The following article will guide you about how to detect vitamin B12 in human body.
Vitamin B12 is a member of the vitamin B complex. Vitamin B12 contains cobalt and therefore is also known as cobalamin. It is a water soluble vitamin. It is exclusively synthesized by bacteria and is found primarily in meat, eggs and dairy products. Vitamin B12 is necessary for the synthesis of red blood cells, the maintenance of the nervous system, and growth and development in children.
Deficiency of vitamin B12 can cause anaemia, vitamin B12 neuropathy, (degeneration of nerve fibres and irreversible neurological damage). Vitamin B12 can be stored in small amounts by the body. Total body store is 2-5mg in adults. Around 80% of this is stored in the liver.
Vitamin B12 was discovered from its relationship to the disease pernicious anemia, which is an autoimmune disease that destroys parietal cells in the stomach that secrete intrinsic factor (IF). Intrinsic factor is crucial for the normal absorption of B12, so a lack of intrinsic factor (as in pernicious anemia) causes a vitamin B12 deficiency.
Reference Nutrient Intakes for Vitamin B12, µg/day. (1000 µg = 1mg) is:
In the upper gastro-intestinal tract (GIT), vitamin B12 binds to a salivary protein (haptocorrin) and then this complex (vitamin B12- haptocorrin) enters the intestine. In the intestine, the pancreatic enzymes digest the haptocorrin and Vitamin B12 is released free. Then Vitamin B12 binds to a glycoprotein (intrinsic factor, IF – molecular weight 50 000) which is produced by the gastric mucosa.
Then this complex (Vitamin B12 – IF) moves to the distal ileum and enters mucosal epithelial cells by binding to the receptors on the surface of these cells. The Vitamin B12 – IF complex then dissociates within the mucosal epithelial cells and the free Vitamin B12 passes into the plasma of the mucosal capillaries and then into the portal vein. Ultimately, Vitamin B12 is stored in the liver and released to the plasma to meet the physiological demands.
We diagnose Vitamin B12 –
(i) To detect the cause of anemia or neuropathy
(ii) To evaluate the nutritional status in patients
(iii) To monitor effectiveness of treatment for Vitamin B12 deficiency
The methods used to diagnose Vitamin B12 are:
1. Competitive protein binding assay
2. Immunometric assay
3. Radio Immunoassay (RIA) – Antibodies are raised in rabbits by immunizing with monocarbonyllic acid (derivative of cyanocobalamin) coupled to human serum albumin containing Vitamin B12. Radioactivity in the Antigen-Antibody precipitate is measured and compared to standard curves.
4. Lactobacillus techmanim assay
5. A self-catalytic carbon paste electrode for the detection of vitamin B12
6. Faradic Impedance Biosensor for the detection of vitamin B12
7. High-performance liquid chromatography with fluorescence detection
8. Methylmalonic acid (MMA) quantitation (marker of metabolic vitamin B12)
Radio Immunoassay (RIA) (See Fig. 5.11):
Antibody (Ab) + Radio-labeled antigen (Ag* – Vitamin B12) → Ag* – Ab Complex → Test sample (Ag – Vitamine B12) Ag of test sample competes with radio-labeled Ag* →Unreacted Ag is removed → Radioactivity measured
Decrease in radioactivity is directly proportional to the concentration of vitamin B12 in the test sample.