Enzymatic microassay of serum bile acids: increased sensitivity with an enzyme amplification technique

A new method for the analysis of bile acids in serum is described. Bile acids are oxidized by the 3α-hydroxysteroid dehydrogenase of Pseudomonas testosteroni and after selective destruction of NAD excess the reduced coenzyme is determined by an enzymatic cycling reaction. This amplifying step uses a single enzyme the 3β- or 17β-hydroxysteroid dehydrogenase of P. testosteroni: the microquantity of NADH (formed in the previous reaction) is used alternatively for the reduction of the 17-oxo group and oxidation of the 3β-OH group of dehydroepiandrosterone giving stoichiometrically after each cycle 17β-OH-3-oxo androst-5-ene. The rat of formation of this product is in direct proportion to the initial NADH concentration and is followed continuously by the enzymatic conversion of the product of the amplifying step into the chromophore testosterone (λ_max 248 nm: M_r = 16,600). This step is catalyzed by the 3-ketosteroid Δ5-isomerase of P.-testosteroni. This step, which is very fast and not rate limiting, has the advantage that it shifts the equilibrium of the amplifying system in the right direction. This rapid and sensitive method is adequate for numerous determinations in routine analysis.