Bowen Raffick A R, Drake Steven K, Vanjani Rachna, Huey Edward D, Grafman Jordan, Horne McDonald K
Department of Laboratory Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892-1508, USA.
Clin Chem. 2006 Nov;52(11):2107-14. doi: 10.1373/clinchem.2006.073882.
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.
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.
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.
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.
据报道，肝病、播散性肿瘤、骨髓增殖性疾病和嗜酸性粒细胞增多综合征患者的血清维生素B12浓度较高。我们最近在一名没有这些基础疾病的患者中发现维生素B12浓度异常升高。
采用亲和色谱和尺寸排阻色谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和ELISA方法，以确定该患者血清中维生素B12浓度升高的原因。
2名明显健康的志愿者和2名近期因贫血接受维生素B12治疗的患者的蛋白G柱洗脱液中维生素B12浓度<74 pmol/L，而该患者蛋白G柱洗脱液中的维生素B12浓度为7380 pmol/L。该患者血清中维生素B12结合蛋白的尺寸排阻色谱洗脱图谱显示存在一种异常的维生素B12结合蛋白。在还原条件下，对蛋白G柱浓缩洗脱液进行SDS-PAGE分析，发现一条额外的条带，表观分子量为76 kDa，对照柱洗脱液中不存在该条带。MALDI-TOF MS鉴定该条带为IgM重链。通过使用改良的ELISA，我们确定患者洗脱液中的IgM与IgG结合形成IgG-IgM免疫复合物。
本病例展示了一名患者因由IgG、IgM和维生素B12组成的免疫复合物导致维生素B12浓度显著升高的罕见情况，并说明了可用于识别这种情况的技术。
