Pancreatic Cholesterol Esterase Research Articles

Lipid-Lowering mechanisms of grape seed extract (Vitis vinifera L) and its antihyperlidemic activity

Grape seed extract (GSE) has been targeted to promote beneficial health effects, especially the prevention of obesity and hyperlipidemia. In present study, we investigated the mechanisms of GSE on inhibition of lipid digestion and absorption. GSE significantly inhibited pancreatic lipase and cholesterol esterase in dose-dependent manner with the IC50 values of 3.71 ± 0.03 and 27.27 ± 4.12 mg/mL, respectively. In addition, GSE also inhibited the formation of cholesterol micelles, and bound to bile acid. The oral administration of GSE significantly decreased serum triglyceride and cholesterol in rat fed high fat emulsion through inhibition of lipid digestion and absorption. GSE may be a feasible therapeutic strategy for prevention and treatment of patients with hyperlipidemia and obesity. Key words: Grape seed extract, bile acid binding, cholesterol micellization, pancreatic cholesterol esterase, pancreatic lipase, mechanisms.

Enzymatic hydrolysis of structurally diverse phthalic acid esters by porcine and bovine pancreatic cholesterol esterases

A weak hydrolyzing activity against bis (2-ethylhexyl) phthalate (DEHP) was discovered in a commercial crude lipase (EC 3.1.1.3) preparation from porcine pancreas. DEHP was hydrolyzed to mono (2-ethylhexyl) phthalate (MEHP) not by a pancreatic lipase but by a cholesterol esterase (CEase, EC 3.1.1.13), a trace contaminant in the crude lipase preparation. Enzymatic hydrolysis of phthalic acid esters (PAEs), suspected to be endocrine-disrupting chemicals, was investigated using CEases from two species of mammals and a microorganism. Eight structurally diverse PAEs, namely diethyl phthalate (DEP), di- n-propyl phthalate (DPrP), di- n-butyl phthalate (DBP), di- n-pentyl phthalate (DPeP), di- n-hexyl phthalate (DHP), DEHP, n-butyl benzyl phthalate (BBP), and dicyclohexyl phthalate (DCHP), were hydrolyzed to their corresponding monoesters by both porcine and bovine pancreatic CEases, while a microbial CEase from Pseudomonas sp. had no hydrolyzing activity against these PAEs. The hydrolysis experiments with bovine pancreatic CEase (50 U) indicated complete hydrolysis of every PAE (5 μmole) except for BBP and DCHP within 15 min; BBP and DCHP were hydrolyzed within 30 min and 6 h, respectively. The rates of PAE hydrolysis could be affected by the bulkiness of alkyl side chains in the PAEs. This study provides important evidence that mammalian pancreatic CEases, such as those from porcine and bovine sources, are potential enzymes for nonspecific degradation of structurally diverse PAEs.

Phosphaisocoumarins as a new class of potent inhibitors for pancreatic cholesterol esterase

Due to the importance of pancreatic cholesterol esterase (CEase) as a potential target in atherosclerosis and for the development of hypocholesterolemic agents, there are increasing interests in designing and synthesizing CEase inhibitors. In the present study, we prepared forty-five isocoumarin phosphorus analogues (i.e., phosphaisocoumarins) and investigated the inhibition of these compounds on the CEase. The results showed that some phosphaisocoumarins could act as potent inhibitors of CEase. The most potent inhibitors, compounds 9d, 10a and 12e give IC 50 values of 4.8 μM, 2.3 μM and 1.9 μM, respectively. The inhibition mechanism and kinetic characterization studies indicate that they are reversible competitive inhibitors.

Plant sterol and stanol substrate specificity of pancreatic cholesterol esterase

Consumption of plant sterols or stanols (collectively referred to as phytosterols) and their esters results in decreased low-density lipoprotein cholesterol, which is associated with decreased atherosclerotic risk. The mechanisms by which phytosterols impart their effects, however, are incompletely characterized. The objective of the present study is to determine if pancreatic cholesterol esterase (PCE; EC 3.1.1.13), the enzyme primarily responsible for cholesterol ester hydrolysis in the digestive tract, is capable of hydrolyzing various phytosterol esters and to compare the rates of sterol ester hydrolysis in vitro. We found that PCE hydrolyzes palmitate, oleate and stearate esters of cholesterol, stigmasterol, stigmastanol and sitosterol. Furthermore, we found that the rate of hydrolysis was dependent on both the sterol and the fatty acid moieties in the following order of rates of hydrolysis: cholesterol>(sitosterol=stigmastanol)>stigmasterol; oleate>(palmitate=stearate). The addition of free phytosterols to the system did not change hydrolytic activity of PCE, while addition of palmitate, oleate or stearate increased activity. Thus, PCE may play an important but discriminatory role in vivo in the liberation of free phytosterols to compete with cholesterol for micellar solubilization and absorption.

Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester

Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.

Phytosterol ester substrate specificity of pancreatic cholesterol esterase

Consuming phytosterols and their esters results in decreased LDL cholesterol, an atherosclerotic risk factor. To more completely characterize how phytosterols impart their effects, the present study aimed to determine if pancreatic cholesterol esterase (PCE; EC 3.1.1.13), the enzyme primarily responsible for cholesterol ester hydrolysis in the duodenum, is capable of hydrolyzing various phytosterol esters, and to compare their rates of hydrolysis in vitro. We found that 1) PCE hydrolyzes palmitate, oleate, and stearate esters of cholesterol, stigmasterol, stigmastanol, and sitosterol; 2) specificity was dependent on both the sterol and the fatty acid moieties in the following order of rates of hydrolysis: cholesterol > (sitosterol = stigmastanol) > stigmasterol, oleate > (palmitate = stearate); 3) addition of free phytosterols did not change hydrolytic activity of PCE, while addition of palmitate, oleate, or stearate increased hydrolytic activity. Thus, PCE may play an important but discriminatory role in vivo in the liberation of free phytosterols to compete with cholesterol for micellar solubilization and absorption.Grant Funding SourceUSDA‐NRI competitive grant no. 2007‐35200‐18298

Characterization of novel cholesterol esterase from Trichoderma sp. AS59 with high ability to synthesize steryl esters

A novel cholesterol esterase with there and throughout to synthesisze steryl ester was obtained from the culture filtrate of a fungal strain Trichoderma sp. AS59 isolated from soil. The extracellular enzyme was a monomeric protein with a molecular mass of approximately 58 kDa and an isoelectric point of 4.3. The optimal temperature was between 35 degrees C and 40 degrees C, and the optimal pH was 7.0. The enzyme retained 75% of the initial activity after 18 h of incubation at 30 degrees C in the pH range of 3.5-7.5. Its relative hydrolytic activities on fatty acid cholesteryl esters were in the following order: butyrate (121%), linoleate (100%), caprylate (79%), myristate (42%), palmitate (38%), caproate (37%), and laurate (35%). Unlike mammalian pancreatic cholesterol esterase that is activated by primary cholates on hydrolysis of long-chain fatty acid cholesteryl esters, the enzyme from Trichoderma sp. AS59 displayed its basal activity and was not affected by cholate up to a concentration of 5 mM. At higher cholate concentrations the activity gradually decreased, but reincreased at about 40 mM to reach more than twice the basal activity at 100 mM. The enzyme exhibited a broad substrate specificity, being capable of hydrolyzing various fatty acid esters of not only cholesterol, but also methanol, glycerol, and p-nitrophenol. When incubated with a mixture of cholesterol and oleic acid of equal amounts, the enzyme achieved stoichiometrical esterification in 5 h, indicating its potential utility in food additives and liquid crystal devices.

Isocoumarin-based inhibitors of pancreatic cholesterol esterase

Pancreatic cholesterol esterase (CEase), which is secreted from the exocrine pancreas, is a serine hydrolase that aids in the bile salt-dependent hydrolysis of dietary cholesteryl esters and contributes to the hydrolysis of triglycerides and phospholipids. Additional roles for CEase in intestinal micelle formation and in transport of free cholesterol to the enterocyte have been suggested. There also are studies that point to a pathological role(s) for CEase in the circulation where CEase accumulates in atherosclerotic lesions and triggers proliferation of smooth muscle cells. Thus, there is interest in CEase as a potential drug target. 4-Chloro-3-alkoxyisocoumarins are a class of haloenol lactones that inhibit serine hydrolases and serine proteases and have the potential to be suicide inhibitors. In the present study, we have developed 3-alkoxychloroisocoumarins that are potent inhibitors of CEase. These inhibitors were designed to have a saturated cycloalkane ring incorporated into a 3-alkoxy substituent. The size of the ring as well as the length of the tether holding the ring was found to be important contributors to binding to CEase. 4-Chloro-3-(4-cyclohexylbutoxy)isocoumarin and 4-chloro-3-(3-cyclopentylpropoxy)isocoumarin were demonstrated to be potent reversible inhibitors of CEase, with dissociation constants of 11 nM and 19 nM, respectively. The kinetic results are consistent with predictions from molecular modeling.

Probing stereoselective inhibition of the acyl binding site of cholesterol esterase with four diastereomers of 2'-N-α-methylbenzylcarbamyl-1, 1'-bi-2-naphthol

BackgroundRecently there has been increased interest in pancreatic cholesterol esterase due to correlation between enzymatic activity in vivo and absorption of dietary cholesterol. Cholesterol esterase plays a role in digestive lipid absorption in the upper intestinal tract, though its role in cholesterol absorption in particular is controversial. Serine lipases, acetylcholinesterase, butyrylcholinesterase, and cholesterol esterase belong to a large family of proteins called the α/β-hydrolase fold, and they share the same catalytic machinery as serine proteases in that they have an active site serine residue which, with a histidine and an aspartic or glutamic acid, forms a catalytic triad. The aim of this work is to study the stereoselectivity of the acyl chain binding site of the enzyme for four diastereomers of an inhibitor.ResultsFour diastereomers of 2'-N-α-methylbenzylcarbamyl-1, 1'-bi-2-naphthol (1) are synthesized from the condensation of R-(+)- or S-(-)-1, 1'-bi-2-naphthanol with R-(+)- or S-(-)-α-methylbenzyl isocyanate in the presence of a catalytic amount of pyridine in CH2Cl2. The [α]25D values for (1R, αR)-1, (1R, αS)-1, (1S, αR)-1, and (1S, αS)-1 are +40, +21, -21, and -41°, respectively. All four diastereomers of inhibitors are characterized as pseudo substrate inhibitors of pancreatic cholesterol esterase. Values of the inhibition constant (Ki), the carbamylation constant (k2), and the bimolecular rate constant (ki) for these four diastereomeric inhibitors are investigated. The inhibitory potencies for these four diastereomers are in the descending order of (1R, αR)-1, (1R, αS)-1, (1S, αR)-1, and (1S, αS)-1. The k2 values for these four diastereomers are about the same. The enzyme stereoselectivity for the 1, 1'-bi-2-naphthyl moiety of the inhibitors (R > S, ca. 10 times) is the same as that for 2'-N-butylcarbamyl-1, 1'-bi-2-naphthol (2). The enzyme stereoselectivity for the α-methylbenzylcarbamyl moiety of the inhibitors is also R > S (2–3 times) due to the constraints in the acyl binding site.ConclusionWe are the first to report that the acyl chain binding site of cholesterol esterase shows stereoselectivity for the four diastereomers of 1.

The in vitro hydrolysis of phytosterol conjugates in food matrices by mammalian digestive enzymes

All fruits, vegetables, and grains contain phytosterols. Numerous clinical studies have documented that phytosterols lower LDL-cholesterol levels and thereby reduce the risk of cardiovascular disease. Most experts believe that the cholesterol-lowering mechanism of phytosterols requires that they be in their "free" form. In addition to their occurrence in the free form, phytosterols also occur as four common phytosterol conjugates: (i) fatty acyl esters, (ii) hydroxycinnamate esters, (iii) steryl glycosides, and (iv) fatty acylated steryl glycosides. This study was undertaken to investigate the extent of hydrolysis of four common phytosterol conjugates by mammalian digestive enzymes (cholesterol esterase and pancreatin, a mixture of pancreatic enzymes) and for comparison purposes, by KOH. Two types of purified hydroxycinnamate esters (sitostanyl ferulate and oryzanol, a mixture of hydroxycinnamate esters purified from rice bran oil) were hydrolyzed by cholesterol esterase and by pancreatin. Both cholesterol esterase and pancreatin hydrolyzed the phytosteryl esters in two functional food matrices, and they hydrolyzed the hydroxycinnamate esters in corn fiber oil. This is the first report to demonstrate that phytostanyl ferulate esters (which are present at levels of 3-6% in corn fiber oil) are hydrolyzed by pancreatic cholesterol esterase. It is also the first report that pancreatin contains enzymes that hydrolyze the fatty acyl moiety of fatty acylated steryl glycoside, converting it to steryl glycoside. Pancreatin had no effect on steryl glycosides. The ability of pancreatin to hydrolyze three other types of lipid conjugates was also evaluated. Phospholipids were completely hydrolyzed. About half of the galactolipids were hydrolyzed, and less than 10% of the polyamine conjugates were hydrolyzed. The extents of hydrolysis of phytosteryl esters by base (saponification) were also studied, and conditions commonly used for the saponification of acyl lipids (1.5 N methanolic KOH, 30 min at 70 degrees C), were found to result in a nearly 100% hydrolysis of TAG but only about 35-45% hydrolysis of the phytosteryl fatty acyl esters or phytosteryl hydroxycinnamate esters.

Ortho effects and cross interaction correlations for the mechanisms of cholesterol esterase inhibition by aryl carbamates

AbstractOrtho‐substituted phenyl‐N‐butyl carbamates (1–11) were synthesized to evaluate the inhibition mechanisms of porcine pancreatic cholesterol esterase. All carbamate inhibitors act as the active site‐directed pseudo substrate inhibitors of the enzymes. The logarithms of dissociation constant (Ki), carbamylation constant (k2) and bimolecular inhibition constant (ki) multiply linearly correlate with the Hammett substituent constant (σ), the Taft–Kutter–Hansch ortho steric constant (ES), and the Swan–Lupton–Hansch ortho polar constant (F). For the –log Ki, log k2 and log ki correlations, the reaction constant for ordinary polar effect (ρ), the intensity factor to ortho steric constant (δ) and the intensity factor to ortho polar constant (f) are 0.7, −0.07, and 0.5; 0.5, 0.04 and −0.5; and 1.1, −0.03 and 0.0, respectively. The cross interaction reaction constant (ρXR) for the –log ki–, log k2– and log ki‐‐σ–ασ*–ασσ* correlations are 3, −2, and 1, respectively. The Ki step may be composed of the following two steps: (1) protonation of carbamates 1–11 and (2) the pseudo‐trans to pseudo‐cis conformation change of protonated carbamates 1–11 due to a large ρXR value of 3 and formation of the enzyme‐protonated carbamates 1–11 tetrahedral intermediate. The k2 step involves departure of the leaving group, which is protonated by the active site histidine of the enzyme, from the tetrahedral intermediate to solution and formation of the carbamyl enzyme. Moreover, the distances between the carbamate and phenyl groups in all transition states of inhibition reactions are relatively short owing to large |ρXR| values. The Ki step shows little ortho steric enhancement effect; moreover, the k2 step shows little ortho steric inhibition effect. Copyright © 2004 John Wiley & Sons, Ltd.

Mechanism of differential inhibition of hepatic and pancreatic fatty acid ethyl ester synthase by inhibitors of serine-esterases: in vitro and cell culture studies

Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri- o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-( o-cresyl)-4 H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di- o-tolyl- o-(∝-hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase–esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase–esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase–esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase–esterase activities.

Mechanism of differential inhibition of hepatic and pancreatic fatty acid ethyl ester synthase by inhibitors of serine-esterases: in vitro and cell culture studies

Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri-o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di-o-tolyl-o-(∝-hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase–esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase–esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase–esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase–esterase activities.

Enzyme-catalyzed hydrolysis of ? -oryzanol

The hydrolysis of γ-oryzanol, a cholesterol-lowering and antioxidative mixture of steryl ferulates (mainly 24-methylenecycloartanyl ferulate 1, cycloartenyl ferulate 2, campesteryl ferulate 3, and β-sitosteryl ferulate 4) occurring in rice, by commercially available enzyme preparations was investigated. Lipase preparations from different sources did not accept γ-oryzanol as substrate. Cholesterol esterases, however, catalyzed a sterol-specific reaction, i.e., β-sitosterol and campesterol were liberated from 3 and 4, whereas 1 and 2 remained unhydrolyzed. The same specificity profile was observed for hydrolysis of γ-oryzanol in artificial pancreatic juice and in reactions catalyzed by pancreatic acetone powders. The data indicate that pancreatic cholesterol esterase is involved in the hydrolysis of γ-oryzanol in vivo. They also provide a perspective for tailoring the composition of steryl ferulate mixtures on a large scale.

Inhibition of pancreatic cholesterol esterase reduces cholesterol absorption in the hamster

BackgroundPancreatic cholesterol esterase has three proposed functions in the intestine: 1) to control the bioavailability of cholesterol from dietary cholesterol esters; 2) to contribute to incorporation of cholesterol into mixed micelles; and 3) to aid in transport of free cholesterol to the enterocyte. Inhibitors of cholesterol esterase are anticipated to limit the absorption of dietary cholesterol.ResultsThe selective and potent cholesterol esterase inhibitor 6-chloro-3-(1-ethyl-2-cyclohexyl)-2-pyrone (figure 1, structure 1) was administered to hamsters fed a high cholesterol diet supplemented with radiolabeled cholesterol ester. Hamsters were gavage fed 3H-labeled cholesteryl oleate along with inhibitor 1, 0–200 micromoles. Twenty-four hours later, hepatic and serum radioactive cholesterol levels were determined. The ED50 of inhibitor 1 for prevention of the uptake of labeled cholesterol derived from hydrolysis of labeled cholesteryl oleate was 100 micromoles. The toxicity of inhibitor 1 was investigated in a 30 day feeding trial. Inhibitor 1, 100 micromoles or 200 micromoles per day, was added to chow supplemented with 1% cholesterol and 0.5% cholic acid. Clinical chemistry urinalysis and tissue histopathology were obtained. No toxicity differences were noted between control and inhibitor supplemented groups.ConclusionsInhibitors of cholesterol esterase may be useful therapeutics for limiting cholesterol absorption.

Purification and characterization of rat pancreatic fatty acid ethyl ester synthase and its structural and functional relationship to pancreatic cholesterol esterase.

Formation of fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) has been implicated in the pathogenesis of chronic pancreatitis. In previous studies, we demonstrated that FAEE synthase, purified from rat liver microsomes, is identical to rat liver carboxylesterase (pI 6.1), and structurally and functionally different than that from pancreas. In this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with rat pancreatic cholesterol esterase (ChE). Since most of the serine esterases express p-nitrophenyl acetate (PNPA)-hydrolyzing activity as well as synthetic activity to form fatty acid esters or amides with a wide spectrum of alcohols and amines, respectively, we used PNPA-hydrolyzing activity to monitor the purification of FAEE synthase during various chromatographic purification steps. Synthesizing activity towards FAEEs, fatty acid methyl esters, and fatty acid anilides was measured only in the pooled fractions. At each step of purification (ammonium sulfate saturation, Q Sepharose XL, and heparin-agarose column chromatographies, and high performance liquid chromatography (anion exchange and gel filtration)) synthetic as well as hydrolytic activities copurified. Using ethanol, methanol, or aniline as substrates, the ester or anilide synthesizing activity of the purified protein was found to be 8709, 13000, and 2201 nmol/h/mg protein, respectively. The purified protein displayed a single band with an estimated molecular mass of approximately 68 kD upon SDS-PAGE under reduced denaturing conditions, cross-reacted with antisera against rat pancreatic ChE and showed 100% N-terminal sequence homology of the first 15 amino acids to that of rat pancreatic ChE. These results suggest that the purified protein has broad substrate specificity towards the conjugation of endogenous long chain fatty acids with substrates having hydroxyl and amino groups and is identical to ChE.

Pancreatic cholesterol esterase, ES-10, and fatty acid ethyl ester synthase III gene expression are increased in the pancreas and liver but not in the brain or heart with long-term ethanol feeding in rats.

Chronic alcohol consumption predisposes susceptible individuals to both acute and chronic pancreatitis. Our hypothesis was that alcohol increases the risk of pancreatitis by disrupting defense mechanisms and/or enhancing injury-associated pathways through altered gene expression. Hence, we studied the expression of pancreatic genes in rats chronically exposed to ethanol. Male Wistar rats were pair-fed liquid diets without and with ethanol for 4 weeks. Total RNA was extracted from rat pancreas and other organs. The mRNA expression patterns among pancreatic samples from ethanol-fed rats and controls were compared with use of mRNA differential display. The differentially expressed cDNA tags were isolated, cloned, and sequenced. One cDNA tag that was overexpressed in the pancreas showed 99% sequence homology to a rat pancreatic cholesterol esterase mRNA (CEL; Enzyme Commission number [EC] 3.1.1.13). The differential expression was confirmed by realtime PCR. Gene expression was also increased in the liver but not in the heart or brain of the alcohol-fed rats. Because CEL has fatty acid ethyl ester (FAEE)-generating activity and FAEEs play a major role in acute alcoholic pancreatitis, we determined the expression of other genes encoding for FAEE-generating enzymes and showed similar organ-specific expression patterns. Our results demonstrate that chronic ethanol consumption induced expression of FAEE-related genes in the pancreas and liver. This upregulation may be a central mechanism leading to acinar cell injury.

Alternate Substrate Inhibition of Cholesterol Esterase by Thieno[2,3-d][1,3]oxazin-4-ones

In a kinetic study, the interaction of bovine pancreatic cholesterol esterase (CEase) with fused 1,3-oxazin-4-ones and 1,3-thiazin-4-ones was investigated, and the compounds were characterized as alternate substrate inhibitors. Inhibition assays were performed in the presence of sodium taurocholate with p-nitrophenyl butyrate as chromogenic substrate. Strong active site-directed inhibition was detected for 2-diethylaminothieno[2,3-d][1,3]oxazin-4-ones with a cycloaliphatic chain at positions 5,6. The most potent inhibitors, compounds 3 and 4, exhibited K(i) values of 0.58 and 1.86 microm, respectively. An exchange of the ring oxygen by sulfur and the removal of the cycloaliphatic moiety as well as the replacement of the thiophene ring by benzene led to a loss of inhibitory potency. CEase has the capability to catalyze the hydrolysis of representatives of fused 1,3-oxazin-4-ones as well as the highly stable 1,3-thiazin-4-ones by using an acylation-deacylation mechanism. Hydrolyses were performed in the presence of a high enzyme concentration, and products were identified spectrophotometrically and by means of high performance liquid chromatography. The kinetic parameters V(max)I and V(max)I/K(m)(I) for the CEase-catalyzed turnover were determined. The compounds whose enzyme-catalyzed hydrolysis followed first-order kinetics (K(m)(I) > 25 microm) failed to inhibit CEase. On the other hand, inhibitors 3 (initial concentration of 25 microm) and 4 (20 microm) were hydrolyzed by CEase under steady-state conditions in the first phase of the reaction. Rate-limiting deacylation was demonstrated in nucleophilic competition experiments with ethanol as acyl acceptor wherein the conversion of compound 3 was accelerated up to an ethanol concentration of 1.5 m. The characterization of these compounds (i.e. 3 and 4) as alternate substrate inhibitors is not only based on the verification of the CEase-catalyzed hydrolysis. It also rests upon the concurrence of corresponding K(i) values obtained in the inhibition assay compared with separately determined K(m)(I) values of their enzyme-catalyzed consumption, as could be predicted from the kinetic model used in this study.

Absorption of intestinal free cholesterol is lowered by supplementation of Areca catechu L. extract in rats

Areca extracts exhibiting a strong inhibitory activity against pancreatic cholesterol esterase (pCEase) in vitro were previously found to lower the absorption of dietary cholesteryl ester. Therefore, to determine whether a combined Areca extract also affects the absorption of intestinal free cholesterol, male rats were fed a diet containing free cholesterol (1%, w/w) either with or without an Areca nut extract supplement (0.5%, w/w). The Areca extract supplement significantly lowered the plasma cholesterol concentration by 25% without any change in the plasma triglyceride concentration, when compared to the control group. The supplement also significantly lowered the small intestinal pCEase activity by 39.1% compared to that of the control group. As regards the hepatic and intestinal ACAT activities, only the intestinal enzyme activity was significantly lowered by the supplement, when compared to the control group. The absorbed cholesterol that appeared in the blood after an oral dose of [1,2(n)– 3H] free cholesterol was significantly lower in the rats supplemented with the Areca nut extract, compared with the control group. These results suggest that the inhibition of intestinal ACAT and possibly pCEase may facilitate the metabolic efficiency of the Areca nut extract as regards the absorption of intestinal free cholesterol. The structure and chemical properties of the active compound in the water-soluble Areca extract remain to be elucidated.

Supplementation of Areca catechu L. Extract Alters Triglyceride Absorption and Cholesterol Metabolism in Rats

Areca extracts have already been found to exhibit a strong inhibitory activity on cholesterol absorption in high-cholesterol-fed rats. Accordingly, this study was performed to determine whether Areca extracts also exert an inhibitory activity on triglyceride absorption in triglyceride-fed rats. Male rats were fed a diet containing corn oil (10%, w/w) with or without an Areca nut extract supplement (0.5%, w/w). The supplementation of the Areca extract significantly lowered the absorption of triglyceride and the plasma lipid concentration. The absorbed triglyceride that appeared in the blood after an oral dose of [9,10(n)-³H] triglyceride was significantly lower in the rats supplemented with the Areca nut extract, compared with the control group. The supplementation also significantly lowered the small intestinal pCEase (pancreatic cholesterol esterase) activity by 22.5% compared to the control group. The hepatic and intestinal ACAT (acyl-CoA:cholesterol acyltransferase) activities were significantly decreased in the Areca group compared with the control group. Hence, further studies are needed to elucidate the structure and chemical properties of the active compound in the water-soluble Areca extract that lowers cholesterol absorption.