Vitamin B12 and methylmalonic acid profile



As vitamins go, B12 is a rather large molecule. One part of its structure is a "corrin" nucleus, which resembles the "haem" of haemoglobin. While the haem moiety in haemoglobin holds an atom of iron, in B12 the corrin group holds an atom of cobalt. In order to be true B12, cobalamin must have one of a number of attachments to the corrin group: depending on the attachment, cobalamin can be cyanocobalamin, hydroxycobalamin, aquacobalamin, nitritocobalamin, methylcobalamin, or adenosylcobalamin. O

Patient Instructions:

Do not consume any vitamin B12 containing nutritional supplements for 24-48 hours prior to testing.

Clinical Indications:

Deficiency states induced by poor dietary intake of B12 (i.e. vegetarian or vegan diets) take up to 20 years to manifest. However, clinical deficiencies as a consequence of abnormalities in one of the multiple steps that regulate cobalamin absorption or its enterohepatic circulation present more rapidly (c. 2 years). Vitamin B12 deficiency is relatively common in the U.K. population and occurs in patients with autoimmune disease (pernicious anaemia), severe primary hypothyroidism and ileal disease, as well as in subjects receiving chronic therapy with antacids, proton pump inhibitors (PPI) or H2 antagonists. B12 deficiency is also seen in chronic malnutrition states, including alcoholism, while other causes include vegetarian diets [3], pregnancy and lactation.

The timely detection, and correction, of vitamin B12 deficiency prevents the development of macrocytic anaemia, as well as elevated homocysteine (which is a thrombotic risk factor), potentially irreversible peripheral neuropathy, memory loss and other cognitive deficits.

Vitamin B12 status assessment

The measurement of serum 'total' levels of vitamin B12 is commonly used as a marker of vitamin B12 status. However, it is not widely appreciated that circulating vitamin B12 is predominately bound to two different proteins (haptocorrin and holotranscobalamin) and that the commonly available laboratory assays are unable to discriminate between the 'Inactive' (haptocorrin-bound) and 'Active' (holotranscobalamin-bound) forms. Although some 80% of circulating vitamin B12 is carried by haptocorrin (holohaptocorrin, abbreviated as HC), extra-hepatic cellular receptors for this form have not been described (hence the term 'Inactive' B12 for this form) [4]. Circulating levels of HC slowly decline in response to the onset of a deficiency state, typically taking 3-6 years to fall below the lower limit of the reference range. If the assessment is done using a B12 assay that cannot discriminate between these two forms, then any decline in the less abundant 'Active' form is masked by the more abundant 'Inactive' variant. Tissue deficiency of vitamin B12 is thus common among patients in whom the serum total ('Inactive' + 'Active') vitamin B12 level is within the reference range for total B12 [5].

The 'Active' Vitamin B12 (holotranscobalamin or holoTC) assay

'Active' vitamin B12 (holo TC) is the metabolically active portion of vitamin B12. This assay measures holotranscobalamin in a form that can be taken up by the receptor-mediated process found in all DNA-producing cells [6]. The holoTC assay is the earliest and most sensitive laboratory marker of a negative vitamin B12 balance and gives a better indication of vitamin B12 deficiency than the previous commonly used markers.

Interpretation of 'Active' Vitamin B12 results using the Guy's and St.Thomas' Nutristasis Unit laboratory algorithm

Patients are defined as deficient if the 'Active' vitamin B12 concentration is < 25 pmol/L.

The status of patients with 'Active' vitamin B12 concentrations between 25 and 50 pmol/L (the intermediate range) requires further investigation and this is best carried out using the serum methylmalonic acid (MMA) assay [7,8,9,10]. When the supply of vitamin B12 is suboptimal production of succinyl CoA is blocked, leading to increased formation of MMA. Circulating levels of MMA are thus a functional indicator of vitamin B12 status. Serum MMA concentrations of <280 nmol/L are considered to be within the reference range for healthy subjects under the age of 65 years. The upper limit of the serum MMA reference range for patients older than 65 years is 360 nmol/L. However, one limitation is that serum MMA levels are not a reliable marker of vitamin B12 status in patients with impaired renal function (which is more common in the elderly).

Patients with 'Active' vitamin B12 levels above 50 pmol/L are replete and require no further investigation for at least 4 months. Please note that the diagnostic utility of this test can be limited immediately after vitamin B12 supplementation [11]. If supplemental B12 has been taken within one month prior to the test, this should be stated on the request form.

There is a transient increase in circulating levels of 'Active' vitamin B12 6-8 hours after oral supplementation. Thus monitoring 'Active' vitamin B12 can identify subjects who are likely to benefit from oral treatment.

See test datasheet for full details.


vitaminB12.pdf (Click to Download)

Sample Requirements:

SST (Gold top tube)

Postal Samples Acceptable:



1. Ehrenpreis E.D. Absorption of vitamin B12. In Herbert V., ed. Vitamin B12 deficiency, pp 15-8. London: Royal Society of Medicine Press, 2002.
2. Schjonsby H. Vitamin B12 absorption and malabsorption. Gut 1989;30:1686-91.
3. Herrmann W, Schorr H, Obeid R, Geisel J. Vitamin B-12 status, particularly holotranscobalamin II and methylmalonic acid concentrations, and hyperhomocysteinemia in vegetarians. American Journal of Clinical Nutrition 2003;78:131-6.
4. Morkbak AL, Poulsen SS, Nexo E. Haptocorrin in humans. Clinical Chemistry and Laboratory Medicine 2007;45:1751-9
5. Herrmann W, Obeid R, Schorr H, Geisel J. The usefulness of holotranscobalamin in predicting vitamin B12 status in different clinical settings. Curr.Drug Metab 2005;6:47-53.
6. Herbert V. Vitamin B12 deficiency - an overview. In Herbert V, ed. Vitamin B12 deficiency, pp 1-8. London: Royal Society of Medicine Press Ltd, 2002.
7. Lloyd-Wright Z, Hvas AM, Moller J, Sanders TA, Nexo E. Holotranscobalamin as an indicator of dietary vitamin B12 deficiency. Clin.Chem. 2003;49:2076-8.
8. Herrmann W, Obeid R, Schorr H, Geisel J. Functional vitamin B12 deficiency and determination of holotranscobalamin in populations at risk. Clin.Chem.Lab Med. 2003;41:1478-88.
9. Obeid R, Jouma M, Herrmann W. Cobalamin status (holo-transcobalamin, methylmalonic acid) and folate as determinants of homocysteine concentration. Clin.Chem. 2002;48:2064-5.
10. Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, Orning L et al. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am.J.Clin.Nutr. 2001;74:233-41.
11. Carmel R, Vasireddy H, Aurangzeb I, George K. High serum cobalamin levels in the clinical setting--clinical associations and holo-transcobalamin changes. Clin.Lab Haematol. 2001;23:365-71.

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