Introduction and Review of literature

            Vitamin B12 is an essential micronutrient that plays a fundamental role in cell division and in one-carbon metabolism (1–4). Chronic vitamin B12 depletion (i.e., prolonged low intake or intestinal malabsorption) results in a state of negative vitamin balance. The nutritional deficiency of vitamin B12 has been linked to many complications including an increased risk of macrocytic anaemia, neuropsychiatric symptoms (5), cardiovascular diseases (6) and the onset of different forms of cancer (7,8). Further to this, biochemical and clinical vitamin B12 deficiency has been demonstrated to be highly prevalent in all the age group individuals as well as among overweight, obese, prediabetic, diabetic patients with diverse clinical manifestations ranging from impaired memory, dementia, delirium, peripheral neuropathy, sub-acute combined degeneration of the spinal cord, megaloblastic anemia and pancytopenia (9). Thirty percent of individuals medicated with metformin for long periods are estimated to have high prevalence of malabsorption such that 20% of which are known to develop low levels of vitamin B12. In another study, low levels of vitamin B12 were found in 17.5% of patients consuming metformin (2 g) taken for at least 2 years (10). Vitamin B12 malabsorption occurs because the absorption of the vitamin B12- intrinsic factor complex is calcium-dependent, and metformin interferes with calcium absorption in the terminal ileum.The very first report by Berchtold et al (11) in 1969 showed evidence of vitamin B12 malabsorption in patients who had been treated with metformin for as little as 3 months for Diabetes Mellitus (DM). Tomkin et al (12) in 1971 recommended that all patients who are on long-term metformin treatment must have screening for serum B12 deficiency. Since then, various studies have reported clinical association between long-term metformin use and vitamin B12 deficiency (13–19).

Meanwhile, metformin, a biguanide drug, is also prescribed during obesity to reduce or reverse the metabolic and endocrine changes associated during puberty. Metformin acts by suppressing the endogenous glucose production in the liver, but may also have an insulin sensitising effect in peripheral tissues through an effect on the key intracellular enzyme AMP kinase (20). Metformin is also known to reduce weight as well as hyperinsulinaemia and hyperglycaemia in type 2 diabetes in adults (21). At the same time, similar benefits on hyperinsulinaemia and BMI have also been reported in non‐diabetic obese adults (22,23) as well as reduction in progression from impaired glucose tolerance to diabetes (24).

The prevalence of obesity in India is high as more than 135 million individuals are affected by obesity as per the recent report (25) and varies due to age, gender, geographical environment, socio-economic status, etc. According to ICMR-INDIAB study 2015, prevalence rate of obesity and central obesity varies from 11.8% to 31.3% and 16.9%–36.3% respectively. Though several studies have shown that there is an association of overweight and obesity with lower serum vitamin-B12 and folate levels (26-28). However, it is currently not clear whether deficiencies of these B-vitamins are a cause or a consequence of obesity (29). It can be proposed that being overweight or obese can alter the absorption, distribution, metabolism and/or excretion of micronutrients, which may cause micronutrient deficiencies. Another possibility is that obesity or overweight could lower serum vitamin B12 levels through decreased dietary intake or absorption, increased catabolism, and sequestration in adipose tissue (30), or changes in the gut microbiota profiles which could affect vitamin B12 metabolism (31). Another study in Danish population showed significant association of lower serum vitamin B12 concentrations with higher BMI, but a genetic risk score related to vitamin B12 concentrations associated variants was not associated with BMI (32). However, the direction of causality is still not confirmed. Since, vitamin B12 intake is overall lower particularly in, India (33) with respect to the suggested daily requirement of 2.4 to 4 µg/day (34) and leading to widespread deficiency in the population (35–37), measuring vitamin B12 bioavailability is important in overweight as well as pernicious anemia population. Moreover, the causal relation between vitamin B12 status and obesity warrants clinical investigation towards bioavailability and effect of short-term metformin drug at different dose régime during obesity that is an alarming pathophysiological condition that needs to be evaluated for underlined mechanism.

Since, pernicious anemia resulting from chronic autoimmune gastritis is highly frequent among patients in diabetes (specifically with T1DM) and chronic autoimmune gastritis and pernicious anemia occurs in about 2% and up to 1% of the general population respectively, the prevalence is increased by 3 to 5 folds among patients with T1DM (38). Vitamin B12 deficiency due to pernicious anemia occurs frequently among patients with T1DM as well. In one of the cross sectional study done in South India among 90 patients with T1DM, low vitamin B12 levels were observed among 45.5% of the participants for a general cutoff point of <180 pg/ml and among 54% using the published cut off point of <200 pg/ml (39). No other positive correlations were noted between low vitamin B12 levels and gender, age, duration of DM and level of glycemic control. Therefore, measuring vitamin B12 bioavailability in such conditions is important.

Need for the study

The absorption of vitamin B12 is classically measured by the Schilling test that uses radioactive isotope, [⁵⁷Co]-cyanocobalamin along with flushing dose of vitamin B12. The use of this test is restricted in general population due to radioactivity (40,41). Alternatively, a modified form of the Schilling’s test; egg-yolk cobalamin absorption test, also uses [⁵⁷Co]-vitamin B12 along with albumin, egg yolk, or chicken in a similar protocol (42,43). However, due to exposure to radioactivity, both tests cannot be performed. Another method that was developed by Carkeet etal (44) demonstrated the use of [¹⁴C]-vitamin B12 (cyanocobalamin) is performed without a flushing dose of vitamin B12 and without 24 hour urine collection, still poses radiation risk (although negligible as compared to [⁵⁷Co]). Other indirect indicative methods such as the use of plasma holotranscobalamin (HoloTC) concentration, also known as active B12, have been developed to estimate the fractional absorption of vitamin B12 (45), but they have other limitations and cannot be performed after subjects/patients have received vitamin B12 supplementation. The need for assessing vitamin B12 bioavailability has been highlighted earlier (46) as well as there was no safe/alternative method available till than. We have recently developed a stable isotope-based test in our laboratory to measure B12 bioavailability in humans by using an oral physiological dose  (1.5-2 µg) of ¹³C-labelled B12, followed by sequential blood sampling for 12h (47). Therefore, we aim to assess the effect of metformin (at different dosages) in overweight participants as well as in participants who are diagnosed with pernicious anemia and validate functional biomarkers of B12 bioavailability.

References:

          1.        Herbert V. Vitamin B-12: plant sources, requirements, and assay. Am J Clin Nutr [Internet]. 1988;48:852–8. Available from: https://academic.oup.com/ajcn/article/48/3/852-858/4716470

          2.        Alexander D, Ball MJ, Mann J. Nutrient intake and haematological status of vegetarians and age-sex matched omnivores. Eur J Clin Nutr. 1994;48:538–46.

          3.        Schneede J, Dagnelie PC, Van Staveren WA, Vollset SE, Refsum H, Ueland PM. Methylmalonic Acid and Homocysteine in Plasma as Indicators of Functional Cobalamin Deficiency in Infants on Macrobiotic Diets. Pediatr Res [Internet]. 1994;36:194–201. Available from: http://www.nature.com/doifinder/10.1203/00006450-199408000-00010

          4.        Herbert V. Staging vitamin B�’12 (cobalamin) status in vegetarians. Am J Clin Nutr. 1994;59:1213S-1222S.

          5.        Lechner K, Födinger M, Grisold W, Püspök A, Sillaber C. [Vitamin B12 deficiency. New data on an old theme]. Wien Klin Wochenschr [Internet]. 2005;117:579–91. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16395986

          6.        Boushey CJ. A Quantitative Assessment of Plasma Homocysteine as a Risk Factor for Vascular Disease. JAMA [Internet]. 1995;274:1049. Available from: http://jama.jamanetwork.com/article.aspx?doi=10.1001/jama.1995.03530130055028

          7.        Arendt JFB, Nexo E. Unexpected high plasma cobalamin/Proposal for a diagnostic strategy. Clin Chem Lab Med [Internet]. 2013;51. Available from: https://www.degruyter.com/doi/10.1515/cclm-2012-0545

          8.        Arendt JFB, Pedersen L, Nexo E, Sørensen HT. Elevated Plasma Vitamin B12 Levels as a Marker for Cancer: A Population-Based Cohort Study. JNCI J Natl Cancer Inst [Internet]. 2013;105:1799–805. Available from: https://academic.oup.com/jnci/article-lookup/doi/10.1093/jnci/djt315

          9.        Kibirige D, Mwebaze R. Vitamin B12 deficiency among patients with diabetes mellitus: is routine screening and supplementation justified? J Diabetes Metab Disord [Internet]. 2013;12:17. Available from: http://jdmdonline.biomedcentral.com/articles/10.1186/2251-6581-12-17

        10.      Stowers JM, Smith OA. Vitamin B 12 and metformin. BMJ [Internet]. 1971;3:246–7. Available from: http://www.bmj.com/cgi/doi/10.1136/bmj.3.5768.246-b

        11.      Berchtold P, Bolli P, Arbenz U, Keiser G. Intestinale Absorptionsst�rung infolge metforminbehandlung (Zur Frage der Wirkungsweise der Bignanide). Diabetologia. 1969;5:405–12.

        12.      Tomkin GH, Hadden DR, Weaver JA, Montgomery DAD. Vitamin-B12 Status of Patients on Long-term Metformin Therapy. BMJ [Internet]. 1971;2:685–7. Available from: http://www.bmj.com/cgi/doi/10.1136/bmj.2.5763.685

        13.      Beulens JWJ, Hart HE, Kuijs R, Kooijman-Buiting AMJ, Rutten GEHM. Influence of duration and dose of metformin on cobalamin deficiency in type 2 diabetes patients using metformin. Acta Diabetol. 2015;52:47–53.

        14.      Pflipsen MC, Oh RC, Saguil A, Seehusen DA, Seaquist D, Topolski R. The Prevalence of Vitamin B12 Deficiency in Patients with Type 2 Diabetes: A Cross-Sectional Study. J Am Board Fam Med. 2009;22:528–34.

        15.      Reinstatler L, Qi YP, Williamson RS, Garn J V., Oakley GP. Association of Biochemical B12 Deficiency With Metformin Therapy and Vitamin B12 Supplements: The National Health and Nutrition Examination Survey, 1999-2006. Diabetes Care [Internet]. 2012;35:327–33. Available from: http://care.diabetesjournals.org/cgi/doi/10.2337/dc11-1582

        16.      Sato Y, Ouchi K, Funase Y, Yamauchi K, Aizawa T. Relationship between metformin use, vitamin B12 deficiency, hyperhomocysteinemia and vascular complications in patients with type 2 diabetes. Endocr J. 2013;60:1275–80.

        17.      Tung ML, Tan LK. Long term use of metformin leading to vitamin B 12 deficiency. Diabetes Res Clin Pract [Internet]. 2014;104:e75–6. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0168822714000151

        18.      Liu Q, Li S, Quan H, Li J. Vitamin B12 Status in Metformin Treated Patients: Systematic Review. Pietropaolo M, editor. PLoS One. 2014;9:e100379.

        19.      Kim J, Ahn CW, Fang S, Lee HS, Park JS. Association between metformin dose and vitamin B12 deficiency in patients with type 2 diabetes. Medicine (Baltimore) [Internet]. 2019;98:e17918. Available from: http://journals.lww.com/10.1097/MD.0000000000017918

        20.      Hundal RS, Inzucchi SE. Metformin. Drugs. 2003;63:1879–94.

        21.      Turner R, Murchison L, Wright AD, Oakley N, Kohner E, Hayes R, Scarpello J, Hadden D, Spathis AG, Yudkin J, et al. United Kingdom Prospective Diabetes Study 24: A 6-Year, Randomized, Controlled Trial Comparing Sulfonylurea, Insulin, and Metformin Therapy in Patients with Newly Diagnosed Type 2 Diabetes That Could Not Be Controlled with Diet Therapy. Ann Intern Med. 1998;128:165.

        22.      Charles MA, Eschw�ge E, Grandmottet P, Isnard F, Cohen J-M, Bensoussan J-L, Berche H, Chapiro O, Andr� P, Vague P, et al. Treatment with metformin of non-diabetic men with hypertension, hypertriglyceridaemia and central fat distribution: the BIGPRO 1.2 trial. Diabetes Metab Res Rev [Internet]. 2000;16:2–7. Available from: http://doi.wiley.com/10.1002/%28SICI%291520-7560%28200001/02%2916%3A1%3C2%3A%3AAID-DMRR75%3E3.0.CO%3B2-G

        23.      Fontbonne A, Charles MA, Juhan-Vague I, Bard JM, Andre P, Isnard F, Cohen JM, Grandmottet P, Vague P, Safar ME, et al. The Effect of Metformin on the Metabolic Abnormalities Associated With Upper-Body Fat Distribution. BIGPRO Study Group. Diabetes Care [Internet]. 1996;19:920–6. Available from: http://care.diabetesjournals.org/cgi/doi/10.2337/diacare.19.9.920

        24.      Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, Nathan DM. Reduction in the Incidence of Type 2 Diabetes with Lifestyle Intervention or Metformin. N Engl J Med [Internet]. 2002;346:393–403. Available from: http://www.nejm.org/doi/abs/10.1056/NEJMoa012512

        25.      Ahirwar R, Mondal PR. Prevalence of obesity in India: A systematic review. Diabetes Metab Syndr Clin Res Rev [Internet]. 2019;13:318–21. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1871402118303655

        26.      Kimmons JE, Blanck HM, Tohill BC, Zhang J, Khan LK. Associations between body mass index and the prevalence of low micronutrient levels among US adults. MedGenMed Medscape Gen Med. 2006;

        27.      Mahabir S, Ettinger S, Johnson L, Baer DJ, Clevidence BA, Hartman TJ, Taylor PR. Measures of adiposity and body fat distribution in relation to serum folate levels in postmenopausal women in a feeding study. Eur J Clin Nutr [Internet]. 2008;62:644–50. Available from: http://www.nature.com/articles/1602771

        28.      Pinhas-Hamiel O, Doron-Panush N, Reichman B, Nitzan-Kaluski D, Shalitin S, Geva-Lerner L. Obese Children and Adolescents. Arch Pediatr Adolesc Med. 2006;160.

        29.      Thomas-Valdés S, Tostes M das G V., Anunciação PC, da Silva BP, Sant’Ana HMP. Association between vitamin deficiency and metabolic disorders related to obesity. Crit Rev Food Sci Nutr. 2017;57:3332–43.

        30.      Krishnaveni G V., Hill JC, Veena SR, Bhat DS, Wills AK, Karat CLS, Yajnik CS, Fall CHD. Low plasma vitamin B12 in pregnancy is associated with gestational ‘diabesity’ and later diabetes. Diabetologia. 2009;52:2350–8.

        31.      Castaner O, Goday A, Park Y-M, Lee S-H, Magkos F, Shiow S-ATE, Schröder H. The Gut Microbiome Profile in Obesity: A Systematic Review. Int J Endocrinol [Internet]. 2018;2018:1–9. Available from: https://www.hindawi.com/journals/ije/2018/4095789/

        32.      Allin KH, Friedrich N, Pietzner M, Grarup N, Thuesen BH, Linneberg A, Pisinger C, Hansen T, Pedersen O, Sandholt CH. Genetic determinants of serum vitamin B12 and their relation to body mass index. Eur J Epidemiol [Internet]. 2017;32:125–34. Available from: http://link.springer.com/10.1007/s10654-016-0215-x

        33.      Tata NIN Centre of Excellence in Public Health Nutrition Dashboard. Overview of food and nutrition in India [Internet]. Available from: https://www.dashboard.nintata.res.in/#/dashboard/nutrient-intake

        34.      Scientific Opinion on Dietary Reference Values for cobalamin (vitamin B12). EFSA J [Internet]. 2015;13. Available from: http://doi.wiley.com/10.2903/j.efsa.2015.4150

        35.      Gonmei Z, Toteja GS. Micronutrient status of Indian population. Indian J Med Res [Internet]. 2018;148:511–21. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30666978

        36.      Singh S, Geddam JJB, Reddy GB, Pallepogula DR, Pant HB, Neogi SB, John N, Kolli SR, Doyle P, Kinra S, et al. Folate, vitamin B12, ferritin and haemoglobin levels among women of childbearing age from a rural district in South India. BMC Nutr. 2017;

        37.      Finkelstein JL, Kurpad A V, Thomas T, Srinivasan K, Duggan C. Vitamin B12 status in pregnant women and their infants in South India. Eur J Clin Nutr [Internet]. 2017;71:1046–53. Available from: http://www.nature.com/articles/ejcn201729

        38.      De Block CEM, De Leeuw IH, Van Gaal LF. Autoimmune Gastritis in Type 1 Diabetes: A Clinically Oriented Review. J Clin Endocrinol Metab [Internet]. 2008;93:363–71. Available from: https://academic.oup.com/jcem/article/93/2/363/2598003

        39.      Kumari Sj, Ayyar V, Kumar P, Koshy A. Evaluation of serum vitamin B12 levels in type 1 diabetics attending a tertiary care hospital: A preliminary cross - sectional study. Indian J Endocrinol Metab. 2012;16:79.

        40.      Zuckier LS, Chervu LR. Schilling evaluation of pernicious anemia: current status. J Nucl Med [Internet]. 1984;25:1032–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/6470805

        41.      Schilling RF. Intrinsic factor studies: II. The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B12. J Lab Clin Med. 2004;144:268–72.

        42.      Carmel R. Malabsorption of food cobalamin. Baillieres Clin Haematol [Internet]. 1995;8:639–55. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0950353605802240

        43.      Carmel R. The Disappearance of Cobalamin Absorption Testing: A Critical Diagnostic Loss. J Nutr [Internet]. 2007;137:2481–4. Available from: https://academic.oup.com/jn/article/137/11/2481/4664489

        44.      Carkeet C, Dueker SR, Lango J, Buchholz BA, Miller JW, Green R, Hammock BD, Roth JR, Anderson PJ. Human vitamin B12 absorption measurement by accelerator mass spectrometry using specifically labeled 14C-cobalamin. Proc Natl Acad Sci [Internet]. 2006;103:5694–9. Available from: http://www.pnas.org/cgi/doi/10.1073/pnas.0601251103

        45.      Nexo E, Hoffmann-Lücke E. Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility. Am J Clin Nutr [Internet]. 2011;94:359S-365S. Available from: https://academic.oup.com/ajcn/article/94/1/359S/4597959

        46.      Green R, Allen LH, Bjørke-Monsen A-L, Brito A, Guéant J-L, Miller JW, Molloy AM, Nexo E, Stabler S, Toh B-H, et al. Vitamin B12 deficiency. Nat Rev Dis Prim [Internet]. 2017;3:17040. Available from: http://www.nature.com/articles/nrdp201740

        47.      Devi etal. Measuring vitamin B12 bioavailability with [13C]-cyanocobalamin in humans. Am J Clin Nutr. 2020;

        48.      Garrod MG, Buchholz BA, Miller JW, Haack KW, Green R, Allen LH. Vitamin B12 added as a fortificant to flour retains high bioavailability when baked in bread. Nucl Instruments Methods Phys Res Sect B Beam Interact with Mater Atoms [Internet]. 2019;438:136–40. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0168583X18303665

        49.      Garrod MG., Rossow HA., Calvert CC., Miller JW., Green R., Buchholz BA., Allen LH. 14C-Cobalamin Absorption from Endogenously Labeled Chicken Eggs Assessed in Humans Using Accelerator Mass Spectrometry. Nutrients [Internet]. 2019;11:2148. Available from: https://www.mdpi.com/2072-6643/11/9/2148