Biochhnica et Bh physica A eta. 1084( 1991) I~)4- 197 "~' 19o! ElsevierScience PuHishersB.V. !)(!05-2760/91/$03.50
194
,-IDONIS 0005276(}91(102067
BBA Report
Effect of pancreatic phospholipase A 2 and gastric lipase on the action of pancreatic carboxyl ester lipase against lipid substrates in vitro M a t s B. L i n d s t r 6 m , J o h a n P e r s s o n , L a r s T h u r n a n d B e n g t Borgstr~Sm Depurtment of Medical and Physudogical CTwmistt3.', U~lirersity of Ltmd, Lurid (Sweden)
Key words: PhospholipaseA2; Gastric lipasc:Carhoxylester lipase: Hydrolysis Preincubation of a triolein/phospholipid/cholesteryl oleate-emulsion in vitro with either pancreatic phospholipase A 2 (PLA 2) or gastric lipase (GL) resulted in hydrolysis (measured by pH.stat-titration) of cholesteryl [SH]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 PLA z 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.
The secretory pancreatic enzyme carboxyl ester lipase (CEL) - a name suggested by Rudd and Brockman [1] - is able to hydrolyse a num~',er of different dietary lipids. The broad spectrum of !ipid substrates includes mono-, di- and triacylglycerols as well as eholesteryl esters, esters of vitamin A, D and E, phospholipids, lysophospholipids etc. CEL has also been shown to accept lipid substrates in different physicochemical forms. The enzyme is most active against cholcstcryl esters when present in mixed micellar aggregates, but CEL is also able to hydrolyse lipid substrates in vesicular or emulsified form. However, bile salts must always be present in order for CEL to be active. Compared to pancreatic lipase (EC 3.1.1.3) the requirements of CEL are relatively unspecific, which explains why the enzyme has obtained names like nonspecific lipase, cholesterol esterase (EC 3.1.1.13), sterol ester hydrolase and carboxyl ester hydrolase (EC 3.1.1.1). Blfickberg et al. [2] have shown that pancreatic CEL is very similar to the bile salt-stimulated lipase of human breast milk. The major physiological role of CEL in the intesti-
Abbreviations: CEL, carboxyl ester lipase; GL, gaslric lipasc; PLA2, phospholipaseA ,. Corrc~,pondcncc: M.B. Lindstr/3m. Department of Medical and PhysiologicalChemistry,Universityof Lund, P.O. Box 94, S-221 110 Lund, Sweden.
hal lumen is not clear, since many of the lipid substrates of CEL can also be hydrolysed by other iipolytic enzymes in the gastro-intestinal tract (gastric/ pregastric lipase, pancreatic lipase and phospholipase A2). Hydrolysis of cholesteryl and retinyl esters is probably the most important function of CEL [1], but it has also been suggested that CEL in combination with pancreatic lipase could drive triacylglyceroi hydrolysis to near completeness since CEL is able to split the 2-ester bond of acylglycerols [3]. According to Rudd and Brockman [1] CEL is dependent on the generation of a lipolytic product phase by the action of pancreatic lipase against triglycerides in the presence of bile salts. They suggested that cholesteryl and vitamin esters are transferred together with the lipolytic products from the oil emulsion droplets to the mixed micellar aggregates of the aqueous phase, where they will be hydrolysed by CEL. This hypothesis was recently confirmed by Lindstr6m et al. [4] who used in vitro incubations of a phospholipid-stabilised triolein emulsion in a buffer containing bile salts to which human CEL and pancreatic lipase were added. Cholesteryl oleate and retinyl palmitate solubilised in the triolein/phospholipid emulsion were hydrolysed by CEL, but only if pancreatic lipase and its protein cofactor, colipase, were present. Thus, a concerted action of the two enzymes was necessary, since CEL alone could hydrolyse neither cholesteryl oleate, retinyl palmitate nor triolein. The aim of this study was to test whether preincubation of a physiologically relevant triolein/phospholipid-emul-
195 sion with two other lipolytic enzymes, i.e. gastric lipase or phospholipase A 2, would make potential lipid substrates available for hydrolysis by CEI in vitro. Results from this study were presented on a poster at the Falsterbo-symposium on "Lipases and Lipid Gastroenterology', May 30-June 1, 1988, Falsterbo. Sweden. Gastric lipase (GL) was purified from human stomach juice by Dr. Stefan Bernb~ick, University of Ume'~, Sweden; phospholipase A , (PLA_,) was purified from porcine pancreas by Dr. Gerard de Haas, Rijksuniversitet Utrecht, the Netherlands; CEL was purified from human pancreatic juice by Dr. Berit Sternby, University of Lund, Sweden. The purity of the different enzymatic preparations was confirmed by measurement of the specific enzymatic activity against relevant lipid substrates. Triolein (TO), cholesteryl oleate, phosphatidylcholine (PC), sodium taurccholate (NaTC) and sodium taurodeoxycholate (NaTDC) were obtained as described earlier [4]. Tri[14C]oleoylglycerol, cholesteryl [3H]oleate and l-palmitoyl,2-[3H]oleoyl-phosphatidyl choline were prepared and purified according to standard methods. All lipids were more than 95% pure by thin-layer chromatography. All other reagents were of analytical grade. Triolein/phosphatidylcholine-emulsion ( T O / P C emulsion) was prepared in buffer containing bile salts (1 mM Tris-maleate, 150 mM NaCI, 1 mM CaCI,, 5 mM NaTC, 5 mM NaTDC, pH 7.0) according to [4]. The lipid composition of the TO/PC-emulsion was 7.53 mM TO, 0.43 mM PC and 0.10 mM cholesteryl oleate. The total vo!ume of the system was 15 ml. Glass vials containing TO/PC-emulsion were allowed to equilibrate for 15 min at 37 ° C on a pH-stat (Mettler) after the pH had been adjusted to pH 7.0. In the PLA2-preineubation experiments 100 p,g of P L A , (from a stock solution with 1 mg PLA_,/ml in buffer) was added after the equilibration period. Buffer without PLA 2 was added to the controls. After 30 min 125 p,g CEL (from a stock solution with 0.5 mg C E L / m l in water: glycerol, 1 : 1, v / v ) was added. The pH was kept constant at 7.0 by titration with 0.1 M NaOH during the whole experiment. Aliquots of 200 ~1 were taken from the incubation system at 10-min .;ntervals, and release of free fatty acids was measured by the extraction procedure of Belfrage and Vaughan [5]. Free fatty acid radioactivity in aliquots from the upper phase of the extraction system and in aliquots from the incubation system was measured in a liquid scintillation spectrometer (Minaxi Tri-Carb, Packard) after addition of 10 ml of scintillator (Emulsifier-Scintillator 299, Packard). Presented data represent the mean value from two pH-stat-incubation experiments. In the GL-preincubation experiments the conditions were the same with a few exceptions. The buffer of the TO/PC-emulsion did not contain any bile salts and the
pH was set to 6.11 from the start of the experiment. When the emulsion-system had equilibrated for 15 min 40 tzg of GL (from a stock solution with 0.2 mg G L / m l in phosphate buffer (pH 6.0), with 150 mM NaCI and ethyleneglycoL 45%, v/v) was added. Buffer alone was added to controls. After 30 min of preincubation with GL the pH was changed to 7.0 and bile salts were added to a finai concentration nf Ill mM (molar ratio of N a T C : N a T D C = 1:1). Then 125 /zg of CEL was added. In another contro! experiment the T O / P C - e m ulsion was preincubated with GL, but after adjustment of pH and addition of bile salts, CEL was omitted. The addition of bile salts and increase of pH was done to mimic the change in physiological conditions that occurs when the gastric content is transferred into the duodenum. During ',he first 30 min of incubation with PLA 2 a rapid hydrolysis of phosphatidylcholine in the TO/PC-emulsion was observed (Fig. IA). Nearly 90% of the [3H]oleic acid at the 2-ester position of the phosphatidytcholine was released during the first 30 min. In the absence of PLA 2 virtually no phosphatidylcholine hydrolysis occurred. Approx. 9 and 15% of the %tty acids were released from cholesteryl[3H]oleate and tri[ t~C]oleoylglycerol, respectively, 60 min after the addition of CEL to incubation systems into which P L A , had previously been added (Fig. IB and C). P L A , itself did not show any activity against these lipids during the first 30 rain of incuba'~lon. Hydrolysis of cholesteryl[3H]oleate and tri[14C]oleoylglycerol was negligible when CEL was added alone. When gastric lipase was added to the T O / P C - e m u l sion at pH 6.0 and with no bile salts present, approx. 3.5% of the fatty acids in tri[14C]oleoylglycerol were released, whereas no appreciable hydrolysis of cholesteryl[3H]oleate occurred during the 30 min of preincubation with GL (Fig. 2). After 30 min pH was increased to 7.0, bile salts were added to give a total concentration of 10 mM, and then CEL was added. During the following 60 min approx. 5.5% of the cholcsteryl[3H]oleate was hydrolysed and release of fatty acids from tri[14C]oleoylglycerol continued to a level of 14%. However, in the absence of gastric lipase the activity of CEL against these two lipids was low or absent. A second control experiment was performed in order to check if the hydrolysis of cholesteryl[3H]oleate and tri[ J4C]oleoylglycerol could be due to activation of GL when pH was raised and bile salts were added. However, no further hydrolysis after the preincubation period was observed when CEL was omitted (Fig. 2). The results ef this study showed that preineubation with either GL or PLA 2 was necessary in order for hydrolysis of cholesteryl oleate and triolein to occur in a TO/PC-emulsion to which CEL was added. Although cholesteryl oleate and triolein are potential substrates for CEL, they were not hydrolysed by CEL
196 100
A
i
-~~ a0 /'~" o = "~ ~., ___o60
-or"o _, g'~ :~.
+PLA2 •
"
-1"
•
•
i
40
o
,o. ~. 20
_PLA2
0~ - ~ g
8
B
°
~
"~
/
6
/
,u
/
ucts from the emulsion droplets into the aqueous phase, where they could form mixed micellar aggregates together with bile salts. In miccllar form cholesteryl oleate became available for hydrolysis by CEL. The mechanism of action of G L was different. G L g e n e r a t e d a lipolytic product phase by hydrolysing triolein. Although the phospholipids were intact, the transfer of cholesteryl oleate together with lipolytic products into mixed micellar aggregates was sufficient to allow C E L - m e d i a t e d cholesteryl oleate hydrolysis. It should be pointed out that the observed effects of P L A 2 or G L on the activity of C E L could not be explained by proteolytic activation of the lipolytic en-
PLA, pH6
~
A
8
;!:
pH7
I"
BS
-PLA 2 D__D~D--O~
g'~c "5 o¢
o-o
i
/'
{
PLA 2
o
,...,i,/'/"
-
~
2 ~o/~o----o--o--o--o
01
~0
0 30 60 90 | Time [min) -+PLA2 CEL Fig. I. Hydrolysis(expressed as % of the total amount of radioactivity) of l-palmitoyl,2-[ 3H]oleoylphosphatidylcholine (A), cholesteryl[3H]oleate (B) and tri[14C]oleoylglycerol(C) in a triolein/ phosphat dy choline emulsion incubated with 100/~g PLA 2 + 125/.¢g CEL (=) or 125/~g CEL alone (O) in the presence of 10 mM bile salts at pH 7.0. Arrows indicate the addition of enzymes. The composition of the emulsion system and the experimental design was the same as in [4]. Data represent the mean of two experiments.
alone u n d e r these conditions. The presence of phospholipids on the surface of the lipid emulsion droplets is probably the factor that inhibits CEL, since the enzyme has b e e n r e p o r t e d to hydrolyse triglycerides when emulsified by other amphiphilic substances than phospholipids [1,2]. The efficient hydrolysis of the phospholipids by PLA2 in our experiments was probably responsible for making the triolein molecules on the surface of the emulsion particles available for hydrolysis by CEL. Thereby a lipolytic product phase ( m a d e up by oleic acid and monoolein) was formed. Cholesteryl oleate was probably transferred together with lipolytic prod-
15
I
o~
_
o
>,~
,/'//
10
=/ =~
I
0 -* GL
30 *- C E L
./ 60
90
TIME Cmin?
Fig. 2. Hydrolysis (exprassed as % of the total amount of radioactivity) of cholesteryl[ 3H]oleate (A) and tri[14C]oleoylglycerol (B) in a triolein/phosphatldylcbollne emulsion incubated with 40 g.g GL+ 125 #g CEL ( | ) , 40 /zg GL (,~) or 125 #g CEL (t~). Arrows indicate addition of enzymes. The composition of the emulsion system and the experimental design was the same as i[ [4], except that pH was shifted from 6.0 to 7.0 and bile salts (BS) were addedto a total concentration of 10 mM after 30 rain of incubation. Data represent the mean of two experiments.
197 zymes used in these experiments. The lipolytic enzymes were purified according to standard procedures. Consequently, impurities in the form of proteolytic enzymes ought to be negligible. Moreover, C E L is according to current knowledge [6] secreted by the pancreas as ,'.'n active enzyme which requires no proteolytic activation. O f the enzymes used in this study, only PI-,k 2 is known to be secreted in an inactive proform. However, the P L A z used had been activated by trypsin unc:er controlled conditions before it was used in the experiments. In conclusion, this study showed that a concerted action of C E L and P L A 2 or C E L and G L makes possible the hydrolysis of cholesteryl esters in vitro. e E L and pancreatic lipase were earlier found to act in a similar way [4]. O t h e r examples of concerted action b e t w e e n lipolytic e n z y m e s have b e e n r e p o r t e d . Borgstr6m [7] showed that hydrolysis of phospholipids by P L A 2 in a p h o s p h o l i p i d / s t a b i l i s e d triglyceride emulsion stimulated the action of pancreatic lipase against triglycerides. Gargouri et al. [8], found that prehydrolysis of a similar emulsion system by gastric lipase also stimulated the activity of pancreatic lipase. Finally, it has b e e n reported by Bernb~ick et al. [9] that a concerted action involving GL, pancreatic lipase and bile salt-stimulated lipase from breast milk is necessary for the complete hydrolysis of milk triglycerides in vitro. The general conclusion from these studies is that one lipolytic enzyme can affect the activity of a n o t h e r lipolytic enzyme. The action of the first enzyme changes the general physico-chemical state of the lipids so that the substrate of the second enzyme becomes av~'!able
for hydrolysis. Thus, a concerted action of the different lipolytic enzymes in the gastro-intestinal tract is probably one important mechanism responsible for the efficient digestion and absorption of the dietary lip:~ds. The authors are grateful to Drs Bernb~ick, de Haas and Sternby for the generous supply of enzymes. Technical assistance was provided by UUa Giilich and Monika Lindstr6m. The study was supported by grants from the Swedish Medical Research Council (B8903X-00071-25A to B.B.), the Medical Faculty at the University of Lund, Albert P~hlsson's Foundation, Maim6, and the Swedish Nutrition Foundation, G6teborg.
References I Rudd, E.A. and H L. Brockman (1984) in Lipases (BorgstriSm,B. and Brockman, H.L., eds.), pp. 185-204, Elsevier, Amsterdam. 2 BI/ickberg, L., D. Lombardo, O. Hernell, O. Guy and T. Olivecrona (1081) FEBS ten. 136, 284-288. 3 Hernell, O. and L. nliickberg (1982) Pediatr. Res, 16, 882-885. 4 Lindstr6m, M.B., B. Sternbv and B. Borgstr(Sm0988) Biochim. Biophys. Acta 959, 178-184. 5 Belfrage, P. and M. Vaughan 0969) J. Lipids Res 10, 341-344. 6 Borgstr/3m,B. (1986) in The Exocrine Pancreas: Biology, Pathobiology and Diseases (Go, V.L.W., Gardner, J.D., Brooks, F.P., Lcbenthal, E.. DiMagno, E.P. and Scheele, G.A., eds.), pp. 361373, Raven Press, New York. 7 Borgstr~m, B. (1980) Gastroenterolclgy "?,SO~4-06~ 8 Gargouri, Y., G. Pieroni, C. Rivi~re, P.A. Lowe, J.-F. Sanni~re, L. Sarda and R. Verger (1986) Biochim. Biophys. Acta 879, 419-423. 9 Bernbiick, S., L. Bl~ickbergand O. Hernell (1990) J. Clin. Invest. 85, 1221-1226.
194
,-IDONIS 0005276(}91(102067
BBA Report
Effect of pancreatic phospholipase A 2 and gastric lipase on the action of pancreatic carboxyl ester lipase against lipid substrates in vitro M a t s B. L i n d s t r 6 m , J o h a n P e r s s o n , L a r s T h u r n a n d B e n g t Borgstr~Sm Depurtment of Medical and Physudogical CTwmistt3.', U~lirersity of Ltmd, Lurid (Sweden)
Key words: PhospholipaseA2; Gastric lipasc:Carhoxylester lipase: Hydrolysis Preincubation of a triolein/phospholipid/cholesteryl oleate-emulsion in vitro with either pancreatic phospholipase A 2 (PLA 2) or gastric lipase (GL) resulted in hydrolysis (measured by pH.stat-titration) of cholesteryl [SH]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 PLA z 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.
The secretory pancreatic enzyme carboxyl ester lipase (CEL) - a name suggested by Rudd and Brockman [1] - is able to hydrolyse a num~',er of different dietary lipids. The broad spectrum of !ipid substrates includes mono-, di- and triacylglycerols as well as eholesteryl esters, esters of vitamin A, D and E, phospholipids, lysophospholipids etc. CEL has also been shown to accept lipid substrates in different physicochemical forms. The enzyme is most active against cholcstcryl esters when present in mixed micellar aggregates, but CEL is also able to hydrolyse lipid substrates in vesicular or emulsified form. However, bile salts must always be present in order for CEL to be active. Compared to pancreatic lipase (EC 3.1.1.3) the requirements of CEL are relatively unspecific, which explains why the enzyme has obtained names like nonspecific lipase, cholesterol esterase (EC 3.1.1.13), sterol ester hydrolase and carboxyl ester hydrolase (EC 3.1.1.1). Blfickberg et al. [2] have shown that pancreatic CEL is very similar to the bile salt-stimulated lipase of human breast milk. The major physiological role of CEL in the intesti-
Abbreviations: CEL, carboxyl ester lipase; GL, gaslric lipasc; PLA2, phospholipaseA ,. Corrc~,pondcncc: M.B. Lindstr/3m. Department of Medical and PhysiologicalChemistry,Universityof Lund, P.O. Box 94, S-221 110 Lund, Sweden.
hal lumen is not clear, since many of the lipid substrates of CEL can also be hydrolysed by other iipolytic enzymes in the gastro-intestinal tract (gastric/ pregastric lipase, pancreatic lipase and phospholipase A2). Hydrolysis of cholesteryl and retinyl esters is probably the most important function of CEL [1], but it has also been suggested that CEL in combination with pancreatic lipase could drive triacylglyceroi hydrolysis to near completeness since CEL is able to split the 2-ester bond of acylglycerols [3]. According to Rudd and Brockman [1] CEL is dependent on the generation of a lipolytic product phase by the action of pancreatic lipase against triglycerides in the presence of bile salts. They suggested that cholesteryl and vitamin esters are transferred together with the lipolytic products from the oil emulsion droplets to the mixed micellar aggregates of the aqueous phase, where they will be hydrolysed by CEL. This hypothesis was recently confirmed by Lindstr6m et al. [4] who used in vitro incubations of a phospholipid-stabilised triolein emulsion in a buffer containing bile salts to which human CEL and pancreatic lipase were added. Cholesteryl oleate and retinyl palmitate solubilised in the triolein/phospholipid emulsion were hydrolysed by CEL, but only if pancreatic lipase and its protein cofactor, colipase, were present. Thus, a concerted action of the two enzymes was necessary, since CEL alone could hydrolyse neither cholesteryl oleate, retinyl palmitate nor triolein. The aim of this study was to test whether preincubation of a physiologically relevant triolein/phospholipid-emul-
195 sion with two other lipolytic enzymes, i.e. gastric lipase or phospholipase A 2, would make potential lipid substrates available for hydrolysis by CEI in vitro. Results from this study were presented on a poster at the Falsterbo-symposium on "Lipases and Lipid Gastroenterology', May 30-June 1, 1988, Falsterbo. Sweden. Gastric lipase (GL) was purified from human stomach juice by Dr. Stefan Bernb~ick, University of Ume'~, Sweden; phospholipase A , (PLA_,) was purified from porcine pancreas by Dr. Gerard de Haas, Rijksuniversitet Utrecht, the Netherlands; CEL was purified from human pancreatic juice by Dr. Berit Sternby, University of Lund, Sweden. The purity of the different enzymatic preparations was confirmed by measurement of the specific enzymatic activity against relevant lipid substrates. Triolein (TO), cholesteryl oleate, phosphatidylcholine (PC), sodium taurccholate (NaTC) and sodium taurodeoxycholate (NaTDC) were obtained as described earlier [4]. Tri[14C]oleoylglycerol, cholesteryl [3H]oleate and l-palmitoyl,2-[3H]oleoyl-phosphatidyl choline were prepared and purified according to standard methods. All lipids were more than 95% pure by thin-layer chromatography. All other reagents were of analytical grade. Triolein/phosphatidylcholine-emulsion ( T O / P C emulsion) was prepared in buffer containing bile salts (1 mM Tris-maleate, 150 mM NaCI, 1 mM CaCI,, 5 mM NaTC, 5 mM NaTDC, pH 7.0) according to [4]. The lipid composition of the TO/PC-emulsion was 7.53 mM TO, 0.43 mM PC and 0.10 mM cholesteryl oleate. The total vo!ume of the system was 15 ml. Glass vials containing TO/PC-emulsion were allowed to equilibrate for 15 min at 37 ° C on a pH-stat (Mettler) after the pH had been adjusted to pH 7.0. In the PLA2-preineubation experiments 100 p,g of P L A , (from a stock solution with 1 mg PLA_,/ml in buffer) was added after the equilibration period. Buffer without PLA 2 was added to the controls. After 30 min 125 p,g CEL (from a stock solution with 0.5 mg C E L / m l in water: glycerol, 1 : 1, v / v ) was added. The pH was kept constant at 7.0 by titration with 0.1 M NaOH during the whole experiment. Aliquots of 200 ~1 were taken from the incubation system at 10-min .;ntervals, and release of free fatty acids was measured by the extraction procedure of Belfrage and Vaughan [5]. Free fatty acid radioactivity in aliquots from the upper phase of the extraction system and in aliquots from the incubation system was measured in a liquid scintillation spectrometer (Minaxi Tri-Carb, Packard) after addition of 10 ml of scintillator (Emulsifier-Scintillator 299, Packard). Presented data represent the mean value from two pH-stat-incubation experiments. In the GL-preincubation experiments the conditions were the same with a few exceptions. The buffer of the TO/PC-emulsion did not contain any bile salts and the
pH was set to 6.11 from the start of the experiment. When the emulsion-system had equilibrated for 15 min 40 tzg of GL (from a stock solution with 0.2 mg G L / m l in phosphate buffer (pH 6.0), with 150 mM NaCI and ethyleneglycoL 45%, v/v) was added. Buffer alone was added to controls. After 30 min of preincubation with GL the pH was changed to 7.0 and bile salts were added to a finai concentration nf Ill mM (molar ratio of N a T C : N a T D C = 1:1). Then 125 /zg of CEL was added. In another contro! experiment the T O / P C - e m ulsion was preincubated with GL, but after adjustment of pH and addition of bile salts, CEL was omitted. The addition of bile salts and increase of pH was done to mimic the change in physiological conditions that occurs when the gastric content is transferred into the duodenum. During ',he first 30 min of incubation with PLA 2 a rapid hydrolysis of phosphatidylcholine in the TO/PC-emulsion was observed (Fig. IA). Nearly 90% of the [3H]oleic acid at the 2-ester position of the phosphatidytcholine was released during the first 30 min. In the absence of PLA 2 virtually no phosphatidylcholine hydrolysis occurred. Approx. 9 and 15% of the %tty acids were released from cholesteryl[3H]oleate and tri[ t~C]oleoylglycerol, respectively, 60 min after the addition of CEL to incubation systems into which P L A , had previously been added (Fig. IB and C). P L A , itself did not show any activity against these lipids during the first 30 rain of incuba'~lon. Hydrolysis of cholesteryl[3H]oleate and tri[14C]oleoylglycerol was negligible when CEL was added alone. When gastric lipase was added to the T O / P C - e m u l sion at pH 6.0 and with no bile salts present, approx. 3.5% of the fatty acids in tri[14C]oleoylglycerol were released, whereas no appreciable hydrolysis of cholesteryl[3H]oleate occurred during the 30 min of preincubation with GL (Fig. 2). After 30 min pH was increased to 7.0, bile salts were added to give a total concentration of 10 mM, and then CEL was added. During the following 60 min approx. 5.5% of the cholcsteryl[3H]oleate was hydrolysed and release of fatty acids from tri[14C]oleoylglycerol continued to a level of 14%. However, in the absence of gastric lipase the activity of CEL against these two lipids was low or absent. A second control experiment was performed in order to check if the hydrolysis of cholesteryl[3H]oleate and tri[ J4C]oleoylglycerol could be due to activation of GL when pH was raised and bile salts were added. However, no further hydrolysis after the preincubation period was observed when CEL was omitted (Fig. 2). The results ef this study showed that preineubation with either GL or PLA 2 was necessary in order for hydrolysis of cholesteryl oleate and triolein to occur in a TO/PC-emulsion to which CEL was added. Although cholesteryl oleate and triolein are potential substrates for CEL, they were not hydrolysed by CEL
196 100
A
i
-~~ a0 /'~" o = "~ ~., ___o60
-or"o _, g'~ :~.
+PLA2 •
"
-1"
•
•
i
40
o
,o. ~. 20
_PLA2
0~ - ~ g
8
B
°
~
"~
/
6
/
,u
/
ucts from the emulsion droplets into the aqueous phase, where they could form mixed micellar aggregates together with bile salts. In miccllar form cholesteryl oleate became available for hydrolysis by CEL. The mechanism of action of G L was different. G L g e n e r a t e d a lipolytic product phase by hydrolysing triolein. Although the phospholipids were intact, the transfer of cholesteryl oleate together with lipolytic products into mixed micellar aggregates was sufficient to allow C E L - m e d i a t e d cholesteryl oleate hydrolysis. It should be pointed out that the observed effects of P L A 2 or G L on the activity of C E L could not be explained by proteolytic activation of the lipolytic en-
PLA, pH6
~
A
8
;!:
pH7
I"
BS
-PLA 2 D__D~D--O~
g'~c "5 o¢
o-o
i
/'
{
PLA 2
o
,...,i,/'/"
-
~
2 ~o/~o----o--o--o--o
01
~0
0 30 60 90 | Time [min) -+PLA2 CEL Fig. I. Hydrolysis(expressed as % of the total amount of radioactivity) of l-palmitoyl,2-[ 3H]oleoylphosphatidylcholine (A), cholesteryl[3H]oleate (B) and tri[14C]oleoylglycerol(C) in a triolein/ phosphat dy choline emulsion incubated with 100/~g PLA 2 + 125/.¢g CEL (=) or 125/~g CEL alone (O) in the presence of 10 mM bile salts at pH 7.0. Arrows indicate the addition of enzymes. The composition of the emulsion system and the experimental design was the same as in [4]. Data represent the mean of two experiments.
alone u n d e r these conditions. The presence of phospholipids on the surface of the lipid emulsion droplets is probably the factor that inhibits CEL, since the enzyme has b e e n r e p o r t e d to hydrolyse triglycerides when emulsified by other amphiphilic substances than phospholipids [1,2]. The efficient hydrolysis of the phospholipids by PLA2 in our experiments was probably responsible for making the triolein molecules on the surface of the emulsion particles available for hydrolysis by CEL. Thereby a lipolytic product phase ( m a d e up by oleic acid and monoolein) was formed. Cholesteryl oleate was probably transferred together with lipolytic prod-
15
I
o~
_
o
>,~
,/'//
10
=/ =~
I
0 -* GL
30 *- C E L
./ 60
90
TIME Cmin?
Fig. 2. Hydrolysis (exprassed as % of the total amount of radioactivity) of cholesteryl[ 3H]oleate (A) and tri[14C]oleoylglycerol (B) in a triolein/phosphatldylcbollne emulsion incubated with 40 g.g GL+ 125 #g CEL ( | ) , 40 /zg GL (,~) or 125 #g CEL (t~). Arrows indicate addition of enzymes. The composition of the emulsion system and the experimental design was the same as i[ [4], except that pH was shifted from 6.0 to 7.0 and bile salts (BS) were addedto a total concentration of 10 mM after 30 rain of incubation. Data represent the mean of two experiments.
197 zymes used in these experiments. The lipolytic enzymes were purified according to standard procedures. Consequently, impurities in the form of proteolytic enzymes ought to be negligible. Moreover, C E L is according to current knowledge [6] secreted by the pancreas as ,'.'n active enzyme which requires no proteolytic activation. O f the enzymes used in this study, only PI-,k 2 is known to be secreted in an inactive proform. However, the P L A z used had been activated by trypsin unc:er controlled conditions before it was used in the experiments. In conclusion, this study showed that a concerted action of C E L and P L A 2 or C E L and G L makes possible the hydrolysis of cholesteryl esters in vitro. e E L and pancreatic lipase were earlier found to act in a similar way [4]. O t h e r examples of concerted action b e t w e e n lipolytic e n z y m e s have b e e n r e p o r t e d . Borgstr6m [7] showed that hydrolysis of phospholipids by P L A 2 in a p h o s p h o l i p i d / s t a b i l i s e d triglyceride emulsion stimulated the action of pancreatic lipase against triglycerides. Gargouri et al. [8], found that prehydrolysis of a similar emulsion system by gastric lipase also stimulated the activity of pancreatic lipase. Finally, it has b e e n reported by Bernb~ick et al. [9] that a concerted action involving GL, pancreatic lipase and bile salt-stimulated lipase from breast milk is necessary for the complete hydrolysis of milk triglycerides in vitro. The general conclusion from these studies is that one lipolytic enzyme can affect the activity of a n o t h e r lipolytic enzyme. The action of the first enzyme changes the general physico-chemical state of the lipids so that the substrate of the second enzyme becomes av~'!able
for hydrolysis. Thus, a concerted action of the different lipolytic enzymes in the gastro-intestinal tract is probably one important mechanism responsible for the efficient digestion and absorption of the dietary lip:~ds. The authors are grateful to Drs Bernb~ick, de Haas and Sternby for the generous supply of enzymes. Technical assistance was provided by UUa Giilich and Monika Lindstr6m. The study was supported by grants from the Swedish Medical Research Council (B8903X-00071-25A to B.B.), the Medical Faculty at the University of Lund, Albert P~hlsson's Foundation, Maim6, and the Swedish Nutrition Foundation, G6teborg.
References I Rudd, E.A. and H L. Brockman (1984) in Lipases (BorgstriSm,B. and Brockman, H.L., eds.), pp. 185-204, Elsevier, Amsterdam. 2 BI/ickberg, L., D. Lombardo, O. Hernell, O. Guy and T. Olivecrona (1081) FEBS ten. 136, 284-288. 3 Hernell, O. and L. nliickberg (1982) Pediatr. Res, 16, 882-885. 4 Lindstr6m, M.B., B. Sternbv and B. Borgstr(Sm0988) Biochim. Biophys. Acta 959, 178-184. 5 Belfrage, P. and M. Vaughan 0969) J. Lipids Res 10, 341-344. 6 Borgstr/3m,B. (1986) in The Exocrine Pancreas: Biology, Pathobiology and Diseases (Go, V.L.W., Gardner, J.D., Brooks, F.P., Lcbenthal, E.. DiMagno, E.P. and Scheele, G.A., eds.), pp. 361373, Raven Press, New York. 7 Borgstr~m, B. (1980) Gastroenterolclgy "?,SO~4-06~ 8 Gargouri, Y., G. Pieroni, C. Rivi~re, P.A. Lowe, J.-F. Sanni~re, L. Sarda and R. Verger (1986) Biochim. Biophys. Acta 879, 419-423. 9 Bernbiick, S., L. Bl~ickbergand O. Hernell (1990) J. Clin. Invest. 85, 1221-1226.
