Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency

Abstract

Vitamin B12 (cobalamin, Cbl, B12) is an indispensable water-soluble micronutrient that serves as a coenzyme for cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MCM). Deficiency of Cbl, whether nutritional or due to inborn errors of Cbl metabolism, inactivate MS and MCM leading to the accumulation of homocysteine (Hcy) and methylmalonic acid (MMA), respectively. In conjunction with total B12 and its bioactive protein-bound form, holo-transcobalamin (holo-TC), Hcy, and MMA are the preferred serum biomarkers utilized to determine B12 status. Clinically, vitamin B12 deficiency leads to neurological deterioration and megaloblastic anemia, and, if left untreated, to death. Subclinical vitamin B12 deficiency (usually defined as a total serum B12 of <200 pmol/L) presents asymptomatically or with rather subtle generic symptoms that oftentimes are mistakenly ascribed to unrelated disorders. Numerous studies have now established that serum vitamin B12 has limited diagnostic value as a stand-alone marker. Low serum levels of vitamin B12 not always represent deficiency, and likewise, severe functional deficiency of the micronutrient has been documented in the presence of normal and even high levels of serum vitamin B12. This review discusses the usefulness and limitations of current biomarkers of B12 status in newborn screening, infant and adult diagnostics, the algorithms utilized to diagnose B12 deficiency and unusual findings of vitamin B12 status in various human disorders.

Keywords: cobalamin; diagnostic algorithm; functional deficiency of B12; holo-transcobalamin; homocysteine; methylmalonic acid; vitamin B12.

Figure 1

Cellular processing and trafficking of dietary vitamin B₁₂. Cells take up holo-TC via TC-receptor-mediated endocytosis (TCR). In the lysosome, cobalamin (Cbl) is liberated and apo-transcobalamin is degraded, while the TCR is recycled back to the cell surface. Cbl exits the lysosome via transporters cblF/cblJ. In the cytosol, Cbl undergoes processing by enzyme cblC, which catalyzes removal of the upper-axial ligand (R), reduction of the cobalt center and conversion into the base-off configuration. The fate of newly processed B₁₂ is dictated by cblC-cblD interactions, which direct the cofactor into either cytosolic methionine synthase (MS, cblG) or mitochondrial methylmalonyl-CoA mutase (MCM, mut). Nutritional and functional deficiencies of B₁₂ lead to the inactivation of client enzymes MS and MCM with elevations of tHcy and MMA.

Figure 2

Pathways for Hcy and MMA metabolism in humans. (A) Homocysteine is a branch-point metabolite at the intersection of either the remethylation or the transsulfuration pathways. Thus, Hcy homeostasis relies on three different biochemical reactions [MS, cystathionine β-synthase (CBS) and S-adenosylhomocysteine hydrolase (SAHH)], two of which (CBS and SAHH) are independent of vitamin B₁₂. In addition to nutritional deficiency of vitamin B₁₂, elevation of Hcy in plasma may arise from reduced function of CBS and MTHFR, as well as nutritional deficiencies of folate. (B) MMA is produced during catabolism of odd-chain fatty acids and amino acids in the mitochondrion. Propionyl-CoA is the precursor of MMA in a reaction catalyzed by propionyl-CoA carboxylase (PCC). Inborn errors of PCC lead to propionic acidemia. Likewise, mutations in AdoCbl-dependent MCM lead to a buildup of MMA-CoA and inhibition of PCC that manifests as increased propionyl-CoA and so of propionic acid the circulation. Propionylcarnitine can also be transported out of the cell to reach systemic circulation. Propionylcarnitine is a first-line test in newborn screening.

Figure 3

Algorithm for the diagnosis of vitamin B₁₂ deficiency in neonates: metabolomic and functional studies. Elevated propionylcarnitine (C3) prompts the analysis of nutritional history with examination of vitamin B₁₂ and its metabolites, tHcy, and MMA. Combined hyperhomocysteinemia and methylmalonic acidemia may be caused by insufficient B₁₂ intake, defective absorption (intrinsic factor, transcobalamin) and genetic mutations affecting uptake (transcobalamin receptor, TCblR), lysosomal exiting (cblF and cblJ), processing (cblC), and trafficking (cblD). Total or incomplete response to a high dose of HOCbl helps to elucidate the cause of the dysfunction. Importantly, mutations in the TCblR gene may present with either the combined phenotype or with isolated methylmalonic acidemia, which should be considered using flow charts (dotted lines). Classical isolated hyperhomocysteinemia and methylmalonic acidemia are typically confirmed by functional assays, except for CBS deficiency that presents with normal to high methionine levels. Mutations affecting methionine synthase (cblG) and its partner reductase (cblE) lead to isolated hyperhomocysteinemia, whereas mutations affecting MCM (mut) and its associated proteins (cblA, cblB) result in isolated methylmalonic acidemia. Final confirmation of the disease is carried out by gene sequencing. This figure was modified from Baumgartner and Fowler (2014).