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Vitamin B12 Testing in General Practice


Vitamin B12 testing remains the most common vitamin investigation in clinical practice and is often included in the investigation of common problems such as anaemia and dementia.
The assessment of Vitamin B12 status using blood tests is imperfect and although a variety of other tests can be used to improve assessment, this can lead to complexity and confusion. In this discussion I hope to share the insights from thousands of analyses and hundreds of clinician’s questions.

Sources of Vitamin B12

Vitamin B12 is a unique cobalt-containing molecule naturally synthesised by bacteria. Some animals, especially herbivores, absorb it from their intestinal microbiome, and build up a store.

Other animals, particularly carnivores, can obtain B12 by eating animals that store B12, or animal-based products such as eggs and milk. Vegetarians consuming milk products and eggs may have low B12 levels, as the B12 content of milk is often low (1mg/L) and even lower if ultra-heat treated. Non-animal sources of B12 are extremely limited, with Nori seaweed containing small amounts and B12 levels in mushrooms and most other plant-based sources reflecting bacterial exposure (eg manure/compost).(1)

Only strict vegetarians are considered at serious risk of dietary B12 deficiency, and even then only after some years. However, vegetarian and vegan diets are becoming increasingly popular. Similarly, breast-fed infants of vegan mothers, if not supplemented, may also be at risk of B12 deficiency.

How common is B12 deficiency?

Vitamin B12 deficiency is relatively common (4- 26%) but difficult to define accurately because of varying definitions.(2)

It is more common in the elderly and significant deficiency is present in up to 23% of elderly Australian populations.(3) Iron deficiency is similarly common, especially in young women, and since low consumption of iron from meat sources correlates with lower B12 intake, B12 deficiency should always be considered when dietary iron deficiency exists.

While pernicious anaemia is often considered as a cause of B12 deficiency, this autoimmune illness has a relatively low prevalence compared to B12 deficiency. In our experience, only 4% of our Intrinsic Factor antibody requests are positive which is a similar result to that described by others.(4) Higher prevalence has been reported when using the less specific parietal antibody test, but even then, less than 20% of B12 deficiency can be attributed to pernicious anaemia.(5)

Less than one in eight patients with positive parietal cell antibodies have pernicious anaemia and this lack of specificity increases in the elderly when the test should be avoided.

Clinical issues

Unexplained anaemia and/or macrocytosis have traditionally been the indications used for suspicion of B12 deficiency. But there are other common reasons for anaemia such as iron deficiency and the anaemia of chronic disease. There are also other common reasons for macrocytosis including liver disease and alcoholism.

Vitamin B12 levels are more likely to be low in a vegetarian (or vegan) than in a patient with anaemia or macrocytosis.(6)

We also find that symptoms of confusion and dementia are just as likely to be associated with low B12 levels as anaemia. And while this may be partly associative due to the higher prevalence of B12 deficiency, it should be concerning because of the neurological sequelae of B12 deficiency that may arise prior to anaemia.

Neurological symptoms of B12 deficiency include paraesthesia of the hands and feet, diminished perception of vibration and position, absence of reflexes, and unsteady gait and balance (ataxia). But the range of symptoms is broad and may include irritability, tiredness, and mild memory and cognitive impairment.

Severe deficiency causes subacute combined degeneration of the spinal cord. In pregnancy, B12 deficiency is associated with some increase in the risk of neural tube defects and in childhood is associated with developmental delay and failure to thrive.

Why is testing so complicated?

Cobalamine is a precious vitamin that is captured and chaperoned around the body. Saliva contains a protein that will capture B12. In the stomach, intrinsic factor is produced to capture B12 released by digestion and transport it into the body.

Within the bloodstream, there are two proteins that bind B12; haptocorrin and transcobalamin. These two proteins seem to have different functions with transcobalamin delivering B12 to the cells whereas haptocorrin correlates with storage. (This is similar to iron where transferrin transports iron to the cells and ferritin reflects storage.)

How to interpret B12 and HoloTC levels.

The amount of B12 attached to transcobalamin (ie holo-transcobalamin or HoloTC) therefore reflects the Vitamin B12 level available to cells.

When there is a cellular deficiency of B12, the reactions dependent on B12 are obstructed and precursors such as homocysteine and methyl-malonic acid (MMA) build up and can be measured as indicators of functional B12 deficiency.

HoloTC correlates better with homocysteine and MMA than the total B12 level of the blood. When total B12 levels are low or equivocal, it is appropriate to follow up with the more specific HoloTC test to help ascertain if there is a functional deficiency.

Pregnant women often deplete their B12 stores during pregnancy, but they uncommonly have B12 deficiency evidenced by their normal HoloTC levels. Conversely, patients with some haematological malignancies may have high B12 stores (eg by tumours producing haptocorrin) but may not mobilise those stores evidenced by a low HoloTC and macrocytic anaemia.


Vitamin B12 deficiency is common and can be associated with neurological symptoms and haematological signs especially in vegetarians, and uncommonly in pernicious anaemia.
HoloTC is more specific for clinical B12 deficiency than total B12 and that is why laboratories reflex test for HoloTC whenever the total B12 is low or equivocal.


General Practice Pathology is a new regular column each authored by an Australian expert pathologist on a topic of particular relevance and interest to practising GPs. The authors provide this editorial free of charge as part of an educational initiative developed and coordinated by Sonic Pathology.



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  3. Flood VM, Smith WT, Webb KL, Rochtchina E, Anderson VE, Mitchell P. Prevalence of low serum folate and vitamin B12 in an older Australian population. Aust N Z J Public Health. 2006 Feb; 30(1): 38-41.
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