Introduction and Review of literature:

Vitamin B₁₂ deficiency is widespread and emerging as a growing public health problem globally. About 75% of Indians have evidence of low B₁₂ levels (1), and from an interventional viewpoint, it is important to have an accurate assessment of vitamin B₁₂ absorption in Indians.

 In developed countries, conservative estimates indicate that 2-3% of this population has or will develop pernicious anemia caused by failure of gastric intrinsic factor production and consequent vitamin B₁₂ malabsorption (2,3). Other estimates suggest that vitamin B₁₂ deficiency may be due to food vitamin B₁₂ malabsorption caused by chronic gastritis, gastric atrophy, and perhaps other unknown causes (4). Vitamin B₁₂ is a compound of significant nutritional and clinical importance (5). The classical manifestations of B₁₂ deficiency include pernicious anemia, a type of megaloblastic anemia, and neurological dysfunction (6).  However, these occur when the deficiency is profound, and there is a need for the development of simpler functional measures of deficiency.  Therefore, there are two areas where vitamin B₁₂ nutrition needs physiological research in India- first in terms of its absorption, and second, in terms of a functional index of its deficiency.

            First, in terms of its absorption, the gold standard Schilling test (7,8) measures vitamin B₁₂ absorption and has been used when deficiency is identified and malabsorption is the suspected cause. The test involves the ingestion of a physiological quantity of B₁₂ labeled with gamma-emitting cobalt, followed by administration of a pharmacological parenteral flushing dose of unlabeled B₁₂ to force urinary excretion of radioactivity, which is measured during a 24-h period. The Schilling test is currently the only accepted method for assessing B₁₂ absorption. Despite of its utility, the method has methodological and practical problems; it is now rarely prescribed, despite the prevalence of B₁₂ malabsorption, for example, in older adults (6, 9).  Besides the Schilling test being a radioactive method, radioactive cobalt is difficult to procure and dispose of.  Equally, studies have been conducted to assess absorption and kinetics of vitamin B₁₂ in humans by using ¹⁴C labeled vitamin B₁₂ (10); however, the use of ¹⁴C-B₁₂ is also rare since it too is a radioactive test. Therefore, we are in the process of developing a modified Schilling test that uses stable isotope (¹³C) labeled vitamin B₁₂, thereby addressing concerns about its safety.

            Second, in terms of a functional assessment, the current assessment of vitamin B₁₂ status either by measuring total vitamin B₁₂ in plasma or serum or by the measurement of concentrations of selected blood metabolites is static. For example, biochemical markers such as plasma homocysteine are used for detection of either vitamin B₁₂ or folate deficiency, while methylmalonic acid (MMA) is used for detection of vitamin B₁₂ deficiency. Recent methods of measuring a physiological relevant fraction (active) vitamin B₁₂ associated with the plasma binding protein transcobalamin (holotranscobalamin) have been developed. Effectively, the detection/confirmation of vitamin B₁₂ deficiency depends on laboratory testing rather than the testing of a function within the body.  We have been considering the development of a measure of the metabolism of stable isotope labeled propionate as a breath test to measure vitamin B₁₂ status in vivo.

Therefore, the aims of this proposal are firstly, to use a modified Schilling test that uses stable ¹³C labeled vitamin B₁₂ to measure the absorption of vitamin B₁₂, and secondly, to develop a simple breath test that will be used to assess vitamin B₁₂ status, based on the metabolism of sodium ¹³C-propionate to ¹³CO₂ which requires B₁₂ as a cofactor. In summary, this proposal seeks to develop simpler methods to assess the status of and understand the absorption of vitamin B_12.

Rationale of the study supported by cited literature

The gold standard Schilling test for vitamin B₁₂ absorption involves the use of gamma-emitting cobalt, which poses methodological and practical problems. Furthermore, the current assessment of vitamin B₁₂ status is done either by measuring total vitamin B₁₂ in plasma or serum or by the measurement of concentrations of metabolites such as methylmalonic acid (MMA), homocysteine (Hcy) and holo transcobalamine (HoloTC) . These are static indices of B₁₂ status and do not test the functionality of B₁₂ per se. Therefore, there are two areas where vitamin B₁₂ nutrition needs physiological research in India- first in terms of its absorption, and second, in terms of a functional index of its deficiency.

We aim to use stable isotopes to assess both absorption and B₁₂ status in a simpler, more accurate manner.

References

1.     Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, Orning L, Guttormsen AB, Joglekar A, Sayyad MG, Ulvik A, Ueland PM. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asia Indians. Am J Clin Nutr. 2001;74:233-41.

2.     Chanarin I.The megaloblastic anemias, 2nd edition. Oxford,UK:Blackwell Scientific Publication, 1979:783pp.

3.     Carmel R. Prevalence of undiagnosed pernicious anemia in the elderly. Archives of Internal Medicine 1996; 156:1097-1100.

4.     Baik HW. Vitamin B₁₂ deficiency in the elderly, Annu Rev Nutr 1999;19:357-77.

5.      Carmel, R. Current concepts of cobalamine deficiency. Annu. Rev. Med. 2000, 51, 357–375.

6.     Green, R. & Kinsella, L. J. Current concepts in the diagnosis of cobalamin deficiency. Neurology 2000, 45, 1435–1440.

7.     Zuckier, L. S. & Chervu, L. R. Schilling evaluation of pernicious anemia: current status. J. Nucl. Med. 1984, 25, 1032–1039.

8.     Schilling, R. 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. 1953, 42, 860–866.

9.     Carmel, R. Prevalence of undiagnosed pernicious anemia in the elderly. Arch. Intern. Med. 1996, 156, 1097–1100.

10.  Carkeet C, Dueker SR, Lango J, Buchholz BA, Miller JW, Green R et al. Human vitamin B12 absorption measurement by accelerator mass spectrometry using specifically labelled (14)C-cobalamin. Proc Natl Acad Sci U S A 2006; 103: 5694–5699.

11.  Wagner DA, Schatz R, Coston R,Curington C, Bolt D, Toskes PP. A new 13C breath test to detect vitamin B12 deficiency: a prevalent and poorly diagnosed health problem. J. Breath Res; 5 (2011) 046001.