Rapid changes in chromatographically determined haemoglobin A1c induced by short-term changes in glucose concentration

Abstract

Chromatographically determined haemoglobin A1c concentration was measured during short-term (1-24h) changes in glucose concentration in vitro and in vivo. In vitro at 37 degrees C the HbA1c concentration increased with glucose concentration and time both in normal and diabetic erythrocytes. In normal erythrocytes incubated in 20--100 mmol/l glucose, the increases in the HbA1c concentration were maximal after 4--6 h and then stable for the next 18--20 h. During the first hour, increases in the HbA1c concentration were linear with time and on average 0.034% HbA1c x h-1 x mmol/l glucose-1. In erythrocytes, after a rapidly produced increase (2 h), HbA1c decreased to preincubation concentrations during a further incubation of the erythrocytes in a glucose-free medium at 37 degrees C for 4--6 h. The mean rate of linear decrease was 0.017% x h-1 x mmol/l glucose-1. After incubation of erythrocytes in 100 mmol/l glucose for 24 h, 1.3% HbA1c remained stable for 6 h in saline. The rapid increase in HbA1c concentration, as determined by chromatography, was not due to stable HbA1c (ketoamine linked glucose) as no increase was found in the HbA1c concentrations determined by the thiobarbiturate method. In juvenile diabetics controlled by an artificial beta-cell, rapid changes of blood glucose concentration (up to 20 mmol/l) resulted in increases in HbA1c concentration of as much as 1.9% within 12 h (mean 1.1%). Rapid in vivo increases in HbA1c concentration were reversible by normalization of the blood glucose concentration. That rapid changes in HbA1c may occur in daily diabetic life was evidenced by differences in HbA1c concentration between blood samples from out-patient diabetics incubated in saline for 16 hours at 4 degrees C and 37 degrees C (range of differences 0.2--1.4% HbA1c). The differences correlated to the blood glucose concentration at the time of sampling blood for HbA1c determination. Thus, incubation of blood at a low glucose concentration prior to determination of the glycosylated haemoglobin concentration may overcome interference from rapidly produced HbA1c.

The measurement of haemoglobin A1c by isoelectric focussing in diabetic patients

“…However a cautionary note should be sounded; unless the method used for the determination of glycosylated haemoglobin can accurately separate HbAlc from its intermediate condensation product, or precautions are taken to remove the intermediate product [2], considerable overestimation of HbAlc may occur. At present we would suggest that isoelectric focussing represents the best method for the direct measurement of HbAac.…”

“…As the kinetics of formation and reversal of the Schiff base are likely to be different to that of the formation of HbAlc, it is likely that any significant concentration of the Schiff base will cause a significant over-estimation in the measurement of HbAlo. However, using an isoelectric focussing method, it is possible to differentiate between HbAlc and this intermediate band [1][2]. This paper reports the results of using this method in investigations in vivo and in vitro of the relationship between HbAlc, the intermediate band and blood glucose levels, and particularly whether short term fluctuations in blood glucose levels can influence significantly HbA~ levels as reported by Svendsen [3].…”

Increase in stable glycosylated haemoglobin after induction of poor glycaemic control

“…The relationship between changes in glycaemic control and HbA1 has not been well characterized in clinical practice. Recent studies have shown changes in HbAt level to occur within hours of changes in blood glucose concentrations [3][4][5]17].…”

“…Rapidly formed HbA1 is labile and can be removed by incubation of the erythrocytes in saline for a few hours [4,5], by dialysis of haemolysates [18,21] or by chemical means [191. The influence of changes in blood glucose level hampers the value of HbA1 as an index of average glycaemia and therefore the labile fraction should be eliminated when the assay is used for this purpose [4][5][6]20].…”

“…The influence of changes in blood glucose level hampers the value of HbA1 as an index of average glycaemia and therefore the labile fraction should be eliminated when the assay is used for this purpose [4][5][6]20]. The resulting stable fraction thus assayed is regarded chemically to be the ketoamine form […”