Hydrolysis of synthetic triacylglycerols by pancreatic and lipoprotein lipase

The stereochemical course of the hydrolysis of synthetic sn-glycerol-1-palmitate-2-oleate-3-linoleate, sn-glycerol-1,2-dipalmitate-3-oleate and their antipodes by pancreatic and milk lipoprotein lipase was investigated by thin layer and gas liquid chromatographies of the diacylglycerol intermediates. The enzymic hydrolyses were made with bile salts or lysolecithin in a 1∶1 molar ratio to the substrate as emulsifiers and were limited to short time intervals which minimized isomerization and the reversal of lipolysis. In all instances, the products of hydrolysis by lipoprotein lipase contained a marked preponderance of the 2,3-diacylglycerols, while the composition of the diacylglycerol intermediates in the products of pancreatic lipase varied with the nature of the fatty acid in the 1 and 3 positions of the triacylglycerol molecule. Pancreatic lipase, but not lipoprotein lipase, gave a preferential release of unsaturated fatty acids. The above results are similar to those obtained with radioactive trioleoylglycerol and conventional stereospecific analyses and suggest that lipoprotein lipase may favor attack on the sn-1 position. It is hypothesized that the small amounts of the 1,2-diacylglycerols present may have arisen from a reversal of lipolysis also catalyzed by this enzyme. Presented in part at the AOCS Fall Meeting, Ottawa, September 1972.     

A colorimetric assay of pancreatic lipase: Rapid detection of lipase and colipase separated by gel filtration

A rapid assay for pancreatic lipase (E.C., glycerol-ester hydrolase 3.1.1.3) is described. The assay is based on the color change of a pH indicator as butyric acid is released from the substrate tributyrin. A mixture made with tributyrin and the water soluble components of the assay is ideally suited for use as a rapid test as, for example, when assaying chromatography fractions. Quantitative data can be obtained by measuring the disappearance of absorbance at 557 nm versus a blank reaction. The assay has been used in the rapid preparation of colipase-free lipase and colipase.     

Is intestinal villus phospholipase A2/lysophospholipase bound pancreatic carboxylester lipase?

Similarities in substrate specificity, localization and molecular weight between villus membrane phospholipase A₂/lysophospholipase and carboxylester lipase of pancreatic origin suggested their possible identity. To test this, a preparation of the phospholipase A₂/lysophospholipase released from brush border vesicles by papain was compared to authentic, pancreatic carboxylester lipase. Susceptibility of both activities to the inhibitor, diisopropylfluorophosphate, was consistent with their identity, but inconclusive. It also indicated that two populations of phospholipase A₂ species may be present in the papain-released preparation. However, comparison of binding of the activities to Sepharose-coupled, anti-carboxylester-lipase IgG indicates that they are immunologically distinct.     

Hydrolysis ofbis-phosphatidic acid from developing soybean by phospholipase A2

bis-Phosphatidic acid from developing soybean was completely hydrolyzed with phospholipase A₂ (lyophilized venom from Ophiophagus hannah) to yield lyso-bis-phosphatidic acid and free fatty acids. It is suggested that soybean bis-phosphatidic acid is derived from sn-3-glycerophospho-sn-3′-glycerol.     

Hydrolysis of used frying palm olein and sunflower oil catalyzed by porcine pancreatic lipase

The enzymatic hydrolysis of frying used vegetable oils with different degrees of alteration were measured using porcine pancreatic lipase (acylglycerol acylhydrolase EC 3.1.1.3). Successive frying of potatoes significantly increased the level of total polar lipid content in the palm olein from 9.3±0.1 mg/100 mg oil to 26.4±0.3 mg/100 mg oil after 90 fryings, and from 4.0±0.1 mg/100 mg oil to 27.7±0.3 mg/100 mg oil in sunflower oil after 60 fryings. Triacylglycerol polymers, triacylglycerol dimers, and oxidized triacylglycerols also increased 37-, 7.9-, and 7.5-times in palm olein, respectively, and 56-, 22-, and 4.7-times in sunflower oil, respectively. However, diacylglycerols and free fatty acid levels related to hydrolytic alteration did not increase with the number of fryings in both oils. The substrate concentration in the reactor was determined by calculating the molecular weight of each oil showing a different degree of alteration. We compared the methodology used by us and that used by other authors. The results show that the methods are reproducible and that the values obtained are in concordance with theoretical values. The kinetic parameters apparent Michaelis-Menten constant (K _M ^app ) and apparent maximum velocity of hydrolysis (V _max ^app ) were different in unused palm olein (5.1±0.7 and 166±7.6, respectively) than in sunflower oil (2.2±0.3 and 62±2.2, respectively). However, changes inK _M ^app andV _max ^app were not related to the degree of alteration of the oils.     

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 μmol/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.     

Enzymatic acyl modification of phosphatidylcholine using immobilized lipase and phospholipase A2

Ethanol‐soluble (ES) lecithin mainly contains phosphatidylcholine (PC). The incorporation of caprylic acid into PC using immobilized phospholipase A₂ (PLA₂) and lipase was investigated. The Rhizomucor meihei lipase and the porcine pancreatic PLA₂ were immobilized on the hydrophobic resin Diaion HP‐20 and the modification was carried out in hexane as solvent. HPTLC with densitometer technique was successfully used for monitoring the production of structured phospholipids (PL) (ML‐type PC, MM‐type PC, and lysophosphatidylcholine; L: long‐chain fatty acid, M: medium‐chain fatty acid). The various parameters such as the effects of reaction temperature, enzyme loading, and the effect of molar proportion of substrate were studied in order to determine the optimum reaction conditions for the acidolysis reaction. The optimal operating conditions for the PLA₂‐catalyzed reaction were obtained as 50°C temperature, 50% (wt/wt of substrate) enzyme loading, and a 1:12 molar proportion of PC/caprylic acid. For the lipase‐catalyzed reaction, the optimized temperature was the same as for PLA₂, but the enzyme loading and molar proportion were slightly lower, i.e., 40 % w/w of substrate and 1:9 PC/caprylic acid, respectively. The effects of these parameters on the production of structured PL were compared. Under these optimal conditions, the ML‐type PC content was higher in the PLA₂‐catalyzed reaction, i.e., 45.29 mol%, and in the lipase‐catalyzed reaction it was 38.74 mol%.     

Transesterification of soy lecithin by lipase and phospholipase

Soy lecithin was modified by enzymatic transesterification in a solvent-free system. 1,3-SpecificRhizomucor miehei lipase was found to be efficient in the transesterification with lauric acid and oleic acid, where oleic acid was more incorporated into soy lecithin. Phospholipase A₂ incorporated lauric acid hardly at all, but it hydrolyzed lecithin efficiently. The mixture of lipase and phospholipase A₂ (1:1, w/w) incorporated lauric acid to the same extent as did 1,3-specific lipase alone at the same total enzyme concentration. The main fatty acids replaced were palmitic and linoleic acids by 1,3-specific lipase and its mixture with phospholipase A₂, and linoleic and linolenic acids by phospholipase A₂ alone, suggesting an improved oxidative stability of the resulting product. Hydrolysis could not be prevented, but it could be regulated by incubation time and by enzyme dosage. The minimal water content for significant incorporation of lauric acid into lecithin was below 0.5% of the weight of the reaction mixture.     

溶胶-凝胶法制备二氧化钛溶胶

采用溶胶-凝胶法,以钛酸四丁酯为主要原料,制备出澄清、透明、均匀稳定的二氧化钛溶胶。讨论了水的用量、乙醇用量、p H值、温度等的影响,确定了n(HAC):(TEOT)=0.6;溶液的p H值调节至2~3时,所成溶胶较稳定均匀;乙醇与钛醇盐的物质的量比取9;水解温度选取30℃左右。制得样品在400℃条件下煅烧的比表面积为126.5m2/g。     

Structural modifications of rat serum high density lipoprotein by pancreatic phospholipase A2

An important and unusual aspect of the high density lipoprotein (HDL) in the rat is its tendency to undergo marked alterations in structure in response to physiological perturbations. In this study, the role of the surface lipids for maintenance of HDL integrity were investigated. Hydrolysis by pancreatic phospholipase A₂ of the phospholipids of rat HDL in the presence of the d>1.21 g/ml fraction of rat serum results in an increase in the particle diameter and an uptake of apo-E and apo A-IV from the lipoprotein-free fraction; augmentation of the albumin concentration in the incubation mixture intensified the observed changes, probably due to enhancement of the compositional changes brought about by phospholipase treatment. Phospholipase A₂ treatment of the d<1.21 g/ml fraction of rat serum produces only minor changes in the properties of the isolated HDL. These data suggest that changes in apoprotein content reflect an uptake of A-IV and E by the rat HDL, rather than a net loss of apo A-I. Likewise, titration of the action of pancreatic phospholipase A₂ on HDL apoprotein composition showed that initially a modest increase in apo A-IV content occurred, but with more extensive phospholipolysis there was a considerably greater increase in the apo-E content of the particle. The data suggest that hydrolysis of phospholipids such as occurs physiologically through the action of lecithin: cholesterol acyl transferase and hepatic lipase may alter the HDL structure independently from changes effected in the neutral lipid core.     

Hydrolysis of lecithin by phospholipase A2 in mixed reversed micelles of lecithin and sodium dioctyl sulphosuccinate

The hydrolysis of egg yolk lecithin was studied as catalysed by porcine pancreatic phospholipase A₂ (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) entrapped in reversed micelles of lecithin and sodium dioctyl sulphosuccinate (AOT) in isooctane. The influence of relevant parameters such as temperature, pH, water content, and buffer, AOT, lecithin, calcium and enzyme concentrations was investigated. The order in which the reactants were mixed showed a strong influence on catalytic activity. Higher activities were obtained if the enzyme, water and calcium were first solubilized and pre-equilibrated in plain AOT reversed micelles before the addition of the substrate. Maximum activity was obtained at 750 mmol dm^?3 calcium in the water pool of the micelles, which is an extremely high concentration when compared with the values reported in the literature. This was related to the formation of divalent chelates of calcium with AOT that compete with the binding of the co-factor to the enzyme. Accordingly, the optimum concentration of calcium in the inner core of the micelles decreased from 750 to 500 mmol dm^?3 when the AOT concentration was lowered from 15 to 10 mmol dm^?3.     

Purification of plasmalogens usingRhizopus delemar lipase andNaja naja naja phospholipase A2

Bovine heart ChoGpl (choline glycerophospholipid) and bovine brain EtnGpl (ethanolamine glycerophospholipid) contain diacyl, alkenylacyl and alkylacyl analogs. Purification of plasmalogens was achieved usingR. delemar lipase andN. naja naja phospholipase A₂ digestion. TheR. delemar lipase hydrolyzes the acyl bond at the 1-position of 1,2-diacyl glycerophospholipids. TheN. naja naja phospholipase A₂ has greater activity with diacyl and alkylacyl than with alkenylacyl glycerophospholipids. These enzymes were mainly used to remove diacyl and alkylacyl analogs respectively. When the diacyl types were removed by double incubation withR. delemar lipase, the plasmalogen content was 94.2%±0.21% (mean±S.E.M., n=4) for PlsCho (plasmenylcholine) and 94.9%±0.19% (mean±S.E.M., n=3) for PlsEtn (plasmenylethanolamine). Recoveries were 74% and 88% respectively. These partially purified plasmalogens were treated withN. naja naja phospholipase A₂. Finally, 97.7%±0.24% (mean±S.E.M., n=4) and 98.8%±0.27% (mean±S.E.M., n=3) pure plasmalogens were obtained for PlsCho and PlsEtn respectively. Plasmalogens were recovered in an overall yield of 7.7%±0.7% (mean±S.E.M., n=4) and 10.2%±1.2% (mean±S.E.M., n=3) for PlsCho and PlsEtn.     

Lipolysis of trivernolin by pancreatic lipase

Vernolic acid (cis-12,13-epoxy-cis-9-oetadece-noic acid) occurs as the triglyceride in the seed ofVernonia anthelmintica. Incubation of the seed produces a 1,3-divernolin. To determine whether the structure of trivernolin is responsible for the apparent secondary ester position specificity of the natural enzyme, trivernolin and tri-olein, were incubated with pancreatic lipase and the free fatty acids and monoglycerides were determined after 5 and 15 min digestion periods. The preponderance of 2-monoglyceride produced by the action of pancreatic lipase was interpreted to indicate that the structure of trivernolin was not solely responsible for the secondary position specificity of theV. anthelmintica lipase toward trivernolin.     

Hydrolysis of a fluorescent substance formed from an oxidized phospholipid and an amino compound by phospholipase A2

Phosphatidylcholine hydroperoxide produced a fluorescent substance (FS-III) through reaction with 1-aminopentane after preincubation with heme methyl ester as a model system. The FS-III was retained at the 2-position of the glycerol backbone of phosphtidylcholine without breakdown into low molecular weight compounds. Phosphatidylcholine oxidized by catalysis with ferrous ion and ascorbic acid also produced the same fluorescent substance (FS-III). Phospholipase A₂ specifically hydrolyzed the FS-III attached to the phospholipid, making it possible to elute the same fluorescent substance (FS-II) as that obtained from oxidized methyl linoleate. The release of FS-II by hydrolysis of FS-III attached to phospholipid increased with greater phospholipase A₂ activity. It is suggested that, with aging, the accumulation of fluorescent lipofuscin pigments in biomembranes may be related to changes in the peroxidized phospholipid content and that phospholipase A₂ may play a role in decreasing the formation and accumulation of fluorescent phospholipids in biomembranes.     

Hydrolysis of neutral lipid substrates by rat hepatic lipase

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). V_max values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The V_max values obtained were 144 μmol/min/mg for 1-oleoyl-sn-glycerol, 163 μmol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 μmol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 μmol/min/mg for PA, 50 μmol/min/mg for PE and 4 μmol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at thesn-1 andsn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereo-selective with respect to 1,3-diacyl-sn-glycerol substrates. To determine if the presence of one lipid could affect the hydrolysis of the other, all possible dual combinations of 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol, in the same micelle were made and the hydrolysis rate of each substrate was determined. Interaction occurred only for the 1,2-dioleoyl-sn-glycerol/1,3-dioleoyl-sn-glycerol mixture where the hydrolysis of 1,2-dioleoyl-sn-glycerol was slightly inhibited and that of 1,3-dioleoyl-sn-glycerol slightly activated compared to the predicted theoretical rate. These findings demonstrate that when presented in similar physical states, the neutral lipid substrates tested were hydrolyzed at a higher rate by hepatic lipase than the phospholipid substrates.     

Hydrolysis of soybean oil by a combined lipase system

The hydrolysis of soybean oil by different lipases was compared, and the results demonstrated that lipases D, N and G hydrolyzed the oil to 44, 42 and 7.2%, respectively, after 10 hr reaction, which represents incomplete hydrolysis. But the combined enzyme systems (lipases (G+N and lipases G+D) hydrolyzed the oil to an extent of 95–98% after 10 hr reaction. Plotting the percentage hydrolysis of soybean oil by combined enzyme systems against the logarithm of the reaction time resulted in essentially straight lines.     

Activation of the phospholipase A1 activity of lipoprotein lipase by apoprotein C-II

The effect of apo very low density lipoprotein (apo VLDL) and apoprotein C-II on the phospholipase A₁ activity associated with lipoprotein lipase (E.C.3.1.1.3) was studied using purified bovine milk lipoprotein lipase. The enzyme degraded¹⁴C phosphatidylcholine (PC) to¹⁴C 2-acyl lysophosphatidylcholine at a rate of 0.28±0.01 nmol/min/ml and triolein at a rate of 20.3±0.4 nmol/min/ml in mixed emulsions of PC and triolein. The phospholipase activity and triacylglycerol lipase activity were both increased by the addition of apo VLDL and apoprotein C-II. After maximal activation, the rate of PC degradation was 1.19±0.02 nmol/min/ml and triolein degradation 64.4±0.4 nmol/min/ml. Activation of phospholipase A₁ activity and triacylglycerol lipase activity occurred in parallel.     

Digestion of butyrate glycerides by pancreatic lipase

The racemic triglycerides, glyceryl-1-palmitate-2,3-dibutyrate (PBB), glyceryl-1-butyrate-2,3-dipalmitate (PPB), glyceryl-2-butyrate-1,3-dipalmitate (PBP), and the diglyceride, racemic glyceryl-1-palmitate-3-butyrate (P-B) were synthesized and digested with pancreatic lipase. Each triglyceride was mixed with equimolar amounts of triolein (OOO) prior to incubation. The following order of digestion rates was observed: PBB>PPB>PBP>P-B. There was no evidence for short-chain fatty acid specificity; however the triglycerides containing butyric acid were hydrolyzed more rapidly than OOO. Based upon the fatty acid composition of partial glycerides, digestion of butyrate glycerides was not a simple phenomenon. For example, in the digestion of PBB, butyric acid accumulated faster than palmitic acid in the diglycerides, and monobutyrin was found to accumulate when the diglyceride, P-B, was digested. As evidenced by the fatty acid composition of the monoglycerides, positional specificity of pancreatic lipase was always maintained. Scientific contribution No. 245, Agricultural Experiment Station, University of Connecticut, Storrs. Presented in part at the AOCS Meeting, Philadelphia, October 1967.     

Hydrolysis of copolyesters containing aromatic and aliphatic ester blocks by lipase

Copolyesters (CPEs) prepared by the transesterification reaction between aromatic and aliphatic polyesters were hydrolyzed by Rhizopus delemar lipase. The susceptibility of CPEs to hydrolysis by this lipase dropped off rapidly during the initial stage of the transesterification reaction and increased gradually as the reaction proceeded. The susceptibility to hydrolysis decreased with increase in aromatic polyester content. It was concluded that the rigidity of the aromatic ring in the CPE chains strongly influenced their susceptibility to hydrolysis by this lipase.