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R E S E A R C H A R T I C L E Open Access
Vitamin B12 deficiency in metformin-
treated type-2 diabetes patients,
prevalence and association with peripheral
neuropathy
Marwan A. Ahmed
1*
, George Muntingh
1
and Paul Rheeder
2
Abstract
Background: The association between long-term metformin use and low vitamin B12 levels has been proven.
However, the prevalence estimates of metformin-induced vitamin B12 deficiency showed considerable variation
among the studies. The potential of the deficiency to cause or worsen peripheral neuropathy in type-2 diabetes
mellitus (T2DM) patients has been investigated with conflicting results. The aim of the study was to investigate:
1) the prevalence of vitamin B12 deficiency in T2DM patients on metformin; 2) the association between vitamin B12
and peripheral neuropathy; 3) and the risk factors for vitamin B12 deficiency in these patients.
Methods: In this cross-sectional study, consecutive metformin-treated T2DM patients attending diabetes clinics of
two public hospitals in South Africa were approached for participation. Participation included measuring vitamin
B12 levels and assessing peripheral neuropathy using Neuropathy Total Symptom Score-6 (NTSS-6) questionnaire.
The prevalence of vitamin B12 deficiency (defined by concentrations <150 pmol/L) was determined. Those with
NTSS-6 scores >6 were considered to have peripheral neuropathy. The relationship between vitamin B12 and
peripheral neuropathy was investigated when the two variables were in the binary and continuous forms. Multiple
logistic regression was used to determine risk factors for vitamin B12 deficiency.
Results: Among 121 participants, the prevalence of vitamin B12 deficiency was 28.1 %. There was no difference in
presence of neuropathy between those with normal and deficient vitamin levels (36.8 % vs. 32.3 %, P=0.209).
Vitamin B12 levels and NTSS-6 scores were not correlated (Spearman’s rho =0.056, P=0.54). HbA1c (mmol/mol)
(OR = 0.97, 95 % CI: 0.95 to 0.99, P=0.003) and black race (OR = 0.34, 95 % CI: 0.13 to 0.92, P=0.033) were risk
factors significantly associated with vitamin B12 deficiency. Metformin daily dose (gram) showed borderline
significance (OR = 1.96, 95 % CI: 0.99 to 3.88, P=0.053).
Conclusions: Close to third of metformin-treated T2DM patients had vitamin B12 deficiency. The deficiency was
not associated with peripheral neuropathy. Black race was a protective factor for vitamin B12 deficiency.
Keywords: Diabetes, Metformin, Peripheral Neuropathy, Vitamin B12
* Correspondence: mrwnwd@yahoo.com
1
Department of Pharmacology, Faculty of Health Sciences, University of
Pretoria, Pretoria, South Africa
Full list of author information is available at the end of the article
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Ahmed et al. BMC Pharmacology and Toxicology (2016) 17:44
DOI 10.1186/s40360-016-0088-3
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
Metformin is the cornerstone medication in the manage-
ment of type 2 diabetes mellitus (T2DM) with estimates
that it is routinely prescribed to 120 million patients with
diabetes around the world [1]. Accumulating evidence
from both observational and interventional studies has re-
vealed the association between long-term use of metfor-
min and vitamin B12 deficiency. It may be surprising
knowing that the first article describing metformin-
associated vitamin B12 malabsorption was published in
1971 [2]. While there is almost a current consensus on the
medication’s potential to lower vitamin B12 levels, four
decades have not been sufficient to clarify other significant
aspects of the topic.
Despite the confirmed association between metformin
and vitamin B12 deficiency, the real size of the problem
is not yet properly quantified. Previous studies have
shown that the prevalence of metformin-induced vita-
min B12 deficiency varied greatly and ranged between
5.8 % and 52 % [3–11]. Such a wide range may be at-
tributed to differences in cut-points chosen to define the
deficiency, participants mean age, study settings, and
metformin dose and duration of use.
Neuropathy may be the only clinical presentation of
vitamin B12 deficiency, without haematological symp-
toms and signs [12]. The clinical picture of vitamin B12
deficiency has revealed salient alterations towards the
predominance of neurological symptoms and signs over
the last few decades [13]. Clinically, vitamin B12 defi-
ciency peripheral neuropathy is indistinguishable from
that of T2DM [14]. Vitamin B12 deficiency-associated
peripheral neuropathy can also remain subclinical [13]
and, possibly, interact with that of T2DM. Such neur-
opathy is thus likely to be misdiagnosed as diabetic
neuropathy. The long-term use of metformin, mediated
by vitamin B12 deficiency, may contribute to increasing
the substantial burden of peripheral neuropathy in
T2DM patients. Several studies have recently tried to
clarify the possible link between long-term metformin
use and its vitamin B12 deficiency-mediated peripheral
neuropathy with conflicting results [7, 8, 14–16]. Research
around the topic is usually faced by many challenges. Vita-
min B12 minimal concentration requirements for proper
neuronal functioning are not yet established [13]. Investi-
gators are also challenged by the fact that neuropathy is a
common complication of T2DM and its attribution to
metformin-induced low vitamin B12 status can never be
certain. Furthermore, it seems difficult to tackle the ques-
tion through randomised controlled trials as the required
study duration, sample size and ethical issues make the
use of such designs impractical. All the currently available
evidence comes from observational studies.
As the first study to explore the topic in African popu-
lations, the aims of our study were to determine the
prevalence of vitamin B12 deficiency in metformin-
treated T2DM patients, to investigate the relationship
between vitamin B12 and peripheral neuropathy and to
identify the risk factors for the vitamin deficiency in
these patients.
Methods
Study design, patient recruitment and selection
This observational cross-sectional study was conducted
at the diabetes clinics of departments of Internal
Medicine at Steve Biko Academic Hospital and Kalafong
Hospital in Pretoria, South Africa between August 2012
and January 2013. A consecutive sampling method was
used. A provisional list of patients eligible to participate
in the study was made from the records. On clinic days,
all eligible patients were approached and asked to par-
ticipate in the study. Those who accepted to participate
signed the informed consent after reading the patient in-
formation leaflet.
Patients who met the following criteria were eligible to
participate: willingness to participate in the study by
signing the informed consent, diagnosed T2DM, use of
metformin for six months or more and ability to read
and write in English. Patients were excluded from the
study if one or more of the following was present:
pernicious anaemia, alcoholism, gastrectomy, gastric by-
pass surgery, pancreatic insufficiency, malabsorption
syndromes, chronic giardiasis, surgery involving small
intestine or HIV infection. Intake of vitamin B12 or any
multivitamin preparation during the past 6 weeks, esti-
mated Glomerular Filtration Rate (e-GFR) < 50 mL/min/
1.73 m
2
based on Cockcroft-Gault formula or diagnosed
vitamin B12 deficiency were also exclusion criteria.
Variables and data collection
The following information was obtained from partici-
pants’records: age, sex, ethnic group, date of diagnosis
of T2DM, most recent HbA1c (glycated hemoglobin)
level, duration of metformin use, dosage of metformin,
smoking status, eGFR, Body-Mass Index (BMI), use of
gastric acid suppressants or low-dose acetylsalicylic acid
(both were linked to vitamin B12 deficiency [17, 18]),
coffee consumption, presence and history of other med-
ical conditions and alcohol consumption or any sub-
stance abuse. Participants were considered alcohol users
when they consumed one drink or more per day. Coffee
consumption was defined as daily consumption of one
cup or more.
Serum vitamin B12 was measured in Ampath Labora-
tories by UniCel DxI 800 instrument (Beckman Coulter,
United States). Vitamin B12 deficiency was defined as
levels <150 pmol/L [3, 5, 6, 8].
Neuropathy Total Symptom Score-6 (NTSS-6) ques-
tionnaire was used to grade peripheral neuropathy. The
Ahmed et al. BMC Pharmacology and Toxicology (2016) 17:44 Page 2 of 10
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investigator asked each participant about frequency and
intensity of six peripheral neuropathic symptoms, and
used the answers to complete the questionnaire. The
sum scores for each patient were obtained (possible
range between 0.00 and 21.96). Grading the frequency
and intensity of symptoms was based on the definitions
of Bastyr et al. [19].
To reduce occurrence of bias, scoring of peripheral
neuropathy was performed by the same investigator
prior to accessing any sort of data that gives information
on metformin dose and/or duration of use. Serum sam-
ples were sent for analysis after completing data extrac-
tion and peripheral neuropathy scoring.
Statistical analysis
Sample size calculations showed that recruiting 120 par-
ticipants would give a 7 % margin of error (95 % CI)
around an estimated 20 % prevalence. STATA version 12
was used for statistical analysis. Two-sided P values
≤0.05 were considered statistically significant. Means
(± standard deviations), actual numbers and percent-
ages were used to describe data.
The prevalence of vitamin B12 deficiency was deter-
mined as the percentage of participants with vitamin
levels <150 pmol/L. Relationship between vitamin B12
and peripheral neuropathy was examined by two
methods. The first utilized chi-square test to investigate
the relationship between the two binary variables created
from the continuous variables of vitamin B12 (deficient
for levels < 150 pmol/L, otherwise normal) and periph-
eral neuropathy (present for NTSS-6 scores > 6, other-
wise absent). The second method used Spearman’s rank
correlation coefficient (rho) to examine bivariate rela-
tionship between the two continuous variables of vita-
min B12 levels and NTSS-6 scores.
Demographic and clinical characteristics of vitamin
B12-deficient participants were compared to those with
normal vitamin levels. Normality of continuous variables
was examined by Shapiro-Wilk test. Unpaired t-test (for
normally distributed data) and Mann–Whitney test (for
non-normally distributed data) were used to compare
the continuous variables. Chi-square test and Fisher’s
exact test (when expected cell frequency was less than
five) were used to compare categorical variables.
Multiple logistic regression was performed where vita-
min B12 status represented the binary dependent vari-
able and all the variables with P values ≤0.25 in the
univariate analysis were taken as independent variables.
The variables metformin duration, metformin cumula-
tive dose (product of total daily dose and duration of
use) and T2DM duration were likely to be highly corre-
lated. Three initial models, each contained one of these
variables, were made to avoid the impact of the multi-
collinearity. Backwards stepwise manner was then used
to reduce initial models to a final model, from which
risk factors significantly associated with vitamin B12 de-
ficiency were determined.
Results
A total of 130 eligible patients were identified. Four
patients did not agree to participate and 126 signed the
informed consent. Of those who signed, 5 patients with
eGFR values less than 50 mL/min/1.73 m
2
were ex-
cluded. The final analysis was performed based on data
obtained from 121 participants.
Table 1 shows participants’demographic and clinical
characteristics. Women represented 66 % of participants.
The sample consisted of 89 (73.5 %) blacks, 19 (15.7 %)
whites, 10 (8.3 %) Indians and 3 (2.5 %) coloured. Indian,
coloured and white ethnic groups were all grouped to-
gether as non-black and represented 26.5 % of the sam-
ple. The sample mean age was 58.5 years and mean
Table 1 Participants’demographic and clinical characteristics
(n=121)
Characteristics Values
Age (years) 58.5 ± 10.5
T2DM duration (years) 11.6 ± 7.5
Duration of metformin use (years) 9.6 ± 6.8
Total daily dose of metformin (gram) 2.4 ± 0.7
Cumulative dose of metformin (gram) 23.7 ± 18.2
eGFR (mL/min/1.73 m
2
) 116.7 ± 44.6
Women, n(%) 80(66)
HbA1c (mmol/mol) [%] 76 ± 27 [9.1 ± 2.5]
Smokers, n(%) 8(6.6)
Insulin use, n(%) 96(79.3)
Alcohol consumption, n(%) 4(3.3)
Coffee consumption, n(%) 23(19)
Race
Black, n(%) 89(73.5)
Non-black, n(%) 32(26.5)
Number of daily metformin doses
One, n(%) 3(2.5)
Two, n(%) 61(51.2)
Three, n(%) 55(46.2)
Acetylsalicylic acid use, n(%) 94(77.7)
PPIs or H2RAs use, n(%) 12(10)
BMI (kg/m
2
) 33.4 ± 6.3
Vitamin B12 levels (pmol/L) 260.6 ± 163.7
NTSS-6 Scores 5.11 ± 3.86
The data are shown as the means ± SD or n (%)
BMI Body-Mass Index, eGFR estimated glomerular filtration rate based on
Cockcroft-Gault formula, PPI Proton Pump Inhibitor, H2RA Histamine 2
Receptor Antagonist, HbA1c Glycated haemoglobin, NTSS-6 Neuropathy Total
Symptom Score-6
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T2DM duration was 11.6 years. The means of metfor-
min duration of use and total daily dose were 9.6 years
and 2.4 g, respectively. The mean cumulative dose of
metformin was 23.7 g. HbA1c, eGFR and BMI had mean
values of 76 mmol/mol (9.1 %), 116.7 mL/min/1.73 m
2
and 33.4 kg/ m
2
, respectively. Ninety six (79.3 %) partici-
pants were on insulin. Twelve (10 %) participants were
on a PPI or H2RA while 92 (77.7 %) were on low-dose
acetylsalicylic acid. The mean of serum vitamin B12 con-
centrations was 260.6 pmol/L and that of NTSS-6 scores
was 5.11.
Thirty four participants, representing 28.1 % of the
sample, were vitamin B12-deficient.
Table 2 shows that 32.3 % of vitamin B12 deficient
participants had neuropathy compared to 36.8 % of
those with normal vitamin levels. Chi square test results
showed a Chi square statistic value of 0.209 with an as-
sociated probability of 0.647, indicating absence of
enough evidence to claim an association between vita-
min B12 status and neuropathy binary variables in the
population. The value of Spearman’s rank correlation co-
efficient (rho) was 0.056 with a P value of 0.54, indicat-
ing that there was no sufficient evidence of association
between vitamin B12 levels and NTSS-6 scores (Fig. 1).
Comparable results of no association were also obtained
when the correlation between vitamin B12 levels and
NTSS-6 scores was examined in those with deficient
(rho = 0.284, P=0.10) (Fig. 2) and normal (rho = 0.057,
P=0.59) (Fig. 3) vitamin B12.
Univariate analysis (Table 3) revealed that vitamin
B12-deficient participants were significantly older than
those with normal vitamin levels (62.3 vs. 57 years, P=
0.012). They also had significantly longer metformin use
duration (11 vs. 8 years, P=0.015) and higher cumula-
tive metformin dose (28.9 vs. 17 g, P=0.009). Those
with deficiency also revealed statistically significant
lower HbA1c (57[7.4 %] vs. 79[9.4 %] mmol/mol, P=
0.001). Duration of T2DM was higher in vitamin-
deficient participants with a P value that almost reached
the statistical significance (12 vs. 9 years, P=0.055).
Table 4 shows ORs, 95 % CIs and P values for vitamin
B12 deficiency risk factors in the three initial models (A,
B and C). The three models ultimately resulted in the
same final reduced model (model D, Table 5). The final
model has shown that the odds of having vitamin B12
deficiency was significantly reduced by being from black
South African descent (OR = 0.34,95 % CI: 0.13 to 0.92,
P=0.033) and by increase in HbA1c value (mmol/mol)
(OR = 0.97, 95 % CI: 0.95 to 0.99, P=0.003). The model
has also shown that metformin total daily dose (gram)
increased the odds of having vitamin B12 deficiency with
an approximately significant P value (OR = 1.96, 95 %
CI: 0.99 to 3.88, P=0.053).
Discussion
Prevalence of vitamin B12 deficiency
The present study has found that the prevalence of vita-
min B12 deficiency, as defined by serum levels <150
pmol/L, among T2DM patients receiving metformin was
28.1 %. Comparing the obtained prevalence with the re-
sults of previous studies is not straightforward and
should consider several factors. Table 6 shows the preva-
lence estimates and certain characteristics of the studies
that used deficiency cutoff points ranging between 145
and 150 pmol/L. The table reveals study-related factors
with potential to affect the obtained prevalence, includ-
ing mean participants age, mean metformin daily dose,
study settings, mean metformin duration of use and
whether participants with renal impairment were ex-
cluded. Our reported prevalence is high relative to previ-
ously reported estimates. It is exactly the same as that of
Beulens et al. study. Mean metformin dose and duration
were higher in our study compared to Beulen et al’s.
Their sample was, however, older. Such non-directional
differences in variables make comparative speculations
on factors behind the high prevalence of the vitamin de-
ficiency in our sample an unattainable task. The present
study had the highest means of metformin dose and dur-
ation of use, possibly explaining the high prevalence
obtained.
Relationship between vitamin B12 and peripheral
neuropathy
The present study found no statistically significant
difference in presence of neuropathy between those
with normal and deficient vitamin levels (36.8 % vs.
32.3 %, P=0.209). The levels of vitamin B12 and the
NTSS-6 scores were not correlated (Spearman’srho=
0.056, P=0.54). Our results are in-line with those of
a recently published study which reported that met-
formin use was not associated with the presence of
diabetic peripheral neuropathy in T2DM patients [20].
Our findings are also in concordance with the results
of the cross-sectional study of Chen et al. which re-
vealed no significant differences between metformin
users and non-users when neuropathy status was
assessed by both objective (monofilament and neu-
rothesiometry) and relatively subjective (question-
naires) measures [14]. Biemans et al. also reported no
Table 2 Cross-tabulation of vitamin B12 status and peripheral
neuropathy
Peripheral Neuropathy
Vitamin B12 status Absent Present Total
Normal 55(63.2 %) 32(36.8 %) 87(100.00 %)
Deficient 23(67.7 %) 11(32.3 %) 34(100.00 %)
Total 78(64.46 %) 43(35.54 %) 121(100.00 %)
Ahmed et al. BMC Pharmacology and Toxicology (2016) 17:44 Page 4 of 10
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differences in occurrence of neuropathy in vitamin
B12-deficient and –normal T2DM patients receiving
metformin [15]. Our results are in contrast with the
case–control study of Wile and Toth who reported
more severe neuropathy among T2DM patients on
metformin compared to non-metformin group [16].
Their results were obtained when the neuropathy
severity was assessed by Toronto Clinical Scoring
System (TCSS) and Neuropathy Impairment Score.
The more objective electrophysiological tests showed
no statistically significant differences among the two
groups. Singh et al. also reported more severe
neuropathy as assessed by TCSS in metformin-treated
T2DM patients [7]. Our results also contrasts with
the data of the cross-sectional study of de Groot-
Kamphuis et al. who reported lower prevalence of
neuropathy in metformin-treated T2DM patients
compared to the non-metformin group [8].
Many animal studies have recently reported the gly-
cemic control-independent neuroprotective impact of
metformin. Mao-Ying et al. found that metformin re-
duced peripheral nerve endings loss and exerted protect-
ive effect against chemotherapy-induced peripheral
neuropathy (CIPN) in mice [21]. Both peripheral
Fig. 1 Scatter plot for vitamin B12 levels vs. NTSS-6 scores among all metformin-treated T2DM patients
Fig. 2 Scatter plot for vitamin B12 levels vs. NTSS-6 scores among metformin-treated T2DM patients with vitamin B12 deficiency
Ahmed et al. BMC Pharmacology and Toxicology (2016) 17:44 Page 5 of 10
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Fig. 3 Scatter plot for vitamin B12 levels vs. NTSS-6 scores among metformin-treated T2DM patients with normal vitamin B12 levels
Table 3 The characteristics of vitamin B12-deficient participants compared to those with normal vitamin B12 levels
Variable Low vitamin B12 (n=34) Normal vitamin B12 (n=87) P value
Age (years) 62.3 ± 10.2 57.0 ± 10.2 0.012
T2DM duration (years) 12(8.75/17) 9(5/16) 0.055
Duration of metformin use (years) 11(6.75/13.25) 8(3/13) 0.015
Total daily dose of metformin (gram) 2.6 ± 0.7 2.4 ± 0.7 0.228
Cumulative dose of metformin (gram) 28.9(14.5/40.8) 17(7.7/31.3) 0.009
eGFR (mL/min/1.73 m
2
) 100.4(78.6/129) 108.5(88/150.7) 0.093
Sex
Women, n(%)
Men, n(%)
21(61.8)
13(38.2)
59(67.8)
28(32.2)
0.530
HbA1c (mmol/mol)
HbA1c (%)
57(45/81)
7.4(6.3/9.6)
79(58/99)
9.4(7.5/11.2)
0.001
Insulin use, yes(%) 28(82.4) 68(78.2) 0.451
Acetylsalicylic acid use, yes(%) 30(88.2) 64(73.5) 0.081
Coffee use, yes(%) 9(26.4) 14(16) 0.191
Race
Black, n(%)
Non-black, n(%)
22(64.7)
12(35.3)
67(75.3)
20(24.7)
0.168
BMI (kg/m
2
) 34.0 ± 6.5 33.1 ± 6.3 0.469
Number of daily doses
One, n(%)
Two, n(%)
Three, n(%)
0(0)
21(63.6)
12(36.6)
3(3.5)
40(46.5)
43(50)
0.198
Use of PPI or H2RA, yes(%) 5(14.7) 7(8) 0.271
NTSS scores 4.16(2/7.25) 4.33(2/8.33) 0.914
Data are expressed as mean ± standard deviation, median (25/75 percentile) or n (%)
Ahmed et al. BMC Pharmacology and Toxicology (2016) 17:44 Page 6 of 10
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neuropathy and CIPN share the glove-and-stocking dis-
tribution nature of sensory symptoms that includes para-
sthesia, dysthesia and pain [21]. Animal studies have
also recently shown that metformin reversed induced
neuropathic pain and protected against nerve injury
[22], protected against neuronal apoptosis induced by
ethanol [23], inhibited neuronal apoptosis in cortical
cells [24], stimulated neurogenesis [25], and promoted
neurogenesis following middle cerebral artery occlusion
in mice [26]. Considering such impact of metformin,
there can be two possible lines through which the medi-
cation affects the neuropathy status, excluding that re-
lated to glycemic control. One involves a positive impact
through neuroprotective mechanisms, while the other
induces neuropathy by enhancing vitamin B12 defi-
ciency. Absence of association between vitamin B12 and
peripheral neuropathy in our study may not thus totally
preclude the potential of the medication to precipitate
or worsen neuropathy through vitamin B12 deficiency.
This theory may also explain the contradictory nature of
results obtained by different studies.
Judicious interpretations of evidence around peripheral
neuropathy as a clinical consequence of metformin-
induced vitamin B12 deficiency may require considering
certain medication-related features with possible signifi-
cant methodological impacts. Being the cornerstone of
the management of T2DM, it should be uncommon to
encounter T2DM patients who are not on metformin. In
observational studies that compare metformin users and
non-users, T2DM patients who are not on metformin
may hence be inherently different from those taking the
medication. Being not on metformin is itself an abnor-
mality with potential to make obtaining similar study
groups a strenuous task. Having a control group may,
therefore, negatively influence the validity in studies
aiming at investigating the impact of metformin-induced
vitamin B12 deficiency on neuropathy. The theory of the
possible neuroprotective impact of metformin commen-
ted above may also lead to the same methodological
conclusion. The possibility exists that neuroprotective
effect of the medication group dilutes or predominates
over its neuropathy-generating impact which is mediated
by vitamin B12 deficiency. Comparing peripheral neur-
opathy in the control and metformin groups may thus
produce distorted results that do not truly reflect the
contribution of the metformin-induced vitamin B12 defi-
ciency to the neuropathy status. From this perspective,
designs that compare peripheral neuropathy among vita-
min B12-deficient and –normal metformin-treated pa-
tients can theoretically result in more valid findings.
Risk factors for vitamin B12 deficiency
Low HbA1c was a significant risk factor for vitamin B12
deficiency in the final (Table 5) (OR = 0.97, 95 % CI: 0.95
to 0.99, P=0.003) as well as the three initial models
(Table 4). Kang et al. reported, but did not explain, simi-
lar results [10]. They reported stronger association be-
tween HbA1c and vitamin B12 deficiency (OR = 0.74, CI:
0.56 to 0.99). Their study included T2DM patients who
were either on metformin plus insulin or metformin plus
sulfonylurea. Such stricter inclusion criterion may ex-
plain the difference in magnitude of OR between the
Table 4 The initial multiple logistic regression models assessing independent predictors of vitamin B12 deficiency in metformin-treated
T2DM participants
Independent variable Model A Model B Model C
OR (95 % CIs) P value OR (95 % CIs) P value OR (95 % CIs) P value
Metformin duration (years) 1.03 (0.96 to 1.10) 0.481 - - - -
Cumulative metformin dose (g) - - 1.01 (0.98 to 1.04) 0.531 - -
T2DM duration (years) - - - - 1.03 (0.97 to 1.10) 0.374
Total daily dose of metformin (g) 1.82 (0.87 to 3.80) 0.111 1.66 (0.71to 3.85) 0.239 1.82 (0.88 to 3.78) 0.107
Age (years) 1.03 (0.96 to 1.10) 0.416 1.03 (0.96 to 1.10) 0.423 1.03 (0.96 to 1.10) 0.429
HbA1c (mmol/mol) 0.98 (0.96 to 0.99) 0.034 0.97 (0.95 to 0.99) 0.036 0.98 (0.95 to 0.99) 0.034
Coffee Consumption (if yes) 1.82 (0.57 to 5.80) 0.310 1.81 (0.57 to 5.74) 0.315 1.86 (0.58 to 5.96) 0.294
Race (if black) 0.30 (0.10 to 0.88) 0.029 0.29 (0.10 to 0.87) 0.028 0.30 (0.10 to 0.89) 0.031
Acetylsalicylic acid use 2.64 (0.73 to 9.58) 0.140 2.63 (0.73 to 9.51) 0.141 2.61 (0.72 to 9.47) 0.144
Number of metformin daily doses 0.84 (0.33 to 2.11) 0.705 0.84 (0.33 to 2.12) 0.707 0.82 (0.32 to 2.06) 0.669
eGFR (mL/min/1.73 m
2
) 0.99 (0.98 to 1.01) 0.703 0.99 (0.98 to 1.01) 0.692 0.99 (0.98 to 1.01) 0.759
Table 5 The reduced multiple logistic regression model for risk
factors of vitamin B12 deficiency in metformin-treated T2DM
patients (Model D). OR > 1 indicates greater risk for vitamin B12
deficiency
Independent variable Odds ratio (95 % CIs) Pvalue
Total daily dose of metformin (gram) 1.96 (0.99 to 3.88) 0.053
HbA1c (mmol/mol) 0.97 (0.95 to 0.99) 0.003
Race (if black) 0.34 (0.13 to 0.92) 0.033
Ahmed et al. BMC Pharmacology and Toxicology (2016) 17:44 Page 7 of 10
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present study and Kang et al’s. We think the association
between HbA1c and vitamin B12 status can be, at least
partly, explained by compliance to metformin treatment.
The well-documented gastrointestinal adverse metfor-
min reactions, adding to the high prevalence of gastro-
intestinal complications in diabetes patients, may
propose higher risk of non-compliance among metfor-
min users. Higher doses of metformin may result in
more prominent gastrointestinal adverse reactions and
higher rates of non-compliance. Our theory is that pa-
tients with poor glycemic control (higher HbA1c) may
have poor compliance to metformin and thus higher
vitamin B12 levels. Raw data from the present study re-
vealed that 21.5 % of participants were on the maximum
daily dose of metformin, possibly supporting our pro-
posed theory on the interaction between dose, non-
compliance, vitamin B12 levels and glycemic control.
Considering the daily dose of metformin and renal
function as direct explanatory factors of the relationship
between HbA1c and vitamin B12 is statistically inappro-
priate as the relationship existed despite adjusting for
metformin dose and eGFR (Table 4).
Black South African descent was a significant protect-
ive factor for vitamin B12 deficiency in the final model
(OR = 0.34, 95 % CI: 0.13 to 0.92, P=0.033) (Table 5) as
well as the three initial models (Table 4). This is the first
study to report ethnic differences in vitamin B12 levels
among metformin-exposed T2DM patients. Reinstatler
et al. found no statistically significant differences in vita-
min levels among black, white and Hispanic metformin-
treated patients in the United States [4]. Higher vitamin
B12 levels in black compared to white general popula-
tions were previously reported [27, 28]. Higher levels of
the vitamin binding proteins transcobalamin II and
haptocorrin in black individuals were described in South
African settings, and explained their relatively elevated
vitamin B12 levels [29].
Daily dose of metformin has shown borderline signifi-
cance as a risk factor for vitamin B12 deficiency in the final
model (OR = 1.96, 95 % CI: 0.99 to 3.88, P=0.053) (Table 5).
The association between the medication daily dose and the
deficiency is sensible, and was previously reported by mul-
tiple regression analyses of many studies [8, 11, 30].
Limitations
Our study was conducted in tertiary academic specialist
clinics, raising the possibility of over-representation of
patients with complicated T2DM. Such patients may
tend to have higher doses and durations of use of met-
formin. The study has only measured serum vitamin
B12 levels to assess the vitamin status. Current recom-
mendations suggest adding methylmalonic acid or homo-
cysteine tests to better assess the intracellular status of the
vitamin [31]. The absence of data on the compliance to
metformin is also a limitation of this study. Compliance
can have an impact on both the response to metformin
and the levels of vitamin B12. To conclusively answer the
secondary aim of investigating the relationship between
peripheral neuropathy and vitamin B12 deficiency, a much
larger sample size would have been needed to show a clin-
ically important difference as small as 10 % to be statisti-
cally significant. NTSS-6 questionnaire, which is relatively
subjective and symptom history-dependent, was the only
used tool to assess neuropathy in the study. However, the
questionnaire has been validated and was found suitable
for evaluating peripheral neuropathy in clinical trials [19].
Table 6 Characteristics of studies that measured the prevalence of metformin-induced vitamin B12 deficiency with diagnostic
cut-points ranging between 145–150 pmol/L
Study Number of
metformin-treated
patients
Prevalence Mean age
(years)
Mean
metformin
dose (gram)
Mean metformin
duration of use
(years)
Study setting Exclusion of renally-
impaired patients
De Jager et al. [3] 196 9.9 %
a
64 2.1 4.3 Outpatient clinics of 3 non-
academic hospitals, The
Netherlands
Yes
Reinstatler et al. [4] 575 5.8 % 63.4 NA 5
b
NHANES sample, United States Yes
Hermann et al. [5] 53 8 % 58.5 2.2 5.2 Outpatient diabetes clinic of a
general hospital, Sweden
Yes
Calvo Romero
and Ramiro
Lozano [9]
81 8.6 % 71.6 1.8 3.6 Internal medicine clinic of a
first level hospital, Spain
No
Beulens et al. [11] 550 28.1 % 61.6 1.3 5.3 4 primary care centers, The
Netherlands
No
De Groot-Kamphuis
et al. [8]
164 14.1 % 62.6 - 4.9
b
Secondary care outpatient
diabetes clinic, The Netherlands
No
This study 121 28.1 % 58.5 2.4 9.6 Outpatient diabetes clinics of 2
tertiary hospitals, South Africa
Yes
NHANES National Health and Nutrition Examination Survey
a
Trial was analysed by Intention To Treat. Using Per Protocol analysis results in 14.5 % prevalence
b
Median value
Ahmed et al. BMC Pharmacology and Toxicology (2016) 17:44 Page 8 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Conclusions and recommendations
This is the first study to address the topic of metformin-
associated vitamin B12 deficiency in African settings. The
study demonstrated that the prevalence of vitamin B12
deficiency, defined by levels <150 pmol/L, in metformin-
treated T2DM patients was as high as 28.1 %. There was
no association between vitamin B12 and peripheral neur-
opathy. A novel finding was the association between black
South African descent and lower odds of vitamin B12 de-
ficiency in metformin-treated patients. We recommend
regular screening for vitamin B12 deficiency in patients
on long-term metformin.
As the first attempt to approach the topic in Africa,
our study reveals the need for setting-specific evidence
to tackle the subject. Further research that judiciously
considers study design issues is warranted to clarify the
possible impact of metformin-induced vitamin B12 defi-
ciency on peripheral neuropathy in T2DM patients.
Abbreviations
CIPN: Chemotherapy-Induced Peripheral Neuropathy; eGFR: Estimated
Glomerular Filtration Rate; H2RAs: Histamine-2 Receptor Antagonists;
HbA1c: Glycated hemoglobin; NHANES: National Health And Nutrition
Examination Survey; NTSS-6: Neuropathy Total Symptom Score-6;
PPIs: Proton Pump Inhibitors; T2DM: Type-2 Diabetes Mellitus; TCSS: Toronto
Clinical Scoring System
Acknowledgements
The authors thank Prof. Danie G. Van Zyl and the Diabetes Clinic of Kalafong
Hospital for assistance in data acquisition. We also thank Ampath
Laboratories for analysing the samples and Eli Lilly and Company
Pharmaceuticals (Lilly) for granting us the permission to use the Neuropathy
Total Symptom Score-6 Questionnaire.
The study was funded by the Department of Pharmacology, University of
Pretoria, without being involved in the design of the study and collection,
analysis, and interpretation of data and in writing the manuscript.
Funding
The study was funded by the Department of Pharmacology, University of Pretoria.
Availability of data and materials
Data will not be shared to ensure patient confidentiality.
Authors’contributions
All authors contributed to the work. MAA conducted the study, interpreted the
results and drafted the manuscript. GM contributed to the study design,
supervised its conduction and critically revised the manuscript. PR conceived
and designed the study, analyzed the data and critically revised the manuscript.
All authors have given approval to the final version of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for Publication
Not applicable.
Ethics approval and consent to participate
This study was conducted in accordance with the Declaration of Helsinki.
The study was approved by the Research Ethics Committee of University of
Pretoria. As a prerequisite, all patients had to sign the informed consent
form to be able to participate in the study.
Author details
1
Department of Pharmacology, Faculty of Health Sciences, University of
Pretoria, Pretoria, South Africa.
2
Department of Internal Medicine, Steve Biko
Academic Hospital, University of Pretoria, Pretoria, South Africa.
Received: 23 April 2016 Accepted: 7 September 2016
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