Impact of metformin treatment on cobalamin status in persons with type 2 diabetes

Abstract Over the last decades, low vitamin B12 status has been reported in individuals with type 2 diabetes mellitus (T2DM). Metformin, the first-line therapy for lowering blood glucose, is the main driving factor behind this association. Although the relationship between vitamin B12 deficiency and metformin is well established, results of studies on the exact effect of the dose and duration of the therapy remain inconsistent. Additionally, a lack of consensus on the definition of vitamin B12 deficiency adds to the conflicting literature. The objectives of this review were to analyze and synthesize the findings on the effects of metformin dose and duration on vitamin B12 status in patients with T2DM and to outline the potential mechanisms underlying metformin’s effect on vitamin B12. Metformin therapy has adversely affected serum vitamin B12 concentrations, a marker of vitamin B12 status. The metformin usage index (a composite score of metformin dose and duration) might serve as a potential risk assessment tool for vitamin B12 screening in patients with T2DM. Considering the health implications of suboptimal vitamin B12 status, vitamin B12 concentrations should be monitored periodically in high-risk patients, such as vegans who are receiving metformin therapy for T2DM. Additionally, it is prudent to implement lifestyle strategies concurrent with metformin therapy in individuals with T2DM, promoting an overall synergistic effect on their glycemic control.


INTRODUCTION
Vitamin B 12 (cobalamin), a hydrophilic vitamer, is derived primarily from foods of animal origin such as dairy, meat, and eggs. 1 Several foods such as breakfast cerelas, bread, and snack bars are fortified with vitamin B 12 , providing an alternate source of vitamin B 12 for vegans and vegetarians.In food, vitamin B 12 is bound to protein and requires cleaving into its free form by hydrochloric acid in the gastric lumen before absorption. 2 After cleavage, haptocorrin, a glycoprotein, binds to vitamin B 12 .This bound form is then cleaved by pancreatic proteases in the duodenum.The resulting free cobalamin binds to a carrier protein called intrinsic factor (IF). IF facilitates cobalamin absorption in the small intestine and its subsequent release into circulation via the intestinal brush border.Vitamin B 12 is delivered to the liver and peripheral tissues attached to the transport proteins haptocorrin or transcobalamin II.Haptocorrin-bound vitamin B 12 is delivered primarily to the liver because of a lack of haptocorrin receptors on peripheral cells, and accounts for 70%-80% of the circulating vitamin B 12 .Transcobalamin II chiefly supplies vitamin B 12 to all cells, though only 20%-30% of serum cobalamin is bound to transcobalamin II as holotranscobalamin in the blood. 3n adults with healthy gastrointestinal function, vitamin B 12 bioavailability ranges from 4.5% to 83%, depending on the food source and saturation of digestive proteins.Bioavailability may be lower in those with less-than-optimal gastric acid, IF, and transport carrier protein. 1,3,4Vitamin B 12 acts as a coenzyme for 2 mammalian enzymes.Methionine synthase remethylates homocysteine to methionine, generating tetrahydrofolate for the synthesis of nucleic acids.It also holds neurological significance through the maintenance of the myelin sheath in nerve cells via methionine and subsequent S-adenosylmethionine production. 5In the mitochondria, methylmalonyl coenzyme A (CoA) is converted to succinyl CoA, which is catalyzed by methylmalonyl CoA mutase.This enzyme is dependent on Sadenosylcobalamin, a vitamin B 12 coenzyme.This biochemical reaction is part of the catabolic pathways of methionine, isoleucine, valine, threonine, cholesterol side chains, and odd-numbered carbon chain fatty acids.][8] The deficiency of cobalamin is diagnosed using serum cobalamin concentrations with varying cutoff values based on the testing laboratory's diagnostic criteria.No gold standard exists for measuring cobalamin status, but serum cobalamin concentrations <148 pmol/L are used for diagnosing vitamin B 12 deficiency. 9Although sensitive, this test has a low specificity. 10Serum vitamin B 12 tests measure total serum cobalamin bound to haptocorrin and transcobalamin II and do not reflect true tissue stores of cobalamin.Tests measuring holotranscobalamin (cobalamin-bound transcobalamin II), the bioactive vitamin B 12 form, can be used as an early indicator of vitamin B 12 malabsorption because of its short half-life. 11Assessing vitamin B 12 status using 1 functional marker of vitamin B 12 deficiency (elevated total homocysteine [tHcy] or MMA) and 1 marker of serum cobalamin (total serum cobalamin or holotranscobalamin) has been suggested to accurately identify cobalamin deficiency in symptomatic individuals with normal serum cobalamin status. 12,13imited dietary intake and absorption, among other causes, may affect vitamin B 12 status and lead to its deficiency (Table 1). 9,14,157][18] The oral hypoglycemic agent metformin has been implicated in vitamin B 12 deficiency.
Metformin is widely used as the first choice medication for the treatment of type 2 diabetes mellitus (T2DM).It is considered safe, economical, and efficacious in improving glycated hemoglobin concentrations. 19T2DM is a global health concern with a prevalence of 462 million cases worldwide. 20In this narrative review, the evidence on cobalamin status in metformin-treated patients with T2DM was analyzed and synthesized.

LITERATURE SELECTION
Relevant studies on the association between metformin and vitamin B 12 status were searched in the PubMed, Cochrane, SCOPUS, and Embase databases.2][23] The search was not limited to study design, though studies with larger sample sizes were given more priority.Studies assessing the specific influence of the dose and duration of metformin were also prioritized.Studies with sample sizes smaller than 250 were excluded.The following search items were used: (((Metformin/) OR (Diabetes Mellitus/)) OR (Diabetes Mellitus, Type 2/)) AND (((Vitamin B 12/) OR (Vitamin B 12 Deficiency/)) OR (Cobalamin)).Some articles were identified through manual searching and reference tracking.

Metformin-associated vitamin B 12 deficiency: analysis of clinical evidence
A summary of selected studies on the relationship between metformin treatment and cobalamin status is presented in Table 2. [24][25][26][27][28][29][30][31][32][33] The relationship between metformin therapy and cobalamin deficiency has long been documented. 34First published in the late 1960s, reports of this relationship demonstrated a decreased absorption of cobalamin in patients who were taking metformin for T2DM. 35,36In a large systematic review of research conducted up until 2013, the majority of included observational studies (59%) revealed significantly lower concentrations of vitamin B 12 in patients with T2DM undergoing metformin therapy compared with those who were not taking metformin. 21In that study, a meta-analysis of 4 intervention trials showed a mean reduction of 57 pmol/L in vitamin B 12 concentrations after 6 weeks to 3 months of metformin use (weighted mean difference, -57.1; 95%CI, -35.5, -78.8). 21ecause of significant heterogeneity between the studies (I 2 ¼ 72%), the authors suggested interpreting the results with caution.5][26][27][28][29][30][31][32][33] However, the focus of the literature began to shift toward determining the impact of metformin dosage and duration of treatment of metformin on serum cobalamin concentrations.One large cross-sectional study in Korea demonstrated that for every 1-mg increase in daily metformin dose, vitamin B 12 concentrations decreased by 0.142 pg/mL (95%CI, -0.169, -0.114). 28When compared with a group taking <1000 mg metformin daily, groups taking 1500-2000 mg or 2000 mg had odds ratios (ORs) of 3.34 (95%CI, 1.95, 5.75), and 8.67 (95%CI, 4.68, 16.06), respectively. 28In another study, the OR for vitamin B 12 deficiency at higher doses of metformin (2000 mg vs 1000 mg) increased to 3.8 (95%CI, 1.82, 7.92). 29On the contrary, after adjustment for confounding factors, some studies found no effect of metformin dose on serum concentrations of vitamin B 12 . 33,37,38imilarly, findings on the effect of metformin duration on vitamin B 12 status remain inconsistent.One large, retrospective cohort of adult patients (n ¼ 13 489) found that the average time to develop vitamin B 12 deficiency after metformin initiation was 5.3 years. 30A post hoc analysis of a randomized control trial found that for every year of metformin use, the OR of cobalamin deficiency increased by 13% (OR, 1.13; 95%CI, 1.06, 1.20). 33In contrast, a cross-sectional study in the Netherlands (n ¼ 298) found that the duration of metformin use had no effect (OR, 0.98; 95%CI, 0.87, 1.11). 24Similarly, another cross-sectional study found no relationship between metformin use and concentration of cobalamin (b ¼ -0.14; 95%CI, -0.44, 0.16) or holotranscobalamin (b ¼ 0.003; 95%CI, -0.09, 0.09), a marker for cellular cobalamin deficiency. 25ecause most studies only examined the impact of the dose and duration of metformin treatment, little is known about their cumulative impact on the circulating concentrations of vitamin B 12 .A recent observational study assessed the additive effect of both metformin dose and metformin duration. 31In that study, authors used a metformin usage index (MUI), calculated as the daily metformin dose (in milligrams) multiplied by the duration of metformin use (in years), then divide the result by 1000.Interestingly, after multivariable adjustment in logistic regression analysis, MUI was determined to be the most significant predictor of deficiency of cobalamin.Furthermore, the risk for cobalamin deficiency proportionally increased with MUI.Compared with non-metformin users, the highest odds for vitamin B 12 deficiency were seen in metformin users with MUI >15 (OR, 6.7; 95%CI, 4.4, 10), followed by those who had an MUI >10 (OR, 5.1; 95%CI, 3.1, 8.5).The OR decreased to 1.37 (95%CI, 0.9, 2.2) in individuals who had MUI <5.Hence, the MUI has been proposed as a valid assessment tool to identify people at high risk for cobalamin deficiency and aid in providing appropriate strategies for interventions.

DISCUSSION
Metformin therapy negatively affected serum vitamin B 12 concentrations in patients with T2DM.However, the findings related to the association between metformin dose and duration with serum vitamin B 12 concentrations are inconsistent.These inconsistent observations can be attributed to several factors.One is that there was no agreement on the definition of cobalamin deficiency, which resulted in variations in the cutoff concentrations, rendering their comparisons difficult.Because of variability in immunoassays and measurement methods, serum cobalamin concentration may not be a reliable biomarker of overall cobalamin deficiency.Some studies lacked comparison groups and others were limited by the absence of sufficient data to compare between metformin users and metformin non-users.Furthermore, other sources of heterogeneity across the studies included wide differences in population characteristics and sample size.

Metformin and vitamin B 12 status: plausible mechanisms
0][41][42] The most plausible mechanism is impaired calcium-mediated uptake of the IF-cobalamin complex to the ileum via the cubilin receptor.Cobalamin absorption occurs in the distal part of the small intestine and is calcium dependent.Here, metformin affects the membrane receptor function by modifying its membrane potential and limiting calcium-dependent vitamin B 12 absorption.Metformin's hydrophobic tail attaches to the cell membrane's hydrophobic core, resulting in a net positive charge that repels calcium cations. 43In 1 study, calcium supplementation was shown to reverse vitamin B 12 malabsorption. 42nother proposed mechanism involves metformininduced impaired motility of the small intestine.Metformin improves the glucose profile through a series of mechanisms, 1 of which is increased intestinal transit time. 44This may alter gut microbiome composition and lead to small intestinal microbial overgrowth.Intestinal bacteria use vitamin B 12 for metabolic processes, and the resulting metabolites compete with cobalamin absorption, inhibiting the binding of the IFcobalamin complex to receptors on the ileal mucosa.Another mechanism is that metformin may lower the secretion of IF by gastric cells and reduce concentrations of cobalamin.Additionally, the distribution and metabolism of cobalamin in the tissues may be altered due to metformin-induced cobalamin accumulation in the liver (Figure 1).

Recommendations
Vitamin B 12 deficiency results in megaloblastic/macrocytic anemia, paresthesia, cognitive impairment, and other neurological manifestations.Elevated circulating tHcy concentration is an indicator of cardiac disease and inflammation and is observed in those with vitamin B 12 deficiency. 8Although vitamin B 12 -associated hyperhomocysteinemia does not increase cardiovascular disease risk, an increase in cardiovascular disease mortality has been reported in those with T2DM and low serum vitamin B 12 concentrations, independent of tHcy concentrations. 45,46n light of the associated health consequences of cobalamin deficiency, it is prudent to periodically screen for vitamin B 12 deficiency in patients who are receiving metformin therapy for T2DM.][49][50][51] Therefore, vitamin B 12 deficiency should be corrected while emphasizing the importance of maintaining a prudent lifestyle to improve glycemic control in patients with T2DM.Furthermore, when screening for deficiency of cobalamin in people with T2DM, it is important to account for both metformin dose and duration.Based on the findings, MUI appeared to be a valid assessment tool that can be incorporated into vitamin B 12 screening for patients with T2DM who are receiving metformin treatment. 31For example, an MUI >5 could serve as the threshold concentration for vitamin B 12 screening in these patients.
3][54][55] Because metformin dose and duration are inversely associated with serum vitamin B 12 concentrations, lifestyle measures focusing on physical activity and prudent dietary practices with concurrent medication use are warranted for a synergistic effect on glycemic control.7][58] Individuals with T2DM should aim for at least 150 minutes of moderate physical activity weekly, with resistance training 2 or 3 times per week, according to the American College of Sports Medicine and the American Diabetes Association. 59iet-based recommendations for patients with diabetes include the consumption of fiber-rich foods (eg, vegetables, fruits, whole grains, legumes), lean meats, and low-fat dairy products, with a focus on foods of low glycemic index or glycemic load. 60,613][64][65][66] Although plant-based diets are beneficial in the management of blood glucose, they are severely deficient in vitamin B 12 . 67Therefore, vegans who are receiving metformin therapy should be tested for vitamin B 12 status periodically.Vegans may also benefit from consuming alternate sources of vitamin B 12 , such as fortified foods, nutritional yeast, fermented nondairy foods, and seaweed.

Limitations
The main limitation of the reserach is the lack of a gold standard for the assessment of cobalamin status.As a result, investigators have used various biomarkers of cobalamin to define cobalamin status.The most commonly used marker is serum vitamin B 12 concentration.Serum vitamin B 12 includes haptocorrin-bound vitamin B 12 and transcobalamin II-bound vitamin B 12 .Haptocorrin-bound vitamin B 12 , an inactive form, constitutes the majority of total serum cobalamin, whereas transcobalamin II-bound vitamin B 12 , an active form capable of delivering cobalamin to tissues, constitutes a minor portion of total serum cobalamin.Because of this, persons with low normal concentration of serum vitamin B 12 may have a tissue deficiency.Another marker of vitamin B 12 status is circulating tHcy.Circulating tHcy concentration lacks specificity because tHcy is elevated not only in vitamin B 12 deficiency but also in folate, riboflavin, and pyridoxine deficiencies.Another marker of cobalamin status is serum MMA concentration.Serum MMA level is also elevated in kidney dysfunction.Perhaps the best marker of cobalamin status is serum transcobalamin II, because it represents the tissue-deliverable vitamin B 12 .However, it also lacks specificity because, in kidney dysfunction, transcobalamin II level is also elevated.Kidney dysfunction is a common comorbidity associated with diabetes, so in patients with diabetes, the cobalamin status may be overestimated if transcobalamin II, MMA, or tHcy concentrations were used.
Another limitation of the research is the lack of data on how much oral vitamin B 12 and at what dosage level of metformin should be increased in patients with T2DM.Therefore, based on the current evidence, it is not possible to recommend a very precise amount of dietary vitamin B 12 for those who are receiving metformin therapy.

CONCLUSIONS
Evidence for the relationship between metformin use and low serum vitamin B 12 concentrations is relatively strong.By and large, the data are somewhat consistent, showing an inverse relationship between the dose and duration of metformin use and serum cobalamin concentrations.The deficiency of vitamin B 12 results in anemia, hyperhomocysteinemia, elevated MMA level, and nerve-related dysfunction, warranting periodic screening for cobalamin deficiency in high-risk groups such as vegans.Healthy lifestyles with medication to improve glycemic control should be emphasized in T2DM management.The potential of lifestyle approaches in lowering metformin dose and improving B 12 status needs to be further explored.
T2DM mainly afflicts persons of middle age or older.As individuals with T2DM age, the risk of developing suboptimal cobalamin status would also increase because of decreased absorption of cobalamin from the aging gut due to increased gastric atrophy resulting from bacterial overgrowth.Therefore, more studies are needed to establish the dietary vitamin B 12 allowance for each metformin dose.
There is sufficient evidence in the literature to recommend the use of the MUI as a tool for risk assessment.Also, because of the lack of a gold standard for the measurement of cobalamin nutritional status, clinicians should consider using the MUI as a marker of cobalamin status when screening for cobalamin deficiency in individuals with T2DM.

Figure 1
Figure 1 Metabolism of vitamin B 12 and proposed mechanisms of metformin-induced vitamin B 12 deficiency.The green arrow depicts the vitamin B 12 accumulation effect of metformin in the liver.The blunted red arrow represents metformin-associated inhibition of calciumdependent vitamin B 12 uptake into the ileum.Abbreviations: B 12 , vitamin B 12 ; Ca 21 , calcium ion; HC, haptocorrin; HCl, hydrochloric acid; IF, intrinsic factor; PP, pancreatic proteases; Pro, protein; TCII, transcobalamin II.

Table 1
Etiology and pathogenesis of vitamin B12 deficiency

Table 2
Summary of studies on vitamin B12 status in relation to metformin therapy in people with type 2 diabetes mellitus