Assessing biomarker status of vitamin B 12 in the laboratory: no simple solution

Cobalamin (vitamin B₁₂) deficiency causes megaloblastic anaemia and irreversible neurological disease leading to death if untreated. Early detection and treatment are therefore essential. Severe deficiency of vitamin B₁₂ arises with pernicious anaemia, an autoimmune gastritis characterized by B₁₂ malabsorption owing to loss of intrinsic factor. ¹ A more subtle depletion of vitamin B₁₂ status can, however, arise from mild atrophic gastritis leading to reduced gastric acid production (hypochlorhydria), thereby diminishing B₁₂ absorption from food because of the essential role of gastric acid in the release of B₁₂ from food proteins. ² Food-bound B₁₂ malabsorption commonly occurs in older adults (reported to affect up to 20%) ¹ and leads to subclinical deficiency, with metabolic evidence of deficient status but without the classical haematological or neurological signs of deficiency. ³ Of note, low B₁₂ status found in older adults is rarely attributable to dietary insufficiency and is typically the result of malabsorption related to atrophic gastritis or use of proton pump inhibitors or other gastric acid suppressant drugs. ⁴ Emerging evidence indicates that low (though not necessarily deficient) biomarker status of B₁₂ is associated with increased risk of various chronic diseases of ageing including cognitive dysfunction, cardiovascular disease and osteoporosis. ⁴ Therefore, assessing B₁₂ status and correction of low/deficient status should be public health priorities.

Vitamin B₁₂ status is assessed using up to four biomarkers, both direct (total B₁₂ and holotranscobalamin; holoTC) and functional (homocysteine and methylmalonic acid; MMA) biomarkers. Measurement of serum total vitamin B₁₂ has been the standard clinical test for many years, with B₁₂ deficiency generally identified as B₁₂ concentrations <148 pmol/L; however, this can vary between laboratories. ⁵ For this purpose, the microbiological assay is generally considered to have good sensitivity for diagnosis of clinical B12 deficiency but, owing to its time consuming and laborious nature, has been replaced in almost all clinical laboratories by automated competitive protein binding assays. ⁶ The diagnostic sensitivity of some assays has caused concern, however, with falsely elevated results reported in patients with pernicious anaemia. ⁶

Measurement of metabolites of vitamin B₁₂-dependent reactions can be used as functional indicators of B₁₂ status. Methionine synthase requires vitamin B₁₂ as a cofactor in the remethylation of homocysteine to methionine. The activity of this enzyme is impaired with vitamin B₁₂ depletion, leading to an elevation of total homocysteine that can be readily measured in plasma using a variety of automated analytical techniques. ⁷ It should be noted, however, that plasma homocysteine is not specific to vitamin B₁₂, as it is influenced by other nutrient (most notably folate) and non-nutrient factors including renal function, greatly limiting its use as a biomarker of B₁₂ status. Vitamin B₁₂ is also a cofactor for methylmalonyl CoA mutase. In vitamin B₁₂ depletion, reduced activity of methylmalonyl CoA mutase leads to an accumulation of the by-product MMA which can be measured in plasma or urine. Measurement of MMA, unlike homocysteine, provides a specific biomarker for vitamin B₁₂. The majority of patients with vitamin B₁₂ deficiency will have elevated serum MMA, and it has also proven to be a useful biomarker in monitoring subclinical deficiency in population-based studies. ³ Limitations of serum MMA as a biomarker of B₁₂ status include the fact that it is greatly influenced by renal dysfunction and genetic variation, along with high running costs. ⁵

Measurement of HoloTC (or ‘active B₁₂’) is theoretically attractive because, unlike serum total B₁₂ (which measures concentrations of the total vitamin, 80% of which is metabolically inert), it represents the metabolically active fraction of vitamin B₁₂ available for cellular processes. HoloTC was reported to be better correlated with tissue stores of vitamin B₁₂ and was found to be superior to serum total B₁₂ and MMA in diagnosing B₁₂ tissue deficiency in an older Irish population. ⁸ Other reports, however, showed that HoloTC was only modestly better than serum total B₁₂ in diagnosing clinical deficiency (as defined using MMA rather than tissue stores of the vitamin).^9,10 Also, HoloTC is found to be elevated in patients with renal insufficiency, liver disease and cancer and is influenced by genetic factors, limiting its use as a first-line diagnostic tool for identifying vitamin B₁₂ deficiency. ¹¹

Given the limitations of individual assays, experts in the field now recommend that more than one biomarker is used to accurately diagnose vitamin B₁₂ deficiency,^3,11 with the recent emergence of approaches that identify deficient status using combinations of two or more biomarkers. The National Health and Nutrition Examination Survey opted to use the combination of serum total vitamin B₁₂ and MMA to monitor B₁₂ status in the United States population. ¹² Furthermore, algorithms have been developed to diagnose vitamin B₁₂ deficiency, including the ‘Fedosov’s Wellness Score’, a combined B₁₂ index which uses two, three or four B₁₂ biomarkers in combination and accounts for age and folate status, with results expressed as probable deficiency, possible deficiency and low, adequate and elevated vitamin B₁₂ status. ¹³ The usefulness of Fedosov’s Wellness Score has been demonstrated in a small number of studies; however, the high costs involved are likely to make this approach prohibitive for the purposes of routine clinical practice. ⁷ More feasible is the approach adopted by a number of clinical laboratories which have developed first- and second-line diagnostic procedures, whereby one biomarker (usually serum total B₁₂ or holoTC) is measured, with additional measurements (usually MMA) performed in samples with indeterminate results. ⁷

In summary, accurate assessment of vitamin B₁₂ is problematic, and there is no consensus as to the best biomarker for use in clinical laboratories. HoloTC shows promise as a reliable biomarker of vitamin B₁₂ status, but the influence of confounding factors needs to be more fully elucidated. The use of a sole biomarker of vitamin B₁₂ status should be avoided where possible, and two or more biomarkers should be used in combination. This will ensure that vitamin B₁₂ deficiency is diagnosed and treated in patients, and vitamin B₁₂ status optimized in older populations generally, to ensure that any adverse health consequences of deficient or low B₁₂ status are prevented.