-
PDF
- Split View
-
Views
-
Cite
Cite
Raffick AR Bowen, Steven K Drake, Rachna Vanjani, Edward D Huey, Jordan Grafman, McDonald K Horne, III, Markedly Increased Vitamin B12 Concentrations Attributable to IgG–IgM–Vitamin B12 Immune Complexes, Clinical Chemistry, Volume 52, Issue 11, 1 November 2006, Pages 2107–2114, https://doi.org/10.1373/clinchem.2006.073882
Close -
Share
Abstract
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.
Vitamin B12 is an essential micronutrient that plays a fundamental role in cell division and 1-carbon metabolism (1). Vitamin B12 in serum is bound to 2 proteins, transcobalamin (TC)1 II and haptocorrin (HC). TC is a 43-kDa nonglycosylated serum protein that is synthesized primarily in the enterocytes and is essential for the transport of vitamin B12 from ileum into the blood and then into most cells via receptor-mediated endocytosis (1)(2). TC saturated with vitamin B12 (holo-TC) constitutes 6%–20% of endogenous circulating vitamin B12, whereas unsaturated TC constitutes ∼90% of TC. In contrast, HC is a glycosylated serum protein of ∼68 kDa that is produced mainly by myeloid cells, binds ∼80%–94% of the endogenous plasma vitamin B12, and is largely saturated with vitamin B12 (1)(2). The physiologic role of HC has not been fully established, but it may play a role in binding potentially harmful vitamin B12 analogs and transporting them to the liver via an asialoglycoprotein receptor for secretion into bile. Thus, it is only vitamin B12 bound to TC that is available for cellular uptake and use as coenzymes by l-methylmalonyl-CoA mutase and methionine synthase for succinyl-CoA and methionine synthesis, respectively (1)(2).
High serum vitamin B12 concentrations frequently occur with myeloproliferative disorders, hypereosinophilic syndromes, and hepatic diseases (3)(4)(5)(6)(7)(8)(9)(10)(11). In these cases, high serum vitamin B12 concentrations may be caused by up-regulation of HC and TC synthesis, increased release of cellular vitamin B12 from the liver, or decreased clearance of cobalamin from plasma (12). We describe here an extraordinarily increased vitamin B12 in a patient with neither myeloproliferative nor hepatic disease. Through the use of several analytical techniques, we determined that the high vitamin B12 concentration in this patient was attributed partially to immune complexes composed of IgG, IgM, and vitamin B12.
case history
The patient was a 46-year-old man with a diagnosis of frontotemporal dementia who was seen at the Warren G. Magnuson Clinical Center at the NIH as part of a research protocol. Amyotrophic lateral sclerosis was diagnosed in this patient while he was enrolled in this protocol. Before being seen at the NIH Clinical Center, the patient had been receiving, for several months, 15 mg of vitamin B12 (methylcobalamin) intramuscularly 3 times per week for his dementia. The patient’s serum vitamin B12 concentration, measured as part of a screening laboratory panel, was 42 600 pmol/L (reference interval, 162–708 pmol/L), a value that was confirmed by another laboratory (Advia Centaur, Mayo Medical Laboratory). The patient’s NIH physicians instructed the patient’s primary caregiver to stop the vitamin B12 injections and contacted the clinical laboratory to investigate the patient’s increased serum vitamin B12. Over the next 5 weeks, the patient’s vitamin B12 gradually decreased to ∼4428 pmol/L and remained stable.
The initial laboratory results were as follows: hemoglobin, 145 g/L (reference interval, 127–167 g/L); mean corpuscular volume, 89.5 fL (reference interval, 79–98 fL); leukocyte count, 10.0 × 109/L (reference interval, 3–9.6 × 109/L); and platelet count, 178 × 109/L (reference interval, 154–343 × 109/L). The absolute counts for lymphocytes, neutrophils, eosinophils, basophils, and monocytes were within the reference intervals. The peripheral blood smear showed normocytic and normochromic cells. The serum vitamin B12 concentration was very high, at 42 600 pmol/L (reference interval, 162–708 pmol/L), whereas erythrocyte folate, at 38.7 nmol/L, was within the reference interval (6.8–70 nmol/L). Tests for intrinsic factor, parietal cell, and rheumatoid factor antibodies were negative. Concentrations of methylmalonic acid (0.25 μmol/L; reference interval, 0–0.30 μmol/L; Mayo Medical Laboratory, Rochester, MN), total homocysteine (6.0 μmol/L; reference interval, 0–13 μmol/L), and holo-TC (110 pmol/L; reference interval, 40–150 pmol/L; Axis-Shield) were normal. The concentration of total TC was 1560 pmol/L (reference interval, 500-1500 pmol/L), and total HC was 626 pmol/L (reference interval, 240–680 pmol/L). The unsaturated vitamin B12 binding capacities of HC (1 pmol/L; reference interval, 70–350 pmol/L) and TC (0 pmol/L; reference interval, 250-1200 pmol/L) were abnormally low, indicating that HC and TC were fully saturated with vitamin B12. Serum urea, electrolytes, hepatic enzymes, glucose, calcium, thyroid function tests, and urinalysis were all normal. Serum IgA was 1.24 g/L (reference interval, 0.91–4.99 g/L), IgG was 6.61 g/L (reference interval, 6.42–17.30 g/L), and IgM was 1.47 g/L (reference interval, 0.34–3.42 g/L). Serum electrophoresis and immunofixation were normal.
Materials and Methods
We obtained blood samples from the case patient, patients with anemia, and apparently healthy volunteers. All specimens were processed within 1 h of collection. Serum was stored at −80 °C until analysis. We obtained written informed consent from the case patient’s guardian and from the other participating patients and volunteers.
heterophile blocking tube
To determine whether the high vitamin B12 concentration observed in our patient was caused by heterophile antibody interference, we assayed his serum for vitamin B12 on an IMMULITE 2000 analyzer (Diagnostic Products Corp.) in duplicate before and after incubating 500 μL of sample in heterophile blocking tubes (Scantibodies) at room temperature for 1 h.
dilution linearity
We checked linearity of the patient’s vitamin B12 result of 42 600 pmol/L by diluting his serum in phosphate-buffered saline (0.14 M NaCl, 0.01 M phosphate, pH 7.4 1:1, 1:2, 1:3, and 1:4) and assaying the diluted serum for vitamin B12.
chromatographic studies
Sephacryl S-200 HR.
To determine the distribution of endogenous vitamin B12 among the vitamin B12-binding proteins, we enriched 1 mL of serum from the patient and from an apparently healthy volunteer with 1 μg of methylcobalamin (Sigma-Aldrich) and applied the samples separately to a Sephacryl S-200 HR filtration column (1.6 × 60 cm) as described by Sviridov et al. (13). Fractions (2 mL) were collected and assayed for albumin, total protein, and vitamin B12.
Superdex 200 HR.
Patient serum was incubated with excess [57Co]cyanocobalamin (10.5 Ci/mL; MP Biomedical) and fractionated with a SMART system (Amersham Biosciences) with a Superdex 200 HR column (1.0 × 30 cm) as described by Nexo et al. (14). Fractions (400 μL) were collected and assayed for vitamin B12 and total TC and HC by ELISA as described previously (14)(15).
Protein G–sepharose.
To determine whether the increased vitamin B12 in the patient’s serum was associated with immune complexes, serum from the patient, 2 apparently healthy volunteers (vitamin B12, 572 and 679 pmol/L), and 2 patients who had recently received vitamin B12 treatment for anemia (vitamin B12, 5991 and 4576 pmol/L) were applied separately to 1-mL, 0.7 × 2.5-cm HiTrap protein G columns (Amersham Biosciences) according to the manufacturer’s instructions. The eluates were concentrated to 500 μL with Vivaspin centrifugal concentrators with a molecular mass cutoff of 3 kDa (Vivascience). The concentrated eluates were assayed in duplicate for vitamin B12 and then stored at −80 °C for further investigation.
Protamine–sepharose.
To concentrate IgM in the eluates from protein G columns, we used a protamine–sepharose column as described by Yilmaz et al. (16). The eluates were concentrated to 500 μL as described above and assayed in duplicate for vitamin B12.
Size-exclusion HPLC.
To assess the possibility that the high vitamin B12 in the patient’s serum was caused by binding to albumin, we loaded a 100-μL aliquot of serum from the patient and a control (anemic patient with recent vitamin B12 treatment; vitamin B12, 5991 pmol/L) onto a Zorbax Bio Series GF-250 column (9.4 × 250 mm) as described previously (13). Fractions (500 μL) were analyzed for albumin and vitamin B12 content. The column was calibrated with IgM, IgG, human serum albumin, and methylcobalamin in a separate assay.
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (sds-page) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms)
Concentrated eluates from the protein G columns were mixed with 4× NuPAGE lithium dodecyl sulfate sample buffer, with or without reducing agent, and heated at 70 °C for 5 min. We then electrophoresed 20 μg of protein from the column eluates along with molecular mass markers (6–200 kDa; Novex) on precast NuPAGE Bis-Tris 4%–12% gradient gels using NuPAGE MOPS buffer (Invitrogen). Gels were stained with SimplyBlue Safestain (Invitrogen). Proteins of interest on the SDS–PAGE gels were excised, digested with porcine trypsin, desalted, concentrated, and analyzed by MALDI-TOF MS as described by Drake et al. (17).
elisa
To test whether the serum protein G and protamine–sepharose eluates contained both IgM and IgG, we assayed the eluates (diluted 1:1000 with phosphate-buffered saline) from the patient and controls with commercially available IgG and IgM ELISAs (Immunotek, Zeptometrix Corporation). To determine whether IgG-IgM immune complexes were present in the eluates from protein G columns, we performed a modified standard solid-phase ELISA as described by Stahl and Sibrowski (18).
Results
heterophile blocking tube and dilution linearity
The vitamin B12 concentration in the original serum sample from the case patient did not differ considerably from the value after heterophile blocking tube treatment (42 600 vs 39 189 pmol/L, respectively). Diluting the original serum with phosphate-buffered saline (1:1, 1:2, 1:3, and 1:4) gave a linear relationship (y = 1.00x − 312; r = 0.99) between the theoretical amounts of vitamin B12(x) and the amounts found by the vitamin B12 assay (y).
chromatographic studies
The Sephacryl S-200 HR elution profile of serum from an apparently healthy volunteer, enriched with methylcobalamin (Fig. 1A1 ) shows that most of the vitamin B12 in the healthy control is in the free form, which is not unexpected, owing to methylcobalamin enrichment. Interestingly, the elution profile of the patient’s serum from a Sephacryl S-200 HR column revealed a large vitamin B12 peak, with a shoulder that eluted at the void volume (Fig. 1B1 ). This large vitamin B12 peak at the void volume was not observed in serum from apparently healthy volunteers enriched with (Fig. 1A1 ) or without (data not shown) methylcobalamin, suggesting that the patient’s serum contained an abnormal vitamin-B12-binding protein. Furthermore, this abnormal vitamin B12 peak in the patient’s serum (pooled fractions 6–19) contained small concentrations of HC (35 pmol/L) and TC (33 pmol/L), demonstrating that the high vitamin B12 in this peak was not composed of HC or TC.
Gel filtration chromatography analysis was also performed with the patient’s serum, as shown in Fig. 22 . The elution profile revealed an abnormal vitamin B12 peak. ELISAs of this broad peak for the 2 major vitamin B12-binding proteins, HC and TC, detected small concentrations of these proteins. Moreover, this peak was not labeled with [57Co]cyanocobalamin, indicating that the vitamin B12-binding protein in this peak was fully saturated with endogenous vitamin B12 (Fig. 22 ). The chromatogram also showed that the patient’s serum contained very little free endogenous vitamin B12. We speculate that this broad vitamin B12 peak on the Superdex 200 column, in contrast to the abnormal vitamin B12-binding protein that eluted at the void volume on the Sephacryl S-200 HR column (Fig. 1B1 ), is attributable to the partial dissociation of vitamin B12 from the abnormal vitamin B12-binding protein caused by repeated freeze and thawing of the patient’s serum sample.
The concentrated protein G eluates from the 2 apparently healthy volunteers, as well as the patients with recent vitamin B12 treatment for anemia, had a vitamin B12 concentration of <74 pmol/L, whereas the vitamin B12 concentration in the concentrated protein G column eluate from the case patient was 7380 pmol/L. These vitamin B12 concentrations in the protein G eluates were confirmed by another laboratory. We also confirmed, by Western blot with chicken antihuman HC antibodies, that the patient and control concentrated protein G eluates did not contain HC (data not shown).
Assays for vitamin B12 in the fractions from the size-exclusion HPLC of the patient showed that ∼80% of endogenous vitamin B12 was present in the fractions that corresponded to the elution time between IgM (7.4 min) and IgG (9.3 min) standards, compared with ∼30% for the anemic patient with recent vitamin B12 treatment (control). In both patient and control samples, however, the albumin fraction (9.7 min) did not contain a significant amount of vitamin B12 (<200 pmol/L) demonstrating that most of the very high vitamin B12 content in the patient’s serum was not bound to albumin. The fractions that corresponded to methylcobalamin standard (free vitamin B12, 13.2 min) had low concentrations of vitamin B12 in the patient serum (<200 pmol/L), whereas the control serum had ∼70% of the total vitamin B12 content in this fraction.
sds-page and maldi-tof ms
Unfortunately, the eluted fractions from the Sephacryl and Superdex 200 HR columns were accidentally discarded, and we were unable to obtain more serum samples because the patient had died. However, SDS–PAGE analysis of the concentrated eluates from the protein G column, under reducing conditions, revealed 2 major bands of ∼50 and 25 kDa for all samples, which corresponded to decreased IgG heavy and light chains, respectively (Fig. 33 , lane 3). MALDI-TOF MS analysis confirmed that the 50- and 25-kDa bands were IgG heavy chain and immunoglobulin light chains, respectively (Table 11 ). Because the streptococcal protein G has affinity to both the Fc region of immunoglobulins and to albumin, it is not surprising that some albumin was also found in the eluate from the protein G column (19) (Fig. 33 ). The presence of the other bands in all the samples on the gel is most likely the result of incomplete reduction of the disulfide bonds of immunoglobulins. In the patient sample, however, an additional band with an apparent molecular mass of ∼76 kDa appeared on the gel (Fig. 33 , lane 7, arrow). Mass spectrometry analysis identified this band as an IgM heavy chain (Table 11 ).
When we loaded the protein G eluates from the patient and controls to determine whether the IgM in the protein G eluates from the patient’s serum was pentameric or monomeric, we did not detect monomeric IgM (∼180–190 kDa) from patient and control protein G eluates when the gel in this molecular mass region was excised, digested with trypsin, and analyzed by MALDI-TOF MS (Fig. 44 ). However, a band at the top of the gel was detected in protein G eluates from the case patient but not the controls. This band was subsequently identified by MALDI-TOF MS to be IgM (Fig. 44 ). Hence, the data show that the IgM molecules in the protein G eluates from the patient are pentameric (∼900 kDa).
To determine whether the IgM in the protein G eluate from the patient contained vitamin B12, we loaded concentrated protein G eluates from the patient and control separately onto a protamine–sepharose column and assayed the eluates from the protamine–sepharose column for vitamin B12. The eluates from the controls had a vitamin B12 concentration of <74 pmol/L, whereas the vitamin B12 concentration in the eluate from the patient was 812 pmol/L.
elisa
The patient’s serum protein G eluate contained IgM (1.51 g/L), but the serum samples from healthy controls and anemic patients treated with vitamin B12 contained no detectable IgM. Furthermore, the protamine–sepharose eluate from the patient contained IgG (1.42 g/L), whereas IgG was not detected in the control eluates. In a modified ELISA, in which the capture antibody was anti-IgM and the detection antibody was anti-IgG, we demonstrated that the patient’s IgG (0.89 g/L), but not control IgG (0 g/L), remained associated with IgM.
Discussion
Heterophile antibodies are common in the population and are known to interfere with immunodiagnostic assay systems (20). Heterophile blocking agents did not decrease the vitamin B12 concentrations in our patient, suggesting that (a) heterophile antibodies were not present in the patient’s serum, (b) the patient had heterophile antibodies that the heterophile blocking tube could not remove, or (c) the very high antibody titers overwhelmed the ability of the blocking agents to remove the heterophile antibodies (20). However, because the patient’s serum was linear on dilution and the high vitamin B12 was confirmed by another laboratory with a different assay, it is unlikely that heterophile antibody interference was the cause of the increased vitamin B12 in our patient.
Although high serum vitamin B12 concentrations have been reported in patients with disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndrome, none of these were present in our patient (3)(4)(5)(6)(7)(8)(9)(10)(11). These clinical conditions are characterized by high myeloid cell mass and turnover associated with maturation (21)(22). The reason for increased serum vitamin B12 in the above-mentioned conditions is the increased synthesis and release of HC from the secondary granules of myeloid cells (21)(22). A correlation between serum unsaturated vitamin B12 binding capacity and total granulocyte pool has been reported (23). In this case patient, however, the high vitamin B12 concentrations should not be caused by increased HC synthesis and release from myeloid cells, because the leukocyte count and differential and total HC were within reference intervals, and total TC was only slightly increased. In addition, it is known that renal failure (24)(25) and hepatic disease (26)(27) are associated with high vitamin B12 concentrations. This patient had normal renal and hepatic function. Previous studies have shown that some patients given cyano- or hydroxycobalamin have developed antibodies to TC, leading to markedly increased serum TC and vitamin B12 (28)(29)(30)(31)(32)(33). In this patient, however, serum holo-TC concentrations were within reference intervals, with a slightly increased total TC, despite a very high vitamin B12 concentration.
The results from this study are consistent with the presence of immune complexes of IgG, IgM, and vitamin B12, contributing to the increased vitamin B12 concentrations in our patient’s serum. Although the extremely high concentrations at presentation were related to very high dosing with vitamin B12, 5 weeks after discontinuation of vitamin B12, serum concentrations remained ∼4428 pmol/L. We found the following evidence for the IgG-IgM-vitamin B12 complex: (a) an abnormal distribution of vitamin B12-binding proteins in the serum from the patient, indicated by the results of 2 types of gel filtration chromatography; (b) high concentrations of vitamin B12 in protein G– and protamine–sepharose eluates from the patient, but not from healthy controls or other patients receiving vitamin B12; (c) pentameric IgM in the protein G eluate from the patient, but not the controls, identified by SDS–PAGE and MALDI-TOF MS; and (d) detection of IgG associated with the protein bound to an anti-μ-chain primary antibody in an ELISA.
Although we can only speculate about why these complexes developed, they may have been acquired by immunization against the extraordinarily high vitamin B12 concentrations (15 mg/week for several months), with subsequent appearance of antivitamin B12 antibodies. Nevertheless, these immune complexes do not appear to compete with the uptake of TC-bound vitamin B12 into the cell, because functional vitamin B12 deficiency was not indicated by holo-TC, homocysteine, or methylmalonic acid concentrations, which were within reference intervals. We hypothesize that the IgG-IgM-vitamin B12 immune complexes caused impaired renal clearance of vitamin B12. As far as we can determine, the high concentrations of vitamin B12 did not affect this patient’s clinical presentation, and discontinuation of the vitamin B12 injections did not improve his symptoms.
To our knowledge, this is the first report of a patient with markedly increased vitamin B12 related to IgG-IgM immune complexes. When an unexpected very high serum vitamin B12 is found, in addition to looking for assay interference, increased HC or TC concentrations, and recent vitamin B12 treatment, immune complexes with vitamin B12 should also be investigated.
Sephacryl S-200 HR gel filtration of serum from an apparently healthy volunteer enriched with 1 μg of methylcobalamin (A) and from the patient (B).
The primary and secondary y axes display the vitamin B12 and protein concentrations from the column fractions, respectively. Elution positions of void volume (Vo), IgG, and methylcobalamin (free vitamin B12) standard protein are indicated by arrows.
Superdex 200 HR gel filtration of serum from the patient.
The primary y axis displays the serum concentration of vitamin B12, total HC, and total TC. The secondary y axis displays the 57Co-cyanocobalamin activity in counts per min (cpm) from the addition of excess 57Cocyanocobalamin in the patient’s serum. The arrows indicate the elution position of standards [in order of elution: void (Vo), IgG, and albumin]. Asterisk denotes the abnormal vitamin B12-binding protein.
Reducing SDS–PAGE of protein G eluates.
Lane 1, molecular mass markers; lane 2, bovine serum albumin standard; lane 3, IgG and bovine serum albumin standards; lanes 4 and 5, protein G eluates from anemic patients treated with vitamin B12; lanes 6 and 8, apparently healthy controls; lane 7, patient. Twenty micrograms of protein were loaded onto each lane. Arrow indicates the band with an apparent molecular mass of ∼76 kDa in the patient’s protein G eluate. Letters indicate bands subjected to digestion followed by MALDI-TOF MS analysis.
Identified proteins in serum from patient and controls by protein G–sepharose chromatography, SDS–PAGE, and MALDI-TOF MS analysis.
| Band1 . | Protein . | Molecular mass, kDa2 . | Masses matched . |
|---|---|---|---|
| a | BSA3 | 66 | 1438.80, 1566.74 |
| b | IgG heavy chain | 50 | 1676.79, 1872.91, 2228.18, 2544.11 |
| c | IgG light chain | 25 | 1676.79, 1872.91, 2227.20, 2543.12, 2800.26 |
| d | IgM heavy chain | 76 | 1028.48, 1248.54, 1385.69, 1773.00 |
| Band1 . | Protein . | Molecular mass, kDa2 . | Masses matched . |
|---|---|---|---|
| a | BSA3 | 66 | 1438.80, 1566.74 |
| b | IgG heavy chain | 50 | 1676.79, 1872.91, 2228.18, 2544.11 |
| c | IgG light chain | 25 | 1676.79, 1872.91, 2227.20, 2543.12, 2800.26 |
| d | IgM heavy chain | 76 | 1028.48, 1248.54, 1385.69, 1773.00 |
Letters refer to Fig. 33 (a, b, c, and d).
Estimated molecular masses from SDS–PAGE gel.
BSA, bovine serum albumin.
Identified proteins in serum from patient and controls by protein G–sepharose chromatography, SDS–PAGE, and MALDI-TOF MS analysis.
| Band1 . | Protein . | Molecular mass, kDa2 . | Masses matched . |
|---|---|---|---|
| a | BSA3 | 66 | 1438.80, 1566.74 |
| b | IgG heavy chain | 50 | 1676.79, 1872.91, 2228.18, 2544.11 |
| c | IgG light chain | 25 | 1676.79, 1872.91, 2227.20, 2543.12, 2800.26 |
| d | IgM heavy chain | 76 | 1028.48, 1248.54, 1385.69, 1773.00 |
| Band1 . | Protein . | Molecular mass, kDa2 . | Masses matched . |
|---|---|---|---|
| a | BSA3 | 66 | 1438.80, 1566.74 |
| b | IgG heavy chain | 50 | 1676.79, 1872.91, 2228.18, 2544.11 |
| c | IgG light chain | 25 | 1676.79, 1872.91, 2227.20, 2543.12, 2800.26 |
| d | IgM heavy chain | 76 | 1028.48, 1248.54, 1385.69, 1773.00 |
Letters refer to Fig. 33 (a, b, c, and d).
Estimated molecular masses from SDS–PAGE gel.
BSA, bovine serum albumin.
Nonreducing SDS–PAGE of protein G eluates from the patient and apparently healthy controls.
Twenty micrograms of protein were loaded onto each lane. Lane 1, molecular mass markers; lane 2, IgM standard; lane 3, control; lane 4, patient; lanes 5 and 6, anemic patients receiving vitamin B12 treatment. The arrow indicates IgM band at the top of the gel in only the IgM standard (lane 2) and patient (lane 4) protein G eluates.
Nonstandard abbreviations: TC, transcobalamin; HC, haptocorrin; holo-TC, TC saturated with vitamin B12; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
We thank Dr. Ebba Nexo for advice and technical assistance and Mark Ruddel, Winnie To, S. Ray Kenney, and Bonnie Meilinger for excellent technical assistance.
Obeid R, Morkbak AL, Munz W, Nexo E, Herrmann W. The cobalamin-binding proteins transcobalamin and haptocorrin in maternal and cord blood sera at birth.
Simons K, Weber T. The vitamin B12-binding protein in human leukocytes.
Carmel R, Herbert V. Vitamin B12-binding proteins of leukocytes as possible major source of third vitamin B12-binding protein of serum.
Carmel R, Coltman CA, Jr. Nonleukemic elevation of serum vitamin B12 and B12-binding capacity levels resembling that in chronic myelogenous leukemia.
Carmel R. Extreme elevation of serum transcobalamin I in patients with metastatic cancer.
Hall CA, Wanko M. Increased transcobalamin I in a leukemoid reaction.
Waxman S, Gilbert HS. A tumor-related vitamin B12 binding protein in adolescent hepatoma.
Nexo E, Olesen H, Norredam K, Schwartz M. A rare case of megaloblastic anemia caused by disturbances in the plasma cobalamin binding proteins in a patient with hepatocellular carcinoma.
Zittoun J, Marquet J, Zittoun R. The intracellular content of the three transcobalamin at various stages of normal and leukemic myeloid cell development.
Carmel R, Herbert V. Vitamin B12-binding protein of leukocytes as a possible major source of the third vitamin B12-binding protein of serum.
Ermens AAM, Vlasveld LT, Lindemans J. Significance of elevated cobalamin (vitamin B12) levels in blood.
Sviridov D, Meilinger B, Drake SK, Hoehn GT, Hortin GL. Coelution of other proteins with albumin during size-exclusion HPLC: implications for analysis of urinary albumin.
Nexo E, Christensen AL, Petersen TE, Fedosov SN. Measurement of transcobalamin by ELISA.
Morkbak AL, Pederson JF, Nexo E. Glycosylation independent measurement of the cobalamin binding protein haptocorrin.
Yilmaz H, Roe JM, Morgan KL. Isolation and preparation of antisera to ovine IgE.
Drake SK, Bowen RA, Remaley AT, Hortin GL. Potential interferences from blood collection tubes in mass spectrometric analyses of serum peptides.
Stahl D, Sibrowski W. Warm autoimmune hemolytic anemia is an IgM-IgG immune complex disease.
Rasmussen LK, Larsen YB, Hojrup P. Characterization of different cell culture media for expression of recombinant antibodies in mammalian cells: presence of containing bovine antibodies.
Kricka LJ. Human anti-animal antibody interferences in immunological assays.
Carmel R, Vasireddy H, Aurangzeb I, George K. High serum cobalamin levels in the clinical setting-clinical associations and holo-transcobalamin changes.
Ghosh K, Mohanty D, Rana KS, Hassan SW, Garewal G, Das KC. Plasma transcobalamins in haematological disorders.
Chikkappa G, Corcino J, Greenerg ML, Herert V. Correlation between various blood blood white cell pools and the serum B12-binding capacities.
Nyberg W, Kuhlback B, Pasternak A, Tallgren AG. Renal failure and serum vitamin B12 concentration.
Sevitt LH, Hoffbrand AV. Serum folate and vitamin B12 levels in acute and chronic renal disease. Effect of peritoneal dialysis.
Schonau JF. Vitamin B12 and its binding proteins in cirrhosis and infectious hepatitis.
Retief FP, Vandenplas L, Visser H. Vitamin B12 binding proteins in liver disease.
Carmel R, Tatsis B, Baril L. Circulating antibody to transcobalamin II causing retention of vitamin B12 in the blood.
Schwartz M, Hom B, Olesen H, Bastrup-Madsen P. An antibody against a vitamin B12-binding serum protein (transcobalamin II): a cause of high vitamin B12 in the serum of patients treated with delayed-action B12 preparations.
Carmel R, Shurafa M. Circulating immunoglobulin-transcobalamin I complex in patients with elevated serum vitamin B12 levels.
Hom BL, Olesen H, Schwartz M. Turnover of 57Co-labelled vitamin B12-transcobalamin II and autologous 131I-labelled IgG in a patient with antibody to transcobalamin II.
Skouby AP, Hippe E, Olesen H. Antibody to transcobalamin II and B12 binding capacity in patients treated with hydroxycobalamin.




