[Measurement of the transport activities of bile salt export pump using chemiluminescence detection method].
Monovalent bile acids, such as taurine- and glycine-conjugated bile acids, are excreted into bile by bile salt export pumps (BSEP, ABCB11). Human BSEP (hBSEP) is physiologically important because it was identified as the gene responsible for the genetic disease: progressive familial intrahepatic cholestasis type 2 (PFIC-2). The evaluation of the inhibitory effect of hBSEP transport activity provides significant information for predicting toxic potential in the early phase of drug development. The role and function of hBSEP have been investigated by the examination of the ATP-dependent transport of radioactive isotopically (RI)-labeled bile acid such as a tritium labeled taurocholic acid, in membrane vesicles obtained from hBSEP-expressing cells. The chemiluminescence detection method using 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) had been developed for a simple analysis of bile acids in human biological fluids. This method is extremely sensitive and it may be applicable for the measurements of bile acid transport activities by hBSEP vesicles without using RI-labeled bile acid. The present paper deals with an application of the chemiluminescence detection method using 3alpha-HSD with enzyme cycling method to the measurement of ATP-dependent transport activities of taurocholic acid (T-CA) in membrane vesicles obtained from hBSEP-expressing Sf9 cells. Calibration curves for T-CA was linear over the range from 10 to 400 pmol/ml. The values of the kinetic parameters for hBSEP vesicles obtained by the chemiluminescence detection method were comparable with the values of that obtained by liquid chromatography-mass spectrometry method. This assay method was highly useful for the measurements of bile acid transport activities.
Yamaguchi
Kana
K
Health Sciences University of Hokkaido, Kanazawa, Japan.
Murai
Tsuyoshi
T
Yabuuchi
Hikaru
H
Hui
Shu-Ping
SP
Kurosawa
Takao
T
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D001692:Biological Transport; D006801:Humans; D007313:Insecta; D008163:Luminescent Measurements / Q000379:methods*; D013656:Taurocholic Acid / Q000378:metabolism
C093491:ABCB11 protein, human; D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; D001647:Bile Acids and Salts; D013656:Taurocholic Acid; D000255:Adenosine Triphosphate; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)