Phospholipase C and diacylglycerol lipase in human gallbladder and hepatic bile

Abstract

A phospholipase C in bile, free of bacterial infection, has recently been identified from cholesterol gallstone patients. Because of the importance of phosphatidylcholine in solubilizing cholesterol in bile, this study further investigates the metabolism of phosphatidylcholine in delipidated gallbladder and common bile duct biles. Phospholipase C activity, as measured by the release of phosphoryl[3H]choline from the substrate 1,2-dipalmitoyl-sn-glycero-3-phospho[N-methyl-3H]choline, was identified in both hepatic and gallbladder biles. Similar levels of activity (nmol · h−1 · mg−1 of delipidated protein) were found in common bile duct (11.25 ± 14.23) and gallbladder bile (19.07 ± 22.24), although per milliliter of bile, the mean gallbladder levels were 6.4 times greater than those found in common duct bile. With the two substrates, 1-palmitoyl-2[9,10-3H] palmitoyl-sn-glycero-3-phosphocholine and 1,2(1-14C) dipalmitoyl-sn-glycero-3-phosphocholine, the majority of organically extracted label, after thin-layer chromatography, was recovered as radiolabeled diglyceride, confirming the presence of phospholipase C. Diglyceride levels were found to be closely correlated with [3H]choline (slope, 0.9820; r = 0.9844). In addition to diglyceride, both radiolabeled free fatty acid and monoglyceride were identified in common bile duct and gallbladder biles, although their levels were an order of magnitude less than measurable phospholipase C activity. To determine whether the free fatty acid release was due to either a diacylglycerol-lipase or a phospholipase A2, the effect of adding unlabeled diglyceride on free fatty acid formation from the substrate [14C]DPPC was examined. As the concentration of unlabeled diglyceride was increased, the amount of free fatty acid and monoglyceride released were both reduced in parallel. Direct measurement of diacylglycerol-lipase activity by incubating the diglyceride, sn-2[3H]dipalmitoyl, resulted in release of both products in a ratio similar to that found with sn-2[3H]DPPC. Finally, no radiolabeled lysolecithin was identified with [3H]choline-DPPC or [14C]DPPC as substrate indicating the free fatty acid was the product of a diacylglycerol-lipase rather than a phospholipase A2. Phospholipase C and diacylglycerol-lipase activities were significantly correlated (P < 0.01). The finding of an active phospholipase C and diacylglycerol-lipase activity in abnormal gallbladder and hepatic biles may be of particular importance in the pathogenesis of cholesterol gallstone disease, particularly in those individuals who are characterized by impaired gallbladder emptying and whose biles would thus be exposed to the enzymes for extended periods of time.