How to Detect Vitamin B12? | Human Body | Biology

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 B_12- 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.

Detection:

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 B_12. 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.