This was a cross-sectional study that was conducted in the outpatient Clinics of Ain Shams University Hospitals in Egypt during September 2019- September 2020. The study recruited 100 type 2 diabetic patients aging from 30 to 70 years old who were divided into 2 groups: the first group included 50 patients receiving metformin therapy and the other group included 50 patients receiving other anti-diabetic therapies.
The recruited patients fulfilled the following inclusion criteria: metformin group that included patients receiving at least 1 g metformin per day and for a duration ≥ 6 months. And non-metformin group that included patients receiving other anti-diabetic drugs than metformin for the last 6 months. While the exclusion criteria included those: who were taking vitamin B12 supplements in the last 3 months; those taking H2 blockers, antacids, or proton pump inhibitors; pregnant females, type 1 diabetes, strictly vegetarian or alcoholic patients. In addition, patients having pernicious anemia, renal insufficiency, HIV infection or malabsorption syndromes or other causes of malabsorption like gastric bypass, gastrectomy or any surgery involving the small intestine were excluded.
The study sample was calculated by Power and Sample Size Program, version 3.1 and based on the results of the recent study of Osama and colleagues, 2016 conducted in Fayoum University Hospital outpatient clinics. The proportion of peripheral neuropathy in patients taking metformin: was 80%, the average of proportion of peripheral neuropathy in patients not taking metformin from two studies was 57% [15, 16]. The provisional sample size in every group was 49 patients to be able to reject the null hypothesis that the exposure rates for the 2 groups are equal, with power 80% and type 1 error 10% . Uncorrected Chi-squared statistic was used to evaluate this null hypothesis.
The collected data included personal and medical history like age, gender, duration of diabetes, type of anti-diabetic medication taken by the patients, its dose and duration. In addition to dietetic history that included frequency of eating food rich in vitamin B12. These food items were selected according to Dietitian Canada Canadian Nutrient file  to evaluate its consumption by the study participants in term of frequency (daily, weekly, monthly and seasonal) and the portion in each time. These data were then analyzed by ESHA (Elizabeth Stewart Hands and Associates) Research Food Processor Nutrition and Fitness Software version 11.7 update in 2019 (Inc., Salem, OR, USA.) to calculate the estimated vitamin B12 daily intake of each participant. Then, it was compared to the RDA of vitamin B12 (2.4 mcg per day) to assess the adequacy of vitamin B12 daily consumption.
Neurological assessment was also done to detect clinical PN among the study participants. It was done using Toronto Clinical Scoring System (TCSS) which is a well validated tool. The score (total = 19) was divided into normal (0–5), mild (6–8), moderate (9–11) or severe (12–19) PN .
Finally, two blood samples were collected to measure HbA1c and serum vitamin B12 levels:
HbA1c assay: Two milliliters of venous blood were collected from each subject under complete aseptic conditions and placed into sterile EDTA vacutainer tube and whole blood was stored at 4 °C until processing (not more than 1 week). HbA1c determination was done by turbidimetric inhibition immunoassay (TINIA) for hemolyzed whole blood on Cobas c311 automated analyzer (Roche Diagnostics GmbH, SandHofer Strasse 116, D-68305 Mannheim, Germany) with reference range of prediabetes 5.7–6.4%, Diabetic ≥ 6.5% and good diabetes control ≤ 7.0%.
Serum vitamin B12 assay: Two milliliters of venous blood were collected from each subject under complete aseptic conditions and placed into sterile serum separation vacutainer tube and was left to clot for 30 min. Samples were centrifuged at 3000 rpm for 20 min for serum separation. The separated serum was stored at − 320 °C until used. Hemolyzed samples were discarded, repeated freezing and thawing was avoided. Vitamin B12 was measured by electrochemiluminescence immunoassay on Cobas e411 automated analyzer (Roche Diagnostics GmbH, SandHofer Strasse 116, D-68305 Mannheim, Germany) with serum reference range 197–771 pg./ml.
The research was conducted according to the declaration of Helsinki, the proposal and conduct of the study was ethically cleared by the Research Ethical Committee at the Faculty of Medicine, Ain Shams University, Cairo, Egypt. Informed written consent to participate in the study was obtained from the participants. All information provided by the participants was kept confidential. In addition, any information leading to identification of study participants was not included in the data collection tool.
The Statistical Package for Social Sciences (SPSS) version 23 for Window (IBM Corp., Armonk, N.Y., USA. 2015) was used for data entry and analysis. Descriptive analyses of qualitative variables were done using frequencies and percentages while for quantitative variables both mean and standard deviation (SD) were applied. Group comparisons were performed using chi square (X2) test for qualitative variables. For quantitative variables Student’s t-test or Mann–Whitney U test when appropriate was done. Correlation coefficient test was performed to correlate between two quantitative variables. To determine the predictors of neuropathy score (TCSS score) and Vitamin B 12, stepwise linear regression model was done. P value < 0.05 was considered as significant and p value < 0.01 as highly significant.
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