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Technical Briefs |
1 Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037;
2 Department of Clinical Laboratories, Omiya Medical Center, Jichi Medical School, Amanuma, Saitama 330-8503, Japan;
3 Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Asahi, Matsumoto, Nagano 390-8621, Japan;
4 Department of Laboratory Medicine, Shinshu University School of Medicine, Asahi, Matsumoto, Nagano 390-8621, Japan;
aauthor for correspondence: fax 858-784-9144, e-mail kyfujit{at}aol.com
Serum fructosamine is used to monitor short-term (1–3 weeks) average glycemia (1)(2), but controversy remains whether it is influenced by the serum concentrations of albumin (3)(4), IgA (5)(6), or monoclonal IgA (7)(8). We recently demonstrated glycation of monoclonal IgA and the presence of IgA-albumin complexes (9), but the relationship between the glycation of monoclonal IgA and the IgA-albumin complexes was not clear.
In this study, we measured immunoglobulin-albumin complexes, serum fructosamine, and the glycation of immunoglobulin in patients with monoclonal proteinemia and in patients with polyclonal hyper-IgA.
We obtained serum samples from 40 patients with M-proteinemia with no history of diabetes and whose plasma glucose concentrations were <1.1 g/L. The M-protein was IgG in 17 of the patients [mean (SD), 29.2 (21.0) g/L], IgA in 13 [18.5 (16.4) g/L], and IgM in 10 [16.8 (11.3) g/L]. We also analyzed sera from 15 nondiabetic patients with hepatitis who had polyclonal hyper-IgA [5.1 (1.1) g/L] as controls.
Serum fructosamine was measured at 37 °C in an automated analyzer (JCA-RX 10 Clinalyzer; Japan Electron Optics Laboratory) with reagents from Roche Diagnostics Corporation.
Serum protein electrophoresis was performed on agarose gels. Immunoelectrophoresis was based on the method of Scheidegger (10). Fructosamine was detected on agarose gels by incubating the gel for 30 min at 37 °C with a 2x concentration of a fructosamine reagent.
IgA was isolated from serum with use of a jacalin-agarose (Funakoshi Corporation) column (11) and 0.8 mol/L galactose to elute the IgA. The IgA fraction was dialyzed against 0.2 mol/L Tris-HCl buffer (pH 8.0), concentrated by ultrafiltration, loaded on a DEAE-Sephacel (Amersham Pharmacia Biotech) ion-exchange column, and eluted with a linear gradient of 0–0.5 mol/L NaCl in the 0.2 mol/L Tris-HCl buffer.
Sera were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), with or without 2-mercaptoethanol (2-ME), and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (12). Immunostaining was performed with rabbit polyclonal antisera (Dako) as first antibodies and peroxidase-conjugated goat anti-rabbit IgG (Dako) as second antibody.
To detect fructosamine on PVDF membranes, we blocked the membranes for 60 min at room temperature in buffer containing 30 g/L bovine serum albumin. After washing, the membranes were incubated for 30 min at 37 °C with a 2x concentration of the fructosamine reagent.
Differences between the groups were verified by the Student t-test. Correlations were studied by means of the Pearson correlation coefficient.
Serum fructosamine concentrations were higher in patients with IgA-type [3.47 (0.93) mmol/L; P <0.05] than with IgG-type [2.55 (0.48) mmol/L] or IgM-type M-proteinemia [2.51 (0.42) mmol/L]. Concentrations of fructosamine and of serum IgA (but not IgG or IgM) in patients with M-proteinemia were significantly correlated (
= 0.698; P <0.05). By contrast, in the 15 patients with hepatitis and polyclonal hyper-IgA, mean (SD) fructosamine was 2.62 (0.23) mmol/L and all values were within our reference interval (1.94–3.16 mmol/L); in addition, fructosamine was not significantly correlated with the IgA concentrations ( = 0.279).
The sera from the patients with IgG- or IgM-type M-proteinemia had fructosamine only at the position of albumin, but 11 of 13 sera with IgA-type M-proteinemia stained for glycoprotein at the position of the M-protein band as well as the albumin band (Fig. 1
in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/Vol49/issue5/). Sera from the patients with polyclonal hyper-IgA did not stain for glycoproteins at the position of IgA but did stain at the position of the albumin band.
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IgA-
-type M-protein also reacted with albumin-specific antiserum (Fig. 1
). The same abnormal precipitin arcs were observed in 11 of 13 sera from patients with IgA-type M-proteinemia that were glycosylated at the position of M-protein band. Moreover, the mobility of the abnormal precipitin arcs corresponded to the position of the glycated M-protein band. The patients’ albumin had a macromolecular mass >300 kDa, as determined by immunostaining using anti-albumin serum after SDS-PAGE without 2-ME.
The abnormal precipitin arcs for albumin disappeared when antisera specific for human albumin or IgA (
-chain specific) were incubated for 37 °C for 30 min with the purified IgA-type M-protein (at a ratio of 6:1 by volume) and supernates were concentrated by ultrafiltration. The immunoelectrophoretic patterns of the sera from patients with IgG- or IgM-type M-proteinemia or with polyclonal hyper-IgA showed no abnormal precipitin arcs with albumin.
After SDS-PAGE with 2-ME, macroalbumin was not detected by immunostaining with anti-albumin serum, and IgA-type M-proteins stained for fructosamine at 
55 kDa, which corresponded to the -chain, and also at 25 kDa, which corresponded to the light chain, in 11 of 13 cases. Sera from patients with IgG- or IgM-type M-proteinemia or with polyclonal hyper-IgA showed fructosamine glycosylation only at 65 kDa, which corresponded to albumin (Fig. 2 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue5/).
To investigate the possibility that monoclonal IgA was glycated in the presence of glucose, we incubated a patient’s serum that had no fructosamine glycosylation at the M-protein band with D-glucose at 10 or 20 g/L. Incubation of the monoclonal IgA serum with 10 or 20 g/L glucose at 37 °C for 60 min produced no fructosamine at the position of the monoclonal IgA band on electrophoresis, but increased the fructosamine at the position of albumin band (Fig. 3 in the Data Supplement).
Glycohemoglobin is an accepted measure of long-term average glycemia. Because nonenzymatic glycation also affects other proteins (13), glycated albumin or total glycated protein (fructosamine assay) can be used as a measure of short-term, average glycemia (1)(2). Wahid et al. (14) stated that serum fructosamine is useful as a maker of 5-year risk of developing diabetes mellitus in patients exhibiting stress hyperglycemia; however, the dependence of fructosamine concentrations on the concentrations of albumin and total protein has been reported (15)(16)(17)(18)(19). Montagna et al. (7) and Nakamura et al. (8) reported that nondiabetic patients with IgA-type M-proteinemia have significantly increased serum fructosamine.
We also found that the fructosamine concentrations in patients with IgA-type M-proteinemia were significantly higher than the fructosamine concentrations in patients with IgG- or IgM-type M-proteinemia. By staining for fructosamine after electrophoresis, we demonstrated that this is the result of an increase in glycated monoclonal IgA, i.e., 84.6% of the patients with IgA-type M-proteinemia had increased fructosamine at the monoclonal IgA bands, and fructosamine was detected in both the heavy and light chains.
The two patients with IgA-type M-proteinemia who did not have fructosamine at the M-protein band had lower IgA values than did the 11 patients with IgA-type M-proteinemia. This suggests that glycation of IgA molecules may be related to the high IgA concentrations [mean (SD), 18.5 (16.4) g/L]. The sera from the patients with polyclonal hyper-IgA had fructosamine only in the albumin band. Thus, glycation of IgA molecules appears to be unique to monoclonal IgA.
It is interesting that the macroalbumin complexes that were composed of IgA and albumin were detected in all sera from patients with IgA-type M-protein containing fructosamine at the M-protein band (11 of 13). The IgA-albumin complex was absent from the sera from patients with IgG- or IgM-type M-proteinemia as well as from the sera from patients with polyclonal hyper-IgA. Moreover, monoclonal IgA that was not associated with albumin was not glycated, although the glucose concentration in the patient’s serum was increased. Thus, a major factor in the glycation of IgA was the binding to albumin. Because the half-life of albumin is longer (15–19 days) (20) than the half-life of human IgA molecules (6 days) (21), the half-life of monoclonal IgA that binds albumin is longer, and this could increase the glycation of the monoclonal IgA.
Why is the monoclonal IgA glycosylated and not the polyclonal IgA? The mean IgA concentrations (18.5 g/L) in patients with monoclonal IgA were clearly higher than the IgA concentrations (5.1 g/L) in patients with polyclonal IgA. Among the patients with IgA concentrations <10 g/L, 3 of the 5 patients with monoclonal IgA had IgA-albumin complexes, but none of the 15 patients with polyclonal IgA had IgA-albumin complexes (Fig. 4 in the Data Supplement). Moreover, serum IgA is present in several polymeric forms, ranging from monomers to pentamers. In normal human serum, the monomeric form usually predominates, but in monoclonal IgA serum, the relative amount of each polymeric form varies. Polymerization of the monoclonal IgA might therefore play an important role in its interaction with albumin. This idea is supported by the fact that the IgA-albumin complexes had a macromolecular mass >300 kDa.
The formation of IgA-albumin complexes has been noted previously (22)(23)(24)(25). Tomasi and Hauptman (23) examined 49 IgA myeloma sera and found that 65% had IgA-albumin complexes, suggesting that albumin was bound predominantly to dimeric IgA.
Why is the abnormal precipitin arc for albumin faster than that for IgA on immunoelectrophoresis? Serum protein electrophoresis usually separates the various components of blood protein into bands or zones according to their electrical charges. The isoelectric points of immunoglobulins are 6.4–7.2, whereas that of albumin is 4.8 (26). It can be considered that the abnormal precipitin arc for albumin in the IgA-albumin complexes moves faster than that for IgA because of the acidity of the albumin.
In conclusion, our findings throw doubt on the clinical utility of fructosamine as a measure of hyperglycemic status in patients with IgA-type M-proteinemia.
Acknowledgments
We thank Minoru Ogawa (Yokote Hospital), Yoshimasa Aoki, and Dr. Mitsuaki Kameko (Nagano Municipal Hospital) for the kind gifts of patient sera.
References
type multiple myeloma. Rinsho Byori 1996;44:85-89.[Medline]
[Order article via Infotrieve]
A-myeloma proteins and Waldenström macroglobulins by disulfide bonds. J Immunol 1967;99:899-906.-1 antitrypsin to human IgA. J Immunol 1974;112:2274-2277.
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light chain (A-