ABSTRACT
Estrogen 2/4-hydroxylase (ESH) and catechol-O-methyltransferase (COMT) activities in mouse liver and uterus were studied. While 2-hydroxyestradiol (2-OHE2) was the predominant product in the liver, equal amounts of 2- and 4-hydroxyestradiol were produced in the uterus. Two-hydroxyestradiol was the preferred substrate for COMT in both tissues, but the level of this enzyme activity was much less in the mouse uterus (17-fold less). Thus, preferential production of 4-hydroxyestradiol (4-OHE2) in the presence of relatively less deactivation provides a mechanism for the local formation of a more chemically active form of estrogen by uterine tissue.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 CYP1A1 , Estrogens, Catechol/biosynthesis , Uterus/metabolism , Animals , Catechol O-Methyltransferase/metabolism , Cytochrome P-450 CYP1B1 , Estradiol/analogs & derivatives , Estradiol/biosynthesis , Female , Mice , Steroid Hydroxylases/metabolism , Time Factors , Uterus/enzymologyABSTRACT
A sensitive, selective, and nonradiometric assay for estradiol 2- and 4-hydroxylase activities using high-performance liquid chromatography with an electrochemical detector (LCEC) is developed. The assay relies on a liquid-solid extraction of the catechol estrogens onto alumina, followed by their elution with dilute acid. Chromatographic separation is accomplished on a C18 reversed-phase column with an isocratic system consisting of 0.05M sodium acetate in methanol/water/acetic acid 34:56:10 (V/V). Using this assay, the estrogen 2- and 4-hydroxylase activities contained in the microsomal fraction of liver from adult female mice is studied. The assay is optimized with regard to pH, substrate or enzyme concentrations, and time. The apparent km and Vmax of the 4- and 2-hydroxylase are 22.5 microM and 35.9 pmol/mg protein/min and 64.7 microM and 206.2 pmol/mg protein/min, respectively.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 CYP1A1 , Microsomes, Liver/enzymology , Steroid Hydroxylases/analysis , Animals , Chromatography, Liquid , Cytochrome P-450 CYP1B1 , Electrochemistry , Female , Kinetics , MiceABSTRACT
Our homogeneous immunoprecipitation inhibition assay (Clin. Chem. 28:659-661, 1982) is applied here to tobramycin, phenobarbital, and theophylline. Only 10-50 microL of test sample is needed. No sample treatment, dilution, or extraction is required. A test serum sample is simultaneously mixed with a drug conjugate and its specific antiserum in a centrifugal analyzer. The subsequent reaction and the measurement are completed in 3 min. Within-run and between-run CVs for clinically relevant concentrations were well below 10%. Results for patients' samples correlated well with those by enzyme immunoassay.
Subject(s)
Anti-Bacterial Agents/blood , Haptens/isolation & purification , Phenobarbital/blood , Theophylline/blood , Tobramycin/blood , Antigen-Antibody Complex/isolation & purification , Centrifugation , Chemical Precipitation , Cross Reactions , Humans , Immunoenzyme Techniques , Kinetics , Spectrophotometry , Time FactorsABSTRACT
An ion-pairing chromatographic method which uses a controlled potential coulometric detector is described. Two coulometric detectors with different electrolytic cell designs have been investigated. The resulting sensitivity can be comparable to the conventional amperometric detector. This technique has been applied to the analysis of catecholamines.
