RESUMO
A method has been developed for the measurement of transport activities in membrane vesicles obtained from Sf9 cells for 3ß-hydroxy-Δ(5)-bile acids by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Calibration curves for the bile acids were linear over the range of 10 to 2000 pmol/mL, and the detection limit was less than 1 pmol/mL for 3ß-hydroxy-Δ(5)-bile acids using selected reaction monitoring analysis. The analytical method was applied to measurements of transport activities in membrane vesicles obtained from human multidrug resistance-associated protein 2-, 3-, and human bile salt export pump-expressing Sf9 cells for conjugated 3ß-hydroxy-Δ(5)-bile acids. The present study demonstrated that human multidrug resistance-associated protein 3 vesicles accepted conjugated 3ß-hydroxy-Δ(5)-bile acids along with common bile acids such as glycocholic acid and taurolithocholic acid 3-sulfate.
Assuntos
Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Animais , Ácidos e Sais Biliares/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Conformação Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células Sf9 , Spodoptera , Espectrometria de Massas em TandemRESUMO
A method for the determination of conjugated and unconjugated 3ß-hydroxy-Δ5-bile acids and related bile acids in human urine and serum has been developed using high-performance liquid chromatography electrospray ionization-tandem mass spectrometry. Calibration curves for the bile acids were linear over the range of 10 2000 pmol/mL, and the detection limit was less than 4 pmol/mL for all bile acids using selected reaction monitoring analysis. The bile acids in urine and serum samples from two patients with severe cholestatic liver disease were measured by this analytical method. Glycine-conjugated 3ß-hydroxy-Δ5-bile acid 3-sulfates were determined to be the major bile acids in the urine and serum from patients with a 3ß-hydroxy-Δ5-C27-steriod dehydrogenase/isomerase deficiency or dysfunction.
