitamins in the daily Indian diet.

iii). Food fortification with folate and vitamin B12 – General considerations. As discussed above, food fortification for Indians warrants an initial rapid replenishment of combined low -folate and -vitamin B12 status with full therapeutic [pharmacological] replacement doses of both folate and vitamin B12 for 4 months prior to institution of a lower dose of both vitamins for long-term supplementation.

Lesson from the US experience: Despite the baseline incidence of NTDs in the USA being nearly one-tenth less than NTDs found across India (i.e., 4.1 cases of NTDs per 1000 births),³ shortly after confirmation that the first occurrence of NTD could be prevented by periconceptional folate supplementation,²⁶ the US Preventive Services Task Force (USPTF) recommended that all women who could become pregnant should take folic acid 0.4-0.8 mg/day (i.e., 400-800 µg/day). Because most young women in the USA do not have a deficit in vitamin B12, this vitamin was not included in USPTF recommendations. Subsequent compilation of several studies on healthy young women from resource-rich countries confirmed that 400- and 800-mcg of folic acid administered daily for 12-weeks led to a mean serum/plasma folate concentrations of 11- and 20-ng/mL, respectively, values that could effectively protect women from having a baby with a NTD,⁸¹ by contrast, merely consuming a lower dose of 140 µg/day took up to three times longer to achieve such optimum protective levels.⁸² This experience is instructive to the Indian context, where a far greater number of women at baseline require full therapeutic replacement of their depleted folate and vitamin B12 stores. Therefore, simply initiating consumption of a low-dose folate and vitamin B12-fortified food at the outset (before replenishing the existing depleted stores first) will not rapidly achieve the target serum folate level of 15- to 20 ng/mL estimated for Indian women to reduce the risk of NTDs.

Because one-half of all pregnancies in the USA were unplanned, and insufficient number of young women were regularly taking daily folic acid tablets, the US Congress mandated that wheat flour be fortified with folic acid at a dose of 140-mcg/100 grams of flour by early 1998. However, as earlier recommended, and even to this day, American women of childbearing age are still encouraged to take a daily supplement of 400- to 800-mcg of folic acid daily.⁶¹ Collectively, this has led to a significant reduction in the incidence of NTDs, near elimination of folate deficiency related anemia,⁸³ and a decrease in the prevalence of serum folate deficiency from 30% to less than 1%.⁸⁴ Importantly, the safety of folate food fortification and supplementation has also been confirmed.

By contrast, there is clinical evidence of frankly low or borderline low-normal folate and/or vitamin B12 status affecting over two-thirds to three-quarters of women in several states across India.^7,12,46,48 Therefore, the immediate need is to reverse the low-folate and -vitamin B12 status with full therapeutic folate and vitamin B12 replacement over 4 months. Once the folate and vitamin B12 status is normalized so that women are at low risk of NTDs, it is then reasonable to reduce the dose of both vitamins to a lower level to supplement just the amounts of vitamins missing in their daily diet to optimize physiological function.

A warning: In this context, a caveat related to slow fortification using physiological low-dose replacement of vitamin B12 among women with vitamin B12 deficiency is worth repeating. A clinical study (from Maharashtra) used physiological daily doses of 2 µg of oral vitamin B12 replacement therapy for women with established vitamin B12 deficiency. Only after 11 months did vitamin B12 levels improve from a baseline median of ∼92 pmol/L [i.e., 124-pg/mL], where normal >150 pmol/L [i.e., >202.5-pg/mL], were restored to ‘normal’ at a median of 161.5 pmol/L [i.e., 218 pg/mL].⁶⁴ However, using the cut-off values for vitamin B12 insufficiency (between 200-300 pg/mL), all that the physiologic oral dose of 2-mcg vitamin B12 daily eventually achieved was a slight movement up from a frankly deficient level to borderline low-normal concentrations, still consistent with incipient vitamin B12 deficiency/insufficiency. Thus, if a large-scale food fortification initiative is introduced to the Indian population using a vitamin-fortified food that only contains small amounts of physiological replacement doses of both vitamins, this approach is not therapeutically appropriate in humans with overt vitamin deficiency. Instead, a full therapeutic dose of vitamins is necessary to rapidly replenish low-vitamin stores that can potentially lead to a clinical deficiency.¹⁰ By contrast, using a pharmacologically based therapeutic dose, a study also from Maharashtra⁷ showed that most women responded to full doses of 1 mg oral folic acid and 0.5 mg vitamin B12 daily within 2 months. But, another caveat learned from women studied in Assam,⁴⁸ where the depth of folate and vitamin B12 deficiency is even more severe than Maharashtra, showed that even 3-months of full therapeutic dosing with 1 mg folate and 1 mg vitamin B12 daily was insufficient and could not sufficiently raise the serum folate to the mid-range of the assay at 15- to 20 ng/mL [to protect their conceptus from a NTD, as discussed above]. So, a higher proven dose of 2 mg dose of oral vitamin B12 for 4 months will also be likely required for those from Assam with vitamin B12 deficiency.⁶⁵

In summary, among Indian women who have a high incidence of combined low-folate and low-vitamin B12 status due to widespread dietary insufficiency of these vitamins,^30,78,85 the imperative to replenish their low stores before instituting a lower dose of vitamin-fortified food is the most efficient method to ensure a rapid rise in serum folate concentrations to overcome the NTD-threshold.

iv). A vitamin-fortified food that initially replenishes the folate and vitamin B12 deficit using full-therapeutic doses over 4 months and then followed by a lower maintenance dose, can significantly improve the future health of all Indians. A key to choosing a food vehicle (wheat, rice, salt, or tea) to fortify with folate and vitamin B12 among Indians, is to ensure that rapid replenishment of the deficient vitamins can be accomplished using a full therapeutic (pharmacological) replacement dose over 4 months, and a much lower fortification dose to supplement the deficit arising from the daily diet [Figure 7].

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Figure 7:

Fortification of candidate food vehicles (wheat, rice, salt) with both vitamins, or vitamin-fortified Tea, and pathways for reaching different groups of women. (Courtesy of RM Vora and Bhavin K Paleja, Sangli)(*Folic acid: [United States Pharmacopoeia (USP)- and Indian Pharmacopoeia (IP)-grade] Folic acid is used clinically because it is a stable synthetic form that is easily absorbed and converted to Folate. Upon solubilization of Folic acid with dilute sodium hydroxide or sodium carbonate, Folic acid is readily converted to sodium Folate (clinically referred to as “Folate”). The alternate name for folic acid, ‘vitamin B9’, is almost never used in clinical parlance. Vitamin B12 is also known as Cobalamin; Cyanocobalamin is the other name for the therapeutically administered form of oral or injectable vitamin B12 suitable for children and adults).

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Measuring the impact of such rapid restoration of optimum folate and vitamin B12 status on several aspects of the health of Indian women of childbearing age, as well as all Indians, warrants additional discussion:

Under optimum circumstances, a pregnant woman with an abundant store of folate and vitamin B12 will pass down her legacy of these vitamins to her newborn baby girls.⁸⁶ By contrast, a pregnant woman with borderline low-normal or frankly deficient stores of these vitamins can only pass down from what little she has to her newborn girls; so, such girls are never ever given an opportunity to begin life with a full quota of optimum vitamin stores. When these girls grow up consuming a similar insufficiently balanced diet at home, they, just like their own mothers, are destined to become pregnant with borderline low to deficient folate and vitamin B12 stores; and they too will likewise confer a depleted quota of these vitamins to their own progeny of girls. This perpetuation of vertical transmission of inter-generational poverty of these micronutrients will continue until a radical change is instituted by rapidly optimizing folate and vitamin B12 nutrition within a single generation.¹ Clinical trials predict that optimization of folate and vitamin B12 status over 4 months can optimize folate and vitamin B12 status among Indian women. Thus, the impact in improving maternal-to-fetal passage of these vitamins will be to stem the scourge of vertical intergenerational transmission of risk of folate and vitamin B12 deficiency from mother to baby at birth. This will also positively influence the overall nutrition and health of the next generation of babies, now born with an abundant legacy of folate and vitamin B12 transmitted from their mothers and allow these girls to likewise bequeath an abundant quota of these vitamins to their own children.

This positive effect on folate and vitamin B12 status vertically over many generations will have long-term benefits. Foremost among these would be a dramatic reduction in the births of babies with NTDs. Assuming 90% of NTD-births can be prevented in India, it is estimated that 115,000 babies will be born without a NTD in every successive year. This will also help avoid predisposing all children to neuropsychiatric disorders and sub-optimal intellectual development that have their origin in utero.¹ Simultaneously, we can also expect a significant reduction in megaloblastic anemia among boys and girls and a reduction in the adverse contribution of anemia to maternal morbidity and mortality among women of childbearing age. Finally, even the elderly will benefit from optimizing their folate and vitamin B12 status by reducing the potential for hyperhomocysteinemia that places them at risk for strokes, premature brain atrophy and dementia. These are important long-term outcomes of the effects of a successful food-fortification program using folic acid and vitamin B12;¹ therefore, it is here that a robust National Birth Defect Surveillance Registry can quantitatively confirm the impact of such successes (see below).

v). Allowance for Indian women to identify a fortified food of their choice at the outset. Given the diverse food preferences in India, and despite the documented appeal of vitamin-fortified tea among most women who have been informally polled across several states of India (see Supplemental Material of Ref ⁷), it is wise to initially assume that no single food vehicle will have universal reach to improve the vitamin B12 and folate status of all Indian women of childbearing age. Therefore, during initial phases of the roll-out of vitamin-fortified foods, we favor the promotion of a variety of candidate foods [wheat flour, rice, salt, and tea] for fortification with folic acid and vitamin B12 provided they have completed clinical trials demonstrating efficacy. Although wheat flour and rice remain strong contenders for fortification with folic acid and vitamin B12 (particularly because they can incorporate iron), these are not uniformly consumed throughout India. Nevertheless, as noted above, a government-sponsored Public Distribution System can reach a larger percentage of the population in smaller cities and towns [and even remote villages] when it is supported by a national educational and awareness campaign. Even the potential drawbacks of vitamin-fortified salt outlined in Annexure 1(j) are not fatal flaws.

The key is to distribute these fortified foods throughout every State of India to reach all women, including those living in the most remote parts of each State. In this way, Indian women can avail of the vitamin-fortified food of their preference. Within a short time, it will be possible to identify one or more foods that is/are most favored among women of childbearing age and are also efficacious in raising vitamin levels; the less popular fortified foods can then be phased out to focus on more widely accepted food(s). We also know from clinical trials that vitamin-fortified tea is a strong candidate as being ideal for rapid full-dose replacement.^1,2,7,48

(c). The third pillar—Development of a national birth defect surveillance registry: Effective monitoring and surveillance of any large-scale public health prevention or intervention scheme warrants an assessment of the numbers of cases including those who are partaking of this initiative and those who are not, and identifying the reasons why. Surprisingly, for the size of her population and with 26 million births each year, India does not have a robust active surveillance system for birth defects; this has resulted in wide variation in the reported prevalence estimates of NTDs, as noted above.

Nevertheless, India has expertise to develop a network across all Indian States: (i), to monitor the efficacy of delivery of vitamin-fortified food; (ii), to monitor the efficacy of consumption of vitamin-fortified food as measured by the extent of optimization of folate and vitamin B12 status in random samples of women from all States in India; and (iii), to monitor the baseline incidence of NTDs across all states of India and to detect the extent of reduction in NTDs and other birth midline defects [following roll-out of folate and vitamin B12 tablets and vitamin-fortified foods].

These registries can be organized along the lines of the National Cancer Registry Programme under the aegis of the Indian Council of Medical Research, which has been routinely collecting data on cancer incidence since 1982. Currently, the network has 28 Population-Based Cancer Registries (PBCRs) and 58 Hospital-Based Cancer Registries (HBCRs). Thus, the development of a robust statewide National Birth Defect Surveillance Registry will be able to document the efficacy of these initiatives at a national scale.

With a well-designed network of National Birth Defect Surveillance Registries to account for the number of NTD- births in each State, India will be in a better position to accurately document the true incidence of NTDs and other birth defects. Importantly, such an Inter-Statewide network will also be able to document the efficacy of folate and vitamin B12 fortified food in reducing the incidence of NTDs; it will also help provide insights as to whether there remain pockets of the population that are not reached. As one instructive example from the USA, it was such information gathered from State Public Health Offices that identified that certain populations in the USA, who subsisted primarily on corn-based products, missed out on the benefits of wheat flour fortified with folic acid. As a result, the US Government included corn flour as another vehicle for [mandatory] fortification with folic acid. Thus, a similar robust Birth Defect Surveillance Registry across India will allow for a continued need for surveillance to inform if additional readjustments in policy are required. Therefore, the GOI must carry out periodic re-assessments of the efficacy of delivery and effectiveness of any dose of tablets or vitamin-fortified food used to ensure improvement in folate and vitamin B12 status and prevention of NTDs in India.⁸⁷

Intrinsic to building a National Birth Defect Surveillance Registry is the development of an Inter-State Teleconferencing Network where all children born with NTDs in each State are systematically enrolled, and an efficient referral system established to refer them to Specialized Birth Defect Centres for comprehensive long-term care and follow-up. Here, genetic counselling can be imparted to mothers, and appropriate rehabilitative, psychological, occupational therapy, and educational opportunities offered to their affected children.

Ideally, involvement of the Federation of Obstetrics and Gynecological Society of India, as well as NGOs like the Spina Bifida Foundation of India, can be recruited to develop a blueprint with the GOI’s own outstanding specialists in computer networking for developing such a National Birth Defect Surveillance Registry network.

Parenthetically, if India develops a robust network of State-wide Birth Defect Registries, it will be possible to determine the impact of the Educational Awareness Campaign, and of vitamin-fortified food (or alternative-equivalent vehicle) on several additional congenital defects involving midline structures (of the lip, palate, heart, omphalocele, epispadias/hypospadias, and etc.). This is how Canada identified that midline cardiac defects were likely to be prevented following mandatory fortification of wheat flour with folate.^88,89 There may yet be unique findings identified in India in the post-fortification period.

For example: It has been determined that some genetic polymorphisms, such as MTHFR C677T, can be negated by reversal of low folate (and vitamin B12) status and by optimization of folate stores by consumption of folic acid at 400-micrograms daily. Therefore, once the Indian population is fully replenished with both vitamins, complications from this polymorphism will not likely be clinically relevant. Nevertheless, there may be genetic factors present that predispose the children to develop NTDs or other midline birth defects that can develop in neural crest cells when there is insufficient folate to support their short bursts of proliferation and/or differentiation during early fetal development, which manifests phenotypically as neurocristopathies.⁴³ The identification of a significant reduction of ventricular septal defects following folate fortification of wheat in Canada is a prime example.^88,89 India may identify other unique preventable developmental defects. In this context, congenital atlanto-axial dislocation (AAD), which is not uncommonly seen by paediatric neurosurgeons in India, involves subluxation between the atlas and axis (the first and second cervical vertebra, respectively); this poses a serious risk of quadriplegia even with mild neck trauma. As with the documented association between MTHFR C677T polymorphisms with NTDs, a similar association has been reported with AAD.⁹⁰ Therefore, if a reduction of AAD is identified following folate and vitamin B12 fortification by the Birth Defect Registry, it may suggest that some types of AAD may also be folate-responsive birth defects, similar to the reduction of ventricular septal defects in Canada.^88,89 In a similar way, additional genetic factors associated with distinct phenotypic birth defects may also be identified within the large population of India. It is therefore conceivable that following widespread replenishment of folate and vitamin B12, some of these midline birth defects could be reduced, thereby pointing to the likelihood that they are also folate- and/or vitamin B12-responsive birth defects.

8) Education of policy leaders

(a). Return on investment. In any Government-sponsored endeavor, comprehensive education designed to help policy leaders understand the medical problem and solution sufficient to engage their ‘political will’ is an essential step. Although an investment case in favor of food fortification with folic acid for the prevention of NTDs has been made,¹⁵ the Indian situation must also address the problem of low vitamin B12 status. The scientific facts that support the need for change from the status quo and its challenges have been presented above in 7,2(D).

The benefits of optimization of folate and vitamin B12 status among women of childbearing age will result in a reduction of approximately 100,000 babies born with NTDs each year in India. This avoids the severe psychological distress and toll incurred by women as primary caretakers of children with NTDs and their families. There is also the added potential in reduction of pregnancy and obstetric complications, reduced megaloblastic anemia of pregnancy, as well as reduction in adverse neuro-psychologic/psychiatric outcomes in childhood that have their origins in utero.^91-95 In addition, the vertical inter-generational transmission of a lower than optimum quota of both vitamins in utero and postnatally via breastmilk, that is perpetuated by daily consumption of an insufficiently low-folate and -vitamin B12 diet, will also be reversed in a single generation.

This impressive benefit for women of childbearing age alone ought not to downplay the fact that reversal of low-folate and vitamin B12 status among all Indians—including children, adults, and the elderly of both genders, will also profoundly improve their overall health and well-being in several additional ways from a medical standpoint. Thus, optimized folate and vitamin B12 status among Indians at all life stages will reduce the risk of [megaloblastic] anemia, improve cognitive health, and independently reduce the risk for vitamin B12 deficiency-induced protean neurological manifestations of subacute combined degeneration of the [spinal] cord. Even middle-aged and elderly Indians will be less likely to suffer from long-term effects of hyperhomocysteinemia, which predisposes to small vessel occlusive vascular disease and strokes, cerebral atrophy, premature dementia, and Alzheimer’s disease. The economic benefits accrued from preventing these medical problems in Indians have yet to be quantified. Nevertheless, it is quite likely that the returns on such an investment in preventive medicine by the GOI on behalf of the health of all Indians are likely to be far greater than those tangible and intangible costs arising from caring for the plethora of medical problems arising from a low-folate and vitamin B12 status.

The consequence of not replenishing folate with vitamin B12 very early in the periconceptual period cannot be overemphasized. Ultimately, the identification of a truly universally acceptable folate and vitamin B12 fortified food will prove to be the most cost-effective method for the prevention of NTDs in India.

(b). Principles for methodically moving forward. Ensuring a positive outcome in Preventive Medicine, requires all great leaders walk the path of illumination and learning about the problem at hand, the crisis of suffering, and its solution; this can trigger self-reflection and lead to an epiphany on their unique role in potentially reversing the root cause, and a determination and focus to make amends. As observed recently: “What is critically needed from India’s leaders is an urgent, clear-eyed reappraisal and act of anagnorisis (an often startling self-recognition and discovery of a profoundly serious error and tragic flaw) in failing to confront this problem for decades. Only when closely followed by a metanoia—(a transformative change of heart that triggers remedial action)—can they help India avoid a catastrophic tryst with destiny¹.”

To this end, we include general principles designed to help coordinate and move this initiative forward:

*The GOI’s Ministry of Health must formally acknowledge that low-folate and -vitamin B12 status and of NTDs is a serious problem and recognize that a comprehensive long term commitment to the health of women and children in India warrants a focus on optimizing their folate and vitamin B12 nutrition across India.^1,2

*The GOI should immediately plan and eventually implement a National Educational Awareness Campaign across India about the consequences of low-folate and -vitamin B12 status on NTDs and other medical problems and the various ways for prevention using this White Paper as a template.

*The GOI should plan a folate and vitamin B12 food fortification program to ensure that all Indians are provided a rapid replacement dose of folate and vitamin B12 for 4 months. Ideally, this should be accomplished using an India-specific, centrally processed, universally acceptable food vehicle that can be fortified so that a daily portion of food contains the full daily replacement doses of both vitamins; an alternative is to use a single full-dose dual vitamin replacement tablet. This will need to be followed by long-term daily supplementation of the existing diet using an acceptable vitamin-fortified food.¹

*Specifically, the GOI can manufacture a tablet containing 2-mg folic acid and 2-mg vitamin B12 (cyanocobalamin) for daily oral consumption over 4-months (primarily for women of childbearing age preparing for pregnancy shortly), or in similar doses of vitamins added to a daily portion of vitamin-fortified -wheat, or -rice, or -salt or -tea for 4-months.^1,2,7 A lower dose tablet containing 1 mg each can be dissolved in sweetened water for children. Following full replenishment, a low-dose daily oral maintenance dose of vitamin-fortified food is recommended for all Indians to compensate for their daily dietary insufficiency of both vitamins.

*The GOI should support research into large-scale factory-based fortification of wheat, rice, salt, and tea with folic acid and vitamin B12. There should be an emphasis on standardization of the method used for each type of folate and vitamin B12-fortified food in GOI-research facilities. In the case of vitamin-fortified tea, the world-renowned GOI’s Tocklai Tea Research Institute, which has its model tea research factory on the campus in Jorhat, Assam, is already participating in such work. The use of standardized large-scale factory-based vitamin-fortified foods for Clinical Trials that are designed to test for efficacy and safety in optimizing folate and vitamin B12 status in women from several Indian States⁷ will allow for the FSSAI to make assessments of the efficacy of each of these vitamin-fortified foods.

*The GOI should register every case of NTD throughout India before and after the initiation of vitamin-fortification of food (or provision of tablets) by developing a robust Inter-State National Birth Defect Surveillance Registry.

*Finally, it should be noted that currently Indian pharmaceutical companies generally rely on the People’s Republic of China for the purchase of vitamin B12.¹ Given the expertise of outstanding microbiologists in India, the GOI should strongly consider development of a fermentation plant for large scale biosynthesis of vitamin B12 so that it does not need to rely on China. Likewise, India, with its expertise, can easily begin to chemically synthesize folic acid rather than rely on China. This is also relevant from a ‘National Pride’ point-of-view, especially since the benefit of folic acid for anemia of pregnancy was originally discovered in Bombay in the late 1920’s by Dr. Lucy Wills. Moreover, because of the Indian diet, Indians will always need these supplemental vitamins.

9) Specifics—The next essential steps prior to initiation of a national campaign

Before large scale deployment across India, each of the following steps must necessarily be taken to identify the optimum approach of delivery of folate and vitamin B12. This must include both basic research — culminating in standardization of large-scale fortification of foods, as well as on clinical trials that demonstrate efficacy of folate and vitamin B12-fortified foods in Indian women from more than one widely separated Indian State.

(a). Pillar one: Education of Indians on the campaig: Indian women need to be educated about why they need to improve their folate and vitamin B12 status; why they need to consume either tablets containing full therapeutic doses of folate and vitamin B12 (2 mg of each for up to 4 months) or to use specific fortified foods that are fortified with similar doses of vitamins; and why they need lower doses of folate and vitamin B12 fortified foods to supplement their diet over the long term.

(b). Pillar two: Availability of folate and vitamin B12 tablets: The GOI must make available pharmacological medications that are prepared in India, as opposed to the current situation where there is a surprisingly heavy reliance on purchase of folate and vitamin B12 from the People’s Republic of China. Each combination tablet should contain either 1 mg folate and 1 mg vitamin B12 (for the young), or combinations of 2 mg folate and 2 mg vitamin B12 for adults. These tablets should be sufficient for a minimum of 4 months for all Indians to rapidly reverse their vitamin status (if this is preferred as an alternative to vitamin-fortified food). In addition, combination 1 mg folate and 1 mg vitamin B12 tablets can be administered to children by their mothers (as a directly observed therapy).

(c). Pillar two: Evaluate candidate food vehicles for fortification: To identify one or more centrally processed food vehicles for large scale fortification with folate and vitamin B12 in full therapeutic doses per day; (later lower maintenance doses of these vitamins can be deployed). Current candidates include wheat and corn flour, rice, salt, and tea; their accessibility and acceptability by all Indians and related logistical, technical, and potential shortcomings/challenges have been discussed in this White Paper.

(d). Pillar two: Logistics of distribution of candidate vitamin-fortified foods across India: Experts within the Government need to help plan and develop logistical pathways to assure equity of distribution to reach all citizens from even remote rural regions across India.

(e). Pillar two: Clinical trials with candidate fortified foods: This is needed to demonstrate the efficacy of consumption of candidate folate and vitamin-fortified foods (prepared in small scale) every day in full therapeutic doses for 4-months in clinical trials among women of childbearing age. Efficacy is defined by the use of robust surrogate biomarker endpoints that can predict prevention of NTDs (i.e., a rise in serum folate concentration to ∼15-to-20-ng/mL and serum vitamin B12 levels to 400-500 pg/mL).

(f). Pillar two: Clinical trials with factory fortified foods: Complete clinical trials using large scale standardized factory fortified foods in widely separated states to demonstrate efficacy in optimizing both serum folate and vitamin B12 concentrations in Indian women. And confirmation of widespread acceptability and potential for equitable distribution across India.

(g). Pillar two: Deployment of vitamin-fortified food across India. There should be a High-Dose version of vitamin fortified foods with full therapeutic doses of both folate and vitamin B12 (2 mg each) per day and a Low-Dose version of Vitamin Fortified Foods based on either the Recommended Daily Allowance (RDA) or an Estimated Average Requirement (EAR) of both vitamins.

(h). Pillar three: Development of a national birth defects surveillance registry: This network can:

• i)

  Evaluate the effectiveness of the distribution of vitamin fortified food in every state of India;

• ii)

  Assess the efficacy of vitamin fortified food in raising the serum folate and vitamin B12 status in multiple states across India among Indians of all life stages, but especially in women at childbirth; and

• iii)

  Document improvement in the folate and vitamin B12 status among random samples of women using vitamin fortified foods every day to confirm the efficacy of the program;

• iv)

  Document baseline incidence of NTDs across India during the first year;

• v)

  Document if there is a reduction of NTDs [and potentially other midline birth defects that are attributable to low folate/vitamin B12 status] following successful deployment of vitamin-fortified foods in year-2 and year-3 onward;

• vi)

  Investigate any potential side effects that are attributed to the food fortification program.

Most of these steps have intrinsic challenges that are discussed in this White Paper.

10) Closing remarks

This White Paper contains significant information [in the Main Text and Annexures] to help point to ways to protect India’s most vulnerable members of society from the consequences of chronic nutritional insufficiency of folate and vitamin B12. The consequences of inaction will continue to allow over 100,000 babies to be born every year with serious NTDs, since most of the ∼26 million Indian women (who become pregnant each year) have these nutritional deficits (see Box 2). In addition, there are likely to be many other babies at risk for long-term neurologic, psychologic, or psychiatric deficits, as well as mother-baby dyads who are burdened with the clinical consequences of low-folate and low-vitamin B12 status over their lifetime. This burden of Hidden Hunger that affects most of the Indian population (women, children, and elderly), saps the vitality of our Nation and prevents Indian babies from reaching their potential for excellence throughout life.

We are acutely aware that persistent reference to large numbers of severely affected individuals in any tragedy can lead to ‘compassion fatigue’ that portends inaction. However, in the realm of Clinical Medicine, Surgery, and Allied Health Care, when even a single loved one [of our own] is seriously ill, we never see ‘compassion fatigue’. Instead, we see firsthand, up-close and personally, the devastating consequences of illness to immediate family members. Likewise, we are always touched to the depths when a tragedy envelops us. Therefore, we are hopeful that following careful review of this issue of ‘Prevention of NTDs in India’, Indian policy leaders will be moved with compassion to reverse this problem by food fortification with both folate and vitamin B12 using established medical practice. This is the only path to ensure that all Indians share in the joy and dignity of striving for excellence in all aspects of their lives.

As members of India’s National Academy of Medical Science’s “Task Force for the Prevention of NTDs” we see the suffering of toddlers and children with NTDs every single day and recognize the magnitude of the problem in India. In the past three decades since a solution to prevent NTD was discovered, there have been over 3 million babies born with NTDs in India. Thus, NTDs remain a problem of epidemic proportions in India that is of great significance and magnitude. In this context, we are aware of the high level of competence the GOI exercised to prevent a recurrence of poliomyelitis in India through the massive PPI program. If India were to devote a similar effort and resolve towards NTDs prevention, it would have accomplished yet another magnificent achievement on behalf of her most precious resource, her women and children. Therefore, we strongly recommend that the Government of India use all its resources to make a concerted effort to expeditiously curb this very serious problem.

Relevant Basic science, preclinical, and clinical information

Annexure 1: (a). How does folate get into the brain and what happens when entry is perturbed?

Cell surface membrane-associated folate receptors were first discovered and characterized from the human placenta.⁹⁷ These folate receptors have been shown to be critical for transport of folate into all proliferating cells, including across the placenta to the fetus⁹⁸ and across the choroid plexus into the human brain.^99,100

Experimental animal studies showed that blocking the entry of folate into specialized cells that comprise the embryonic neural tube in mice [by disrupting the function of folate receptors^97,98,101] predictably leads to NTDs.^102,103 Simultaneous interference of the function of folate receptors on developing neural crest cells also gave rise to neurocristopathies (i.e., midline defects of neural crest migration and differentiation), which include cleft-lip, cleft palate, and disorganized heart development.⁴³

Human ‘experiments of nature’ also point to adverse consequences of sustained postnatal interference in brain folate transport.¹⁰ Thus, congenital mutation of folate receptors can lead to early neurodegeneration and a variety of neuropsychiatric behaviors arising from reduced delivery of folate to the postnatal brain.¹⁰ There is also a clinical syndrome where post-natal development of antibodies to cow’s milk folate-binding proteins, which are structurally related to human folate receptors,^98,104 can also inhibit folate receptor function in infants, and lead to cerebral folate deficiency with severe neuropsychiatric and behavioral manifestations.¹⁰

Other experimental animal studies seeking to identify the effects of nutritional folate deficiency in pregnant mice,¹⁰⁵ [using one-third of optimum dietary folate, which is similar to the dietary availability of folate among women in North India⁸], resulted in profoundly abnormal development of white matter in the brains of fetal mice, which indicated interference with axonal development. There was also reduced fetal grey matter, reflecting a 20% cortical cell loss in the brains of fetal mice.¹⁰⁵ These changes were not reversed postnatally, and the striking finding was that mice that experienced folate deficiency in utero exhibited anxiety in young adulthood.¹⁰⁶

Following closely upon these animal experiments,^105,106 clinical studies in women and children from both the West and the East—United Kingdom,^1,107 The Netherlands, Norway, Nepal and India^91-95—confirmed that low maternal folate status early in pregnancy profoundly affects the fetal brain and predisposes affected children to psychological-neuropsychiatric disorders in early childhood.¹⁰⁷ The spectrum of abnormal behavior is dramatic and included hyperactivity-inattention and peer problems;¹⁰⁷ the children were either emotionally-reactive or aggressive, or withdrawn, anxious, or depressed, with learning deficits and somatic complaints. By contrast, women who consumed folic acid in early pregnancy had very positive outcomes on their child’s intellect, emotional intelligence, cognition, language, and academic performance.^92,93,108

Thus, there is substantial clinical evidence that in utero deficiency of folate in a pregnant woman can adversely affect in utero neurodevelopment of her baby, and lead to either NTDs or postnatal neurologic, psychologic, or psychiatric consequences in childhood. Collectively, these studies make clear the point that when we improve the folate status of women with an intention to reduce the risk of NTDs, we will also simultaneously reduce the incidence of several other postnatal psychological-neuropsychiatric manifestations in childhood.

Annexure 1: (b). How does insufficient folate lead to midline defects?

Experimentally perturbing folate receptors in mice during development,^102,103 leads to a spectrum of congenital birth defects involving neural crest and neural tube-derived structures.^43,102 This highlights the primacy of folate receptors in the delivery of folate to the developing brain and spinal cord. Two related hypotheses involving the developing neural tube and the neural crest can explain what accounts for the wide spectrum of folate-responsive dysmorphogeneses.^43,102 An underlying precept is that folate is critically important to enable proliferating cells to double their DNA. Because embryonic neural tube or neural crest cells exhibit high mitotic activity at very precise times during development, there is a critical need for an abundant supply of folate to enable such highly proliferative bursts in these cells.⁴³ Without sufficient folate present to support DNA synthesis and cell doubling, these cells cannot double and are arrested in the S phase and G2/M phases of the cell cycle. Therefore, depending on when in pregnancy various cohorts of these highly proliferative neural tube/crest cells are deprived of folate, the failure of these affected cells to reach their eventual destination will determine the type of developmental dysmorphogenesis. Therefore, a relative maternal folate deficiency leading to reduced delivery of folate to neural tube cells around the 4^th week of conception, can interfere with neural tube closure in the midline, and thereby predispose to various forms of NTDs.⁴³ Similar interference during midline closure of neural crest cell-derived structures that are referred to collectively as ‘neurocristopathies,’ such as the palate or the lip, or cardiac midline structures, can, in theory, give rise to cleft palate, cleft-lip, and some congenital heart diseases (CHD). This hypothesis has been confirmed in part following the introduction of folate fortification of food in Canada.^88,89

Annexure 1: (c). A serum folate level required to overcome the risk of NTD—the NTD-threshold

Not all cases of nutritional folate deficiency or folate insufficiency in women place them at risk for having a baby with a NTD or neurocristopathy. Research in the past decade has clarified that women with sufficient vitamin B12 stores but a RBC folate concentration below the threshold of 400-ng/mL (i.e., 906 nmol/L), which corresponds to a plasma folate level of 11.3-ng/mL (i.e., 25.5 nmol/L), remain at risk for having a baby with a NTD.^18,41 Therefore, in practical terms, most women must ideally achieve a higher serum folate concentration, well beyond the NTD-threshold of 11.3 ng/mL, to reduce their risk of having a baby with an NTD. Parenthetically, in clinical medicine, most women would not be considered to have either folate deficiency (normal value to prevent megaloblastic anemia is usually 2.4 to 3 ng/mL) with a serum folate value of 11.3 ng/mL. This is a function of the exquisite sensitivity of embryonic neural tube-derived cells and tissues that require far more folate to meet their high proliferative bursts in response to specific developmental signals; this is also a testament to the robust nature of hematopoietic cells that have developed mechanisms to overexpress sufficient folate receptors^109,110 to meet the high demands of hematopoietic cells during fetal life. These studies reinforce the critical importance of ensuring abundant folate is present in the woman prior to conception, and the need for adequate nutritional preparation for the ordeal of pregnancy.

Annexure 1: (d). Embryonic and foetal tissues are much more sensitive to small reductions in maternal folate and vitamin B12 than hematopoietic cells

All discussions related to the ‘optimum’ folate and vitamin B12 status in a woman of childbearing age to protect against NTDs must be tempered by recent findings pointing to the minimum serum folate concentration that must be achieved prior to pregnancy. It is now established that a woman with normal vitamin B12 status and a serum/plasma folate concentration below the ‘NTD-threshold’ of 11.3 ng/mL before pregnancy places her at risk for having a baby with NTDs.¹⁸ This points to the fact that the serum folate concentration must be significantly over 11.3 ng/mL, which is a far higher value than is required to induce megaloblastic anemia; therefore, based on these population based data, a value of serum folate less than 11.3 ng/mL (using a microbiological assay) can adversely impact the normal closure of the neural tube. This observation implies that embryonic neural tissues are exceedingly sensitive to even mild reduction of maternal serum folate levels that would not normally impact maternal hematopoietic cells.

Similar findings have been identified in experimental animals where a diet that is one-third of optimum leads to profound changes in neural tissue of the fetus.¹⁰⁵ Similar findings have been confirmed clinically in the West and India,^91-95 leading to the caution that a low-folate status places the fetus in utero at additional risk for neuro-psychological-psychiatric problems in childhood. Given the important role of normal vitamin B12 status in supporting the intracellular function of folate, these studies also point to the importance of ensuring that maternal vitamin B12 status is also optimized prior to pregnancy. Indeed, since most Indian women have low-vitamin B12 status, it is imperative to replenish vitamin B12 to optimize the function of folate in cells.

Annexure 1: (e). Do we need more evidence to justify widespread vitamin B12 and folate fortification of food to prevent NTDs in India?”

Many leading epidemiologists in recent years have published analytical reviews pointing towards a more comprehensive understanding of the traditional and modern approach to define “evidence” in topics that affect public health assessments and to help decision-making in the realm of public health. These more nuanced assessments are not the same as what has previously been deemed essential to document advances in the practice of clinical medicine. This modern way of looking at “evidence” was developed because a common concern raised by policy leaders in several countries has been to categorically insist that “traditional gold standards of evidence (RCT’s and systematic reviews of these RCTs), which have been used to make decisions in clinical medicine, should likewise be used to inform public health policies.”

Therefore, learning ‘why and how’ modern epidemiologists have analyzed and identified the pitfalls and drawbacks of traditional RCTs and then identified alternative methods to generate “evidence” in large populations in India is germane and eminently worthy of serious study. This is elaborated below:

Clinical medicine has long relied on evidence based on RCTs providing the best evidence for establishing causality for medical decision-making in both the diagnosis of disease and in clinical therapeutics. However, such an approach has not been so relevant in guiding decisions in the realm of Public Health, which is often fraught with limitations related to variables and biases and cannot possibly be controlled between large populations. As a result, there are very few large-scale population-based studies involving hundreds of thousands of individuals.

Sir Austin [Bradford Hill], Professor Emeritus of Medical Statistics, University of London, in his Presidential Address to The Royal Society Medicine identified a set of key criteria,¹¹¹coherence, plausibility, temporality, consistency, the extent or magnitude of effect, and dose-response gradient, as a distinct form of evidence that can be used to strengthen and substantiate causality from a plurality of observational studies. In the past 2 decades, many other leaders in epidemiology have further clarified how observational studies can strengthen evidence and collectively reignited discussion on limitations of RCTs in public health policy decisions.

In 2017, the former director of the US CDC¹¹² wrote a scholarly review entitled “Evidence for health decision making - beyond RCT’s.” This review, published in the most prestigious US journal in all of Medicine, pointed out several instances and in many areas of clinical medicine (e.g., vaccination, directly observed therapy for tuberculosis, salt sensitive hypertension, and hormonal therapy for improved women’s cardiovascular health), where RCTs have proved insufficient and occasionally less relevant and even misleading when compared to other ‘apparently less robust’ forms of evidence for making practical decisions from a public health standpoint.¹¹² This review was designed to also challenge us to think ‘out of the box’ in matters of public health policy, and to allay resistance from those policy-makers who remain skeptical of implementing fortification because of “insufficient evidence” from their country.

With regard to evidence extolling the virtue of prevention of NTDs by folic acid, there is a substantial wealth of data from a variety of studies: These range from several RCTs on the prevention of first occurrence of NTDs²⁶ to prevention of recurrence of NTDs,^25,35 non-randomized intervention trials,^113,114 and observational studies.^115,116 This constellation of studies finally led to a strong recommendation in the USA that “all women capable of being pregnant should consume 400-micrograms of folic acid daily to lower their risk of NTDs.”

What has not been sufficiently stressed is the potential ethical breach that can occur when the use of a placebo is used in a “confirmatory trial” when it has already been shown that the use of a placebo will result in either death or very significant morbidity in children born with NTDs. Indeed, there is such a little-publicized and methodologically flawed trial from India (only available in abstract form and not easily accessible on the internet).³⁹

There are continued strong recommendations from the US Preventive Services Task Force⁶¹ for the supplementation of folic acid to prevent NTDs. This document continues to extol the rationale and supports continued use of periconceptional folic acid to prevent NTDs, while highlighting its demonstrated safety for the entire US and Canadian population. Of added significance, this paper has referenced similar recommendations by other major US organizations involved in the care of pregnant women (American College of Obstetricians and Gynecologists, American Academy of Family Physicians) and their children (American Academy of Pediatrics).⁶¹

Thus, the requirement for traditional evidence-based medicine-related criteria using meta-analyses and systematic reviews (of multiple RCTs) as the ‘gold standard’ for clinical decision making is now being challenged by a distinct form of evidence.¹¹¹ Rather than categorically rejecting non-randomized studies, Frieden¹¹² argued on the significant [and invaluable] merit of using the data from such studies as being complementary to traditional randomized controlled studies to help make real-time decisions. The preeminent factor was the rigor with which each study was conducted, including how well potential biases were controlled to reduce ambiguity of the data, and quality of the data collection/retrieval. Additional evidence from Hill’s criteria includes coherence, plausibility, temporality, consistency, the extent or magnitude of effect, and dose-response gradient.¹¹¹ Collectively, these can help establish a cause-and-effect relationship of a variable (sufficient/optimal periconceptional folate status) and an outcome (NTDs) from less-than-stellar evidence-based criteria that have been traditionally used in clinical medical decision-making.¹¹¹

Therefore, taking together the criteria proposed by Hill¹¹¹, and following the lead of Frieden,¹¹² other experts in epidemiology and birth defects research³⁰ have made a strong argument on the available extensive evidence in favor of the role of folic acid in the prevention of NTDs.^{25,26,35,113-116} The gist of their argument is as follows: Rather than insisting on “traditional evidence evaluation schema, which are more applicable to clinical decisions but less relevant in guiding public health decisions,”³⁰ there is an urgent need to follow the lead of modern epidemiology which favors a paradigm shift in evidence required for making public health decisions.

Finally, it should be noted that these recommendations are primarily given for folate because the US population does not have a significant number of women in the childbearing age with vitamin B12 deficiency. However, there is also extensive basic science-related evidence that sufficient intracellular vitamin B12 is essential to enable intracellular folate to participate in the very complex interrelated pathways that are collectively referred to as One-Carbon Metabolism.⁴⁰ Moreover, these myriad functions of vitamin B12 are quite apart from its essential role in maintaining the integrity of the central and peripheral nervous system in children and adults.¹⁰ So whenever vitamin B12 status is borderline low-normal, or frankly deficient, it must be rapidly replenished to ensure optimal function of both folate and vitamin B12 in human physiology.

These facts make it imperative that in a population like India with a high percentage of women with low vitamin B12 status, vitamin B12 must be administered with folate to optimize the essential physiological function of the latter, and to optimize all the unique functions of vitamin B12 that are separate from folate.

Annexure 1: (f). Key basic science related interrelationships between folate and vitamin B12

(i). How are folate and vitamin B12 related to each other from a metabolic standpoint? Both folate deficiency and vitamin B12 deficiency can cause hematological abnormalities arising from impaired DNA synthesis, which manifests clinically as megaloblastic anemia.^10,11 Folate deficiency can also lead to several adverse neurological effects on embryonic development (as NTDs) and fetal neurodevelopment that can manifest postnatally as severe neuropsychiatric syndromes (as discussed above).¹⁰ In addition, perturbation of the function of choroid plexus folate receptors (by congenital mutation or anti-folate antibodies to folate receptors) in children can also lead to neuropsychological-neuropsychiatric syndromes.¹⁰

There is no clinical evidence in children or adults that pure nutritional deficiency of folate can independently lead to neurological manifestations (except for a ‘mask-like facies’). By contrast, nutritional vitamin B12 deficiency in children and adults can uniquely and will predictably result in neurological and neuropsychiatric manifestations from severe combined degeneration of the cord.^10,73

When serum folate (as 5-methyl-tetrahydrofolate) enters cells, it interacts with a critically important vitamin B12-dependent enzyme called methionine synthase.¹⁰ When there is sufficient 5-methyl-tetrahydrofolate and vitamin B12 in cells, methionine synthase transfers the methyl group of 5-methyl-tetrahydrofolate to homocysteine to form methyl-homocysteine (i.e., methionine) and tetrahydrofolate; this allows tetrahydrofolate to participate in a complex series of intracellular enzyme reactions [referred to collectively as ‘One-Carbon Metabolism’⁴⁰] that eventually allows for DNA synthesis and key methylation reactions. Conversely, with a deficiency of vitamin B12, the methyl-tetrahydrofolate that enters cells cannot be processed intracellularly. As a result, 5-methyltetrahydrofolate (and homocysteine) leaks out of cells into blood. This leads to a functional cellular folate deficiency that develops over time into a bona fide tissue folate deficiency. Parenthetically, this explains why individuals with nutritional vitamin B12 deficiency can have normal-to-high serum folate levels (that can mask an associated mild-to-moderate folate deficiency), and high serum homocysteine levels.¹⁰

Low intracellular vitamin B12 status can also increase markers of genome instability, such as chromosomal abnormalities^10,117,118, and DNA strand breaks.¹¹⁹ Vitamin B12 depletion leads to depressed rates of de novo nuclear thymidylate (deoxythymidine monophosphate; TMP) biosynthesis, which is critical for DNA synthesis and genomic stability.^10,117,118 While some of these could involve dysfunction of neuronal cells via impaired DNA synthesis,¹¹⁷ there are likely many other interrelationships of folate and vitamin B12 that can influence neurodevelopment. One example is related to the long-term intracellular elevation of homocysteine, which is increased in serum and cells during either folate- or vitamin B12 deficiency, and is now clinically linked to cerebral atrophy, cognitive decline, dementia, and Alzheimer’s disease.¹⁰ Homocysteine can covalently bind in a dose-dependent manner to an intracellular protein known as heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) to open its cryptic mRNA-binding site.^120,121 Homocysteinylated-hnRNP-E1 has a high affinity for a common signal sequence found in a unique group of over a hundred mRNAs; such mRNA-protein interactions can trigger either the activation or suppression of many of these mRNAs, which collectively belong to a single nutrition-sensitive posttranscriptional RNA operon. Some of these mRNAs code for proteins that are involved in human neurodevelopment.^10,99 Two examples should suffice: (a), axonal integrity depends on a perfect retention of the stoichiometric balance between three distinct neurofilaments [high, middle, and low-molecular mass] that are critical components of neurofilaments that are found in all axons. This balance is severely disrupted when homocysteinylated-hnRNP-E1 binds to the middle molecular mass-neurofilament mRNA, leading to abnormal net axonal development, which also interferes with the need to form millions of dendritic synapses that are critical for brain function, memory, and allowing for neuronal plasticity. (b), Homocysteinylated-hnRNP-E1 also interacts with opioid μ-mRNA to alter neurotransmitter levels in the fetal brain in utero, which predicts post-natal behavioral changes. In like fashion, there could be many as-yet-unidentified mechanisms where low maternal vitamin B12 status can adversely influence the integrity of in utero neurodevelopment that can continue in the postnatal human nervous system.

(ii). On the absolute need for the addition of vitamin B12 to folic acid in India: Vitamin B12 is independently essential for the integrity of the postnatal and adult central and peripheral nervous system. Therefore, if left untreated in either children or adults, vitamin B12 deficiency can lead to severe neuropsychiatric and neuropathic manifestations arising from subacute combined degeneration of the cord.^10,52 Vitamin B12 also has a critical role in folate metabolism. Another important fact is that vitamin B12 deficiency is a worldwide problem^52,59 and India is no exception;^1,5,9,10,59 But a more ominous problem is that the supermajority (over two-thirds) of Indian women have a low-folate status as well as a low-vitamin B12 status.^{1,7,12,46,48-50}

Apart from megaloblastic anemia and neuropsychiatric manifestations of subacute combined degeneration of the spinal cord, chronic vitamin B12 insufficiency also gives rise to hyperhomocysteinemia; in adults, this is associated with the potential for catastrophic neurological events arising from longstanding untreated vitamin B12 (and folate) deficiency; this is causally related to small vessel strokes, cognitive dysfunction, premature dementia and Alzheimer’s disease in late middle age to elderly Indians.^1,10 Therefore, it would be highly unethical not to attempt to treat all such individuals with vitamin B12. Indeed, an axiom in clinical medicine is to [first and foremost] reverse fundamental deficiencies of key vitamins and minerals to avoid serious long-term clinical effects.

There is a well-established biological (i.e., molecular, biochemical, cellular, and physiological) functional relationship between folate and vitamin B12. In the presence of vitamin B12, serum 5-methyltetrahydrofolate that enters cells must first be converted by the vitamin B12-dependent enzyme, methionine synthase, to form tetrahydrofolate and methionine. Only then does tetrahydrofolate become polyglutamylated and thereby retained within cells to further participate in “One-Carbon Metabolism,⁴⁰“ multiple enzymatic pathways that eventually lead to purine, pyrimidine, and DNA synthesis. Therefore, in the absence of vitamin B12, methionine synthase cannot be activated, so 5-methyltetrahydrofolate cannot get converted to tetrahydrofolate and is thereby unable to optimally participate in ‘One-Carbon Metabolism.’⁴⁰ In addition, after methionine is generated [by the vitamin B12-dependent methionine synthase reaction], it can be converted to a methyl donor through its adenosylation to S-adenosylmethionine (SAM). SAM is a universal donor of methyl groups for critically important biologic methylation reactions involving over 80 proteins, membrane phospholipids, the synthesis of neurotransmitters, RNA, DNA, and histones. Thus, these critical reactions, some of which also contribute to the integrity of the nervous system, cannot function optimally without vitamin B12.

Note: Therapeutic administration of folic acid can, however, enter cells through folate receptors and then be converted intracellularly in two steps by dihydrofolate reductase to also form tetrahydrofolate. This can allow the cell to bypass the vitamin B12-dependent methionine synthase reaction thereby allowing for perpetuation of “One-Carbon Metabolism”⁴⁰ and DNA biosynthesis sufficient to avoid megaloblastic anemia. However, this cannot substitute for other critical independent functions of vitamin B12 in maintaining its neuroprotective role for a pregnant woman and her embryonic-fetal and postnatal newborn. Nor can it compensate for many additional functions of vitamin B12 in maintaining the nervous system of Indians at various life stages.

Based on studies from the United Kingdom,¹⁰⁷ Norway, Sweden, the Netherlands, Nepal, and India,^91-95 a low-folate status in mothers during pregnancy can lead to serious adverse problems in embryonic and fetal neurological development as well as in post-natal life.^1,2 This indicates the crucial role of optimizing folate by ensuring that the vitamin B12 status in both mother and child is optimized. This has led to the grave concern as to whether a low vitamin B12 status [sufficient to predictably induce a functional folate deficiency] in Indian women of childbearing age can also adversely affect embryonic-and-fetal neurodevelopment in utero that continues in the breastfed infant and beyond as the child continues to subsist on the mother’s inadequate diet.

Based on these considerations, there is no scientifically valid clinical role for any suggestion⁷⁸ that folic acid given alone at the outset can substitute for the lack of documented nutritional low vitamin B12 status across India.^{1,7,12,46,48-50} Therefore, the GOI must ensure from the outset that sufficient folic acid and vitamin B12, are administered to optimize both folate and vitamin B12 status. In this context, the FSSAI has a mandate for ensuring that science-based evidence informs policy. Therefore, clinical trials should urgently confirm the efficacy of a large-scale food vehicle fortified with folate and vitamin B12 in rapidly raising the serum levels of both folate and vitamin B12 status among Indian women of childbearing age.^122,123

Some have inferred that treating the elderly with folate-fortified folate alone (without vitamin B12) in the USA has aggravated the issue of vitamin B12-induced cognitive defects and dementia. However, this hypothesis remains unproven. Even if true, this issue can easily be rectified by diagnosing and treating such individuals with vitamin B12, which should reduce hyperhomocysteinemia-associated neuropsychiatric problems. This issue is not relevant to the Indian context, since we recommend administration of both folate and vitamin B12 in full therapeutic doses at the outset to eliminate persistent low folate/vitamin B12 status in Indians at all life stages.

To summarize, there is evidence from a variety of studies on the critically important value of restoring low vitamin B12 status to normal. This is based on the key independent functions of vitamin B12 on human brain development from embryonic and fetal development, through childhood and adolescence, among women of childbearing age, and now even among those of middle-to-old age; this captures the entire population of India. Moreover, in the context of NTD prevention, there is evidence of the importance of vitamin B12 to optimize folate function in all aspects of normal human development. Finally, because low vitamin B12 and folate status are intrinsically linked in maternal-to-fetal intergenerational transmission of folate and vitamin B12, as noted elsewhere in this White Paper, there is a strong reason to suggest that despite a lack of evidence from large scale public health interventions, we can intelligently use data from understanding of the physiology of vitamin B12 and folate in cells,¹⁰ to advocate rapid replacement of both these vitamins.

Annexure 1: (g). Distinction between folate- or vitamin B12- “deficiency” versus “insufficiency”

(i). Distinction between “folate deficiency” versus “folate insufficiency”: Shortly after measurements of serum folate concentration were clinically established by the 1970s, patients with megaloblastic anemia were classified as having “folate deficiency” when serum folate values fell below ∼2.4-3 ng/mL (the lower cut-off levels of the assay used) in patients with megaloblastic anemia.¹²⁴ However, after metabolite tests for serum homocysteine and methylmalonic acid were developed in the 1980s to help distinguish folate deficiency from vitamin B12 deficiency,^52,124-127 several individuals with borderline low-normal serum folate concentration between 2.4 to -4 or -5 ng/mL (depending on the laboratory assay) had metabolic evidence of early folate deficiency; this was manifest by high serum/plasma homocysteine values (without an increase in methylmalonic acid) that also responded to folate replenishment with a drop to within normal values of homocysteine. While this reflected the greater sensitivity of metabolite studies, it also pointed out that subjects with borderline low-normal serum folate values had an incipient (i.e., a very early stage of) folate deficiency; this has usually been referred to as “folate insufficiency.” Such subjects with “folate insufficiency” have the potential to easily become transformed to frank folate deficiency under either the physiological stress of pregnancy, or during pathological states involving hemolysis, when an increase in folate demand is essential to support either the growth of maternal-fetal tissues in pregnancy or to support compensatory erythropoiesis, respectively.

Parenthetically, use of the term ‘folate insufficiency’ has now also recently entered the clinical literature in reference to the risk of NTDs. Thus, folate insufficiency in the context of risk of NTDs points to a serum folate concentration of less than 11.3 ng/mL (using a microbiological assay, in a subject without associated vitamin B12 deficiency). Therefore, the obvious ambiguity between use of a single term “folate insufficiency” for two distinct but related issues can be easily avoided by qualifying whether “folate insufficiency” refers to either (1) megaloblastic anemia (a value reflecting borderline low-normal value between a lower cut-off value to a higher upper value of 5-ng/mL or even higher (depending on the type of laboratory assay used); or (2) folate insufficiency can refer to risk for NTDs, a value less than 11.3 ng/mL using the microbiological assay. This approach can avoid confusion between the historical use of folate insufficiency in relation to megaloblastic anemia, and the newer use of the same term in describing the risk for NTDs. An example of the clinical distinction made between ‘folate deficiency’ from ‘folate insufficiency’ can be found in recent literature from India.^{7,10,12,46,48} In this document, unless specified, the combination of frank folate deficiency plus borderline-low normal values indicative of folate insufficiency (in relation to megaloblastic anemia) is referred to as a low-folate status.

(ii). Distinction between “vitamin B12 deficiency” versus “vitamin B12 insufficiency”: The same nomenclature can be applied to distinguish ‘vitamin B12 deficiency’—defined as a serum vitamin B12 concentration less than 200 pg/mL [148 pmol/L]) and ‘vitamin B12 insufficiency’. Thus, the National Institutes of Health (NIH, USA) fact sheet for health professionals uses terminology for “vitamin B12 insufficiency (assessed as serum vitamin B12 <300 pg/mL [221 pmol/L]) is more prevalent, affecting approximately 12.5% of all adults age 19 and older, and 12.3% of those age 60 and older ^128,129“ This document also adds: “However, low or marginal vitamin B12 status (200–300 pg/mL [148–221 pmol/L]) without classic hematologic or neurological symptoms associated with vitamin B12 deficiency is much more common, at up to 40% in Western populations, especially in those with low intake of vitamin B12-rich foods^{7,10,12,46,48,128,130,131}. In this document, the combination of vitamin B12 deficiency and vitamin B12 insufficiency is referred to as a low vitamin B12 status.

Annexure 1: (h). What is the basis for the use of full therapeutic doses of folate and vitamin B12 to ensure rapid replenishment of folate and vitamin B12 among Indian women?

(i). Therapeutic versus maintenance dosing: Since folate and vitamin B12 were independently discovered as the cause of megaloblastic anemia, it has been an over 50-year dictum to rapidly replenish depleted folate and/or vitamin B12 stores with full therapeutic (pharmacological) doses of 1-2 mg/day orally with each vitamin. Although developed empirically by pioneering clinical hematologists, there is now growing evidence from clinical trials that this is the only clinically appropriate treatment for low folate [and vitamin B12] status [that reflects low vitamin stores].^1,10,43

Parenthetically, this approach is based on an axiom in Hematology^10,11 and Internal Medicine⁷³ and Nutrition^11,14 (based on pharmacological principles of medication dosing in humans), to rapidly achieve steady state drug levels (of antibiotics or any other drug, including replenishment of a deficient vitamin or a mineral) safely over the shortest time. This invariably warrants the use of pharmacological doses of the micronutrient. Only after this first step is accomplished will it be necessary to administer smaller amounts of these agents to supplement a daily diet that is an inadequate source of these vitamins/materials.

As noted below in Annexure 1:(i) “Why is the NTD threshold not valid for the majority of Indian women?”, the population-based NTD threshold is not strictly valid for use in India because most Indian women have combined low-folate and vitamin B12 status (and the NTD threshold was derived from those who did not have vitamin B12 deficiency). Nevertheless, during discussions related to rapid replenishment of Indian women with both folate and vitamin B12, and assuming that low-vitamin B12 status is also rapidly normalized, knowledge of the NTD threshold can provide a general guide for Indian women to be replenished, i.e., until they achieve a serum folate value much higher than [the NTD threshold of 11.3 ng/mL], preferably to a serum folate range between 15-20 ng/mL. This provides context for determining how best to vault over the NTD threshold in the shortest time.

(ii). Vaulting the NTD threshold in the shortest possible time: Shortly after mandatory food fortification with folic acid was instituted in 1998 in the USA, it was confirmed that the increase in circulating folate concentration was linearly related to folic acid intake over the range of 100-1000 μg/d (r = 0.984, P < 0.0001).⁶⁶ Subsequent compilation of data from 11 intervention studies on healthy adults from high-income countries confirmed the consistent relationship between chronic folate intervention and changes in steady-state serum folate concentrations.⁸¹ In these studies, intervention periods of between 3-4 months were sufficient to achieve plateau serum folate concentrations. However, and very significantly, there was a dose-dependent rise in the serum/plasma folate: For example, on average, following 12-weeks intervention with 100-, 200-, 400-, 600-, 800- 1000-μg/day of oral folic acid, the improvement in mean serum/plasma folate concentrations progressively increased from 2-, 5-, 11-, 15-, 20-, to 24- ng/mL, respectively [See Table 1 and Figure 1 in Ref. 81].

In a RCT among [non-folate deficient] healthy women from New Zealand, the long-term effect of low-dose folic acid intake of 140-μg/day led to a safe RBC folate concentration of over 1068 nmol/L only after 36 weeks.⁸² By contrast, it took only 12 weeks of daily supplementation with a higher (400-μg folic acid/day) to achieve this RBC folate concentration. This dose [of 400-μg folic acid/day taken 12 weeks before pregnancy until the end of the first trimester] corresponds to the recommendation for women to prevent NTDs in the USA. This provides added assurance that use of full therapeutic doses of folic acid (1-2 mg/day) is appropriate to provide a brisk response in women with low folate status who require rapid optimization of folate status. However, in contrast to vitamin-replete women from New Zealand,⁸² the majority of Indian women have low folate and vitamin B12 status. Therefore, the duration to achieve a rise of serum folate between 15-to-20 ng/mL and serum vitamin B12 between 400-500 pg/mL may be longer than 12 weeks (and closer to 16 weeks); this is under active study.

(iii). How long will existing FSSAI guidelines for folate and vitamin B12 fortification of foods take to optimize the folate and vitamin B12 status of Indian women (based on published data?): If we are to employ either the 2016 FSSAI approval of WHO recommendations for fortification of a food vehicle with approximately 130-μg/100 grams, or the 2018 revision to a significantly lower folic acid concentration of 7.5- to 12.5-μg per 100 g plus a vitamin B12 concentration of 0.074- to 0.125 μg/100 g food,¹³² it would take considerably more time than 9-months to optimize the folate and vitamin B12 status of Indian women. And during this slow accumulation of blood folate and vitamin B12 concentrations, at least 25 million Indian women of childbearing age (destined to become pregnant during this time) would continue to remain at continued risk for having a baby with a NTD; this could add yet another 100,000 babies with NTDs for each year of delay in optimization of folate and vitamin B12 status.

This conclusion is based on studies in an Indian population of women of childbearing age from Maharashtra⁷ and Assam⁴⁸ where the supermajority had low-folate and low-vitamin B12 status. Thus, even after daily consumption of 1 mg of folic acid daily for 2- and 3 months, respectively, most women failed to register serum/plasma folate values well beyond the borderline low-normal range. By contrast, healthy Western women easily registered serum folate values of up to 24 ng/mL within 3 months at a dose of 400-μg folate daily.^81,82 This comparison between Western^81,82 and Indian women^7,48 clearly illustrates the importance of first rapidly filling the serious deficit of folate [and vitamin B12] stores in young Indian women of childbearing age with full doses of folate [and vitamin B12] using a minimum of 1-2 mg daily over at least 3-4 months initially. Only after this step would it be appropriate to deploy smaller maintenance doses of folate and vitamin B12 to compensate for the existing chronic dietary deficiency of folate and vitamin B12 in the daily diet from most regions in India. Studies to identify the optimum dose of both vitamins are ongoing.

Annexure 1: (i). Why is the NTD-threshold not valid for most Indian women?

For Chinese women of childbearing age without vitamin B12 deficiency, a population-level RBC folate concentration [measured by a microbiological assay] that fell below the threshold of 906 nmol/L (or 400 ng/mL) pointed to a state of relative folate insufficiency that predicted suboptimal NTD prevention.⁴¹ This corresponds to a plasma folate threshold of 25.5 nmol/L (or 11.3 ng/mL) for optimal NTD prevention.¹⁸ However, attempting to extrapolate such information from Chinese women to Indian women is hazardous because of the unusually high prevalence of longstanding dietary insufficiency of both folate and vitamin B12 in India, which results in combined low-folate and low-vitamin B12 status. Since Chinese women with vitamin B12 deficiency were excluded from analysis,¹⁸ no formal assessment has been made for populations of women with combined low-folate and low-vitamin B12 status. This limits the usefulness of the NTD threshold for most Indian women.

An important related question is: “How and why does vitamin B12 deficiency alter the NTD-threshold of serum folate concentration that is needed to protect against having a baby with NTD?” As noted above, the concentration of serum folate of ≥11.3 ng/mL (using a microbiological assay) correlates with the likelihood of protection against NTDs in those without an associated vitamin B12 deficiency.¹⁸ This is likely, in part, because normally vitamin B12 activates methionine synthase and helps facilitate the conversion of 5-methyltetrahydrofolate [monoglutamates] to tetrahydrofolate polyglutamates; this helps retain this form of polyglutamylated folate in immature erythroid precursors. However, in the absence of vitamin B12, intracellular 5-methyltetrahydrofolate [monoglutamates] fail to be converted to tetrahydrofolate (and thence to tetrahydrofolate polyglutamates for their retention in cells). This allows 5-methyltetrahydrofolate monoglutamates to leak out of erythroid precursor cells, while also raising serum folate levels disproportionately higher than what should be reflected based on the tissue folate stores.^1,6 Since RBC folates were used to originally define the NTD-threshold, the expected loss of RBC folates from red cell precursors in the presence of associated low-vitamin B12 status should invalidate this approach for the supermajority of Indian women who have an associated low vitamin B12 status.

Therefore, the upshot [and ‘intuitively safe’ solution] from a clinical standpoint is to attempt to reverse a low-folate and -vitamin B12 status by rapid full dose replacement of both vitamins, and aim for an even higher serum folate and vitamin B12 concentration—i.e., in the mid-range of both assays, at 15-20 ng/mL and 400-500 pg/mL, respectively, which are more generous targets than the original NTD threshold serum folate value of 11.3 ng/mL.

Annexure 1: (j). The track record of the Indian Government’s studies on vitamin-fortification of food: Arguments in favor and against fortification of wheat, rice, salt, and tea

In 2017, the Ministry of Women and Child Development of the GOI put out “a notice calling for suggestions, views, comments on the consultation paper on food fortification from the general public and other stakeholders.”¹³³ This document was designed to highlight the importance of fortification of a contextually appropriate Indian food (wheat flour) to improve micronutrient deficiency of folates and vitamin B12.

The value of this document is that the GOI’s advisors, in large part, appear to understand the importance of food fortification in general.

We will reproduce portions from this document¹³³ and then provide a critique as a prelude to further discussion of fortification.

Begin Quote from Reference¹³³

India has a very high burden of micronutrient deficiency diseases, such as anemia, vitamin A deficiency, iodine deficiency disorders, etc. Micronutrient deficiencies are prevalent in all age groups and socio-economic groups, but the consequences are more severe when it affects children below 24 months of age, since they are largely irreversible. Micronutrient deficiency disorders (MNDs) have many adverse effects on human health, not all of which are clinically evident.

Indian diets are typically cereal-based and the consumption of micronutrient-rich foods like pulses/legumes and vegetables/fruits is low, leading to deficiencies of multiple micronutrients. The National Nutrition Monitoring Bureau data and the Household Consumer Expenditure Surveys reveal that the intake of foods rich in micronutrients is far less than the recommended intakes (RDA) in all age groups. Common micronutrients deficient in Indian diets are Iron, Iodine, Vitamin A, folate/folic Acid, vitamin B12, and Vitamin D, and their absence has serious consequences.

Fortification fills the gaps in nutrition needs and is one of the most cost-effective strategies available.¹³³ It also had the added advantage of not requiring any behaviour modification or compliance that is expected in supplementation at the end-user level… [It] offers a promising opportunity to deliver micronutrient-rich foods to large populations.

Fortification is a cost-effective and reliable means of reducing micronutrient malnutrition. Fortification is a preventive measure for micronutrient malnutrition. The goal is not to provide 100% daily requirements of micronutrients but rather “fill the gap” between intake from other sources and daily micronutrient needs. The cost of micronutrients is clearly small on a per-person-per-year basis, and its success requires active collaboration among several sectors. Fortification has a great potential of enriching the nutritional quality of food and in turn, enriching the lives of millions of children; by giving them a healthy start to life, which they rightly deserve.¹³³

ANNEXURE 1 of this document (Reference¹³³) entitled: “Fortification of Wheat Flour with Iron, folic acid and vitamin B12” contains the following sentences:

[Fortification experts estimate that] with folic acid, changes in folate status may be observed within 3 to 4 months after fortification is fully implemented. It will take at least 12 months to see an impact on NTDs because women need to be consuming folic acid at the time they conceive to prevent birth defects.

Level of Fortification: The flour fortification standard as established by FSSAI recommends addition of iron, folic acid and B12 to ‘atta’ or ‘maida’ at the level of 20 mg/Kg Iron, 1300 micrograms/Kg folic Acid and 10 micrograms/Kg vitamin B12.

Constraints:

• There are about 1200 Roller Flour Mills in India; about 800 automated ‘Chakkis’ (stone grinding mills) and approximately 2.8 million small ‘Chakkis’. These are two different segments. Large automated ‘Chakkis’ that produce ‘atta’ that is sold as branded ‘Atta’ in the market. Wheat that is milled in small ‘Chakkis’ is difficult to fortify. Only the ‘Atta’ that is produced by organized sector is fortifiable. Organized sector produces ‘atta’ only for sale as branded ‘atta’ in urban and periurban markets. This is actually a miniscule portion of the whole.

• This will require time to scale up as the industry has to install the necessary capacity of automated ‘chakkis’ to handle the huge volume.

End quote from reference¹³³

Commentary: The above quote is from a document by the GOI’s Ministry of Women and Child Development.¹³³ This quote correctly highlights the importance and benefits of fortification in general terms. However, with regard to use of wheat flour fortification, the section on Constraints notes: Only the ‘Atta’ that is produced by organized sector is fortifiable. Organized sector produces ‘atta’ only for sale as branded ‘atta’ in urban and periurban markets. This is actually a miniscule portion of the whole.

This document¹³³ is also overly optimistic and in error with respect to expectations for widespread implementation in reducing NTDs within a year for several reasons:

(1). The extant low-folate and low-vitamin B12 status among the majority of Indian women of childbearing age requires full therapeutic replacement with pharmacologic doses of daily oral folic (1-2 mg) and vitamin B12 (1-2 mg) for up to 4 months to ensure restoration of optimal folate and vitamin B12 status.^1,10,11 This is because of the depth of low-folate and -vitamin B12 status found among Indian women, However, the concentration of folate (with or without vitamin B12) planned for fortification of wheat flour in India — without plans for rapid replacement of existing deficiency with pharmacologically-appropriate full doses of both folate and vitamin B12 — will not likely achieve optimization of folate and vitamin B12 status until well past one year.^1,10,11

(2). The more serious issue with this choice of wheat flour (or rice) for fortification is that it will not reach all Indian women of childbearing age (this is elaborated below); therefore, the entire enterprise for fortification of food for prevention of folate and vitamin B12 deficiency anemia and NTDs and neurological defects is likely to fail. However, there is a salvage pathway in deploying vitamin-fortified food using the Public Distribution System.

(3) Thus, in 2017, they estimated that folate status would improve so much within 3-4 months that within 12 months they could see a reduction of NTDs. However, we have seen no such success documenting the feasibility of this approach in India using 1 mg of folic acid and 1 mg of vitamin B12 daily for 3 months.

To their credit, they correctly recommended 130 micrograms per 100 g wheat flour (1300 micrograms/kg), which is close to what is being done in the USA. Unfortunately, this was inexplicably reduced later without supporting evidence to a sub-therapeutic [i.e., but near homeopathic] dose of folic acid.

(4) Their recommendation to use vitamin B12 at 10 µg per kg of flour (a dose of 1 µg of vitamin B12 per 100 g flour is also seriously sub-therapeutic since it is now estimated that most individuals need ∼7 µg vitamin B12 per day;¹ (note however, that this estimate is based on Western diets that generally contain more vitamin B12 than an average Indian diet; therefore it is likely that Indians need even more than the estimated amount of ∼7 µg vitamin B12 per day).¹ Given the fact that the national average consumption of wheat is 160 g/day/capita, it would give barely only 1.6 µg of vitamin B12 per day. This value ought to be increased to a much more liberal amount of 5-10 µg vitamin B12 per 100 g of wheat flour to ensure the higher amount is available when per capita daily intake drops due to poverty and food insecurity in India.

Both these levels of fortification are meant to “fill in the gap” arising from dietary insufficiency. However, this amount will not be adequate for women who already have low-folate and -vitamin B12 status (low stores); this warrants a full therapeutic replenishment with pharmacological doses of these vitamins as is usually indicated in clinical medicine; this can be provided by 4 months intake of a daily intake of 1-2 mg folic acid and 1-2 mg vitamin B12 in the form of either a combination tablet or a vitamin-fortified food vehicle.¹

(i). Fortification of wheat – Pros and cons

The commonest staple in the Western diet is wheat flour, which is usually generated in large roller mills by grinding the wheat kernel after extracting its bran and germ; a desired amount of folic acid can easily be added during this process. Because such ready-made wheat flour is widely distributed in the USA, wheat flour is an appropriate fortification for that country. However, deployment of vitamin-fortified wheat flour in India poses several problems, as follows:

While a goal of fortifying wheat flour with folic acid and vitamin B12 in large roller mills and large-automated stone grinding mills can be theoretically achieved in cities across India, this is the home of only one-third of the Indian population. Ensuring there is successful deployment of centrally processed folate-fortified flour to reach all Indian women living in the smallest towns, villages, and hamlets throughout India is a major logistical problem. However, there are yet other stumbling blocks. For example, in most small towns and villages, cereal grain is more often grown and purchased locally, and flour is manually milled from this grain by small-scale millers; this bypasses any need for centrally processed folate-fortified flour.

Indian wheat-derived ’atta’ flour, which is processed by grinding the whole wheat kernel, contains a higher fiber content than conventional [white] wheat flour. As discussed above, there are approximately 1200 large roller mills and 800 automated stone-grinding mills (known as Chakkis) in India, which constitute the ‘organized sector’. Unfortunately, however, “only the Atta that is produced by [the] organized sector is fortifiable…” and such branded [fortified] atta is sold [only] in urban and periurban markets. The experts at the GOI admit: “This is actually a minuscule portion of the whole.¹³³“ This stems from the fact that there are 2.8 million manual stone-grinding mills (small-scale chakkis), that are not part of the organized sector, but who serve the greater majority of Indians living in towns and villages. This serves a longstanding custom for most Indians to purchase locally produced grain and get it milled into flour by a local miller; (it has not been a tradition to purchase pre-milled ready-made flour from a shop). Indeed, many families living in cities also purchase local grains and take them to a small-scale miller within the city to prepare customized flour.

Thus, using wheat flour for fortification with folate is not likely to be an easy task for India because of some [admittedly] unique and interrelated stumbling blocks that have coalesced to thwart progress in the prevention of NTDs in India.

Notwithstanding these obstacles, the use of fortified wheat flour (‘atta’) can be widely distributed via India’s vast Public Distribution System — but will require a significant investment by the Government designed to convince Indians to preferentially make a move away from traditional practice; this will be a significant problem, challenge, and risk, since maintaining a tradition (परंपरा; parampara) in India, is itself a powerful unifying force against change. Thus, the challenge the GOI remains to achieve a robust vitamin-food fortification program solely using wheat flour as a food-vehicle sufficient to reach every single Indian every day.

(ii). Fortification of rice – Pros and cons

Because 70% of Indians consume rice, this has been explored as an option. Even though rice is processed to remove husk, a common preparatory step to boiling rice in India is to subject it to a thorough rinsing, which will effectively wash out any micronutrient that has been added merely to cover the surface of rice grains. Therefore, a much more viable option has been to first pulverize rice flour, followed by the addition of a premix containing vitamins and minerals. Next, fortified rice kernels are produced from this mixture using an extruder machine. The end-product (looks like a rice grain) and can remain camouflaged among other grains of rice. While cumbersome, this method requires significant GOI investment and support; the advantage of fortifying rice is that it is purchased ready-made so it can be widely distributed by the Public Distribution System to reach the urban poor, as well as the remaining two-thirds in all towns and villages throughout India. However, this still leaves 30% of Indians (over 300 million) without a contextually appropriate fortified food.

(iii). Fortification of salt – Pros and cons

Currently, in addition to folate-fortified wheat flour and rice, sea salt is also being examined as a food vehicle. Adding folic acid and vitamin B12 fortified salt to Indian cooking will not pose a problem from the organoleptic standpoint since any color is easily camouflaged in traditional Indian cuisine. However, adding folic acid and vitamin B12 to this vehicle for use at the table can pose a challenge; this relates to the fact that the pure white color of sea salt can be tainted by visual [organoleptic] properties conferred by the mixture of light yellow colored folic acid and red-colored vitamin B12. This may pose a problem when full-therapeutic doses of folate and vitamin B12 are required, which can potentially lead users to question the acceptance (palatability) and esthetic aspects of vitamin-fortified salt. This may even lead to rejection by the poorer members of society who usually prefer the use of the cheaper form of ‘rock salt’. However, a recent publication suggested that low-dose fortification with these vitamins did not confer changes in visual appeal or taste to fortified salt.^74,75

In addition, given the high incidence of hypertension associated with Metabolic Syndrome-X in India, there can be problems with salt-sensitive hypertension with the use or overuse of vitamin-fortified salt. Salt is also usually kept exposed to sunlight (in a salt-shaker or an open dish), which can pose a problem with the stability of folic acid and vitamin B12 over the long term. Therefore, an immediate concern is the need for the addition of vitamin B12 as well as clinical testing for efficacy. In places along the coastline of India (especially during monsoons) when humidity is high, sea salt absorbs moisture and forms a moist cake that is impossible to sprinkle out of a saltshaker and requires a spoon; this can lead to non-uniformity in the delivery of such salt with meals.

Another important question relates to the extent of distribution and acceptability of another related additive added to salt, namely iodine. The extent to which iodized salt has consistently reached all Indians can provide insight into how efficiently folate and vitamin B12-fortified salt will likewise reach all Indians. There is a significant disagreement here: Although a declaration favoring the implementation of universal salt iodization in India by the Central Council of Health was enacted in 1983, a recent review notes that iodine deficiency still remains a significant public health problem in India, a full 36 years later.¹³⁴ Not unexpectedly, studies led by the Indian Government paint a more rosy picture, pointing to near-universal coverage (of 90-95%) of India. However, an illustrative example comes from Rajasthan, which is the second-largest producer of iodized salt in India; yet, Rajasthan is among the many States having the lowest household coverage of iodized salt (65.5%).¹³⁵ Even if the truth lies between these opposing points of view, there should be concern over the potential lack of effective penetration of vitamin-fortified salt across India to prevent NTDs. Nevertheless, fortification of salt with both iodine and folic acid is yet another avenue for countries where large-scale milling infrastructure for grain fortification does not reach everyone.⁷⁵ Therefore, more clinical research to define the efficacy of vitamin-fortified salt to optimize the folate and vitamin B12 status among Indian women is needed.

(iv). Fortification of tea – Pros and cons

Indian Black Tea meets all criteria¹³⁶ as an ideal, perfect, contextually appropriate, food vehicle for full therapeutic replenishment with folate and vitamin B12 [and by implication, for long-term fortification] in India for several reasons^1,2,7: Besides water, tea is the most common and cheap beverage in India (and worldwide),¹³⁷ and is largely grown and processed in the highlands of 4 [out of 28] states of India, Assam, West Bengal, Tamil Nadu, and Kerala, from where it is efficiently distributed to people of every geographic location and every socio-economic group throughout India. Importantly, the vast majority of adult Indians [men women and adolescent children] from every geographic area within India, from mega-cities to small towns, villages, and hamlets, consume at least one cup of tea from one of these regions every day; thus, tea retains a unique role by being a common ‘food’ that rises above the otherwise extremely diverse food preferences of Indians.^1,2,7 Tea is also easily accessible in tea stalls across India and is also affordable by the poorest of the poor.

All major tea companies in India have centralized tea-producing factories close to large tea plantations where tea can be fortified. Folate and vitamin B12 are both water-soluble, resistant to near-boiling hot water used in brewing tea, so the entire dose of both vitamins is immediately delivered into infused tea. (Parenthetically, both these vitamins can tolerate being in the heat of an oven sufficient to make bread). Finally, both vitamins are non-toxic with no UL for folate¹³⁸ or any upper limit for vitamin B12.^72,139 Fortification with folic acid- and vitamin B12 can be carried out during the penultimate step of the processing of tea, just prior to drying and packaging. Once fortified, the dried tea leaves retain the folate and vitamin B12, which finely coats these tea leaves. Loose tea is usually stored in closed tin or cardboard box containers so fortified tea is protected from direct sunlight [which can be detrimental to the stability of folate and vitamin B12].^140,141 Folate and vitamin B12-fortified tea is stable for storage at room temperature for extended periods. The vitamins in tea are easily excreted from the body in urine and stool when taken in excess. The yellow color of folic acid and the red color of vitamin B12 are completely camouflaged when added to the amber color of infused tea, and neither the taste nor color of the tea is affected by added folate and vitamin B12. Importantly, the addition of folate or vitamin B12 as therapeutic agents to tea is in keeping with the FSSAI regulations for nutraceuticals ¹²² and the spirit of the FSSAI Joint Declaration to identify additional vehicles for fortification.¹²³

Recent research using vitamin-fortified Teabags,¹ demonstrated efficacy among Indian women of childbearing age from Sangli, Maharashtra.⁷ Thus, daily consumption of 2 grams of tea fortified with 1 mg folic acid and 0.5 mg vitamin B12 for 2-months significantly increased the mean difference in pre- versus post-intervention serum folate of 8.37 ng/mL (95% CI 5.69 to 11.04, p<0.05), and two-thirds of women exhibited increases in serum vitamin B12 levels over 300 -pg/mL. A significant post-interventional increase in mean hemoglobin concentration up to 1.45 g/dL (95% CI 0.64 to 2.26, p=0.002), reflected a bona fide clinical response to vitamin-fortified tea.^1,2,7 However, most Indians do not use teabags, and instead enjoy a hot-brewed cup of factory-processed ‘loose’ black-tea prepared by the ‘crush, tear, curl’ (CTC) method.

Accordingly, the efficacy of directly fortifying ‘loose’ black-CTC-Tea-[granules] with folate and vitamin B12 was assessed in young women of childbearing age in Dibrugarh, Assam, using a placebo-controlled interventional trial.⁴⁸ At baseline, 89% of women (n=60) had low-folate status and 72% had low vitamin B12 status. Two clusters of women studying nursing and pharmacy at Assam Medical College consumed a daily cup of hot tea prepared from 2 g of either unfortified-CTC-Tea (Control-group) or vitamin-fortified-CTC-Tea containing 1 mg folate and 1 mg vitamin B12 (Experimental-group) under a directly observed therapy format for 90 days. The control group had a clinically insignificant mean post-interventional increase in serum folate of 1.3 ng/mL and serum vitamin B12 of 1 pg/mL. By contrast, the experimental group experienced a mean rise in serum folate of 5.3-ng/mL (95% CI 3.9 to 6.8; p<0.001) and serum vitamin B12 of 194.6-pg/mL (95% CI 154.7 to 234.5; p<0.001). This trial also assessed the concentration of folate and vitamin B12 recovered per cup of vitamin-fortified CTC-Tea at the GOI’s flagship, Tocklai Tea Research Institute, Jorhat, Assam.⁴⁸ The results compared favorably with the concentration of vitamins originally added to CTC-Tea. This demonstrated that vitamin-fortified-CTC-Tea is an efficacious, scalable, food-vehicle to replenish Indian women with combined low folate-and-vitamin B12 status. However, Assamese women could not be optimally replenished even after 3 months of daily full therapeutic doses of vitamin-fortified tea, signifying a much greater depth of low-folate and low-vitamin B12 status when compared to Maharashtrian women, who were largely replenished in just 2 months.⁷

Assuming a daily cup of vitamin-fortified tea is required for 90 to 120 days to restore optimum folate-and-vitamin B12 status, this translates to a total cost of vitamins per person of Rs. 18-24. This is the cost of a single cup of tea from most tea stalls across India. Moreover, the annual cost of adding folic acid [0.5 mg] and vitamin B12 [100-µg] to a daily cup of tea to supplement an Indian diet that is intrinsically low in these vitamins will cost less than Rs. 10 per person.¹

Therefore, tea meets the challenge in identifying a common food-based vehicle that is acceptable to all Indian women of every geographic, social, economic, cultural, and ethnic distinction, whose diet is dictated by these very differences.^1,2,7 (Even South Indians who may generally prefer coffee in the morning will still consume a cup of tea at tea-time (i.e., ∼4 PM). Thus, although there are several potential vehicles developed around the world, fortification of a universally accepted Indian beverage that is consumed all across India — a daily cup of vitamin-fortified tea — has appeal and a decisive advantage in being affordable and able to reach single woman (including pre-pubertal and adolescent girls) living in the most remote parts of India.^1,2,7

One theoretical downside to the use of tea warrants discussion: Consumption of tea can inhibit iron absorption and potentially aggravate existing iron deficiency; it is believed that tannins in tea act as chelators to bind iron. However, the fact is that daily consumption of a cup of tea is deeply ingrained within Indian culture and any such effects of tea on iron have clearly not swayed Indians away from drinking their morning cup of ‘bed tea,’ which is often an hour before breakfast. A recent controlled trial in the UK among healthy women documented that a 1-hour interval between a meal containing iron and consumption of tea attenuates the inhibitory effects of iron absorption.¹⁴² The trial from Assam confirmed that despite a borderline low-normal iron status in most women, there was no adverse impact of CTC-Tea—consumed 2 hours between meals, on either percent-transferrin saturation or hemoglobin concentration.⁴⁸ Thus, the superior benefit of NTD prevention using vitamin-fortified tea far outweighs any negative effect on iron absorption.

The Tea Board of India’s Director of Tea Research and Director of Tea Development for the massive tea growing and processing enterprise in India have both strongly supported the use of vitamin-fortified tea for India as a ‘win-win’ situation. Collaborative research to optimize methods for large-scale generation of folate-and-vitamin B12 fortified CTC-Tea in the model tea factory at the Tocklai Tea Research Institute, and documentation of its efficacy with clinical trials are currently underway.

Annexure 1: (k). Potential for folate fortification to unmask other midline congenital defects

Following folate-fortification of flour in Canada in 1998, there is now evidence that aligns with the hypothesis that, apart from periconceptional prevention of NTDs, folate fortification can also help to unmask other midline congenital defects in the realm of congenital heart diseases (CHD).¹⁴³ CHD, which occurs in ∼1% of all live births, involves dysmorphogenesis of groups of neural crest cells that contribute to midline structures during heart development. Specifically, evaluating the potential role of folic acid fortification on the incidence of CHD before and after fortification that was introduced to Canada (in 1998) has been instructive on what can be expected in India. A population-based cohort study involving nearly 6 million live births and stillbirths (including late-pregnancy terminations) delivered at ≥20 weeks’ gestation in Canada from 1990 to 2011 revealed that folic acid food fortification was associated with a 27% reduction in conotruncal defects, a 23% reduction in coarctation of the aorta, a 15% reduction in ventricular septal defects, and an 18% reduction in atrial septal defects.^88,89 Because the prevalence of CHD is approximately 10-fold higher than NTDs (in the West), even a small percentage reduction of CHD following folate-fortification can lead to significant public health benefits.⁸⁹ Therefore, if India can develop a robust network of Birth Defect Surveillance Registries in each State, it will be possible to prospectively quantify the effect of vitamin-repletion on all congenital defects involving midline structures.

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