Vitamin B12 (Cyanocobalmin)

Sample 

1. B12 estimation is done on the serum of the patient.
2. A fasting blood sample is needed.
3. The specimen is stable for 8 weeks at 8 °C.
4. 24 hours of urine samples are collected.

Precautions

1. Avoid hemolysis.
2. Protect the sample from the light.
3. Avoid heparin, fluoride, and ascorbic acid.
4. Drugs like chloral hydrate increase the B12 level.
5. Drugs that decrease the level are alcohol, anticonvulsants, colchicine, oral contraceptives, and aminoglycosides.

Pathophysiology

1. Vit.B12 is a complex molecule and this is also called Cyanocobalamine.
2. This is a water-soluble and hematopoietic vitamin.
3. This has a central cobalt nucleus surrounded by porphyrins like a ring to which are attached nucleotide and aminopropanol.
4. This was also called an extrinsic factor.
5. For absorption intrinsic factor (glycoprotein) secreted in saliva, and gastric juice. This is produced by the parietal cells of the gastric mucosa.

6. Cyanocobalamin is a stable compound that forms dark red needle-like crystals.
   1. The physiological form of Cobalamin in the serum is Methylcobalamin.
7. The deficiency of the Intrinsic factor leads to pernicious anemia.
   1. Vitamin B12 deficiency may take place due to parasitic infestation like Diphyllobothrium latum which competes with B12.

Vit.B12 functions

8. Food produces Haptocorrin (transcobalamin, or R-protein) from the salivary glands.
   1. It binds the B12 and protects from the acid of the stomach.

9. Dietary source of Vitamin B12:
   1. The diet source is meat, eggs, milk, and milk products.
      1. This is not found in the vegetable.
10. The deficiency of Vit.B12 is due to the lake of intrinsic factor (IF) by the stomach and intestinal malabsorption.
    1. In the absence of intrinsic factor, the patient will have pernicious anemia and Vit.B 12 is not absorbed.
    2. In case partial gastrectomy Vit.B12 deficiency may occur.
    3. Intestinal malabsorption of Vit.B12 is caused by gastrectomy or ileal resection.
    4. Malabsorption may be due to tropical sprue, inflammatory bowel disease, intestinal stasis, and overgrowth of bacteria in the colon.
    5. This may occur by the intestinal parasitic infestation like Dophyllobothrium latum that competes for the vitamin.
11. Signs and symptoms of Vit.B12 deficiency:
    1. There is megaloblastic anemia (pernicious anemia).
       1. Patients with pernicious anemia may have atrophic gastritis.
       2. There is an increased incidence of gastric carcinoma.
    2. There may be neuropathy.
       1. Sometimes there is an irreversible neurological loss like burning pain or loss of sensation in the extremities.
       2. There may be weakness, paralysis, or spasticity.
       3. The patient may develop dementia, confusion, and disorientation.

 Normal

Source 1

Age	pg/mL
Newborn	160 to 1300
Adult	200 to 835
60 to 90 years	110 to 770

Other sources

• Newborn  = 160 to 1300 pg/mL, (118 to 959 pmol/L)
• Adult  = 200 to 835 pg/mL (148 to 616 pmol/L).
• Critical value :
  • Decreased B12 level = <100 pg/mL.
  • Increased B12 level = >700 pg/mL.

• Some references use:
  • Normal value as  200 to 900 pg/mL OR
    • Other sources =  206 to 678 ng/L.
  • Another reference = 110 to 800 pg/mL.

Diagnosis

1. The indirect method measures the urinary and serum levels of methylmalonic acid and homocysteine.
2. The Patient deficient in vitamin B12 secrete an excess amount of methylmalonic acid in the urine. Methylmalonic acid can be measured by gas chromatography.
3. Homocysteine level is increased in patients deficient in vitamin B12. Because their cells can not metabolize homocysteine to methionine at a normal rate.
4. Vitamin B 12 absorption test known as the Schilling test.
   1. This test can differentiate:
      1. B12 deficiency (Pernicious anemia)
      2. Intestinal malabsorption.
      3. Diet.

The procedure of the Schilling test:

1. This is a two-step test.
   1. If the first step is normal then no need to do the second step.
   2. Ask the patient to empty the bladder.
2. Step 1: In this test vitamin B12 is radiolabeled with 57^′Co or 58^′Co-vitamin B12 is given.
   1. An oral dose of 0.5 to 1.0 µg radiolabelled vitamin B12 is given.
3. Within 2 hours the patient is injected with a flushing dose of 1000 µg of unlabelled vitamin B12.
4. Radioactivity is measured in the urine, feces, and serum.
   1. Start collecting 24 hours of urine after both doses.
   2. Measure the number of radiolabelled B12 in the urine.
   3. If the amount of radiolabelled B12 measured in the urine exceeds 7.5% of the oral dose.
      1. Absorption is considered normal and the test is complete.
      2. The deficiency is considered dietary.
5. Step 2: This will differentiate between pernicious anemia and other causes of malabsorption.
   1. Repeat the doses of vitamin B12 from step 1, but add the intrinsic factor along with the oral dose of labeled B12.
   2. Collect again 24 hours urine and count the radiolabelled B12.
   3. If the B12 labeled in urine from step 2 is >7.5% then,
      1. Malabsorption is corrected by the addition of intrinsic factors and the diagnosis is pernicious anemia.
      2. If the result is <7.5%, it indicates other causes of malabsorption like Diphyllobothrium latum or celiac sprue.
6. Schilling test normal value:
   1. 15 to 40% of 0.5 µg dose.
   2. 5 to 40% of 1.0  µg dose.
   3. 24 hours of urine results are:
      1. Radiolabeled B12 at the first step:
      2. >7.5% indicates dietary deficiency.
      3. The result in step 2:
         1. >7.5% = indicates pernicious anemia.
         2. <7.5% = suggest another cause of malabsorption.
7. Intrinsic factor (IF) antibodies can be detected in the serum of patients with pernicious anemia.
   1. 50% of the patient shows these antibodies. While these are absent in other conditions.

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