Summary
A competitive radioimmunoassay for the quantitative determination of glycated haemoglobin was developed. The antiserum, obtained by immunizing guinea pigs with reduced glycated human albumin, was capable of identifying and quantitating the glucitollysine residues of glycated Hb after reduction with sodium borohydride. To simplify the sample preparation we introduced trichloroacetic acid precipitation to remove unreacted sodium borohydride instead of using dialysis or gel filtration. Using this procedure, our radioimmunoassay became relatively simple and provided satisfactory within- and between-run (1.3–2.8% and 1.9–5.4% coefficient of variation, respectively). The radioimmunoassay method was compared to the measurement of HbA 1c by high performance liquid chromatography which is the most widely used method for quantitating glycated Hb. For this purpose glycated Hb was measured in normal glucose tolerance, impaired glucose tolerance, and diabetes mellitus groups based on WHO criteria. Both assays were able to discriminate between the normal and diabetic groups. In addition, while the determination of glycated Hb by the radioimmunoassay method was able to clearly discriminate between the normal and impaired glucose tolerance groups, the determination of HbA 1c by the high performance liquid chromatography method failed to discriminate between these two groups. Moreover, 15 of the 20 impaired glucose tolerance patients exceeded the upper normal range (mean normal values + 2 SD) in radioimmunoassay. But all 20 patients with impaired glucose tolerance were within the upper normal range in HbA 1c values.
These results demonstrate that the measurement of glycated Hb by radioimmunoassay is more sensitive than the measurement of HbA 1c by high performance liquid chromatography since it can discriminate between the normal and impaired glucose tolerance groups.
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Koenig RJ, Blobstein SH, Cerami A (1977) Structure of carbohydrate of hemoglobin A1c. J Biol Chem 252: 2992–2997
Nathan DM, Singer DE, Hurxthal K, Goodson JD (1984) The clinical information value of the glycosylated hemoglobin assay. N Engl J Med 310: 341–346
Jovanovic L, Peterson CM (1981) The clinical utility of glycosylated hemoglobin. Am J Med 70: 331–338
Bunn HF (1981) Nonenzymatic glycosylation of protein: relevance to diabetes. Am J Med 70: 325–330
Goldstein DE, Little PR, Wiedmeyer HM, England JD, Mckenzie E (1986) Glycated hemoglobin: methodologies and clinical applications. Clin Chem 32: B64-B70
Baynes JW, Burn HF, Goldstein D, Harris M, Martin DB, Peterson CM, Winterhalter K (1984) National Diabetes Data Group: report of the expert committee on glycosyated hemoglobin. Diabetes Care 7: 602–607
Peterson CM, Formby B (1985) Glycosylated proteins. In: Alberti KGMM, Krall LP (eds) The Diabetes Annual, 1. Elsevier, Amsterdam, pp 178–197
Bunn HF, Shapiro R, McManus M, Garrick L, McDonald MJ, Gallop PM, Gabbay KM (1979) Structural heterogeneity of human hemoglobin A due to nonenzymatic glycosylation. J Biol Chem 254: 3892–3898
Shapiro R, McManus MJ, Zalut C, Bunn HF (1980) Sites of nonenzymatic glycosylation of human hemoglobin A. J Biol Chem 255: 3120–3127
Nakayama H, Makita Z, Kato M, Taneda S, Yoshida H, Yanagisawa K, Nakagawa S (1987) Quantitative enzyme-linked immunosorbent assay (ELISA) for non-enzymatically glycated serum protein. J Immunol Methods 99: 95–100
Nakayama H, Taneda S, Manda N, Aoki S, Komori K, Kuroda Y, Misawa K, Tsushima S, Nakagawa S (1986) Radioimmunoassay for nonenzymatically glycated protein in human serum. Clin Chim Acta 158: 293–299
Trüeb B, Hughes GJ, Winterhalter KH (1982) Synthesis and quantitation of glucitollysine: a glucosylated amino acid elevated in proteins from diabetics. Anal Biochem 119: 330–334
Schwartz BA, Gray GR (1977) Proteins containing reductively aminated disaccharides. Arch Biochem Biophys 181: 542–549
Nathan DM, Avezzano ES, Palmer JL (1981) A rapid chemical means for removing labile glycohemoglobin. Diabetes 30: 700–701
Abraham EC, Huff TA, Cope ND, Wilson JB, Bransome ED, Augusta DS (1982) Determination of the glycosylated hemoglobins (HbA1c) with a new microcolumn procedure. Diabetes 27: 931–937
WHO Expert Committee on Diabetes Mellitus (1980) Second Report. Geneva. World Health Organization (Tech. Rep. Ser. 646)
Nakayama H, Manda N, Komori K, Aoki S, Ono Y, Makita Z, Nakagawa S (1982) Studies on the determination of glucosylated albumin using affinity chromatography. J Jpn Diabetes Soc 25: 963–968
Lowry O, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275
Ma A, Naughton MA, Cameron DP (1981) Glucosylated plasma protein: a simple method for the elimination of interference by glucose in its estimation. Clin Chem Acta 115: 111–117
Curtiss LK, Witzum JL (1983) A novel method for generating region-specific monoclonal antibodies to modified proteins. J Clin Invest 72: 1427–1438
Schleicher E, Scheller L, Wieland OH (1981) Quantitation of lysine-bound glucose of normal and diabetic erythrocyte membranes by HPLC analysis of furosine. Biochem Biophys Res Commun 99: 1011–1019
Schleicher E, Wieland OH (1986) Kinetic analysis of glycation as a tool for assessing the half-life of proteins. Biochim Biophys Acta 884: 199–205
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Makita, Z., Nakayama, H., Taneda, S. et al. Radioimmunoassay for the determination of glycated haemoglobin. Diabetologia 34, 40–45 (1991). https://doi.org/10.1007/BF00404023
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DOI: https://doi.org/10.1007/BF00404023