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Some competitive enzymatic bile acids methods are NBT-based and their methodology is essentially derived from the work of Mashige F, Tanaka N, Maki A, Kamei S and Yamanaka M. Direct spectrophotometry of total bile acids in serum. Clin Chem 24:1150; 1978. In this classic method, bile acids from the patient's serum are enzymatically oxidized by the enzyme 3-α-hydroxysteroid dehydrogenase with the concurrent reduction of NAD to NADH. In the fasting state and early stages of disease, levels of bile acids are low so relatively few molecules of bile acid are available for the enzyme to act on so only a small amount of NAD is reduced to NADH. Since a small amount of NADH is formed, there is not a significant absorbance change when this reaction is measured directly at 340 nm. Conventional enzymatic, colorimetric bile acids assays couple the NADH produced to the formazan dye nitro blue tetrazolium <http://www.dclchem.com/Bile_Acids/Bile_NBT.html> (NBT/Formazan) read at 540nm to increase test sensitivity. While NBT produces far more color (absorbance change) per molecule than NADH its use introduces a new set of analytical problems limiting the method to an endpoint procedure with concomitant interference and low end sensitivity issues Current total bile acids method
In the fasting state, levels of bile acids are low so that very few molecules of bile acid are available to reduce NAD to NADH in vitro making it analytically disadvantageous to directly measure the change in absorbance at 340 nm with NADH. This need for increased sensitivity is why Mashige et al coupled nitro blue tetrazolium (NBT) to NADH. Unfortunately, this scheme has several shortcomings almost all of which are a direct result of having to use NBT for increased sensitivity:
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