B12

Question 1

Functions of B12

Answer 1

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)

Question 2

Describe the structure of vitamin B12

Answer 2

Tetrapyrrole ring (also known as a corrin) surrounding a central cobalt atom (corrin + cobalt = cobalamin) with various side chains appended.

Question 3

List a few different types of cobalamin based on their side chain(s)

Answer 3

Methylcobalamin - methyl side chain
Aquacobalamin/vitamin B12b - water side chain
Cyanocobalamin - cyanide side chain
Cobalamin/coenzyme B12 - adenosyl side chain
Hydroxocobalamin - hydroxyl side chain

Question 4

What is the reference compound for measuring serum cobalamin? Why?

Answer 4

Cyanocobalamin - a stable, red, needle-like crystal - has stability in aqueous solutions and a distinct absorption spectrum, with maxima at 278, 361 and 550nm.

Question 5

What is the main physiologic form of cobalamin in serum?

Answer 5

Methylcobalamin

Question 6

What is the main physiologic form of cobalamin in the cytosol?

Answer 6

Adenosylcobalamin

Question 7

Cyanocobalamin is gradually destroyed on exposure to light

Answer 7

True

Question 8

Which two vitamin deficiencies can cause hyperhomocysteinaemia?

Answer 8

Vitamin B6 and Vitamin B12

Question 9

How do PPIs cause vitamin B12 deficiency?

Answer 9

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.

Question 10

What is the role of intrinsic factor in Vitamin B12 absorption?

Answer 10

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.

Question 11

What is the difference between total B12 and holotranscobalamin?

Answer 11

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

Question 12

What is the function of holohaptocorrin?

Answer 12

Unknown!

Question 13

How is B12 excreted?

Answer 13

In the urine and faeces (via bile)

Question 14

Methods for B12 determination

Answer 14

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

Question 15

Pros and cons of microbiological assays for B12

Answer 15

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

Question 16

Pros and cons of competitive protein binding assays for B12

Answer 16

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)

Question 17

Pros and cons of automated assays for B12 measurement

Answer 17

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

Question 18

Pros and cons of serum MMA as an indirect test of B12

Answer 18

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

Question 19

Pros and cons of homocysteine as an indirect test for B12

Answer 19

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

Question 20

Pros and cons of holotranscobalamin measurement for B12

Answer 20

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

Question 21

Pros and cons of deoxyuridine suppression test for B12

Answer 21

Cons: time consuming; not widely available

Question 22

What is the Schilling test?

Answer 22

A test of vitamin B12 absorption. It permits differentiation of causes of vitamin B12 deficiency.

Question 23

Describe the Schilling test

Answer 23

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

Question 24

Pre-analytical variables to consider with B12 measurement

Answer 24

1. Fasting preferred
2. Stable up to 72 hours without freezing
3. EDTA, serum, citrated or heparinised samples all acceptable
4. Centrifuge within 1 hour or keep on ice until centrifugation

Question 25

Causes of a low B12

Answer 25

1. Deficiency
2. Low haptocorrin due to low total granulocyte mass (benign neutropenia, MM, leukaemic reticuloendotheliosis, hypoplastic bone marrow)

Question 26

Functions of folate

Answer 26

Interconversion of amino acids eg homocysteine to methionine.
Purine and pyrimidine (DNA) synthesis

Question 27

Folate methods

Answer 27

Red cell folate
Serum folate
Competitive protein binding
Microbiological
Homocysteine

Question 28

Folate deficiency causes

Answer 28

Megaloblastic anaemia
Neural tube defects

Question 29

Causes of folate deficiency

Answer 29

Gut sterilization
Malabsorption
Decreased intake
Increased requirements (eg, pregnancy)
Medications (eg, methotrexate, anticonvulsants)

Question 30

Predominant form of folate in blood

Answer 30

5-methyltetrahydrofolate

Question 31

What does serum folate tell you?

Answer 31

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

Question 32

Problems with competitive protein binding assays for folate

Answer 32

Method-method variability
Lack of standardisation
Radioactivity

Question 33

Stability of folate in serum and plasma?

Answer 33

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.