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Inhibitory effect of lysophosphatidylcholine on pancreatic lipase-mediated hydrolysis in lipid emulsion
Biochimica et Biophysica Acta 1684 (2004) 1 – 7 www.bba-direct.com
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Inhibitory effect of lysophosphatidylcholine on pancreatic lipase-mediated hydrolysis in lipid emulsion Wakako Tsuzuki a,*, Akemi Ue a, Akihiko Nagao a, Miyuki Endo b, Masahiko Abe b b
a National Food Research Institute, Kannondai, 2-1-12, Tsukuba, Ibaraki 305-8642, Japan Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
Received 29 August 2003; received in revised form 25 May 2004; accepted 25 May 2004 Available online 17 June 2004
Abstract In the lipid metabolism pathway, dietary lipid emulsified with bile salts and phospholipids is mainly digested by pancreatic lipase into free fatty acids and monoacylglycerols. In order to study substrate recognition mechanism of a pancreatic lipase, we investigated its catalytic property toward the lipid emulsion prepared with long- or intermediate-chain acylglycerols and several physiological surfactants. When lysophosphatidylcholine (LysoPC), rather than bile salts or phospholipid, was incorporated into the lipid emulsion, it caused an increase in the Km(app) and a decrease in the Vmax(app) values in the interactions between the lipase and triacylglycerol (triolein or tricaprin). This indicated that LysoPC inhibited hydrolysis by decreasing both the substrate affinities and the catalytic activity of this lipase. Interestingly, further addition of taurodeoxycholic acid sodium salts or phospholipid completely restored the inhibitory effect of LysoPC on hydrolysis by lipase. On the other hand, the change in these kinetic values between the lipase and two 1-monoacylglycerols (1-monocaprin and 1-monoolein) were not particularly large when LysoPC was added. Particle size analysis of the lipid emulsion composed of LysoPC and triacylglycerols showed that most of the particles were less than 200 nm in size, which was smaller than the particle size in the triacylglycerol emulsions containing bile salts or phospholipid. The composition of the emulsion would affect its surface characteristics and thus contribute to changing lipase activity. D 2004 Elsevier B.V. All rights reserved. Keywords: Pancreatic lipase; Hydrolysis; Lysophsophatidylcholine; Inhibition
1. Introduction In the metabolism of dietary lipids, pancreatic lipase, along with bile salt conjugates, plays the most important role: 50– 70% of oral-mediated fat is hydrolyzed by this enzyme to produce monoacylglycerols and free fatty acids for absorption in the duodenum [1]. Because pancreatic lipase is one of the surface-active enzymes, it attacks lipid molecules at the interface of the emulsion particles (the oil – water interface) [2,3]. In general, the enzymatic process of the lipolytic enzyme is comprised of two dependent steps: binding to the emulsion, and then catalysis of the substrate [4]. The latter step is common to other enzymes. The former step is specific to lipolytic enzymes and is a rate-limiting step through the entire catalytic process [5]. Thus, the
* Corresponding author. Tel.: +81-29-838-8039; fax: +81-29-838-7996. E-mail address: [email protected] (W. Tsuzuki). 1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2004.05.002
emulsification of lipids is of great importance as an essential lipolytic reaction by these enzymes. In other words, the kinetics of the lipolytic enzyme is a function of the surface characteristics of the emulsion offered to the enzyme [6,7]. In the digestion pathway of lipids in vivo, the major constituents of the emulsion offered to a pancreatic lipase are dietary lipids, bile salts and phosphatidylcholine. Thus the lipase activity is mainly modulated by these two physiological surfactants. Therefore, previous studies have investigated the catalytic behaviors of lipase at the interface of the lipid emulsion [3,4,6 –9]. These works have led to general agreement that the surface composition, the lateral lipid distribution, and the physicochemical nature of its interface significantly influence the lipid digestion by lipase. For example, lipase activity was induced by a small increase in the substrate mole fraction on the surface of the emulsion [3]. In another study, the lag time duration was directly related to the phospholipid content of the interface of the emulsion [8]. Several researchers have approached the
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problem of the interfacial quality using various detergents [4,9]. However, the substrate recognition mechanism of this enzyme has not been characterized in detail. In particular, the mechanism of the inhibitory effect of lipid emulsion on lipase hydrolysis remains poorly understood. By exploiting the fact that the catalytic site of pancreatic lipase is composed of the same amino acids as the catalytic sites of serine proteases, researchers have succeeded in identifying several inhibitors of pancreatic lipase. Most of these inhibitors covalently bond to the serine residue located at the catalytic site in the lipase, thereby reducing enzyme activity. In particular, the mechanisms of the inhibition of lipase activity by tetrahydrolipstatin (THL) have been well characterized [10 –12]. Recently, Saito et al. [13,14] demonstrated that a lipid emulsion containing sphingomyelin inhibited lipoprotein lipase activity. They provided conclusive evidence that the inhibition by sphingomyelin mainly occurred via changes in the emulsion surface structure, rather than by a direct interaction between sphingomyelin and the active site of the enzyme. Further, they demonstrated that the specific interaction between sphingomyelin and substrates in the emulsion would inactivate the catalysis by the lipoprotein lipase. Although pancreatic lipase, like lipoprotein lipase, is a surface-active enzyme, researchers have not yet identified a lipid emulsion that has an inhibitory effect on this enzyme. One reason for this is that it is very difficult to measure the lipase activity toward the long-chain acylglycerols. The lipase assay is usually conducted using short-chain acylglycerols such as triacetin and tributylin as substrates. However, long-chain acylglycerols should also be considered in the kinetic study of pancreatic lipase in order to estimate the digestive behaviors of dietary lipid. We previously developed an assay method with high sensitivity for detecting lipase hydrolysis of long and intermediate-chain acylglycerols [15]. In the present study, we use this assay to investigate the influence of lipid emulsions prepared with long- and intermediate-chain acylglycerols and physiological surfactants on lipase hydrolytic activity. We presumed that the physiological nature of the lipid emulsion would play a critical role in the lipid metabolism, and particularly in pancreatic lipase-mediated hydrolysis. The purpose of this study was to identify the factors regulating the lipase activity of the lipid emulsion, and to obtain information on the role of physiological surfactants in the metabolism of dietary lipids.
corresponding to colipase. Colipase, essentially salt-free lyophilized powder, was obtained from Sigma. Taurodeoxycholic acid sodium salt (Tau), phosphocholine, 1oleoyl-2-palmitoyl-sn-glycerol-3-(PC) and L-a-monopalmitoyl lecithin (LysoPC) were obtained from Sigma. Several acylglycerols as substrates for hydrolysis by the lipase were purchased from Funakoshi Co. (Tokyo, Japan). Chloroform and methanol of analytical grades for extracting enzymatic reactive products from an aqueous solution were obtained from Wako Pure Chemicals (Tokyo, Japan). All other chemicals of analytical grade were purchased from Wako Pure Chemicals or Kanto Chemicals (Tokyo, Japan). For fluorescent labeling, reagents of fatty acid, 9-bromomethylacridine (9BMA) and tetraethylammonium carbonate (TEAC) were prepared according to the method described previously [16]. 2.2. Preparation of enzymatic reactive solution For routine lipase assay, the lipid emulsion was prepared according to the previously described methods, with slight modifications [17]. An appropriate volume of stock substrate solution of triacylglycerol or monoacylglycerol was transferred to a glass vial, and the organic solvent was removed under a steam of nitrogen. The residue was dissolved in 10 mM HEPES buffer (pH 7.0, about 500 Al, 37 jC) with or without Tau or LysoPC and repeatedly sonicated using a micro-equipped Astrason ultrasonic processor W-380 (Heat-System Ultrasonics Inc. Farmingdale, NY) until achieving a predefined particle size distribution. The weight-averaged particle size of each emulsion was determined from quasielastic light scattering measurements (NICOMP model 380 ZLS; Particle Sizing System Co. (Santa Barbara, CA). A 195 Al volume of the lipid emulsion solution was transferred to a small reactive vial. The final concentrations of each component in the reaction medium were as follows: 0.2 mM substrate, 0 –10 mM Tau, and 0– 10 mM LysoPC. Because the commercially available porcine pancreas lipase did not contain proteins corresponding to colipase, the latter was added to the lipase solution (lipase, 0.1 mg/ml; colipase, 0.16 mg/ml). The mole ratio between lipase and colipase was within physiological range at 1:5 and it kept content throughout all experiments. The reaction was started by the addition of the pancreatic lipase/ colipase solution (5 Al). The mixture (200 Al) was incubated at 37 jC for 30 min with constant stirring (150 rpm). Hydrolysis was terminated by adding 400 Al of chloroform to the reaction solution and mixing vigorously.
2. Materials and methods 2.3. Measurement of lipase activity 2.1. Materials Highly purified lipase from porcine pancreas (Type VI-S) was purchased from Sigma Chemicals Co. (St. Louis, MO) and used without further purifications. Electrophoresis of this lipase fraction indicated that it contained no proteins
Free fatty acids released by the lipase were extracted by the method of Folch et al. [18]. Briefly, following addition of chloroform (400 Al), methanol (200 Al) was added to the enzymatic solution, which was then mixed vigorously for 5 min. After centrifugation at 15,000 rpm for 10 min, the
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chloroform layer was isolated. After evaporating chloroform, the pellet was dissolved in a mixture of 200 Al (5 mM) 9BMA in dimethyl sulfoxide (DMSO) and 200 Al (2.5 mM) TEAC in DMSO. The TEAC solution contained myristic acid (0.1 mM) as an internal standard fatty acid. The mixture was then allowed to stand for more than 40 min for fluorescent labeling. The quantitative determination of free fatty acid by HPLC was conducted according to a previously reported method [15]. The HPLC peak area of 9BMA labeled fatty acid produced during the lipase assay was converted into units of Amol of fatty acid generated per mg of protein by using the peak of an internal standard myristic acid. Most experiments were repeated six times, and the independent data were statistically analyzed to confirm the reliability of the results. In addition, the amounts of free fatty acids released during the hydrolysis were measured using an enzymatic colorimetric reagent, NEFA-C (Wako Pure Chemicals). Although several lipases showed their activity even in chloroform, the pancreatic lipase used in this study lost its activity completely by addition of chloroform. No increase of fatty acid released by lipase was detected after chloroform was added to the enzymatic reactive solution. The efficiency of the extraction of the fatty acid from an aqueous solution into chloroform phase was determined as follows. Instead of the substrates for hydrolysis, free capric acid or free oleic acid at a known concentration was dispersed in the enzymatic solution and they were extracted by the method of Folch. The extraction efficiencies of the fatty acids from Tau or LysoPC solution decreased slightly depending on the concentrations of the coexisting surfactants. 95 F 5%, 90 F 6% and 87 F 5% of the original free capric acid was extracted to the chloroform layer from buffer (pH 7.0), LysoPC (1 mM) and Tau (10 mM) solutions, respectively. The calculated extraction efficiencies were used for determining the types and amounts of fatty acids extracted from the enzymatic reaction solutions. We also investigated the fluorescent-labeling efficiency with 9BMA. Myristic acid contained in TEAC solution,
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which was used as an internal standard, was a good indicator of the labeling efficiency of free fatty acids produced by the lipase reactions. Monitoring of the labeling efficiency of myristic acid revealed that Tau or LysoPC coexisting in the enzymatic solution did not affect the labeling efficiency of any fatty acids. To confirm the results of the lipase hydrolysis evaluation, the quantitative measurements of fatty acid by fluorescentHPLC were followed by measurements using the NEFA-C method. Although the NEFA-C method has been widely used for estimating enzymatic activities, some modifications were needed before using NEFA-C to measure lipase activity under our experimental condition. Since the NEFA-C method uses successive enzymatic processes to color the free fatty acids, the Tau and LysoPC contained in the lipase reactive solution used here affected the NEFA-C coloring process. This caused an excessive overestimation of the amounts of fatty acids in the Tau or LysoPC solutions. However, the corrected NEFA-C results were coincident with those calculated from the fluorescence-HPLC method.
3. Results and discussions In vivo, lysophosphatidylcholine (LysoPC) is produced from phosphatidylcholine (PC) by pancreas phospholipase A2 and plays an important role in the absorption of lipid digestives into intestinal cells. In general, PC but not LysoPC mainly interacts with a dietary lipid in its digestive process. This is one of the reasons why LysoPC has not been considered in lipolysis. In this study, LysoPC was first introduced to the lipid hydrolysis system in order to investigate the effect of LysoPC in the lipid emulsion on lipase accessibility. As the substrates for hydrolysis by lipase, four acylglycerols were used. The hydrolysis activity of the lipase toward both triacylglycerols (tricaprin and triolein) was drastically decreased by the addition of LysoPC, as shown in Fig. 1. LysoPC inhibited triolein more strongly than tricaprin. On the other hand, the inhibitory effect of
Fig. 1. Influence of LysoPC on the hydrolysis of tricaprin (open circles in panel A), 1-monocaprin (closed circles in panel A), triolein (open squares in panel B) and 1-monoolein (closed squares in panel B) by pancreatic lipase.
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LysoPC on the hydrolysis by lipase using monoacylglycerols (1-monocaprin and 1-monoolein) as substrates was small (Fig. 1). To identify the interaction of LysoPC with substrates or with enzyme, the particle size distribution of the enzymatic solution was investigated. Without LysoPC, the particle size of tricaprin dispersed in the solution was mainly distributed in the range between 140 and 800 – 2000 nm (data not shown). When LysoPC was added, the distribution of the particle size of the tricaprin solution changed to a range of 30 –220 nm, with most of the particles being < 200 nm in diameter (Fig. 3C). The difference in particle size distribution between the trial with and that without LysoPC indicated that tricaprin was incorporated into LysoPC. In the case of 1monocaprin, no particles could be detected with or without LysoPC in the examined concentration range. Because of the strong hydrophobicity, triacylglycerols would be subject to interaction with LysoPC, and would form emulsions, whose interface would hinder the enzyme molecules from approaching. In contrast, the hydrophobicity of monoacylglycerols was not strong enough to form any micelles or emulsion with LysoPC. These results demonstrated that LysoPC has an inhibitory effect on lipase-mediated hydrolysis by way of the formation of lipid emulsion. The effect of colipase on inhibition of the lipase by LysoPC was investigated using a lipase solution containing no colipase. It revealed that no differences were detected in the inhibitory effect of LysoPC on lipase activity with or without colipase in the enzymatic solution. This finding suggested that inhibition by LysoPC would occur even without the participation of colipase in the enzyme (data not shown). It is considered that LysoPC could regulate lipolysis by several mechanisms: inhibition of lipase in binding lipid emulsion, inhibition at the emulsion surface, or a combination of the above. To investigate the inhibition mechanism by LysoPC, a kinetics was analyzed and it revealed that LysoPC decreased the apparent maximum velocity (Vmax(app)) and increased the apparent Michaelis – Menten constant (Km(app)) (Table 1). The results indicated that LysoPC Table 1 Apparent kinetic parameters of hydrolysis by lipase toward various lipid emulsions Substrate
Lyso (mM)
Tau (mM)
Kmapp (mM)
Vmaxapp (Amol/min mg)
Triolein
0 0.2 0 0.2 0 0.2 0 0.2 0 0.2 0 0.2
0 0 2 2 0 0 5 5 0 0 0 0
0.36 F 0.07 1.25 F 0.10 0.28 F 0.06 0.29 F 0.04 0.47 F 0.05 1.53 F 0.13 0.35 F 0.07 0.34 F 0.06 0.86 F 0.07 1.73 F 0.18 1.17 F 0.13 2.35 F 0.28
1.88 F 0.16 0.22 F 0.05 1.90 F 0.17 1.87 F 0.18 2.18 F 0.18 0.18 F 0.04 2.21 F 0.23 2.19 F 0.21 1.02 F 0.09 0.87 F 0.10 1.25 F 0.14 1.02 F 0.15
Tricaprin
1-Monoolein 1-Monocaprin
Fig. 2. Influence of Tau on hydrolysis of the lipid emulsion composed of LysoPC and tricaprin (open squares) and of LysoPC and triolein (open circles).
decreased both the catalytic activity of the pancreatic lipase and its affinity for the substrates, thereby reducing the lipolysis rates of two triacylglycerols. Next, the effect of Tau on the inhibition of hydrolysis by LysoPC was investigated. In the presence of 0.2 mM LysoPC, the hydrolytic activities toward two triacylglycerols were predominantly depressed, as shown in Fig. 1. When Tau was added to such solutions, the lipase activity was restored in relation to an increase of Tau concentration (Fig. 2). Finally, the depressed hydrolyses of tricaprin and triolein by LysoPC were completely recovered at Tau concentrations of 2 and 5 mM, respectively. When PC was added to the LysoPC solution, the hydrolyses of tricaprin and triolein were restored at PC concentrations of 0.75 and 2.5 mM, respectively (data not shown). These results suggested that the inhibition by LysoPC was reversible and that Tau and PC were able to restore the depressed activity. The rates of lipase-mediated hydrolysis of triacylglycerols in the presence of both LysoPC and Tau were close to those in only Tau solution. The apparent kinetic parameters, Km(app) and Vmax(app), of two triacylglycerols in LysoPC (0.2 mM) and Tau (5 mM) solution were almost the same as those in Tau solution (5 mM) (Table 1). These results can be explained as follows. In respect to the hydrolysis by lipase, triacylglycerol emulsion in both Tau and LysoPC solutions is similar to that composed of triacylglycerols and Tau rather than that composed of triacylglycerols and LysoPC. To confirm the change of triacylglycerol-containing emulsions in various solutions, the particle sizes of the emulsions were compared. The particle size distribution of each enzymatic reactive solution was measured before lipase solution was added, as described in Materials and methods. In the case of the emulsion of tricaprin and surfactants, the representative distribution pattern of the
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Fig. 3. Weight-mean emulsion droplet size distributions of the lipid emulsion prepared with 0.2 mM tricaprin and 2 mM Tau (A), those prepared with 0.2 mM tricaprin and 0.8 mM PC (B), those prepared with 0.2 mM tricaprin and 0.2 mM LysoPC (C), and those prepared with 0.2 mM tricaprin, 0.2 mM LysoPC and 2 mM Tau (D).
emulsion was complicated, as shown in Fig. 3. Comparing with the emulsion of tricaprin and Tau (Fig. 3A) or PC (Fig. 3B), the emulsion composed of tricaprin and LysoPC distributed in a rather small range (40 – 200 nm), and no emulsion was detected in the particle size around 1 Am (Fig. 3C). Further addition of Tau to the emulsion of LysoPC and tricaprin induced the appearance of particles in the range of 1 Am in diameter, and the typical small particles of LysoPC and tricaprin disappeared (Fig. 3D). The distribution of tricaprin emulsion in both LysoPC and Tau solution was similar to that in Tau solution rather than to that in LysoPC solution. Changes in the distribution of particle size in various solutions indicated the diversity in the constituents of the emulsion. In particular, the small particles of tricaprin and LysoPC would be reconstructed by further addition of Tau. Finally, the inhibitory effect of LysoPC on lipase hydrolytic activity was examined using other microbial lipases. Tricaprin was hydrolyzed by lipase originating from Aspergillus oryzae, Mucor miehei, Candida cylindracea and Pseudomonas fluorescens in various concentrations of LysoPC (0– 0.2 mM). As shown in Fig. 4, the activities of all of the examined microbial lipases were diminished according to the LysoPC concentration, although a small difference was found in the degree of the inhibitory effect of
LysoPC on hydrolysis by these four lipases. The substrate specificity and hydrolysis properties of these lipases are clearly different from each other [20 – 23]. But the inhibitory effect of LysoPC on hydrolysis by these enzymes occurred
Fig. 4. Influence of LysoPC on hydrolysis of tricaprin by A. oryzae (open circles), M. miehei (open squares), C. cylindracea (open triangles) and P. fluorescens (closed circles).
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in the same manner as that shown in Fig. 4. This strongly suggests that the inhibitory effect of LysoPC is caused by the formation of an inactive complex of LysoPC and substrate rather than by specific interaction between LysoPC and the active site of each lipase. This suggested that inhibition by LysoPC was not unique to a pancreatic lipase but was general to other lipases from microorganisms. In previous works, the particle sizes of emulsions containing bile salts, phospholipids, and dietary lipids were measured [9,19,23 –25]. The particle size distribution of the emulsion prepared in our experiments was not contradictory to those reported previously. However, no information is available about the relation between the particle size of the emulsion and the inhibition of hydrolysis by lipase. This study demonstrated that the change in the particle size of the emulsion is dependent on the variety of the emulsion’s components. Composition of the emulsion would be one of the factors affecting the surface nature of the emulsion and would result in the change of lipase activity. The most significant finding in this work was that LysoPC had an inhibitory effect on the lipolysis of longand intermediate-chain acylglycerols by lipase. This inhibition had two characteristic features. First, it was strongly dependent on the physical properties, especially the hydrophobicity, of the substrates used, as shown in Fig. 1; and second, the inhibitory effect of LysoPC on hydrolysis by lipase was reversible. As shown in Fig. 3, the reduced activity by LysoPC was restored by the addition of either of two other surfactants, Tau or PC. These two features of the inhibition by LysoPC were considered to have been due to the interaction between lipase molecules and the emulsified substrates. Previous studies on lipoprotein lipase revealed that sphingomyelin induced an inhibitory effect on the lipoprotein lipase activity by way of its incorporation into the emulsified substrates [13,14]. In those works, inhibition by sphingomyelin was strongly related to the hydrophobicity of the substrate, because the length of acyl chains in the substrate was responsible for the strength of the hydrophobic interaction between a surfactant and a substrate in the emulsion, such as the lipid – lipid interaction. The authors of these previous studies concluded that the strong interaction between sphingomyelin and substrate would retard the transfer of the substrate to the catalytic pocket of the enzyme. The same explanation would apply to the inhibition of the lipase by LysoPC. We are currently studying the effect of the hydrophobic interaction of acyl chains in the lipid emulsion on the hydrolysis by lipase, using several surfactants and acylglycerols with chains of various lengths. In addition to the lipid – lipid interaction in the lipid emulsion, other factors affecting lipase activity should be considered. Analysis of the particle size distribution of the emulsion conducted in this study cannot directly reveal the interfacial properties of the lipid emulsion. However, it is at least possible to conclude that the triacylglycerols emulsified with LysoPC formed an inactive complex toward lipase.
When Tau was added, the lipid emulsion was reconstructed with additional Tau, accompanied by a change in the particle size of the emulsion. Substrates in the reformed emulsion were subject to hydrolysis by lipase. The differences in the constituents of the emulsion must affect not only the particle size but also the structure of the surface offered to the enzyme molecule, which was conclusively influenced by the hydrolytic activity of lipase. To analyze the relationship between the surface characteristics of the lipid emulsion and enzymatic activity, a physiological study of the interface of the emulsion is currently underway in our laboratory. As described above, the hydrolytic activity depressed by LysoPC was recovered by the addition of a second surfactant (Tau or PC in this experiment), which hindered the formation of an inactive emulsion of the substrate and LysoPC. Such recovery of lipase activity has not been reported even for the other lipolytic enzymes. This is the first study to demonstrate that a combination of added surfactants can regulate the lipase activity. Further investigations will be needed to investigate the use of mixtures of various surfactants to control the activity of surface-active enzymes. In the duodenum, dietary fats are emulsified by bile salts and PC secreted from the liver, then hydrolyzed by pancreatic lipase. The secreted PC is then broken down into LysoPC by the action of phospholipase A2. Even when LysoPC is produced, the presence of bile salts protects against a reduction in digestive activity by lipase in the duodenum. Thus, our findings suggest an additional physiological role of bile salts in the digestive organs.
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Rapid report
Inhibitory effect of lysophosphatidylcholine on pancreatic lipase-mediated hydrolysis in lipid emulsion Wakako Tsuzuki a,*, Akemi Ue a, Akihiko Nagao a, Miyuki Endo b, Masahiko Abe b b
a National Food Research Institute, Kannondai, 2-1-12, Tsukuba, Ibaraki 305-8642, Japan Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
Received 29 August 2003; received in revised form 25 May 2004; accepted 25 May 2004 Available online 17 June 2004
Abstract In the lipid metabolism pathway, dietary lipid emulsified with bile salts and phospholipids is mainly digested by pancreatic lipase into free fatty acids and monoacylglycerols. In order to study substrate recognition mechanism of a pancreatic lipase, we investigated its catalytic property toward the lipid emulsion prepared with long- or intermediate-chain acylglycerols and several physiological surfactants. When lysophosphatidylcholine (LysoPC), rather than bile salts or phospholipid, was incorporated into the lipid emulsion, it caused an increase in the Km(app) and a decrease in the Vmax(app) values in the interactions between the lipase and triacylglycerol (triolein or tricaprin). This indicated that LysoPC inhibited hydrolysis by decreasing both the substrate affinities and the catalytic activity of this lipase. Interestingly, further addition of taurodeoxycholic acid sodium salts or phospholipid completely restored the inhibitory effect of LysoPC on hydrolysis by lipase. On the other hand, the change in these kinetic values between the lipase and two 1-monoacylglycerols (1-monocaprin and 1-monoolein) were not particularly large when LysoPC was added. Particle size analysis of the lipid emulsion composed of LysoPC and triacylglycerols showed that most of the particles were less than 200 nm in size, which was smaller than the particle size in the triacylglycerol emulsions containing bile salts or phospholipid. The composition of the emulsion would affect its surface characteristics and thus contribute to changing lipase activity. D 2004 Elsevier B.V. All rights reserved. Keywords: Pancreatic lipase; Hydrolysis; Lysophsophatidylcholine; Inhibition
1. Introduction In the metabolism of dietary lipids, pancreatic lipase, along with bile salt conjugates, plays the most important role: 50– 70% of oral-mediated fat is hydrolyzed by this enzyme to produce monoacylglycerols and free fatty acids for absorption in the duodenum [1]. Because pancreatic lipase is one of the surface-active enzymes, it attacks lipid molecules at the interface of the emulsion particles (the oil – water interface) [2,3]. In general, the enzymatic process of the lipolytic enzyme is comprised of two dependent steps: binding to the emulsion, and then catalysis of the substrate [4]. The latter step is common to other enzymes. The former step is specific to lipolytic enzymes and is a rate-limiting step through the entire catalytic process [5]. Thus, the
* Corresponding author. Tel.: +81-29-838-8039; fax: +81-29-838-7996. E-mail address: [email protected] (W. Tsuzuki). 1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2004.05.002
emulsification of lipids is of great importance as an essential lipolytic reaction by these enzymes. In other words, the kinetics of the lipolytic enzyme is a function of the surface characteristics of the emulsion offered to the enzyme [6,7]. In the digestion pathway of lipids in vivo, the major constituents of the emulsion offered to a pancreatic lipase are dietary lipids, bile salts and phosphatidylcholine. Thus the lipase activity is mainly modulated by these two physiological surfactants. Therefore, previous studies have investigated the catalytic behaviors of lipase at the interface of the lipid emulsion [3,4,6 –9]. These works have led to general agreement that the surface composition, the lateral lipid distribution, and the physicochemical nature of its interface significantly influence the lipid digestion by lipase. For example, lipase activity was induced by a small increase in the substrate mole fraction on the surface of the emulsion [3]. In another study, the lag time duration was directly related to the phospholipid content of the interface of the emulsion [8]. Several researchers have approached the
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problem of the interfacial quality using various detergents [4,9]. However, the substrate recognition mechanism of this enzyme has not been characterized in detail. In particular, the mechanism of the inhibitory effect of lipid emulsion on lipase hydrolysis remains poorly understood. By exploiting the fact that the catalytic site of pancreatic lipase is composed of the same amino acids as the catalytic sites of serine proteases, researchers have succeeded in identifying several inhibitors of pancreatic lipase. Most of these inhibitors covalently bond to the serine residue located at the catalytic site in the lipase, thereby reducing enzyme activity. In particular, the mechanisms of the inhibition of lipase activity by tetrahydrolipstatin (THL) have been well characterized [10 –12]. Recently, Saito et al. [13,14] demonstrated that a lipid emulsion containing sphingomyelin inhibited lipoprotein lipase activity. They provided conclusive evidence that the inhibition by sphingomyelin mainly occurred via changes in the emulsion surface structure, rather than by a direct interaction between sphingomyelin and the active site of the enzyme. Further, they demonstrated that the specific interaction between sphingomyelin and substrates in the emulsion would inactivate the catalysis by the lipoprotein lipase. Although pancreatic lipase, like lipoprotein lipase, is a surface-active enzyme, researchers have not yet identified a lipid emulsion that has an inhibitory effect on this enzyme. One reason for this is that it is very difficult to measure the lipase activity toward the long-chain acylglycerols. The lipase assay is usually conducted using short-chain acylglycerols such as triacetin and tributylin as substrates. However, long-chain acylglycerols should also be considered in the kinetic study of pancreatic lipase in order to estimate the digestive behaviors of dietary lipid. We previously developed an assay method with high sensitivity for detecting lipase hydrolysis of long and intermediate-chain acylglycerols [15]. In the present study, we use this assay to investigate the influence of lipid emulsions prepared with long- and intermediate-chain acylglycerols and physiological surfactants on lipase hydrolytic activity. We presumed that the physiological nature of the lipid emulsion would play a critical role in the lipid metabolism, and particularly in pancreatic lipase-mediated hydrolysis. The purpose of this study was to identify the factors regulating the lipase activity of the lipid emulsion, and to obtain information on the role of physiological surfactants in the metabolism of dietary lipids.
corresponding to colipase. Colipase, essentially salt-free lyophilized powder, was obtained from Sigma. Taurodeoxycholic acid sodium salt (Tau), phosphocholine, 1oleoyl-2-palmitoyl-sn-glycerol-3-(PC) and L-a-monopalmitoyl lecithin (LysoPC) were obtained from Sigma. Several acylglycerols as substrates for hydrolysis by the lipase were purchased from Funakoshi Co. (Tokyo, Japan). Chloroform and methanol of analytical grades for extracting enzymatic reactive products from an aqueous solution were obtained from Wako Pure Chemicals (Tokyo, Japan). All other chemicals of analytical grade were purchased from Wako Pure Chemicals or Kanto Chemicals (Tokyo, Japan). For fluorescent labeling, reagents of fatty acid, 9-bromomethylacridine (9BMA) and tetraethylammonium carbonate (TEAC) were prepared according to the method described previously [16]. 2.2. Preparation of enzymatic reactive solution For routine lipase assay, the lipid emulsion was prepared according to the previously described methods, with slight modifications [17]. An appropriate volume of stock substrate solution of triacylglycerol or monoacylglycerol was transferred to a glass vial, and the organic solvent was removed under a steam of nitrogen. The residue was dissolved in 10 mM HEPES buffer (pH 7.0, about 500 Al, 37 jC) with or without Tau or LysoPC and repeatedly sonicated using a micro-equipped Astrason ultrasonic processor W-380 (Heat-System Ultrasonics Inc. Farmingdale, NY) until achieving a predefined particle size distribution. The weight-averaged particle size of each emulsion was determined from quasielastic light scattering measurements (NICOMP model 380 ZLS; Particle Sizing System Co. (Santa Barbara, CA). A 195 Al volume of the lipid emulsion solution was transferred to a small reactive vial. The final concentrations of each component in the reaction medium were as follows: 0.2 mM substrate, 0 –10 mM Tau, and 0– 10 mM LysoPC. Because the commercially available porcine pancreas lipase did not contain proteins corresponding to colipase, the latter was added to the lipase solution (lipase, 0.1 mg/ml; colipase, 0.16 mg/ml). The mole ratio between lipase and colipase was within physiological range at 1:5 and it kept content throughout all experiments. The reaction was started by the addition of the pancreatic lipase/ colipase solution (5 Al). The mixture (200 Al) was incubated at 37 jC for 30 min with constant stirring (150 rpm). Hydrolysis was terminated by adding 400 Al of chloroform to the reaction solution and mixing vigorously.
2. Materials and methods 2.3. Measurement of lipase activity 2.1. Materials Highly purified lipase from porcine pancreas (Type VI-S) was purchased from Sigma Chemicals Co. (St. Louis, MO) and used without further purifications. Electrophoresis of this lipase fraction indicated that it contained no proteins
Free fatty acids released by the lipase were extracted by the method of Folch et al. [18]. Briefly, following addition of chloroform (400 Al), methanol (200 Al) was added to the enzymatic solution, which was then mixed vigorously for 5 min. After centrifugation at 15,000 rpm for 10 min, the
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chloroform layer was isolated. After evaporating chloroform, the pellet was dissolved in a mixture of 200 Al (5 mM) 9BMA in dimethyl sulfoxide (DMSO) and 200 Al (2.5 mM) TEAC in DMSO. The TEAC solution contained myristic acid (0.1 mM) as an internal standard fatty acid. The mixture was then allowed to stand for more than 40 min for fluorescent labeling. The quantitative determination of free fatty acid by HPLC was conducted according to a previously reported method [15]. The HPLC peak area of 9BMA labeled fatty acid produced during the lipase assay was converted into units of Amol of fatty acid generated per mg of protein by using the peak of an internal standard myristic acid. Most experiments were repeated six times, and the independent data were statistically analyzed to confirm the reliability of the results. In addition, the amounts of free fatty acids released during the hydrolysis were measured using an enzymatic colorimetric reagent, NEFA-C (Wako Pure Chemicals). Although several lipases showed their activity even in chloroform, the pancreatic lipase used in this study lost its activity completely by addition of chloroform. No increase of fatty acid released by lipase was detected after chloroform was added to the enzymatic reactive solution. The efficiency of the extraction of the fatty acid from an aqueous solution into chloroform phase was determined as follows. Instead of the substrates for hydrolysis, free capric acid or free oleic acid at a known concentration was dispersed in the enzymatic solution and they were extracted by the method of Folch. The extraction efficiencies of the fatty acids from Tau or LysoPC solution decreased slightly depending on the concentrations of the coexisting surfactants. 95 F 5%, 90 F 6% and 87 F 5% of the original free capric acid was extracted to the chloroform layer from buffer (pH 7.0), LysoPC (1 mM) and Tau (10 mM) solutions, respectively. The calculated extraction efficiencies were used for determining the types and amounts of fatty acids extracted from the enzymatic reaction solutions. We also investigated the fluorescent-labeling efficiency with 9BMA. Myristic acid contained in TEAC solution,
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which was used as an internal standard, was a good indicator of the labeling efficiency of free fatty acids produced by the lipase reactions. Monitoring of the labeling efficiency of myristic acid revealed that Tau or LysoPC coexisting in the enzymatic solution did not affect the labeling efficiency of any fatty acids. To confirm the results of the lipase hydrolysis evaluation, the quantitative measurements of fatty acid by fluorescentHPLC were followed by measurements using the NEFA-C method. Although the NEFA-C method has been widely used for estimating enzymatic activities, some modifications were needed before using NEFA-C to measure lipase activity under our experimental condition. Since the NEFA-C method uses successive enzymatic processes to color the free fatty acids, the Tau and LysoPC contained in the lipase reactive solution used here affected the NEFA-C coloring process. This caused an excessive overestimation of the amounts of fatty acids in the Tau or LysoPC solutions. However, the corrected NEFA-C results were coincident with those calculated from the fluorescence-HPLC method.
3. Results and discussions In vivo, lysophosphatidylcholine (LysoPC) is produced from phosphatidylcholine (PC) by pancreas phospholipase A2 and plays an important role in the absorption of lipid digestives into intestinal cells. In general, PC but not LysoPC mainly interacts with a dietary lipid in its digestive process. This is one of the reasons why LysoPC has not been considered in lipolysis. In this study, LysoPC was first introduced to the lipid hydrolysis system in order to investigate the effect of LysoPC in the lipid emulsion on lipase accessibility. As the substrates for hydrolysis by lipase, four acylglycerols were used. The hydrolysis activity of the lipase toward both triacylglycerols (tricaprin and triolein) was drastically decreased by the addition of LysoPC, as shown in Fig. 1. LysoPC inhibited triolein more strongly than tricaprin. On the other hand, the inhibitory effect of
Fig. 1. Influence of LysoPC on the hydrolysis of tricaprin (open circles in panel A), 1-monocaprin (closed circles in panel A), triolein (open squares in panel B) and 1-monoolein (closed squares in panel B) by pancreatic lipase.
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LysoPC on the hydrolysis by lipase using monoacylglycerols (1-monocaprin and 1-monoolein) as substrates was small (Fig. 1). To identify the interaction of LysoPC with substrates or with enzyme, the particle size distribution of the enzymatic solution was investigated. Without LysoPC, the particle size of tricaprin dispersed in the solution was mainly distributed in the range between 140 and 800 – 2000 nm (data not shown). When LysoPC was added, the distribution of the particle size of the tricaprin solution changed to a range of 30 –220 nm, with most of the particles being < 200 nm in diameter (Fig. 3C). The difference in particle size distribution between the trial with and that without LysoPC indicated that tricaprin was incorporated into LysoPC. In the case of 1monocaprin, no particles could be detected with or without LysoPC in the examined concentration range. Because of the strong hydrophobicity, triacylglycerols would be subject to interaction with LysoPC, and would form emulsions, whose interface would hinder the enzyme molecules from approaching. In contrast, the hydrophobicity of monoacylglycerols was not strong enough to form any micelles or emulsion with LysoPC. These results demonstrated that LysoPC has an inhibitory effect on lipase-mediated hydrolysis by way of the formation of lipid emulsion. The effect of colipase on inhibition of the lipase by LysoPC was investigated using a lipase solution containing no colipase. It revealed that no differences were detected in the inhibitory effect of LysoPC on lipase activity with or without colipase in the enzymatic solution. This finding suggested that inhibition by LysoPC would occur even without the participation of colipase in the enzyme (data not shown). It is considered that LysoPC could regulate lipolysis by several mechanisms: inhibition of lipase in binding lipid emulsion, inhibition at the emulsion surface, or a combination of the above. To investigate the inhibition mechanism by LysoPC, a kinetics was analyzed and it revealed that LysoPC decreased the apparent maximum velocity (Vmax(app)) and increased the apparent Michaelis – Menten constant (Km(app)) (Table 1). The results indicated that LysoPC Table 1 Apparent kinetic parameters of hydrolysis by lipase toward various lipid emulsions Substrate
Lyso (mM)
Tau (mM)
Kmapp (mM)
Vmaxapp (Amol/min mg)
Triolein
0 0.2 0 0.2 0 0.2 0 0.2 0 0.2 0 0.2
0 0 2 2 0 0 5 5 0 0 0 0
0.36 F 0.07 1.25 F 0.10 0.28 F 0.06 0.29 F 0.04 0.47 F 0.05 1.53 F 0.13 0.35 F 0.07 0.34 F 0.06 0.86 F 0.07 1.73 F 0.18 1.17 F 0.13 2.35 F 0.28
1.88 F 0.16 0.22 F 0.05 1.90 F 0.17 1.87 F 0.18 2.18 F 0.18 0.18 F 0.04 2.21 F 0.23 2.19 F 0.21 1.02 F 0.09 0.87 F 0.10 1.25 F 0.14 1.02 F 0.15
Tricaprin
1-Monoolein 1-Monocaprin
Fig. 2. Influence of Tau on hydrolysis of the lipid emulsion composed of LysoPC and tricaprin (open squares) and of LysoPC and triolein (open circles).
decreased both the catalytic activity of the pancreatic lipase and its affinity for the substrates, thereby reducing the lipolysis rates of two triacylglycerols. Next, the effect of Tau on the inhibition of hydrolysis by LysoPC was investigated. In the presence of 0.2 mM LysoPC, the hydrolytic activities toward two triacylglycerols were predominantly depressed, as shown in Fig. 1. When Tau was added to such solutions, the lipase activity was restored in relation to an increase of Tau concentration (Fig. 2). Finally, the depressed hydrolyses of tricaprin and triolein by LysoPC were completely recovered at Tau concentrations of 2 and 5 mM, respectively. When PC was added to the LysoPC solution, the hydrolyses of tricaprin and triolein were restored at PC concentrations of 0.75 and 2.5 mM, respectively (data not shown). These results suggested that the inhibition by LysoPC was reversible and that Tau and PC were able to restore the depressed activity. The rates of lipase-mediated hydrolysis of triacylglycerols in the presence of both LysoPC and Tau were close to those in only Tau solution. The apparent kinetic parameters, Km(app) and Vmax(app), of two triacylglycerols in LysoPC (0.2 mM) and Tau (5 mM) solution were almost the same as those in Tau solution (5 mM) (Table 1). These results can be explained as follows. In respect to the hydrolysis by lipase, triacylglycerol emulsion in both Tau and LysoPC solutions is similar to that composed of triacylglycerols and Tau rather than that composed of triacylglycerols and LysoPC. To confirm the change of triacylglycerol-containing emulsions in various solutions, the particle sizes of the emulsions were compared. The particle size distribution of each enzymatic reactive solution was measured before lipase solution was added, as described in Materials and methods. In the case of the emulsion of tricaprin and surfactants, the representative distribution pattern of the
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Fig. 3. Weight-mean emulsion droplet size distributions of the lipid emulsion prepared with 0.2 mM tricaprin and 2 mM Tau (A), those prepared with 0.2 mM tricaprin and 0.8 mM PC (B), those prepared with 0.2 mM tricaprin and 0.2 mM LysoPC (C), and those prepared with 0.2 mM tricaprin, 0.2 mM LysoPC and 2 mM Tau (D).
emulsion was complicated, as shown in Fig. 3. Comparing with the emulsion of tricaprin and Tau (Fig. 3A) or PC (Fig. 3B), the emulsion composed of tricaprin and LysoPC distributed in a rather small range (40 – 200 nm), and no emulsion was detected in the particle size around 1 Am (Fig. 3C). Further addition of Tau to the emulsion of LysoPC and tricaprin induced the appearance of particles in the range of 1 Am in diameter, and the typical small particles of LysoPC and tricaprin disappeared (Fig. 3D). The distribution of tricaprin emulsion in both LysoPC and Tau solution was similar to that in Tau solution rather than to that in LysoPC solution. Changes in the distribution of particle size in various solutions indicated the diversity in the constituents of the emulsion. In particular, the small particles of tricaprin and LysoPC would be reconstructed by further addition of Tau. Finally, the inhibitory effect of LysoPC on lipase hydrolytic activity was examined using other microbial lipases. Tricaprin was hydrolyzed by lipase originating from Aspergillus oryzae, Mucor miehei, Candida cylindracea and Pseudomonas fluorescens in various concentrations of LysoPC (0– 0.2 mM). As shown in Fig. 4, the activities of all of the examined microbial lipases were diminished according to the LysoPC concentration, although a small difference was found in the degree of the inhibitory effect of
LysoPC on hydrolysis by these four lipases. The substrate specificity and hydrolysis properties of these lipases are clearly different from each other [20 – 23]. But the inhibitory effect of LysoPC on hydrolysis by these enzymes occurred
Fig. 4. Influence of LysoPC on hydrolysis of tricaprin by A. oryzae (open circles), M. miehei (open squares), C. cylindracea (open triangles) and P. fluorescens (closed circles).
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in the same manner as that shown in Fig. 4. This strongly suggests that the inhibitory effect of LysoPC is caused by the formation of an inactive complex of LysoPC and substrate rather than by specific interaction between LysoPC and the active site of each lipase. This suggested that inhibition by LysoPC was not unique to a pancreatic lipase but was general to other lipases from microorganisms. In previous works, the particle sizes of emulsions containing bile salts, phospholipids, and dietary lipids were measured [9,19,23 –25]. The particle size distribution of the emulsion prepared in our experiments was not contradictory to those reported previously. However, no information is available about the relation between the particle size of the emulsion and the inhibition of hydrolysis by lipase. This study demonstrated that the change in the particle size of the emulsion is dependent on the variety of the emulsion’s components. Composition of the emulsion would be one of the factors affecting the surface nature of the emulsion and would result in the change of lipase activity. The most significant finding in this work was that LysoPC had an inhibitory effect on the lipolysis of longand intermediate-chain acylglycerols by lipase. This inhibition had two characteristic features. First, it was strongly dependent on the physical properties, especially the hydrophobicity, of the substrates used, as shown in Fig. 1; and second, the inhibitory effect of LysoPC on hydrolysis by lipase was reversible. As shown in Fig. 3, the reduced activity by LysoPC was restored by the addition of either of two other surfactants, Tau or PC. These two features of the inhibition by LysoPC were considered to have been due to the interaction between lipase molecules and the emulsified substrates. Previous studies on lipoprotein lipase revealed that sphingomyelin induced an inhibitory effect on the lipoprotein lipase activity by way of its incorporation into the emulsified substrates [13,14]. In those works, inhibition by sphingomyelin was strongly related to the hydrophobicity of the substrate, because the length of acyl chains in the substrate was responsible for the strength of the hydrophobic interaction between a surfactant and a substrate in the emulsion, such as the lipid – lipid interaction. The authors of these previous studies concluded that the strong interaction between sphingomyelin and substrate would retard the transfer of the substrate to the catalytic pocket of the enzyme. The same explanation would apply to the inhibition of the lipase by LysoPC. We are currently studying the effect of the hydrophobic interaction of acyl chains in the lipid emulsion on the hydrolysis by lipase, using several surfactants and acylglycerols with chains of various lengths. In addition to the lipid – lipid interaction in the lipid emulsion, other factors affecting lipase activity should be considered. Analysis of the particle size distribution of the emulsion conducted in this study cannot directly reveal the interfacial properties of the lipid emulsion. However, it is at least possible to conclude that the triacylglycerols emulsified with LysoPC formed an inactive complex toward lipase.
When Tau was added, the lipid emulsion was reconstructed with additional Tau, accompanied by a change in the particle size of the emulsion. Substrates in the reformed emulsion were subject to hydrolysis by lipase. The differences in the constituents of the emulsion must affect not only the particle size but also the structure of the surface offered to the enzyme molecule, which was conclusively influenced by the hydrolytic activity of lipase. To analyze the relationship between the surface characteristics of the lipid emulsion and enzymatic activity, a physiological study of the interface of the emulsion is currently underway in our laboratory. As described above, the hydrolytic activity depressed by LysoPC was recovered by the addition of a second surfactant (Tau or PC in this experiment), which hindered the formation of an inactive emulsion of the substrate and LysoPC. Such recovery of lipase activity has not been reported even for the other lipolytic enzymes. This is the first study to demonstrate that a combination of added surfactants can regulate the lipase activity. Further investigations will be needed to investigate the use of mixtures of various surfactants to control the activity of surface-active enzymes. In the duodenum, dietary fats are emulsified by bile salts and PC secreted from the liver, then hydrolyzed by pancreatic lipase. The secreted PC is then broken down into LysoPC by the action of phospholipase A2. Even when LysoPC is produced, the presence of bile salts protects against a reduction in digestive activity by lipase in the duodenum. Thus, our findings suggest an additional physiological role of bile salts in the digestive organs.
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