CHOLYLGLYCINE HYDROLASE AND 7-ALPHA-DEHYDROXYLASE OPTIMUM ASSAY CONDITIONS IN-VITRO AND CECAL ENZYME-ACTIVITIES EX-VIVO

Citation
La. Thomas et al., CHOLYLGLYCINE HYDROLASE AND 7-ALPHA-DEHYDROXYLASE OPTIMUM ASSAY CONDITIONS IN-VITRO AND CECAL ENZYME-ACTIVITIES EX-VIVO, Clinica chimica acta, 268(1-2), 1997, pp. 61-72
Citations number
32
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Medical Laboratory Technology",Biology
Journal title
ISSN journal
0009-8981
Volume
268
Issue
1-2
Year of publication
1997
Pages
61 - 72
Database
ISI
SICI code
0009-8981(1997)268:1-2<61:CHA7OA>2.0.ZU;2-3
Abstract
Increasing evidence implicates deoxycholic acid (DCA) in the pathogene sis of cholesterol-rich gallbladder stones. However, relatively little is known about the activities of the two intestinal bacterial enzymes (cholylglycine hydrolase and cholic acid 7 alpha-dehydroxylase) respo nsible for the deconjugation and subsequent dehydroxylation of conjuga ted cholic acid (CA), to form DCA. We, therefore, established optimal reaction conditions for measuring the activities of these two enzymes in vitro, and applied these conditions to the determination of the enz ymes in caecal aspirates from six subjects undergoing clinically-indic ated colonoscopy. With respect to cholylglycine hydrolase activity: ze ro order kinetics were found over 20 min at 37 degrees C (pH optimum 4 .0), with K-m and V-max values of 1.66 mmol/l and 0.90 mmol CA min(-1) mg prot(-1), respectively. For cholic acid 7 alpha-dehydroxylation ze ro order kinetics were found over 7.5 min at 37 degrees C, under anaer obic conditions (pH optimum 8.0), with K-m and V-max values of 5.23 X 10(-8) mol/l and 1.88 x 10(-7) mol DCA min(-1) mg prot(-1), respective ly. Applying these reaction conditions to the caecal aspirates, endoge nous cholylglycine hydrolase activities ranged from 0.49 to 2.43 units (mg protein(-1) min(-1)) and CA 7 alpha-dehydroxylase activities from 1.75 to 5.82 X 10(-7) units (mg protein(-1) min(-1)). This study is u nique in assaying quantitatively both the deconjugation and dehydroxyl ation enzyme activities in human caecal samples - an essential first s tep to further studies of intestinal bacterial enzymes in the pathogen esis of cholesterol gallstone disease. (C) 1997 Elsevier Science B.V.