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Trisialo- and disialo-transferrin
Re: Trisialo- and disialo-transferrin
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John Whitfield, Biochemist Royal Prince Alfred Hospital, Sydney
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johnwhit{at}bioc.rpa.cs.nsw.gov.au John Whitfield
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The paper in the February issue by Wuyts and colleagues, 'Determination of Carbohydrate-deficient Transferrin Using Capillary Zone Electrophoresis' presents an alternative to chromatographic and isoelectric focusing methods for quantitation of individual transferrin isoforms, and offers a combination of high throughput with improved precision. One of the opportunities which such a method provides is the possibility of studying and comparing the use of individual isoform measurements or their combinations (e.g. asialo only, monosialo only, asialo + monosialo + disialo), and coming to a conclusion about which is the best marker of excessive alcohol intake. The reported differences in test performance between the original and modified Pharmacia CDTect methods may be related to the isoforms included or excluded as CDT by the two methods. The authors mention the clinical performance of separate fractions as an area for further work. However, the capillary zone electrophoresis method described does not separate the disialo and trisialo transferrins. This is unfortunate because such a method cannot be used to evaluate the diagnostic power of these two important isoforms. It is evident that the method is well able to separate isoforms differing by one terminal sialic acid, such as asialo from monosialo, or (with poorer resolution) tetrasialo from pentasialo. The differences in retention time appear (from Figure 1 in Wuyts et al) to be about 0.16 and 0.09 minutes, respectively. Despite this, the disialo and trisialo isoforms appear as one peak, with no obvious broadening or asymmetry (Figure 1 and Table 1). The authors do not discuss this issue and it would be interesting to know whether they think it can be overcome by modified capillary zone electrophoresis methods or whether there is some feature of the carbohydrate structures which makes the two forms indistinguishable by this technique. Both isoelectric focusing and HPLC methods are able to resolve the disialo and trisialo isoforms. |
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J Delanghe University Hospital Gent, Belgium
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joris.delanghe{at}rug.ac.be J Delanghe
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To the Editor: We appreciate the comments made by Dr. Whitfield on our paper 'Determination of Carbohydrate-deficient Transferrin Using Capillary Electrophoresis' in the February issue. The capillary zone electrophoresis method described in our study was developed to present a fully automated and standardized method for CDT quantification, allowing detection of transferrin variants. We chose to perform the iron saturation of transferrin on-analysis. This produced not only high resolution, demonstrated by the high number of theoretical plates and attributable to the sample stacking, but also an improvement of standardization leading to improved reproducibility. Unfortunately, it also meant the comigration of the disialo- and trisialotransferrin isoforms due to need of modifications of the ionic compounds of the buffer system. Indeed, it is possible to separate the disialo- and trisialotransferrins using a different buffer system, however with the disadvantage to loose turn around time. Currently, we are evaluating a capillary electrophoresis method that allows full resolution of all the transferrin isoforms. However, we had to refrain from on-analysis iron saturation to achieve it. Pre-analysis iron saturation implies an increased risk of pre-analytical errors, standardization problems and sample dilution. Although all transferrin isoforms could be resolved, also a decrease in number of theoretical plates was observed. Furthermore, reproducibility of transferrin isoform quantification in equal peak areas decreased due to the sample dilution. Our results with the buffer system without on-column iron saturation are comparable with those of Crivellente et al. (1). Until now, development of a capillary electrophoresis method combining both on-column iron saturation and resolution of all transferrin isoforms has not been achieved. We made a choice for on-column iron saturation, since in routine laboratory management better standardization and user-friendly automation leading to higher reproducibility are extremely important. 1. Crivellente F, Fracasso G, Valentini R, Manetto G, Riviera AP, Tagliaro F. Improved method for carbohydrate deficient transferrin determination in human serum by capillary zone electrophoresis. J Chromatogr B Biomed Sci Appl 2000; 739; 81-93. B. Wuyts |
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