Long term treatment with metformin in patients with type 2diabetes and risk of vitamin B-12 deficiency: randomisedplacebo controlled trial
Jolien de Jager, resident,1,2 Adriaan Kooy, internist,2,3 Philippe Lehert, professor of statistics,4 Michiel GWulffele´, general practitioner,2,3 Jan van der Kolk, biochemical engineer,5 Danie¨l Bets, program manager,6 JoopVerburg, chief laboratory attendant,5 Ab J M Donker, professor of internal medicine,7 Coen D A Stehouwer,professor and chair8
vitamin B-12) for patients with a normal vitamin B-12
Objectives To study the effects of metformin on the
incidence of vitamin B-12 deficiency (<150 pmol/l), low
Conclusions Long term treatment with metformin
concentrations of vitamin B-12 (150-220 pmol/l), and
increases the risk of vitamin B-12 deficiency, which
Centre, Bethesda GeneralHospital, Hoogeveen, Netherlands
folate and homocysteine concentrations in patients with
results in raised homocysteine concentrations. Vitamin
type 2 diabetes receiving treatment with insulin.
B-12 deficiency is preventable; therefore, our findings
Design Multicentre randomised placebo controlled trial.
suggest that regular measurement of vitamin B-12
Setting Outpatient clinics of three non-academic
concentrations during long term metformin treatment
Participants 390 patients with type 2 diabetes receiving
Trial registration Clinicaltrials.gov NCT00375388.
Intervention 850 mg metformin or placebo three times a
Metformin is considered a cornerstone in the treat-
Main outcome measures Percentage change in vitamin B-
ment of diabetes and is the most frequently prescribed
12, folate, and homocysteine concentrations from
first line therapy for individuals with type 2 diabetes.1
Development, Merck Netherlands,Amsterdam, Netherlands
baseline at4, 17, 30, 43, and 52 months.
In addition, it is one of a few antihyperglycaemic
Results Compared with placebo, metformin treatment
agents associated with improvements in cardio-
was associated with a mean decrease in vitamin B-12
vascular morbidity and mortality,2 3 which is a major
−19% (95% confidence interval −24% to
cause of death in patients with type 2 diabetes.4
−14%; P<0.001) and in folate concentration of −5% (95%
There are few disadvantages to the use of metformin.
CI −10% to −0.4%; P=0.033), and an increase in
Metformin does, however, induce vitamin B-12 malab-
homocysteine concentration of 5% (95% CI −1% to 11%;
sorption, which may increase the risk of developing
P=0.091). After adjustment for body mass index and
vitamin B-12 deficiency5-7—a clinically important and
smoking, no significant effect of metformin on folate
treatable condition. In addition, metformin treatment
concentrations was found. The absolute risk of vitamin
has been reported to be associated with decreased
B-12 deficiency (<150 pmol/l) at study end was 7.2
folate concentration, although the mechanism of this
percentage points higher in the metformin group than in
effect has not been elucidated.8 Finally, decreases in
the placebo group (95% CI 2.3 to 12.1; P=0.004), with a
both folate and vitamin B-12 concentrations might, in
number needed to harm of 13.8 per 4.3 years (95% CI
turn, result in an increase in homocysteine concentra-
43.5 to 8.3). The absolute risk of low vitamin B-12
tions (web figure A), an independent risk factor for
concentration (150-220 pmol/l) at study end was 11.2
cardiovascular disease, especially among individuals
percentage points higher in the metformin group (95% CI
4.6 to 17.9; P=0.001), with a number needed to harm of
All current evidence on vitamin B-12 deficiency in
8.9 per 4.3 years (95% CI 21.7 to 5.6). Patients with
metformin treatment comes from short term
vitamin B-12 deficiency at study end had a mean
studies.5-712-14 No long term, placebo controlled data
homocysteine level of 23.7 µmol/l (95% CI 18.8 to 30.0
on the effects of metformin on concentrations of vita-
µmol/l), compared with a mean homocysteine level of
min B-12 in patients with type 2 diabetes have been
18.1 µmol/l (95% CI 16.7 to 19.6 µmol/l; P=0.003) for
reported. In addition, placebo controlled data on the
patients with a low vitamin B-12 concentration and 14.9
effects of metformin on homocysteine concentrations
µmol/l (95% CI 14.3 to 15.5 µmol/l; P<0.001 compared
in type 2 diabetes are sparse,12 15 and again no long term
with vitamin B-12 deficiency; P=0.005 compared with low
Blood samples for this study were drawn at baseline
and after 4, 17, 30, 43, and 52 months, and stored at
−80°C until analysis. Concentrations of vitamin B-12,
folate, and homocysteine were measured in serum.
Vitamin B-12 and folate concentrations were deter-
mined by an electrochemiluminescence immunoassay(ECLIA) using the competition principle. The meanintra-assay coefficients of variation for vitamin B-12
and folate were 2.3% and 3.5%, respectively. Themean inter-assay coefficients were 2.9% and 4.7%,
We studied the effects of metformin treatment on
serum concentrations of vitamin B-12, folate, and
Total homocysteine concentration was measured
homocysteine in patients with type 2 diabetes in a
using a kit from Chromsystems (Martinsried, Ger-
many). The results were corrected against two typesof “consensus plasma samples” (SKML, Nijmegen,
the Netherlands) that had concentrations of 13 µmol/
l and 55 µmol/l. The correction factor found was 0.90.
This study was part of the Hyperinsulinaemia: the Out-
The intra-assay coefficients of variation were 2.2% and
come of its Metabolic Effects (HOME) randomised
1.8% at 12.8 µmol/l and 72.2 µmol/l, respectively. The
trial investigating the effects of metformin on meta-
inter-assay coefficients of variation were 6.1% and
bolism and on microvascular and macrovascular dis-
5.2% at 9.8 µmol/l and 21.1 µmol/l, respectively.
ease in type 2 diabetes. The trial included 390 patients
In the HOME trial, vitamin B-12, folate, and homo-
aged 30-80 years with type 2 diabetes who were receiv-
cysteine concentrations had been measured previously
ing treatment with insulin, as previously described.3 16
in samples obtained at baseline and after 16 weeks oftreatment.12 To investigate the stability of the assay
procedures, we compared the previously obtainedvalues with values obtained for the present investiga-
The HOME trial was conducted in the outpatient
tion. The correlation between old and new measure-
clinics of three non-academic hospitals in the Nether-
ments of vitamin B-12 was 0.58 for baseline
lands: Bethesda General Hospital, Hoogeveen;
measurements and 0.91 for measurements taken after
Diaconessen Hospital, Meppel; and Aleida Kramer
16 weeks; for folate these values were 0.90 and 0.83,
respectively, and for homocysteine 0.99 and 0.99,
assigned by a computer program to receive either
respectively. The relatively low correlation for vitamin
850 mg of metformin three times a day or 850 mg of
B-12 values obtained at baseline was caused by five
placebo thrice daily, which were provided in identical
cases for which a large discrepancy existed between
old and new values; these cases were subsequently
The trial consisted of three phases: the 12 week
excluded from analyses involving vitamin B-12.
pre-randomisation phase, in which patients were trea-ted with insulin only and concomitant medication was
discontinued; the 16 week short term treatment phase,
Sample size calculations were based on expected dif-
at the beginning of which patients were randomised to
ferences in the occurrence of disease related end
receive either metformin or placebo in addition to
points, as described previously.3 With the sample size
insulin therapy; and the four year (48 month) long
obtained, however, a decrease in vitamin B-12 concen-
term treatment phase (fig 1). An interim analysis was
tration of 5% in the metformin group compared with
conducted at the end of the short term treatment phase,
the placebo group according ANCOVA tests should
during which the treatment codes were not disclosed to
be detectable at a two sided 95% confidence level,with a power of 0.82.
We log transformed data on vitamin B-12, folate,
and homocysteine concentrations before analysis
because their distribution was skewed. Data are given
Patients visited the clinics at the start of the pre-rando-
as geometric means with 95% confidence intervals.
misation phase (three months before randomisation), at
Given that log values are not directly interpretable,
baseline (for randomisation to metformin or placebo),
the antilogs are reported instead. These values are the
and one month after baseline (to check the tolerance of
geometric mean percentage change from baseline.
the drug titration), then subsequently every three
The end point of interest was the percentage change
months until the end of the trial. During these visits, a
of each variable from baseline at 4, 17, 30, 43, and
physical examination was carried out, a medical history
52 months, which was calculated from baseline values
was taken, and laboratory investigations were per-
and the summary mean. The differences between the
formed. At baseline, and after 10 and 52 months, dietary
metformin and the placebo group were tested by a cen-
counselling was given to all patients.
tral t test on log transformed values. We also calculated
more extensively elsewhere.3 Only two participants
were lost to follow-up (at 33 and 26 months, respec-tively), both of whom were in the metformin group.
Excluded (n=355)Did not give informed consent
The actual mean dose in the metformin group was
2050 mg a day. At the final visit, laboratory samples
were available for 256 patients (127 metformin, 129placebo). The main outcomes of this trial have beenreported previously.3
Table 1 shows baseline characteristics of all patients
analysed. Five randomised patients were excluded
from the analysis because of poor correlations between
old and newly measured vitamin B-12 values (see
Methods). Patients randomised to metformin were
older than those randomised to placebo (64±10 years
v 59±11 years), and were more likely to have a history
of cardiovascular disease and less likely to be a smoker(30 (19%) v 59 (30%)). The other characteristics were
comparable between the two treatment groups.
Vitamin B-12, folate, and homocysteine concentrationsDuring the 52 months of placebo treatment, vitamin B-12 concentration increased from baseline by 0.2 pmol/
the hazard ratio for developing vitamin B-12 defi-
l (0% change, 95% confidence interval −3% to 4%),
ciency, which was defined as a vitamin B-12 concentra-
folate increased by 1.01 nmol/l (8%, 95% CI 4% to
tion below the value of 150 pmol/l, and of having low
12%), and homocysteine increased by 1.60 μmol/l
vitamin B-12 levels, which was defined as a vitamin B-
(20%, 95% CI 16% to 25%; fig 3). During metformin
12 concentration below 220 pmol/l but above 150
treatment, vitamin B-12 decreased by 89.8 pmol/l
pmol/l.17 All analyses were by intention to treat and
(−19%, 95% CI −22% to −15%) from baseline, whereas
used the last observation carried forward. To test
folate concentration increased by 0.21 nmol/l (3%,
whether results obtained were robust, we also used
95% CI −1% to 6%) and homocysteine concentration
mixed models analysis to impute missing data. Patients
increased by 3.26 μmol/l (26%, 95% CI 21% to 31%).
with vitamin B-12 concentrations below 150 pmol/l at
Compared with placebo, metformin treatment was
baseline, at the interim analysis, or at both time points
associated with a 19% decrease in vitamin B-12 con-
were supplemented at 16 weeks (n=8) and, therefore,
centration (95% CI −24% to −14%; P<0.001) and a5% decrease in folate concentration (95% CI −10% to
excluded from analyses after 16 weeks.
−0.4%; P=0.033), and a 5% increase in homocysteine
We used linear mixed models to explore the effects
concentrations (95% CI −1% to 11%; P=0.091). The
of metformin on concentrations of vitamin B-12, folate,
effects of metformin on concentrations of vitamin B-
and homocysteine. We also investigated whether met-
12, folate, and homocysteine were re-analysed follow-
formin associated changes in homocysteine concentra-
ing adjustment for age, previous metformin treatment,
tions, if any, could be explained by changes in the
duration of diabetes, gender, insulin dose, and smok-
concentrations of folate, vitamin B-12, or both, and, if
ing habits. None of these variables materially changed
so, whether the changes were independent of age, gen-
the results for vitamin B-12 and homocysteine (data
der, duration of diabetes, smoking, body mass index,
not shown), but they did have an effect on the results
insulin dose, serum creatinine, high density lipopro-
for folate. After adjustment for body mass index and
tein cholesterol, or glycated haemoglobin. The good-
smoking, no significant effect of metformin on folate
ness of fit between alternative models was compared
concentration was found (change in concentration
using the maximum likelihood technique.
compared with placebo −0.1%; P=0.57).
At baseline, three patients (1.6%) in the metformin
group and four (2.2%) in the placebo group had vita-
min B-12 deficiency (vitamin B-12 concentration
We screened the medical files of all three participating
<150 pmol/l), whereas 14 patients (7.3%) and 14
outpatient clinics and identified 745 eligible patients.
patients (7.5%), respectively, had a low vitamin B-12
All were approached to enrol into the trial and 390
concentration (150-220 pmol/l). At the end of the
individuals gave written informed consent. A total of
study period, 19 patients (9.9%) in the metformin
196 patients were randomised to receive metformin
group and five (2.7%) in the placebo group had vita-
and 194 to receive placebo. Out of the 390 included
min B-12 deficiency, whereas 35 patients (18.2%) and
patients, 277 individuals (72%) were still receiving met-
13 patients (7.0%), respectively, had a low vitamin
formin or placebo at the end of the trial (fig 2). A total of
46 patients (30 metformin, 16 placebo) discontinued
The risk for vitamin B-12 deficiency at study end was
because of adverse effects, which have been described
7.2 percentage points higher in the metformin group
Table 1 | Baseline characteristics of all patients analysed
The interaction between treatment and time was a sig-
nificant determinant of vitamin B-12 concentration
(P=0.023)—that is, the lowering effect of metformin
on vitamin B-12 concentrations increased with time.
Body mass index and smoking were strong inverse
determinants of folate concentration (P=0.003 and
P<0.0001, respectively). There was no relation
between time and folate concentration. After adjust-
ment for body mass index and smoking, treatment
with metformin was not a significant determinant of
folate concentration, nor was the interaction between
treatment and time (P=0.57 and P=0.23, respectively).
Vitamin B-12 and folate levels were strong determi-
nants of homocysteine concentration (P<0.0001).
Homocysteine concentration increased with age at
baseline (P<0.0001). There was no significant inter-
action between treatment and time for homocysteine
Per protocol analysis using only available data for those
patients who remained in the trial until the final visit
Low density lipoprotein cholesterol (mmol/l)
(n=256) yielded similar results to our original intention
to treat analysis (data not shown). General mixed
High density lipoprotein cholesterol (mmol/l)
model analysis yielded similar results to analysis using
last observation carried forward (data not shown).
Our study on the long term effects of metformin treat-
ment on serum concentrations of vitamin B-12, folate,
and homocysteine in patients with type 2 diabetes trea-
ted with insulin had three main findings. Firstly, met-
formin significantly reduced concentrations of vitamin
Values are mean (standard deviation) unless otherwise stated.
B-12, in accordance with findings from previousstudies.131819 Importantly, our study shows that thisdecrease is not a transitory phenomenon, but persists
than in the placebo group (95% CI 2.3 to 12.1;
and grows over time. Secondly, a small, significant
P=0.004), with a number needed to harm of 13.8 per 4.
decrease in folate concentrations was found in the met-
3 years (95% CI 43.5 to 8.3). The risk difference at
formin group compared with the placebo group; how-
study end for a low vitamin B-12 concentration was
ever, this reduction was not statistically significant after
11.2 percentage points higher in the metformin group
adjustments for body mass index and smoking.
(95% CI 4.6 to 17.9; P=0.001), with a number needed
Thirdly, the decrease in vitamin B-12 concentrations
to harm of 8.9 per 4.3 years (95% CI 21.7 to 5.6). The
was associated with an increase in homocysteine levels,
hazard ratio for developing vitamin B-12 deficiency
which was not statistically significant. Further analyses,
when treated with metformin was 5.5 (95% CI 1.6 to
however, showed that homocysteine concentrations
19.1; P=0.01), and the hazard ratio for a low vitamin B-
did increase in individuals in whom vitamin B-12 levels
12 concentration was 3.0 (95% CI 1.3 to 6.6; P=0.007).
decreased below the concentration generally consid-
Patients with a vitamin B-12 deficiency at the end of
ered to indicate clinical deficiency—that is, 150 pmol/l.
the study had a mean homocysteine level at study end
The finding of decreases in vitamin B-12 concentra-
of 23.7 µmol/l (95% CI 18.8 to 30.0 µmol/l), compared
tion during metformin treatment is not novel and has
with 18.1 µmol/l (95% CI 16.7 to 19.6 µmol/l;
been reported before. A novel finding here, however,
P=0.003) for patients with a low vitamin B-12 concen-
is that the decrease in vitamin B-12 levels is progres-
tration and 14.9 µmol/l (95% CI 14.3 to 15.5 µmol/l;
sive. Furthermore, concentrations in some patients
P<0.001 compared with vitamin B-12 deficiency;
drop to the level at which most authorities agree vita-
P=0.005 compared with low vitamin B-12) for patients
min substitution is required. This is also a novel find-
with a normal vitamin B-12 level (>220 pmol/l; fig 4).
ing, because although earlier trials in well fed, middle
Homocysteine concentrations did not differ signifi-
aged patients showed that metformin decreases vita-
cantly between treatment groups when stratified for
min B-12 concentrations, levels recorded remained
end of treatment vitamin B-12 concentration.
Metformin is thought to induce malabsorption of
vitamin B-12 and intrinsic factor in the ileum, an effect
that can be reversed by increasing calcium intake.618
The consequences of clinically important decreases invitamin B-12 concentrations—such as macrocytic
anaemia, neuropathy, and mental changes—can be
profound. We note that there is no consensus on the
issue of whether “asymptomatic” vitamin B-12 defi-ciency should be treated.20 However, studies show
that some symptoms of vitamin B-12 deficiency are dif-
ficult to diagnose and can be irreversible, and treatment
of vitamin B-12 deficiency is relatively easy, cheap,
safe, and effective,21-24 in effect arguing in favour of
treatment. In addition, although the necessity of treat-
ing “spontaneous” vitamin B-12 deficiency may be
debated, one should be more easily inclined to treatdrug induced vitamin B-12 deficiency, as a key princi-
ple of drug prescription is to do no harm. On the otherhand, our study shows that it is reasonable to assume
harm will eventually occur in some patients with met-
formin induced low concentrations of vitamin B-12.
Folate concentration increased in both the metfor-
min group and the placebo group, possibly as a result
of dietary counselling received by all patients through-out the trial. Our short term interim analysis showed a
significantly larger increase in folate concentration in
the placebo group,12 a finding that was initially repli-
cated in the current analysis but that disappeared after
adjustment for body mass index and smoking.
Previous studies have shown either no or small
effects of metformin treatment on concentrations of
homocysteine.13 14 25 26 We clearly show that homocys-
teine concentrations do increase with decreasing levels
of vitamin B-12 (fig 4). The finding that metformintreatment significantly lowered concentrations of vita-
min B-12 but did not significantly alter levels of homo-
cysteine probably reflects the relatively low incidence
of vitamin B-12 deficiency in the entire study popula-
tion. As treatment with metformin continues, however,
we expect that vitamin B-12 levels will continue to
decrease, making increases in homocysteine concen-trations inevitable in time.
Strengths of our study include the randomised, pla-
Number of available samplesMetformin group
cebo controlled, double blind design and its relatively
long follow-up of 4.3 years, as well as frequent serum
collection. Furthermore, the study was conducted in a
non-academic setting and, therefore, the findings have
Fig 3 | Concentrations of vitamin B-12, folate, and
homocysteine with 95% confidence intervals. Solid lines
Another strength is that we used last observation car-
represent the metformin group, dotted lines the placebo
ried forward in this analysis because this method is con-
group. Number of available samples for the metformin and
sidered more conservative than general mixed model
placebo group is indicated. Five patients were excluded from
analysis, “freezing” any observed divergence between
the analysis because of poor correlations between old and
two groups by retaining the last observation made. In a
newly measured vitamin B-12 values (see Methods). In
mixed model analysis with missing data, estimations of
addition, some patients had vitamin B-12 levels below 150pmol/l at baseline, at interim analysis, or at both stages, and
future observations are made on the basis of observa-
were supplemented; these individuals were also excluded.
tions made earlier in the trial, thereby reflecting adivergence more accurately but less conservatively.
Limitations of our study include the fact that we mea-
sured only total vitamin B-12 levels and not levels of
treated with metformin had a seven percentage pointgreater absolute risk of vitamin B-12 deficiency than
those treated with placebo during 4.3 years of follow-
up. In addition, the reduction in vitamin B-12 concen-
tration associated with metformin increased with time.
Current guidelines indicate that metformin is a cor-
nerstone in the treatment of type 2 diabetes, but make
no recommendations on the detection and preventionof vitamin B-12 deficiency during treatment. Our data
provide a strong case for routine assessment of vitaminB-12 levels during long term treatment with metformin.
Contributors: AK, AJMD, and CDAS are responsible for the study concept
and design. JdJ, AK, MGW, and DB collected the data, and statisticalanalysis was conducted by PL and JdJ. JdJ, AK, PL, and CDAS undertook
Fig 4 | Homocysteine concentrations with 95% confidence
analysis and interpretation of the data. JdJ, AK, and CDAS drafted the
intervals for patients with a normal vitamin B-12
manuscript, and AK, AJMD, and CDAS undertook critical revisions of themanuscript for important intellectual content. Administrative, technical or
concentration (>220 pmol/l), a low vitamin B-12 concentration
material support was provided by JvdK, DB, JV, MGW, JdJ, and AK. JdJ, AK,
(150-220 pmol/l), and vitamin B-12 deficiency (<150 pmol/l)
and PL had full access to all of the data in the study and take responsibility
after 4.3 years. The number of patients in each treatment
for the integrity of the data and the accuracy of the data analysis. JdJ, AK,
and CDAS accept full responsibility for the work and the conduct of thestudy, had access to the data, and controlled the decision to publish, and
holotranscobalamin II or methylmalonic acid, which
as such act as guarantors for the study. Funding: Hyperinsulinaemia: the Outcome of its Metabolic Effects
may have been more precise indicators of vitamin
(HOME) trial was supported by grants from Altana, Lifescan, Merck Santé,
B-12 status. Finally, it is likely that, if anything, we
Merck Sharp & Dohme, and Novo Nordisk. The sponsors had no role in the
underestimated the impact of metformin treatment on
design and conduct of the study; in the collection, analysis, and
the risk of clinically important vitamin B-12 deficiency.
interpretation of the data; or in the preparation, review, or approval of themanuscript.
We showed that metformin treatment was associated
Competing interests: All authors have completed the Unified Competing
not only with a raised risk of developing vitamin B-12
Interest form at www.icmje.org/coi_disclosure.pdf (available on request
concentrations below 150 pmol/l but also with an ele-
from the corresponding author) and all authors want to declare: (1)
vated risk of developing vitamin B-12 levels between
Financial support for the submitted work from Merck Sharp & Dohme. All
150 and 220 pmol/l, which is likely to represent clini-
authors also declare: (2) No financial relationships with commercialentities that might have an interest in the submitted work; (3) No
cally important vitamin B-12 deficiency in at least some
spouses, partners, or children with relationships with commercial entities
individuals.27 A further reason that we may have some-
that might have an interest in the submitted work; and (4) No non-
what underestimated the adverse effects of metformin
financial interests that may be relevant to the submitted work.
is that all participants in our trial received frequent diet-
Ethical approval: The medical ethical committees of the threeparticipating hospitals approved the trial protocol. The trial has been
ary counselling, which may have attenuated the impact
conducted in accordance with the Committee for Medicinal Products for
of metformin treatment on vitamin status and may not
Human Use note for guidance on good clinical practice (CPMP/ICH/135/
be available in routine clinical practice.
95), dated 17 July 1996, and in accordance with the Declaration of Helsinki(revised version of Hong Kong in 1989 and Edinburgh in 2000). Allpatients gave written informed consent.
Data sharing: No additional data available.
In conclusion, we showed that in patients with type 2diabetes being treated with insulin, those additionally
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id2198171 pdfMachine by Broadgun Software - a great PDF writer! - a great PDF creator! - http://www.pdfmachine.com http://www.broadgun.com NORMA INTERNACIONAL DE AUDITORíA 250 CONSIDERACIÓN DE LEYES Y REGLAMENTOS EN UNA AUDITORÍA DE ESTADOS FINANCIEROS (En vigor para auditorías de estados financieros por periodos que comiencen en o después del 15 de diciembre de 2004)* CONTENI