Abstract
Due to the unusual presence of several different hemoglobin components in the rat, determination of glycated hemoglobin (Hb) has been considered difficult and often unreliable in this animal species. In the present study, we compare a fully automated high-performance liquid chromatographic (HPLC) method of analysis of glycated hemoglobin that has been assessed for clinical use with an affinity chromatography technique using boronate micro-columns; we used blood samples taken from Sprague-Dawley rats of various ages and streptozotocin-diabetic rats. In nondiabetic rats, the sum of HbA1c and other minor glycated hemoglobins separated by the HPLC method is close to the total glycated hemoglobin obtained by affinity chromatography for each age group of animals. In diabetic rats, the glycated hemoglobins measured by whatever method show a linear increase during the first 3 weeks following streptozotcin administration, with the difference that glycated hemoglobin values obtained by affinity chromatography are markedly higher than those obtained by HPLC technique. Interestingly, a comparative determination of glycated hemoglobin in diabetic patients gives the same results with both methods. Therefore, it appears that in the rat, unlike man, at high glucose concentrations glycation occurs preferentially at the amino groups of hemoglobin components, which are not separated by the HPLC method. Our results indicate that while affinity chromatography should be used to detect the total extent of hemoglobin glycosylation in diabetic rats, the utilization of rapid and automatized HPLC procedures can be a very convenient alternative for the determination of glycated hemoglobin in both euglycemic and hyperglycemic rats.
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References
Blanc MH, Rhie FH, Dunn PJ, Soeldner JS (1981) The determination of glycosylated hemoglobins in rats using high pressure liquid chromatography. Metabolism 30:317–322
Bunn HF, Gabbay KH, Gallop PM (1978) The glycosylation of hemoglobin: relevance to diabetes mellitus. Science 200:21–34
Datta MC, Gilman JG (1981) Rat hemoglobin heterogeneity: postnatal changes in proportions of multiple components and effects of erythropoietin on marrow cell cultures. Hemoglobin 5:701–714
Fluckiger R, Woodtli T, Berger W (1984) Quantitation of glycosylated hemoglobin by boronate affinity chromatography. Diabetes 33:73–76
Garrick LM, Sloan RL, Ryan TW, Klonowski TJ, Garrick MD (1978) Primary structure of the major beta-chain of rat hemoglobins. Biochem J 173:321–330
Gonen B, Rubenstein A (1979) Glycosylated hemoglobin in diabetes: a reappraisal. Diabetes Care 2:451–453
Higgins PJ, Garlick RL, Bunn HF (1982) Glycosylated hemoglobin in human and animal red cells. Diabetes 31:743–748
Klenk DC, Hermanson GT, Krohn RI, Fujimoto EK, Mallia AK, Smith PK, England JD Wiedmeyer HM, Little RR and Goldstein DE (1982) Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences. Clin Chem 28:2088–2094
Kondo N, Shibayama Y, Toyomaki Y, Yamamoto M, Ohara H, Nakano K and Ienaga K (1989) Simple method for determination of A1c-type glycated hemoglobin(s) in rats using high performance liquid chromatography. J Pharmacol Toxicol Methods 21:211–221
Masoro EJ, Katz MS and McMahan CA (1989) Evidence for the glycation hypothesis of aging from the food restricted rodent model. J Gerontol 44:B20-B22
Odetti P, Borgoglio A, De Pascale A, Rolandi R and Adezati L (1990) Prevention of diabetes-increased aging effect on rat collagen-linked fluorescence by aminoguanidine and rutin. Diabetes 39:796–801
Shapiro R, McManus MJ, Zalut C and Bunn HF (1980) Sites of nonenzymatic glycosylation of human hemoglobin A. J Biol Chem 255:3120–3127
Stein S, Cherian MG, Mazur A (1971) Preparation and properties of six rat hemoglobins. J Biol Chem 246:5287–5293
Trivelli LA, Ranney HM, Lai H (1971) Hemoglobin components in patients with diabetes mellitus. N Engl J Med 284:353–357
Vialettes B, Vovan L, Simon MC, Lassmann V, Altomare E, Vague P (1982) Kinetics of fast haemoglobin in diabetic rats. Diabetologia 22:264–268
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De Tata, V., Novelli, M., Bombara, M. et al. Determination of glycated hemoglobins in the rat: comparison between two different chromatographic methods and application in experimental diabetology. Res. Exp. Med. 196, 9–16 (1996). https://doi.org/10.1007/BF02576824
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DOI: https://doi.org/10.1007/BF02576824