Most of the bile acids in body fluid are conjugated with glycine and taurine, and some of them are esterified as sulfate and glucuronide. Because of poor volatility of amino acid conjugated bile acids, these bile acids can not be determined directly by gas chromatography. Therefore, it is necessary to separate these bile acids and deconjugate amino acids from them prior to determination of the bile acid in gas chromatography. In order to improve this difficulty, the authors developed a new analytical method for conjugated 3β-hydroxy bile acids and its glucuronides by high-performance liquid chromatography (HPLC) combined with immobilized 3β-hydroxy steroid dehydrogenase (3β-HSD) and NAD^+ as a system for detection without pretreatment of fractionation and deconjugation. However, this method requires a simple ethanol extraction of bile acids from biological samples and enzymatic hydrolysis of glucuronides by β-glucuronidase as pretreatment. It is concluded that this method is useful for the elucidation of pathophysiological significance of 3β-hydroxy bile acids in hepatobiliary disease, because of high accuracy, high sensitivity and high specificity of this method.

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We present a new analytical method for sulfated individual bile acids in serum by high-performance liquid chromatography, immobilized 3α- and β-hydroxysteroid dehydrogenase in a column form and NAD^+ as a coenzyme, using enzymatic hydrolysis by sulfatase The principle of the enzyme reaction is as follows: 3α(β)-sulfated bile acid+[numberical formula]-hydroxy bile acid +SO_4^<2->+H^+ 3α(β)-hydroxy bile acid+[numberical formula]-keto bile acid+NADH NADH produced was determined fluorometrically. The optimum conditions for the enzymatic hydrolysis was an incubation for 48 hours at 37℃ with 0.5ml of 0.2M acetate buffer (pH5.0), 0.4ml of 25×10^<-7>M D-saccharic acid-1,4-lactone solution, and 0.1ml of enzyme solution (32units/ml sulfatase in 0.2% NaCl). It is considered that the new analytical method for sulfated individual bile acids, which is much easier than solvolysis, is capable of giving reliable and reproducible results, and can be applied clinically to patients with hepatobiliary diseases for the study of the metabolism of sulfated bile acids.

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