High-performance liquid chromatographic assay of serum glycated albumin

Abstract

A method for determination of serum glycated albumin by high-performance liquid chromatography is presented. The system involves anion exchange chromatography to separate albumin and consecutive boronate affinity chromatography to separate glycated and nonglycated albumin. The method is rapid (20 min), precise (coefficient of variation, 0.7-4.9%), requires only a small sample (5 microliters), and can be automated. Assay of glycated albumin by this method is not influenced by the protein concentration of the sample or the presence of glucose. The variation in glycated albumin values in consecutive samples obtained within a day from diabetic patients (coefficient of variation, 2.02 +/- 0.65%) was significantly smaller (p less than 0.001) than that of values for fructosamine (coefficient of variation, 4.33 +/- 2.0%). The values of glycated albumin in normal subjects (20.2 +/- 1.6%) were clearly less than those in diabetic patients [39.6 +/- 5.4% in 40 Type 1 (insulin-dependent) and 39.4 +/- 5.9% in 25 Type 2 (non-insulin-dependent) patients]. The serum glycated albumin level was well correlated with HbA1c in 65 diabetic patients (r = 0.60). Because the life span of albumin in the circulation is short, measurement of glycated albumin should be useful as a short-term index of glycaemic control.

Association between Markers of Glycemic Control, Cardiovascular Complications and Survival in Type 2 Diabetic Patients with End-Stage Renal Disease

“…(5,13). Some previous studies showed a weak correlation between HbA1c levels and mean blood glucose levels in diabetic ESRD patients (11,13,15 (13,14).…”

“…HbA1c level is influenced by erythrocyte lifespan or erythropoietin therapy (12,13) (14). GA levels may reflect blood glucose levels more correctly in ESRD patients (15).…”

Tests of Glycemia in Diabetes

“…Alternatively, albumin can be separated first by Affi-Gel Blue affinity chromatography or anion-exchange HPLC (174), and boronate affinity chromatography can then be used to determine the GSA fraction. Boronate affinity chromatographic methods for measurement of GSP or GSA are commercially available in the Technical Review form of minicolumns and automated HPLC.…”

Caution: interpretation of results of HPLC assay for serum glycated albumin

“…The exceedingly high values for glycated albumin (normal, 20.2 _+ 1.6 %, diabetic, 39.6 _+ 5.4 %) reported with the high performance liquid chromatographic (HPLC) assay described by Shima et al [1] are far in excess of values established with any other method, including boronate affinity chromatography (normal range, 1.5-2.6 % [2]), densitometric scanning of agarose gel electrophoresis (normal range, 0.4-2.0% [3]), or monoclonal antibody based enzyme linked immunosorbent assay (normal, 2.3 + 0.29 % [4,5]). Careful examination of the principles underlying the HPLC assay and the interpretation of its results are therefore necessary in order to avoid erroneous assignment of the acceptable range of values for non-diabetic populations and for assessment of glycaemic control.…”

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Caution: interpretation of results of HPLC assay for serum glycated albuminDear Sir,The exceedingly high values for glycated albumin (normal, 20.2 _+ 1.6 %, diabetic, 39.6 _+ 5.4 %) reported with the high performance liquid chromatographic (HPLC) assay described by Shima et al [1] are far in excess of values established with any other method, including boronate affinity chromatography (normal range, 1.5-2.6 % [2]), densitometric scanning of agarose gel electrophoresis (normal range, 0.4-2.0% [3]), or monoclonal antibody based enzyme linked immunosorbent assay (normal, 2.3 + 0.29 % [4, 5]). Careful examination of the principles underlying the HPLC assay and the interpretation of its results are therefore necessary in order to avoid erroneous assignment of the acceptable range of values for non-diabetic populations and for assessment of glycaemic control.

The principal determinants of the extent of nonenzymatic glycation of any protein are the glucose concentration to which it is exposed, the half-life of the involved protein, and the accessibility of free amino groups within the protein for condensation with glucose, the latter in turn dependent on local factors such as pKa and structural considerations.

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