The prevalence of vitamin B12 deficiency in the general population is greater than previously thought; early diagnosis is crucial because of the possible risk of irreversible neurological damage. The widely used total serum B12 assay, however, does not reliably indicate vitamin B12 status. To improve the accuracy of diagnosis, the concept of measuring holotranscobalamin (Active B12) has aroused great interest. This article introduces newer methods for Active B12 measurement, explores the issues and discusses the clinical utility of Active B12.
by E. Valente
Vitamin B12 deficiency
Vitamin B12 deficiency is widespread and a major public health issue.. Among the population groups at risk are the elderly, pregnant women and their offspring, vegetarians and patients with intestinal diseases. The classic haematological signs and symptoms may be absent or non-specific. Neurological symptoms may be the only manifestation of deficiency and can be irreversible if treatment is delayed. Early and accurate detection of vitamin B12 deficiency is therefore important. The current widely used analyte, total serum B12, is now recognised as being a late marker, with sub-optimal sensitivity and specificity. The diagnostic challenge associated with this has lead to the search for earlier and more accurate biochemical markers for vitamin B12 deficiency. Holotranscobalamin, or Active-B12, has been shown to be an early marker of vitamin B12 deficiency and may fulfill this role.
Active-B12 (holotranscobalamin)
Intrinsic Factor (IF), transcobalamin (TC) and haptocorrin (HC) are three binding proteins involved in the assimilation and transport of vitamin B12. These binding proteins ensure the efficient uptake of the very small amounts of dietary vitamin B12. All of these proteins have extremely high affinity for vitamin B12. with dissociation constants around 5fM [1]. When transcobalamin and haptocorrin bind vitamin B12 the resulting complexes are known as holotranscobalamin (Active-B12 or holoTC) and holohaptocorrin (holoHC).
The function of transcobalamin is to transport vitamin B12 from its site of absorption in the ileum to tissues and cells that express specific receptors. The vitamin is then internalised as the Active-B12 (holoTC) complex. Once in the cell vitamin B12 is an important co-factor for two enzymes. As adenosyl cobalamin it catalyses the formation of succinyl-CoA from methylmalonyl-CoA; methyl cobalamin is the co-factor for the conversion of homocysteine to methionine by methionine synthase.
Circulating vitamin B12
Normally, less than 30% of the vitamin B12 in plasma circulates as Active-B12 (holoTC). The remaining ~70% is bound to haptocorrin, whose function is uncertain. It is thought that haptocorrin transports surplus B12 to the liver and may also be involved in clearing B12 analogues (corrinoids) from the circulation [2]. Absent or dysfunctional haptocorrin is relatively rare, clinically benign and usually discovered accidentally [3]. On the other hand, absent or dysfunctional transcobalamin manifests as the typical haematological and neurological pathologies of vitamin B12 deficiency [4]. This condition is usually discovered shortly after birth and aggressive therapy is required to avoid irreversible neurological damage. Frequently, in such cases serum analysis shows misleading total vitamin B12 levels (within the normal range).
Diagnosis of vitamin B12 deficiency
Accurate assays for determining vitamin B12 status are needed because of the high prevalence and serious complications of deficiency. Typically, vitamin B12 deficiency is suspected only when an individual presents with haematological manifestations of megaloblastic anaemia, which tends to occur only in the most severely vitamin B12 depleted individuals [5].
Determination of total plasma vitamin B12 concentration is the current standard clinical test for vitamin B12 deficiency. Depending on the method and population under investigation, total vitamin B12 concentrations of less than ~150pmol/L (200pg/mL) are generally considered indicative of deficiency. However, a proportion of individuals with vitamin B12 levels that would be considered deficient exhibit no clinical or biochemical evidence of deficiency [6]. Conversely, neuropsychiatric and metabolic abnormalities can occur with plasma vitamin B12 concentrations well within the normal reference range [6,7].
Limitations of total vitamin B12 assays
The measurement of total plasma vitamin B12 suffers from a number of limitations, most particularly that the majority of vitamin B12 that is measured is the fraction bound to haptocorrin. As Active-B12 (holoTC) has a shorter circulating half-life compared to holohaptocorrin, the earliest change that occurs on entering negative vitamin B12 balance is very likely to be a decrease in plasma Active -B12 (holoTC) concentration [8].
Total vitamin B12 assay indeterminate zone
There are many publications attesting to a significant indeterminate zone when using the total vitamin B12 assay. Around 30-40% of all requested B12 determinations would fall within an indeterminate zone of 151-300pmol/L.
It has been estimated that vitamin B12 deficiency can occur up to total B12 levels of 300pmol/L and even beyond, and that 45% of vitamin B12 deficient subjects would remain undiagnosed if screening only measured vitamin B12 level [9].
This suggests an initial clinical utility for Active-B12 (holoTC) in order to resolve indeterminate vitamin B12 results. It is expected however that Active-B12 (holoTC) will eventually supplant total serum B12 as the initial diagnostic test for suspected vitamin B12 deficiency and may also have utility for screening.
Measuring Active-B12 (holoTC)
The use of Active-B12 (holoTC) as an accurate marker of vitamin B12 status was first proposed by Herzlich and Herbert in 1988 [8]. The cloning of the transcobalamin gene paved the way for new assay methods based on specific transcobalamin antibodies [10,11]. Very recently a novel monoclonal antibody was developed with at least 100-fold specificity for Active-B12 (holoTC) as compared to transcobalamin apo-protein (TC), which has allowed the development of simple, direct immunoassays for the quantitation of Active-B12 (holoTC) [12,13]. Such assays avoid the need for the various reduction/extraction/conversion steps employed by most vitamin B12 assays, thus removing a possible cause of pre-analytical variability. Axis-Shield have been in the vanguard in developing methods for estimating Active B12, having developed the first EIA on the Abbott AxSYM analyser and having further automated methods in development. Additionally, there will soon be a new microwell EIA, available from Axis-Shield.
The clinical utility of Active-B12
As expected, Active-B12 (HoloTC) levels are low in patients with biochemical signs of vitamin B12 deficiency [14]. Low values have been reported in vegetarians [15,16], vegans [17], and populations with a low in-take of vitamin B12 [18]. Some comments regarding the use of Active B12 are quoted below:
• “HoloTC can be used as a first line parameter in detecting cobalamin deficiency” [19].
• “..we report that the decline in cobalamins during pregnancy is caused by alteration in cobalamins attached to HC (haptocorrin) rather than in alterations in holoTC. Our data suggest that holoTC, rather than cobalamins can be used as a marker for vitamin B12 deficiency during pregnancy” [20].
• “Cobalamin status (ie, holotranscobalamin) remained significantly lower in breastfed than in non-breastfed infants. We also observed that low serum cobalamin during breastfeeding was explained by a low holoTC concentration, whereas the holohaptocorrin concentration did not change” [21].
• “There was almost a tripling of risk for neural tube defects in the presence of low maternal B12 status, as measured by holoTC” [22].
• “This longitudinal cohort study showed that low serum concentrations of holoTC..were..independently and significantly associated with a more rapid cognitive decline during a 10-y period” [23].
References
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20. Morkbak AL et al. Haematologica 2007; 92: 1711-1712
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22. Ray JG et al. Epidemiol 2007; 18: 362-366
23. Clarke R et al. Am J Clin Nutr 2007; 86: 1384-1391
The author
Edward Valente, Marketing Manager
Axis-Shield Diagnostics
The Technology Park
Dundee, DD2 1XA, UK
Tel. +44 1382 422000
e.mail: Edward_valente@uk.axis-shield.com
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