20 February / March 2019
Heterogeneity of Glycated Haemoglobin as Revealed by Chromatography, Isoelectric Focusing and Mass Spectrometry: The A1c Test Standardisation and Assessment of Individual Variabilities in Haemoglobin Glycation
1 Alexander Stoyanov, 1 1
2 3 Uzma Khan, 2 Gerard Rozing 3 Tiemin Huang, 1
University of Missouri, School of Medicine, Columbia MO. PharmaFludics, Zwijnaarde 82B-9052 Ghent, Belgium.
Advanced Electrophoresis Solutions Ltd., Cambridge ON, N3H4W5, Canada. Correspondence:
Alexander V. Stoyanov
Department of Pathology and Anatomical Sciences, University of Missouri, 1 Hospital Dr, MSB Room-M575, Columbia, MO 65212, USA. Phone: 1 573 884 6830. Fax: 1 573 884 4748. Email:
stoyanovA@health.missouri.edu.
Glycated Haemoglobin or A1c is used as a marker of long-term glucose control in diabetes patients. For decades, different A1c tests are employed in healthcare for diabetes diagnosis and monitoring patient progress in therapy. The heterogeneous nature of glycated haemoglobin results from the multiple possible glycation sites on each haemoglobin chain and this presents a serious challenge for A1c test standardisation, since different approaches to measuring A1c - such as electrophoretic, chromatographic, or affinity approaches - essentially measure different parts of the total glycated haemoglobin population. High resolution separation techniques such as ion exchange chromatography and electrophoresis, in particular isoelectric focusing, followed by MS - can deliver a complete picture of the glycated haemoglobin which can be of clinical interest. MS based approaches in A1c quantitation also provide an opportunity of considering the individual variabilities in haemoglobin glycation that potentially allows for personalised approach to diabetes patients.
Glycated Haemoglobin
Glycation is a non-enzymatic reaction involving the addition of glycose and/or other reducing sugars to the native protein. Glucose in its aldehyde configuration reacts with primary amines (specifically the side chains of lysine and arginine as well as the N-terminus). The glycated haemoglobin test, referred to as the A1c test, serves as an indicator of long-term glycemic control in diabetes patients since it correlates with the average blood glucose over the life-time of erythrocytes (red blood cells), which is by normal conditions is approximately 120 days. Despite wide use of A1c test in clinical practice, a complex, highly heterogeneous nature of glycated haemoglobin can potentially generate issues in both test standardisation and the results interpretation. As stated previously, the different A1c tests measure different
parts of the total population of glycated haemoglobin. Another, perhaps more important issue, consists in the existence of so-called biologic variability in haemoglobin glycation, which may result in different A1c numbers for patients with the same average blood glucose.
Experimental
The chromatography instrument used was Shimadzu Prominence with a VICI Valco switching valve. The absorbance was monitored at 415 nm. The column was Mono S GL 5/50, Supelco (Bellefonte, PA). For capillary isoelectric focusing, an IEF instrument with whole column imagine detection WCID was used (Advanced Electrophoresis Solutions Ltd, Cambridge, Canada, ON).
Agilent 3100 OFFGEL Fractionator was also used to collect haemoglobin fractions which were then further analysed using a Sciex API 4000.
Sample preparation: Erythrocytes were isolated from whole blood. Boronate Affinity Chromatography was performed on Glyco- Tec columns (Helena Laboratories, Beaumont, TX, USA), to separate the initial haemoglobin sample into two fractions: boronate reactive (PHB+) and non-reactive (PHA-).
Differential Glycation Index Approach
Patients mean blood glucose was measured with continued blood glucose monitors (Medtronic) by a five day continuous recording. Mass-spectrometric A1c quantitation was used [1]. Finally, the Differential glycation index, DGIHB
, was Dmitiry Shin.
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