Abstract
We have established a highly sensitive simultaneous determination of the individuals of human serum 3α-hydroxy bile acids, using high performance liquid chromatography (HPLC) combined with enzymatic fluorimetric method. The separation of thirteen components of bile acids, free and glycine- or taurine-conjugated bile acids, was satisfactorily attained by HPLC on a μ Bondapak phenyl column, using 0.3% ammonium carbonate solution mixed with acetonitrile (the volume ratio is 14:4 and 8:4) as mobile phase and 3α-hydroxysteroid dehydrogenase as reagent.
A significant linear correlation was obtained between the peak height and the amount of each bile acid (unconjugated bile acids; 0-125ng, glycine-conjugated bile acids; 0-187.5ng and taurine-conjugated bile acids; 0-250ng). The minimum detectable amount of each bile acid was ranged from 6.3 to 31.0ng.
The recovery rate of each bile acid added to human serum was approximately from 81.7 to 106.6%.
CV value of each bile acid in human serum was ranged 0.72-9.09% (intra-assay) and 1.39- 9.09% (inter-assay).
The concentration of bilirubin (0-40mg/dl) did not interfere to the determination by this method.
A present method made it available to separate thirteen components of bile acids simultaneously within one hour, and to determine free and conjugated bile acids inlml of even healthy human serum.
In the cases of intrahepatic cholestasis and extrahepatic cholestasis, the levels of glycocholic acid and taurocholic acid were markedly elevated and especially in the cases of intrahepatic cholestasis the level of cholic acid was significantly higher than that of extrahepatic cholestasis.
The patients with liver cirrhosis have increases of every bile acid components. Especially the level of conjugated chenodeoxycholic acid in the non-compensative stage was significantly higher than that in the compensative stage.
In the patients with acute hepatitis glycocholic acid and glycochenodeoxycholic acid was increased.
In the cases of chronic hepatitis, the level of total serum bile acids was elevated in parallel with the severity of liver cell damage, and glycochenodeoxycholic acid and taurochenodeoxycholic acid were higher in the cases of active type than that of inactive type.
Patients with a silent stone were shown to have normal patterns of bile acid components except for a slightly increased level of lithocholic acid. These results showed characteristic patterns of bile acid components in sera of patient with various diseases and might indicate their liver function or dynamic state of enterohepatic circulation.
