Studies of the electrophoretic determination of serum amylase isoenzymes

Abstract

This study deals with the separation and detection of alpha-amylase isoenzymes in serum by adapting the electrophoretic method of Legaz and Kenney to Helena Laboratories' Titan III cellulose acetate isoenzyme plates for use in the clinical laboratory. The existing techniques have involved long eletrophoretic times, time-consuming assay methods, or cumbersome electrophoretic media which made them unsuitable for a clinical setting.

Experiments were preformed to determine the optimum conditions for the electrophoresis of alpha-amylase isoenzymes using cellulose acetate. The optimized conditions included use of a discontinuous buffer system, a high voltage, and a relatively short electrophoresis time. Agar gel plates were studied for their suitability as an electronphoretic medium. They were not as suitable as the cellulose acetate plates due to difficulty in detecting the bands after electrophoresis.

Several detection systems were considered including Phadebas, Dyamyl*-L, amylopectin anthranilate, and amylopectin azure. All of these were incorporated in molten agarose and the electrophoresed cellulose acetate plate layered on top. Phadebas and amylopectin anthranilate were the two detection systems preferred.

Three salivary and three pancreatic isoamylases were detected. Electrophoretic patters were correlated to various disease states. A pattern typical for pancreatitis was discerned as well as a pattern showing predominate salivary gland involvement.

Various sources of alpha-amylase were studied as controls for the isoenzyme assays. No commercially prepared reference serum was found to be suitable but fresh, frozen extracts from the pancreas and parotid gland can be used.

A study was made to determine with which serum protein fraction the alpha-amylase isoenzymes migrate. The results of this study pointed to the gamma-globulin fraction but were inconclusive. The glycoprotein content of the isoamylase bands was studied; however, the glycoproteins detected may have been from other sources. Also, an effort was made to resolve the question of the source of alpha-amylase isoenzymes and the location of bands from each of these sources on the electrophoresed and developed cellulose acetate plates. There were no isoamylase bands found in liver extract which did not correspond to those in the salivary or pancreatic extracts.