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.

Abstract

An enzymatic fluorometric microassay using 3a-or 7a-hydroxysteroid dehydrogenase was devised to determine serum total or primary bile acids. The present microassay using 50 ul 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 3a-or 7a-hydroxy bile acids, sensitive (a detection limit of 1.5 ,u M with a linear range of 1.5-80 p M), reproducible (CV = less than 5%) and inexpensive. As measured by this microassay, the ratio of serum primary to total bile acids (P/T ratio)

Age- and sex-related profiles of serum primary and total bile acids in infants, children and adults.

“…The present enzymatic fluorometric microassay using a small volume of serum was developed based on a modified method of Barnes and Spenney (1980) and showed a high sensitivity and precision (Ikawa et al 1985). Serum bile acid levels during the fasting state are mainly dependent upon hepatic and intestinal handling of bile acids.…”

“…Serum concentrations of PBA and TBA were measured in each serum by an enzymatic fluorometric microassay described previously (Ikawa et al 1985).…”

“…The present study examined the relationship of age and sex to serum primary (PBA) or total bile acid (TBA) concentrations in a large population of normal infants, children and adults by an enzymatic fluorometric microassay (Ikawa et al 1985) which has a high sensitivity and precision with the use of a small volume of serum.…”

Κινητικη Μελετη Και Αναλυτικες Εφαρμογες Ενζυμικων Συστηματων Για Τον Προσδιορισμο Χολικων Οξεων Σε Βιολογικα Υγρα

“…Pseudomonas testosteroni κ.ά. και η 7a-HSD που παράγεται κυρίως από τους μικροοργανισμούς Escherichia coli, Bacteroides fragil is κ.ά.. Τα ένζυμα αυτά διατίθενται στο εμπόριο και χρησιμοποιούνται ευρύτατα σε ενζυμικές αναλυτικές μεθόδους προσδιορισμού των χολικών οξέων (108,114,115,(144)(145)(146)(147)(148)(149)(150)(151)(152)(153)(154)(155)(156)(157)(158)(159)(160)(161). (215,307).…”

“…Οι κλασσικές ενζυμικές μέθοδοι προσδιορισμού των χολικών οξέων στον ορό βελτιώθηκαν (156)(157)(158). Το κύριο όμως μειονέκτημα των ενζυμικών μεθόδων αναλύσεως αποτελεί το σχετικά υψηλό κόστος των ενζύμων.…”