B12, folate and iron Flashcards
What two carrier proteins bind B12 in the blood?
○ 10-30% bound to transcobalamin.
- “Holotranscobalamin”
- Metabolically active form of cobalamin.
- > 90% of absorbed cobalamin will bind to transcobalamin and will be rapidly cleared from the circulation
○ 70-90% bound to haptocorrin
- Metabolically inactive form, may represent the storage form of B12.
- Low turnover/ slow clearance from the plasma.
In man, B12 is required as a cofactor/ coenzyme for which two reactions?
1) As methylcobalamin as a cofactor for methionine synthatase.
(conversion of homocysteine to methionine)
2) As adenosyl cobalamin as a cofactor for the enzyme methylmalonyl-coA mutase.
(conversion of MMA to succinyl co-A)
How is homocysteine converted to methionine?
- 5-MTHR donates a methyl group to cobalamin to form methylcobalamin
- Methylcobalamin used as a cofactor for methionine synthetase to enable conversion of homocysteine to methionine.
- This pathway is a vital source of methyl groups critical for a series of methylation reactions involving proteins, phospholipids, neurotransmitters and both RNA and DNA.
What does folate do in the body?
• Folates participate in 1-carbon metabolism and are therefore essential for DNA synthesis.
○ Necessary for dTMP synthesis (thymidine).
○ In the absence of folic acid, dUMP (uracil in RNA) cannot be converted to dTMP
How does vitamin B12 deficiency cause a functional deficiency in folate?
- While 5-MTHF is the metabolically active form of folate, it needs to be converted to 5-10, methylene THF for pyrimidine synthesis.
- This conversion requires B12.
- Cobalamin deficiency traps folate in the 5-MTHF state leading to a reduction in 5,10 MTHF.
- This causes a block in the rate limiting step of DNA synthesis: production of dTMP.
- 5-MTHF is a very poor substrate of the enzyme responsible for folate polyglutamation which is necessary to keep folate in the cells.
- Folate therefore leaves the cells and enters the serum resulting in a high serum folate and low red cell folate in B12 deficiency.
What are some of the issues with B12 assays?
• Issues with “grey zone” or mildly reduced B12 levels.
- Generates a high number of false positive results
- Poor positive predictive value
- Significant limitations in pregnant patients: cobalamin levels fall by ~50% at term (haemodilution, fall in haptocorrins).
- False negative can occur in those with a high level of anti-IF antibodies
• Difficulty defining sensitivity and specificity due to difficulty in defining a truly deficient standard.
○ MMA often used as a gold standard.
What are some of the issues with folate assays?
• Altered by acute dietary change and interruption of enterohepatic circulation.
- Serum levels can be low without tissue deficiency.
- Red cell folate better reflects folate levels over the lifespan of the red cell (although there is little to be gained in testing red cell folate in the majority of people)
- Haemolysis will cause a significant increase in serum folate (folate stored in RBCs released into the serum).
- MTX and folinic acid interfere with folate measurement (cross react with folate binding proteins).
- High triglycerides and bilirubin also interfere with the assay.
What are the steps of the B12/ cobalamin assay?
1) Alkaline hydrolysis (NaOH) used to release cobalamin from binders (haptocorrin and transcobalamin)
2) Synthetic cobalamin (complexed to a chemiluminescent or fluorescent substrate or enzyme) and IF added.
3) Patients serum B12 competes with labelled B12 for binding on IF.
4) IF is bound to a solid phase or coated onto magnetic beads.
5) Unbound ligand washed away
6) Conjugate to the labelled B12 added
7) Substrate added which binds the conjugate and creates chemiluminescence that is proportional to the amount of labelled B12 present.
- Inverse relationship between the quantity of patient B12 present and the amount of luminescence generated.
How is the serum folate assay performed?
1) Alkaline denaturation to remove binders
2) Synthetic folate (complexed to a chemiluminescent or fluorescent substrate or enzyme) and B lactoglobin (a folate binding protein) added.
3) Patients serum folate competes with chemiluminescent or enzyme labelled folate for binding sites on FBP
4) FBP is bound to a solid phase or coated onto magnetic beads.
5) Unbound ligand washed away
6) Conjugate to the labelled folic acid added
7) Substrate added which binds the conjugate and creates chemiluminescence that is proportional to the amount of labelled folate present.
- Inverse relationship between the quantity of patient folate present and the amount of luminescence generated.
Discuss MMA measurement
- Quantified by gas chromatography- mass spectrometry. High cost.
- Plasma or serum MMA is extracted, purified and using derivatives of MMA, measured by gas chromatography mass spec.
- A stable isotype of MMA is used as an internal standard.
• Not specific. Also elevated with renal failure and small bowel bacterial overgrowth
Is probably still more useful than homocysteine.
Discuss Homocysteine measurement
- Sample must be kept cool and centrifuged within two hours of collection
- Can be measured by an immunoassay, competitive binding assay or enzyme immunoassay.
•Not specific. Also elevated with folate deficiency, B6 deficiency, renal failure, hypothyroidism and certain genetic abnormalities.
Discuss holotranscobalamin “Active B12”Immunoassay
• Two step quantitative immunoassay
○ Sample and anti-holotranscobalamin coated beads combined. Holotranscobalamin in the sample binds to the beads.
○ Sample washed
○ Anti-transcobalamin labelled conjugate added
○ Sample washed
○ Substrate added
○ Resulting chemiluminescent reaction measured.
• Appears to perform better than serum B12 based on MMA levels
○ Smaller grey zone
○ Better sensitivity and specificity (when MMA used as a gold standard)
○ Not affected by pregnancy, the OCP
○ Place still not clearly established
How are the tests of B12 deficiency affected by pregnancy?
· Spuriously low serum vitamin B12 when total B12 is measured (reduced haptocorrin in pregnancy). Levels fall as pregnancy progresses and are the lowest just before delivery.
· Holotranscobalamin measures the metabolically active form of transcobalamin. Better sensitivity and specificity than total B12, not affected by pregnancy (or oral contraceptive use)
· Plasma homocysteine and methylmalonic acid levels:
- Plasma MMA more specific. Homocysteine elevated in a variety of conditions.
- Levels of both can be affected by pregnancy with a gradual increase seen in both levels as pregnancy proceeds to term.
- Haemodilution may affect homocysteine results
What are the chemical symbols for ferric and ferrous iron?
- Ferric Fe3+
- Ferrous Fe2+
How is serum iron measured?
○ Measured via a colourimetric assay.
- Elemental iron is removed from transferrin in the presence of acid.
- The released Fe3+ is then reduced with ascorbic acid to Fe2+
- Fe2+ reacts with an added chromogen to form a coloured complex.
- This complex is then measured. The colour change is directly proportional to the amount of Fe2+.
