ORIGINAL INVESTIGATION

Risk Factors of Vitamin B12 Deficiency

in Patients Receiving Metformin
Rose Zhao-Wei Ting, MBBS; Cheuk Chun Szeto, MD, FRCP; Michael Ho-Ming Chan, FRCPA, FHKCPath;
Kwok Kuen Ma, MBBS; Kai Ming Chow, MRCP

Background: Identification of risk factors for metformin-
related vitamin B12 deficiency has major potential impli-
cations regarding the management of diabetes mellitus.
Methods: We conducted a nested case-control study from
a database in which the source population consisted of
subjects who had levels of both serum vitamin B12 and
hemoglobin A1c checked in a central laboratory. We iden-
tified 155 cases of diabetes mellitus and vitamin B12 de-
ficiency secondary to metformin treatment. Another 310
controls were selected from the cohort who did not have
vitamin B12 deficiency while taking metformin.
Results: A total of 155 patients with metformin-related
vitamin B12 deficiency (mean ± SD serum vitamin B12 con-
centration, 148.6 ± 40.4 pg/mL [110 ± 30 pmol/L]) were
compared with 310 matched controls (466.1 ± 330.4
pg/mL [344 ± 244 pmol/L]). After adjusting for con-
founders, we found clinically important and statisti-
cally significant association of vitamin B12 deficiency with
dose and duration of metformin use. Each 1-g/d metfor-
min dose increment conferred an odds ratio of 2.88 (95%
confidence interval, 2.15-3.87) for developing vitamin
B12 deficiency (P⬍.001). Among those using metformin
for 3 years or more, the adjusted odds ratio was 2.39 (95%
confidence interval, 1.46-3.91) (P=.001) compared with
those receiving metformin for less than 3 years. After ex-
clusion of 113 subjects with borderline vitamin B12 con-
centration, dose of metformin remained the strongest in-
dependent predictor of vitamin B12 deficiency.
Conclusions: Our results indicate an increased risk of
vitamin B12 deficiency associated with current dose and
duration of metformin use despite adjustment for many
potential confounders. The risk factors identified have
implications for planning screening or prevention strat-
egies in metformin-treated patients.
Arch Intern Med. 2006;166:1975-1979

Author Affiliations:
Departments of Medicine and
Therapeutics (Drs Ting, Szeto,
and Chow) and Chemical
Pathology (Dr Chan), Prince of
Wales Hospital, The Chinese
University of Hong Kong,
Sha Tin; and Department of
Surgery, Queen Mary Hospital
(Dr Ma), Hong Kong, China.
M
ETFORMIN HAS GREATLY
improved the progno-
sis of diabetic pa-
tients by improving in-
sulin sensitivity and
protection against vascular complica-
tions.1 The United Kingdom Prospective
Diabetes Study (UKPDS)2 confirmed the
long-term benefit of metformin in decreas-
ing diabetes-related end points, diabetes-
related death, and all-cause mortality in
overweight patients with diabetes melli-
tus, and with less weight gain and fewer
hypoglycemic attacks than occurred with
insulin and sulphonylureas treatment.2
Evidence from early clinical observa-
tion, however, indicated a prevalence of
30% for vitamin B12 malabsorption among
patients undergoing long-term metformin
treatment.3 Subsequent studies reported
that metformin decreased serum vitamin B12
level by 14% to 30%.4-6 The findings of met-
formin-related vitamin B12 deficiency have
caused us to question whether this ad-
verse effect is predictable among patients
with type 2 diabetes mellitus who receive
metformin. To date, knowledge of the risk
factors of this adverse event of metformin
is still limited. From a clinical standpoint,
characterization of risk factors for metfor-
min-related vitamin B12 deficiency is the key
to better patient care. First, there is likely
to be an improved yield of detecting vita-
min B12 deficiency if high-risk individuals
can be identified. Second, subjects identi-
fied as having substantial risk for metfor-
min-related vitamin B12 deficiency might
benefit from empirical screening or pri-
mary prevention with other means such as
calcium supplementation.6
METHODS
We undertook a nested case-control study in the
New Territories East Cluster region, Hong Kong,
between January 2003 and November 2005. A

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3987 Source Patients With Both
Hemoglobin A1c and Vitamin B12
Checked for Clinical Reasons
355 Subjects With 3632 Subjects With Serum
Vitamin B12 Deficiency Vitamin B12 Level
(≤150 pmol/L) >150 pmol/L
200 Cases Did Not
Fulfill Definition of Exclusion of Subjects
Metformin-Related Not Taking Metformin
Vitamin B12 Deficiency
155 Recruited as Cases 310 Matched Controls
Figure 1. Patient profile of the nested case-control study. Cases refer to
subjects with diabetes mellitus and vitamin B12 deficiency during metformin
therapy, whereas the controls had metformin-treated diabetes mellitus but
no vitamin B12 deficiency. Cases and controls were matched according to the
date of blood sampling. To convert vitamin B12 to picograms per milliliter,
divide by 0.738.
database was generated by a central chemical pathology labora-
tory that provides vitamin B12 assay service to cover 19% (1.3 mil-
lion) of the population in Hong Kong. In other words, we nested
the case-control study within a source cohort population, as de-
fined by patients who had levels of both hemoglobin A1c and se-
rum vitamin B12 being checked for clinical reasons within the study
period. To ensure complete data extraction, we also restricted the
subjects to have at least 1 year of continuous medical history re-
corded on computer. Serum vitamin B12 and folate concentra-
tions were determined by electrochemiluminescent immunoas-
say (E170 Analytic; Roche Diagnostics, Indianapolis, Ind). The
mean±SE lower limit of normal by our vitamin B12 assay was
253.4±50.1 pg/mL (187 ± 37 pmol/L); therefore, we chose a cut-
off point of 203.3 pg/mL (150 pmol/L) for vitamin B12 defi-
ciency. Serum methylmalonic acid concentration evaluation was
not routinely performed.
We defined a case as a Chinese subject with diabetes mellitus
and vitamin B12 deficiency during metformin treatment after ex-
cluding subjects who had pernicious anemia (positive Schilling
test result or anti-intrinsic factor antibodies), pancreatic exo-
crine insufficiency, and/or history of gastrectomy or small bowel
resection. To avoid potentially confounding causes of vitamin B12,
patients receiving oral or parenteral vitamin B12 supplementa-
tion within 3 months prior to study initiation were excluded. Two
controls were selected for each case, matched according to the
date of blood sampling. Eligible controls for each case were in-
dividuals within our database with diabetes mellitus who did not
have vitamin B12 deficiency (serum concentration ⬎203.3 pg/mL
[150 pmol/L]) while taking metformin.
Standardized data collection forms were used to abstract in-
formation from computerized medical records and pharmacy
records. Data collected included demographic information, con-
centrations of serum vitamin B12, folate, and blood hemoglo-
bin, white blood cell and platelet counts, mean corpuscular vol-
ume, documentation of concomitant histamine H2 receptor
antagonist or proton pump inhibitor therapy, vegetarian diet,
smoking history, and alcohol consumption. Detailed data of met-
formin daily dose and duration of metformin therapy at the time
of blood collection were also collected.
Mean and standard deviation values for continuous variables
were calculated to characterize our study population. The asso-
ciation between discrete variables and metformin-related vita-
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min B12 deficiency was assessed by cross-tabulation and the ␹2
test or Fisher exact test, whereas continuous variables between
groups were compared with unpaired t test or the nonparamet-
ric Wilcoxon rank sum test as appropriate. To estimate the strength
of association, a binary logistic regression model was used for both
univariate and multivariate analysis to calculate the odds ratio (OR)
of metformin-related vitamin B12 deficiency. Our model was con-
structed with stepwise selection for relevant variables by previ-
ous studies and those associated with metformin-related vita-
min B12 deficiency at an ␣ level of ⬍.1 in our univariate analysis.
Because of the lack of a precise standard and uncertainty of defi-
nition of vitamin B12 deficiency without supplementary methyl-
malonic acid measurement, we repeated the regression analysis
after excluding cases with borderline vitamin B12 concentration
(⬎203.3 and ⱕ298.1 pg/mL [⬎150 and ⱕ220 pmol/L])7,8 to mini-
mize misclassification error. Confidence intervals (CIs) re-
ported are likelihood based. All P values were 2-sided, and we
regarded P⬍.05 as significant. All statistical analyses were per-
formed using The Statistical Package for the Social Sciences (Win-
dows, version 13.0; SPSS Inc, Chicago, Ill).
RESULTS
Between January 2003 and November 2005, a total of 3987
source subjects were evaluated for the study. Figure 1
summarizes the trial profile. Of 355 cases with a labora-
tory finding of vitamin B12 deficiency (serum vitamin B12
concentration ⱕ203.3 pg/mL [ⱕ150 pmol/L]), 155 cases
fulfilled the case selection criteria. We therefore identi-
fied 155 cases (mean±SD serum vitamin B12 concentra-
tion, 148.6±40.4 pg/mL [110 ± 30 pmol/L]; range, 50.1-
203.3 pg/mL [37-150 pmol/L]) and 310 matched controls
(serum vitamin B12 concentration, 466.1±330.4 pg/mL
[344 ± 244 pmol/L]; range, 204.6-2000.0 pg/mL [151-
1476 pmol/L]) from our database.
Characteristics of the cases and controls are listed in
Table 1. There were no significant differences in se-
rum folate concentration between these 2 groups. Sex dis-
tribution, drinking, and smoking habits were similar
among cases and controls. Vegetarians appeared to be
more common among cases than controls, with border-
line statistical significance (prevalence 2.6% vs 0.3%;
P =.04). There was no substantial difference in age, with
a mean ± SD age of 72.5 ± 9.3 years among cases vs
71.4±11.2 years among controls (P =.24).
In the univariate analysis, vegetarians (OR, 8.19; 95%
CI, 0.91-73.9) had a borderline significant association with
the risk of metformin-related vitamin B12 deficiency. As sum-
marized in Table 2, the unadjusted analysis showed a sig-
nificant increase in risk associated with the current dose
and duration of metformin medication. The metformin-
treated patients with vitamin B12 deficiency had a mean±SD
daily dose of 2.0±0.7 g (interquartile range, 1.5-3.0 g),
whereas the control group received a daily metformin dose
of 1.4±0.7 g (interquartile range, 1.0-2.0 g). The crude OR
for metformin-related vitamin B12 deficiency was 2.37 (95%
CI, 1.60-3.52) for a history of metformin use longer than
3 years. There was a significant increased risk of metformin-
related vitamin B12 deficiency associated with each addi-
tional 1-g/d dose increment of metformin (OR, 2.61; 95%
CI, 2.00-3.42) (P⬍.001).
Once adjustments were made for potential confound-
ing variables (Table 2), the most significant OR was as-
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 Table 1. Characteristics of Cases of Metformin-Related Vitamin B12 Deficiency and Matched Controls Identified From a
Population-Based Cohort*

Cases Controls
Characteristic (n = 155) (n = 310) P Value
Age, y 72.5 ± 9.3 71.4 ± 11.2 .24
Men , No. (%) 57 (37) 127 (41) .42
Serum vitamin B12, pg/mL 148.6 ± 40.4 (110 ± 30 pmol/L) 466.1 ± 330.4 (344 ± 244 pmol/L) ⬍.001
Serum folate, ng/mL (nmol/L) 10.8 ± 5.9 (24.4 ± 13.4) 10.7 ± 6.0 (24.2 ± 13.5) .87
Blood hemoglobin, g/dL 11.6 ± 8.6 11.3 ± 4.8 .66
Mean corpuscular volume, fL 89.0 ± 14.9 87.6 ± 9.7 .32
Cigarette use, No.†
Current 18 37
Former 26 49 .68
Never 68 152
Moderate to heavy alcohol use (⬎1 drink daily, No.)†
Current 5 17
Former 18 23 .28
Never 80 174
Vegetarian, No. (%) 4 (2.6) 1 (0.3) .04‡
Use of histamine H2-blocker or proton pump inhibitor therapy, No. (%) 19 (12) 37 (12) .92
Daily dose of metformin, g 2.0 ± 0.7 1.4 ± 0.7 ⬍.001
Duration of metformin use, median (IQR), y 4 (2-5) 2 (1-4) ⬍.001

Abbreviation: IQR, interquartile range.

*Unless otherwise indicated, data are reported as mean ± SD.
†Data of smoking and alcohol consumption were missing in 25% and 32% of the study population, respectively.
‡Fisher exact test.

Table 2. Risk Factors Associated With Development of Metformin-Related Vitamin B12 Deficiency, Including Borderline Deficiency

Crude OR Adjusted OR
Risk Factor (95% CI) P Value (95% CI) P Value
Age, per 10-y increment 1.10 (0.92-1.33) .27 1.36 (1.08-1.69) .01
Vegetarian 8.19 (0.91-73.9) .06 16.2 (1.69-154.00) .02
Use of histamine H2 receptor antagonist or 1.03 (0.57-1.86) .92 1.13 (0.58-2.17) .72
proton pump inhibitor
Daily dose of metformin, per 1-g increment 2.61 (2.00-3.42) ⬍.001 2.88 (2.15-3.87) ⬍.001
Use of metformin for more than 3 y 2.37 (1.60-3.52) ⬍.001 1.99 (1.30-3.05) .001

Abbreviations: CI, confidence interval; OR, odds ratio.

sociated with current dose of metformin. This was fol-
lowed by duration of metformin use and patient age. Each
1-g/d dose increment conferred a more than 2-fold in-
creased risk of developing vitamin B12 deficiency with met-
formin (adjusted OR, 2.88; 95% CI, 2.15-3.87) (P⬍.001).
Compared with metformin users of less than 3 years, the
adjusted OR was 2.39 (95% CI, 1.46-3.91) (P =.001) for
users of metformin for 3 years or more. Increased age ap-
peared to be positively related to metformin-related vi-
tamin B12 deficiency albeit with negligible clinical sig-
nificance (adjusted OR, 1.36 for each 10-year increment
in age). Vegetarian diets were also associated with vita-
min B12 deficiency, but the confidence interval was wide
(OR, 16.2; 95% CI, 1.69-154.00). We found no signifi-
cantly increased risk for concurrent use of histamine H2
receptor antagonist or proton pump inhibitor.
We repeated the analyses after excluding 113 subjects
(24%) with borderline vitamin B12 concentration between
203.3 and 298.1 pg/mL (150 and 220 pmol/L). This did
not significantly influence the results (Table 3); current
dose of metformin remained the strongest independent pre-
dictor of vitamin B12 deficiency (OR, 3.75 for each 1-g/d
dose increment; 95% CI, 2.63-5.35) (P⬍.001). Of the 113
subjects excluded, their characteristics, including dose and
duration of metformin use, were similar to the control sub-
jects with serum vitamin B12 concentration exceeding 298.1
pg/mL (220 pmol/L) (details not shown).
We also investigated the effects of metformin dose on
the serum vitamin B12 concentration among the cases.
Cases were divided into 3 groups according to their cur-
rent daily metformin doses: 1.0 g or less (n=29), more
than 1.0 g to 2.0 g (n=76), or more than 2.0 g to 3.0 g
(P⬍.001 by analysis of variance) (Figure 2). Post hoc
analysis performed by the Bonferroni-adjusted pairwise
comparisons revealed significantly lower vitamin B12 con-
centration in cases receiving more than 2.0 to 3.0 g/d than
in those receiving more than 1.0 to 2.0 g/d (P =.01) and
those receiving 1.0 g/d or less (P⬍.001). Conversely, when
all the cases and controls in this study were included in
the post hoc analysis, there was also a significant differ-
ence in the proportion of subjects with deficient vita-
min B12 concentrations according to the daily metfor-
min doses. The distribution of subjects (Figure 3) varied
with the metformin doses with respect to the vitamin B12

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 Table 3. Risk Factors Associated With Development of Metformin-Related Vitamin B12 Deficiency, Excluding Borderline Deficiency

Crude OR Adjusted OR
Risk Factor (95% CI) P Value (95% CI) P Value
Age (per 10-y increment) 1.21 (0.99-1.47) .06 1.60 (1.24-2.04) ⬍.001
Vegetarian 5.19 (0.57-46.9) .14 10.9 (1.09-109.00) .04
Use of histamine H2-receptor antagonist or proton pump inhibitor 0.88 (0.47-1.65) .69 1.33 (0.63-2.79) .45
Daily dose of metformin (per 1-g increment) 3.06 (2.25-4.16) ⬍.001 3.75 (2.63-5.35) ⬍.001
Use of metformin for more than 3 y 2.98 (1.92-4.64) ⬍.001 2.39 (1.46-3.91) .001

Abbreviations: CI, confidence interval; OR, odds ratio.

100
P <.001

P = .34 P = .01
200 <220 pmol/L
80
Serum Vitamin B12 Concentration, pmol/L

160
60

Patients, %
120 ≤150 pmol/L

40

80

20
40

0
0 0 - 1.0 >1.0 - 2.0 >2.0 - 3.0
0 - 1.0 >1.0 - 2.0 >2.0 - 3.0 (n = 186) (n = 191) (n = 88)
Current Metformin Dose, g/d Dose of Metformin, g/d

Figure 2. Box-and-whisker plot shows the serum vitamin B12 concentration Figure 3. Effect of daily metformin dose on the proportion of all subjects
for cases of metformin-related vitamin B12 deficiency according to the with serum vitamin B12 concentration up to 203.3 pg/mL and up to 298.1
different daily doses of metformin received. The lower and upper bounds of pg/mL. Error bars indicate standard error of the mean. To convert vitamin B12
the boxes denote the 25th and 75th percentiles, respectively, and the to picograms per milliliter, divide by 0.738.
horizontal lines in the boxes correspond to the median value. The lower and
upper error bars indicate the 10th and 90th percentiles, respectively. To
convert vitamin B12 to picograms per milliliter, divide by 0.738. ficiency may result from disorders in intestinal mobility
and/or bacterial overgrowth.7 However, more recent evi-
dence has demonstrated that metformin administration
cutoff points of either 203.3 or 298.1 pg/mL (P⬍.001 for
neither alters the intestinal motility9 nor causes bacte-
both).
rial overgrowth.6 On the other hand, metformin may dis-
rupt the ileal vitamin B12 absorption.10,11 The vitamin B12-
COMMENT intrinsic factor complex is dependent on the luminal
calcium concentration to facilitate uptake by the ileal cell
In our nested case-control study of metformin-related vi- surface receptor, whereas metformin is believed to give
tamin B12 deficiency, metformin dose and treatment du- a positive charge to the surface of the membrane, which
ration emerged as the most consistent risk factors of vi- would act to displace divalent cations such as calcium.
tamin B12 deficiency within a Chinese population with Impaired calcium availability due to metformin activity
diabetes mellitus. Of special interest is that their asso- would therefore interfere with the calcium-dependent pro-
ciation remained stable after adjustment for potential con- cess of vitamin B12 absorption.6,10-12 It should be noted
founding factors in the multivariate analysis, thus rein- that our study design precluded a full assessment of the
forcing our conclusion that higher metformin dose and dietary or supplementary intake of calcium.6 Practi-
longer treatment duration are independent risk factors. cally, our data and the graded relationship of metformin
There is also evidence from our post hoc analysis that dose with serum vitamin B12 concentration (Figure 2) sup-
serum vitamin B 12 concentration showed a dose- port the notion of a causal relation between metformin
dependent decrease with increasing dose of metformin. administration and vitamin B12 deficiency rather than sug-
It is impossible to deduce from our case-control study gesting any particular mechanism(s).
the mechanism of metformin-related vitamin B12 defi- Unlike previous studies,13-15 our study demonstrated
ciency. The literature has reported that vitamin B12 de- no excess risk of vitamin B12 deficiency among metfor-

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 min users who concurrently use H2-blockers or proton
pump inhibitors. Theoretically, these medications can pro-
duce malabsorption of dietary protein-bound vitamin B12.
The lack of association in the current study may stem from
imprecise hospital-based medication records. We were
unable to track the use of H2-blockers or proton pump
inhibitors that are nowadays commonly prescribed by fam-
ily practitioners or purchased over the counter. Such mis-
classification of exposure status (to H2-blockers or pro-
ton pump inhibitors), if present and nondifferential, would
have had the effect of biasing the OR toward 1.
To minimize the risk of overmatching, no matching
(other than the date of blood sampling) was used in this
study, which might have hindered informative results or
introduced confounding if the matching factor were linked
with the risk of vitamin B12 deficiency. Subjects in our
study, nevertheless, needed to have at least 1 serum vi-
tamin B12 sample checked for a clinical purpose before
being eligible as controls. Patients with certain charac-
teristics may therefore have been more likely to be re-
cruited as controls. Clinicians are in general more in-
clined to screen for vitamin B 12 deficiency among
vegetarians or the geriatric population, for instance, when
a hematologic, neuropsychiatric, or cognitive disorder is
either detected or suspected. If this had been the case,
we would have expected the strength of association be-
tween metformin-related vitamin B12 deficiency and el-
derly16-18 or vegetarian subjects, if any, to be negated. This
is a genuine possibility in the current study because sta-
tistically significant relationships between increased age,
vegetarianism, and the development of vitamin B12 defi-
ciency are apparent in the multivariate but not the uni-
variate analysis.
Our results have to be interpreted with caution in light
of several other limitations. One potential limitation of
our study was the use of serum vitamin B12 concentra-
tion alone to define vitamin B12 deficiency, without me-
tabolite (plasma homocysteine or methylmalonic acid)
measurements for confirmation.7,8,16 Controversy exists
as to whether serum measurement accurately reflects stor-
age levels of vitamin B12. The possibility exists that bias
may have been introduced by misclassifying cases and
controls based on serum vitamin B12 measurement. None-
theless, repeated analysis after our careful exclusion of
subjects with borderline serum vitamin B12 data did not
qualitatively affect the findings (Table 3), which sug-
gests that any biases thus introduced were likely to be
small. Furthermore, the retrospective nature of informa-
tion retrieval for our subjects led to predictable limita-
tions of data completeness. Another potential bias arises
from the unblinded data acquisition method. Finally, the
data from this study cannot be used to assess the inci-
dence of vitamin B12 deficiency with metformin use.
In conclusion, this nested case-control study in a Chi-
nese population showed that the risk of vitamin B12 de-
ficiency is magnified in patients who have received both
a higher dose and longer course of metformin treat-
ment, independent of other clinical variables. Although
this observational study may be subject to residual con-
founding that cannot be fully corrected for, we believe
our findings should reinforce the heightened vigilance
about vitamin B12 deficiency. Enough concerns exist to
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1979
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call attention to the value of vitamin B12 screening,19 par-
ticularly among at-risk patients receiving metformin. Our
data underscore the need for monitoring subjects un-
dergoing high-dose and/or prolonged-course metfor-
min therapy.
Accepted for Publication: June 27, 2006.
Correspondence: Kai Ming Chow, MRCP, Department
of Medicine and Therapeutics, Prince of Wales Hospi-
tal, The Chinese University of Hong Kong, Sha Tin, Hong
Kong, China (Chow_Kai_Ming@alumni.cuhk.net).
Author Contributions: Study concept and design: Ting and
Chow. Acquisition of data: Ting, Chan, and Chow. Analy-
sis and interpretation of data: Ting, Szeto, Ma, and Chow.
Drafting of the manuscript: Ting and Chow. Critical revi-
sion of the manuscript for important intellectual content:
Szeto, Chan, and Chow. Statistical analysis: Szeto and
Chow. Study supervision: Chow.
Financial Disclosure: None reported.
Acknowledgment: We thank Clara Law for her contri-
bution to data retrieval.
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