Acid lipase and carboxylesterases in EBV-transformed lymphoid cell line from Wolman's disease: influence of fatty acid structure of substrate

Lymphoid cell lines established by Epstein-Barr virus transformation of blood B lymphocytes from a patient with Wolman's disease exhibited the acid lipase deficiency characteristic for this disease. Comparison of hydrolysis by normal and Wolman's cells of 4-methylumbelliferyl-acyl esters with variable chain length demonstrates that: (1) the best substrates for acid lipase were characterized by an acyl chain length of 12-18 carbon atoms; (2) the acid residual activity in Wolman's cells showed a slightly different substrate specificity and this is probably due to an acid carboxylesterase different from the lysosomal acid lipase, and (3) the 'nonspecific' carboxylesterases (at pH 6.0 and 8.0) not inhibited by taurocholate showed a characteristic substrate specificity for short-chain fatty acids. In the used assay conditions (optimal for acid lipase), methylumbelliferyl-palmitate, -elaidate and -lignocerate are the most accurate synthetic substrates for the diagnostic of Wolman's disease.     

New spectrophotometric assays of acid lipase and their use in the diagnosis of Wolman and cholesteryl ester storage diseases

Trinitrophenylaminolauric acid (TNPAL) was linked to glycerol or cholesterol and the resulting yellow compounds were used as substrates for several lipases and cholesteryl esterase in cells from normal individuals and patients with Wolman's or cholesteryl ester storage diseases. Normal cells (lymphoid cell lines or skin fibroblasts) showed two peaks of lipase or cholesteryl esterase activity at about pH 4.0 and 6.0 each. The activity of the most acidic enzyme, which hydrolyzed natural or synthetic triacylglycerols as well as cholesteryl esters, was considerably reduced in cells derived from patients with Wolman's or cholesteryl ester storage diseases. Simple spectrophotometric procedures were developed for using tri-TNPAL glycerol or TNPAL cholesterol to identify homozygotes of these two respective diseases.     

Effect of atherosclerosis on lysosomal cholesterol esterase activity in rabbit aorta.

Radiolabeled cholesteryl oleate, when incorporated into phospholipid vesicles, was hydrolyzed at acid pH by an enzyme present in rabbit aortic homogenates. In contrast, cholesteryl oleate presented as an acetone dispersion was not effectively hydrolyzed at acid pH under identical conditions. Using the vesicle preparation as substrate, a sensitive assay system for the acid hydrolase was developed in which hydrolysis was proportional to protein concentration and incubation time, and was independent of substrate concentration. The physical state of the vesicles was apparently not altered by the assay conditions, and no hydrolysis of the vesicle-associated phospholipid was detected. Acid cholesterol esterase activity in atherosclerotic aortic tissue was 2.5-fold greater than that of control tissue, and even greater increases were observed in the activities of other lysosomal enzymes (N-acetyl-beta-d-glucosaminidase and beta-glucuronidase). Glucose-6-phosphatase activity was also increased in aortas from cholesterol-fed animals while 5' nucleotidase activity remained unchanged. Labeled triolein also was incorporated into phospholipid vesicles and was hydrolyzed by an acid lipase in aortic tissue. Similarities between triolein and cholesteryl oleate hydrolysis existed with respect to pH optimum and the effect of cholesterol feeding on activity, suggesting that a single enzyme may hydrolyze both lipids.     

Inhibitors of sterol synthesis. 15-oxygenated steryl ester hydrolase activity of rat liver at neutral and acid pH

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one (15 ketosterol) is a potent inhibitor of cholesterol biosynthesis with significant hypocholesterolemic activity. The results of a recent study (Schroepfer, G.J., Jr., Christophe, A., Chu, A.J., Izumi, A., Kisic, A. and Sherrill, B.C. (1988) Chem. Phys. Lipids 48, 29-58) have indicated that, after intragastric administration of the 15-ketosterol in triolein to rats, most of the compound in intestinal lymph occurs in the form of the oleate ester, which is associated with chylomicrons. Moreover, after intravenous administration of chylomicrons containing the oleate ester of 15-[2,4-3H]ketosterol, rapid and selective uptake of 3H by liver was observed, which was associated with the rapid and substantial appearance of labeled free 15-ketosterol in liver. The present study concerns the capabilities of rat liver fractions to catalyze the hydrolysis of 15-ketosteryl oleate. Efficient hydrolysis was observed at acid pH with a digitonin-solubilized extract of rat liver, with a rate similar to that for the hydrolysis of cholesteryl oleate. The distribution of acid 15-ketosteryl oleate hydrolase of whole liver homogenate on a metrizamide isopycnic density gradient was similar to that of acid cholesteryl oleate hydrolase and acid phosphatase, suggesting that the lysosomal acid lipase is the enzyme responsible for the hydrolysis of the 15-ketosteryl oleate at acid pH. At neutral pH, 15-ketosteryl oleate and cholesteryl oleate was hydrolyzed at similar rates by the microsomal fraction of liver homogenate, whereas the 15-ketosteryl oleate was hydrolyzed at a much lower rate than cholesteryl oleate by the cytosolic fraction. The distribution of neutral 15-ketosteryl oleate hydrolase activity of whole liver homogenate on a metrizamide isopycnic density gradient was most correlated to a microsomal esterase, whereas cholesteryl oleate hydrolase activity was most correlated to a cytosolic enzyme. Both 15-ketosteryl oleate and cholesteryl oleate hydrolase activities were correlated to a mitochondrial marker enzyme.     

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells

(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types. (2) With cholesteryl[1-14C]oleate as substrate both parenchymal and non-parenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties. (3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.     

Cholesteryl ester and triglyceride hydrolysis by an acid lipase from rabbit aorta.

The properties of the triglyceride- and cholesteryl ester-hydrolyzing activity by an acid lipase from rabbit aortic tissue were compared under different experimental conditions. Radiolabeled cholesteryl oleate or triolein was incorporated into phospholipid vesicles by sonication and the resulting preparations were used for in vitro studies. No distinction was observed between triglyceride lipase and cholesterol esterase activity in the aortic cytosol fraction following either thermal inactivation, inhibition by a mercurial, fractionation by ammonium sulfate or acid precipitation, or DEAE-cellulose chromatography. Addition of rabbit lipoproteins to the assay system resulted in inhibition of both cholesterol esterase and triglyceride lipase activity. Parallel changes in the hydrolysis of both substrates also were observed when exogenously added lipids were added to the incubation system in various physical states. Specificities of the enzyme system towards different cholesteryl esters were examined. No differences in the rate of hydrolysis were observed between cholesteryl oleate, palmitate and linoleate. The data suggest that a single acid lipase, presumably of lysosomal origin, has broad specificity towards triglycerides and cholesteryl esters, and may play a role in the hydrolysis of these lipids during intralysosomal degradation of lipoproteins.     

Lysosomal acid lipase deficiency in rats: lipid analyses and lipase activities in liver and spleen

We report the biological characterization of an animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected rats accumulated cholesteryl esters (13.3-fold), free cholesterol (2.8-fold), and triglycerides (5.4-fold) in the liver, as well as cholesteryl esters (2.5-fold) and free cholesterol (1.33-fold) in the spleen. Triglycerides did not accumulate, and the levels actually decreased in the spleen. Analysis of the fatty acid composition of the cholesteryl esters and triglycerides showed high percentages of linoleic acid (18:2) and arachidonic acid (20:4) in both organs, especially in the liver. No accumulation of phospholipids, neutral glycosphingolipids, or gangliosides was found in the affected rats. Acid lipase activity for [14C]triolein, [14C]cholesteryl oleate, and 4-methyl-umbelliferyl oleate was deficient in both the liver and spleen of affected rats. Lipase activity at neutral pH was normal in both liver and spleen. Heterozygous rats showed intermediate utilization of these substrates in both organs at levels between those for affected rats and those for normal controls, although they did not accumulate any lipids. These data suggest that these rats represent an animal counterpart of Wolman's disease in humans.     

Metabolism of high density lipoprotein lipids by the rat liver: evidence for participation of hepatic lipase in the uptake of cholesteryl ester.

In order to determine the role of hepatic lipase in the hepatic uptake and metabolism of high density lipoprotein (HDL) triglycerides, cholesteryl esters, and phospholipids, isolated rat livers were perfused with a reconstituted HDL (rHDL) radiolabeled with [3H]triolein and [14C]cholesteryl oleate or palmitoyl-[14C]linoleoyl phosphatidylcholine. A bolus of radiolabeled rHDL was injected into the portal vein and livers were perfused for 5 min using a nonrecirculating perfusion system. Recovery of rHDL triolein in the liver as intact triolein was used to determine the amount of unmetabolized rHDL remaining in the liver. After correcting for the amount of unmetabolized rHDL remaining in the liver, about 30% of the rHDL triolein was hydrolyzed of which 19% was recovered in the liver and 11% in the perfusate. Moreover, about 7% of the rHDL phosphatidylcholine was hydrolyzed to lysophosphatidylcholine, all of which was recovered in the perfusate. Although there was no hydrolysis of rHDL cholesteryl oleate, about 30% of the cholesteryl oleate was taken up by the liver. Preperfusion of the liver with heparin to deplete the liver of hepatic lipase resulted in about a 70% reduction in rHDL triolein hydrolysis and about a 75% reduction in rHDL cholesteryl oleate uptake. Although hepatic lipase hydrolyzes both triglycerides and phosphatidylcholines, elimination of the triolein from rHDL had no effect on the uptake of rHDL cholesteryl oleate, but replacement of the rHDL phosphatidylcholine with a nonhydrolyzable phosphatidylcholine diether resulted in an 87% reduction in cholesteryl oleate uptake. These results indicate that hepatic lipase is necessary for the hepatic uptake of both HDL triglycerides and cholesteryl esters and that the uptake of cholesteryl esters is not dependent on the hydrolysis of HDL triglycerides but is dependent on the hydrolysis of HDL phospholipids.     

Rat liver retinyl palmitate hydrolase activity. Relationship to cholesteryl oleate and triolein hydrolase activities

Studies were conducted to explore relationships in rat liver between retinyl palmitate hydrolase activity and the hydrolytic activities against cholesteryl oleate and triolein. Previous studies have shown positive correlations between these three lipid ester hydrolase activities. In order to extend this work, the hydrolase activities were further purified and characterized. The activities against cholesteryl oleate and triolein resembled retinyl palmitate hydrolase activity in showing great variability from rat to rat as assayed in vitro. The relative levels of the three activities were highly correlated with each other over a 50-fold range of activity in a series of 66 liver homogenates. Partial purification (approx. 200-fold) in the absence of detergents was achieved by sequential chromatography of an acetone powder extract of liver on columns of phenyl-Sepharose, DEAE-Sepharose and heparin-Sepharose. The three hydrolase activities copurified during each of these chromatographic steps. The properties of the three copurifying activities were similar with regard to stimulation of activity by trihydroxy bile salts, pH optimum (near 8.0), and observance of Michaelis-Menten-type saturation kinetics. The three activities were different in their sensitivity towards the serine esterase inhibitors diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and in their solubility properties in 10 mM sodium acetate, pH 5.0. Thus, triolein hydrolase activity was much less sensitive than the other two activities to the two inhibitors. In addition, the activity against cholesteryl oleate could be separated from the other two activities by extraction of an acetone powder with acetate buffer, pH 5.0. These results indicate that the three lipid hydrolase activities are due to at least three different catalytically active centers, and at least two distinct and separable enzymes. It is likely that three separate but similar enzymes, that appear to be coordinately regulated, are involved.     

Effect of pancreatic phospholipase A2 and gastric lipase on the action of pancreatic carboxyl ester lipase against lipid substrates in vitro.

Preincubation of a triolein/phospholipid/cholesteryl oleate-emulsion in vitro with either pancreatic phospholipase A2 (PLA2) or gastric lipase (GL) resulted in hydrolysis (measured by pH-stat-titration) of cholesteryl [3H]oleate only after human pancreatic carboxyl ester lipase (CEL) was added to the system. No appreciable hydrolysis was observed when CEL was added alone. Consequently, a concerted action either of PLA2 and CEL or of GL and CEL made the substrate cholesteryl oleate available for hydrolysis by CEL. This was the case when cholesteryl oleate was solubilised in a phospholipid-stabilised triglyceride emulsion, which is the physico-chemical form in which the major part of dietary cholesteryl esters are presented to the gastro-intestinal tract of man.     

Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases

Fluorescent pyrene-methyl lauryl ester (PMLes) was synthesized and used for the determination of cellular lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or cholesteryl ester storage diseases (both exhibiting a deficiency of the lysosomal acid lipase). The hydrolysis of PMLes by acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use PMLes as a fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (i.e. pyrene-methanol) was read in the lower aqueous-ethanolic phase, at 378 nm. PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or cholesteryl ester storage diseases. This demonstrates that the fluorescent PMLes is hydrolyzed by the lysosomal acid lipase and can be used as a very sensitive fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.     

Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture

Lysosomal acid lipase was purified to near homogeneity in a yield of 25-30% from secretions of human fibroblasts grown on microcarriers in spinner culture. Ammonium chloride was added to the serum-free medium to stimulate production of extracellular enzyme and minimize modifications, including proteolytic processing and destruction of the mannose 6-phosphate recognition marker, that have been associated with packaging and maturation of acid hydrolases in lysosomes. Chromatography of secretions by decyl-agarose, hydroxylapatite, phenylboronate-agarose, and gel filtration resulted in greater than 1500-fold purification of the lipase, representing a 10,000-fold increase above the specific activity of intracellular enzyme. The apparent molecular weight of approximately 49,000, estimated for the lipase by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was similar to that determined for the native enzyme by gel filtration (Mr approximately 47,000). By contrast, a smaller molecular weight (Mr approximately 41,000) was estimated for the intracellular enzyme. The purified enzyme was susceptible to hydrolysis by endo-beta-N-acetylglucosaminidase H, which resulted in at least two new forms, reduced in apparent molecular weight by approximately 4,000-6,000. Treatment with the endoglycosidase did not alter the catalytic activity or heat stability of the acid lipase. However, the treated enzyme was no longer internalized by fibroblasts via the mannose 6-phosphate receptor and thereby had lost the capacity to correct cholesteryl ester accumulation in cultured lipase-deficient cells. Acid fatty acyl hydrolase activity for cholesteryl oleate, triolein, and methylumbelliferyl oleate co-purified. All three esters were hydrolyzed optimally at pH 4.0, but the pH profile was altered by addition of salts or albumin to the phospholipid-bile salt substrate mixtures. In a series of saturated fatty acyl esters of 4-methylumbelliferone, a derivative with an intermediate chain length (9 carbons) was the best substrate and was hydrolyzed at a rate comparable to that of the oleate ester at pH 4. The optimal pH for hydrolysis of the intermediate and shorter chain length esters was higher by about 2 pH units than that for the longer chain esters (pH approximately 4). The activity of the purified lipase was stimulated by several different proteins. The relationship of this effect to the possible requirement for a natural activator substance has not been determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and properties of human placental acid lipase

Two peaks of lysosomal acid lipase activity were purified from normal human placenta. Acid lipase I, with an estimated molecular weight of 102 500, was purified 1016-fold while acid lipase II, with an estimated molecular weight of 30 600, was purified 3031-fold. The final yields of enzyme activity for acid lipase I and II were 0.9% and 2.2% respectively. The purity of the final preparations was documented by demonstration of a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both preparations of the purified enzyme demonstrated activity towards triolein, cholesteryl oleate and the artificial substrate 4-methylumbelliferyl oleate. Examination of Km values, thermal stability, pH optima, and electrophoretic mobility revealed similar properties for the two enzyme peaks. The response of the two enzyme preparations to inhibitors was similar with both being significantly inhibited by 0.2 M NaCl, 0.2 M KCl, 5 mM HgCl2 and 5 mM p-chloromercuribenzoate. The activity of the two preparations as assayed with either triolein or cholesterol oleate was not significantly affected by the addition of bovine serum albumin. In contrast, the 4-methylumbelliferyl oleate activity of both preparations was significantly inhibitred by albumin. These findings support the hypothesis that the same enzyme or enzymes are responsible for the intralysosomal hydrolysis of triacylglycerols and cholesterol esters in human tissues.     

Separation and characterization of the acid lipase and neutral esterases from human liver.

Electrophoresis of human liver homogenates followed by reaction with 4-methylumbelliferyl palmitate reveals the presence of two major electrophoretic forms with esterase (lipase) activity toward this substrate. The two enzymes were isolated and partially purified based on their solubility differences and their relative affinities for the lectin column concanavalin A-Sepharose 4B. Lipase A was particulate with an acidic pH optimum (5.2) and could be solubilized with the non-ionic surfactant Triton X-100. Lipase B was soluble and had a more neutral pH optimum (6.3--6.6). Both forms bound to immobilized concanavalin A and could be specifically eluted. Buffers containing alpha-methylmannoside eluted lipase B, and buffers with alpha-methylmannoside and Triton X-100 eluted lipase A, giving a 22- and 257-fold purification, respectively, over whole-tissue homogenates. Cholesterol oleate, trioleoylglycerol, and 4-methylumbelliferyl palmitate were substrates for solubilized lipase A. Lipase B hydrolyzed 4-methylum-belliferyl palmitate but not trioleoylglycerol or cholesterol oleate. Lipase B was more thermolabile than lipase A, and it was selectively inhibited by diethyl-p-nitrophenyl phosphate at low concentrations. We conclude that lipase A and B are distinctly different enzymes and that they are probably not related polymorphic forms of one another.     

Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa,with its application to cleaning lipid-stained contact lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.     

Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.     

Defective plasma clearance of chylomicron-like lipid emulsions in Watanabe heritable hyperlipidemic rabbits

Human patients with familial hypercholesterolemia (FH) and Watanabe heritable hyperlipidemic rabbits (WHHL), while lacking normal receptors recognizing low-density lipoproteins (LDL), are said to have normal clearance of chylomicrons. In the present study, emulsions with a similar lipid composition to chylomicrons were injected intravenously in homozygous WHHL rabbits and normal control rabbits fed diet with low or high cholesterol. Radioactive labels tracing emulsion triolein and cholesteryl oleate were both removed rapidly from the bloodstream, with the removal rate of triolein always faster than that of cholesteryl oleate. This pattern was similar to the clearance of normal chylomicrons in rabbits or rats, and was consistent with the formation of remnant lipoproteins after hydrolysis of emulsion triolein by lipoprotein lipase, followed by hepatic uptake of the remnants. The removal of cholesteryl oleate was significantly slower in WHHL rabbits than in normal controls, suggesting that the absence of LDL receptor function led to impaired remnant clearance. Measured in post-heparin plasma, the activity of lipoprotein lipase was decreased in WHHL rabbits, but this was not associated with clear evidence of defective lipolysis of emulsion triolein. Apolipoprotein E did not appear to be deficient in WHHL rabbits. Plasma devoid of lipoproteins less than 1.006 g/ml from WHHL and normal control rabbits transferred similar amounts of apolipoprotein E to chylomicron-like emulsions after incubation. Impaired clearance of chylomicron remnants possibly contributes to the hypertriglyceridemia of WHHL rabbits and to accelerated atherogenesis when the function of LDL receptors is defective.     

Acid lipase cross-reacting material in Wolman disease and cholesterol ester storage disease

Material cross-reacting with antibodies to acid lipase was demonstrated in fibroblasts of three patients with Wolman disease and three with cholesterol ester storage disease. Quantitation of the immunologically cross-reacting material (CRM) by a single radial immunodiffusion method revealed normal levels in both mutant cell types. CRM specific activity toward triolein and cholesteryl oleate was reduced about 200-fold in the Wolman disease fibroblasts and 50- to 100-fold in the cholesterol ester storage disease cells when compared to normal.     

Enzymatic assay of cholesterol by reaction rate measurements

A dynamic method for free and total cholesterol assay based on the oxidation of cholesterol by cholesterol oxidase, and conversion of cholesteryl oleate to cholesterol by cholesterol esterase is discussed in this article. The reaction conditions for total cholesterol assay were a temperature of 310 K and pH of 7.4. For conversion of cholesteryl oleate to cholesterol, the samples were incubated with 0.6 unit/mL of cholesterol esterase and 0.02 g/mL of taurocholate. The determination of initial reaction rates in the oxidation of free cholesterol, which is directly related to the cholesterol concentration, was found to be rapid, reliable, and inexpensive. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 391-396, 1997.     

Pancreatic cholesterol esterases. 3. Kinetic characterization of cholesterol ester resynthesis by the pancreatic cholesterol esterases

The ability of cholesterol esterase to catalyze the synthesis of cholesterol esters has been considered to be of limited physiological significance because of its bile salt requirements for activity, though detailed kinetic studies have not been reported. This study was performed to determine the taurocholate, pH, and substrate requirements for optimal cholesterol ester synthesis catalyzed by various pancreatic lipolytic enzymes, including the bovine 67- and 72-kDa cholesterol esterases, human 100-kDa cholesterol esterase, and human 52-kDa triglyceride lipase. In contrast to current beliefs, cholesterol esterase exhibits a bile salt independent as well as a bile salt dependent synthetic pathway. For the bovine pancreatic 67- and 72-kDa cholesterol esterases, the bile salt independent pathway is optimal at pH 6.0-6.5 and is stimulated by micromolar concentrations of taurocholate. For the bile salt dependent synthetic reaction for the 67-kDa enzyme, increasing the taurocholate concentration from 0 to 1.0 mM results in a progressive shift in the pH optimum from pH 6.0-6.5 to pH 4.5 or lower. In contrast, cholesterol ester hydrolysis by the 67-, 72-, and 100-kDa enzymes was characterized by pH optima from 5.5 to 6.5 at all taurocholate concentrations. Optimum hydrolytic activity for these three enzyme forms occurred with 10 mM taurocholate. Since hydrolysis is minimal at low taurocholate concentrations, the rate of synthesis actually exceeds hydrolysis when the taurocholate concentration is less than 1.0 mM. The 52-kDa enzyme exhibits very low cholesterol ester synthetic and hydrolytic activities, and for this enzyme both activities are bile salt independent. Thus, our data show that cholesterol esterase has both bile salt independent and bile salt dependent cholesterol ester synthetic activities and that it may catalyze the net synthesis of cholesterol esters under physiological conditions.