An enzymatic cycling method for the determination of serum total bile acids with recombinant 3alpha-hydroxysteroid dehydrogenase.
A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay. We constructed a prokaryotic expression system to prepare the recombinant 3alpha-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay. The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm. The enzymatic cycling method could detect 0.22 micromol/L total bile acids in serum. Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively. The calibration curve for total bile acids in serum was linear between 0.5 and 180 micromol/L. This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase. In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3alpha-hydroxysteroid dehydrogenase as the tool enzyme.
Zhang
Guo-Hua
GH
Department of Clinical Laboratory, Peking University First Hospital, Beijing, China.
Cong
Ai-Ri
AR
Xu
Guo-Bing
GB
Li
Chuan-Bao
CB
Yang
Rui-Feng
RF
Xia
Tie-An
TA
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001647:Bile Acids and Salts / Q000097:blood*; D001774:Blood Chemical Analysis / Q000379:methods*; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001647:Bile Acids and Salts; D011994:Recombinant Proteins; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)