111
Biochimicu et Biophysics Acta, 1044 (1990) 111-117 Elsevier
BBALIP 53387
Effects of human pancreatic lipase-colipase and carboxyl ester lipase on eicosapentaenoic and arachidonic acid ester bonds of triacylglycerols rich in fish oil fatty acids Qi Chen ‘, Berit Sternby
*, Bjijrn Akesson
* and Ake Nilsson ’
’ Department of Medicine and 2 Department of Physiological Chemistry, University of Lund (Sweden) (Received 12 October 1989)
Key words: Lipase; Colipase; Carboxyl ester lipase; Icosapentaenoic acid; Arachidonic acid; (Fish oil)
Fish oil chylomicrons, obtained from mesenteric duct chyle of rats fed 13H]20: 5 and [ 14C]20: 4 or 13H]20: 5 and [ 14C]18: 2 in a fish oil emulsion, were incubated with human pancreatic lipase-colipase, human carboxyl ester lipase (CEL) and human duodenal contents. With duodenal contents, the triacylglycerols labelled with 13H]20:5 and [‘4C]20:4 were rapidly converted to free fatty acids (FFA) and monoacylglycerols. Also during incubation with lipase-colipase the [ 3H]- and [14C]triacylglycerols disappeared completely and at equal rates, but in this case much [ 3H]20: 5 and [ 14C]20 : 4 accumulated in diacylglycerols. When CEL was also added, the rate of disappearance of [ 3H]and [‘4C]triacylglycerols increased and the radioactivity of diacylglycerols decreased markedly. During incubation of chylomicrons labelled with [ 3H]20: 5 and [ 14C]18: 2 with lipase-colipase, the rates of hydrolysis of [ 3H]- and [ “C]triacylglycerols were similar, but more [ 3H]20: 5 than [ “C]18: 2 accumulated in diacylglycerols. The accumulation of [ 3H]diacylglycerol was reduced by adding CEL. Also when fatty acids were analyzed by gas chromatography, 20 : 5 was enriched in remaining triacylglycerol and in diacylglycerol after incubation with lipase-colipase alone. The data thus indicate that both lipase-colipase and CEL participate in the hydrolysis of 20 : 5 and 20: 4 ester bonds of dietary triacylglycerol.
Introduction Diets rich in marine oils lower plasma triacylglycerols and alter platelet function and eicosanoid formation (for references see Ref. 1). These effects may be related to the epidemiological findings, showing an inverse relationship between coronary heart disease and consumption of a diet high in n - 3 fatty acids (see Ref. 2, for other references see Refs. 1 and 3). Despite the large number of studies conducted in this area, relatively little is known about the digestion and absorption of the fish oil triacylglycerol and the subsequent tissue uptake of dietary polyenoic C&,-C,, fatty acids. Studies by Bottino et al. [4] indicated that the polyenoic 20-22 carbon fatty acid ester bonds of fish oil triacylglycerol may be poorer substrates for pig pancreatic lipase than the C,,-C,, fatty acid esters.
Abbreviations: CEL, carboxyl ester lipase; 20: 5, eicosapentaenoic acid; 20 : 4, arachidonic acid; FFA, free fatty acid. Correspondence: A. Nilsson, Department of Medicine, University of Lund, S-221 85 Lund, Sweden. 0005-2760/90/%03.50
Recently it was reported that the postprandial rise in EPA of plasma triacylglycerol in man was larger when fish oil fatty acids were fed as unesterified fatty acid than as triacylglycerol or ethyl esters [5,6]. It was suggested that this was due to the slow digestion of the fish oil fatty acid esters by pancreatic lipase. In a previous study [7] we incubated [3H]20 : 4- and [14C]18 : 2-labelled, linoleate-rich chylomicrons with human pancreatic lipase-colipase and carboxylester lipase (CEL). During the hydrolysis with lipase-colipase alone, the amount of 3H appearing in 1,2-X-diacylglycerol markedly exceeded that of 14C. When also CEL was added, this [ 3H]diacylglycerol was efficiently hydrolyzed. If acylglycerols containing polyenoic 20- and 22-carbon fatty acids exhibit a relatively resistance to pancreatic lipase, a concerted action of this enzyme and CEL, which has a broad substrate specificity, may be necessary to rapidly completely the hydrolysis of fish oil triacylglycerol in the intestinal lumen. In this study we used mesenteric duct chylomicrons, which were rich in n - 3 fatty acids and labelled with [14C]20 : 4 and [3H]20 : 5 or [14C]18 : 2 and [3H]20 : 5, as model substrates to study the effects of human lipase-
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
112 colipase and CEL on the triacylglycerol these fatty acids.
ester bonds
of
Materials and Methods Human lipase and colipase were purified from human pancreatic glands [8,9] and CEL from pancreatic juice [lo]. Human duodenal contents were collected as described earlier [7]. The contents of lipase, colipase and CEL in the duodenal contents were determined by radioimmunoassay [11,12]. The fish oil emulsion was prepared as follows: 50 PCi of [5,6,8,9,11,12,14,15,17, 18-3H]20: 5 and 50 PCi of [1-‘4C]20: 4, or 50 PCi of [3H]20: 5 and 50 PCi of [l-i4C]18: 2 (New England Nuclear Corporation) were mixed with 1 ml of a chloroform solution containing 1 mg egg phosphatidylcholine per ml. The mixture was blown to dryness with N, and immediately dispersed in 0.9% NaCl by buzzing. The dispersion was then mixed with a 10% (v/v) fish oil triacylglycerol (MAX-EPA, Naturprodukter, Grebro, Sweden) emulsion, which had been prepared by ultrasonication for 2 min in 0.9% NaCl containing 1% gum arabic. The fish oil contained as major n - 3 fatty acids, 20 : 5 (16.5%) and 22 : 6 (12.3%). The mesenteric lymph duct of male white SpragueDawley rats, weighing 200-280 g, was cannulated and a gastric fistula was inserted with the tip placed in the proximal duodenum [13]. The rats were treated postoperatively as described earlier [14]. 24 h after surgery, 2 ml of the radioactive fish oil emulsion was fed through the gastric fistula and chyle was then collected on ice in the presence of Na,EDTA (final concentration 2 mM) and stored at 4°C. Chylomicrons were isolated as described earlier [7]. The composition of the incubation medium was 10 mM Hepes buffer (pH 7.4), 2.5 mM CaCl,, 0.12 M NaCl and 3.5 mM sodium taurodeoxycholate [7]. Incubations were performed at 37 o C in a total volume of 3 ml. After preincubating the medium with the enzymes for 5 min at 37O C the chylomicrons labelled with [3H]20 : 5 and [14C]20 : 4 were added. In another series of incubations, with chylomicrons labelled with [3H]20 : 5 and [14C]18 : 2, the same conditions were used as described above, except that the mass of chylomicron triacylglycerol, the amount of enzyme and the total volume (30 ml) were increased, to make gas chromatographic analyses of fatty acids possible. Aliquots of the mixture were taken at the time intervals indicated in the figures and were immediately mixed with 8 ~01s. of chloroform/methanol (1 : 1, v/v) containing 0.005% butylated hydroxytoluene. The lipids were extracted and separated by thin-layer chromatography as described earlier [7]. The spots were visualized by staining with iodine. Tri-, di- and monoacylglycerols and FFA fractions were scraped into counting vials into which 1 ml methanol/water (1 : 1, v/v) and 10 ml Instagel/ toluene (1 : 1) were added. Radioactivity was
determined in a Packard 460 CD liquid scintillation system using the computerized external standard for quench correction. In the incubation series using a larger amount of chylomicrons labelled with [ 3H]20 : 5 and [i4C]18: 2 as substrate, the mass distribution of different fatty acids into various lipid classes was analyzed by thin-layer chromatography and gas chromatography as described by Ekstrom et al. [ll]. The triacylglycerol content of chylomicrons was determined by a calorimetric enzymatic kit method (BoehringerMannheim, F.R.G.).
Results When a constant concentration of lipase-colipase was incubated with the [ 3H]20 : 5 and [14C]20 : 4 labelled chylomicrons the time-courses for the disappearance of [ 3H]20 : 5- and [14C]20 : 4-labelled triacylglycerol were very similar, the conversion to FFA and partial acylglycerols being almost complete in 30 min (Fig. 1). At 5-30 min, the amounts of 3H and 14C appearing in diacylglycerols markedly exceeded those in monoacylglycerol and reached a maximum at 20 min. At 60 min the radioactivity of monoacylglycerol was similar to that of diacylglycerol. As the 3H and 14C in diacylglycerol declined between 30 and 60 min, the radioactivity of FFA, but not of monoacylglycerol, increased correspondingly (Fig. 1). The hydrolysis of both the [ 3H]- and [‘4C]triacylglycerol was faster when CEL was added in addition to lipase-colipase (Fig. 1). The amount of 3H and 14C in diacylglycerol was also markedly decreased by the addition of CEL. At the high CEL concentration, virtually no 14C and only 2% of the 3H remained in diacylglycerols after 60 min. The radioactivity of the FFA formed increased with increasing hydrolysis of tri- and diacylglycerols by CEL, whereas the CEL had little or no influence on the amount of [ 3H]20 : 5 and [14C]20 : 4 in monoacylglycerols. The 14C radioactivity of monacylglycerols exceeded that of 3H, the amounts of 14C and 3H appearing in monoacylglycerols after 30-60 min being 10 and 6%, respectively (Fig. 1). When [ 3H]20 : 5 and [14C]20 : 4 labelled chylomicrons were incubated with duodenal contents the initial rate of hydrolysis of both [3H]- and [‘4C]triacylglycerol was fast, the hydrolysis being completed within 5 min (Fig. 2). The clearance of intermediary [‘4C]diacylglycerol was complete in less than 10 min, whereas small amounts of 3H radioactivity appeared in diacylglycerol also at the longer time intervals. The diacylglycerol radioactivity appeared mainly in the 1,2-X-isomer in the incubations with lipase-colipase. The disappearance of [ 3H]- and [‘4C]diacylglycerol when CEL was also added must thus reflect the action of CEL on the 1,2-X-diacylglycerol isomer. The radioactiv-
113 * :, > f
L
3H =TG 80
I: 60 40 20 0
IO
20
60
30
TIME
”
10
20
(min)
10
60
30
20
60
30
14 C*MG
3H4i3G
L
5 n
ii
0
10
30]
20
30
p\
60
10 30
3H-DG
1
20
30
A
60 14C.DG
20
10 :R' 10
20
30
60 TIME
0'
10
20
30
60
(min)
Fig. 1. Effects of CEL on the time-course for hydrolysis of [3H]20 : 5- and 1’4C]20 : Clabellcd chylomicron triacylglycerol by lipase-colipase. 100 ~1 of chylomicrons containing 477 pg of triacylglycerol were added after 5 min preincubation with the enzymes, the total radioactivity of chylomicrons added being 6.0 X lo4 dpm of ‘H and 8.3.10’ dpm of 14C. The enzymes added are indicated as follows: lipase-colipase (1 : 1, w/w) 0.25 pg without CEL, (0);
and with CEL 1 pg, (A); or 10 pg, (I).
114 100
TG
201
jpk+
F
80 60
DG
10 FFA
TIME
20
1
20 MG
(min)
Fig. 2. Time-course for the hydrolysis of [3H]20: 5- and [t4C]20: 4-labelled chylomicron triacylglycerol by human duodenal content. 10 pl duodenal content containing 4.3 ng lipase, 0.1 pg colipase and 6.7 ng CEL was incubated with 100 ~1 of the chylomicron preparation described in the legend to Fig. 1, using the conditions described in Materials and Methods. The open symbols are [ 3H]20 : 5 and closed symbols are [14C]20 : 4.
ity of the 1,3- was far less than that of the 1,2-X-diacylglycerol (data not shown). In the experimental series with chylomicrons labelled with [3H]20 : 5 and [i4C]18 : 2 as a substrate, the hydrolysis of [‘4C]triacylglycerol by lipase-colipase after 60 and 90 min was somewhat more extensive than that of [3H]triacylglycerol (Fig. 3). The amount of 3H radioactivity in 1,2-X-diacylglycerol, however, markedly exceeded that of i4C. About 25% of the 3H was in diacylglycerol at 60 min as compared to less than 10% of the 4C. When CEL was added to the incubation with lipase-colipase, the accumulation of 3H in diacylglycerol was reduced to the same level as [‘4C]diacylglycerol (Fig. 3). The data on fatty acid composition confirmed that when triacylglycerol was degraded into di- and monoacylglycerol, and FFA during incubation with lipasecolipase, the distribution of individual fatty acids between different lipid classes varied (Table I). The changes in the distribution of the C,,-C,, fatty acids that had occurred after 90 min incubation differed from those of the C&-C,, fatty acids. For instance, the proportion of 20 : 4, 20: 5, 22: 5 and 22: 6 in diacylglycerol increased to 23.6-30.0%, whereas the proportions of the C16-Cl8 fatty acids in diacylglycerol increased to 5.9-15.1%. Correspondingly, the increase in the proportion found in FFA was less for the polyenoic C,,-C,, fatty acids than for the C,,-C,, acids (Table I). There was also an overrepresentation of 20: 5 in remaining triacylglycerol. Discussion The lipase-colipase hydrolyzed both the [ 3H]20 : 5and [ 14C]20 : 4-triacylglycerol to partial acylglycerols and FFA completely and at equal rates. There was thus
no evidence for the existence of a triacylglycerol pool containing [ 3H]20 : 5 or [14C]20 : 4, that was resistant to the initial step of the lipase action, i.e., the conversion of tri- to diacylglycerols. Pancreatic lipase is specific for the ester bonds of primary alcohols, its main substrate being the l- and 3-esters of acylglycerols (for references see Ref. 16). In the present study, more than 75% of both the [ 3H]20 : 5 and [14C]20 : 4 could be released as FFA by lipase-colipase, most of the remaining parts being in a diacylglycerol pool that underwent only a slow hydrolysis. The amount of [ 3H]20 : 5 (Fig. 1) and of 20 : 5 mass (Table I) in monoacylglycerol for the longest time intervals (60 and 90 min) did not exceed 10% indicating that the 20 : 5 esters were located mainly at primary ester bonds. [3H]20 : 5 and [14C]20 : 4 exhibited a similar lipolysis pattern (Fig. l), indicating that the susceptibility to lipase action, as well as the positional location of the two fatty acids were similar. Older studies by Bottino et al. [4] as well as recent findings of Lawson and Hughes [5] indicate that 20 : 5 is located primarily at the 1 and 3 position of whale fish oil, although there may be variations between different marine oils [17,18]. A recent study indicated that the docosahexaenoate in the triacylglycerol of fish oil was esterified primarily to the ~2-2 position of glycerol, whereas eicosapentaenoate had a more random distribution over all glycerol positions [19]. In the present study, the amount of 22 : 6 accumulation in MG after incubation with lipase-colipase exceeded that of 20 : 5 (Table I), yet much more was released as FFA, indicating that the major portion also of 22: 6 was esterified to the primary positions in the chylomicrons. CEL had two effects on the lipolysis course when added together with lipase-colipase. First, it increased the rate of elimination of both [14C]- and [3H]triacylglycerol and second, it markedly decreased the accumu-
115
TIME (min)
3H.1.2-X-DG
14C .l.P-X-DG
40 i
10
30
60
10
90
30
60
90
TIME (min) Fig 3. Time-course for the hydrolysis of [3H]20: S- and [‘4C]18: 2-labelled chylomicron triacylglycerol by hpase-colipase with and without CEL present. 0.5 ml of [3H]20:5- and [14C]18:2-labell~ ~hylo~crons containing 5.1 mg triacylglycerol and 2.0 ml of unlabelied chylomicrons containing 25 mg triacylglycerol were preincubated with 32 ml buffer at 37’ C, 15 mm before enzymes were added. The enzyme was then added, and aliquots analyzed at the time intervals indicated. The open symbols are incubations with lipase-colipase alone 15 ~1 (2: 1) and the closed symbols are with hpase-colipase pfus CEL (80 ~1).
lation of [r4C]20 : 4 and f3H]20 : 5 in diacylglycerol. In the previous study [7], using [3H]20 : 4- and [14C]18 : 2labelled, linoleate-rich chylomicrons as substrate, CEL had little effect on the disappearance-rate of radioactive triacyI~ycero1, ~thou~ it markedly increased the elimination of the [ 3H]20 : 4-labelled diacylglycerol formed. The presence in the fish oil chylomicrons of a
large proportion of polyenoic fatty acid esters being a relatively poor substrate for lipase-colipase, may be the reason why CEL increased the [14C]- and [3H]triacylglycerol clearance in the present experiments, but not in the earlier experiments with linoleate-rich chylomicrons. In the latter incubations, [14C]triacylglycerol was almost completely hydrolyzed to monoa~yl~ycerol and FFA
116 TABLE I Fairy acid composition of chylomicron lipids (a) and drstribution of individual fatty acids among neutral lipids (b) after incubation with pancreatic lipase-colipase Data arc means of duplicate analyses. (a) Fatty acid
14:o 16:O 16:l 18:0 18:l 18:2 18:3 20:3 20 : 4 20:5 2215 22~6
Fatty acid composition (% of total fatty acids) Omin
90 min
TG
TG
DG
MG
FFA
9.0 23.6 8.3 3.1 15.6 1.1 1.3 0.4 2.0 15.9 2.0 11.7
5.4 19.8 6.6 2.5 15.3 5.1 1.1 0.4 2.9 24.7 2.2 13.9
6.7 17.5 6.2 1.4 10.7 6.0 1.3 0.3 3.4 26.6 2.7 17.1
10.2 26.7 7.9 1.1 12.8 10.0 1.2 0.3 2.7 9.9 2.4 14.9
8.5 26.5 8.4 5.9 20.4 5.9 1.3 0.4 1.6 10.6 1.8 8.7
(b) Fatty acid
Distribution of fatty acid (% of each fatty acid) at 90 min TG
DG
MG
FFA
14:o 16:0 16:l 18 :0 18 : 1 18:2 18:3 20:3 20:4 20:5 22:5 2216
16.9 18.6 19.5 15.2 19.2 19.1 19.1 22.3 25.6 30.0 23.6 24.8
13.6 11.8 12.8 5.9 10.2 15.1 16.3 10.6 22.9 26.3 19.5 22.0
16.5 14.2 13.5 4.0 9.1 20.1 12.7 11.3 15.8 9.0 15.5 17.6
53.0 55.5 54.2 74.9 60.9 45.7 51.9 55.8 35.7 34.7 41.3 35.6
Total fatty acids
21.4
15.7
13.2
49.7
by lipase-colipase alone, whereas the [ 3H]20 : 5 esters exhibited a lipolysis pattern similar to that observed in the incubations with [ 3H]20 : 5 and [14C]20 : 4. Also in comparison to [14C]18 : 2, the lipolysis pattern of the [3H]20 : 5 esters observed in the present study was thus similar to that observed for 20: 4 in the earlier study. After incubation of the fish oil chylomicrons with lipase-colipase, the gas-liquid chromatographic analysis of the individual lipid classes showed differences in the fatty acid distribution between remaining triacylglycerols, partial acylglycerols and FFA (Table I). The fatty acid composition of the chylomicrons, which were obtained from rats fed the fish oil emulsion was very similar to that of the fish oil used, in which the proportion of 18:2, 20:4, 20: 5 and 22:6 was 1.1. 0.8, 16.5 and 12.3%, respectively. After incubation with lipase-
colipase, 20 : 4 and 20 : 5 were enriched in diacylglycerol, thus confirming the findings made with the isotope-labelled substrates both in the present and the previous study [7]. Also the proportion of docosahexaenoic acid (22 : 6) in remaining tri- and diacylglycerol was higher than in the original triacylglycerol, suggesting that the 22 : 6 esters exhibit a lipolysis pattern similar to that of 20 : 4 and 20 : 5 esters. At the high substrate concentration, hydrolysis was slowly initiated by lipase-colipase (Fig. 3). This may be due to the presence of a phospholipid surface coat on the chylomicrons, which is not easily removed to facilitate the binding of the lipase-colipase to the oil/water interface under the condition used. Other studies [4] have indicated that some long-chain polyenoic 20-carbon fatty acid esters of marine oil triacylglycerol are hydrolyzed at a slower rate by porcine pancreatic lipase than the C,,-C,, fatty acids. The steric configuration of the fatty acid induced by the occurrence of a double bond near the carboxyl group, particularly at position 5, may be an important cause of this relative resistance [20]. CEL has a broader substrate specificity than lipase-colipase and will hydrolyze a large number of lipid substrates, such as acylglycerols (including the 2-ester bond), cholesteryl ester and vitamin A esters (for references see Ref. 21). The present study shows that CEL is active also against 20 : 4 and 20 : 5 esters of 1,2-X-diacylglycerol and triacylglycerol. The triacylglycerols rich in marine oil fatty acid esters may therefore be most efficiently hydrolyzed by the concerted action of the lipase-colipase and CEL. Acknowledgements This study was supported by grants from the Swedish Medical Research Council (Project No. 3969,3968), the Medical Faculty, University of Lund, the Pahlsson Foundation, the Nutrition Foundation of the Swedish Margarine Industry, the Johanna Andersson Foundation, the Research Foundations of the Hospital of Lund, the Swedish Medical Association and the National Association against Heart and Lung Diseases. References 1 2 3 4 5 6 7 8
Harris, W.S. (1989) .I. Lipid Res. 30, 785-807. Bang, H.O. and Dyerberg, J. (1972) Acta Med. Stand. 192, 85-89. Leaf, A., Weber, P.C. (1988) N. Engl. J. Med. 318, 549-557. Bottino, N.R., Vanderburg, G.A. and Reiser, R. (1967) Lipids 2, 489-493. Lawson, L.D. and Hughes, B.G. (1988) B&him. Biophys. Res. Commun. 152, 328-335. Boustani, ES., Colette, C., Monnier, L., Descomps, B., Paulet, A.C.D. and Mendy, F. (1987) Lipids 22, 711-714. Chen, Q., Stemby, B. and Nilsson, A. (1989) B&him. Biophys. Acta 1004, 372-385. Sternby, B. and Borgstrlim, B. (1981) Comp. B&hem. Physiol. 68B, 15-18.
117 9 Stemby, B. and Borgstrom, B. (1979) Biochim. Biophys. Acta 572, 235-243. 10 Lombardo, D., Guy, 0. and Figarella, C. (1978) B&him. Biophys. Acta 527, 142-149. 11 Erlanson, C. and Borgstrom, B. (1972) B&him. Biophys. Acta 271, 400-412. 12 Sternby, B. and Akerstrom, B. (1984) B&him. Biophys. Acta 789, 164-169. 13 Warshaw, A.L. (1972) Gut 13, 66-67. 14 Flortn, C.H. and Nilsson, A. (1977) Eur. J. B&hem. 77, 23-30. 15 Ekstrom, B., Nilsson, A. and Akesson, B. (1989) Eur. J. Clin. Invest. 19, 259-264. 16 Verger, R. (1984) in Pancreatic Lipase (Borgstrom, B. and Brockman, H.L., eds.), pp. 84-150, Elsevier, Amsterdam.
17 Brockerhoff, H., Hoyle, R.J. and Huang, P.C. (1966) Can. J. B&hem. 44,1519-1525. 18 Brockerhoff, H., Hoyle, R.J., Huang, P.C. and Litchfield, C. (1968) Lipids 3, 24-29. 19 Chernenko, G.A., Barrowman, J.A., Kean, K.T., Heraberg, G.R. and Keough, K.M.W. (1989) Biochem. Biophys. Acta 1004,95-102. 20 Heimermann, W.H., Holmann, R.T., Gordon, D.T., Kowalyshyn, D.E. and Jensen, R.G. (1973) Lipids 8, 45-47. 21 Rudd, E.A. and Brockman, H.L. (1984) in Pancreatic Carboxyl Ester Lipase (Borgstrom, B. and Brockman, H.L., eds.), pp. 185204, Elsevier, Amsterdam.
Biochimicu et Biophysics Acta, 1044 (1990) 111-117 Elsevier
BBALIP 53387
Effects of human pancreatic lipase-colipase and carboxyl ester lipase on eicosapentaenoic and arachidonic acid ester bonds of triacylglycerols rich in fish oil fatty acids Qi Chen ‘, Berit Sternby
*, Bjijrn Akesson
* and Ake Nilsson ’
’ Department of Medicine and 2 Department of Physiological Chemistry, University of Lund (Sweden) (Received 12 October 1989)
Key words: Lipase; Colipase; Carboxyl ester lipase; Icosapentaenoic acid; Arachidonic acid; (Fish oil)
Fish oil chylomicrons, obtained from mesenteric duct chyle of rats fed 13H]20: 5 and [ 14C]20: 4 or 13H]20: 5 and [ 14C]18: 2 in a fish oil emulsion, were incubated with human pancreatic lipase-colipase, human carboxyl ester lipase (CEL) and human duodenal contents. With duodenal contents, the triacylglycerols labelled with 13H]20:5 and [‘4C]20:4 were rapidly converted to free fatty acids (FFA) and monoacylglycerols. Also during incubation with lipase-colipase the [ 3H]- and [14C]triacylglycerols disappeared completely and at equal rates, but in this case much [ 3H]20: 5 and [ 14C]20 : 4 accumulated in diacylglycerols. When CEL was also added, the rate of disappearance of [ 3H]and [‘4C]triacylglycerols increased and the radioactivity of diacylglycerols decreased markedly. During incubation of chylomicrons labelled with [ 3H]20: 5 and [ 14C]18: 2 with lipase-colipase, the rates of hydrolysis of [ 3H]- and [ “C]triacylglycerols were similar, but more [ 3H]20: 5 than [ “C]18: 2 accumulated in diacylglycerols. The accumulation of [ 3H]diacylglycerol was reduced by adding CEL. Also when fatty acids were analyzed by gas chromatography, 20 : 5 was enriched in remaining triacylglycerol and in diacylglycerol after incubation with lipase-colipase alone. The data thus indicate that both lipase-colipase and CEL participate in the hydrolysis of 20 : 5 and 20: 4 ester bonds of dietary triacylglycerol.
Introduction Diets rich in marine oils lower plasma triacylglycerols and alter platelet function and eicosanoid formation (for references see Ref. 1). These effects may be related to the epidemiological findings, showing an inverse relationship between coronary heart disease and consumption of a diet high in n - 3 fatty acids (see Ref. 2, for other references see Refs. 1 and 3). Despite the large number of studies conducted in this area, relatively little is known about the digestion and absorption of the fish oil triacylglycerol and the subsequent tissue uptake of dietary polyenoic C&,-C,, fatty acids. Studies by Bottino et al. [4] indicated that the polyenoic 20-22 carbon fatty acid ester bonds of fish oil triacylglycerol may be poorer substrates for pig pancreatic lipase than the C,,-C,, fatty acid esters.
Abbreviations: CEL, carboxyl ester lipase; 20: 5, eicosapentaenoic acid; 20 : 4, arachidonic acid; FFA, free fatty acid. Correspondence: A. Nilsson, Department of Medicine, University of Lund, S-221 85 Lund, Sweden. 0005-2760/90/%03.50
Recently it was reported that the postprandial rise in EPA of plasma triacylglycerol in man was larger when fish oil fatty acids were fed as unesterified fatty acid than as triacylglycerol or ethyl esters [5,6]. It was suggested that this was due to the slow digestion of the fish oil fatty acid esters by pancreatic lipase. In a previous study [7] we incubated [3H]20 : 4- and [14C]18 : 2-labelled, linoleate-rich chylomicrons with human pancreatic lipase-colipase and carboxylester lipase (CEL). During the hydrolysis with lipase-colipase alone, the amount of 3H appearing in 1,2-X-diacylglycerol markedly exceeded that of 14C. When also CEL was added, this [ 3H]diacylglycerol was efficiently hydrolyzed. If acylglycerols containing polyenoic 20- and 22-carbon fatty acids exhibit a relatively resistance to pancreatic lipase, a concerted action of this enzyme and CEL, which has a broad substrate specificity, may be necessary to rapidly completely the hydrolysis of fish oil triacylglycerol in the intestinal lumen. In this study we used mesenteric duct chylomicrons, which were rich in n - 3 fatty acids and labelled with [14C]20 : 4 and [3H]20 : 5 or [14C]18 : 2 and [3H]20 : 5, as model substrates to study the effects of human lipase-
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
112 colipase and CEL on the triacylglycerol these fatty acids.
ester bonds
of
Materials and Methods Human lipase and colipase were purified from human pancreatic glands [8,9] and CEL from pancreatic juice [lo]. Human duodenal contents were collected as described earlier [7]. The contents of lipase, colipase and CEL in the duodenal contents were determined by radioimmunoassay [11,12]. The fish oil emulsion was prepared as follows: 50 PCi of [5,6,8,9,11,12,14,15,17, 18-3H]20: 5 and 50 PCi of [1-‘4C]20: 4, or 50 PCi of [3H]20: 5 and 50 PCi of [l-i4C]18: 2 (New England Nuclear Corporation) were mixed with 1 ml of a chloroform solution containing 1 mg egg phosphatidylcholine per ml. The mixture was blown to dryness with N, and immediately dispersed in 0.9% NaCl by buzzing. The dispersion was then mixed with a 10% (v/v) fish oil triacylglycerol (MAX-EPA, Naturprodukter, Grebro, Sweden) emulsion, which had been prepared by ultrasonication for 2 min in 0.9% NaCl containing 1% gum arabic. The fish oil contained as major n - 3 fatty acids, 20 : 5 (16.5%) and 22 : 6 (12.3%). The mesenteric lymph duct of male white SpragueDawley rats, weighing 200-280 g, was cannulated and a gastric fistula was inserted with the tip placed in the proximal duodenum [13]. The rats were treated postoperatively as described earlier [14]. 24 h after surgery, 2 ml of the radioactive fish oil emulsion was fed through the gastric fistula and chyle was then collected on ice in the presence of Na,EDTA (final concentration 2 mM) and stored at 4°C. Chylomicrons were isolated as described earlier [7]. The composition of the incubation medium was 10 mM Hepes buffer (pH 7.4), 2.5 mM CaCl,, 0.12 M NaCl and 3.5 mM sodium taurodeoxycholate [7]. Incubations were performed at 37 o C in a total volume of 3 ml. After preincubating the medium with the enzymes for 5 min at 37O C the chylomicrons labelled with [3H]20 : 5 and [14C]20 : 4 were added. In another series of incubations, with chylomicrons labelled with [3H]20 : 5 and [14C]18 : 2, the same conditions were used as described above, except that the mass of chylomicron triacylglycerol, the amount of enzyme and the total volume (30 ml) were increased, to make gas chromatographic analyses of fatty acids possible. Aliquots of the mixture were taken at the time intervals indicated in the figures and were immediately mixed with 8 ~01s. of chloroform/methanol (1 : 1, v/v) containing 0.005% butylated hydroxytoluene. The lipids were extracted and separated by thin-layer chromatography as described earlier [7]. The spots were visualized by staining with iodine. Tri-, di- and monoacylglycerols and FFA fractions were scraped into counting vials into which 1 ml methanol/water (1 : 1, v/v) and 10 ml Instagel/ toluene (1 : 1) were added. Radioactivity was
determined in a Packard 460 CD liquid scintillation system using the computerized external standard for quench correction. In the incubation series using a larger amount of chylomicrons labelled with [ 3H]20 : 5 and [i4C]18: 2 as substrate, the mass distribution of different fatty acids into various lipid classes was analyzed by thin-layer chromatography and gas chromatography as described by Ekstrom et al. [ll]. The triacylglycerol content of chylomicrons was determined by a calorimetric enzymatic kit method (BoehringerMannheim, F.R.G.).
Results When a constant concentration of lipase-colipase was incubated with the [ 3H]20 : 5 and [14C]20 : 4 labelled chylomicrons the time-courses for the disappearance of [ 3H]20 : 5- and [14C]20 : 4-labelled triacylglycerol were very similar, the conversion to FFA and partial acylglycerols being almost complete in 30 min (Fig. 1). At 5-30 min, the amounts of 3H and 14C appearing in diacylglycerols markedly exceeded those in monoacylglycerol and reached a maximum at 20 min. At 60 min the radioactivity of monoacylglycerol was similar to that of diacylglycerol. As the 3H and 14C in diacylglycerol declined between 30 and 60 min, the radioactivity of FFA, but not of monoacylglycerol, increased correspondingly (Fig. 1). The hydrolysis of both the [ 3H]- and [‘4C]triacylglycerol was faster when CEL was added in addition to lipase-colipase (Fig. 1). The amount of 3H and 14C in diacylglycerol was also markedly decreased by the addition of CEL. At the high CEL concentration, virtually no 14C and only 2% of the 3H remained in diacylglycerols after 60 min. The radioactivity of the FFA formed increased with increasing hydrolysis of tri- and diacylglycerols by CEL, whereas the CEL had little or no influence on the amount of [ 3H]20 : 5 and [14C]20 : 4 in monoacylglycerols. The 14C radioactivity of monacylglycerols exceeded that of 3H, the amounts of 14C and 3H appearing in monoacylglycerols after 30-60 min being 10 and 6%, respectively (Fig. 1). When [ 3H]20 : 5 and [14C]20 : 4 labelled chylomicrons were incubated with duodenal contents the initial rate of hydrolysis of both [3H]- and [‘4C]triacylglycerol was fast, the hydrolysis being completed within 5 min (Fig. 2). The clearance of intermediary [‘4C]diacylglycerol was complete in less than 10 min, whereas small amounts of 3H radioactivity appeared in diacylglycerol also at the longer time intervals. The diacylglycerol radioactivity appeared mainly in the 1,2-X-isomer in the incubations with lipase-colipase. The disappearance of [ 3H]- and [‘4C]diacylglycerol when CEL was also added must thus reflect the action of CEL on the 1,2-X-diacylglycerol isomer. The radioactiv-
113 * :, > f
L
3H =TG 80
I: 60 40 20 0
IO
20
60
30
TIME
”
10
20
(min)
10
60
30
20
60
30
14 C*MG
3H4i3G
L
5 n
ii
0
10
30]
20
30
p\
60
10 30
3H-DG
1
20
30
A
60 14C.DG
20
10 :R' 10
20
30
60 TIME
0'
10
20
30
60
(min)
Fig. 1. Effects of CEL on the time-course for hydrolysis of [3H]20 : 5- and 1’4C]20 : Clabellcd chylomicron triacylglycerol by lipase-colipase. 100 ~1 of chylomicrons containing 477 pg of triacylglycerol were added after 5 min preincubation with the enzymes, the total radioactivity of chylomicrons added being 6.0 X lo4 dpm of ‘H and 8.3.10’ dpm of 14C. The enzymes added are indicated as follows: lipase-colipase (1 : 1, w/w) 0.25 pg without CEL, (0);
and with CEL 1 pg, (A); or 10 pg, (I).
114 100
TG
201
jpk+
F
80 60
DG
10 FFA
TIME
20
1
20 MG
(min)
Fig. 2. Time-course for the hydrolysis of [3H]20: 5- and [t4C]20: 4-labelled chylomicron triacylglycerol by human duodenal content. 10 pl duodenal content containing 4.3 ng lipase, 0.1 pg colipase and 6.7 ng CEL was incubated with 100 ~1 of the chylomicron preparation described in the legend to Fig. 1, using the conditions described in Materials and Methods. The open symbols are [ 3H]20 : 5 and closed symbols are [14C]20 : 4.
ity of the 1,3- was far less than that of the 1,2-X-diacylglycerol (data not shown). In the experimental series with chylomicrons labelled with [3H]20 : 5 and [i4C]18 : 2 as a substrate, the hydrolysis of [‘4C]triacylglycerol by lipase-colipase after 60 and 90 min was somewhat more extensive than that of [3H]triacylglycerol (Fig. 3). The amount of 3H radioactivity in 1,2-X-diacylglycerol, however, markedly exceeded that of i4C. About 25% of the 3H was in diacylglycerol at 60 min as compared to less than 10% of the 4C. When CEL was added to the incubation with lipase-colipase, the accumulation of 3H in diacylglycerol was reduced to the same level as [‘4C]diacylglycerol (Fig. 3). The data on fatty acid composition confirmed that when triacylglycerol was degraded into di- and monoacylglycerol, and FFA during incubation with lipasecolipase, the distribution of individual fatty acids between different lipid classes varied (Table I). The changes in the distribution of the C,,-C,, fatty acids that had occurred after 90 min incubation differed from those of the C&-C,, fatty acids. For instance, the proportion of 20 : 4, 20: 5, 22: 5 and 22: 6 in diacylglycerol increased to 23.6-30.0%, whereas the proportions of the C16-Cl8 fatty acids in diacylglycerol increased to 5.9-15.1%. Correspondingly, the increase in the proportion found in FFA was less for the polyenoic C,,-C,, fatty acids than for the C,,-C,, acids (Table I). There was also an overrepresentation of 20: 5 in remaining triacylglycerol. Discussion The lipase-colipase hydrolyzed both the [ 3H]20 : 5and [ 14C]20 : 4-triacylglycerol to partial acylglycerols and FFA completely and at equal rates. There was thus
no evidence for the existence of a triacylglycerol pool containing [ 3H]20 : 5 or [14C]20 : 4, that was resistant to the initial step of the lipase action, i.e., the conversion of tri- to diacylglycerols. Pancreatic lipase is specific for the ester bonds of primary alcohols, its main substrate being the l- and 3-esters of acylglycerols (for references see Ref. 16). In the present study, more than 75% of both the [ 3H]20 : 5 and [14C]20 : 4 could be released as FFA by lipase-colipase, most of the remaining parts being in a diacylglycerol pool that underwent only a slow hydrolysis. The amount of [ 3H]20 : 5 (Fig. 1) and of 20 : 5 mass (Table I) in monoacylglycerol for the longest time intervals (60 and 90 min) did not exceed 10% indicating that the 20 : 5 esters were located mainly at primary ester bonds. [3H]20 : 5 and [14C]20 : 4 exhibited a similar lipolysis pattern (Fig. l), indicating that the susceptibility to lipase action, as well as the positional location of the two fatty acids were similar. Older studies by Bottino et al. [4] as well as recent findings of Lawson and Hughes [5] indicate that 20 : 5 is located primarily at the 1 and 3 position of whale fish oil, although there may be variations between different marine oils [17,18]. A recent study indicated that the docosahexaenoate in the triacylglycerol of fish oil was esterified primarily to the ~2-2 position of glycerol, whereas eicosapentaenoate had a more random distribution over all glycerol positions [19]. In the present study, the amount of 22 : 6 accumulation in MG after incubation with lipase-colipase exceeded that of 20 : 5 (Table I), yet much more was released as FFA, indicating that the major portion also of 22: 6 was esterified to the primary positions in the chylomicrons. CEL had two effects on the lipolysis course when added together with lipase-colipase. First, it increased the rate of elimination of both [14C]- and [3H]triacylglycerol and second, it markedly decreased the accumu-
115
TIME (min)
3H.1.2-X-DG
14C .l.P-X-DG
40 i
10
30
60
10
90
30
60
90
TIME (min) Fig 3. Time-course for the hydrolysis of [3H]20: S- and [‘4C]18: 2-labelled chylomicron triacylglycerol by hpase-colipase with and without CEL present. 0.5 ml of [3H]20:5- and [14C]18:2-labell~ ~hylo~crons containing 5.1 mg triacylglycerol and 2.0 ml of unlabelied chylomicrons containing 25 mg triacylglycerol were preincubated with 32 ml buffer at 37’ C, 15 mm before enzymes were added. The enzyme was then added, and aliquots analyzed at the time intervals indicated. The open symbols are incubations with lipase-colipase alone 15 ~1 (2: 1) and the closed symbols are with hpase-colipase pfus CEL (80 ~1).
lation of [r4C]20 : 4 and f3H]20 : 5 in diacylglycerol. In the previous study [7], using [3H]20 : 4- and [14C]18 : 2labelled, linoleate-rich chylomicrons as substrate, CEL had little effect on the disappearance-rate of radioactive triacyI~ycero1, ~thou~ it markedly increased the elimination of the [ 3H]20 : 4-labelled diacylglycerol formed. The presence in the fish oil chylomicrons of a
large proportion of polyenoic fatty acid esters being a relatively poor substrate for lipase-colipase, may be the reason why CEL increased the [14C]- and [3H]triacylglycerol clearance in the present experiments, but not in the earlier experiments with linoleate-rich chylomicrons. In the latter incubations, [14C]triacylglycerol was almost completely hydrolyzed to monoa~yl~ycerol and FFA
116 TABLE I Fairy acid composition of chylomicron lipids (a) and drstribution of individual fatty acids among neutral lipids (b) after incubation with pancreatic lipase-colipase Data arc means of duplicate analyses. (a) Fatty acid
14:o 16:O 16:l 18:0 18:l 18:2 18:3 20:3 20 : 4 20:5 2215 22~6
Fatty acid composition (% of total fatty acids) Omin
90 min
TG
TG
DG
MG
FFA
9.0 23.6 8.3 3.1 15.6 1.1 1.3 0.4 2.0 15.9 2.0 11.7
5.4 19.8 6.6 2.5 15.3 5.1 1.1 0.4 2.9 24.7 2.2 13.9
6.7 17.5 6.2 1.4 10.7 6.0 1.3 0.3 3.4 26.6 2.7 17.1
10.2 26.7 7.9 1.1 12.8 10.0 1.2 0.3 2.7 9.9 2.4 14.9
8.5 26.5 8.4 5.9 20.4 5.9 1.3 0.4 1.6 10.6 1.8 8.7
(b) Fatty acid
Distribution of fatty acid (% of each fatty acid) at 90 min TG
DG
MG
FFA
14:o 16:0 16:l 18 :0 18 : 1 18:2 18:3 20:3 20:4 20:5 22:5 2216
16.9 18.6 19.5 15.2 19.2 19.1 19.1 22.3 25.6 30.0 23.6 24.8
13.6 11.8 12.8 5.9 10.2 15.1 16.3 10.6 22.9 26.3 19.5 22.0
16.5 14.2 13.5 4.0 9.1 20.1 12.7 11.3 15.8 9.0 15.5 17.6
53.0 55.5 54.2 74.9 60.9 45.7 51.9 55.8 35.7 34.7 41.3 35.6
Total fatty acids
21.4
15.7
13.2
49.7
by lipase-colipase alone, whereas the [ 3H]20 : 5 esters exhibited a lipolysis pattern similar to that observed in the incubations with [ 3H]20 : 5 and [14C]20 : 4. Also in comparison to [14C]18 : 2, the lipolysis pattern of the [3H]20 : 5 esters observed in the present study was thus similar to that observed for 20: 4 in the earlier study. After incubation of the fish oil chylomicrons with lipase-colipase, the gas-liquid chromatographic analysis of the individual lipid classes showed differences in the fatty acid distribution between remaining triacylglycerols, partial acylglycerols and FFA (Table I). The fatty acid composition of the chylomicrons, which were obtained from rats fed the fish oil emulsion was very similar to that of the fish oil used, in which the proportion of 18:2, 20:4, 20: 5 and 22:6 was 1.1. 0.8, 16.5 and 12.3%, respectively. After incubation with lipase-
colipase, 20 : 4 and 20 : 5 were enriched in diacylglycerol, thus confirming the findings made with the isotope-labelled substrates both in the present and the previous study [7]. Also the proportion of docosahexaenoic acid (22 : 6) in remaining tri- and diacylglycerol was higher than in the original triacylglycerol, suggesting that the 22 : 6 esters exhibit a lipolysis pattern similar to that of 20 : 4 and 20 : 5 esters. At the high substrate concentration, hydrolysis was slowly initiated by lipase-colipase (Fig. 3). This may be due to the presence of a phospholipid surface coat on the chylomicrons, which is not easily removed to facilitate the binding of the lipase-colipase to the oil/water interface under the condition used. Other studies [4] have indicated that some long-chain polyenoic 20-carbon fatty acid esters of marine oil triacylglycerol are hydrolyzed at a slower rate by porcine pancreatic lipase than the C,,-C,, fatty acids. The steric configuration of the fatty acid induced by the occurrence of a double bond near the carboxyl group, particularly at position 5, may be an important cause of this relative resistance [20]. CEL has a broader substrate specificity than lipase-colipase and will hydrolyze a large number of lipid substrates, such as acylglycerols (including the 2-ester bond), cholesteryl ester and vitamin A esters (for references see Ref. 21). The present study shows that CEL is active also against 20 : 4 and 20 : 5 esters of 1,2-X-diacylglycerol and triacylglycerol. The triacylglycerols rich in marine oil fatty acid esters may therefore be most efficiently hydrolyzed by the concerted action of the lipase-colipase and CEL. Acknowledgements This study was supported by grants from the Swedish Medical Research Council (Project No. 3969,3968), the Medical Faculty, University of Lund, the Pahlsson Foundation, the Nutrition Foundation of the Swedish Margarine Industry, the Johanna Andersson Foundation, the Research Foundations of the Hospital of Lund, the Swedish Medical Association and the National Association against Heart and Lung Diseases. References 1 2 3 4 5 6 7 8
Harris, W.S. (1989) .I. Lipid Res. 30, 785-807. Bang, H.O. and Dyerberg, J. (1972) Acta Med. Stand. 192, 85-89. Leaf, A., Weber, P.C. (1988) N. Engl. J. Med. 318, 549-557. Bottino, N.R., Vanderburg, G.A. and Reiser, R. (1967) Lipids 2, 489-493. Lawson, L.D. and Hughes, B.G. (1988) B&him. Biophys. Res. Commun. 152, 328-335. Boustani, ES., Colette, C., Monnier, L., Descomps, B., Paulet, A.C.D. and Mendy, F. (1987) Lipids 22, 711-714. Chen, Q., Stemby, B. and Nilsson, A. (1989) B&him. Biophys. Acta 1004, 372-385. Sternby, B. and Borgstrlim, B. (1981) Comp. B&hem. Physiol. 68B, 15-18.
117 9 Stemby, B. and Borgstrom, B. (1979) Biochim. Biophys. Acta 572, 235-243. 10 Lombardo, D., Guy, 0. and Figarella, C. (1978) B&him. Biophys. Acta 527, 142-149. 11 Erlanson, C. and Borgstrom, B. (1972) B&him. Biophys. Acta 271, 400-412. 12 Sternby, B. and Akerstrom, B. (1984) B&him. Biophys. Acta 789, 164-169. 13 Warshaw, A.L. (1972) Gut 13, 66-67. 14 Flortn, C.H. and Nilsson, A. (1977) Eur. J. B&hem. 77, 23-30. 15 Ekstrom, B., Nilsson, A. and Akesson, B. (1989) Eur. J. Clin. Invest. 19, 259-264. 16 Verger, R. (1984) in Pancreatic Lipase (Borgstrom, B. and Brockman, H.L., eds.), pp. 84-150, Elsevier, Amsterdam.
17 Brockerhoff, H., Hoyle, R.J. and Huang, P.C. (1966) Can. J. B&hem. 44,1519-1525. 18 Brockerhoff, H., Hoyle, R.J., Huang, P.C. and Litchfield, C. (1968) Lipids 3, 24-29. 19 Chernenko, G.A., Barrowman, J.A., Kean, K.T., Heraberg, G.R. and Keough, K.M.W. (1989) Biochem. Biophys. Acta 1004,95-102. 20 Heimermann, W.H., Holmann, R.T., Gordon, D.T., Kowalyshyn, D.E. and Jensen, R.G. (1973) Lipids 8, 45-47. 21 Rudd, E.A. and Brockman, H.L. (1984) in Pancreatic Carboxyl Ester Lipase (Borgstrom, B. and Brockman, H.L., eds.), pp. 185204, Elsevier, Amsterdam.
