di Tomaso, Emmanuelle; (1994) Analysis of isoenzymes and isoforms of human alkaline phosphatase, hexosaminidase and transferrin by micro column chromatography. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The main objective of this study was to develop a micro column chromatographic system (MCC) for the separation, detection and quantitation of isoenzymes and isoforms of proteins in human plasma. Three proteins (hexosaminidase, alkaline phosphatase and transferrin) were chosen, as they presented different clinical and analytical problems. Developmental work showed that a 5 mm x 4.6 mm column, packed with a strong polymer-based anion exchanger, used with a step-gradient and a slow flow rate (< 0.55 ml/min) was capable of resolving the different forms of the three proteins. A rapid post-column detection system was designed for the analysis of hexosaminidase and alkaline phosphatase. Transferrin was detected at 460 nm which is specific for iron. The chromatography was found to be reproducible; the coefficient of variation (CV) for the retention times of the various forms of the three proteins generally being approximately 2%. The precision for the within-batch and between-batch quantitation of individual isoenzymes and isoforms varied greatly from one protein to another (from a CV of 1.2% for hexosaminidase to 48.56% for alkaline phosphatase). This appeared to result principally from problems with the software of the commercial integration system. When MCC was compared with appropriate reference methods such as agarose electrophoresis and isoelectric focusing (IEF), the following conclusions were made. The percentage contribution of the various forms of hexosaminidase and transferrin following MCC was comparable with that obtained using electrophoresis and IEF respectively. MCC therefore, provided a rapid and easy method for the clinical analysis of these proteins. For alkaline phosphatase, however, MCC and electrophoresis gave poor agreement, particularly in neonatal samples, which may have resulted from different method specificities. The optimised methods (MCC, IEF and electrophoresis) for the analysis of hexosaminidase and transferrin were applied to a study of patients with Carbohydrate Deficient Glycoprotein Syndrome.

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