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 Table of Contents  
Year : 2022  |  Volume : 14  |  Issue : 2  |  Page : 89-93

Decreased serum B12 due to antidiabetic drugs: Is it a potential contributor to inflammation in Type II diabetes mellitus

1 Department of Physiology, Apollo Institute of Medical Sciences and Research, Hyderabad, Telangana, India
2 Professor of Medicine, Saveetha Medical College Hospital, Chennai, Tamil Nadu, India

Date of Submission21-Mar-2022
Date of Decision09-Jun-2022
Date of Acceptance13-Jun-2022
Date of Web Publication19-Jul-2022

Correspondence Address:
Divya Kanchi
Apollo Institute of Medical Sciences and Research, Hyderabad, Telangana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajprhc.ajprhc_30_22

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Background: The first-line medication, metformin, can decrease hepatic gluconeogenesis and improve insulin sensitivity. Metformin inhibits the mitochondrial respiratory chain at the molecular level, which leads to an increase in adenosine monophosphate-activated protein kinase, which enhances insulin sensitivity. With no known renal or hepatic disease, updated recommendations recommend using metformin as a first-line glucose-lowering medication in conjunction with lifestyle changes. Metformin has been demonstrated to lower vitamin B12 levels. Vitamin B12 is a vitamin that is necessary for proper hemopoietic, neurocognitive, and cardiovascular function. Vitamin B12 deficiency has been shown to be quite common in people with type 2 diabetes mellitus, both biochemically and clinically. Diabetes is thought to be an oxidative stress and chronic inflammatory disease. Vitamin B-12 is an antioxidant, as per recent research, and a low vitamin B-12 level might be a contributing factor to oxidative stress, particularly in diabetic patients. Vitamin B-12 is an antioxidant and anti-inflammatory substance that may influence oxidative stress responses, including inflammatory reactions. As a result, the goal of this research was to study at possible associations between vitamin B-12 status and inflammation in Type 2 diabetes mellitus who are on metformin and on metformin with other associated anti-diabetic drugs. Aims and Objectives: In our study we tried to see the association between serum B12 levels and proinflammatory cytokines which lead to many macrovascular complications like cardiovascular diseases, renal failure etc. Materials and Methods: The present study recruited 500 participants between 30 and 65 years of age. patient pool who has been recently diagnosed as type 2 diabetics on metformin usage for more than 12 to 18 months. The study design was divided into 3 groups: A, B, and C. Group A consisted of 200 subjects with type 2 diabetes only on metformin with a daily dosage of 500 mg/day, and Group B consisted of 200 subjects with type 2 diabetes who were on both metformin with a daily dosage of 500 mg/day and other anti-diabetic drugs of around 2 mg/day. Both groups A and B had no other complications from diabetes. Group C included controls, consisting of 100 healthy individuals with no history of diabetes. Results: The percentages of Serum B12, in both male and female groups were significantly decreased in cases and consequently, inflammatory markers like leptin, IL-6, hsCRP, TNF-α were significantly increased who were on metformin than who were on metformin and other associated drug group. Conclusions: Findings from this study clearly indicates the link between decrease in B12 and progression of inflammation. the role of metformin in vitB12 deficiency levels, uncontrolled glucose levels, and chronic inflammatory responses in T2DM patients, gradually leading to the dire consequences of B12 deficiency.

Keywords: Cobalamine, diabetes mellitus, inflammation, oxidative stress

How to cite this article:
Kanchi D, Rajendran K, Vijayan V, Dutta M, Ramreddy B. Decreased serum B12 due to antidiabetic drugs: Is it a potential contributor to inflammation in Type II diabetes mellitus. Asian J Pharm Res Health Care 2022;14:89-93

How to cite this URL:
Kanchi D, Rajendran K, Vijayan V, Dutta M, Ramreddy B. Decreased serum B12 due to antidiabetic drugs: Is it a potential contributor to inflammation in Type II diabetes mellitus. Asian J Pharm Res Health Care [serial online] 2022 [cited 2022 Sep 27];14:89-93. Available from: http://www.ajprhc.com/text.asp?2022/14/2/89/351374

  Introduction Top

Globally, the number of people with diabetes has quadrupled in the past 30 years and is the ninth leading cause of death. Currently, about 1 in 11 adults worldwide has diabetes, and 90% of them have type 2 diabetes (T2DM). India and China are the two major epicentres of the global T2DM epidemic in Asia.[1] Diabetes is a multifactorial disease and metformin is the first-line treatment. Metformin is a widely used drug with many benefits related to glucose metabolism and complications related to diabetes by improving peripheral insulin sensitivity. Physiologically, metformin is not to explain all effects by this mechanism, only by reducing the production of glucose across glucose. At the molecular level, the conclusion depends on the dose and treatment period of metformin. By inhibiting the mitochondrial glycerophosphate dehydrogenase, and amplifying the mechanisms associated with the lysosomes by inhibiting the use of AMP activated protein kinase (AMPK) dependent and AMP activated protein kinase AMPK-independent mechanisms.[2],[3],[4],[5] Vitamins have a significant impact on diabetes, a significant impact on mellitus risks, development, and results. The best offer to provide acceptable food condition is to consume a significant amount of products that include appropriate levels of vitamins.[6] Vitamin B12 is an essential vitamin for proper haematopoiesis, neurocognitive and cardiovascular function. The symptoms of Vitamin B12 deficiency can sometimes be confused, as the effects of Vitamin B12 deficiency can have multiple functions. The vitamin has antioxidant properties that can help people with diabetes stay healthy. B12 deficiency is associated with oxidative stress and hyperhomocysteinemia in diabetic patients.[7] People taking metformin have reduced B12 absorption and lower blood levels of total Vitamins B12 and TCIIB12.[8],[9],[10] Metformin use is associated with biochemical B12 deficiency over time. Individuals receiving metformin should have their Vitamin B12 levels checked on a regular basis.[11],[12],[13],[14] Low B12 levels (203 pg/mL) at 5 years (4.3% vs. 2.3%; P = 0.02) were more common in the metformin group but not at 13 years (7.4 vs. 5.4%, P = 0.12). At age 5 (19.1 vs. 9.5%; P = 0.01) and age 13 (20.3% vs. 15.6%; P = 0.02), the metformin group had a higher prevalence of combined low B12 and borderline low B12 (298 pg/ml) associated with an increased risk of B12 deficiency (odds ratio, B12 deficiency/years of metformin use, 1.13, 95% confidence interval [CI], 1.06–1.20). The prevalence of anemia was higher in the metformin group, but there was no difference in the B12 status. The prevalence of neuropathy was higher in the low B12 metformin group.[15],[16],[17]

  Materials and Methods Top

This study was conducted through collaboration between the Physiology department, Medicine OPD and the Genetics Lab. Ethical approval for this study was received 007/02/2019-/IEC/SMHC-and the study was conducted from March 2019 to February 2021. Based on WHO and ADA guidelines for the screening of type 2 diabetes mellitus, the plasma glucose criteria, the fasting plasma glucose (FPG) value or the 2-h plasma glucose value during a 75-g oral glucose tolerance test, or A1C criteria, the present study recruited 500 participants between 30 and 65 years of age. Patient pool who has been recently diagnosed as type 2 diabetics on metformin usage for more than 12 to 18 months. The study design was divided into three groups: A, B, and C. Group A consisted of 200 subjects with type 2 diabetes only on metformin with a daily dosage of 500 mg/day, and Group B consisted of 200 subjects with type 2 diabetes who were on both metformin with a daily dosage of 500 mg/day and other anti-diabetic drugs of around 2 mg/day. Both Groups A and B had no other complications from diabetes. Group C included controls, consisting of 100 healthy individuals with no history of diabetes. The study included type 2 diabetics on metformin for more than a year as cases and age-matched nondiabetic healthy volunteers as controls. People who are on Vitamin B12 supplements, no complications from liver diseases, renal diseases, gastrointestinal disorders, thyroid diseases, or parathyroid disorders. Strict vegetarians, alcoholics, and smokers were excluded. A detailed clinical history, drug history, dosage of the drug, and B12 supplementation were documented in a structured pro forma. The blood samples were collected from the subjects through vein puncture for FPG and glycated hemoglobin (HbA1c) were determined and quantified. Whole blood samples were collected into EDTA-coated vacutainers for quantification of HbA1c in the blood. Fasting and postprandial plasma glucose was estimated using the glucose oxidase-peroxidase method. Vitamin B12 was measured with the Chemiluminescent Microparticle Immuno Assay, which is a modified and advanced form of the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique. We ensured that the study complies with international ethical norms according to the Helsinki Declaration-Ethical Principles for Medical Research Involving Human Subjects (World Medical Association et al. 1964).

Estimation of inflammatory markers

Interleukin 6

Interleukin 6 (IL6) levels were quantified in the serum of the subjects by the ELISA method. A highly specific IL-6 antibody (1:1000 dilution) was coated on the microtiter plate overnight at 40°C. The IL-6 present in the sample (1:500 dilution) would bind to the specific antibody and subsequently bind to the biotinylated anti-IL-6 secondary antibody (1:8000 dilution), later incubated with Streptavidin-HRP (1:10,000 dilution) solution for 60 min. The plate was developed by incubating with TMB substrate solution for 15 min and stopped by adding 1.25 mM H2SO4 and the OD was read at 540 nm.[18]

Leptin and highly sensitive C-reactive protein

Leptin and hs-CRP in the serum were measured using the standard kit (Cal Biotech) by adopting the ELISA principle.

Tumor necrosis factor alpha

Tumor necrosis factor alpha (TNF α) was measured in subjects' serum using a commercial kit (Diaclone SAS), and the protocol was the same as described in the section (IL-6).

All data were statistically analyzed and expressed as mean standard deviation. The mean was analysed by one-way ANOVA (with a Student's t-test for comparison with controls). A Pearson correlation test was done to see the relationship between control subjects and diabetics.

  Results Top

Biochemical assays

Observations made indicate the long-term side effects of metformin alone and with metformin-other antidiabetic drugs in diabetic subjects. There is a significant rise in the blood glucose levels and inflammatory biomarkers (leptin, hsCRP, TNF-a and IL6) in both metformin alone and metformin with other diabetic drugs in comparison to the control subjects. Among them, subjects with metformin alone show a little aggravated condition of Vitamin B12 loss, which would lead to various pathies in the future due to vitB12 deficiency.

  Discussion Top

The current research on animals and clinical trials suggests that the key function of metformin is to decrease hepatic glucose production, mainly by suppressing gluconeogenesis.[19],[20] The inhibitory effects on hepatic gluconeogenesis can be due to changes in enzyme activities or suppressed hepatic uptake of gluconeogenic substrates supported by research evidence.[21] Metformin has been shown to act via both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms; by inhibition of mitochondrial respiration and also perhaps by inhibition of mitochondrial glycerol phosphate dehydrogenase.[22],[23] A rational accumulation of metformin in the liver could be higher compared to other tissues, leading to high micromolar concentrations in the per portal area. Research over a certain time period indicates metformin's action is targeted around the intestines, by reducing the net glucose uptake and enhancing anaerobic glycolysis in enterocytes, causing an increased release of lactic acid in the liver. The molecular level findings vary depending on the dosage of metformin and the duration of treatment, with some differences between acute and chronic administration. Some studies showed T2DM patients on metformin treatment had lower levels of B12. Similar findings were reported by Roy et al. that patients on metformin had a lower level of B12.[24] According to the DPPOS study, an increased risk of B12 deficiency is shown to have an association with metformin.[25] A recent study from India has shown an association between metformin use and B12 levels.[26] Similar studies by Den Elen et al. on B12 deficiency in older adults with prolonged proton-pump inhibitors and H2 blockers have also reported hypocobalinemi. A meta-analysis was done by Chapman et al. on the B12 lowering effect of metformin, which took approximately 6 weeks to 3 months after commencing metformin. All these studies were in correlation with our studies showing decreased B12 levels with oral anti-diabetic drug usage. We found B12 levels had significantly decreased by about 82% in the metformin group and 62% in the associated anti-diabetic drug group when compared with controls. According to Reinstatler et al., metformin therapy is associated with a higher prevalence of biochemical B12 deficiency. Absorption of Vitamin B12 is a multifactorial process and depends on factors like Haptocorrin (HC), also commonly known as the R-protein secreted by the salivary glands of the oral cavity, which binds to Vitamin B12, which is an intricate and necessary mechanism to protect against the acidic environment of the stomach. In this complex, HC attaches to Vitamin B12 to create a complex, and this HC–B12 complex passes via the pylorus to the duodenum, where HC is cleaved from the complex in the presence of pancreatic proteases. Intrinsic factor (IF) is a glycoprotein secreted by parietal cells of the gastric mucosa and the IF binds to the free Vitamin B12 to form the Vitamin B12–IF complex. Then, the Vitamin B12 is transported to the ileum by the ileal receptor (i.e., Cubam) which comprises the Cubilin and Megalin complex, and takes them up into the circulation via endocytosis-mediated absorption. Hence, in our study, patients with type 2 diabetes developed a marked reduction in serum Vitamin B12 levels in patients treated with metformin alone for T2DM. We have also witnessed that there is an association between pro inflammatory markers with the levels of fasting blood sugar, postprandial blood sugar, HbA1C, and serum B12 in subjects with metformin alone and also with other diabetic drugs [Table 1]. There is upregulation of pro inflammatory markers in the metformin group when compared with Other associated drug group and control group.[27],[28] Further research is required to find the association between antidiabetic drugs and levels of proinflammatory markers in T2DM [Table 2].
Table 1: Mean±SD values of different variables by groups

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Table 2: The standard reference range

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  Conclusion Top

Vitamin B12 is a pivotal micronutrient required for maintaining essential functions such as hematopoiesis, neuro-cognition, and cardiovascular functions. Our retrospective study has given us important proof against the antidiabetic drugs commonly advised to T2DM patients for controlling their blood glucose levels, but instead they are facing the inevitable consequence of being Vitamin B12 deficient and its associated manifestations. The strength of our study is the strong association between B12 levels and inflammation. Type 2 diabetes itself is a chronic inflammatory disease the first-line drug is metformin which decreases serum B12 as a major side effect. Which itself is a potent stimulator for inflammation which leads to microvascular and macrovascular complications like retinopathy, nephropathy, microvascular neuropathy, ischemic heart disease, peripheral vascular disease, and cerebrovascular disease due to decreased scavenging of reactive oxygen species, by increasing homocysteine induced oxidative stress when compared between both the drug groups, serum B12 has significantly decreased in metformin group than other antidiabetic drug group. Future large and well-designed studies on screening for Vitamin B12 deficiency, Vitamin B12 supplementation, and optimal supplementation dose among type 2 diabetic patients are warranted to help guide formulation of guidelines in diabetes clinical care.

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Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2]


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