1. Measurement of the Transport Activities of Bile Salt Export Pump Using Chemiluminescence Detection Method
- Author
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Kana Yamaguchi, Takao Kurosawa, Tsuyoshi Murai, Shu-Ping Hui, and Hikaru Yabuuchi
- Subjects
Taurocholic Acid ,Taurine ,Insecta ,medicine.drug_class ,Pharmaceutical Science ,law.invention ,Bile Acids and Salts ,chemistry.chemical_compound ,Adenosine Triphosphate ,law ,medicine ,Animals ,Humans ,ABCB11 ,ATP Binding Cassette Transporter, Subfamily B, Member 11 ,Chemiluminescence ,Pharmacology ,Chromatography ,Bile acid ,Vesicle ,Progressive familial intrahepatic cholestasis ,Biological Transport ,3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) ,medicine.disease ,Taurocholic acid ,Bile Salt Export Pump ,chemistry ,Luminescent Measurements ,ATP-Binding Cassette Transporters - Abstract
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.
- Published
- 2010