Comparison of assays for catechol estrogen synthase activity: product isolation vs radioenzymatic catechol-O-methyltransferase-coupled procedures.
Reported values for the activity of enzymes mediating catechol estrogen formation by hamster kidney and liver, measured by catechol-O-methyltransferase-coupled radioenzymatic assay, have been uniformly low and there have been marked discrepancies in values reported from different laboratories. Therefore, we examined the validity of the radioenzymatic assay used in these studies. NADPH-dependent estrogen 2- and 4-hydroxylase activity of hamster liver microsomes measured by radioenzymatic assay was comparable to that reported in the literature but at least one order of magnitude lower than that obtained with a direct product isolation assay. Several features of the radioenzymatic assay were identified which, together, contribute to the underestimation of enzyme activity. They include, incomplete protection from oxidative degradation of both the catechol estrogens generated and of the catechol-O-methyltransferase and assay conditions which are suboptimal for O-methylation of the catechol estrogens. We conclude that results obtained using the catechol-O-methyltransferase-based radioenzymatic assay can only be considered valid if a consistent stoichiometric relationship can be demonstrated between the amounts of catechol estrogens and their O-methylated products.
Roy
D
D
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston 77550.
Bui
Q D
QD
Weisz
J
J
Liehr
J G
JG
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002021:Buffers / Q000494:pharmacology; D002394:Catechol O-Methyltransferase / Q000378:metabolism*; D006224:Cricetinae; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D002393:Estrogens, Catechol / Q000096:biosynthesis*; D066298:In Vitro Techniques; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D011237:Predictive Value of Tests; D011854:Radiochemistry
D000975:Antioxidants; D002021:Buffers; D002393:Estrogens, Catechol; D004958:Estradiol; C001390:2-hydroxyestradiol; C014036:4-hydroxyestradiol; D002394:Catechol O-Methyltransferase