The determination of lipase and phospholipase activities in gut contents of turbot (Scophthalmus maximus) by fluorescence-based assays

To study the potential of fluorescence based assays in the study of lipid digestion in fish, acyl esters of 4-methylumbelliferone and 1-acyl-2-[6 (7 nitro-1,3 benzoxadiazol-4-yl)amino]caproyl labeled phosphatidylcholine compounds (NBD-PC) were used as substrates for the assay of neutral lipase and phospholipase, respectively, in the gut contents of turbot. 4-Methylumbelliferyl hepatanoate (4-MUH) was hydrolysed at a higher rate than the butyrate or oleate esters whilst the hexanoic (C6) ester of NBD-PC was a more convenient substrate for the phospholipase assay than the dodecanoic (C12) ester. Neutral lipase activity was almost 10% higher when 50 mm potassium phosphate buffer pH 7.8 was used instead of 0.01 m citrate/sodium phosphate buffer pH 7.2. Both assays were very sensitive: neutral lipase and phospholipase activities were detectable at a minimum protein concentration in the digesta of 0.04 and 1.25 mg/ml, respectively. When the variations in lipolytic activities with gut segment and with size of fish were examined neutral lipase activity was always found to be higher in the hindgut and rectum segments than in the foregut. Although phospholipase activity was also found to be highest in the hindgut of the largest fish examined (av. wt. 182.3g), in fish of average weight 8g fish the activity was similar in all three segments. In the digesta from the whole gut of smaller fish (av. wt. 0.2, 0.6 and 1.43g) neutral lipase and phospholipase activities increased with increasing body mass when expressed as per ml of digesta. It is concluded that fluorescence-based assays are applicable to the study of lipid digestion in fish of different size.