23
Figure 2. Experimental IEF steady-state profi le for the human haemoglobin sample in the narrow pH- gradient 4-8. Run conditions: 1 min at 1500 V and 6 min at 3000V. A resolution close to ion exchange chromatography as achieved. Please note that the IEF electropherogram does not resemble the ion exchange chromatogram, since the two separations are based on different mechanisms.
chain analysis provides the easiest way to quantify A1c, even if does not identify a single component. It should be noted, however, that the use of collision induced dissociation techniques would allow for this to happen. Many commercial A1c methods do not isolate a single component, (as it is clearly visible from Figure 1, p.e.) that results in all the A1c standardisation efforts having some certain limits. This is also valid when an MS reference method is going to be used.
Differential Glycation Index concept as a tool for evaluation of individual variability in haemoglobin glycation
It has been shown that individual variability in haemoglobin glycation between diabetes patients occurs. It would, therefore, be benefi cial for physicians to have a parameter which can quantify such a variability in order to modify the therapy accordingly.
The Differential Glycation Index (DGI) is the difference between the experimentally measured A1c minus the expected A1c, normalised by A1c measured. Here, the expected A1c is theoretically calculated based on average blood glucose level [2]. The index is dimensionless and it directly shows how any given individual differs from an average patient. In contrast to previous attempts to connect the average blood glycose and A1c [7-9], the approach is based on simultaneous mass-spectrometric A1c quantitation and continuous glucose monitoring CGM for average blood glucose measuring, providing the highest possible precise evaluation of the individual variability of haemoglobin glycation in diabetes patients. While blood glucose - A1c relationship is a result of multiple gene infl uencing both glycose metabolism and erythrocytic pathways [10], the DGIHb
, which
connects the same values, can be obtained with two easily measurable biochemical parameters. Based on the index value the physician becomes able to modify the therapy according to individual patient needs, in particular, to set the optimal A1c goals [11]. The A1c goals, should be essentially different, for example, for the two patients with high negative and high positive DGIs.
Conclusions
Glycated haemoglobin represents essentially heterogeneous populating circulating in human blood. Mass spectrometry provides an opportunity for better A1c test standardisation and quantitative evaluation of the individual variabilities in haemoglobin glycation. Further progress in electrophoresis and chromatography can potentially facilitate the establishment of the complete pattern of glycated haemoglobin. This goal can be achieved only with
extensive pre-fractionation, since the concentration range of different glycated haemoglobin isoforms varies essentially. Mass spectrometric A1c quantitation methods combined with continuous glucose monitoring allows for evaluation of the individual variability in haemoglobin glycation that can lead to personalised therapy for diabetes patients.
References
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Figure 3. MS data for selected hemoglobin fractions separated with preparative isoelectric focusing (Agilent OFFGel Fractionator). The three collected fractions represent different parts of a narrow pH gradient 4-7. The upper panel on each fi gure corresponds to a Total Ion Count (TIC), or continuous density profi le. Lower panels correspond to the individual peaks as pointed by vertical lines on the TIC curve. MS analysis was performed using an API-4000 triple quadrupole mass spectrometer (AB Sciex) coupled to a Shimadzu Prominence LC system integrated with a switching valve (VICI Valco). Reverse-phase chromatography was performed using a Jupiter 5µ C18 column (Phenomenex). Two mobile phases were used for elution: (A) 0.4% formic acid in water and (B) 0.4% formic acid in acetonitrile. The total fl ow rate was 0.5 ml /min. Each fraction represents an essentially heterogeneous populations including modifi ed and partially truncated haemoglobin chains as revealed by MS.
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