1

IRB Proposal:

Can supplementation improve vitamin low B12 serum levels in vegans?

Research question(s): Can a nutrition intervention program using nutrition education

and supplement over a 12 weeks period improve blood serum levels in vegans with

vitamin B12 deficiency?

Hypotheses:

 H0 (null hypothesis): There is no difference in B12 blood serum levels in vegans

that receive nutrition intervention consisting of nutrition education and a B 12

supplement.

 H1 (alternate hypothesis): There is a difference in B12 blood serum levels in

vegans that receive nutrition intervention consisting of nutrition education and a

B12 supplement?

Goals and Objectives:

Primary objective: To determine whether an intervention involving the use of nutrition

education and vitamin B12 supplement produces changes in blood serum levels in a

sample of vegan Seventh Day Adventist.

Secondary objectives: To determine blood concentrations of B12 serum levels at

baseline and post-intervention.

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Introduction

The nutrient of interest for the institutional review board (IRB) proposal is vitamin

B12. The purpose of the research study is to assess vitamin B 12 levels in Seventh Day

Adventists who have been vegans for more than five years. Studies show that

vegetarians and vegans are vulnerable to vitamin B 12 deficiency.1,2 Vegetarians and

vegans are particularly vulnerable in becoming deficient because of their dietary intake.

Vitamin B12 is only found in foods from animals and vegans do not eat animal flesh. 3

There are vegans who may be living with a vitamin B 12 deficiency of which they are not

aware. Vitamin B12 can be stored in the body for years.4 If one does ingest the vitamin

over a period the nutrient will be depleted. The deficiency could be present with non-

specific symptoms.5

It is established that B12 deficiency among vegetarians is prevalent.6,7 Persons

who are vegan may be deficient in this vitamin because they refuse to take vitamin B 12.

The argument for refusing to take supplements is that they can have adverse side

effects.8 The recommended daily allowance (RDA) for vitamin B 12 is 2.4 mg daily for

adults therefore, it should not be difficult to ingest that recommended daily allowance.

If vitamin B12 is lacking in vegetarian diets hence vegans will be at greater risk for

having B12 deficiency.5 Many vegans do not have nutrition knowledge, this prevent them

from planning their meals to include vitamin B 12 enriched foods and the taking of

supplements. If vegans do not get vitamin B12 from fortified foods or supplements their

health will suffer.9 It is recommended that oral intake of vitamin B 12 supplement be used
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in the treatment of the vitamin B12 deficiency.10 Vitamin B12 fortified foods and milk

substitutes that contain vitamin B12 can be successfully used to treat the deficiency.11,12

Vitamin B12 is a water -soluble vitamin required for many bodily functions. Vitamin

B12 assists in making red blood cells, which transports oxygen throughout the body. 13 It

is essential for the function and development of brain and nerve cells. 14 It helps to break

down a protein called homocysteine Vitamin B12 must bind with the foods we eat. In the

stomach hydrochloric acid unbinds it, turning it into free form. It then mixes with intrinsic

factor protein it is then absorbed into the body via the small intestines.

The symptoms of vitamin B12 deficiency can be irreversible if they are left

untreated for a long time. It is important that vegans get regularly testing for the

deficiency. To confirm if there is a vitamin B12 deficiency lab work is necessary. Tests

can be conducted to measure the serum vitamin B 12 level.15 One test the MMA gives the

best result for vitamin B12 because it explains the extent of the deficiency. 16 Lowered

serum holoTC concentration another test can show early deficiency. The result of this

test shows that the body does not have enough available vitamin B12. 17

Review of current literature

The number of persons following a vegetarian diet has increased worldwide.

There are 375 million vegetarians worldwide. 18 With growing vegetarian levels, the

prevalence of individuals who has vitamin B12 will increase. There could be many

persons walking around with undiagnosed vitamin B 12 deficiency. Based on research

persons who exclude meat from their diet and do not take supplements are at risk for

developing vitamin B12 deficiency.3

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Vitamin B12 deficiency is a highly researched area. 5,8 In 2014 Mearns and

colleagues 18 conducted a 6-month randomized controlled trial to examine the

effectiveness, acceptability and sustainability of interventions to reduce vitamin B12

(B12) deficiency in South Asian women before conception. South Asian women, 18–50

years old) were stratified by dietary practices, then randomized to three treatment

groups: B12 Supplement (oral cyanocobalamin 6μg/day) (n=21), Placebo (n=21), or B 12

Dietary Advice (n=20).18 Primary outcome measures were changes in B12 biomarkers

(serum B12 and holotranscobalamin (holoTC)) at 6 months. A sample size of 62

participants was determined to detect a mean change in serum B 12 of 66±71pmol/lover

6 months.19

From the analysis, half of the women of childbearing age in the study had either

deficient or insufficient B12 stores. Women who were vegetarians were found to be of

higher risk for developing B12 deficiency.19 The findings support low-dose oral

cyanocobalamin supplementation for improving B 12 status for women with insufficient

dietary B12 intake. The researchers suggest closer monitoring and prevention of B12

deficiency in women where low or non-meat dietary practices are common. 19

Vitamin B12 deficiency happens because of poor dietary intake. 20 Since animal

foods are the only source of vitamin B12 is common in diets with little or no meat. 20

Inadequate intake of folate and vitamin B12 leads to low serum or plasma

concentration.20 It is recommended that patients who are susceptible to B 12 deficiency

take supplement.8
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A cross-sectional study was conducted in 2018 to determine dietary factors that

are associated with plasma concentrations of vitamin B 12.21 The aim was to identify the

dietary sources of vitamin B12 intake from supplements, fortified foods, and animal

source foods (that include meat, fish, milk, and eggs) in a health-conscious population. 21

A total of 728 participants were assessed. The median age was 58 years and Serum

vitamin B12 was measured by a microplate enzyme immunoassay for quantitative

determination of vitamin B12 in human serum.20 Diet was assessed using 24 h dietary

recalls (24 HDR) and a previously validated food frequency questionnaire. Intake of

vitamin B12 was estimated from two sets of three 24HDRs, which included information

on all foods, beverages, and supplements consumed by each subject during the

previous 24 h.21

From the findings it was ascertained that most of the respondents got their B 12

intake from supplements. multiple Although not all subjects in the current study obtained

vitamin B12 from supplements, multiple linear regressions showed that supplements

were the strongest predictor of holoTC and serum vitamin B 12.21 The writers concluded

that vitamin B12 supplements are important in a vegetarian or vegan diet to prevent

vitamin B12 deficiency.21

There is a potential risk of that vitamin B12 deficiency could be present without

any symptoms, the question remains if some groups are more prevalent to the

deficiency?22 One study conducted in 2019 found that, elderly people, pregnant women,

and vegans are more susceptible to B12 deficiency. These groups may have symptoms

and signs can be non-specific, particularly in the early stages. 22

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In a recent study published in 2017, it is recommended that laboratory evaluation

be conducted for patients with suspected vitamin B 12 deficiency, including blood count

and serum level. 22 A level of less than 150 pg per mL (111 pmol per L) is diagnostic for

deficiency.23 Patients in high risk groups should be evaluated to find out if they have

vitamin B12 deficiency.

Persons who prescribe to vegetarian diets are at risk for vitamin B 12 deficiency

based on a study done in 2002.24 The current work was undertaken to investigate some

hematological parameters in relation to vitamin B 12 and iron status in vegetarians.

twenty‐nine vegans, 64 lacto‐ and lacto‐ovo‐vegetarians, in addition to 20 occasional

meat eaters, were enrolled for this study.24

Subjects were stratified into three groups, based on their habitual dietary pattern.

Twenty‐nine subjects reported a plant‐based diet (vegans), 64 subjects were lacto‐or

lacto‐ovo‐vegetarians, and 20 subjects had a reduced intake of meat (once weekly). 24

Total serum vitamin B12 levels were assayed by chemiluminescence immunoassay

(Bayer). Complete blood count was performed, using a hematology analyzer (Sysmex

XE‐2100TM). Based on the findings of the Eighty‐five percent of the subjects had

metabolic evidence that indicates vitamin B12 deficiency.24 The lower platelet count, and

the giant platelet form were accompanied by metabolic evidence that indicated vitamin

B12 deficiency.

Foods fortified with vitamin B12 and vitamin B12 supplements have been used to

treat deficiency of vitamin B12.

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In a randomized controlled study conducted in 2014 a study was conducted to examine

how B12 status could be improved over 6 months in a group of South Asian women of

childbearing age by using either tailored dietary advice or a daily B 12 supplement.24

Sixty-two South Asian women aged 18–50 years living in Auckland, NZ participated in

the 6-month trial. Fasting blood samples, anthropometric measurements and

estimations of dietary B12 intake were collected at baseline and at 2 and 6 months.

Participants fasted overnight for 12 hours before blood test sampling.

Participants in the Placebo and B12 Supplement groups were advised to swallow

either one cyanocobalamin or placebo capsule with a full glass of water each day for 6

months.25 Repeat bottles of capsules were delivered to participants at 2 and 4 months.

Sustainability and adherence to the treatment were assessed from the number of

capsules returned over the six-month period.24 Dietary Advice group participants

received verbal and written guidelines on how to increase or maintain consumption of

B12-containing foods in order to meet the 2.4μg minimum recommended daily intake

(RDI) for B12.24

Based on the findings, at baseline B12FFQ and serum B 12 measurements, B12

status was moderately correlated with dietary B 12 intake (r=0.5, 95% CI (0.3–0.7)) and

44% women reported insufficient dietary intake (o2.4μg/day). 24 The B12 supplement

treatment was associated with substantial increases in both serum B 12 (geometric mean

increase of 30%, 95% CI (11–48)) and holoTC (42% (12–72)) over 6 months.
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The VitB12 study findings support low-dose oral cyanocobalamin supplementation as

being effective in improving B12 status for women with insufficient dietary B12 intake.24

Vitamin B12 deficiency can happen if persons do not eat food from meat source,

eat fortified foods or take supplements.8,10,11, 19,20 Persons who are vegans are at serious

risk of developing B12 deficiency, so they require fortified foods and supplements to

meet their nutritional needs. It is possible to improve blood serum level with

supplementation.24 The aim of this proposal is to examine if a nutritional intervention

including nutrition education and an oral supplement changes low B 12 serums levels in

Seventh Day Vegans.

Significance of Study:

The aim of this study is to determine if oral supplement can improve low serum B 12

levels in Seventh Day Adventist vegans. Vegans are at risk for vitamin B 12 deficiency

because their diets do not contain meat and many of them do not take supplements.

Contribution to Field:

This research will add to the body of knowledge and will also provide information for

Seventh Day Adventist who are vegans. This will help them to plan their diet to have

adequate amounts of vitamin B12 in their diets.

Discoveries/Interventions

There are interventions that are associated with this research. Participants are expected

to attend nutrition education class, once per week for the duration of the study. This

nutrition education is to provide information about vitamin B 12.

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Participants will learn about foods which are fortified with the vitamin. They will also

learn about functions of the vitamin and importance of supplementation. The second

intervention is the use of supplements to improve B 12 serum levels.

Subject Recruitment:

The participants of the study will be male and female members of the Seventh Day

Adventist church in DeKalb County, Georgia. To qualify for the study, participants must

be between 50 and 65 years of age and must meet the following requirements: The

screening of the participants will be done through advertisement at various churches in

DeKalb County, Georgia. The participants’ eligibility will be assessed by a medical

doctor through physical examination and the use of a health questionnaire. This health

questionnaire will give information on the general health of the participants. Persons will

be excluded from the study if they have diabetes, renal insufficiencies, gastrointestinal

disorders, have had bariatric or stomach resection, take medications, smoke, drink or

take supplements. Persons will be included in the study if they meet the following

requirements:

Methods and Procedures:

Study Activities

Participants will be entered into the study if they fulfill all the requirements. The study is

expected to last for three months. Once the study is completed, data will be compiled

and analyzed. Participants will have their blood drawn a week before the study begins.

This procedure will be done by a phlebotomist. These blood samples will be sent to the

lab to test for serum B12 blood levels.

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The purpose of the labs is to find out if the participants have low levels of serum B 12.

The best indicator of vitamin B12 deficiency is a laboratory analysis which provides

accurate assessment.25 Measurement of serum vitamin B12 levels is a common and

low-cost method of assessing vitamin B12 status. 25 Serum B12 levels by themselves may

not show reserves in the participants. A serum holotranscobalamin II (TC2) test will be

conducted along with the serum levels test. TC2 is a protein that carries vitamin B 12 in

the blood, if its levels are reduced then there is a vitamin B 12 deficiency.26

Persons who are assigned to the study must attend a nutrition education class as part

of the research. The purpose of the nutrition education class is teaching the participants

about the importance of eating foods fortified with vitamin B 12 and taking supplements.

This class will also provide information about the functions of the vitamins, the effects of

its deficiencies, common symptoms, dietary needs and supplementation. Patients will

learn that it is necessary to take supplements for their duration of their lives because

they do not eat meat.

Each class will last for one hour, once per week for the duration of the study. As an

incentive for attending the class the participants will get a snack that is fortified with

vitamin B12. After completing the first two weeks of the class the participants will receive

supplements. The supplements will be administered for 10 weeks. The

supplementation for the study is 50 mcg per day. Toxicity of this vitamin is rear and

those participants who have low serums levels need to take a higher dosage to increase

their serum levels. Compliance will be checked by counting the unused capsules

remaining in the capsule dispensers and by verifying pill counts in the participants’

diaries.
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All participants will be asked to complete TrackMyStack to record their daily intake of

capsules.27

Provisions to Maintain Confidentiality of Data:

The patients’ information will be kept confidential for the duration of the study. The study

will take place at the Seventh Day Adventist conference center. The Health Insurance

Portability and Accountability Act (HIPAA) Privacy Rule will be applied to the data. 28 The

HIPAA ensures that medical records are protected such as medical history. When

working with research subjects, additional steps will be taken to ensure confidentiality.

Study codes will be used on data documents instead of recording identifying

information. All identifiable data for the participants in the study will be encrypted. All

records will be kept online using the researcher’s computer system.

Only the researcher will have access to confidential records such as medical records.

There will be no paper records associated with the study. Patients will be provided

information about the HIPAA when they enter the study. 27 Signatures of the patients are

mandatory for the researcher’s team to have access to medical information related to

the study and this is part of the informed consent.

The research team will not have access to all the participants records. They will be able

to view information names, date of birth and certain medical history. All the participants

will have an identification number instead of a name to protect their identity. Clinical

staff will make documentation based on the number assigned to each participant.
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On the completion of the study and collection of data a study identification number will

be used to represent the participants. Participants will be issued copies of the result of

the study.

Research Setting:

The study will be conducted at the Mountainside Seventh Day Adventist church in

Dekalb County, Georgia. This church is ideal because it has a health and wellness

department which offers privacy. Data will be collected from five churches in the Dekalb

County. The medical doctor and phlebotomist who are part of the study attend the

Mountainside church. All interactions and appointments concerning the participants will

be undertaken at the facility. A phlebotomy station will be temporary erected at the

church and the blood works will be safely stored and transported for testing.

Consent/Assent Process

For this research the Belmont report will be utilized, and this entails respect, justice, and

beneficence for all participants. As soon as the participants are accepted in the study

the consent process will come on stream.

All the participants will meet with the researcher and the study will be discussed at

length with them, this is to decide if the participants want to be part of the study. The

information presented to participants will be at the grade eight reading level to make

sure that they understand what the study is all about and what is required of them.
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All information regarding the research team will be available to the participants and their

contact number. When the participants meet with the research team, they can sign the

document for the study at that time. If they do not sign at that time, then they will have a

week to make their decision and sign the consent.

Risks/Benefits to Participants

The participants of this research study could benefit directly, and their B 12 serums levels

could increase. However, the possibility exists that there could be risks associate with

the study. The following are foreseeable risks that may happen with this study.

Participants B12 serum levels may not increase to expected levels after the ten weeks

interventions with supplements. They could have allergic reactions to the supplement.

To prevent harm to the patients the doctor will check each participant after one week of

taking the supplement to see if there are any allergic reactions. All participants will have

a visit with the doctor every two weeks after that. If the participants develop allergies at

any time, they will get medical attention and will stop taking the supplement. Patients

will be educated on how to identify allergies.

In the same way there are risks associated with the study there are also benefits to be

gained from it. Taking a vitamin B12 supplement can increase blood serum levels B 12

and this will decrease the risk of deficiency. The nutrition education will help the patients

to plan and prepare meals that have vitamin B 12 fortified foods. The participants will get

valuable information about the functions of vitamin B 12 in the body. This will help them to

improve their health and well-being. They may also be able to educate other vegans

about the importance of eating fortified foods or taking supplements to reduce the risk of

deficiency.
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Data Analysis:

Once the research is completed the quantitative data will then be assessed. Statistical

analysis of the research data will be performed; analyses will include standard

descriptive statistics. The significance level was chosen as 0·05. SAS version 8.02

(SAS Institute, Inc., Cary, NC, USA) will be used for all statistical analyses. Data that

will be assessed are vitamin B12 serum levels and frequency of intake of oral B 12

supplement. A blood test will be conducted before the study commences. Another blood

test will be conducted at the end of the study. Each blood test will be recorded as soon

as results are available. The purpose of the aggregated data is to explain the summary

descriptive statistics for the study.

Data will be presented as means and standard deviations or medians and ranges, as

appropriate. Categorical variables in the study will be presented in terms of frequencies

and percentage. Categorical values will be assigned to the various serum B 12 levels of

the participants. The Spearman (R) test will be used to estimate correlation between the

changes in serum vitamin B12 levels and consumption of supplements.

A logistic regression analysis will be undertaken to test for the probability of

experiencing a reduction in vitamin B12 serum levels after three months of oral intake of

B12 supplements. The study data could prove if vitamin B 12 supplementation is enough

to maintain vitamin B12 stores in vegans who have low B12 serum levels. The data will

help to ascertain if there are any relationships in the variations of serum levels and

adherence to the taking of supplements over the three months period.

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The information obtained from each subject will be anonymous and be combined so that

the findings will a group. The summarized quantitative data will include lab values and a

Track-My-Stack chart.

The Track-My-Stack chart will provide information on how often the subjects ingest the

supplement. The study data will show if supplementation is enough to increase blood

serum levels in vegans who have low B12 serum levels. The nutrition education aspect

of the study will provide information as to how much the participants took the

supplements. If the supplements were taken as directly on all the days, then the

educational aspect was successfully.

The expected results are that the prevalence of low serum level vitamin B 12 will be

higher among subjects who are vegans. Some of the participants will have very low

vitamin B12 serum levels because they have been vegan for more than ten years. These

vegans may already have been experiencing deficiency of the vitamin. The B 12

supplement treatment should cause substantial increases in serum B 12 over three

months. The findings will encourage vegans who did not take support low-dose oral

cyanocobalamin supplementation as being effective in improving B 12 status for vegans

who are tested and have low serum levels.

Discussion:

Assessing the impact of vitamin B12 supplement in vegans’ diet is very important. It will

provide more insight on the importance of supplementation to assist in maintaining

vitamin B12 stores in vegans.

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It will allow the research team to see how supplementation can help in preventing

vegans from having vitamin B12 deficiency from a diet that is free from meat.3 As noted

from the literature, persons who subscribe to vegan diets are at greater risk for getting

vitamin B12 deficiency. The deficiency is also prevalent in this group. 5,6 If vegans can

meet their B12 needs through supplementation then the deficiency can be avoided. 28

There are many potential risks for the study population. Some of the subjects may not

be able to take the supplement orally and as such their B 12 serum level might not

increase. Also, there is a risk that they may not meet their nutritional needs through

supplementation. The subjects may not take supplements as recommended or do a

daily recording of the supplements on the Track-My-Stack chart and this could be a set-

back for those subjects.

Participants must not be taking any form of supplementation before the intervention. If

any participant having extremely low lab values at the pre-assessment will be monitored

and counselled by the medical doctor.

Since there are possible risks involved with the study, the patients will be monitored

closely by clinicians during the study. This will prevent the subjects from having a lower

risk of becoming deficient. The group will be provided with supplements which is an

added benefit and decreases the burden of extra costs of purchasing supplements
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LIMITATIONS OF THE STUDY

The small sample size and the study being confined to a small geographical location

means that the result cannot be generalized for the larger population. The use of Track-

My-Stack chart is self-reported data and participants may not be truthful which can

cause bias to the research and thus affect the results. The intervention will only 12

weeks long and longer interventions may be needed to address the long-term effects on

determinants of dietary behaviors. Serum B12 levels of participants may remain the

same post intervention because of confounding factors such as failure of supplement to

work in such short length of time.

FUTURE OPPORTUNITIES

The possible results of this study can contribute to this area of research by underlining

the validity of supplementation for vitamin B 12 deficiency treatment. Other future studies

could build off these results by increasing the intervention timeline or population to

further support the alternative hypothesis. This study could be the first step for future

studies in the Greater Atlantic Conference of Seventh Day Adventist Church. These

studies could look at the importance of vegans in the church getting nutrition

intervention to increase their vitamin B12 serum levels. Other groups of vegans like the

Rastafarians could benefit from this information.

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Appendix 1.

Track my Stack. https://trackmystack.com/

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