Pancreatic Carboxylic Ester Hydrolase Research Articles

In Vitro Digestion of the Self-Emulsifying Lipid Excipient Labrasol® by Gastrointestinal Lipases and Influence of its Colloidal Structure on Lipolysis Rate

Labrasol(®) is a self-emulsifying excipient used to improve the oral bioavailability of poorly water-soluble drugs. It is a mixture of acylglycerols and PEG esters, these compounds being substrates for digestive lipases. The characterization of Labrasol(®) gastrointestinal lipolysis is essential for understanding its mode of action. Labrasol(®) lipolysis was investigated using either individual enzymes (gastric lipase, pancreatic lipase-related protein 2, pancreatic carboxyl ester hydrolase) or a combination of enzymes under in vitro conditions mimicking first the gastric phase of lipolysis and second the duodenal one. Specific methods for quantifying lipolysis products were established in order to determine which compounds in Labrasol(®) were preferentially hydrolyzed. Gastric lipase showed a preference for di- and triacylglycerols and the main acylglycerols remaining after gastric lipolysis were monoacylglycerols. PEG-8 diesters were also hydrolyzed to a large extent by gastric lipase. Most of the compounds initially present in Labrasol(®) were found to be totally hydrolyzed after the duodenal phase of lipolysis. The rate of Labrasol(®) hydrolysis by individual lipases was found to vary significantly with the dilution of the excipient in water and the resulting colloidal structures (translucent dispersion; opaque emulsion; transparent microemulsion), each lipase displaying a distinct pattern depending on the particle size. The lipases with distinct substrate specificities used in this study were found to be sensitive probes of phase transitions occurring upon Labrasol(®) dilution. In addition to their use for developing in vitro digestion models, these enzymes are interesting tools for the characterization of self-emulsifying lipid-based formulations.

Analysis of the discriminative inhibition of mammalian digestive lipases by 3-phenyl substituted 1,3,4-oxadiazol-2(3H)-ones

We report here the reactivity and selectivity of three 5-Methoxy-N-3-Phenyl substituted-1,3,4-Oxadiazol-2(3H)-ones (MPOX, as well as meta and para-PhenoxyPhenyl derivatives, i.e.MmPPOX and MpPPOX) with respect to the inhibition of mammalian digestive lipases: dog gastric lipase (DGL), human (HPL) and porcine (PPL) pancreatic lipases, human (HPLRP2) and guinea pig (GPLRP2) pancreatic lipase-related proteins 2, human pancreatic carboxyl ester hydrolase (hCEH), and porcine pancreatic extracts (PPE). All three oxadiazolones displayed similar inhibitory activities on DGL, PLRP2s and hCEH than the FDA-approved anti-obesity drug Orlistat towards the same enzymes. These compounds appeared however to be discriminative of HPL (poorly inhibited) and PPL (fully inhibited). The inhibitory activities obtained experimentally in vitro were further rationalized using in silico molecular docking. In the case of DGL, we demonstrated that the phenoxy group plays a key role in specific molecular interactions within the lipase's active site. The absence of this group in the case of MPOX, as well as its connectivity to the neighbouring aromatic ring in the case of MmPPOX and MpPPOX, strongly impacts the inhibitory efficiency of these oxadiazolones and leads to a significant gain in selectivity towards the lipases tested. The powerful inhibition of PPL, DGL, PLRP2s, hCEH and to a lesser extend HPL, suggests that oxadiazolone derivatives could also provide useful leads for the development of novel and more discriminative inhibitors of digestive lipases. These inhibitors could be used for a better understanding of individual lipase function as well as for drug development aiming at the regulation of the whole gastrointestinal lipolysis process.

Hydrolysis of tocopheryl and retinyl esters by porcine carboxyl ester hydrolase is affected by their carboxylate moiety and bile acids

The objective of this study was to examine the in vitro hydrolysis of vitamin E esters (α-tocopheryl acetate, α-tocopheryl succinate and α-tocopheryl nicotinate) by pancreatic carboxyl ester hydrolase (CEH) at the concurrent presence of different bile acids at different concentrations. The assay was performed by measuring the amount of α-tocopherol released by porcine pancreatic juice upon addition to different solutions of α-tocopheryl esters, which were dispersed in bile acid mixed micelles at 37°C, pH 7.4. The CEH activity was 10 U in the final assay, and the optimal concentration of cholate in this in vitro-system was determined to 30 mM for the hydrolysis of α-tocopheryl acetate. The hydrolysis of α-tocopheryl esters required presence of pancreatic juice and bile acids, and the results showed furthermore that the ability of pancreatic CEH towards hydrolysis of different α-tocopheryl esters increased with increasing lipophility, irrespective of the type or concentration of bile acid present in the assay. Likewise, retinyl palmitate was hydrolyzed at a faster rate than retinyl acetate. The structure of the bile acid influenced the rate of hydrolysis. Thus, cholate followed by glycodeoxy- and glycochenodeoxycholate were the most effective activators of CEH among the bile acids tested in this assay. The presence of γ-tocopherol or all- trans-retinyl acetate in the assay showed a non-competitive inhibition of the hydrolysis rate of α-tocopheryl acetate.

All-rac-α-Tocopherol Acetate Is a Better Vitamin E Source than all-rac-α-Tocopherol Succinate for Broilers

The difference in bioavailabilities of the acetate and succinate esters of all-rac-α-tocopherol was investigated in a feeding experiment with broilers. The experiment was initiated with 96 12-d-old male Cobb broilers and lasted for 4 wk. The two sources of vitamin E were fed to eight groups of broilers at four different dietary levels (50, 100, 150 and 200 mg/kg feed, including the naturally occurring α-tocopherol). A total collection of droppings for determination of apparent tocopherol absorption were performed at two separate time periods (d 28–34 and d 35–41). There were no differences among the eight experimental groups with respect to animal performance or feed intake. At all dietary levels, the apparent absorption coefficient for all-rac-α-tocopherol succinate was significantly lower than that of the acetate ester. The mean (± SD) apparent absorption coefficient for all-rac-α-tocopherol succinate was 58.0 ± 5.4 compared with 70.8 ± 5.6 for all-rac-α-tocopherol acetate. Furthermore, the apparent absorption coefficients for both esters was significantly lower in the first collection period (d 28–34) than in the second collection period (d 35–41). This difference in the apparent absorption coefficient between the succinate and the acetate ester was accompanied by significant differences in α-tocopherol concentrations in plasma, breast muscle, liver and adipose tissue of the broilers, which were lower in those fed the succinate ester. Based on a comparison of plasma and tissue responses, the succinate ester was utilized only 69–76% as efficiently as the acetate ester. In vitro studies showed a significantly higher capacity of pancreatic carboxyl ester hydrolase to hydrolyze α-tocopherol acetate compared to α-tocopherol succinate. This difference in intestinal hydrolysis of the two vitamin E sources may explain the observed differences in biopotency.

Carboxylic ester hydrolase and amylase in ischemic pancreatitis in the guinea pig.

The observation that an elevated level of pancreatic carboxylic ester hydrolase (CEH) in serum is a more sensitive and specific marker of acute pancreatitis than is elevated serum amylase activity prompted us to explore whether these findings could be confirmed in an experimental model and, if so, to find the explanation behind this difference. We therefore developed a model for ischemic pancreatitis in the guinea pig and a sandwich enzyme-linked immunosorbent assay for determination of CEH in this species. There was a strong correlation between duration of ischemia and severity of pancreatic inflammation and between severity of inflammation and serum CEH level. In contrast, serum amylase was elevated only in animals with the most severe grade of inflammation. Amylase was, however, increased in urine in animals with mild inflammation, but the level did not increase with severity of inflammation. Only one of 31 animals had detectable CEH in urine. In animals with intermediate serum CEH levels the serum and biliary concentrations correlated, indicating that CEH may be cleared by the liver. Amylase was detectable in bile only in animals with high serum levels. The results confirm our observations made in previous clinical studies. A likely explanation for differences in serum levels of CEH and amylase is clearance from the circulation at different rates and, at least partly, via different routes, e.g., the liver and kidney, respectively.

Recombinant human milk bile salt-stimulated lipase. Catalytic activity is retained in the absence of glycosylation and the unique proline-rich repeats.

Human milk bile salt-stimulated lipase ensures efficient utilization of triacylglycerol by breast-fed infants. Cloning and sequencing of cDNA have revealed that the peptide chain consists of 722 amino acid residues showing only little homology to typical lipases. The sequence is identical to that of pancreatic carboxylic-ester hydrolase. The COOH-terminal part contains 16 proline-rich repeats of 11 residues with O-linked carbohydrate. The only N-linked sugar chain is situated close to the active-site serine. Using C127 cells and a bovine papilloma virus vector, high and stable expression of full-length lipase and of several variants, obtained by site-directed mutagenesis, was achieved. The produced proteins were purified and further characterized. Variants lacking all, or all but two, repeats were active with similar specific activity and the same bile salt dependence as the native milk enzyme. Changing the asparagine necessary for N-glycosylation gave the same principal results. Active recombinant full-length lipase was also produced in a bacterial system. We conclude that neither glycosylation (N- or O-linked) nor the proline-rich repeats are essential for catalytic activity or bile salt activation of human milk bile salt-stimulated lipase.

CDNA cloning of human‐milk bile‐salt‐stimulated lipase and evidence for its identity to pancreatic carboxylic ester hydrolase

We have isolated and sequenced cDNA clones covering the entire coding sequence of human-milk bile-salt-stimulated lipase, as well as 996 nucleotides of the 3' end of the pancreatic enzyme carboxylic ester hydrolase. The deduced amino acid sequence of the lipase starts with a 23-residue leader peptide. The open reading frame continues with 722 amino acid residues. The sequence contains in the C-terminal part a proline-rich repeat, 16 repeats of 11 amino acid residues each. The mRNA was estimated to be approximately 2500 nucleotides from Northern blot and of similar size in mammary and pancreatic tissues. Data obtained indicate that the lipase and the carboxylesterase are identical and coded for by the same gene. The cDNA is 2428 bases long, which indicates that a near full-length copy of the transcript has been isolated. Comparisons with other enzymes show that the lipase is a new member of the supergene family of serine hydrolases. It is not only closely related (and in its N-terminal half virtually identical) to lysophospholipase from rat pancreas and cholesterol esterase from bovine pancreas, but also shows a high degree of similarity to several esterases, e.g. acetylcholine esterase. In contrast, no such similarity could be found to typical lipases.

Hydrolysis of fluorescent pyrene‐acyl esters by human pancreatic carboxylic ester hydrolase and bile salt‐stimulated lipase

Fluorescent esters containing pyrenedecanoic acid (P10) or pyrenebutanoic (P4) acid (P4cholesterol, P10cholesterol, P4- and P10-containing triacylglycerols) were synthesized and used as substrates for human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase from human milk. Both enzymes were purified by immunoaffinity chromatography. All fluorescent pyrene derivatives were hydrolyzed by pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase, but at different rates. The hydrolytic rates of the "short" acyl esters (P4-containing esters) were higher than those of the "long" ones (P10-containing esters). Conditions were optimized for sensitivity of the assay using fluorescent cholesteryl esters. The pH optimum was 7.5-8.0. Sodium cholate exhibited a stronger activating effect than taurocholate or taurodeoxycholate (maximal activation was achieved with 5 mmol/L cholate and with a molar ratio cholesteryl ester/cholate around 1:10). Both pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase from milk were strongly inhibited by the other amphiphiles tested, namely phosphatidylcholine and Triton X-100, and were inactivated by low concentrations (10 mumol/L) of the serine-reactive diethyl-paranitrophenyl phosphate (E600). Both enzymes were strongly inhibited by relatively low concentrations of plasma low density lipoproteins. These studies indicate that the fluorescent esters containing pyrene fatty acids can be used as substrates for assaying and investigating the properties of pancreatic carboxylic ester hydrolase as well as bile salt-stimulated lipase from milk.

Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase

A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).

Immunocytochemical localization of pancreatic carboxylic ester hydrolase in human paneth cells.

The protein-A gold method using specific rabbit sera directed against pure human pancreatic chymotrypsinogen and carboxylic ester hydrolase was applied to locate these (pro)enzymes in human pancreatic acinar cells and intestinal Paneth cells. Quantitative evaluation of the labelling indicated that both (pro)enzymes are present in pancreatic acinar secretory granules. In Paneth cell secretory granules, only carboxylic ester hydrolase was present in significant amounts, although the labelling for this enzyme was less intense than that observed in pancreatic zymogen granules. The results obtained support the view that Paneth cells represent a "diffuse exocrine gland" scattered along the intestine, whose role is either to act as a substitute in the event of a deficient pancreas or to regulate the intestinal flora.

Implication of a tyrosine residue in the unspecific bile salt binding site of human pancreatic carboxylic ester hydrolase

Tyrosine residues of the human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) (also referred to as cholesterol-ester hydrolase, EC 3.1.1.13) were nitrated in the ortho-position by the use of tetranitromethane. The specificity of the reaction has been verified and the inhibition observed was shown to be unrelated to the weak polymerizaton of the protein. Among the 27 tyrosines present in the enzyme, seven or eight were nitrated but only one residue, wth a p K of 8.3, seems to be responsible for the loss of activity. This decrease in enzyme activity appears only in assays which were performed in the presence of bile salts, suggesting that of the two bile salt binding sites postulated on the enzyme, only one, referred to as the ‘unspecific site’ (Lombardo, D. and Guy, O. (1980) Biochim. Biophys. Acta 611, 147–155), was modified. This is in agreement with the similar loss of enzyme activity observed on emulsified and soluble substrate. The most important result is the difference observed in experiments of the protective effects of bile salts. The protection with sodium taurodeoxycholate is independent of its critical micellar concentration, showing that monomers protect this site, whereas the protection observed in experiments with sodium cholate appears only for supramicellar concentrations of bile salt. Since this latter bile salt promotes the dimerization of the enzyme, we can conclude that a premicellar bile salt binding site (protected by monomers) is transformed in a functional micellar binding site (protected by micelles). This conformational transformation seems to be consecutive to the dimerization, as has been recently proposed.

On the probable involvement of arginine residues in the bile-salt-binding site of human pancreatic carboxylic ester hydrolase.

Modification of arginine residues with 2,3-butanedione inhibits the carboxylic-ester hydrolase activity on soluble and emulsified substrates when assayed with bile salts. The alpha-dicarbonyl reagent modifies seven of the nineteen arginine residues present per enzyme molecule. Nevertheless the inactivation with butanedione is greatly diminished when the protein is in the presence of negatively charged micellar bile salt. In these conditions we observe the protection of one arginine residue by sodium taurodeoxycholate and of two arginine residues by sodium cholate. This suggests that the carboxylic-ester hydrolase from human pancreatic juice contains at least two arginine residues essential for the activation by bile salts. All our data confirm the presence of two bile-salt-binding sites on the enzyme in which one arginine per site is involved and plays the general role of an anionic binding site. This study provides evidence that arginine residues may play an essential role in the interaction between bile salts and protein.

Catalytic properties of modified human pancreatic carboxylic-ester hydrolase

Human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) modified by specific reagents (diisopropyl phosphorofluoridate, diethyl p-nitrophenyl phosphate, Woodward's K reagent and ethoxyformic anhydride) was studied for its activity. The three residues probably implicated in the active site acting on soluble substrate are shown to be essential for the activity on emulsified substrates such as tributyrin or cholesterol esters solubilized by bile salts (hydrolyzing and synthetizing activities). The modified enzyme is still able to bind to interfaces. Nevertheless, one supplementary carboxylic acid group is responsible for the stability of the enzyme bound to interfaces.

Modification of the essential amino acids of human pancreatic carboxylic-ester hydrolase

Chemical modifications of human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) were performed using organophosphorus compounds, ethoxyformic anhydride and Woodward's K reagent. It has been shown that: (1) the inhibition of the enzyme activity by organophosphorus compounds is due to the phosphorylation of only one alcohol residue, probably a serine residue which may represent the acylable group of the enzyme: (2) 35–36 free carboxyl groups are modified by Woodward's K reagent but only one is responsible for the loss of enzyme activity. This carboxyl group has a p K a of 5.2.: (3) carbethoxylation of histidine residues leads to the inhibition of the enzyme activity. All nine histidine residues are reactive but only one is essential for activity. Taking into account the probable formation of an acyl-enzyme intermediate (Lombardo, D. and Guy, O. (1981) Biochim. Biophys. Acta 657, 425–437) during substrate hydrolysis and the present data we discuss a possible mechanism for carboxylic-ester hydrolase catalysis, a mechanism similar to that described for chymotrypsin.

Binding of human pancreatic carboxylic ester hydrolase to lipid interfaces

Human pancreatic carboxylic ester hydrolase (EC 3.1.1.1), usually characterized by its activity on water-soluble substrates, is shown to catalyze reactions taking place at a lipid/water interface. The inhibition of tributyrin hydrolysis by 1-alcohols follows the pattern of a Langmuir adsorption isotherm. Experiments peformed with siliconized glass beads show that human pancreatic carboxylic ester hydrolase is adsorbed on this artificial (or substitute) interface with a dissociation constant for the enzyme-glass beads complex equal to 1.5 · 10 −8 M. The average molecular area at saturation is 4375 Å 2. Tripropionin hydrolysis is increased by the presence of glass beads. These results are strong arguments in favor of the interfacial activity of pancreatic carboxylic ester hydrolase. The activation of the enzyme bound to the interface is very weak. Bile salts do not prevent the adsorption of carboxylic ester hydrolase on siliconized glass beads and increase strongly the hydrolysis rate of emulsified tributyrin.

Effect of alcohols on the hydrolysis catalyzed by human pancreatic carboxylic-ester hydrolase

Transfer reactions catalyzed by human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) were studied in the presence of methanol and butanol as nucleophiles. The addition of alcohols produced an increase in the total rate of 4-nitrophenyl acetate and n-propylthiol acetate disappearance and a concomitant slow decrease of the hydrolysis rate. These results indicate a competitive partitioning of an acyl-enzyme intermediate between water and nucleophile. Moreover, a strong inhibition of the rates of hydrolysis of methyl butyrate and triacetin by nucleophiles is in agreement with a rate-limiting acylation step. The kinetic data and a trans-ester characterization argue in favor of the formation of an acyl-enzyme intermediate and a two-step reaction mechanism, acylation and deacylation both being rate-limiting. The experiments performed with 4-nitrophenyl acetate show the existence of a nucleophile binding site.

Esterification of cholesterol and lipid-soluble vitamins by human pancreatic carboxyl ester hydrolase

Human pancreatic carboxyl ester hydrolase is shown to catalyse the esterification of cholesterol and lipid-soluble vitamins A, E and D3 with oleic acid. The acitivity requires the presence of bile salts, and the trihydroxylated or the 3 alpha, 7 alpha dihydroxylated bile salts are better activators than the 3 alpha, 12 alpha dihydroxylated bile salts. The hydrolyzing and synthetizing activities of human pancreatic carboxyl ester hydrolase are separated by a large pH range since the synthesis of cholesterol esters is optimal at pH 5.25 and the hydrolysis of cholesterol and vitamin E esters is optimal at pH 8.0. From the comparison of the catalytic constants determined for the hydrolyzing and synthetizing activities and from the pH dependence of the two activities, it appears that human carboxyl ester hydrolase plays an important part in the intestinal lumen. The role of the enzyme in the esterification of cholesterol and lipid-soluble vitamins is questionable.

Isolation of a specific acyl-chymotrypsin intermediate

Chemical modifications of human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) were performed using organophosphorus compounds, ethoxyformic anhydride and Woodward's K reagent. It has been shown that: (1) the inhibition of the enzyme activity by organophosphorus compounds is due to the phosphorylation of only one alcohol residue, probably a serine residue which may represent the acylable group of the enzyme: (2) 35–36 free carboxyl groups are modified by Woodward's K reagent but only one is responsible for the loss of enzyme activity. This carboxyl group has a pKa of 5.2.: (3) carbethoxylation of histidine residues leads to the inhibition of the enzyme activity. All nine histidine residues are reactive but only one is essential for activity. Taking into account the probable formation of an acyl-enzyme intermediate (Lombardo, D. and Guy, O. (1981) Biochim. Biophys. Acta 657, 425–437) during substrate hydrolysis and the present data we discuss a possible mechanism for carboxylic-ester hydrolase catalysis, a mechanism similar to that described for chymotrypsin.