Hexosaminidase A and Total Hexosaminidase, Serum
Method Description Describes how the test is performed and provides a method-specific reference
The hexosaminidases are among the more active of the lysosomal enzymes, which hydrolyze derivatives of beta-D-N-acetylglucosamine and beta-D-N-acetylgalactosamine. Natural substrates are certain sphingolipids (ie, GM2) in which acetylgalactosamine is the terminal monosaccharide. The two hexosaminidase isoenzymes, A and B, differ in their electrophoretic mobility and heat stability. Hexosaminidase A moves toward the anode and is heat labile, while hexosaminidase B moves toward the cathode and is heat stable.
The procedure is performed using an automated pipetting station and a spectrophotometer.. The substrate used is 4-methylumbelliferyl-N-acetyl-beta-D-glucopyranoside (4-MUF-acetamido-2-deoxy-beta-D-glucopyranoside) from which the fluorescent compound, 4-methylumbelliferone, is liberated by both hexosaminidases.
The sample is mixed with citrate phosphate buffer and mixture is separated into 2 tubes. One tube stays at ambient temperature and the other is heated at 51.5 degrees C for 15 minutes. On the automated pipetting station, sample and substrate are pipetted into a microtiter test tube located in a 37 degree C waterbath. The hexosaminidase A fraction is destroyed in the heated sample, leaving only hexosaminidase B to react with the substrate. The unheated sample provides the total hexosaminidase (A and B). The reaction is stopped with glycine after the 30-minute incubation. Sample intensities are compared to that of a 4-methylumbelliferone standard curve to quantitate both the total and the B fraction. The percentage of the A fraction that was inactivated by heating is calculated based on these results. The difference in heat inactivation is used to fractionate hexosaminidase activities.(O'Brien JF: Lysosomal storage diseases. In Tietz Textbook of Clinical Chemistry. Edited by CA Burtis, ER Ashwood. Second edition. Philadelphia, PA, WB Saunders Company, 1994 pp 2149-2160)
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Varies; 10 a.m.
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