Efficacy of tea as an ideal vehicle for therapeutic vitamin replacement in women with low folate and vitamin B12 status in Assam: a placebo-controlled interventional clinical trial

Abstract

Background

Most Indian women have a low folate and vitamin B₁₂ status that can progress to clinical deficiency and contributes to over 100 000 births annually with neural tube defects (NTDs). A common, widely accepted, Indian food vehicle for fortification with folate and vitamin B₁₂ has long remained elusive. Earlier, we employed folate and vitamin B₁₂-fortified teabags to brew a daily cup of black tea and documented improvement in serum folate and vitamin B₁₂ concentrations among women from Maharashtra state within 60 days. Because most Indian households prefer hot-brewed (‘loose’) black tea prepared by the ‘crush, tear, curl’ (CTC) method in large commercial tea factories, we tested the feasibility of directly fortifying CTC tea with folate and vitamin B₁₂ and assessed its efficacy in improving the folate and vitamin B₁₂ status among women from Assam state within 90 days.

Methods

Two groups of women studying nursing (n=30) or pharmacy (n=30) at Assam Medical College and Hospital 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) for 90 days. We then compared their pre-interventional versus post-interventional changes in serum folate and vitamin B₁₂ status, iron status and haemoglobin concentration.

Results

At baseline, 89% of all women had low folate status (serum folate ≤5 ng/mL) and 72% had low vitamin B₁₂ status (serum vitamin B₁₂ ≤300 pg/mL). After 90 days daily consumption of unfortified CTC tea, the control group had clinically insignificant mean increases in serum folate of 1.3 ng/mL and serum vitamin B₁₂ of 1 pg/mL. By contrast, the experimental group consuming vitamin-fortified CTC tea exhibited 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). Moreover, 28/30 women receiving vitamin-fortified CTC tea had a post-interventional rise in mean serum folate of 9.2±3.6 (SD) ng/mL, and 25/30 women normalised their serum vitamin B₁₂ ≥300 pg/mL. However, only a minority achieved the higher serum folate concentration required to reduce their risk of NTDs. Despite borderline low normal iron status in most women, there was no adverse impact of CTC tea—consumed ~2 hours between meals—on either per cent-transferrin saturation or haemoglobin concentration.

Conclusions

Consumption of a daily cup of hot brewed folate and vitamin B₁₂-fortified CTC tea is feasible, efficacious, and safe to clinically normalise the folate and vitamin B₁₂ status of Indian women within 90 days in Assam. Therefore, CTC tea is an ideal and eminently scalable food vehicle for fortification with these vitamins. The next challenge is to determine the optimum duration and/or dose of vitamins required to fortify CTC tea sufficient to lower the risk of NTDs among women across India.

Trial registration number

CTRI/2022/10/046289.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• A daily hot cup of black tea—a quintessentially Indian beverage consumed across India—showed promise as an ideal food vehicle for fortification with therapeutic doses of folate and vitamin B₁₂. Therefore, we tested the feasibility of fortifying the preferred form of black tea used throughout India (2 g (loose) CTC (crush, tear, curl) tea/cup) with 1 mg each of folate and vitamin B₁₂ among women in Assam state, where 52% of India’s tea is grown and processed.

WHAT THIS STUDY ADDS

• When compared with unfortified CTC tea that had negligible clinical benefits, vitamin-fortified CTC tea normalised the low folate status of >90% women and low vitamin B₁₂ status of >80% women within 90 days. However, most women could not rise above the ‘NTD threshold’—a serum folate ≥11.3 ng/mL—that can help lower the risk of neural tube defects (NTDs); this warrants additional clinical study using an increase in duration and/or higher dose of vitamins needed to fortify CTC tea.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• The next steps involve the standardisation of large scale vitamin fortification of CTC tea in the Government of India’s flagship, Tocklai Tea Research Institute, and confirmation of the efficacy of such tea to enable Indian women to predictably overcome the ‘NTD threshold’.

• This work dovetails with recent approval by the Indian National Academy of Medical Sciences (NAMS)—the key advisory body to the Government of India on National Health Policy—to disseminate a comprehensive NAMS Task Force’s White Paper that contains strategic and pragmatic plans for ‘Prevention of NTDs in India’ under the aegis of the Government of India’s Ministry of Health and Family Welfare.

Introduction

Since childhood, most Indians consume a diet that fails to provide sufficient daily requirements of folate and vitamin B₁₂.1,4 The resulting low folate and vitamin B₁₂ status increases the risk of developing megaloblastic anemias⁴ and vitamin B₁₂ deficiency-induced neuropsychiatric and neurological deficits from subacute combined degeneration of the cord.⁴ In addition, a low folate and vitamin B₁₂ status among Indian women before pregnancy predisposes to intergenerational transmission of a combined low vitamin status to their progeny in utero and during lactation; over 100 000 babies with serious—but largely preventable⁵—neural tube defects (NTDs) every year3,7; an as-yet-uncharacterised number with congenital heart defects⁸; and postnatal psychological–psychiatric problems.^{1 4} Finally, a chronically low folate and/or vitamin B₁₂ status among older Indians can result in occlusive small vessel cerebrovascular disease-related strokes,⁹ accelerated brain atrophy, dementia and Alzheimer’s disease.^{3 10}

Informal surveys among educated young college-aged women across several Indian states consistently point to their unanimous preference for the inconspicuous incorporation of vitamin(s) into a commonly consumed food (Ref.,¹¹ online supplement). A similar situation in USA led to centralised folate fortification of wheat flour in 1998, which improved serum folate levels,¹² and reduced first occurrence of NTDs.^{13 14} Today, >80 countries employ folate fortification of industrially-milled cereal grain.¹⁵ There are however, inherent, intractable, logistical obstacles in attempting a similar approach to deploying centralised folate-fortified wheat flour in India.^{1 3 11 16} In addition, the low vitamin B₁₂ status in up to three-quarters of Indians,24 17,19 mandates the added replacement of vitamin B₁₂—to sustain intracellular ‘One-Carbon Metabolism,’ optimise endogenous folate function⁴ and is critical for the integrity of the nervous system.

The identification of a centrally-processed, food vehicle that is universally acceptable to all Indian women, is currently consumed every day, and which rises well above the diversity posed by social, religious, ethnic and cultural food preferences across India, has been an elusive challenge for over two decades.¹⁶ This vitamin-fortified food vehicle must also exhibit no organoleptic differences from the unfortified food vehicle and be capable of rapidly reversing the extant low folate and vitamin B₁₂ status among several hundred million Indian women.2,4

Earlier, we posited that a daily cup of black tea, which is widely consumed every day throughout India, could qualify as a food vehicle to deliver full replacement doses of both folate and vitamin B₁₂ to women with combined low folate and vitamin B₁₂ status.^{3 11 16} In a preliminary study from Sangli, Maharashtra state, we determined that a daily cup of Indian black tea (Camellia sinensis)—(brewed using 2 g teabags containing a proprietary blend of CTC (crush, tear, curl) tea, that were individually spiked with 1 mg folate and 0.5 mg vitamin B₁₂) and consumed for 2 months, significantly reversed the extant low folate and vitamin B₁₂ status of most young women, when compared with negligible effects in a small number of women using mock-fortified teabags.¹¹ Thus, black tea possessed all characteristics of a universally acceptable food vehicle capable of delivering daily full replacement doses of folate and vitamin B₁₂ to Indian women.

However, an important practical issue is that rather than using relatively expensive teabags, most Indian households use large-scale factory-generated ‘loose’ CTC tea (see online supplemental material: BOX_ 1) to brew their hot tea. Thus, a decisive advantage in focusing on direct vitamin fortification of CTC tea was the potential for vitamin fortification during the large-scale processing of CTC tea in commercial factories that can eventually reach all Indians.

Therefore, we first sought to test the feasibility of manual fortification of 2 g of CTC tea with 1 mg each of folate and vitamin B₁₂, followed by laboratory confirmation that brewing such vitamin-fortified CTC tea (in 100 mL of hot water at 95°C for 4 mins) yielded significant recovery of both vitamins. And second, we determined the clinical efficacy of daily consumption of a cup of hot tea brewed from 2 g of vitamin-fortified CTC tea (when compared with unfortified CTC tea) over 90 days to improve the generally low folate and low vitamin B₁₂ status among young women studying in Assam. In addition, because the majority of women from Assam²⁰ like most Indian women²¹ do not regularly consume Government-provided iron folate tablets despite their low iron status, and polyphenols in tea are potent inhibitors of non-haem iron absorption,²² we also addressed the legitimate concern of whether daily consumption of tea between meals adversely interfered with iron status.³

Methods

Primary and secondary outcomes of the clinical trial

The primary outcome was to assess the role of daily consumption of a single cup of folate-fortified and vitamin B₁₂-fortified CTC tea for 90 days in significantly improving the folate and vitamin B₁₂ status of women in the experimental group, when compared with those in the control group who received unfortified CTC tea. A related key outcome was to identify how many women at baseline had a serum folate concentration ≥15 ng/mL and serum vitamin B₁₂ concentration ≥400 pg/mL, and whether consuming vitamin-fortified CTC tea daily for 90 days allowed them to attain these “trial-specific optimum targets”—(see online supplemental material, Part_1, Clarification of Terminology used in the Clinical Trial). The secondary outcome assessed the potential inhibitory effects of unfortified and vitamin-fortified CTC tea on iron status, and the effects of vitamin-fortified CTC tea on haemoglobin concentration.

This trial was conducted between November 2022 and February 2023.

Eligibility criteria

Any woman 18–30 years, who was either a nursing or pharmacy student and resided in her respective Assam Medical College and Hospital (AMCH) Nursing Hostel or AMCH Pharmacy Hostel was eligible. She should not have had a medical history of fever or other chronic illness in the past 3 months. She should not be pregnant and was not planning pregnancy in the coming year. She had not used multivitamins containing folate or vitamin B₁₂ (including iron folate tablets) in the past 6 months. She would also agree not to consume multivitamins during the trial. If iron tablets were initiated during the trial, she would inform the research team. She would adhere to consuming a cup of trial-assigned CTC tea at the preset hour each day (including weekends) over 90 days. She did not have excess menstrual bleeding (warranting gynaecological consultation). Finally, each woman was requested to carefully read, understand, sign, and date the informed consent form.

Sample size estimate

Because the COVID-19 pandemic was ongoing when the study was initiated, we did not have an opportunity to carry out preliminary studies using manual vitamin-fortified CTC tea to accurately estimate sample size among the population to be studied. Accordingly, we used estimates from our earlier vitamin-fortified teabag study (completed before COVID-19)¹¹ of the differences in folate between the control (placebo) arm versus experimental arm (Cohen’s d effect size of 1.24), alpha of 0.016, and power of 80%; the sample size per group required to detect differences in folate levels was 13 per group (control and experimental group at a 1:1 randomisation).

However, we could not use these estimates for the following reasons: First, there is a fundamental difference between use of vitamin-fortified teabags (containing a blend of CTC tea) and ‘loose’ vitamin-fortified CTC tea. This stems from the fact that in comparison to the precise amounts of vitamins manually added to teabags using an accurate Eppendorf pipette, (which engendered confidence that each cup of brewed tea contained the precise amount of vitamins added to each teabag), there are multiple steps involved with direct vitamin fortification of CTC tea; this, in turn, could potentially lead to a net greater loss of vitamins with each added fortification step. This reduced confidence on the precise amount of vitamin in each 2 g of CTC tea, and of the validity of using the calculated sample size estimates solely based on our previous study with teabags.¹¹ Second, there are unique problems related to dropouts of subjects from clinical trials carried out in India—these have been bundled under the rubric of ‘India specific factors’ by Desai.²³ Third, it was also necessary to anticipate the adverse influence of COVID-19 infection on nursing and pharmacy students entered in the trial. This study was conducted between November 2022 and February 2023, when the Omicron variant of COVID-19 was dominant nationwide, while another variant ‘Arcturus’ was an impending threat to India. Since these women resided under crowded hostel conditions, and very few were vaccinated against COVID-19 (and none vaccinated against COVID-19 variants), it was possible that shortly after entry into the trial, several subjects could fall sick enough to be hospitalised and drop out—sufficient to threaten the viability of the study. Fourth, it was also anticipated that enrolled women could temporarily leave the study for 2 weeks or more to care for sick relatives, or to mourn those who succumb to COVID-19-related complications.

Based on these considerations, in anticipation of significant losses of women during the trial, and because the cost of entering additional subjects into the trial was not overwhelmingly expensive, we elected to err on the side of enrolling more women into the trial (than estimated based on the earlier teabag trial).¹¹ Accordingly, we enrolled a total of 30 women into each arm.

Methods for fortification of CTC tea

See online supplemental material, Part_1, Rationale for Dose of Vitamins Used for Replacement; Indian Pharmacopoeia (IP) Grade Folic Acid and Vitamin-B12; and Manual Fortification of CTC-Tea.

Trial design and venue, recruitment, allocation and blinding

There were several reasons to avoid conventional randomisation of a cohort of women into experimental versus control groups on the campus of AMCH. This stemmed from logistical difficulty in preparation of a daily batch of freshly brewed vitamin-fortified CTC tea and unfortified CTC tea to a large group of assembled women from different disciplines at a particular time of the day between meals or on weekends, and the real potential for mix-up in assignment where women could receive the wrong form of ‘trial-assigned CTC tea’ leading to a risk of contamination.

These issues were circumvented by ensuring that women of each control or experimental group were physically separated from each other by virtue of studying in different buildings, and where two dedicated individuals only prepared vitamin-fortified CTC, and two other individuals only prepared unfortified CTC tea. The two groups of women were thereby provided with tea during classes in different buildings during weekdays and at their respective hostels during the weekend. Thus, the daily dose of vitamins added to CTC tea, the timing of consumption of CTC tea between meals, and the duration of treatment was controlled. As a result, this simple placebo-controlled interventional study could allow for causal conclusions regarding the exposure to vitamin-fortified CTC tea or unfortified CTC tea and the primary outcome shown by a change in the serum folate and vitamin B₁₂ concentration in either group.

We made a priori assumptions that two groups of nursing or pharmacy students at AMCH would have a comparable age, diet, educational status, and general health, as well as parity in basic blood counts, and folate, vitamin B₁₂ and iron status.

This clinical trial was open to nursing and pharmacy students at AMCH, who resided in separate nursing and pharmacy hostels and had classes in separate buildings on the >300 acre AMCH campus. All 27 women in the second-year pharmacy class and all 29 women from the first-year nursing class who attended the initial informational lecture volunteered to enter the trial. To ensure there were 30 women in each group, the first of three additional women who volunteered from the first-year pharmacy class following a lecture were recruited. We also recruited the nursing hostel warden to complete 30 women in the nursing group. All these women were administered their daily trial-assigned CTC tea at the same time as the larger group to which they belonged.

This allowed us to employ a simple placebo-controlled interventional clinical trial design—based on a single coin-toss, with results known only to HKD and PG—where one cohort of women (either pharmacy or nursing students) received a daily cup of unfortified CTC tea (control group) whereas the other cohort of women received vitamin-fortified CTC tea containing 1 mg folate and 1 mg vitamin B₁₂ (experimental group) for 90 days. Comparison of pre-intervention and post-intervention blood test results in each group allowed us to compare the effects of unfortified CTC tea and vitamin-fortified CTC tea on the serum folate and vitamin B₁₂ concentrations, iron status, and complete blood count. (A format of the trial design is included with the online supplement).

Cluster allocation used in this study was to enhance feasibility and reduce contamination, as women from the Pharmacy Institute and Nursing School were grouped within their respective classrooms and hostels. While such allocation can introduce statistical concerns, including reduced sample efficiency and potential baseline imbalances, these were addressed by ensuring comparable demographic distribution across clusters and by applying appropriate statistical methods to adjust for intracluster correlation. Although individual randomisation would maximise statistical power, it was not practical in this setting. Cluster allocation ensured intervention fidelity, minimised crossover between groups and allowed for a realistic evaluation of outcomes within the natural social and living environment of participants.

We used key items from the CONSORT (Consolidated Standards of Reporting Trials) checklist when writing our report.²⁴

None of the women participating in the trial from either the School of Nursing Group or Pharmacy Institute Group were aware if they belonged to the control or experimental group. Even the dedicated workers who prepared and delivered trial-assigned CTC tea to women were not aware of the nature of the trial or the trial-assigned CTC tea administered.

To keep assessors of the trial data free from bias, the code linked to each woman and the group to which she belonged was only known to HKD and PG. No other co-investigators, including the primary assessors of outcomes (MPB and ACA), were aware of details on the identity of the experimental and control groups under study.

Only after completion of the data analysis, the group that received unfortified CTC tea was informed after the trial and advised to take the vitamin tablets provided daily for 3 months (at a minimum).

Logistics of tea delivery, consumption and safeguards against contamination, and monitoring of attendance

Because of significant physical separation of the location of Nursing College versus Pharmacy Institute with regards to both their classes and hostels, this reduced the risk of inadvertently serving trial-assigned CTC tea to the wrong group. Each control and experimental group had their trial-assigned CTC tea separately prepared by two dedicated individuals (per group) throughout the study in a different building from where tea was consumed by each group. However, the preparation of trial-assigned CTC tea for both groups was in a similar location, but at a different time to avoid mix-up of tea assignments. Identical 3 litre capacity thermos flasks labelled ‘Trial-assigned CTC tea’ was then carried by these individuals back to the respective classrooms in the Nursing College building and the Pharmacy Institute building (both on the AMCH campus) on weekdays and Saturday. Tea was served to the nursing cohort at 14:30, 2 hours after lunch. The pharmacy cohort received trial-assigned CTC tea at 11:00 on weekdays and Saturday, over 3 hours after breakfast (and ~1.5 hours before lunch). On Sundays, trial-assigned CTC tea was sequentially prepared and delivered to each hostel canteen at 09:30, ~2 hours after breakfast.

Each subject was provided a single daily cup of hot tea from either 2 g of unfortified CTC tea or vitamin-fortified CTC tea that was brewed in 100 mL hot water at 95°C for ~4 min followed by addition of milk and sugar.

During the first 3 weeks of the trial, with each cup of trial-assigned CTC tea, both cohorts were served two Britannia Marie Gold ‘tea biscuits’ that contained a total of 30 µg folate and 0.27 µg vitamin B₁₂. This small fraction of the recommended daily allowance (RDA) for folate (400 µg)²⁵ and vitamin B₁₂ (2.4 µg)²⁵ was unlikely to have significantly contributed to the final post-interventional serum folate or vitamin B₁₂ concentration in either group. Subsequently, other brands of tea biscuits containing no supplemental folate or vitamin B₁₂ was used for both cohorts.

Both cohorts were not prohibited from their usual pattern of daily tea consumption of three (and often more) cups of unfortified CTC tea daily, which is culturally the norm in Assam. Apart from consumption with breakfast, tea is usually consumed in-between meals.

Compliance was maintained by the dedicated workers (who prepared and delivered their trial-assigned CTC tea) to their assigned group of women. These workers took a daily ‘tea attendance’ of women in their group. This ensured both groups were monitored for consumption of trial-assigned CTC tea using a ‘Directly Observed Therapy’ format.

For subjects who inadvertently missed their tea for 5 days or more, additional catch-up trial-assigned CTC tea was prepared, so each woman completed a full 90 days of trial-assigned CTC tea. For those who anticipated missing several days, small packets containing 2 g of trial-assigned CTC tea for each day missed was given to each woman to self-prepare their tea every day.

Trial compliance assurance and report

The overall compliance among women was good. Attendance to document compliance with consumption of a single cup of trial-assigned CTC tea on-site under ‘Directly Observed Therapy’ was taken every day by two individuals for each group and a group monitor. Women did not complain of any organoleptic differences (ie, in appearance, aroma, malty taste or mouthfeel) or side effects from consumption of either unfortified CTC tea or vitamin-fortified CTC tea. Informal questioning of trial subjects indicated that they could not tell which group they belonged to by virtue of the trial-assigned CTC tea they were served.

In the experimental group, one woman required sick leave for two 8-day periods (she was eventually diagnosed with hypothyroidism) and another went home for 9 days to attend a family function.²³ They were given ‘catch-up’ days of tea (upon return to AMCH) to eventually receive 90 days of vitamin-fortified tea. Six other women gave notice they would miss between 1 and 3 days when they went home for holidays; they were provided with 2 g packets of vitamin-fortified CTC tea for each day missed and asked to prepare their trial-assigned CTC tea at home. As a result, all 30 women in the experimental group received a full 90 days of vitamin-fortified tea.

In the control group, 18 women went home for holidays for between 1 and 5 days; all were given ‘catch-up’ days of trial-assigned CTC tea (upon return to AMCH) to eventually receive 90 days of trial-assigned CTC tea. A few women who anticipated missing over 5 days were given sufficient 2 g packets of unfortified CTC tea for each day absent with instructions for self-brewing their trial-assigned CTC tea at home; among this control group, six women missed between 6 and 10 days, and three others missed 12, 15 and 18 days for a variety of reasons.²³

Blood tests related to the clinical trial

See online supplemental material, Part_1, Pre- and Post-Interventional Blood Tests and Test-Kits Used for Analysis of Blood Samples of Women.

Statistical analysis

Descriptive statistical analysis was used to assess the comparability of the experimental group (vitamin-fortified CTC tea, n=30) and control group (unfortified CTC tea, n=27). Between-group mean differences, as well as within-group mean differences in levels of pre-intervention versus post-intervention serum folate, serum vitamin B₁₂, per cent-transferrin saturation and haemoglobin—(at baseline, and after 90 days on trial-assigned CTC tea)—was evaluated using suitable parametric or non-parametric tests. For selecting the proper statistical test, normality of the data was evaluated using graphics methods, such as, boxplot, q-q plot and statistical tests, namely, Kolmogorov-Smirnov test. The between-group mean was tested using independent sample t-test or its non-parametric counterpart Mann-Whitney based on the normality of the data. Within-group differences were calculated using paired t-test or Wilcoxon signed-rank tests based on normality of the data.

Informed consent

Three authors (HKD, PG and MJK) participated in providing an initial talk to women who resided in either the General Nursing and Midwifery (GNM) Nursing School Hostel or the Institute of Pharmacy Hostel, who were potential candidates for entry into the study. This lecture highlighted the problem of dietary folate and vitamin B₁₂ deficiency in India, as well as the consequences of such deficiencies for their overall health, including the potential for adverse pregnancy complications and outcomes of women who remained folate-deficient and/or vitamin B₁₂-deficient. The general plan and key elements of the planned trial was also discussed. Both groups of women were informed that all of them from one hostel (nursing or pharmacy) would be randomly assigned to one intervention and receive either unfortified CTC tea or vitamin-fortified CTC tea, whereas women from the other hostel would receive the other intervention. Moreover, neither group would be informed of their assignment to the control group or experimental group of the study. The women were also informed that the group that received unfortified CTC tea would be provided full doses of vitamin replacement (at least 1 mg tablets each of vitamin B₁₂ and folate daily for 3 months) at the end of the study. Women were also encouraged to report any new symptoms they experienced during the trial. Ample time was allotted for answering questions. Those women expressing an interest in this study were provided informed consent forms, which highlighted details of the study. On receipt of the informed consent form, all women were requested to carefully read and discuss the study with their family for at least 1 week before agreeing to sign consent to enter the study. Because of the power imbalance that existed between the AMCH faculty investigators and students, the wardens in-charge of the students in the nursing and pharmacy hostels were encouraged to emphasise to the women that entry into the study was entirely voluntary, and there was to be no coercion, as emphasised in the informed consent form.

Ensuring post-trial equity between trial participants

On completion of the trial, all women in the control group who received unfortified CTC tea were provided tablets containing at least 1 mg folic acid and 1 mg vitamin B₁₂ for 3 months. This ensured equity in that both groups received folate and vitamin B₁₂ for at least 3 months. Other women in the experimental group with persistently low serum folate and/or low serum vitamin B₁₂ concentrations in post-intervention blood samples were also advised to take similar tablets daily for up to 3 months.

Results

Trial design

Figure 1 depicts the general format of this interventional trial that assessed the efficacy of response among two groups of women to either unfortified CTC tea (control group) or vitamin-fortified CTC tea (experimental group): One group included 30 women residing in the hostel of GNM School of Nursing on the AMCH campus, whereas the other group included 30 women studying at the Institute of Pharmacy on the AMCH campus, but residing in the AMCH Pharmacy Hostel located 4 km outside the AMCH campus.

Figure 1. Flow diagram of the progress through the phases of the intervention trial of two groups (from enrolment, intervention allocation, follow-up, to data analysis).

Thus, the risk for contamination was reduced by choosing students from different disciplines, since they had classes in different buildings on the AMCH campus, and there was a significant distance between their respective hostels. This avoided the potential for physical contact between the pharmacy and nursing students; moreover, by not randomising individuals within the same hostel, this precluded students in the experimental group from easily sharing information, behaviours or benefits with students in the control group. The logistics and feasibility of serving the ‘trial-assigned CTC tea’ to either Group (at a different time and place) was far more practical and manageable than to deliver to an entire class/hostel at one time instead of separating individuals within the same living environment. This also allowed easier monitoring, follow-up and ensuring compliance at the different class/hostel level.

Although all 30 women in the control group provided pre-interventional blood, only 27 women provided post-interventional blood; of three who opted out, two indicated feeling unwell, but the third declined to provide a reason. Therefore, in subsequent comparisons between pre-intervention and post-intervention of several parameters studied, we sequestered data of these 3 women from the original control group, and used the remainder (n=27), to allow for comparison of paired observations. All women (n=30) in the experimental group provided both pre-interventional and post-interventional blood (figure 1).

There was no evidence that daily consumption of unfortified or vitamin-fortified CTC led to any adverse clinical side effects. This is not surprising because (after water) consumption of a cup of tea is the second most common beverage in the world with a long history of having no adverse effect. Moreover, the mere addition of therapeutic doses of Indian Pharmacopoeia-grade folic acid and vitamin B₁₂ have not detected organoleptic or other adverse effects on consumption for over 50 years.

Table 1 shows a comparison of baseline data between women in the control group and experimental group. The cohort receiving unfortified CTC tea (n=30) had an average age of 21.5±1.1 (SD) years whereas the cohort receiving vitamin-fortified CTC tea (n=30) had an average age of 24.4±2.5 (SD) years. Other parameters related to both the serum folate and vitamin B₁₂ concentrations, haemoglobin and a parameter of iron status appeared generally comparable between both groups of women at the outset.

Table 1. Baseline values of the control group and the experimental group.

Parameter	Control group (n=30)			Experimental group (n=30)
	Mean	±SD	95% CI	Mean	±SD	95% CI
Age (years)	21.60	±1.54	21.05 to 22.15	25.20	±1.68	24.60 to 25.80
Haemoglobin (g/dL)	12.00	±0.98	11.65 to 12.35	10.70	±1.34	10.22 to 11.18
Serum folate (ng/mL)	2.73	±1.21	2.29 to 3.16	3.84	±1.89	3.16 to 4.52
Serum Vitamin B12 (pg/mL)	280	±111.9	240.0 to 320.0	269.6	±101.32	233.4 to 305.9
Transferrin saturation (%)	18.63	±9.48	15.09 to 22.16	14.98	±7.86	12.05 to 17.91

See online supplemental materials, Part_1, Clarification of Terminology [Defining Women with a ‘Low-Folate Status’ and a ‘Low-Vitamin-B12 Status’, and Reassessment of an ‘Optimum’ Serum Concentration of Folate and Vitamin-B12 among Indian Women].

Baseline serum folate and vitamin B₁₂ concentrations among all women

As specified in online supplemental material, Part_1, Clarification of Terminology used in the Clinical Trial, because many subjects with either borderline low normal serum folate (≤5 ng/mL) or borderline low normal serum vitamin B₁₂ (≤300 pg/mL) can also have metabolic evidence of folate and/or vitamin B₁₂ deficiency using more sensitive tests,^{4 26} all women with such values in either group were characterised as those with a ‘low folate status’ and ‘low vitamin B₁₂ status’, respectively. Therefore, at baseline (table 1), the control group was fairly-well matched with the experimental group with respect to the number of women with low folate status and low vitamin B₁₂ status. For example, 26/27 (96%) women in the control group had a low folate status (only 1 woman had a value of 7.5 ng/mL), while in the experimental group, 27/30 (90%) women had similar low folate values (the 3 women with serum folate values >5 ng/mL had values of 5.2, 5.6, 10.7 ng/mL). In addition, 20/27 (74%) women in the control group had baseline serum vitamin B₁₂ of ≤300 pg/mL consistent with a low vitamin B₁₂ status; although 6/7 women had baseline vitamin B₁₂ values between 300 and 400 pg/mL, only 1/27 (4%) women had a robust serum vitamin B₁₂ value >400 pg/mL. Likewise, in the experimental group, 21/30 (70%) women had low vitamin B₁₂ values; but among the 9 women with serum vitamin B₁₂ values >300 pg/mL, only 2/30 (7%) women had robust serum vitamin B₁₂ values >400 pg/mL.

Figure 2 shows pre-intervention serum folate and serum vitamin B₁₂ concentrations in all 57 women who completed the trial; this revealed many women with combined low folate and low vitamin B₁₂ status. Whereas folate deficiency was detected in 26/57 (43%) women, a full 51/57 (89%) women had serum folate ≤5 ng/mL—consistent with low folate status. While vitamin B₁₂ deficiency was found in 29/57 women (51%), a full 41/57 (72%) women had serum vitamin B₁₂ ≤300 pg/mL—consistent with low vitamin B₁₂ status.

Figure 2. Distribution of the baseline pre-intervention concentration of serum folate (A) and serum vitamin B₁₂ (B) among 57 women of childbearing-age in Dibrugarh, Assam, India. The bars for serum folate and serum vitamin B₁₂ concentration for each of the 30 subjects in the experimental group followed by each of the 27 subjects in the control group are aligned from left to right in A and B, respectively. The horizontal dashed line across A (----) depicts the cut-off level of 2.8 ng/mL, below which the serum folate level is consistent with frank folate deficiency. The horizontal dashed line across B (----) depicts the cut-off level of 239 pg/mL, below which the serum vitamin B₁₂ level is consistent with frank vitamin B₁₂ deficiency.

Of significance, none of the 27 women in the control group at baseline met our ‘trial-specific optimum target’ value of ≥15 ng/mL for serum folate, and only 1/27 women had a serum vitamin B₁₂ ≥400 pg/mL. Likewise, in the experimental group at baseline, none of the 30 women met our ‘trial-specific optimum target’ values of ≥15 ng/mL for serum folate, but 1/30 women had a serum vitamin B₁₂≥400 pg/mL.

Comparison of pre-interventional versus post-interventional changes in mean serum folate and vitamin B₁₂ concentration

Figure 3A shows pre-interventional vs post-interventional changes in mean serum folate concentration in the control group and experimental group who received a daily cup of either unfortified CTC tea or vitamin-fortified CTC tea, respectively, for 90 days. Although there was no measurable folate (or vitamin B₁₂) detected in unfortified CTC tea (presented below), the control group had a pre-intervention versus post-intervention mean serum folate of 2.8±1.3 (SD) ng/mL and 4.1±2.5 (SD) ng/mL, respectively. This small unexplained rise of mean serum folate was statistically significant, but of questionable clinical significance, since the net rise remained under the borderline low-normal cut-off of ≤5 ng/mL—reflecting a persistent low folate status; indeed, most women receiving unfortified CTC tea remained at low folate status after 90 days. By contrast, among women in the experimental group who received vitamin-fortified CTC tea, the mean pre-interventional serum folate was 3.8±1.9 (SD) ng/mL, but the corresponding post-interventional value was 9.2±3.6 (SD) ng/mL, reflecting a statistically significant and clinically meaningful difference (p<0.001).

Figure 3. Comparison of mean serum folate concentration (A) and serum vitamin B₁₂ concentration (B) in women at baseline (pre-intervention; saffron bars) and after 3 months (post-intervention; green bars) consumption of a daily 100 mL cup of tea brewed from either 2 g of unfortified CTC tea (control group, n=27), or 2 g of vitamin-fortified CTC tea (containing 1 mg folate plus 1 mg vitamin B₁₂) (experimental group, n=30). Data is shown as mean±SE. The double asterisks shown in the figure indicates a statistically significant difference (p<0.001) in the pre-intervention versus post-intervention in either serum folate or serum vitamin B₁₂ concentrations within the groups indicated. CTC, crush, tear, curl.

When the extent of differences in mean serum folate levels of control and experimental groups before and after daily consumption of unfortified and vitamin-fortified CTC tea for 90 days, respectively, were directly compared, the magnitude of change was more clearly defined. Thus, there was a small mean increase in serum folate in the control group of 1.3 ng/mL (95% CI 0.6 to 1.9); by contrast, the mean increase in serum folate in the experimental group was 5.4 ng/mL (95% CI 3.9 to 6.8; p<0.001). This rise in mean serum folate level following daily consumption of vitamin-fortified CTC tea for 90 days was highly significant when compared with results with use of unfortified CTC tea, where a small rise in serum folate did not have a scientific rationale and can be ascribed to chance.

Figure 3B shows pre-interventional and post-interventional changes in mean serum vitamin B₁₂ levels in both cohorts. The control group value of <1 pg/mL (95% CI −22.1 to 24.1) revealed no differences; by contrast, the corresponding mean increase in serum vitamin B₁₂ in the experimental group was highly significant at 194.6 pg/mL (95% CI 154.7 to 234.5; p<0.001).

Magnitude of individual changes in pre-interventional and post-interventional vitamin levels

Serum folate concentration

In the control group (unfortified CTC tea) 23/27 women experienced a small post-interventional rise in serum folate (figure 4). Whereas four women had a larger unexplained post-interventional rise in serum folate >5 ng/mL, only one with a baseline serum folate >7 ng/mL who inexplicably doubled her serum folate, had an extended stay for 15 days at home (to attend a festival²³), where she may have consumed a folate-rich diet; the other three women spent less than 5 days at home. None of the other 23 women experienced a rise of serum folate >5 ng/mL (the cut-off that defined a ‘low folate status’], and 4/27 women dropped their serum folate levels.

Figure 4. Comparison of the individual responses of serum folate concentration (left panels) and serum vitamin B₁₂ concentration (right panels) among women at baseline (pre-intervention) and after 3 months (post-intervention) consumption of a daily 100 mL cup of tea brewed from either 2 g unfortified CTC tea (control group, n=27), (upper panels), or 2 g vitamin-fortified CTC tea (containing 1 mg folate plus 1 mg vitamin B₁₂) (experimental group, n=30), (lower panels). Each coloured line denotes a single woman’s pre-intervention to post-intervention response. The lower limit of normal for the serum folate concentration corresponds to a value of 2.8 ng/mL in left panels. The lower limit of normal for serum vitamin B₁₂ concentration corresponds to a value of 239 pg/mL in right panels. CTC, crush, tear, curl.

By contrast, 28/30 women in the experimental group (vitamin-fortified CTC tea) had a clear-cut rise in post-intervention mean serum folate of 9.2±3.6 (SD) ng/mL. Among these responders, one-woman had a suboptimal response from 1.9 ng/mL to 4.4 ng/mL, suggesting she was more severely folate deficient than the others. Two women experienced a drop in serum folate: one, who dropped from 7.8 ng/mL to 6.0 ng/mL, also had a baseline low serum vitamin B₁₂ of 228 pg/mL that rose to 383 pg/mL after vitamin-fortified CTC tea; such a drop in serum folate has been observed among Indians with vitamin B₁₂ deficiency who were replenished with vitamin B₁₂.^{3 18} However, the other woman with a drop of serum folate from 10.7 ng/mL to 8.3 ng/mL, had a normal pre-intervention serum vitamin B₁₂ of 377 pg/mL that subsequently rose to 637 pg/mL; so in her case, the observed drop in serum folate remains unexplained.

Of added significance, despite 90 days of daily vitamin-fortified CTC tea, only 2/30-women achieved a rise in serum folate that cleared our ‘trial-specific optimum target’ of ≥15 ng/mL to help lower their risk of NTD.^{3 27} Parenthetically, neither of these 2 women (or any others) had obvious haemolysis in their processed serum, which can raise the serum folate.¹⁸

Thus, for almost all women in the experimental group, there was a significant rise in mean serum folate values >5 ng/mL; however, the magnitude of rise in serum folate was insufficient to clear our ‘trial-specific optimum target’ serum folate concentration of ≥15 ng/mL.

Serum vitamin B₁₂ concentration

In the control group, 7/27 women had pre-interventional serum vitamin B₁₂ levels >300 pg/mL, and after 90 days of unfortified CTC tea, 6 women maintained levels >300 pg/mL (and 3 of them even exceeded ≥400 pg/mL), whereas 1 dropped below this value. Only 2 women exhibited a pre-interventional to post-interventional rise from <300 pg/mL to just over this value. Thus, despite a small rise in serum vitamin B₁₂ among 20/27 women, the magnitude was both statistically and clinically insignificant.

By contrast, in the experimental group the magnitude of post-interventional rise in serum vitamin B₁₂ among most women was both highly statistically significant and clinically meaningful. All but 2/30 women consuming vitamin-fortified CTC tea exhibited a brisk rise of serum vitamin B₁₂ >239 pg/mL; the 2 women with subnormal responses had low baseline values of serum vitamin B₁₂ levels of 173 pg/mL and 176 pg/mL that rose to 228 pg/mL and 188 pg/mL, respectively, suggesting they had severe vitamin B₁₂ deficiency that warranted additional vitamin B₁₂ replacement. Although 25/30 women exhibited post-interventional increased serum vitamin B₁₂ ≥300 pg/mL,^{4 26} there were 18/30 women with serum vitamin B₁₂ values ≥400 pg/mL reflecting a good response, and 11/30 women even had excellent values >500 pg/mL. Despite these encouraging results, there remained 12/30 women who failed to achieve our empirically developed ‘trial-specific optimum target’ of serum vitamin B₁₂ levels ≥400 pg/mL (that has been associated with lowered risk of NTD).²⁸

Thus, for the supermajority of women from Assam with baseline low folate and low vitamin B₁₂ status, consumption of a daily cup of CTC tea fortified with 1 mg of folate and 1 mg vitamin B₁₂ led to a statistically significant rise in mean serum folate and vitamin B₁₂ values. Although this would avoid the risk of folate-induced and/or vitamin B₁₂-induced megaloblastic anaemia, the magnitude of rise in serum folate concentration was insufficient to clear our ‘trial-specific optimum target’ of ≥15 ng/mL; moreover, nearly one-third of women needed additional vitamin B₁₂ to clear our ‘trial-specific optimum target’ of a serum vitamin B₁₂ value of ≥400 pg/mL. Taken together, these data suggest that the depth of deficiency of both vitamins in women on our trial was unexpectedly severe, which warrants either an increase in duration of daily vitamin-fortified CTC tea beyond 90 days, or an increase in the dose of vitamins used to fortify CTC tea.

Recovery of folate and vitamin B₁₂ from random samples of 100 mL tea brewed from 2 g of unfortified CTC tea and vitamin fortified CTC tea used in the interventional trial

See online supplemental material, Part_2: ‘Analysis of key chemicals in vitamin-fortified-CTC-Tea’. High performance liquid chromatography analysis of freshly brewed CTC tea from random samples of 2 g of either unfortified CTC tea or vitamin-fortified CTC tea used in our trial, yielded the following results (table 2): There was no folate or vitamin B₁₂ detected in 2 g of unfortified CTC tea per 100 mL of brewed tea. However, when compared with the amount of 1 mg folate and 1 mg vitamin B₁₂ originally added to fortify 2 g of vitamin-fortified CTC tea, we recovered 0.92±0.16 (SD) mg folate and 0.79±0.04 (SD) mg vitamin B₁₂ per 100 mL brewed tea. This indicated that the net release of folate and vitamin B₁₂ from brewed vitamin-fortified CTC tea was good.

Table 2. Estimation of folate and vitamin B₁₂ per 100 mL cup of tea brewed from 2 g samples of either unfortified CTC tea or vitamin-fortified CTC tea used in the clinical trial^*.

Sample	Folate (mg/100 mL cup of tea)	Vitamin B12 (mg/100 mL cup of tea)	Theaflavins (%)	Thearubigins (%)	pH	Moisture (%)
Unfortified CTC tea	Not detected	Not detected	0.72	12.28	4.54	6.0
Vitamin-fortified CTC tea	0.92±0.16 (SD)	0.79±0.04 (SD)	0.69	12.30	4.69	6.4

^*See online supplemental material for additional details on the chemical analysis of both clinical trial samples of CTC tea. SD based on three sets of replicated analyses.

CTC, crush, tear, curl.

These results suggest that vitamin loss incurred during manual vitamin fortification of CTC tea can be compensated by the addition of 10% and 20% more folate and vitamin B₁₂, respectively, to each 2 g of vitamin-fortified CTC tea. A similar analysis during the initial phases of large-scale factory generation of vitamin-fortified CTC tea, can clarify the amount of folate and vitamin B₁₂ that needs to be added (as ‘overage’) to compensate for expected losses during large-scale factory processing of vitamin-fortified CTC tea.

Comparison of pre-interventional versus post-interventional changes in iron status

To detect any cumulative adverse effects of trial-assigned CTC tea—consumed by women ~2 hours after meals for 90 days—on iron absorption,²⁹ we evaluated for changes in per cent-transferrin saturation as a reflection of iron status. The normal reference range for transferrin saturation is 15–50%. We first examined within-group values of the transferrin saturation of women before and after 90 days daily consumption of either unfortified CTC tea or vitamin-fortified CTC tea (figure 5). In the control group at baseline, 10/27-women had transferrin saturation values of <15%, consistent with iron deficiency. The pre-intervention mean transferrin saturation in the control group was 19±10% (SD), whereas the corresponding post-interventional value was 22±10% (SD). Likewise, in the experimental group at baseline, 16/30-women had transferrin saturation values consistent with iron deficiency, with a pre-interventional mean transferrin saturation of 15±8% (SD) and post-intervention value of 15±9% (SD).

Figure 5. Comparison of the serum per cent-transferrin saturation in women at baseline (pre-intervention; saffron bars) and after 3 months (post-intervention; green bars) consumption of a daily 100 mL cup of tea brewed from either 2 g of unfortified CTC tea (control group, n=27), or 2 g of vitamin-fortified CTC tea (containing 1 mg folate plus 1 mg vitamin B₁₂) (experimental group, n=30). CTC, crush, tear, curl.

The mean difference in per cent-transferrin saturation pre-intervention versus post-intervention for the control group was only 2.7 (95% CI −0.7 to 6.1), when compared with the corresponding value of 0.3 (95% CI −2.5 to 3.1) for the experimental group. These results reflected insignificant changes in per cent-transferrin saturation among women consuming either unfortified CTC tea or vitamin-fortified CTC tea (p=0.4) and confirmed a lack of adverse effect of trial-assigned CTC tea on both cohorts for this parameter of iron status.

Women with haemoglobin E trait had more severe basal folate deficiency than women with hemoglobin A

Haemoglobin fractionation uncovered a fortuitous imbalance between both cohorts. In the control group, 11/27 women had haemoglobin E trait, whereas in the experimental group only 1/30 women had haemoglobin E trait. In addition, among the 11 women with haemoglobin E trait in the control group, 8/11 had baseline serum folate values of <2.8 ng/mL, consistent with frank folate deficiency. This raised the possibility that the imbalance in distribution of many more women with haemoglobin E trait in the control group may have influenced their lower pre-interventional serum folate values compared with the corresponding pre-interventional serum folate values in the experimental-group (compare saffron bars in figure 3A). Indeed, the mean pre-interventional serum folate concentration of all women in the trial with haemoglobin E trait (n=12) was 2.6±0.7(SD) ng/mL, which was significantly lower than the mean serum folate of 3.6±1.9 (SD) ng/mL among those with haemoglobin A (n=42); p<0.01.

Comparison of pre-interventional versus post-interventional haemoglobin

Figure 6A shows that 38/57 (67%) women had baseline anaemia with low haemoglobin <12.0 g/dL. However, 25/30 (83%) women in the experimental group had baseline anaemia compared with 13/27 (48%) women in the control group. Such data highlights the general problem of anaemia among young women from Assam. As shown in figure 6B, the pre-interventional haemoglobin in the control group was also significantly higher at 12.0 g/dL (95% CI 11.7 to 12.3) than the experimental group’s value of 10.7 g/dL (95% CI 10.2 to 11.2; p<0.001). This difference can be partly explained by slightly higher pre-interventional mean per cent-transferrin saturation in the control group of 19±10(SD), whereas the experimental group had a lower corresponding mean per cent-transferrin saturation of 15±8 (SD). The control group inexplicably experienced a significant rise in mean pre-interventional haemoglobin from 12.0±0.8(SD) g/dL to a post-interventional haemoglobin of 12.5±0.9 (SD) g/dL; (p<0.001); despite the statistical significance, these changes were small and clinically not meaningful. Moreover, there was no statistical difference in pre-intervention vs post-intervention haemoglobin in the experimental group. Importantly, vitamin-fortified CTC tea did not have any adverse effect in decreasing the haemoglobin concentration of women.

Figure 6. (A) Baseline haemoglobin concentration of the first 27 women in the control group followed by all 30 women in the experimental group who completed the trial. (B) Haemoglobin concentration in response to unfortified CTC tea or vitamin-fortified CTC tea. Haemoglobin concentration at baseline (pre-intervention haemoglobin; saffron bars) and after 3 months (post-intervention haemoglobin; green bars) consumption of a daily 100 mL cup of tea brewed from either 2 g of unfortified CTC tea (control group, n=27), or 2 g of vitamin-fortified CTC tea (containing 1 mg folate plus 1 mg vitamin B₁₂) (experimental group, n=30). Data is shown as mean±SE. The double asterisks signify a statistically significant difference (p<0.001) in the pre-intervention versus post-intervention haemoglobin concentration within the group indicated. The horizontal dashed line (set at 12 g/dL) in both panels signifies the cut-off between normal haemoglobin concentration and anaemia at sea level. CTC, crush, tear, curl.

Discussion

Feasibility and efficacy of vitamin-fortified CTC tea among Indian women

Our previous preliminary trial using vitamin-fortified teabags confirmed the feasibility of using a daily cup of tea as an ideal ‘food-vehicle’ to deliver therapeutic doses of folate and vitamin B₁₂ to women with combined low-folate and low-vitamin B₁₂ status.¹¹ However, because (loose) CTC tea generated in large commercial factories in India is commonly used in the supermajority of Indian households, we tested the feasibility of direct fortification of every 2 g cup of CTC tea with 1 mg folate and 1 mg vitamin B₁₂, and assessed the efficacy of such a daily brewed cup of hot tea to replenish women from Assam with pre-interventional low folate status and low vitamin B₁₂ status over 90 days.

The need among our educated population of women for folate and vitamin B₁₂ replenishment was significant. At baseline, 14/30 women in the control group and 16/30 women in the experimental group had normal serum vitamin B₁₂ concentrations >239 pg/mL; however, none of these women had a sufficiently high serum folate concentration ≥11.3 ng/mL to overcome the ‘NTD-threshold’ and lower their risk for having a baby with NTD.²⁷ This underscored the urgent need to improve both folate and vitamin B₁₂ status among this population of young women in Assam.

In this first-of-its-kind placebo-controlled interventional trial, we observed that whereas daily consumption of unfortified CTC tea (control group) led to minimal clinical impact on both serum vitamin levels, vitamin-fortified CTC tea (experimental group) led to highly significant and clinically meaningful improvement in both serum folate and vitamin B₁₂ concentrations within 90 days.

These results were achieved because biochemical analysis of random samples of 2 g CTC tea that were manually fortified with 1 mg each of folate and vitamin B₁₂ (used in our trial) allowed for recovery of 92% of folate and 79% of vitamin B₁₂ from each 100 mL brewed cup of vitamin-fortified CTC tea. These results confirmed the feasibility of direct fortification of CTC tea and suggest the high likelihood of success in moving forward to initiate research on large-scale vitamin fortification of CTC tea at a tea research factory.

Specifically, 93% of women receiving vitamin-fortified CTC tea exhibited a rise of mean serum folate concentration up to ~9 ng/mL, which effectively moved them out of a ‘low folate status’. However, after 90-days of vitamin-fortified CTC tea, only 2/30 women attained our empirically determined stringent ‘trial-specific optimum target’ serum folate concentration of ≥15 ng/mL. Likewise, although 2/30-women in the experimental group had pre-interventional serum vitamin B₁₂ concentration ≥400 pg/mL—set by our ‘trial-specific optimum target’ for serum vitamin B₁₂—after a daily cup of vitamin-fortified CTC tea for 90 days, 16/30 women achieved a robust rise in serum vitamin B₁₂ concentration ≥400 pg/mL; however, the remaining 12/30 (40%) women could not attain this target.

At this juncture, it behoves us to compare our stringent post-interventional ‘trial-specific optimum target’ serum folate concentration to the population-based ‘NTD-threshold’ target serum folate concentration of ≥11.3 ng/mL, which can lower a woman’s risk for having a baby with NTD.^{27 30 31} This ‘NTD-threshold’ was developed from a database of Chinese women with normal serum vitamin B₁₂ concentration.^{27 30 31} By contrast, most of our women studied in Assam had a pre-trial low vitamin B₁₂ status, which precluded use of the ‘NTD-threshold’ value of serum folate concentration as the gold-standard in our trial; moreover, we did not have pre-trial assurance that women in the experimental group could achieve a normal serum vitamin B₁₂ concentration after 90 days of vitamin-fortified CTC tea. Therefore, we opted to set a higher bar for (our) ‘trial-specific optimum targets’ for each vitamin—(ie, serum folate ≥15 ng/mL; serum vitamin B₁₂ ≥400 pg/mL). Although only 2/30 women in the experimental group achieved post-interventional serum folate concentrations of ≥15 ng/mL, we also discovered that the post-interventional serum vitamin B₁₂ concentration was unambiguously normalised (≥300 pg/mL) in 25/30 women. Therefore, this post-interventional cohort of 25 women (with normal vitamin B₁₂ status) could be reasonably assessed for insight into the number of women who overcame the population-based ‘NTD-threshold’^{27 30 31} through daily consumption of vitamin-fortified CTC tea over 90 days. However, we noted that only 5/25 women had achieved a serum folate concentration of ≥11.3 ng/mL after 90 days of vitamin-fortified CTC tea; this inability of the (other) 20 women to clear this bar likely reflects the significantly greater depth of their pre-existing low-folate stores.

Thus, CTC tea is an excellent vehicle for fortification with full therapeutic replacement doses of folate and vitamin B₁₂, and in delivering this in a daily cup of CTC tea for 3 months. This is sufficient to normalise the serum folate (>5 ng/mL) and serum vitamin B₁₂ (>300 pg/mL) and thereby prevent megaloblastic anaemia and neurological manifestations of these vitamin deficiencies. However, determination of the optimum dose or duration of folate and vitamin B₁₂ fortified CTC tea to protect Indian women from the risk of having babies with NTDs or related birth defects involving other midline structures remains to be determined. Therefore, additional study is warranted to determine if an increase in the duration of daily consumption of vitamin-fortified CTC tea to 120 days, or a further increase in the therapeutic dose of folate and vitamin B₁₂ incorporated into vitamin-fortified CTC tea can enable many more women to overcome the ‘NTD-threshold’.^{27 30 31} An increase in dose of both vitamins to 1.5 mg or 2 mg per cup of tea per day would not be considered out of the ordinary since (traditionally) a full therapeutic oral replacement dose of folic acid has been 1 to 5 mg daily⁴ and an oral replacement dose of vitamin B₁₂ is 2 mg per day for 120 days.³²

Lack of toxicity with long-term use of folate and vitamin B₁₂ fortified CTC tea

There was no evidence that either unfortified CTC tea or vitamin-fortified CTC led to any adverse side effects. This is because the body does not store excess amounts of folic acid and vitamin B₁₂, which are both highly water-soluble, and exceedingly well excreted in the urine²⁵; this accounts for a lack of toxicity using full therapeutic doses of both vitamins. Indeed, in the realm of haematology, internal medicine and nutrition,⁴ there is an over 50-year history of safety with use of both folic acid and vitamin B₁₂ given in full therapeutic doses over the long term to rapidly replenish folic acid (at 1 to 2 mg daily orally) for chronic haemolytic states like sickle cell anaemia and thalassaemia, and vitamin B₁₂ (at 1 to 2 mg daily orally) for pernicious anaemia as well as gastric food vitamin B₁₂ malabsorption that is found in a high percentage among the elderly.⁴

There has also been over 25 years of experience following the institution of a mandatory programme of folic acid fortification in USA and Canada under the safeguard of a careful surveillance system to identify early evidence of adverse effects. Thus, the safety of folic acid when used in food fortification and clinically in other conditions, as well as vitamin B₁₂, has been confirmed through several years of experience, through careful study and reinforced by authoritative reviews.133,35

Women with haemoglobin E trait had significantly lower serum folate levels

The finding that women with haemoglobin E Trait had a statistically significantly lower serum folate concentration when compared with women with haemoglobin A was an unexpected result that was only uncovered by our use of haemoglobin typing of all women on the study.

Hemoglobin E, involving substitution of lysine for glutamic acid at position-26 of the beta-globin chain, is a common haemoglobin variant in Southeast Asia.³⁶ Haemoglobin E trait is usually considered an asymptomatic carrier state, without anaemia despite minimal microcytosis and hypochromia. However, some evidence suggests that the haemoglobin E chain can become unstable at increased body temperatures—during febrile infectious diseases³⁶—resulting in haemolysis.³⁷ Therefore, one plausible hypothesis to explain the observed lower serum folate concentration in women with haemoglobin E trait when compared with haemoglobin A is that periodic bouts of fever-induced haemolysis would trigger increased demand from folate stores to support compensatory erythropoiesis⁴; however, in the context of chronic dietary insufficiency of folate, progressive depletion of folate from stores would eventually result in a lower serum folate concentration in women with haemoglobin E trait.

Thus, although authorities³⁸ believe that ‘Hemoglobin E trait has no clinical significance’, our results suggest that in women from Assam with haemoglobin E trait, there is a greater increase in demand for folate that is not met by the existing diet. As a consequence, those with haemoglobin E trait remain at a greater risk for adverse consequences of folate deficiency^{3 4} than those with haemoglobin A.

Does CTC tea have an adverse effect on iron status? The inhibitory effect of polyphenols in tea on non-haem iron absorption can be attenuated by maintaining a 1-hour interval between consumption of tea and meals.²⁹ Our results align with these findings²⁹—both cohorts waited over 1 hour between meals before consuming trial-assigned CTC tea over the 3-month trial, and neither post-interventional transferrin saturation nor haemoglobin concentration exhibited a significant drop compared with pre-intervention values.²⁹

Why did haemoglobin not improve with vitamin-fortified CTC tea? The lower haemoglobin by 1 g/dL in the experimental group versus control group at baseline was a random event. But the reason why vitamin-fortified CTC tea failed to raise haemoglobin in the experimental group (despite significant rises in serum folate and serum vitamin B₁₂), was because 53% had frank iron deficiency.^{3 4} In addition, many with borderline low normal iron status may have still had iron deficiency³⁹; indeed, up to one-quarter of young women with absent stainable bone marrow iron—the gold standard test for diagnosis of iron deficiency—had such borderline low normal iron status.³⁹ Following replenishment of folate and vitamin B₁₂ in women with normal iron stores, a peak rise in haemoglobin can be detected up to 2 months later.^{4 26} Therefore, had we delayed post-interventional blood testing (for haemoglobin) by ~2 months after 90 days of vitamin-fortified CTC tea, more women with improved haemoglobin might have been detected.

Limitations

This was the first study to assess the feasibility of directly fortifying CTC tea to define the superior efficacy of vitamin-fortified CTC tea over unfortified CTC tea in improving low folate and vitamin B₁₂ status in women from Assam. Because we did not have the opportunity to carry out preliminary studies using manual vitamin-fortified CTC tea during early stages of the COVID-19 pandemic in India, we estimated the sample size estimates from our earlier study using vitamin-fortified teabags; this study had been completed just before the pandemic. Accordingly, we relied on documenting significant improvements in serum folate and vitamin B₁₂ concentrations as surrogate objective markers of improved vitamin nutrition. However, given the unexpected greater pre-existing depth of the low folate status of subjects, we have yet to define the optimum dose and duration of vitamin-fortified CTC tea required for women from Assam.

The use of our study population of nursing and pharmacy students, who could likely have distinctly different dietary habits, lifestyles and better health status than the general population, precludes generalisation of our results at this time.

During the first 21 days of the 90-day trial, women in both groups were inadvertently given ‘tea biscuits’ containing 30 µg of folate per day, which comprised 7.5% of the RDA for folate (of 400 µg),²⁵ as well as 0.27 µg of vitamin B₁₂ per day, that amounted to 11.25% of the RDA for vitamin B₁₂ (of 2.4 µg)²⁵ in the USA. Thus, the net contribution of this additional folate and vitamin B₁₂ to the post-interventional serum folate or vitamin B₁₂ concentration in either control group or experimental group after 90 days consumption of trial-assigned CTC tea is unlikely to have had a significant influence on the results.

This study has highlighted the absence of a simple, reliable, non-invasive measurement of the depth of deficiency of folate stores in the presence of a low vitamin B₁₂ status in women. It may, however, be possible to indirectly estimate the relative depth of this parameter by determining the net dose of folate and or vitamin B₁₂ required to optimise the folate and vitamin B₁₂ status among different populations of women in future studies.

Although the present study using manual methods for preparation of vitamin-fortified CTC tea provides valuable information, this approach cannot be used for larger populations. Hence, we need to standardise methods for the large-scale factory fortification of CTC tea; document that every 2-gram cup of such factory-processed vitamin-fortified CTC tea yields the expected amount of folate and vitamin B₁₂; and we will need to document that such large-scale vitamin-fortified CTC tea is also efficacious under real-life conditions among Indian women.

Because the basis for our empirical ‘trial-specific optimum targets’ for folate and vitamin B₁₂ concentrations were crude estimates derived from prior studies, they cannot be construed as being reliable predictors of a lowered risk of NTD. However, because an ‘India-specific NTD-threshold’ is unlikely to be developed from among Indian women with combined low folate and low vitamin B₁₂ status, these ‘trial-specific optimum targets’ can continue to help to estimate the comparative efficacy of vitamin-fortified CTC tea among women from diverse Indian states.

Analysis of folate and vitamin B₁₂ content in random samples of 2 g vitamin-fortified CTC tea used in our trial indicated an 8% and 21% reduction of folate and vitamin B₁₂, respectively, related to losses incurred during manual vitamin fortification of CTC tea. Although this deficit can be reduced by the addition of 10% folate and 20% vitamin B₁₂ as permissible overage, it is unlikely that this (ie, addition of overage) would have significantly improved the number of women from Assam who met our ‘trial-specific optimum targets’ for both vitamins.

Because of low iron status among many women in Assam, we could not evaluate the independent effects of vitamin-fortified CTC tea in improving their haemoglobin concentration. Therefore, a pragmatic solution for replenishment of iron must necessarily be included with vitamin-fortified CTC tea to reduce nutritional anemias that contribute to the high number of obstetrical deaths each year in India.^{3 4}

Potential benefits of vitamin-fortified CTC tea in India

The benefits of optimisation of folate and vitamin B₁₂ status go far beyond prevention of NTD. Indeed, normalisation of these vitamin values would prevent nutritional megaloblastic anaemia⁴; decisively interrupt the vertical (maternal-to-fetal) intergenerational passage of a low folate and vitamin B₁₂ status^{3 40}; prevent a variety of postnatal neuropsychiatric syndromes in childhood that have origins in utero^{3 4}; reduce the risk of chronic hyperhomocysteinaemia that is associated with an increased incidence of occlusive small vessel strokes, cerebral atrophy, cognitive dysfunction, dementia and even Alzheimer’s disease^{3 4}; reduce the risk of cancer^{4 34}; reduce other congenital diseases involving midline structures (some congenital heart diseases).⁸ Thus, collectively, it is easy to see how the cost of comparatively inexpensive micronutrients³ added to fortify a beverage like tea can have such a powerful impact on the health of the entire Indian population.

Of significance to India, an Expert Committee³³ recommended that ‘in countries with a high prevalence of [vitamin] B₁₂ deficiency, consideration of [vitamin] B₁₂ inclusion with folic acid should be a priority in neural tube defect [NTD] prevention efforts…’ and ‘it is important to invest in further research of potential new vehicles to enhance scale-up of the delivery of folic acid’.³³ Moreover, as noted earlier,¹¹ the use of vitamin-fortified CTC tea is concordant with the Food Safety Standards Authority of India regulations for nutraceuticals.

Conclusion and significance

This study from Assam state confirms that among women with combined low folate and low vitamin B₁₂ status, full therapeutic daily doses of 1 mg each of folate and vitamin B₁₂ can be conveniently delivered via a daily cup of vitamin-fortified CTC tea, which exhibits all the characteristics of an ideal food vehicle among Indians. This study has established that (1 mg each of) folate and vitamin B₁₂-fortified CTC tea consumed daily over 90 days enabled 93% women to normalise their serum folate concentration and 83% women to normalise their vitamin B₁₂ concentration and prevent clinical manifestations of deficiency. However, among those women who achieved a normal serum vitamin B₁₂ concentration, only a minority achieved a high enough serum folate concentration to reduce their risk for NTDs. Therefore, efforts to reduce folate-responsive NTDs using vitamin-fortified CTC tea require additional study. Consumption of brewed CTC tea ~2 hours before or after meals had little adverse effects on their iron status. Finally, because of pre-existing low iron status in most women, there was no impact of vitamin-fortified CTC tea in improving haemoglobin levels; this highlights the axiom^{3 4} that replacement of folate and vitamin B₁₂ cannot reverse anaemia until associated iron deficiency is also addressed.

There is now significant progress on several fronts: The present study sets the stage for initiation of randomised clinical trial(s) designed to define whether increasing the duration of daily consumption of a cup of vitamin-fortified CTC tea (containing 1 mg folate and 1 mg vitamin B₁₂ per cup) for up to 120 days versus daily consumption of vitamin-fortified CTC tea (containing 2 mg folate and 2 mg vitamin B₁₂ per cup) for 90 days is the better approach for Indian women in Assam and Maharashtra to lower their risk for NTDs.

Plans are also underway to determine the most efficient method for large-scale vitamin fortification during the processing of CTC tea at the Model Tea Factory of the Tocklai Tea Research Institute (led by coauthors: PPS, PC and SS). These studies will assess the amount of overage required to compensate for expected losses during large-scale factory fortification, assess the stability of vitamin-fortified CTC tea under various conditions and document the efficacy of daily consumption of a cup of tea prepared from large-scale vitamin-fortified CTC tea in women.

Finally, a comprehensive White Paper containing concrete plans for the prevention of NTDs in India¹ has been endorsed by the Indian National Academy of Medical Sciences for publication in the Academy’s Journal that is under the advocacy of the Government of India’s Ministry of Health and Family Welfare. This document contains the blueprint for a massive project to optimise the folate and vitamin B₁₂ status among several hundred million pre-adolescent and adolescent girls and women of childbearing age in India within the shortest possible time. In this context, because the dietary intake of an Indian woman exerts a profound influence on what her family consumes over many decades at home, it is quite likely that vitamin-fortified CTC tea may be required to help protect well over 1 billion Indians from serious adverse consequences of a chronically low folate and vitamin B₁₂ status.^{3 4}

Data availability statement

Data are available upon reasonable request.