Measurement of the ratio of primary to total bile acids in serum by enzymatic fluorometric microassay and its clinical significance in patients with liver disease.

An enzymatic fluorometric microassay using 3 alpha- or 7 alpha-hydroxysteroid dehydrogenase was devised to determine serum total or primary bile acids. The present microassay using 50 microliter of a serum sample was run as a two-step method; the first step is the reaction of substrates with NAD+ and the hydroxysteroid dehydrogenase to produce as much NADH as possible in the incubation system and the second step is that of NADH with resazurin coupled with 1-methoxy phenazinemethosulfate to produce fluorescent resorfin. The assay is specific for 3 alpha- or 7 alpha-hydroxy bile acids, sensitive (a detection limit of 1.5 microM with a linear range of 1.5-80 microM), reproducible (CV = less than 5%) and inexpensive. As measured by this microassay, the ratio of serum primary to total bile acids (P/T ratio) in 10 normal subjects was 62.1 +/- 0.7% and was significantly elevated in the early phase of acute hepatitis (p less than 0.01), followed by return to the normal level after 4 weeks.