Combined sensitive analytical methods for cholecystokinin levels and tryptic activity in individual fish larvae

Abstract

A protocol for combined analysis of cholecystokinin (CCK) levels based on radioimmunoassay (RIA) and fluorescence tryptic enzyme activity (FTA) was developed in order to accomplish a sensitive analysis of individual bodies and gut segments of fish larvae. Methanol was used for CCK extraction. The gut of herring larvae contained 8.9±1.2 fmol CCK/mg dry weight and in the post-larval Atlantic halibut the CCK levels varied significantly ( p<0.05) from 20.9±15.6 to 101.8±56.7 fmol/mg dry weight for separated intestinal and pyloric segments, respectively. Acid solution, 0.02 mol/l HCl–CaCl 2 (pH 1.8), and alkaline solution, 0.1 mol/l Tris–0.02 mol/l CaCl 2 (pH 8.0) were tested to prepare crude trypsin extracts from Coregonids and Atlantic halibut larvae. The tryptic activity of crude extracts prepared with acid solution was enhanced by a factor of 3.19±0.52 compared to the tryptic activity of crude extracts prepared with alkaline solution. The larval trypsins (from yolk sac larvae) were stable in methanol, preserving 88% of its starting activity after 6 days of storage. Based on the results, the method of extraction using methanol and acid solution (pH 1.8) was suitable for the combined analysis for CCK levels and FTA in gut segments or single larvae. The potential application of these analytical tools may allow a better understanding of the individual variability of gut functionality, nutritional condition and the feeding activity of developing fish based on their content of CCK and tryptic activity.