Bile acids bioluminescence

Clinical Analysis. A wide range of clinically important substances can be detected and quantitated using chemiluminescence or bioluminescence methods. Coupled enzyme assay protocols permit the measurement of kinase, dehydrogenase, and oxidases or the substrates of these enzymes as exemplified by reactions of glucose, creatine phosphate, and bile acid in the following ... 

As well as the enzyme 3a-hydroxysteroid dehydrogenase, a 7a-enzyme has been isolated from E. coli and a 12a enzyme from a strain of Clostridium (Ml). These group-specific enzymes can be used to measure the amounts and ratios of the three main bile acids (cholic, chenodeoxycholic, and deoxy-cholic acids) in bile specimens. Bioluminescent assays suitable for serum bile acid analysis have been described using 7a-hydroxysteroid dehydrogenase (R6) and 12a-hydroxysteroid dehydn enase (S12), as well as for the 3a enzyme (S13, S44). 

It is still too early to tell whether serum bile acid determinations will be added to the currently available combination of liver function tests or replace individual tests, such as the measurement of bilirubin (H19). In correctly diagnosing patients with histologically defined liver disease, serum bile acids appear to slightly improve the results from conventional liver tests, if used in combination with these tests (F3). Perhaps when sensitive analytical methods such as bioluminescence, which can be applied to serum bile acid analysis, become available and established in diagnostic laboratories, serious consideration will be given to routine measurement of serum bile acid levels to... 

Roda A, Girotti S, Ghini S. Continuous-flow determination of primary bile acids by bioluminescence with use of nylon-immobilized bacterial enzymes. Clin Chem 1984 30 206-10. 

The marine bacterial bioluminescent system when coupled to 7-a-hydroxysteroid dehydrogenase provides a very sensitive (0.5 pmol) and precise method for serum bile acids according to the analytical scheme shown in reaction [XI], This assay is typical of bioluminescent coupled enzyme assays for either a dehydrogenase enzyme or its substrate. 

Sensitive methods for SBA based on bioluminescence measurement have recently been deve p using hydroxysteroid dehydrogenase (3o( or 12d or 7d -HSD). The HSD catalyzes the conversion of the bile acid hydroxy g up (3o( or 7d or 12 c. ) to a keto-group in the presence of NAD(P). The resulting NAD(P)H, in presence of NAD(P)H FMN oxydoreductase (OXRED), converts FMN to its reduced form (FMNH ). This in turn reacts with decanal and oxygen in the presence dr bacterial luciferase to produce light. The intensity of the light emitted is proportional to the amount of bile acids in the initial raction. The overall scheme,for 7 -HSD for instance, is this ... 

Table 4. Characteristics of the Enzymatic Bioluminescent Methods for Serum Bile Acids with Bacterial Luciferase...

The first assay developed by one of us was for primary bile acids using cy-HSD and bioluminescent enzymes immobilized on Sepharose 4B (Table 4). A linear response in the range 1.5 to 50 mol/l was obtained for SBA on serum heated at 70 °C for 15 min. The use of a Sepharose-immobilized enzyme system increases the catalytic activity per unit enzyme and gives more stable analytical reagents. 

Automated bioluminescent assay for NADH, glucose 6-pho-sphate, primary bile acids and ATP, Anal. Biochem. 129 392 (1983). 

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