Two reactions are combined in the new generation enzymatic cycling method. In the presence of Thio-NAD, the enzyme 3-α-hydroxysteroid dehydrogenase (3-α-HSD) converts bile acids to
3-ketosteroids and Thio-NADH. The reaction is reversible, and 3-α-HSD converts 3-ketosteroids and NADH to bile acids and NAD. In the presence of excess NADH, the enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring the specific change of absorbance at 405 nm. This cycling reaction leads to significant signal amplification [29].