Improved preparation of hepatic microsomes for in vitro diagnosis of inherited disorders of the glucose-6-phosphatase system.
Disruption of microsomal membranes after freezing liver samples can undermine the reliability of in vitro enzymatic diagnosis of the type 1 glycogen storage diseases. However, freezing of biopsy material is necessary if biopsy samples are to be safely transported to the place of assay. We have therefore examined several different methods (each of which could easily be carried out in routine hospital laboratories) of preparing and freezing liver tissue before analysis for glucose-6-phosphatase (EC 3.1.3.9) enzyme activity, and determination of microsomal intactness. Our study showed that homogenizing fresh liver, and centrifuging the homogenate at 10,000 x g for 10 min at 4 degrees C, followed by freezing the resulting supernatant material at -80 degrees C, provided the optimum source of material for subsequent preparation of microsomes for analysis of glucose-6-phosphatase activity. We also demonstrated that 1-naphthol UDP glucuronosyltransferase (EC 2.4.1.17) activity could be used to assess microsomal intactness in cases of type 1a glycogen storage disease, where mannose-6 phosphatase activity cannot be used.
Coughtrie
M W
MW
Department of Biochemical Medicine, University of Dundee, Scotland, U.K.
Blair
J N
JN
Hume
R
R
Burchell
A
A
D000818:Animals; D005260:Female; D005952:Glucose-6-Phosphatase / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism; D005953:Glycogen Storage Disease Type I / Q000175:diagnosis* / Q000473:pathology; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013048:Specimen Handling / Q000379:methods
D014453:Glucuronosyltransferase; D005952:Glucose-6-Phosphatase