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Markedly Increased Vitamin B₁₂ Concentrations Attributable to IgG–IgM–Vitamin B₁₂ Immune Complexes

¹ Department of Laboratory Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD.
² Cognitive Neuroscience Section, National Institutes of Neurological Disorders and Stroke, Bethesda, MD.

^aAddress correspondence to this author at: Department of Laboratory Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bldg. 10, Rm. 2C-306, 10 Center Dr., Bethesda, MD 20892-1508. Fax 301-402-2046; e-mail: mhorne{at}mail.cc.nih.gov.

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.