1. VITAMIN B12 and HYPERHOMOCYSTEINEMIA
BY MONA HOSSAIN
I will be presenting my research protocol for the interaction between vitamin b12 and hyperhomocysteinemia in patient undergoing hemodialysis for endstage renal disease.

2. InTRODUCTION Key Points:
Hyperhomocysteinemia is a consequence of end stage renal disease (ESRD).
exact mechanism by which ESRD leads to hyperhomocysteinemia is unclear
inverse correlation between increased levels of protein bound homocysteine and reduced glomerular filtration rate (GFR) (Maron, B. A., & Loscalzo., 2009).
renal insufficiency resulting from renal parenchymal loss leads to extreme elevations in homocysteine levels when compared to other morbidities or conditions (Wu et al., 2012).
Patients with renal diseases often have altered metabolism and impaired excretion which can account for extreme rates of elevated plasma homocysteine (Wu et al., 2012).
Dialysis Patients have a much higher rate compared to the general population (Wu et al., 2012).
About % of hemodialysis patients have hyperhomocysteinemia (Azadibakhsh N, Hosseini RS, Atabak S, et al, 2009).
Hyperhomocysteinemia is a manifestation of ESRD but the mechanism is unclear.. There is definitely an aspect of altered metabolism, impared excretion since the liver and kidneys are the site of homocysteine metabolism.
The American Heart Association considers 5-15 μmol/L as normal so anything above that could be considered hyperhomocysteinemia.
In terms of renal insuffiency as in the case of ESRD it seems the rates of elevations homon cysteine fall in the extremes almost above the 95th percentile of normal.
Studies have shown a link between circulating proteinbound homocysteine and reduced (GFR).
In comparison with the general population, dialysis patients have a much higher rate; almost % of hemodialysis patients have it.

3. What is homocysteine? HOMOCYSTEINE Two CATABOLIC PATHWAYS:
sulfur containing non-essential amino acid intermediate of methionine
associated with oxidative stress and cellular toxicity (Maron & Loscalzo, 2009
Two CATABOLIC PATHWAYS:
REMETHYLATION: Folic acid as substrate; VITAMIN B12 as Cofactor
Transsulfuration: req vitamin b6
Primary concern with impaired metabolism: HYPERHOMOCYSTEINEMIA as an INDEPENT RISK FACTOR FOR CARDIOVASUCULAR DISEASE
atherothrombotic coronary artery disease (CAD), peripheral vascular disease, myocardial infraction (MI) and stroke) (Maron & Loscalzo., 2009
Image
In case you forgot about homocysteine, it is associated with cellular toxicity and oxidative stress.
The diagram shows the two major breakdown pathway of homocysteine– remethylation and the transsulfuration pathway where b vitamins are the key players.
Folic acid serves as the substrate for production of tetrahydrofolate (THF), a precursor to 5-methyl-THF that is required for normal methionine synthase enzyme activity where vitamin b12 serves as the cofactor which breaks down homocysteine to methionine.
When metabolism is impared it leads to cardiovascular disease and increase the chance of thrombotic diseases. CAD, MI, and Stroke are the primary concerns
Image

4. Image https://www. researchgate
I like this image a little better because it shows the nutritional implications that can affect the pathway.
For example patients at risk for achlorhydria, low intrinsic factor levels, pancreatic exocrine insufficiency, and inflammatory bowel disease history, or who have a history of gastric surgery may be at increased risk for b12 deficiency which can be a concern for your geriatric ESRD patients
Image

5. NEED FOR FURTHER RESEARCH
Epidemiologic studies
the link between elevated homocysteine as the independent risk factor for development of cardiovascular diseases (CVD)
increased levels of morbidity and mortality especially in patients with kidney failure/ESRD (Maron & Loscalzo., 2009).
randomized control studies and clinical trials
unable to establish normalization of homocysteine levels through homocysteine lowering therapy in hemodialysis patients
vitamin supplementation of folic acid, vitamin B12, alone and in combination with other B vitamins (B6) with varying and inconsistent results (Biniaz, V., Tayebi, A., Savari, S et al. 2016).
Recommendations based on varying dosages, route of administration (oral or parenteral) and duration of intervention
Epidemiological studies have linked hyperhomocysteinemia as an independent risk factor for CVD but this also presents as increased levels of morbidity in mortality in ESRD patients
Randomized control studies and clinical trials have considered using vitamin b12, folic acid and vitamin b6 as homocysteine lowering therapy but the main concern is inconsistency in reaching normal levels. Results do show a reduction in homocysteine levels but with varying degree of success in terms of the effect.
So more research is recommended based on varying dosages, using parenteral treatment versus oral and increasing the duration of treatment based on the literature I’ve reviewed.

6. Search PLAN Inclusion Criteria
PubMed’s Medline Complete (EBSCO) database
Major keywords “vitamin B12”AND “hemodialysis” AND “homocysteine”
132 results
32 articles after limiting to full text articles between the years and modifying based on criteria inclusion and exclusion criteria
27 articles were excluded
other B-vitamins apart from vitamin B12, folate
patients under the age of 18,
sample groups with only known MTHFR genotypes
other morbidities or disease states apart from hyperhomocysteinemia as a consequence of renal insufficiency
Three RCTs, one clinical trial and one review article were identified as satisfying the eligibility criteria.
Inclusion Criteria
Age: Adult patients aged 18 and over
Setting: In patient or outpatient centers
Health Status: undergoing hemodialysis treatment
Nutrition Related Problem/Condition: stable patients undergoing hemodialysis without significant comorbidities that may affect drastic changes in homocysteine levels or health
Study design Preference: Randomized Control Trials, Clinical Study Trials, Crossover Trials, review articles
Size of study groups: Atleast 10 individuals in each study group
Study dropout rate: <10%
Year range:
Authorship: Most recent research from the same author if primary research. Secondary sources from the same author will be considered.
Language: Only articles published in English
Nutrient: Vitamin b12, folic acid
Free full text
Exclusion Criteria
Age: Anyone under 18 years of age
Setting: anything other than inpatient or outpatient settings, home based settings
Health Status: anyone undergoing peritoneal dialysis treatment or not undergoing hemodialysis
Nutrition Related Problem/Condition: patients with multiple comorbidities or critically ill hospitalized unstable condition, pregnant
Study design Preference: metaanalysis, opinion pieces
Study dropout rate: >10%
Year range: before 1998
Authorship: No new research presented.
Language: Articles published in other articles other than English, doesn’t address question topics
Nutrient: any other vitamins or combinations of b vitamins
Only abstracts or free full text not available for viewing
Using the inclusion and exclusion criteria listed I searched Pubmeds Medline Complete database using keywords vitamin b12, hemodialysis and homocysteine.
I got 132 hits and it was narrowed down to 32 articles after modifying to full text articles usinf the criteria catergories.
Article titles and abstracts were scanned for relevancy of the interaction between vitamin B12, folate and levels of homocysteine in patients undergoing hemodialysis for end stage renal disease (ESRD).
27 pf them had to be excluded for reasons like if the interventions considered other vitamins and if it didn’t like the research to renal disease.
Only randomized control trials (RCT), clinical study trials, crossover trials and systematic review articles were considered for analysis
3 rcts, 1 clinical trial and 1 review article were indentified as meeting the criteria.

7. LITERATURE SUMMARY OF EVIDENCE ANALYSIS
Title: Parenteral vitamin B12 reduces hyperhomocysteinemia in end-stage renal disease.
Year: 2001
Author: Ludmila N. Kaplan, MD Orval A. Mamer, PhD L. John Hoffer, MD, PhD
Study Design: Open Phase II clinical trial
Purpose: Assess the efficacy of parenterally administered vitamin B12 on clinically stable HD pts on lowering levels of tHcy.
Population: 14 clinically stable HD pts already receiving 5mg/d FA supplementation
Intervention: three parenteral injections of 1mg Vitamin b12 given on a 4-wk interval basis
Outcomes: plasma tHcy and serum b12 on before, during and 7months following therapy
Results: tHcy values 13% reduction from baseline values p < month after intervention; increase in serum b12 levels p<0.05
Limitations: confounding variables (eg. Daily FA/ multivitamin supplements pt already receiving) not completely accounted for
Title: Efficacy of folate and vitamin B12 in lowering homocysteine concentrations in hemodialysis patients
Year: 2009
Author: Nassim Azadibakhsh, Rahebeh Shaker Hosseini, Shahnaz Atabak, Navid Nateghiyan, Banafsheh Golestan, Anahita Hooshyar Rad
Study Design: Double-blind randomized control trial
Purpose: Study the effect of low and high doses of Folic Acid (FA) with or without vitamin B12 on lowering levels of homocyteine (tHcy) in hemodialysis (HD) pts and effect of dietary intake on levels
Population: 40 HD pts previously receiving 12 hours HD/ wk with 5mg/d FA supplementation for atleast 3 months
Intervention: GROUP I: 5 mg/day FA only; GROUP II: 5 mg/day FA + 1 mg/day b12; GROUP III: 15 mg/day FA only; GROUP IV: 15 mg/day FA + 1 mg/day b12 for a period of 8 weeks
Outcomes: plasma tHcy; serum FA; serum b12, dietary intakes of energy, macronutrients and vitamins that may effect Hcy (FA and vitamins B12, B6 and B 2)
Results: decreased by Group II- 6.99%,Group II14.54% and Group IV 30.09% (p<0.05) from baseline
Limitations: * population not representative of those on HD that may not be receiving 5mg/d FA; b12 and FA interact on the same pathway so may interact with serum levels
The main outcome of all my research studies was total circulating homocysteine in comparison to baseline values as well as examining the effect of serum levels of vitamin b12 and/or folic acid on reducing total homocysteine.
The studies differed in the mode of treatment such as route of administration whether is was oral or intravenous treatment, how long the intervention was, and whether it was only vitamin b12 alone or in combination with folic acid because they are known to have the greatest effect with a synergistic combination but the research showing vitamin b12 alone is effective is low.
Some of the limitations were the confounding variables, duration of treatment or the dosage selected was not enough to establish clinically significant result.

8. LITERATURE SUMMARY OF EVIDENCE ANALYSIS
Title: Pharmacological dose of vitamin B12 is as effective as low-dose folinic acid in correcting hyperhomocysteinemia of hemodialysis patients.
Year: 2009
Author: Yi-Wen Chiu, Jer-Ming Chang, Shang-Jyh Hwang, Jer-Chia Tsai, and Hung-Chun Chen
Study Design: Prospective Randomized Control Trial
Purpose: analyze the effects of low-dose folinic acid or a pharmacologic dose of Vit B12, or a combination of both as weekly supplementation, on the plasma Hcy levels of hemodialysis (HD) patients
Population: 75 chronic HD patients undergoing HD 3x a week previously receiving 5 mg/d oral folic acid supplement
Intervention: FNA group- intravenous (IV) Lfolinic acid calcium 3 mg weekly (n = 24); • B12 group-IV hydroxocobalamin 1 mg weekly (n = 23);• combination group- both agents
Outcomes: serum levels of HCY, folic acid, vit b12
Results: Decreases in tHcy from baseline FNA- group 16.4%, Vitamin B12 group- 29.3%, and Combination Group- 38.9% all p<0.05
Limitations: baseline folic acid levels were already high; dosage of folinic acid possibly not enough to yield clinically significant decrease in HcY
Title: Vitamin B12 supplementation in end stage renal diseases: a systematic review
Year: 2015
Author: Maryam Amini, Maryam Khosravi, Hamid Reza Baradaran, and Rasha Atlasi
Study Design: Systematic Review
Purpose: Assess effects of Vitamin B12 alone on tHCy concentrations in ESRD patients
Population: Dialysis patients with ESRD to receive vitamin B12 supplementation orally or intravenously
Intervention: Vitamin B12 injected or taken orally
Outcomes: plasma tHcy
Limitations: differences in design, dosage of supplements, method of application, status of other supplements and treatments made it difficult to form a conclusion about efficacy
The 2015 systematic review by Amini et al analyzed 5 articles, concluded that the best mode of treatment is the IV route rather than the oral route, there are more efficient results with the combination of both vitamin b12 and folic acidd and the need for more RCT designed studies with sufficient sample size and on patients with normal level of folate and B12 is highly recommended to clarify the effect of B12 supplementation on tHcy concentrations

9. LITERATURE SUMMARY OF EVIDENCE ANALYSIS
Title: Effect of Vitamin B 12 supplementation on serum homocysteine in patients undergoing hemodialysis: A randomized controlled trial
Year: 2016
Author: Tayebi A, Biniaz V, Savari S, Ebadi A, Shermeh MS, Einollahi B, Rahimi A.
Study Design: Randomized Control Trial
Purpose: Study the effect of vitamin B12 supplementation on serum homocysteine levels in hemodialysis patients
Population: 140 HD patients (receiving HD ≥ 3mo, 3x weekly) aged 18 and older
Intervention: Control Group: No intervention; Experimental Group: intravenous 100 μg/ml vitamin b12 for 8 weeks
Outcomes: Incidence of hyperhomocysteinemia; serum hemoglobin and hematocrit; relationship between variables; relationship between vitamin b12 and homocysteine levels
Results: IV B12 intervention significantly lowered tHcy from baseline (p=0.006 ,p<0.05) the control group increased.
Limitations: Baseline folate levels not measured as it may affect homocysteine- didn't distinguish folate deficiency in samples; duration may not be long enough to produce clinically significant results
Tayebis Study compared the B12 IV supplementation group which received 100 μg/ml vitamin b12 for 2x a week for 8 weeks to the control which received no intervention showed a significant decrease in Homocysteine compared baseline while the control group actually increased in values.

10. RESEARCH QUESTION AND HYPOTHESIS
How effective is varying doses (1 mg and 0.5 mg) of parenterally administered hydoxocobalamin (vitamin B12) in reducing levels of homocysteine either administered alone or in combination with L folinic acid (active form of folic acid) in hemodialysis patients with end stage renal disease?
Null Hypothesis: There will be no difference in the mean serum homocysteine (tHcy) levels when comparing doses of either low or high vitamin B12 (alone or on combination with folic acid) supplementation from baseline or there is will be an increase.
Alternative hypothesis: The mean levels of serum homocysteine (tHcy) should decrease from baseline for patients undergoing hemodialysis with a high dose vitamin b12 supplementation (either alone or in combination with folic acid).
So based on the data presented my research was aimed at determining how effective 0.5 mg or 1 mg of vitamin b12 administered in the form of an IV would be in reducing levels of homocysteine in hemodialysis patients either alone or in combination with folic acid so this is a quantitative study.
The null hypothesis is that there will be no difference from baseline or there will be an increase in homocysteine levels.
The desired alternative is that the mean serum level of homocysteine will significantly decrease from baseline with high dose of vitamin b12 either alone or in combination with folic acid.
So the aims of the study are to establish the effectiveness of the route of intervention (intravenous vs. oral), the amount of dosage, and whether it is more effective in vitamin B12 supplementation alone or requires the synergistic effect with folic acid.

11. Sampling and Recruitment
Methods
Sampling and Recruitment
60 folate and vitamin B12 replete hemodialysis
three hemodialysis centers throughout New York, US
All study participants have been on maintenance HD with three 4-hr sessions per week with oral 5mg/d folic acid supplementation.
Patients without vitamin B12 deficiency will be identified by serum levels >200 pg/mL (Tayebi A, Biniaz V, Savari S, et al., 2016)
inclusion criteria
aged 18 and over
tHcy levels > 16μmol/L defined as hyperhomocysteinemia (Azadibakhsh N, Hosseini RS, Atabak S, et al., 2009).
duration of dialysis should be ≥ 3 months with 3 sessions per week totaling 12 hours (4 hours per session).
no other B-vitamin supplementations or multivitamins
Except folic acid (5mg/d) which is given as standard protocol upon hemodialysis initiation as an attempt to normalize homocysteine levels (Azadibakhsh N, Hosseini RS, Atabak S, et al., 2009).
The exclusion criteria (Azadibakhsh N, Hosseini RS, Atabak S, et al., 2006.)
under 18 years of age,
without having at least 3 months of dialysis and undergoing any maintenance hemodialysis treatment other than 12 hours a week with three 4-hour sessions
having acute exacerbations of other disease states
homocysteine interacting drugs
being severely malnourished (assessed by BMI)
having a history of graft rejection in the last 6 months or history of liver disease.
As part of the study procedure, a sample of 60 folate and vitamin b12 replete hemodialysis patients will be obtained from 3 hemodialysis centers throughout new York. They will all have been on hemodialysis for atleast for 3 months with 3 4hour sessions per week. They will be assessed for lack or vitamin b12 deficiency and presence of hyperhomocysteinemia. They shouldn’t be taking any other vitamin supplementation although a common practice in all my literature showed that the participants take 5mg of folic acid/d to attempt to normalize levels of homocysteine which is ineffective. It will exclude patients with severe malnourishment or acute exacerbations of disease or having history of graft rejection or homocysteine drugs

12. STUDY PROTOCOL STUDY DESIGN: randomized control trial
simple random sampling via a computer-generated number assignment
c0ntrol group (N=12)
four treatment groups administered intravenous (IV) solutions (N=12).
QUALITATIVE MEASURES:
Baseline characteristics -demographic questionnaire
age, sex, education, marital status employment status, smoking, etiology of CKD, duration and session of dialysis (Tayebi A, Biniaz V, Savari S, et al., 2016).
Quantitative MEasureS
Total homocysteine levels (tHCY)
serum vitamin B12
folate
The study will be a randomized control trial; participants will be assigned via a computer generated number assignment to either the control group or one of the IV treatment groups based on the dosage which will serve as the independent variable. And the main dependent variable will be the total homocysteine level along with serum vitamin b12 and folic acid as secondary outcomes because they will affect the level of homocysteine. .

13. Study Treatment Groups
STUDY PROTOCOL
Study Treatment Groups
Control Group (N=12):
Continue normal hemodialysis treatment with 5mg/d oral supplementation
Intravenous (IV) Treatment Groups (N=12):
Group I mg of hydroxocobalamin
Group II- 0.5 mg of hydroxocobalamin + 5 mg of L folinic acid calcium,
Group III- 1 mg of hydroxocobalamin,
Group IV- 1 mg hydroxocobalamin + 5 mg of L folinic acid calcium
Prior to beginning IV supplementation treatment, the participants in the assigned to the IV treatment groups will suspend their standard 5mg/d oral supplementation of folic acid as previously part of their hemodialysis regimen.
administered at biweekly intervals for 16 weeks after hemodialysis sessions.
Serum levels for tHCY, vitamin B12 and folic acid will be reassessed monthly
once again 2 months after study period following similar protocol as described by Chiu Y, Chang J, Hwang S, et al., 2009.
. Each of the study groups will have 12 participants. The control group will continue hemodialysis treatment with no IV intervention and the IV treatment groups will receive either the high or low dose of vitamin b12 with or without folic acid.
The participants in the IV group will suspend all other vitamin treatments before beginning the study
The intervention will be administered on a bi weekly basis for 16 weeks after their hemodialysis sessions.
Serum Levels will be measured monthly and again 2 months following the intervention for a duration of study period for a total of 6 months.

14. DATA COLLECTION AND ANALYSIS
LABORATORY ANALYSIS
DATA ANALYSIS
Blood samples
baseline
monthly through duration of 16-wk intervention period
two months following the study period
Collection will be performed by a trained Registered Nurse (RN) before dialysis session on collection days
levels of tHCY, vitamin b12 and folic acid status.
Samples will be preserved at −20°C until analysis after being centrifuged within 1 hour of collection.
Plasma Hcy levels
fluorescence polarization immunoassay using an IMx® Hcy kit (Solid Phase No Boil Dualcount®, Abbott Laboratories, Abbott Park, Illinois, USA)
levels of folic acid and vitamin B12
radioimmunoassay obtained from Diagnostic Products Corporation from Los Angeles, California, USA following laboratory assay (Chiu Y, Chang J, Hwang S, et al., 2009).
statistical analysis- SPSS 10.0 Software.
descriptive statistics
mean ± standard deviation for baseline characteristics
level of significance p <0.05.
Differences between and within groups
chi square
Analysis of Variance (ANOVA)
Kruskal Wallis test
Chiu Y, Chang J, Hwang S, et al., 2009
The analysis from measurements collected will answer the main research question when compared to baseline values for both the control and IV treatment groups.
It will establish which dosage was more effective (vitamin b12 alone or in combination with folic acid) and
whether the intervention groups were more successful at reducing tHcy levels compared to the control group which received no IV intervention.
The analysis will reveal how administration of the independent variable of varying dosages affected levels of these vitamins in addition to measuring whether the values significantly affects the main dependent variable tHcy.

15. LIMITATIONS AND DELIMITATIONS
large sample size
Difficult to Match in clinical parameters.
confounding variables
Sample participants selected will be vitamin replete
unlikely to be represented in ESRD patients in the larger population
Possible dropouts due to death may obscure results; downfall of long term intervention
Delimitations:
MTHFR genotype MUTATION were not considered as a parameter
negligible in dialysis patients if they are folate replete (Friedman, A. N., Bostom, A. G., Selhub, J. et al., 2001).
The current study considers a rather large sample size which may be hard to match in clinical parameters.
Additionally, confounding variable may not be completely accounted for.
Sample participants selected will be vitamin replete which is unlikely to be represented in ESRD patients in the larger population.
One concern with dropout rates were death in participants which may obscure data if occurring in rates greater than the expected limitation within study groups.
Basically my study did not consider the MTHFR genotype mutation encodes a cytosine to thymine mutation at nucleotide 677 (C677T) of N5,N10-methylenetetrahydrofolate reductase (MTHFR).
MTHFR facilitates conversion of homocysteine to methionine by methionine synthase; this mutation is linked with increased homocysteine levels by ∼25%, and may occur in up to 40% of some U.S. subpopulations but it can be considered negligible if the patients are folate replete.

16. REFERENCES
Amini, M., Khosravi, M., Baradaran, H. R., & Atlasi, R. (2015).Vitamin B12 supplementation in end stage renal disease: a systematic review. Medical Journal of the Islamic Republic of Iran, 29, 167. Retrieved November 12, 2017.
Azadibakhsh, N., Hosseini, R. S., Atabak, S., Nateghiyan, N., Golestan, B., & Rad, A. H. (2009). Efficacy of Folate and Vitamin B12 in Lowering Homocysteine Concentrations in Hemodialysis Patients. Saudi Journal of Kidney Diseases and Transplantation, 20(5), Retrieved November 12, 2017.
Biniaz, V., Tayebi, A., Savari, S., Ebadi, A., Shermeh, M., Einollahi, B., & Rahimi, A. (2016). Effect of Vitamin B12 supplementation on serum homocysteine in patients undergoing hemodialysis: A randomized controlled trial. Saudi Journal of Kidney Diseases and Transplantation, 27(2), 256. doi: /
Chiu, Y., Chang, J., Hwang, S., Tsai, J., & Chen, H. (2009). Pharmacological Dose of Vitamin B12 Is as Effective as Low-Dose Folinic Acid in Correcting Hyperhomocysteinemia of Hemodialysis Patients. Renal Failure, 31(4), doi: /
Friedman, A. N., Bostom, A. G., Selhub, J., Levey, A. S., & Rosenberg, I. S. (2001, March 24). The Kidney and Homocysteine Metabolism. Retrieved November 28, 2017, from
Kaplan, L. N., MD, Mamer, O. M., PhD, & Hoffer, L. J., MD, PhD. (2001). Parenteral Vitamin B12 reduces hyperhomocysteinemiain end-stage renal disease. Clinical Investigative Medicine, 24(1), Retrieved November 12, 2017.
Maron, B. A., & Loscalzo, J. (2009). The Treatment of Hyperhomocysteinemia. Retrieved November 28, 2017, from
NIH. (2016, February 16). Vitamin B12 Dietary Supplement Fact Sheet. Retrieved November 14, 2017, from
Wu, C. C., Zheng, C. M., Lin, Y. F., Lo, L., Liao, M. T., & Lu, K. C. (2012, November). Role of homocysteine in end-stage renal disease. Retrieved November 28, 2017, from