Vitamin B12 deficiency in metformin-treated type-2 diabetes patients, prevalence and association with peripheral neuropathy

Prevalence of vitamin B12 deficiency

The present study has found that the prevalence of vitamin B12 deficiency, as defined
by serum levels 150 pmol/L, among T2DM patients receiving metformin was 28.1 %. Comparing
the obtained prevalence with the results of previous studies is not straightforward
and should consider several factors. Table 6 shows the prevalence 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, including mean participants
age, mean metformin daily dose, study settings, mean metformin duration of use and
whether participants with renal impairment were excluded. Our reported prevalence
is high relative to previously 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
deficiency in our sample an unattainable task. The present study had the highest means
of metformin dose and duration of use, possibly explaining the high prevalence obtained.

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

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’s
rho?=?0.056, P?=?0.54). Our results are in-line with those of a recently published study which reported
that metformin 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 revealed no significant differences between metformin users and
non-users when neuropathy status was assessed by both objective (monofilament and
neurothesiometry) and relatively subjective (questionnaires) measures 14]. Biemans et al. also reported no 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 glycemic control-independent neuroprotective
impact of metformin. Mao-Ying et al. found that metformin reduced peripheral nerve endings loss and exerted protective
effect against chemotherapy-induced peripheral neuropathy (CIPN) in mice 21]. Both peripheral neuropathy and CIPN share the glove-and-stocking distribution nature
of sensory symptoms that includes parasthesia, 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 medication affects the neuropathy status, excluding that related to glycemic control.
One involves a positive impact through neuroprotective mechanisms, while the other
induces neuropathy by enhancing vitamin B12 deficiency. 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 significant 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 abnormality 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 commented 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 neuropathy in the
control and metformin groups may thus produce distorted results that do not truly
reflect the contribution of the metformin-induced vitamin B12 deficiency to the neuropathy
status. From this perspective, designs that compare peripheral neuropathy among vitamin
B12-deficient and –normal metformin-treated patients 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, similar results 10]. They reported stronger association between 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
explain the difference in magnitude of OR between the 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 metformin reactions, adding to the high prevalence of gastrointestinal complications
in diabetes patients, may propose higher risk of non-compliance among metformin users.
Higher doses of metformin may result in more prominent gastrointestinal adverse reactions
and higher rates of non-compliance. Our theory is that patients with poor glycemic
control (higher HbA1c) may have poor compliance to metformin and thus higher vitamin
B12 levels. Raw data from the present study revealed that 21.5 % of participants were
on the maximum daily dose of metformin, possibly supporting our proposed 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 inappropriate as
the relationship existed despite adjusting for metformin dose and eGFR (Table 4).

Black South African descent was a significant protective 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 vitamin levels among black, white
and Hispanic metformin-treated patients in the United States 4]. Higher vitamin B12 levels in black compared to white general populations 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 significance 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 multiple 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 metformin. The study has only measured serum
vitamin B12 levels to assess the vitamin status. Current recommendations suggest adding
methylmalonic acid or homocysteine 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 clinically important difference as small as 10 %
to be statistically 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].