Abstract: A brief review is presented of the development of chemi-bioluminescent flow injection analysis and its applications, especially in the determination of steroids and cholic acid in biological materials.

Abstract: To determine bile acid, sample was injected into a carrier stream (1-1.2 ml/min) containing 0.2 mM NAD+ in 0.05 M pyrophosphate buffer of pH 9.5 and passed through a column containing 3α-hydroxysteroid dehydrogenase immobilized on glass beads (prep. described). The column effluent was merged with a stream (0.55 ml/min) of 50 µM-1-methoxy-5-methylphenazinium methyl sulfate and, after passing through a reaction coil, with a 1:1 mixture of 0.2 mM isoluminol in 0.8 M phosphate buffer of pH 9.5 and 1 µM-microperoxidase (0.55 ml/min); the chemiluminescent intensity was then measured. Glucose was determined as above using 1 mM NAD+ in 0.1 M phosphate buffer of pH 7 as carrier stream and a column containing immobilized glucosedehydrogenase. ATP was determined using a carrier stream of NADP+, glucose and Mg2+ in 0.1 M Tris hydrochloride buffer of pH 8 and a co-immobilized hexokinase/glucose-6-phosphate dehydrogenase column; in this instance the flow rate of all the reagent streams was 0.5 ml/min. The calibration graphs were rectilinear for 5-100 pmol of bile acid, 0.5-5 nmol of glucose and 0.1-10 µM-ATP; corresponding RSD were 4.1, 2.3 and 5.3%. The method was used to analyze serum. We have developed a chemiluminescent flow injection method for analysis of bile acid, glucose and ATP using the chemiluminescent assay of NADH using 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS)/isoluminol(IL)/microperoxidase (m-POD) system and immobilized enzyme reactors such as 3 α-hydroxysteroid dehydrogenase, glucose-dehydrogenase, hexokinase and glucose-6-phosphate dehydrogenase. The standard curves were obtained in the range of 5-100 pmol for bile acid, 0.5-5.0 nmol for glucose and 10^-7-10^-5 mol/L for ATP. The coefficient of variation for each assay was not more than 4.1% for bile acid, 2.3% for glucose and 5.3% for ATP, respectively.