Effects of dietary intervention on vitamin B12 status and cognitive level of 18-month-old toddlers in high-poverty areas: a cluster-randomized controlled trial

Background The local diet in high-poverty areas in China is mainly vegetarian, and children may be more vulnerable to vitamin B12 deficiency. Objective The aims of this study were to explore the vitamin B12 status of toddlers living in high-poverty areas of China and to observe the effects of different complementary foods on the vitamin B12 status and cognitive level of these toddlers. Methods The study was nested within a cluster-randomized controlled trial implemented in 60 administrative villages (clusters) of Xichou County in which infants aged 6 months old were randomized to receive 50 g/d of pork (meat group), an equi-caloric fortified cereal supplement (fortified cereal group) or local cereal supplement (local cereal group) for one year. At 18 months, a subsample of the 180 toddlers (60 from each group) was randomly tested for serum vitamin B12 and total homocysteine (tHcy) levels, and their neurodevelopment was evaluated. Results The median serum concentrations of vitamin B12 and tHcy were 360.0 pg/mL and 8.2 μmol/L, respectively, in children aged 18 months. Serum vitamin B12 concentrations less than 300 pg/mL were found in 62 (34.4%) children, and concentrations less than 200 pg/mL were found in 30 (16.7%) children. The median vitamin B12 concentration was significantly different among the three groups (P < 0.001). The highest vitamin B12 level was demonstrated in the fortified cereal group (509.5 pg/mL), followed by the meat group (338.0 pg/mL) and the local cereal group (241.0 pg/mL). Vitamin B12 concentration was positively correlated with the cognitive score (P < 0.001) and the fine motor score (P = 0.023) of the Bayley Scales of Infant Development, 3rd Edition (BSID III) screening test. Compared to the local cereal group, children in the meat group had higher cognitive scores (P < 0.05). Conclusion In poor rural areas of China, vitamin B12 deficiency in toddlers was common due to low dietary vitamin B12 intake. Fortified cereal and meat could help improve the vitamin B12 status of children and might improve their cognitive levels. Trial registration The larger trial in which this study was nested was registered at clinical trials.gov as NCT00726102. It was registered on July 31, 2008.

effective separation of test and control clusters will be assured by location of clusters in separate administrative villages and supervision of intervention/control feeds 7 days each week in the community doctors' homes for each administrative village.
Infectious disease morbidity will be monitored by community doctors daily (including presence of diarrhea, monitoring of fever when febrile illness appears clinically possible and respiratory rate when participant has cough). Anthropometry and quantitative 24-hr diet will be performed at ages 6,7,9,12,15 and 18 mo;Bayley Scales of Infant Development, III (BSID) will be taken at 12 and 18 mo; biomarker and ancillary assays will be performed in Xi-Chou Women and Children's Hospital (CBCs) or in Shanghai Jiao Tong University from samples obtained at 18 mo.
Meanwhile, studies of zinc absorption and gut function tests will be performed in a sub-group of 40 subjects each from meat intervention, micronutrient fortified intervention or control clusters, total 120 subjects. Training and supervision of these assessment teams will be the responsibility of collaborative faculty at Jiao Tong University, working closely with the staff at Xi-Chou hospital. Data will be collected using paper/pencil forms and transferred daily to Xi-Chou Women and Children's Hospital after entering into local data management system; data will be transferred weekly to the data manager at Shanghai Jiao Tong University. The DSMB and the Colorado research team will receive 3-monthly reports. Data will be analyzed by the project statistician at UCDHSC in Denver using SAS software (SAS Institute Inc., Cary, NC) and outcome-specific methods.

Study rationale:
Undernutrition during the first two years of life is recognized as a major preventable cause of mortality in children aged one mo to 5 y. In contrast to the intensity of efforts to promote exclusive breast feeding until age 6 mo, optimization of complementary feeding (CF) starting at age 6 mo is only recently receiving equivalent attention with, for example, publication of WHO guidelines. Though these guidelines include feeding of animal source foods (ASF) at least by age 9 mo, the great majority of poor children in developing countries receive little or no ASF in their CF. Except in times of emergency food shortages, adequate macronutrients are typically available from plant foods, provided caregivers have adequate knowledge of how to feed their babies. Recent attention has, therefore, focused on micronutrient deficiencies which are widely considered major contributory factors to the rapid onset of stunting later in the first and in the second years of life, with its associated increased risk of Third version May 2009 impaired neurocognitive development, infectious disease morbidity and mortality.
Results of recent randomized placebo controlled trials of zinc supplements have provided persuasive evidence of the public health importance of zinc deficiency in young children as a major cause of stunting and of infectious disease morbidity/mortality. Treatment of putative zinc deficiency with supplements, however, presents major logistical problems as evidenced by the challenges and costs of step-up campaigns to enact the WHO recommendation for zinc supplementation in the management of acute diarrhea. Supplements may also have adverse effects as evidenced by the effects of iron supplements in non-anemic young children in falciparum malarial areas.
Sprinkles, with delayed release of micronutrients, have some advantage, but this has been confirmed only for iron. Fortification of food staples is increasingly promoted, but it is difficult to achieve the optimal dose of fortification for all segments of any population. Moreover, there have been some unexpected failures of fortified foods to achieve the hypothesized effects. Perhaps the most compelling reasons for optimizing nutrient intakes from non-fortified, affordable, locally grown foods is either lack of access or very uncertain access to fortified foods or supplements. This emphasizes the dependence on locally grown foods which require major attention to food diversity, especially the inclusion of ASF and specifically meat in order to meet requirements for key micronutrients, notably zinc and iron. Though the benefits of ASF, including meat, in complementary feeding have been recognized, this recognition has been insufficient to inspire programmatic action at any national or global level.
Two ounces (50g) of lean pork a principal source of meat in China where this study will be located, provides 2.5 mg bioavailable zinc. Minced meat mixed with a little polished rice, which has a negligible phytate content, has favorable bioavailability (approximately 0.4 fractional absorption) and the lean pork alone will more than meet physiologic requirements for zinc in young children. Additional zinc will continue to be absorbed from breast milk for most of the duration of this project as well as small quantities from other complementary foods. The quantity of iron provided by 2 oz (50g) lean pork is 1.0 mg. Though the latter is modest compared with the Estimated Average Requirement (EAR), especially that between 6-11 mo of age, the EAR is based on absorption of inorganic iron. No estimates of dietary requirements have been published based on a diet providing mainly heme iron.
Absorption of heme iron is very favorable and it is hypothesized that the iron derived from 2 oz (50g) lean pork will significantly improve parameters of iron status. At least one WHO expert considers it likely that meat will be found to be more efficacious than micronutrient-fortified plant foods (R Bahl, MD, PhD, personal communication).
However, investigations of the efficacy of meat as a first and regular complementary food, a likely scenario in hunter-gatherer cultures have been extraordinarily limited, partly, no doubt, because of the assumed high cost of production in terms of global resources. These assumptions and calculations do not appear to account for the ability of foraging, scavenging animals to survive and grow on waste around human habitations, especially those of the rural poor, making availability of affordable meat a realistic goal. In China, pork is a principal animal meat consumed and pigs can scavenge/forage in rural communities at a cost of $0.30/lb lean meat. If the remaining parts of the animal are sold, this reduces the cost of production of lean meat to approximately $0.15/lb or $0.02/2 oz daily serving.
Alternative meat from local foraging/scavenging birds/animals, e.g. poultry, rabbits, and goats, can be substituted elsewhere on a global basis if efficacy is demonstrated for lean pork in this project. In terms of DALYS and of Net Present Value it is hypothesized that regular intake of meat as a complementary food will compare favorably with alternative strategies. Moreover, this approach offers potential for microfinance initiatives thus empowering women in rural communities, an approach taken by the ENAM project in Ghana. Recent studies that encouraged greater use of meats in young children include especially those of Neumann and colleagues in Kenya and the successful complementary feeding education study in Peru with enhanced growth associated with messages that included the regular use of animal source foods, including liver.

Third version May 2009
Section 2: Research Ethics

Ethics
Before beginning any research, the protocol has to be approved by the ethics committee. This protocol has been reviewed and approved by the Xin-Hua Hospital Institutional Review Boards (IRBs) and COMIRB, including: • Nutrition monitoring design; • Participants recruitment, enrollment and interventions; • Daily interviews, biological measures and specimen collections; • Data analysis and publication of findings.
Senior staffs of this project have been certified in "Ethics in Research" by the Office of Human Protection in Research. Field personnel will be trained in the most important aspects of ethics in research by senior staff.

Principles of human research ethics
A. Respect for persons • The autonomy, self-determination.
• Protection of vulnerable groups (those with limited education, the poor, those with difficult access to heath services, women). • The informed consent: Respect for persons is embodied in the Informed consent process.
Informed consent is designed to empower the individual to make a voluntary informed decision regarding participation in the research.
Potential research participants must fully comprehend all elements of the informed consent process.

B. Beneficence
• Physical, mental and social well-being. Risks reduced to a minimum.

Confidentiality
A. All information obtained from participants must be kept confidential. Data about participant cannot be associated with her/him as an individual.
B. Monitoring staff must maintain confidentiality of all information gathered from or about a participant: • Information collected on the data forms; • Contact information; • Medical record abstractions.
C. Do not discuss a participant with anyone except other project staff: • Information about a participant could be repeated to someone who could identify her/him; • Monitoring result and other data collection materials like official medical records.

Safeguards to ensure confidentiality and protection of data
A. Materials with identifying information kept in a secure place.
B. Evaluation interviews conducted in privacy and at a convenient time for participant.
C. Participant's name can not connected to results when the data have been analyzed.
The participant's ID number: • Created by data management system; • Identifies a study participant; • On all forms and data files to replace name; • Guarantees anonymity while allowing tracking of activities.

A. Research description
• Description of the nature of study, the objectives, expected responsibilities, procedures involved, study duration and explanation of randomization or placebo.
B. Description of risks • Anticipated or foreseeable risks, physical, social and psychological risks, and culturally appropriate.
C. Description of benefits • Reasonably expected, no exaggeration of benefits once research is ended.

D. Description of available alternatives
• Alternative procedures or treatment, advantages and disadvantages and availability.
E. Description of confidentiality • Indicate persons or organizations who may have access to the information.

F. Compensation
• It is permissible to compensate participants for their time, travel and inconvenience; however this should not be so high as to unduly influence a potential participant's decision in the study.

G. Contacts
• Contact for research-related questions.
• Contact for concerns about rights as a participant, realistic and viable.
• Voluntary Participation, absolutely voluntary, right to discontinue at any time and no penalty for refusal.

Protocol
• The protocol provides the scientific basis for the study. The study objectives, rationale, study design, methodology, and statistical considerations of a clinical trial.

Consent form
• The consent form describes the study procedures that participants are expected to take part in and adhere. • Compensation and contact information and other information regarding participation are included.

Manual of operation
• The manual of operation describes in detail the procedures necessary to implement the study protocol. • The manual describes the study organization, preparations, recruitment and enrollment, baseline assessments, follow-up data collection, outcome ascertainment, and standard operating procedures.

Data forms
Data forms are used to acquire information (by interview, abstraction, or observation) that will be used to support the implementation of the trial and test the study hypotheses. A copy of each data form is included in the manual of operation.

Section 5: Identification, Recruitment and Enrollment of Participants
Identify potential participants (mother/child), and mobilization, recruitment and enrollment of the participants. Complete data form NIM 1 (Birth and Screening Log), NIM 2 (Screening Form), and NIM 3 (Enrollment Form).

Recording births
Complete data form NIM 1 (Birth and Screening Log), and identify potential participants.
• Available birth registry data will be obtained from the local health center or other available registry by coordinator staff of Xi-Chou monitoring team. • NIM 1 (Birth and Screening Log) will be completed by core staff of Xi-Chou monitoring team. Coordinator staff of Shanghai monitoring team will provide ID number. ID number will be used in other data forms, for substituting the name of the participant.
• Organized by coordinator staff of Xi-Chou monitoring team, community doctors will identify and recruit potential participants, and contact to enroll once a month.
• NIM 1 (Birth and Screening Log) will be completed by core staff of Xi-Chou monitoring team and coordinator staff of Shanghai monitoring team together.

Screening of participants
Complete data form NIM 2 (Screening Form), according to NIM 1 (Birth and Screening Log).
• On the monthly meetings for screening and enrollment of participants, complete data form NIM 2 (Screening Form).
Inclusion criteria (all three required for inclusion): + The infant must be between 3 and 5.5 months of age at the time of enrollment.

+
The mother must be breastfeeding the child as a primary source of food. But, it is not required that the child be exclusively breastfed in order to be eligible for the study.

+
The mother should be intending to breastfeed the child to at least 12 months. Again, it is not required that the mother exclusively breastfeed the child through 12 months.
Exclusion criteria (if any one of these is present, infant is not eligible):

+
The mother feeds the infant formula daily or most days.

Section 6: Study Food Dispensing and Storage
Study food dispense and requirements for appropriate storage.

Fresh lean pork
• Pork will be provided by assigned supplier in local farm product market; • Handi-wrap will be used to package pork, 50g per portion; • According to daily requirements, coordinator staff of Xi-Chou monitoring team will supervise the purchase of lean pork and deliver to each community doctor daily or at least twice of week; • Meat must be stored in refrigerator as soon as possible. The meat was been keep in cooler no more than one day, freeze no more than one week.
• Mother will bring her child to community doctors' home to eat it, or community doctors will distribute it to each participant through daily home visit and supervise mother feeding it to child.
• Remaining cooked meat can be fed again up to 2 hrs later provided it is covered and stored in cool place. If infant is only just starting to eat meat, mother may chose to save half of meat to cook and feed later in the day.
• Initially minced pork should be fed alone or with minimum of rice cereal.
When infant is a little older and eats 2oz of pork/day, other foods can be added to the meal in progressively increasing quantities.

Fortified cereal
Regulations do not allow addition of a separate micronutrient mix to cereal grain flour, therefore: • Nestle fortified cereal will be purchased by Shanghai monitoring team from the market.
• Fortified cereal will be packaged into 20g bag.
• According to daily requirements, core staff of Xi-Chou monitoring team will deliver fortified cereal to each community doctor weekly through coordinator staff of Xi-Chou monitoring team.
• Cereal must be stored at room temperature; room should also be well ventilated and protected from flood, leakage of water, rodents or other vermin.
• Mother will bring her child to community doctors' home to eat it, or community doctors will distribute it to each participant through daily home visit and supervise mother feeding it to child.
• Save small samples of fortified cereals for analysis at time of each purchase.

Ordinary cereal
• Product is produced by assigned store in local market and supervised by Xi-Chou monitoring team. • The ordinary cereal is made of sticky rice and sugar, with the ratio of 2:1.
• The ordinary cereal will be packaged into 20g bag.
• According to next month requirements, core staff of Xi-Chou monitoring team will notify the store two weeks in advance. Cereal will be stored less than 2 months.
• According to daily requirements, core staff of Xi-Chou monitoring team will deliver it to each community doctor weekly through coordinator staff of Xi-Chou monitoring team.
• Cereal must stored at room temperature; room should also be well ventilated and protected from flood or leakage of water, rodents or other vermin.
• Mother will bring her child to community doctors' home to eat it, or community doctors will distribute it to each participant through daily home visit and supervise mother feeding it to child.
• Save small samples of ordinary cereals for analysis at time of each purchase.

Section 7: Assessment Visits
Assessment visits of nutrition monitoring will be performed at ages 6, 7, 9, 12, 15 and 18 months, including short diet records, sub-sample 24-hr diet records, and anthropometry. At 7 months, only short diet record required to determine meat consumption. Developmental testing performed only at ages 12 and 18 months. Blood sample only at 18 months (except for those in metabolic study); stool specimens at ages 6, 9, 12 and 18 months.
24-hr diet records, anthropometry, blood and stool specimen collections will be accomplished within a two week window (i.e. ± 1 week) around each age; Developmental testing will be accomplished within a four week window (i.e. ± 2 week) around each age.

A Questionnaire
To be completed at ages 6, 7, 9, 12, 15 and 18 moths by assessment team by questioning primary caregiver. Fill in data form NIM 6, Section B. This part will be accomplished by coordinator staff of Shanghai monitoring team with assistance of Xi-Chou monitoring assessment team.
B 24-hr diet records 24-hr dietary recalls will be obtained at ages 6, 9, 12 and 18 months during the assessment visits. These records to be collected on a convenience sample of 100 participants in each group, with repeat data collections in 30 participants per group in order to determine intra-individual variation.

Anthropometry
Anthropometry will be obtained at 6, 9, 12, 15 and 18 mo during the assessment visits, and then complete data form NIM 6, Section C. This part will be accomplished by Xi-Chou monitoring assessment team.
A. Measuring weight 1. Remove the infant's clothing to a dry diaper. 14. Two measurements must be taken and recorded on Sheet 6. 15. If the two measurements do not agree within 0.4cm, a third measurement must be taken.

Developmental testing
The Bayley Scales of Infant Development, 3nd Edition, (BSID III) will be used to assess the infant's development at 12 and 18 months of age.
All participants will accept the test by appropriately trained and experienced testers with assistance of Xi-Chou monitoring assessment team.
This test should be accomplished within a four week window (i.e. ± 2 week) for each participant around each age.

Blood and stool specimen collections
Blood specimen should be collected at 18 months of age, total 5ml. This part will be completed by Shanghai & Xi-Chou Laboratory assistant with assistance of Xi-Chou Monitoring Assessment research worker.
A small stool sample will be collected at 6, 9, 12 and 18 month. This sample can be stored at room temp for 6 days.

Section 8: Home Visits by Community Doctor
Home visits by community doctor including: potential participants' home visits before 5-6 m at least once; participants' weekly home visits for monitoring, food preparation & feeding, morbidity monitoring, and nutrition education messages (rationale and delivery). For the meat group, the community doctor is responsible for daily delivery of uncooked meat if mother is unable to collect from doctor's home..

Potential participants home visits (Interim Home Visit)
• At least one interim visit will be made between enrollment (age 3-5.5months) and before the infant reaches 6 months of age. • Purpose of the visit: Remind the mother of the Nutrition Monitoring and assessment visits; Encourage continued breastfeeding, including exclusive breastfeeding as much as possible Encourage responsive feeding; Encourage mother to plan to start complementary foods when the infant is 6 months old A. Delivery of study foods • Coordinator staff of Xi-Chou monitoring team will distribute meat, fortified cereal or ordinary cereal to community doctors; Meat will be distributed at least once a week. • The community doctors will be responsible for study food dispensing to each participant daily, or provide to the mother if she comes to the doctor's home.
The doctor may also choose to feed the participants at his/ her home.
• Cereal can be delivered to participants' homes weekly by the community doctors • The community doctors will be in charge of monitoring the feeding process weekly.
• Complete data form NIM 4, and submit it to Coordinator staff of Xi-Chou monitoring team weekly.

B. Study food preparation & feeding
• When the study food is prepared by community doctors or mothers, please notice: Wash your hands with clean soap and water before preparing food and feeding.
Wash baby's hands with soap and water before feeding Use clean water for preparing food Feed the baby with clean spoon, cup or bowl, and never use a feeding bottle Give the food soon after preparing • Lean pork One 50g packet of lean pork should be fed to the child each day; Lean pork can be steamed, boiled, or stewed, and a paste or puree is made, then be provided to the infant straight; The lean pork should be fed as a single food to begin with; After the infant has learned to eat it well, the infant can eat other foods as ages; After the infant has learned to eat it well, it may be added to a small amount of other foods.
• Fortified cereal Boiling water, and keep 5 minutes longer; Put the boiling water into the plastic box, and add the full 20 g packet of fortified cereal; Stir until it thickens and a purée consistency is achieved; Cool and feed to child (mothers should be encouraged to test the cereal before feeding it to the child); The fortified cereal should be fed as a single food to begin with; After the infant has learned to eat it well, the infant can eat other foods as ages; • Ordinary cereal Boiling water, and keep 5 minutes longer; Put the boiling water into the plastic box, and add the full 20 g packet of ordinary cereal; Stir until it thickens and a purée consistency is achieved; Cool and feed to child (mothers should be encouraged to test the cereal before feeding it to the child); The ordinary cereal should be fed as a single food to begin with; After the infant has learned to eat it well, the infant can eat other foods as ages; • Feeding the study foods For young infants, the lean pork, fortified or ordinary cereal should be the only food (in addition to breast milk) the infant eats until he or she can eat it all.
At the beginning (around 6 month of age), mothers should encourage, but not force, the child to eat the entire packet each day.
At the beginning, the young infants eat only a small proportion at one feed, the mother may split the daily ration in two to three feeds to assure that the entire packet is eaten during the day.
If the daily cooked (or moistened) portion is split into 2 feedings, the unused portion should be stored in the study container until use. The food should be used within ~ 2 hr, or be heated to feed again, never feed food which is overnight to child.
For the older infant (e.g. after 7 months), if the study is added to another food item(s), instruct the mother to first feed the study food.
For 12-18 month olds, encourage self-feeding (e.g. picking up meat cubes, using spoon for cereal or purees). Guide the mother help her child eat the entire packet, and avoid spatter and waste.

C. Monitoring Morbidity
Awareness and report of study food eating and infectious disease morbidity will be greatly facilitated by the weekly home visits from 6 to 18 mo of age. Fill in data form NIM 4 /5.
• Monitoring respiratory illness, diarrhea and any febrile or severe illness the community doctors should conduct the weekly home visits with participants, and record on data form NIM 4 faithfully. Give your baby at least three meals a day, every day Give your baby many different foods Be wise in how frequently you repeat the same advice depending on your assessment of each household and the apparent nutritional status of the child. Give time to respond to questions.
Module 12: Study Management 1. The PI will go to Xi-Chou to supervise and direct the implementation of the monitoring once a month.

2.
The core staff of Xi-Chou monitoring team will meet with coordinator staff of Xi-Chou monitoring team at least once a week.
• Deliver the completed data forms to the core staff of Xi-Chou monitoring team; • Discuss problems encountered in implementing monitoring procedures; • Identify and provide solutions; • Plan activities for the forthcoming weeks: home visits, assessments and delivery of study food. offer foods by spoon. This might be in the morning, in the middle of the day, and in the evening or afternoon.
Maximize local food diversity ("Give your baby many different foods") • No single food provides everything the baby needs.
• Other foods must be eaten with the "staple" (maize, rice, roots, etc) to fill the energy and nutrient gaps. • By 6 months babies are able to digest all foods.
• Babies may be surprised by new tastes and textures; this does not mean they do not like the food.
• They need practice each day to become familiar with the food.
• Types of foods that are best to fill the gaps: Pulses (beans, peas, groundnuts) and oil seeds (sesame seeds) Foods from animals (eggs, cheese, milk)

Dark-green leaves and orange-colored fruits and vegetables
Oils and fats

12-18 months old children
Continue to reinforce same messages, but recognize that as the infant grows and develops, the specific implementation of the messages will change.
• As the baby gets older, increase the amount and the variety of foods (as possible); an older baby needs to eat more to meet his/her nutrition needs. • The child should be allowed to hold the spoon and begin to feed him/herself. At 12 months, the mother will still need to assist some, but the child should be encouraged to practice using the spoon. The child will also enjoy picking up pieces of food (e.g. the meat cubes, or for the cereal group, other soft foods, such as fruits or cooked vegetables) • Family foods can be mashed to make thick puree/mixture; • Continue to offer at least 3 meals per day in addition to breastfeeding as the child wants. • Give the child many different foods.