Effect of Vitamin B12 Supplement in Metformin Treated Diabetic Patients and it’s Correlation to Peripheral Neuropathy

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Int J Pharma Res Health Sci. 2018; 6 (2): 2394-00
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IIIIIIIII© International Journal of Pharma Research and Health Sciences. All rights reserved
DOI:10.21276/ijprhs.2018. 2018. 02.09
Ismaeel G L et al.CODEN (USA)-IJPRUR, e-ISSN: 2348-6465
Original Article
Effect of Vitamin B12 Supplement in Metformin
Treated Diabetic Patients and it’s Correlation to
Peripheral Neuropathy
Satish B Bhise1,Yogesh D. Kadam 2, Ghufran L Ismaeel 3, *
Director of LNBC Institute of pharmacy, Satara-415004, India.
Honorary Associate Professor of Medicine, B. J. Medical College, Pune-411001, India.
Research student at Sinhgad Institute of pharmaceutical science, lonavale, Pune-410401, India.
A RT IC LE IN FO A B S T R A C T
______
1. INTRODUCTION
Diabetes mellitus (DM) is a disease associated with risk of
cardiovascular diseases, which cannot be fully justified by
important risk factors such as hyperglycemia, hypertension,
and dyslipidemia 1.The incidence of diabetes in the world is
showing an increasing trend with each passing day 2.
International Journal of Pharma Research and Health Sciences
Available online at www.pharmahealthsciences.net
Received: 23 Feb 2018
Accepted: 14 Mar 2018
Diabetes is an increasingly prevalent disorder with a range of systemic complications including
diabetic peripheral neuropathy (DPN), which occurs in up to 50% of diabetic patients and causes
sensory, motor, and/or autonomic dysfunction. Metformin is considered a cornerstone in the
treatment of diabetes and is the most frequently prescribed first line therapy for individuals with
type 2 diabetes. In addition, it is one of a few antihyperglycaemic agents associated with
improvements in cardiovascular morbidity and mortality, which is a major cause of death in
patients with type 2 diabetes. Metformin does, however, induce vitamin B12 malabsorption, which
may increase the risk of developing vitamin B12 deficiency. Hence it is of interest to investigate
interrelation between metformin consumption and peripheral neuropathy in the context of vitamin
B12 administration.
The objectives of this research project are: 1. To assess the correlation between vitamin B 12 level
with nutritional status and metformin use (dose relation). 2. To find out the correlation between
vitamin B12 deficiency and development of neuropathy in diabetic patients who are taking
metformin. 3. To do an intervention in patients who’s having vitamin B 12 deficiency and analyze
improvement of neuropathy status by B12 replacement oral versus parenteral (i.m).
The results of this research show that there is a significant correlation between vitamin B12
deficiency and dose of metformin. Also there is a significant association between vitamin B12
deficiency and diet of the patient. It also shows an important connection between vitamin B 12
deficiency and status of neuropathy. It was observed that when there is severe vitamin B 12
deficiency, the symptoms of peripheral neuropathy worsen. The supplementation of vitamin B 12,
i.e. oral tablet and parenteral (i.m injection), results in a significant improvement of peripheral
neuropathy symptoms.
Keywords: Diabetes, Vitamin B12 deficiency, Metformin and Peripheral Neuropathy.
Corresponding author *
Mrs Ghufran L Ismaeel
Sinhgad Institute of pharmaceutical science, lonavale, Pune-
410401, India
Email: ghufranlutfi@gmail.com

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Type 2 diabetes mellitus (T2DM) is a chronic metabolic
disorder that is increasingly becoming a pandemic in
developed and developing world 3.
T2DM is nowadays becoming a public health concern. The
disease is associated with a variety of systemic
macrovascular and microvascular complications. Diabetic
peripheral neuropathy (DPN) is the most common
complication, and it may eventually develop in up to 50% of
patients 4, causing sensory, motor and/or autonomic
dysfunction 5.
Metformin is the first-line treatment for patients with type 2
diabetes due to its low cost and low incidence of
hypoglycemia. It has low rate of drug-drug interactions
because of renal excretion and improvement of
cardiovascular morbidity and mortality 6.
The major mechanisms of metformin action, as an insulin
sensitizer, are the following: inhibiting hepatic glucose
production, increasing peripheral tissues sensitivity (i.e.,
muscle and fat) to insulin, and thus, decreasing insulin
secretion and also reducing the absorption of blood glucose
in the intestine 7. However, metformin causes reduction in
vitamin B12 absorption leading to vitamin B12 deficiency
which is a clinically important condition.
Vitamin B12, also called cobalamin, is a water-soluble
vitamin involved in the optimal functioning of the
hemopoetic, neuro-cognitive and vascular systems. It is
involved in DNA synthesis, fatty acid metabolism and
energy production 8.
The B12-intrinsic factor complex uptake by ileal cell
membrane receptors is known to be calcium-dependent, and
metformin affects calcium-dependent membrane action(9).
The resulting B12 deficiency can be reversed by
administering calcium, and this seems to be the clearest
mechanism of action of Metformin 9.
Metformin has a good safety profile and limited side effects
although early discontinuations due to gastro-intestinal
intolerance occur in up to 20% of cases. Malabsorption of
vitamin B12 under metformin treatment has been known for
decades 10.
Vitamin B12 deficiency causes an increase in homocysteine
level. This may negatively impact patient’s health, as
elevated homocysteine levels are associated with an
increased risk of cardiovascular disease 11.
Also, Vitamin B12 plays a crucial role in the nervous system.
It is a coenzyme for methyl malonyl-CoA mutase, the action
of which is required for myelin synthesis 12. Impaired myelin
formation can lead to neuropathy, neuropsychiatric
abnormalities, myelopathy, and optic nerve atrophy 12.
Clinical evidence of vitamin B12 deficiency-related
neuropathy includes loss of vibratory sensation, diminished
proprioception, and loss of cutaneous sensation in the lower
limbs 13.
The National Health and Nutrition Examination Survey
(NHANES) data reported that oral B12 supplementation
reduced the rate of B12 deficiency by two-thirds in those
without diabetes, but there was no association seen in those
taking metformin 14.
The aim of this study is first to find out the relation between
metformin dose, vitamin B12 deficiency and severity of
peripheral neuropathy. It has also an objective to find out
whether oral and injectable vitamin B12 supplementation are
a possible methods to assess the efficacy of these
supplements in reducing the symptoms of peripheral
neuropathy.
2. MATERIALS AND METHODS
Subjects:
Total (200) type 2 diabetic patients with metformin therapy
and symptoms of neuropathy of different sex and age had
been enrolled in the study which was conducted at Poona
Diabetes centre, East Street, Pune, India. Adults aged more
than 30 years on treatment of oral metformin for at least 6
months and suffering from pain / subjective symptoms
(subjective symptoms including pain, numbness,
hyperesthesia, coldness in the extremities, muscular
weakness, dizziness, and orthostatic fainting) were included
in the study.
Exclusion criteria:
Exclusion criteria included following categories of patients:
1. Non diabetic peripheral neuropathy patients (alcoholic
neuropathy, carpal tunnel syndrome, sequelae of
cerebrovascular disease, etc).
2. Unstable glycemic control (HbA1C ≥ 10).
3. If the patients had severe hepatic or renal disorder,
history of alcoholism, ongoing pregnancy and history of
malabsorption.
4. If the patients were receiving other experimental
medications for diabetic neuropathy or any other
medication that affects symptoms of diabetic
neuropathy like tetracycline antidepressant, SSRI etc.
5. If the patients were participating in other interventional
studies.
Informed consent and ethical approval:
Ethical approval (ECR/354/INST/MH/2013/100/2013-
2014) was obtained from Institutional Ethics Committee
(IEC), Inamdar Multispeciality Hospital, Pune, India. The
participants in the study were informed about the details of
the study and written consents were obtained from each of
them.
Observational phase:
After taking the written consent of each patient, the patients
had undergone observational phase of the study. They were
screened and divided into different groups according to
different parameters.
Screening involved case history: demographic, health,
diabetic and treatment history were recorded. Serum
vitamin B12 level test for each patient was recorded so as to
check whether the patient can be included in the study and
also to keep those parameters as initial reading.

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The study was designed according to vitamin B12 level of the
patients. Patients were divided in to three groups: sufficiency
or normal vitamin B12 level (where B12 level was ≥ 400
pg/ml), insufficiency vitamin B12 level (where B12 level was
between 251-400 pg/ml) and deficiency or low vitamin B12
level (where B12 level was < 250 pg/ml).
After checking of serum vitamin B12 level of the patients,
they were compared with different parameters:
The patients were arranged according to their duration
of diabetes. Patients were divided in to: patients who
were diabetic for (≤ 60 months), patients who were
diabetic for (61 –120) months and patients who were
diabetic for (>120) months.
The patients were arranged according to their metformin
treatment. Patients were divided as per their metformin
dose in to: patients who were taking metformin (≤1000
mg/day), patients who were taking metformin between
(1000 –1500 mg/day) and patients who were taking
metformin (>1500 mg/day).
Duration of metformin was also included where patients
should be on metformin treatment for not less than six
months. Patients were divided according to their
metformin duration in to: patients who were on
metformin treatment for (≤12 months), patients who
were on metformin treatment for (13 –24) months and
patients who were on metformin treatment for (25 –36)
months.
The patients were categorized according to their diet
status. Indian individuals were divided into vegetarians
and non-vegetarians.
Mean corpuscular volume and hemoglobin level were
also included. Patients were divided into normal level
and abnormal level.
Toronto Clinical Scoring System (TCSS) and
Neuropathy Total Symptoms Score-6 questionnaire
(NTSS-6) for evaluation of the severity of peripheral
neuropathy of each patient were recorded and kept as
initial reading. These scoring systems gave information
about the patient and decision made whether he/she
neuropathic or non-neuropathic. Patients were
classified according to their neuropathy status in to
neuropathic patients and non-neuropathic patients.
Interventional phase:
After completion of the observational phase, second phase
was started (i.e. the interventional phase). The interventional
phase was including selection of patients who fit into the
inclusion criteria i.e. these patients were checked for their
serum vitamin B12 level. The patients who were B12 deficient
(serum vitamin B12 is ≤250 pg/ml) followed supplement of
vitamin B12 (methylcobalamin).
The treatment of vitamin B12 deficient patients was divided
in two types of treatment:
1. Oral treatment where the patients took oral
methylcobalamin tablet (1500 mg/day) for 180 days
and
2. Parenteral treatment in that methylcobalamin injection
(1000 umg/week I.M) for six months as follows: one
injection per week for five weeks, one injection on
alternative week for five weeks and then one injection
per month for three months.
After completion of treatment duration, the patients were
subjected for evaluation of neuropathy status through
Toronto Clinical Scoring System (TCSS) and Neuropathy
Total Symptoms Score-6 questionnaire (NTSS-6) so that the
effect of this treatment on improvement of neuropathy was
correlated.
Statistical analysis:
Data analysis was done by using SPSS (Statistical Package
for Social Sciences) version 20:0. Qualitative data variable
was expressed by using frequency and percentage (%). Chi-
sequare year/ Fisher’s exact test used to find the association
of vitamin B12 level with various risk factors. P-value <0.05
was considered as significant.
3. RESULTS AND DISSCUSION
The study results give us an idea about the correlation
between vitamin B12 level and various parameters in diabetic
patients. Observations in table 1 indicate that there is a
correlation between vitamin B12 level and duration of
diabetes mellitus. It was found that there is no significant
difference between vitamin B12 level and duration of the
disease (p-value = 0.154) i.e. diabetes itself doesn’t cause
vitamin B12 deficiency, (see table1). However other studies
have shown that vitamin B12 deficiency is more common
among patients with type 2 diabetes mellitus 15-17. One recent
study confirmed that higher vitamin B12 deficiency with
greater duration of diabetes was seen 18, which is not in
conformity with our findings.
Table 2 presents the correlation between vitamin B12 level
and metformin dose. Among 200 patients, 99 patients were
taking metformin dose of ≤1000 mg/day where 55 patients
were having normal vitamin B12 level and 24 patients were
suffering from deficiency in vitamin B12 level. At higher
dose of metformin (>1500 mg/day), out of 61 patients 19
patients only were having normal vitamin B12 level and 24
patients were suffering from deficiency in vitamin B12 level.
It confirmed that there is a significant correlation between
vitamin B12 level and dose of metformin (p-value = 0.014).
Several studies confirmed that vitamin B12 deficiency in type
2 diabetes mellitus is related to long exposure to metformin
19-21.
Table 3 shows the correlation between vitamin B12 level and
duration of metformin treatment. It was found that there is
no significant correlation between vitamin B12 level and
duration of metformin treatment (p-value = 0.768).
However, it is found that: long-term follow-up data support
the evidence that metformin is associated with vitamin B12

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deficiency, and routine measurement of vitamin B12 for
metformin-treated individuals should be considered 22.
The study results show that most of the patients were taking
metformin for less than one year i.e. 127 patients out of 200
were taking metformin for less than one year. The maximam
duration for metformin treatment presented in our study is
three years and it shows insignificant difference to cause
vitamin B12 deficiency. It means that metformin treatment
may require more than three years to cause deficiency in
vitamin B12 level. (See table 3)
Correlation between vitamin B12 level and diet of the patients
is presented in table 4. It was found that there is a significant
difference between vegetarian and non-vegetarian patients.
(p-value = 0.001). Among 56 vegetarian patients, there were
13 patients having deficiency in vitamin B12 level and out of
144 non-vegetarian patients there were 46 patients having
deficiency in vitamin B12 level. Thus our observation
indicates that non-vegetarian diabetic patients are more
prone for vitamin B12 deficiency. Probably processing of
non-vegetarian food might be destroying vitamin B12
content.
Some recent studies indicate that deficiency of vitamin B12
was more common among vegetarian population on
prolonged metformin therapy than non-vegetarian
population 23, 24, which is not in conformity with our
findings.
Table 5 presents the correlation between vitamin B12 level
and MCV (Mean corpuscular volume). It was found that
there is no significant correlation between vitamin B12 level
and MCV (p-value = 0.823).
The earlier studies show that there was no correlation
between vitamin B12 levels and MCV 25, 26, which is in
conformity with our findings.
Correlation between vitamin B12 level and hemoglobin level
showed that there is no significant difference between
vitamin B12 level and hemoglobin level (p-value = 0.288).
These results are probably raised because 128 patients out of
200 patients were having normal hemoglobin level. (See
table 6).
A recent review presented that increased incidence of
anaemia among patients treated using metformin was
noticed. However, vitamin B12 deficiency was also observed
with lack of anemia 27, which is in conformity with our
findings.
Table 7 shows the correlation between the scores of Toronto
Clinical Scoring System (TCSS) at baseline i.e. initially
before starting supplementation of vitamin B12 and after
following up of patients for evaluation of the severity of
neuropathy. Our results indicate that there is a significant
difference (p-value <0.05) between the severity symptoms of
neuropathy. Scoring showed that the result at baseline was
(mean ± SD) 8.64+1.13 while after follow up and
completion of six months of vitamin B12 treatment was
2.53+1.50. It indicates that vitamin B12 supplement reduces
the severity of peripheral neuropathy in diabetic patients.
An earlier research paper presented that the patients with
type 2 DM on metformin therapy had lower serum vitamin
B12 levels and a greater incidence of neuropathy by Toronto
clinical scoring system as compared to non-metformin group
28.
Table 8 shows results about the correlation between the
scores of Neuropathy Total Symptoms Score-6 questionnaire
(NTSS-6) at baseline i.e. initially before starting
supplementation of vitamin B12 and after following up of
patients for evaluation of the severity of neuropathy. Our
results indicate that there is a significant difference (p-value
<0.05) between the severity symptoms of neuropathy.
Scoring showed that the result at baseline was (mean ± SD)
8.88+1.75 while after follow up and completion of six
months of vitamin B12 treatment was 3.08+2.39. It indicates
that vitamin B12 supplement reduces the severity of
peripheral neuropathy in diabetic patients. (See figure 2).
One study confirmed that a statistically significant difference
in clinical neuropathy scoring systems between the groups,
with the metformin-exposed group having higher scores,
indicating clinically more severe peripheral neuropathy 29.
NTSS-6 scores are also compared between capsule and
injection for supplementation of vitamin B12. The results
presented that there is no significant difference (p-value =
0.988) between the two scores. It indicates that both capsule
and injection of vitamin B12 supplement are responsible for
effective reducing of symptoms of peripheral neuropathy.
(See figure 3).
There are sublingual tablets and oral spray of vitamin B12
supplement which need to be investigated and may have
better efficacy than oral tablet and injection as their
destination are away from metformin pathway.
Table 1: Vitamin B12 count in correlation to duration of diabetes
mellitus
Duration of
DM
Vitamin B12 count
Total
p-value
≤ 250
251 –400
> 400
≤ 60 month
23
12
38
73
0.154
61- 120 month
22
13
23
58
> 120 month
14
18
37
69
Total
59
43
98
200
Table 2: Vitamin B12 count in correlation to metformin dose
Metformin
dose
Vitamin B12 count
Total
p-value
≤ 250
251 –400
> 400
≤ 1000
24
20
55
99
0.014
1000 –1500
11
5
24
40
> 1500
24
18
19
61
Total
59
43
98
200
Table 3: Vitamin B12 count in correlation to the duration of metformin
Duration of
Metformin
Vit B12_group
Total
p-value
≤ 250
251 –400
> 400
≤ 12 month
35
32
60
127
0.768
13 - 24 month
12
5
16
33
25 - 36 month
5
2
7
14
> 36 month
7
4
15
26
Total
59
43
98
200

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Fig 1: Flow chart for the proposal work
Table 4: Vitamin B12 count in correlation to diet of patient
Diet
Vitamin B12 count
Total
p-value
≤ 250
251 –400
> 400
Vegetarian
13
4
39
56
< 0.001
Non Vegetarian
46
39
59
144
Total
59
43
98
200
Table 5: Vitamin B12 count in correlation to MCV (Mean corpuscular
volume)
MCV group
Vit B12_group
Total
p-value
≤ 250
251 - 400
> 400
Normal
39
29
61
129
0.823
Abnormal
20
14
37
71
Total
59
43
98
200
Table 6: Vitamin B12 count in correlation to hemoglobin level
Hemoglobin group
Vit B12_group
Total
p-value
≤ 250
251 - 400
> 400
Normal
36
32
60
128
0.288
Abnormal
23
11
38
72
Total
59
43
98
200
Table 7: Correlation between TCSS scores at baseline and after follow
up
Variables
TCSS Baseline (n=58)
TCSS At
follow up (n=58)
Min
6
1
Max
11
6
Mean
8.64
2.53
SD
1.13
1.50
Median
9
2
Table 8: Correlation between NTSS-6 scores at baseline and after
follow up
NTSS 6 score
At Baseline (n=29)
At Follow up (n=29)
p-value
Mean
8.88
3.08
< 0.001
SD
1.75
2.39
Median
8.99
3.33
Table 9: Correlation of NTSS-6 scores in capsule and injection
treatment
NTSS Score
Capsule (n=16)
Injection (n=13)
p-value
Mean
3.06
3.10
0.966
SD
2.24
2.66
Fig 2: Mean of NTSS-6 score at baseline and after follow up

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Fig 3: Mean of NTSS-6 score in capsule and injection treatment
4. CONCLUSION
This study indicates that diabetic patients on metformin
therapy are prone for vitamin B12 deficiency. Probably
vitamin B12 deficiency is related to metformin and not to the
disease itself.
Vitamin B12 deficiency can cause elevation of symptoms of
peripheral neuropathy, this study shows that
supplementation of either tablet or injection of cobalamin is
an effective method to reduce peripheral neuropathy
symptoms in diabetic patients on metformin therapy.
Thus our study indicates that metformin induced vitamin B12
deficiency and peripheral neuropathy can be corrected by
oral / parenteral of cobalamin (vitamin B12).
5. ACKNOWLEDGEMENT
The authors would like to acknowledge Principal of Sinhgad
institute of pharmaceutical sciences and staff members for
their help and valuable instruction to the study. Also we
would like to acknowledge Inamdar multispecialty hospital,
pune and Poona diabetes centre for providing facilities to
conduct the work. Finally, thanks to all the participants of
the study.
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Conflict of Interest: None
Source of Funding: Nil