Markedly increased vitamin B₁₂ concentrations attributable to IgG-IgM-vitamin B₁₂ immune complexes

Background: High serum vitamin B₁₂ concentrations have been reported in patients with hepatic disease, disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndromes. We recently discovered an extraordinarily increased vitamin B₁₂ concentration in a patient without these underlying conditions. Methods: Affinity and size-exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and ELISA methods were used to determine the cause of the increased vitamin B₁₂ concentrations in this patient's serum. Results: The protein G column eluates from 2 apparently healthy volunteers and 2 patients with recent vitamin B₁₂ treatment for anemia had vitamin B₁₂ concentrations of <74 pmol/L, whereas the vitamin B₁₂ concentration in the protein G column eluate from the patient was 7380 pmol/L. The elution profile from size-exclusion chromatography of vitamin B₁₂-binding proteins in the patient's serum revealed an abnormal vitamin-B₁₂-binding protein. SDS-PAGE analysis of the concentrated eluates from the protein G column, under reducing conditions, revealed an additional band with an apparent molecular mass of 76 kDa, which was not present in control column eluates. MALDI-TOF MS identified this band as an IgM heavy chain. By use of a modified ELISA, we determined that the IgM present in the patient's eluates was associated with the IgG to form IgG-IgM immune complexes. Conclusions: This case demonstrates the unusual circumstance of a patient with markedly increased vitamin B₁₂ concentrations attributed to immune complexes composed of IgG, IgM, and vitamin B₁₂ and illustrates techniques that can be used to identify this occurrence.