Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl‐dithio‐5‐(2‐nitrobenzoic acid)

Abstract

We studied the covalent inhibition of lipases by the monolayer technique. We report the inactivation of porcine pancreatic and human and rabbit gastric lipases, acting on mixed monomolecular films of dicaprin containing tetrahydrolipstatin or new hydrophobic disulfide compounds, which can be described as a 'poisoned-interface' system.A kinetic model is presented for depicting the covalent inactivation of lipolytic enzymes at a lipid/water interface. The stoichiometry of the interfacial situation can be described as follows: one lipase molecule embedded among lo5 substrate molecules will be inactivated to half its initial velocity by the presence of 10 tetrahydrolipstatin molecules. This inactivation was independent of the surface pressure.When tested in the form of mixed films, all the disulfide compounds investigated specifically reduced the hydrolysis of 1,2-didecanoyl-sn-glycerol films by gastric lipases, but did not affect hydrolysis by pancreatic lipase. With this poisoned-interface system, tetrahydrolipstatin was found to be the most potent inactivator, whereas disulfide compounds showed a higher degree of selectivity than tetrahydrolipstatin.Gastric lipase differs from pancreatic lipase in several major respects. Under acidic pH conditions, gastric lipase have been found to be remarkably stable and active, whereas pancreatic lipase irreversibly loses its lipolytic capacity. The optimum pH for gastric lipase activity is around 5.4 [l, 21, compared to 8-9 in.the case of pancreatic lipase [3]. The partial hydrolysis of alimentary triacylglycerols which occurs at the pH levels prevailing in the stomach rapidly triggers pancreatic lipase activity in the intestine [4].We have shown that some amphiphiles (bile salts, alimentary proteins and phospholipids), which inhibit pure PPL [3], activate gastric lipase, since they can prevent the irreversible denaturation of the latter enzymes at substrate/water interfaces [l, S].Human gastric lipase (HGL) and rabbit gastric lipase (RGL) are enzymes which have been found to be inactivated by classical sulfhydryl reagents [5,S'-dithiobis(2-nitrobenzoic acid), NbS, ; 4,4'-dipyridyl disulfide) and by a hydrophobic disulfide compound: dodecyl-dithio-S-(2-nitrobenzoic acid) (12: 0-S-SNb) [6, 71. More recently, we reported that ajoene, derived from ethanol extracts of garlic, specifically inactivated HGL and RGL and had no effect on porcine pancreatic lipase With emulsified systems, it is not possible to control the 'interfacial quality' and to easily assess the distribution of soluble versus adsorbed amphiphilic molecules. This prompted us to use the monolayer technique, based upon surface-pressure decrease due to lipid-film hydrolysis [16 -181. This technique is applicable to those cases where the lipid forms a stable monomolecular film at the air/water interface and where reaction products are freely soluble and diffuse away rapidly into the aqueous phase.We studied, for the first time, the covalent inhibition of lipases by the monolayer technique. We report the inactivati...

Advances in Structural Understanding of Lipases

“…These data suggest that, despite the importance of a sulphydryl group, these enzymes are probably serine hydrolases. This is supported by the results of Moreau et al (1991), who showed that the essential sulphydryl group and the E600-label1ed serine function independently.…”

Antihyperlipidemic and hypolipidemic properties of Tacca leontopetaloides (L.) Kuntze (Dioscoreales: Dioscoreaceae) tuber’s aqueous extracts in the rats

“…Reduction of lipids blood levels observed could be explained by saponins, polyphenols and alkaloids containing in extract. In fact, saponins, polyphenols and alkaloids could form complex with cholesterol and bile acids and cause theirs excretion in feces (Oakenfull and Hood, 1979). Creatinin levels showed no changed in the group fed with HFD + aqueous tuber extract, but it is increased in negative control group which fed with HFD only.…”

“…Since food intake is low in rats treated with extracts, the weight loss could be due to the effect of aqueous tuber extract which would reduce appetite. The inhibition of intestinal lipase and absorption of fatty acid by polyphenols could also induce loss of weight as explain by Ransac et al (1991) on their study.…”

“…Studies of Ndouyang et al (2009) showed that the tubers of T. leontopetaloides were rich in saponins (10.78%). Several studies have shown hypolipidemia properties of saponins (Francis et al, 2002;Reynolds et al, 2006;Afrose et al, 2010). In spite of the fact that T. leontopetaloides tubers content saponins, no work has been interest on their effect on lipidemia.…”

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

“…In order to validate a possible application of the PHI-BLA assay in the screening of lipase inhibitors, THL, a well-known covalent inhibitor of digestive lipases ( 56,57 ), was used. Because the inhibition by rHPL and RGL by THL has been extensively studied with other methods (pH-stat technique, monomolecular fi lms), these lipases were chosen for testing the PHIBLA assay.…”