Prevalence of Vitamin B12 Deficiency in Type 2 Diabetes Mellitus Patients on Metformin Therapy

Background Long-term metformin treatment in individuals with type 2 diabetes mellitus causes vitamin B12 insufficiency, which is typically neglected, undetected, and under-treated. A severe deficit may cause life-threatening neurological problems. This study assessed the prevalence of vitamin B12 deficiencies among T2DM patients and its factors at a tertiary hospital in the Tamil Nadu district of Salem. Materials and Methods This is an analytical cross-sectional study conducted in a tertiary care hospital in the Salem district, Tamil Nadu, India. Patients with type 2 diabetes mellitus who were prescribed metformin at the outpatient department of general medicine took part in the trial. Our research instrument was a structured questionnaire. We used a questionnaire containing information on sociodemographic characteristics, metformin use among diabetic mellitus patients, diabetes mellitus history, lifestyle behaviors, anthropometric measurement, examination findings, and biochemical markers. Prior to administering the interview schedule, each participant's parents provided written informed consent. A thorough medical history, physical exam, and anthropometric examination were performed. Data were entered in Microsoft Excel (Microsoft Corporation, Redmond, WA) and analyzed using SPSS version 23 (IBM Corp., Armonk, NY). Results Among the study participants, we diagnosed nearly 43% of diabetes cases in participants between the ages of 40-50 years, while we diagnosed 39% aged under 40 years. Nearly 51% had diabetes for 5-10 years, while only 14% had diabetes for over 10 years. In addition, 25% of the study sample had a positive family history of type 2 diabetes. Nearly 48% and 13% of the study group had been on metformin for 5-10 years and >10 years, respectively. The majority, 45%, were found to take 1000 mg of metformin per day, whereas just 15% take 2 g per day. In our study, the prevalence of vitamin B12 insufficiency was 27%, and nearly 18% had borderline levels. The duration of diabetes mellitus, the duration of metformin intake, and the dose of metformin were statistically significant (p-value = 0.05) among the variables associated with diabetes mellitus and vitamin B12 deficiency. Conclusion The results of the study show that a deficiency in vitamin B12 increases the likelihood that diabetic neuropathy would worsen. Therefore, individuals with diabetes who take larger dosages of metformin (more than 1000mg) for an extended period must have their vitamin B12 levels monitored often. Preventative or therapeutic vitamin B12 supplementation can mitigate this issue.


Introduction
Hyperglycemia is a symptom of the metabolic disorder diabetes mellitus, which is characterized by difficulties in the breakdown of carbohydrates, lipids, and proteins. Patients with diabetes are deficient in absolute or relative insulin. Diabetes mellitus is a significant noncommunicable disease whose prevalence has exploded during the past two decades [1]. In the technologically advanced world of the twenty-first century, diabetes mellitus has arisen as a significant threat to public health, and with time it has become an epidemic that cannot be ignored [2,3].
According to a recent report by the World Health Organization (WHO), India has the highest number of diabetics in the world, surpassing China [4]. Diabetes mellitus affects around 88 million Southeast Asians, a number that is projected to rise to 153 million by 2045. Diabetes mellitus affects around 77 million people in India between the ages of 20 and 79, accounting for 8.9% of the population [5].
In the "biguanide" class, metformin is an anti-hyperglycemic medication. Metformin decreases blood glucose levels and is the cornerstone of treatment for type 2 diabetes [6]. Metformin decreases the insulin resistance characteristic of T2DM by enhancing insulin sensitivity. If glycemic control is insufficient, we may use metformin alone or in combination with other oral hypoglycemic medications [7].
Cobalamin is an important B vitamin that is water soluble. Vitamin B12 plays a significant role in normal brain function (both central nervous system and peripheral nervous system), DNA synthesis, and red blood cell (RBC) synthesis [8]. Vitamin B12 is naturally abundant in non-vegetarian diets, and the recommended daily requirement (RDA) is 2.4 micrograms per day. A normal vitamin B12 concentration exceeds 221 pmol/L in the human body [9]. Nearly 50% of subclinical vitamin B12-deficient patients have normal vitamin B12 levels [10].
Metformin inhibits calcium-dependent channels in the ileum, hence preventing the absorption of vitamin B12 in the ileum. Several studies have shown that extended use of metformin causes vitamin B12 insufficiency through this mechanism [11][12][13][14][15][16]. A severe deficit may cause significant neurological problems [17]. According to the literature, 30% of T2DM patients on long-term metformin exhibited vitamin B12 malabsorption, and multiple studies conducted in various regions of the world suggest that metformin medication reduces serum vitamin B12 levels by 14% to 30% [18][19][20][21].
The literature on vitamin B12 deficiency is also devoid of findings, and they have undertaken few investigations on the frequency of vitamin B12 deficiency in Tamil Nadu. This study assessed the frequency of vitamin B12 deficiencies among T2DM patients and its causes at a tertiary hospital in the Tamil Nadu district of Salem.

Study setting and study period
We conducted this study as an analytical cross-sectional study in a hospital of tertiary care in the Tamil Nadu district of Salem. Participants in the study were type 2 diabetics using metformin who visited the General Medicine outpatient department. We conducted the study from November 2020 to April 2021, six months.

Sample size estimation
We estimated the sample size based on a 2012 study by Reinstatler et al. in the United States, which found a prevalence of vitamin B12 deficiency of 5.8% [22]. We calculated the sample size using the formula, N = 3.84*p*q/ d2. The sample size was 83, allowing for a non-response rate of 15%. We computed the final sample size to be 96, rounded up to 100.

Selection criteria
The inclusion criteria were patients having type 2 diabetes for at least two years, aged between 30 and 75 years, free of urinary tract infection complaints, taking metformin up to the maximum daily dose of 2,000 mg, and willing to participate in the study. Patients with type 1 diabetes, type 2 diabetes mellitus patients on insulin therapy or mixed therapy (insulin with metformin or other hypoglycemic drugs), pregnant women, patients with pernicious anemia or a history of malabsorption syndrome after gastrointestinal surgery, patients with chronic hepatitis, and those with hypothyroid disorders were excluded from the study.

Ethics clearance
Before conducting the research, we got approval from the Institutional Ethics Committee of Medical College and Hospital (No: VMKVMC&H/ IEC/20/08). The informed consent was drafted in the local language and was based on ICMR criteria. Before beginning the study, we received approval from our institution's Ethics Committee.

Data collection procedure
We adopted a semi-structured questionnaire to get data from study participants through interviews. We used a questionnaire containing information on sociodemographic characteristics, metformin use among diabetes mellitus patients, diabetes mellitus history, lifestyle behaviors, anthropometric measurement, examination findings, and biochemical markers. After receiving consent, a single investigator performed the interview, and their responses were documented on the questionnaire. Each participant was interviewed for 10 to 15 minutes. A thorough medical history was taken and after collecting blood samples, biochemical analyses were conducted.

Data analysis
Data were entered in Microsoft Excel (Microsoft Corporation, Redmond, WA) and analyzed using SPSS v. 23 (IBM Corp., Armonk, NY). Descriptive and analytic statistics were used for our data analysis. We carried out a descriptive analysis and presented qualitative data as frequencies and percentages as well as means and standard deviations for qualitative data. The odds ratio was determined, and the chi-square test was employed for statistical analysis (a p-value of less than 0.05 is considered as significant).

Results
About 47% of samples were of patients aged 40-50 years, while only 12% were over the age of 60. The respondents' average age was 49.93 ± 9.58 years. In our survey, there was a preponderance of men (55%), and only 45% were females. Around 16% of the population was illiterate, while 31% had completed middle school and 22% had completed high school. Regarding occupation, 25% were unemployed, while 40% and 24% were engaged in unskilled and semi-skilled occupations, respectively ( Table 1).  About 22% of the individuals in the study were smokers, while 37% of the participants consumed alcohol regularly. Only 12% were hypertensive, 6% had chronic obstructive pulmonary disease (COPD), and 4% had coronary artery disease.
Around 43% of diabetic patients were diagnosed between the ages of 40 and 50, whereas 39% of diabetic patients were diagnosed before the age of 40. Nearly half had diabetes mellitus for 5-10 years, while only 14% had diabetes for beyond 10 years. In addition, 25% of the study sample had a positive family history of type 2 diabetes. Estimated 48% and 13% of the study group had been on metformin for 5-10 years and > 10 years, respectively. Around 45% of patients were taking 1000 mg of metformin per day, whereas just 15% were taking 2 g per day. About 45% of responders on metformin report taking 500 mg twice daily, while 39% were taking 500 mg three times daily.
In our study, 27% of participants had vitamin B12 deficiency, approximately 18% had borderline levels, and the mean vitamin B12 level was 308.6 ± 6115.58 pmol/L. Around 27% of respondents were deficient in vitamin B12 (148 pmol/Litre), whereas 55% were normal (> 221 pmol/litre) ( Table 2).  In our study, the duration of diabetes mellitus, duration of metformin intake, and dose of metformin were statistically significant (p-value <0.05) among the variables related to diabetes mellitus and vitamin B12 deficiency. We described the association between variables related to diabetes mellitus and vitamin B12 deficiency among the study respondents in Table 3.
In our study, the mean duration of metformin medication was 6.42 ± 3.37 years, whereas, in studies by Nearly 45% were taking 1000 mg/day of metformin and just 15% were taking 2 g/day in the present study. In the study by Krishnan et al.,83.9% of patients were taking over 1000 mg of metformin per day [34]. In research conducted by Ko et al., about 62.5% were on a daily metformin dose of 1000 mg, and 12% were on a daily metformin dose of 2000 mg [35]. The study by Chattopadhyay et al. showed that 57% of participants took over 1000 mg to 2 g of metformin daily, and 41% took over 500 mg to 1000 mg daily [27].
Using proton pump inhibitors and H2 blockers, as well as the duration of metformin therapy, was found to be significantly associated with vitamin B12 deficiency in the Damiao et al. study [39]. In the Krishnan et al. study, non-Malay race and diabetes duration were significantly associated with vitamin B12 deficiency [34]. HbA1c, homocysteine levels, metformin duration, and dose were all associated with vitamin B12 deficiency in the Miyan et al. study [26].
In our study, vitamin B12 deficiency was negatively correlated with the duration and daily dose of metformin therapy and positively correlated with hemoglobin values. We observed comparable results in studies by Sato

Limitations
Our study had a few limitations. First, the study was conducted with a smaller number of samples because of a lack of resources. However, the results might be generalized to a comparable population in a comparable setting. We did not perform nerve conduction investigations and neuropathy screening because of logistical difficulties. A cross-sectional examination of causal relationships and risk estimates is not feasible. A future analytic investigation (case-control or cohort study) in the study population can circumvent this constraint. Patients on combined therapy (metformin plus other medicines) were excluded, which restricts the generalizability of the study. These patients may experience a higher incidence of B12 deficiency and/or neuropathy (or lower).

Conclusions
According to the findings of the study, a large proportion of the sample group was deficient in vitamin B12. This study also demonstrates that there are gaps in the identification and treatment of vitamin B12 deficiency in the study area, as it is frequently undiagnosed. The longer duration and higher daily dose of metformin were the risk factors for vitamin B12 insufficiency. A vitamin B12 shortage increases the likelihood that diabetic neuropathy may worsen. Therefore, monthly vitamin B12 measurement is required for diabetic patients on long-term, high-dose metformin. Preventative or therapeutic vitamin B12 supplementation can mitigate this issue.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Institutional Ethics Committee of Kirupananda Variyar Medical College and Hospitals issued approval VMKVMC&H/ IEC/20/08. Before conducting the research, we got approval from the Institutional Ethics Committee of Medical College and Hospital. The informed consent was drafted in the local language and was based on ICMR criteria. Before beginning the study, we received approval from our institution's Ethics Committee. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.