B12 Flashcards
Functions of B12
Erythropoiesis (DNA synthesis)
Methylation processes necessary for metabolism
Cofactor for enzymes involved in the metabolism of MMA ie conversion of methyl malonyl CoA to succinyl CoA (deficiency causes methylmalonic aciduria)
Cofactor for the enzyme metabolising homocysteine to methionine (deficiency causes hyperhomocysteinaemia)
Describe the structure of vitamin B12
Tetrapyrrole ring (also known as a corrin) surrounding a central cobalt atom (corrin + cobalt = cobalamin) with various side chains appended.
List a few different types of cobalamin based on their side chain(s)
Methylcobalamin - methyl side chain
Aquacobalamin/vitamin B12b - water side chain
Cyanocobalamin - cyanide side chain
Cobalamin/coenzyme B12 - adenosyl side chain
Hydroxocobalamin - hydroxyl side chain
What is the reference compound for measuring serum cobalamin? Why?
Cyanocobalamin - a stable, red, needle-like crystal - has stability in aqueous solutions and a distinct absorption spectrum, with maxima at 278, 361 and 550nm.
What is the main physiologic form of cobalamin in serum?
Methylcobalamin
What is the main physiologic form of cobalamin in the cytosol?
Adenosylcobalamin
Cyanocobalamin is gradually destroyed on exposure to light
True
Which two vitamin deficiencies can cause hyperhomocysteinaemia?
Vitamin B6 and Vitamin B12
How do PPIs cause vitamin B12 deficiency?
Dietary vitamin B12 is tightly bound to proteins. The first step to enable absorption of B12 is to break its bonds to protein by acid and pepsin. PPIs inhibit gastric acid formation and therefore the release of vitamin B12 from protein.
What is the role of intrinsic factor in Vitamin B12 absorption?
Vitamin B12 liberated from protein by acid and pepsin binds to intrinsic factor in the stomach. The vitamin B12-IF complex is transported to the distal ileum where IF binds its receptor - cubam - on mucosal epithelial cells and is internalised.
What is the difference between total B12 and holotranscobalamin?
B12 circulates in blood bound to haptocorrin (holohaptocorrin) or transcobalamin (holotranscobalamin). Only the transcobalamin bound fraction has receptor-mediated cellular uptake and is therefore the bioactive fraction
What is the function of holohaptocorrin?
Unknown!
How is B12 excreted?
In the urine and faeces (via bile)
Methods for B12 determination
Direct and indirect (functional) methods are available.
Indirect tests: urinary and serum assays for concentrations of MMA, plasma homocysteine, deoxyuridine suppression test, vitamin B12 absorption test.
Ancillary methods: cytochemical staining of RBC precursors, test for IF blocking antibodies
Direct: microbiological, competitive protein binding, immunometric assays, automated/semiautomated systems using, for example, chemiluminescence as a signal
Pros and cons of microbiological assays for B12
Pros: still the reference method for determination of biologically active B12
Cons: require 24 hours to grow the microorganisms, microorganisms susceptible to inhibition by antibiotics/other drugs in patient serum.
Offsets: use of microtitre enzyme-linked immunosorbent assay plate technology has enhanced the utility of some microbiological assays
Pros and cons of competitive protein binding assays for B12
Cons: Binder used is often non-human IF - if not highly purified, it may contain R proteins that bind other substances and cause falsely increased values. Some methods require a preliminary step in which sample is boiled in buffer solution.
Offsets: highly purified IF, or addition of cobinamide to saturate all R protein binding sites (cobinamide does not bind IF)
Pros and cons of automated assays for B12 measurement
Pros: higher precision, better turn around times, less labour intensive
Cons: spurious increases in B12 due to IF-blocking antibodies in patients with pernicious anaemia
Pros and cons of serum MMA as an indirect test of B12
Pros: sensitive - the first test to be raised in subclinical vitamin B12 deficiency; unaffected by folate deficiency
Cons: need G/LCMS, MMA increased by renal insufficiency and age >65 years
Pros and cons of homocysteine as an indirect test for B12
Pros: Sensitive; reliably measured by HPLC with fluorescent or electrochemical detection and with enzymatic and capillary gas GCMS
Cons: not specific - elevated in deficiencies of folate, B2 and B6 as well as B12; preanalytical rises with if sample left standing
Offsets: analysis soon after venipuncture or use of EDTA tubes
Pros and cons of holotranscobalamin measurement for B12
Pros: the only B12 moiety specifically available for uptake by all cells; has the best diagnostic accuracy for B12 deficiency
Cons: not widely available; sensitivity and specificity of assays in the diagnosis of B12 deficiency yet to be established
Pros and cons of deoxyuridine suppression test for B12
Cons: time consuming; not widely available
What is the Schilling test?
A test of vitamin B12 absorption. It permits differentiation of causes of vitamin B12 deficiency.
Describe the Schilling test
The proportion of B12 absorbed from orally administered 57 or 58-Cobalt labelled vit B12is measured by determining radioactivity in faeces, urine or serum, or by externally scanning the liver. Usual procedure - measure radioactivity in 24 hour urine sample collected after oral administration of 0.5mcg radioactive Co-labelled vit B12 after overnight fast
Pre-analytical variables to consider with B12 measurement
- Fasting preferred
- Stable up to 72 hours without freezing
- EDTA, serum, citrated or heparinised samples all acceptable
- Centrifuge within 1 hour or keep on ice until centrifugation
Causes of a low B12
- Deficiency
- Low haptocorrin due to low total granulocyte mass (benign neutropenia, MM, leukaemic reticuloendotheliosis, hypoplastic bone marrow)
Functions of folate
Interconversion of amino acids eg homocysteine to methionine.
Purine and pyrimidine (DNA) synthesis
Folate methods
Red cell folate
Serum folate
Competitive protein binding
Microbiological
Homocysteine
Folate deficiency causes
Megaloblastic anaemia
Neural tube defects
Causes of folate deficiency
Gut sterilization
Malabsorption
Decreased intake
Increased requirements (eg, pregnancy)
Medications (eg, methotrexate, anticonvulsants)
Predominant form of folate in blood
5-methyltetrahydrofolate
What does serum folate tell you?
Recent intake, not stores. Whole blood and RBC folate concentrations are more indicative of tissue stores over the lifetime of RBCs and are therefore a better indicator of longer term folate status than serum folate
Problems with competitive protein binding assays for folate
Method-method variability
Lack of standardisation
Radioactivity
Stability of folate in serum and plasma?
EDTA preserves well - stable at room temp up to 48 hours. Gradual decline in serum folate over 72 hours at RT - if delayed analysis, spin, separate and freeze.
