Biochimica et Biophy~wu ,'Iota. I075(t'~cH)25c~-26fl 1991 Elsevier Science Publishers B.V. All
rights reser'.'cd03~14.-llt~5/t~I/S0350
259
ADONIS 031)44165910t)253G
BBAGEN 2361H1
Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase T o r M e l i n t, C h e n Q i ~, G u n i l l a o B e n g t s s o n - O l i v e c r o n a -~ B j 6 r n ,'Xkesson 3 and Akc Nilsson t Department o[.~h'dicim,. Um,er~'it~" of I.umL I.,ml I.~'w,,dcn~ " D,'l,artm~'nt of Mcdi~'al Bir~'l:¢,m.~trv and Biovhy~ic~" Unit er~-it~"o[ Ume~7 Umt'l[ (5,,rdcnl aml ~[h'partmt'nt ~t Ptn ~tohJ.~.,t¢al ('lwmistt3.. Uni* t'r~ttr o[ I.und I.und [Sn'cth'nl
(Rccei'.cd 23 Janua~ ItltH) IRc'.'tsed manuscript rccct'.cd 24 June 1'¢91) Key words: Arachldonic;reid: ('hyhmficron:Docosahcxaenoicacid: [!ict~,apcntanoicacid: }lep*diclipa~,c:[.ip~ptotein lipast: The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and h u m a n hepatic lipase was examined in vitro. Chylomierons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentacnoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the Ct.t-Cts acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eieosatrienoic, docosatrlenoic and docosahexanoic acids exhibited an intermediate llpolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 2 0 : 4 esters of both triand diaoJIRlycerols. Addition of NaCI (final concentration l M) during the eot, rse of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylg~jceral lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 2 0 : 4 and 20:5 esters thus exhibit a relative resistance to lilmprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids ma~" accumulate at the surface of the r e m n a n t particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase. Introduction Much attention has been paid to the biological effects of marine oils, mainly due to the epidemiological evidence that ischemic heart disease is inversely correlated to consumption of fish products. Dietary marine oils rich in n - 3 fatty acids have been shown to lower very low density lipoprotein (VLDL) triacylglycerol levels in plasma, decrease the secretion of VLDL triacylglycerol, and to alter platclet function and eicosanoid production ffor references see Ref 1).
Abbreviations: GC, gas chromatography;HDL, high density iipoprorein; TLC. thin layer chromatography; VLDL. very tow density lipoprotein: 14:0, myristicacid: 16:0. palmiticacid: 16: I palmitolcic acid; 18:0, stearic acid; 18: I, oleic acid; 18:2, linoleic acid; 18:3, linolenic acid; 20:3, eicosatricnoicacid; 20:4, arachidonicacid; 20:3, cicosapentaenoic acid: 22:5. docosapentaenoic acid; 22:6, doco.,;ahexaenoicacid. Correspondence: ,A Nilsson, Department of Medicine, University Hospital. S-221 85 Lund, Sweden.
The pathways by which the polyenoic 20-22 carbon fatty acids of the fish oil triacylglycerol reach the tissue sites at which they exert their effects are, however, poorly characterized. Earlier studies indicated that arachidonic acid (20:4) esters of triacylglycerol in rat chylomicrons [2] and VLDL from hypothyreotic rats [3] exhibited a relative resistance to llpoproteln llpase and were thus enriched in triacylglycerol and diacylglycerol of the remnants formed. During incubation in vitro w!th postheparin plasma which contains both lipoprorein lipase and hepatic lipase, part of the hydrolysis of the triacylglycerol-20:4 esters and the further hydrolysis of diacylglycerol-20:4 esters was blocked by antiserum against hepatic lipase, indicating that the 20:4 containing triacylglycerol and X-l,2-diacylglycerol were substrates for rat postheparin plasma hepatic lipase [2]. In accordance with these observations the [3H]20:4 of the intravenously injected chylomicron triacylglycerol was cleared from blood at a slower rate than the [14C]18:2. More [3H]20:4 appeared in the liver and less in the adipose tissue as compared to the [t':C]18:2 [4]. Since eicosapcntaenoic acid (20:5) is an alternative
260 precursor of cicosanoids and since it has not been studied if 20:5 occurring in fish oil chylomicrons is metabolized in a similar way a,', 20:4. we examined the action of lipoprotein lipase and hepatic lipase on 2(1:5 and 2(1:4 esters of chylomlcrons obtained after feeding fish oil to rats. Lip0protein lipase and hepatic lipase are related enzymes which both participate in lipoprotein metabolism. Both hydrolyze primary ester bonds in triacylglyccrols and phospholipids with some prefercncc for the s n - I fatty acid in triacylglycerol. An important difference between the two lipases is that they prefer different lipoproteins as substrates (for rcfcrcnees see Ref. 5). Lipoprotein lipase acts primarily on large triacylglycerol-rich lipoproteins as chylomicrons and VLDL. Hepatic lipase on the other hand scems to prefer denser lipoproteins and remnants formed by the action of lipoprotein lipase on chylomicrons and VLDL. The action of the two enzymes can thus be seen as complementary. This is in accordance with the recent demonstration that they have evolved from a common ancestral gene [6]. The physiological purpose served by this specialization is not fully clear. Wc are here exploring the possibility that one aspect may be a different handling of long-chain polyunsaturated fatty acids by the two enzymes, resulting in a metabolic channeling of these fatty acids different from that of the more common Ci4-Cls fatty acids. For this purpose we have examined the hydrolysis of 20:4 and 20:5 ester bonds in chylomicrons with lipoprotein lipase with and without the simultaneous addition of hepatic lipase.
18-~H(N)]20:5 (79.0 m C i / m m o l ) and [l-taC]20:4, (52.0 m C i / m m o l ) were obtained from New England Nuclear Research Products. Boston. Different types of radioactive chyle were produced. In some experimental series the animals were given 5{I tzCi of [3H]20:4 and 25 #Ci of [~4C]18:2. The solutions of the radioactive fatty acids were blown to dryness with nitrogen, together with 1 mg egg phosphatidylcholinc and then dispersed by shaking in l ml 0.9% NaCI and I ml of 20% Intralipid (KABI Vitrum AB, Stockholm, Sweden). This lipid emulsion contains 200 mg soybean triacylglycerol and 12 mg egg yolk phospholipid/ml. The labelled emulsions were then given to the anim.ds through the gastric fistula 18-24 h after the operation. as described eadier [7]. In another series 10 ~tCi [~'~C]20:4 and 5t) /aCt [~H]20:5 were dispersed with egg phosphatidylcholine in the same way. The dispersion was then mixed with a 10% emulsion of fish oil triacylglycerol (Max-EPA, R.P. Scherer, Swindon. U.K.). This emulsion was prepared by sonicating the fish oil for 1 min in 0.15 M NaCI containing 1% gum arabic.
Collection of chile and isolation of chylornicrons
Materials and Methods
The ehyle from the mesenteric duct fistul~ was collected in glass tubes on ice. N a . E D T A was added to a final concentration of 2 mM and the ehyle was then stored at 4 ° C . The chyle was defibrlnated by filtration through a nylon mesh. diluted in 0.15 M NaCI, and the chylomierons were isolated by ultraeeutrifugation at 25000 rpm for 2 h using a Beckman ultracentrifuge and a SW 41 rotor. Triacylglycerol mass of the ehylomicroas was determined by an enzymatic kit method (Boehringer-Mannheim, Germany).
Anbnal p:'eparation
hwubation with lipoprotein lipase and hepatic lipase
Male white Sprague-Dawley rats (ALAB, Stockholm, Sweden), weighing about 250 g, fed a commercial standard pellet diet, were used. The food was withdrawn for 24 h, but the rats had free access to a water solution containing 2.5% glucose, 0.5% NaCI, and 0.05% KCI. The rats were then operated under light diethyl ether anesthesia. A polyvinyl tube (0.5 × 0.8 ram) draining fistula was inserted into the mesentcric lymphatic duct [7], and a feeding fistula of the same diameter and material was im;erted into the fundus part of the stomach. After the operation the rats were kept at 27 ° C in restraining cages and infused through the gastric fistula with the solution mentioned above at a rate of 2.5 m l / h . The infusion was interrupted 1 h before the fat meal and started again l h after the meal.
Bovine milk lipoprotein lipase was prepared according to [8] and h u m a n hepatic Iipase according to [9]. The chylomicrons were incubated with enzymes in a medium consisting of 2 ml 10% bovine serum albumin, 1 ml rat serum, 2 ml 10 mM Hepcs containing 2 mM CaCI z (pH 7 4). The reaction was started by adding the ch~,lomicrons to the medium which had a temperature of 37°C. At the time intervals indicated, aliquouts were taken from the incubations and the reaction was stopped by immediately adding 8 vol. of chloroform/ methanol 1:1 containing 0.005% butylated hydroxytoluene as an antioxidant. The protein was precipitated by centrifugation and the supernafaut was poured into new tubes. After adjusting the proportions to m e t h a n o l / w a t e r / c h l o r o f o r m l : 1:2 by adding chloroform and 0.1 M KI-I_,PO.~, the upper phase of methanol-water containing salts and Hepes was discarded and the lower phase containing the lipids was washed twice with m e t h a n o l / w a t e r / c h l o r o f o r m 48:47 : 3. After drying with nitrogen the lipid extracts
Preparation of radioactive enteral feeding emulsions [1-1~C]18:2 (59.0 mCi/mmol), [5,6,8,9,11,14.153H(N)]20:4 (51.3 mCi/mmol), [5,6.8,9,11,12,I4,15,17,
261
were dissolved in chlorofi)rm, and the lipids wcrc separated by thin layer c h r o m a t o g r a p h y (TLC) on silica gel G plates, which were developed in light p e t r o l e u m / d i e t h y l / e t h e r / a c e t i c a c i d / m c t h a n o l 80 : 2ll : 1 : 2 (v/v). The lipid spots were stained by iodine ~,apour and the lipid zones were scraped into counting vials. 1 ml of methanol/water 1:1 (v/v) and 9 ml of l n s t a g e l / t o l u c n e I : 1 ( v / v ) was a d d e d and radioactivity was d e t e r m i n e d in a Packard 4011 C D liquid scintillation system using the c o m p u t e r i z e d a u t o m a t i c external s t a n d a r d for quench correction.
Separation o f partially lipolyzed lipoproteins by ultr~K'eHtri[ugation O n e e x p e r i m e n t a l series was performed to determine to what extent the diacylglycerel was transferred from the r e m n a n t particle to o t h e r serum c o m p o n e n t s d u r i n g the lipolysis. Fish oil ehvlomicrons labelled with [J4120:4 and [3H]20:5 were hydrolyzed with lipoprotein lipase. After different time intervals aliquots of the reaction mixture were mixed with sodium bromide (density 1.35 g / r o l l , thereby adjusting the density to i.21. T h e mixture was overlayered with sodium bromide solution (density 1,019 g / r o l l . The tubes were then centrifuged for 48 h at 351100 rpm at 4°C. Thus three fractions with d < 0 . 1 9 g / m l ( r e m n a n t s and VLDL), 1 . 0 1 9 < d < 1.21 g / m l ( H D L ) and d > 1.21 g / m l ( a l b u m i n ) could be collected and analyzed by T L C as described above.
by analyzing the distribution of fatty acids between lipid classes by capillary gas c h r o m a t o g r a p h y [10]. In this experiment the incubation conditions described above were used, except that the volume, and the mass of llpoprotein lipase and ehylomieron triacylglycerol wcrc scaled up 20-fold, to get enough lipid mass for analysis of fatty acid composition in all lipid cla~ses. The T L C zones c o r r e s p o n d i n g to triacylglycerol, diacylglycerol, monoacylglycerol, free fatty acids and phospholipid identified after spraying with dichlorofluorescoin (0.2e,~ in ethanol), were scraped and transesterificd in 2 ml 2% sulphurlc acid in methanol ",tt. 6 5 ° C for 4 h. P e n t a d e c a n o i e acid ( ( I . l - I rag) was added as internal standard. After extraction, the methyl esters were analyzed by gas c h r o m a t o g r a p h y using a 25 m capillary, column c o a t e d with SP 10O0 in a Sigma 3B instrument, e q u i p p e d with a flame ionization detector (Perkin-Elmcr, Norwalk, CT, U.S.A.). The analysis wits run at 20{I ~ C for 7 rain, and then the t e m p e r a t u r e was increased by 2 ° C / r a i n up to 2 4 0 ° C where it was m a i n t a i n e d for 3 rain. The p e a k s were quantified with a Sigma 15 i n t e g r a t o r (Perkin E l m e r ) a n d identified by comparison with a methyl ester s t a n d a r d (68 A, NUCheck-Prep, Elysian, MN, U.S.A.). This m e t h o d was also used for analysis of the chylomicron lipid composition in fish oil chyle. Results
Compositiorz o f chylomicrons Fatty acid analysis by gas chromatography In one e x p e r i m e n t a l series the course 9f lipolysis of fish oil chylomicrons by lipoprotein [ipas~: was followed
The fatty acid composition and the distribution of each fatty acid in the chylomicrons from rats fed fish oil are given in T a b l e I. Long-chain polyunsaturated
TABLE I
Chylomicron lipid composilion in fish oil chyle unuly,'cdby gu.~('hrtnnatography Means ± S.E. of
Fatty acid
three samples Percent of total triacylglycerol
Percent of total phospholipid
Percent of fatty acid mass in
Percent of fatty acid ma~s in
fatty acid m a ~ J
fatty acid mass "
triacylglycerol h
phtxspholipid h
14:0 16:11 16: I
9.9±0.6 24.4_4- I.I 8.8+0.3
11.9+_0.1 24.9_+0.~ 11.9_+11.1
t~4.6+-11.5 76.8 _+!.2 94.0+_114
1.9.+.11.1 18.1 ±0,9 2.2_+11.2
18:0 18: I 18:2
2.8_+11.2 14.5 +-(1.5 9.6 + 0.1
22.4+04 12.5 ±11.3 17.0_+0.2
31.3±2.5 78.9+- 1.9 67.8+_2.11
57.q ± 28 15.7± 1.4 27.h+_ 1.6
18 : 3 20:3 20:4
1.5 + 0.0 0.3+_0.0 1.8±0.1
(1.7+.11.I 1.9+-0.1 7.3.+.0.3
87.7 _+2. I 35.6+_ 1.3 49.1 ±3.5
8.7 +_2.0 58.4_+2,1 46.1 +_3.2
20:5 22:5 2.~:6
!4.4 +_.0.5 1.7±11.2 10.3 ~().8
7.1 ~-0.2 I.I ±0.1 3.344-1).3
~h.5 + 1.2 83.2 .t 1.5 88.3± 1.0
13.2± 1.5 6.4 ~-115
a Values show for each fatty acid its percentage of total triacylglycerol and p h o s p h o l i p i d fatty acid ma~', (molC/~).
h Values show for each fatty acid its distribution in mol% between triacylglycerol and phospholipid.
9.9+- 1.0
262
r ~ 10'
o,oj oo
30'
.L
°"1,o~>--~ 60'
10'
..~]
30"
60"
~,
exhibiting the slowest d i s a p p e a r a n c e rate o f ' a l l fatty acids. 2 0 : 4 and 2 0 : 5 also a c c u m u l a t e d in diacylglycerol and monoacylglycerol to a larger extent than the other fatty acids. The 2 0 : 3 , 2 2 : 5 and 2 2 : 6 esters exhibited an i n t e r m e d i a r y lipolysis pattern. The c h a n g e s d u r i n g incubation in the content of different fatty acids in the phospholioid fraction were s small that they could not account for any i m p o r t a n t portion of the F A being formed.
Pattern of lipolysis with lipoprotebt lipase attd hepatic lipase using radioactive "hy.',omic:ons
10' ~0" 60 10" 30" 60" Fig. I. Fatty acid patterns in lipolysis products from fish oil chylomicram, hydrolyzed with lipoprotcin lipase. The added chylomicrons contained 34.2 mg triacylglyccrol. The activity of the lipoprotcin lipi~s.2 preparation, measured at pH 8.5, 25 °C [81 was 0.36 v.mol free fatty acid/vl pet min and the added volume was 25 V,I. Values indicate for each fatty acid the distribution of percent among nonpolar llpids. Valu,,'sarc means of three incubations with the same chyle. S.E.M. is not indicated everywhere to make the figures less complicated. The symbols indicate: c,. 20:4; • 20:5; t2.22:5; U. 22:6; o, 2(1: 3: and O. 14-18 carbon filtty acids. Siguificances of differences between the individual 20-22 carbon fatty acids as compared to the 14-18 carbon fatty acids as a group were estimated by Student's t-test, and arc indicated * P
rights reser'.'cd03~14.-llt~5/t~I/S0350
259
ADONIS 031)44165910t)253G
BBAGEN 2361H1
Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase T o r M e l i n t, C h e n Q i ~, G u n i l l a o B e n g t s s o n - O l i v e c r o n a -~ B j 6 r n ,'Xkesson 3 and Akc Nilsson t Department o[.~h'dicim,. Um,er~'it~" of I.umL I.,ml I.~'w,,dcn~ " D,'l,artm~'nt of Mcdi~'al Bir~'l:¢,m.~trv and Biovhy~ic~" Unit er~-it~"o[ Ume~7 Umt'l[ (5,,rdcnl aml ~[h'partmt'nt ~t Ptn ~tohJ.~.,t¢al ('lwmistt3.. Uni* t'r~ttr o[ I.und I.und [Sn'cth'nl
(Rccei'.cd 23 Janua~ ItltH) IRc'.'tsed manuscript rccct'.cd 24 June 1'¢91) Key words: Arachldonic;reid: ('hyhmficron:Docosahcxaenoicacid: [!ict~,apcntanoicacid: }lep*diclipa~,c:[.ip~ptotein lipast: The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and h u m a n hepatic lipase was examined in vitro. Chylomierons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentacnoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the Ct.t-Cts acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eieosatrienoic, docosatrlenoic and docosahexanoic acids exhibited an intermediate llpolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 2 0 : 4 esters of both triand diaoJIRlycerols. Addition of NaCI (final concentration l M) during the eot, rse of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylg~jceral lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 2 0 : 4 and 20:5 esters thus exhibit a relative resistance to lilmprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids ma~" accumulate at the surface of the r e m n a n t particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase. Introduction Much attention has been paid to the biological effects of marine oils, mainly due to the epidemiological evidence that ischemic heart disease is inversely correlated to consumption of fish products. Dietary marine oils rich in n - 3 fatty acids have been shown to lower very low density lipoprotein (VLDL) triacylglycerol levels in plasma, decrease the secretion of VLDL triacylglycerol, and to alter platclet function and eicosanoid production ffor references see Ref 1).
Abbreviations: GC, gas chromatography;HDL, high density iipoprorein; TLC. thin layer chromatography; VLDL. very tow density lipoprotein: 14:0, myristicacid: 16:0. palmiticacid: 16: I palmitolcic acid; 18:0, stearic acid; 18: I, oleic acid; 18:2, linoleic acid; 18:3, linolenic acid; 20:3, eicosatricnoicacid; 20:4, arachidonicacid; 20:3, cicosapentaenoic acid: 22:5. docosapentaenoic acid; 22:6, doco.,;ahexaenoicacid. Correspondence: ,A Nilsson, Department of Medicine, University Hospital. S-221 85 Lund, Sweden.
The pathways by which the polyenoic 20-22 carbon fatty acids of the fish oil triacylglycerol reach the tissue sites at which they exert their effects are, however, poorly characterized. Earlier studies indicated that arachidonic acid (20:4) esters of triacylglycerol in rat chylomicrons [2] and VLDL from hypothyreotic rats [3] exhibited a relative resistance to llpoproteln llpase and were thus enriched in triacylglycerol and diacylglycerol of the remnants formed. During incubation in vitro w!th postheparin plasma which contains both lipoprorein lipase and hepatic lipase, part of the hydrolysis of the triacylglycerol-20:4 esters and the further hydrolysis of diacylglycerol-20:4 esters was blocked by antiserum against hepatic lipase, indicating that the 20:4 containing triacylglycerol and X-l,2-diacylglycerol were substrates for rat postheparin plasma hepatic lipase [2]. In accordance with these observations the [3H]20:4 of the intravenously injected chylomicron triacylglycerol was cleared from blood at a slower rate than the [14C]18:2. More [3H]20:4 appeared in the liver and less in the adipose tissue as compared to the [t':C]18:2 [4]. Since eicosapcntaenoic acid (20:5) is an alternative
260 precursor of cicosanoids and since it has not been studied if 20:5 occurring in fish oil chylomicrons is metabolized in a similar way a,', 20:4. we examined the action of lipoprotein lipase and hepatic lipase on 2(1:5 and 2(1:4 esters of chylomlcrons obtained after feeding fish oil to rats. Lip0protein lipase and hepatic lipase are related enzymes which both participate in lipoprotein metabolism. Both hydrolyze primary ester bonds in triacylglyccrols and phospholipids with some prefercncc for the s n - I fatty acid in triacylglycerol. An important difference between the two lipases is that they prefer different lipoproteins as substrates (for rcfcrcnees see Ref. 5). Lipoprotein lipase acts primarily on large triacylglycerol-rich lipoproteins as chylomicrons and VLDL. Hepatic lipase on the other hand scems to prefer denser lipoproteins and remnants formed by the action of lipoprotein lipase on chylomicrons and VLDL. The action of the two enzymes can thus be seen as complementary. This is in accordance with the recent demonstration that they have evolved from a common ancestral gene [6]. The physiological purpose served by this specialization is not fully clear. Wc are here exploring the possibility that one aspect may be a different handling of long-chain polyunsaturated fatty acids by the two enzymes, resulting in a metabolic channeling of these fatty acids different from that of the more common Ci4-Cls fatty acids. For this purpose we have examined the hydrolysis of 20:4 and 20:5 ester bonds in chylomicrons with lipoprotein lipase with and without the simultaneous addition of hepatic lipase.
18-~H(N)]20:5 (79.0 m C i / m m o l ) and [l-taC]20:4, (52.0 m C i / m m o l ) were obtained from New England Nuclear Research Products. Boston. Different types of radioactive chyle were produced. In some experimental series the animals were given 5{I tzCi of [3H]20:4 and 25 #Ci of [~4C]18:2. The solutions of the radioactive fatty acids were blown to dryness with nitrogen, together with 1 mg egg phosphatidylcholinc and then dispersed by shaking in l ml 0.9% NaCI and I ml of 20% Intralipid (KABI Vitrum AB, Stockholm, Sweden). This lipid emulsion contains 200 mg soybean triacylglycerol and 12 mg egg yolk phospholipid/ml. The labelled emulsions were then given to the anim.ds through the gastric fistula 18-24 h after the operation. as described eadier [7]. In another series 10 ~tCi [~'~C]20:4 and 5t) /aCt [~H]20:5 were dispersed with egg phosphatidylcholine in the same way. The dispersion was then mixed with a 10% emulsion of fish oil triacylglycerol (Max-EPA, R.P. Scherer, Swindon. U.K.). This emulsion was prepared by sonicating the fish oil for 1 min in 0.15 M NaCI containing 1% gum arabic.
Collection of chile and isolation of chylornicrons
Materials and Methods
The ehyle from the mesenteric duct fistul~ was collected in glass tubes on ice. N a . E D T A was added to a final concentration of 2 mM and the ehyle was then stored at 4 ° C . The chyle was defibrlnated by filtration through a nylon mesh. diluted in 0.15 M NaCI, and the chylomierons were isolated by ultraeeutrifugation at 25000 rpm for 2 h using a Beckman ultracentrifuge and a SW 41 rotor. Triacylglycerol mass of the ehylomicroas was determined by an enzymatic kit method (Boehringer-Mannheim, Germany).
Anbnal p:'eparation
hwubation with lipoprotein lipase and hepatic lipase
Male white Sprague-Dawley rats (ALAB, Stockholm, Sweden), weighing about 250 g, fed a commercial standard pellet diet, were used. The food was withdrawn for 24 h, but the rats had free access to a water solution containing 2.5% glucose, 0.5% NaCI, and 0.05% KCI. The rats were then operated under light diethyl ether anesthesia. A polyvinyl tube (0.5 × 0.8 ram) draining fistula was inserted into the mesentcric lymphatic duct [7], and a feeding fistula of the same diameter and material was im;erted into the fundus part of the stomach. After the operation the rats were kept at 27 ° C in restraining cages and infused through the gastric fistula with the solution mentioned above at a rate of 2.5 m l / h . The infusion was interrupted 1 h before the fat meal and started again l h after the meal.
Bovine milk lipoprotein lipase was prepared according to [8] and h u m a n hepatic Iipase according to [9]. The chylomicrons were incubated with enzymes in a medium consisting of 2 ml 10% bovine serum albumin, 1 ml rat serum, 2 ml 10 mM Hepcs containing 2 mM CaCI z (pH 7 4). The reaction was started by adding the ch~,lomicrons to the medium which had a temperature of 37°C. At the time intervals indicated, aliquouts were taken from the incubations and the reaction was stopped by immediately adding 8 vol. of chloroform/ methanol 1:1 containing 0.005% butylated hydroxytoluene as an antioxidant. The protein was precipitated by centrifugation and the supernafaut was poured into new tubes. After adjusting the proportions to m e t h a n o l / w a t e r / c h l o r o f o r m l : 1:2 by adding chloroform and 0.1 M KI-I_,PO.~, the upper phase of methanol-water containing salts and Hepes was discarded and the lower phase containing the lipids was washed twice with m e t h a n o l / w a t e r / c h l o r o f o r m 48:47 : 3. After drying with nitrogen the lipid extracts
Preparation of radioactive enteral feeding emulsions [1-1~C]18:2 (59.0 mCi/mmol), [5,6,8,9,11,14.153H(N)]20:4 (51.3 mCi/mmol), [5,6.8,9,11,12,I4,15,17,
261
were dissolved in chlorofi)rm, and the lipids wcrc separated by thin layer c h r o m a t o g r a p h y (TLC) on silica gel G plates, which were developed in light p e t r o l e u m / d i e t h y l / e t h e r / a c e t i c a c i d / m c t h a n o l 80 : 2ll : 1 : 2 (v/v). The lipid spots were stained by iodine ~,apour and the lipid zones were scraped into counting vials. 1 ml of methanol/water 1:1 (v/v) and 9 ml of l n s t a g e l / t o l u c n e I : 1 ( v / v ) was a d d e d and radioactivity was d e t e r m i n e d in a Packard 4011 C D liquid scintillation system using the c o m p u t e r i z e d a u t o m a t i c external s t a n d a r d for quench correction.
Separation o f partially lipolyzed lipoproteins by ultr~K'eHtri[ugation O n e e x p e r i m e n t a l series was performed to determine to what extent the diacylglycerel was transferred from the r e m n a n t particle to o t h e r serum c o m p o n e n t s d u r i n g the lipolysis. Fish oil ehvlomicrons labelled with [J4120:4 and [3H]20:5 were hydrolyzed with lipoprotein lipase. After different time intervals aliquots of the reaction mixture were mixed with sodium bromide (density 1.35 g / r o l l , thereby adjusting the density to i.21. T h e mixture was overlayered with sodium bromide solution (density 1,019 g / r o l l . The tubes were then centrifuged for 48 h at 351100 rpm at 4°C. Thus three fractions with d < 0 . 1 9 g / m l ( r e m n a n t s and VLDL), 1 . 0 1 9 < d < 1.21 g / m l ( H D L ) and d > 1.21 g / m l ( a l b u m i n ) could be collected and analyzed by T L C as described above.
by analyzing the distribution of fatty acids between lipid classes by capillary gas c h r o m a t o g r a p h y [10]. In this experiment the incubation conditions described above were used, except that the volume, and the mass of llpoprotein lipase and ehylomieron triacylglycerol wcrc scaled up 20-fold, to get enough lipid mass for analysis of fatty acid composition in all lipid cla~ses. The T L C zones c o r r e s p o n d i n g to triacylglycerol, diacylglycerol, monoacylglycerol, free fatty acids and phospholipid identified after spraying with dichlorofluorescoin (0.2e,~ in ethanol), were scraped and transesterificd in 2 ml 2% sulphurlc acid in methanol ",tt. 6 5 ° C for 4 h. P e n t a d e c a n o i e acid ( ( I . l - I rag) was added as internal standard. After extraction, the methyl esters were analyzed by gas c h r o m a t o g r a p h y using a 25 m capillary, column c o a t e d with SP 10O0 in a Sigma 3B instrument, e q u i p p e d with a flame ionization detector (Perkin-Elmcr, Norwalk, CT, U.S.A.). The analysis wits run at 20{I ~ C for 7 rain, and then the t e m p e r a t u r e was increased by 2 ° C / r a i n up to 2 4 0 ° C where it was m a i n t a i n e d for 3 rain. The p e a k s were quantified with a Sigma 15 i n t e g r a t o r (Perkin E l m e r ) a n d identified by comparison with a methyl ester s t a n d a r d (68 A, NUCheck-Prep, Elysian, MN, U.S.A.). This m e t h o d was also used for analysis of the chylomicron lipid composition in fish oil chyle. Results
Compositiorz o f chylomicrons Fatty acid analysis by gas chromatography In one e x p e r i m e n t a l series the course 9f lipolysis of fish oil chylomicrons by lipoprotein [ipas~: was followed
The fatty acid composition and the distribution of each fatty acid in the chylomicrons from rats fed fish oil are given in T a b l e I. Long-chain polyunsaturated
TABLE I
Chylomicron lipid composilion in fish oil chyle unuly,'cdby gu.~('hrtnnatography Means ± S.E. of
Fatty acid
three samples Percent of total triacylglycerol
Percent of total phospholipid
Percent of fatty acid mass in
Percent of fatty acid ma~s in
fatty acid m a ~ J
fatty acid mass "
triacylglycerol h
phtxspholipid h
14:0 16:11 16: I
9.9±0.6 24.4_4- I.I 8.8+0.3
11.9+_0.1 24.9_+0.~ 11.9_+11.1
t~4.6+-11.5 76.8 _+!.2 94.0+_114
1.9.+.11.1 18.1 ±0,9 2.2_+11.2
18:0 18: I 18:2
2.8_+11.2 14.5 +-(1.5 9.6 + 0.1
22.4+04 12.5 ±11.3 17.0_+0.2
31.3±2.5 78.9+- 1.9 67.8+_2.11
57.q ± 28 15.7± 1.4 27.h+_ 1.6
18 : 3 20:3 20:4
1.5 + 0.0 0.3+_0.0 1.8±0.1
(1.7+.11.I 1.9+-0.1 7.3.+.0.3
87.7 _+2. I 35.6+_ 1.3 49.1 ±3.5
8.7 +_2.0 58.4_+2,1 46.1 +_3.2
20:5 22:5 2.~:6
!4.4 +_.0.5 1.7±11.2 10.3 ~().8
7.1 ~-0.2 I.I ±0.1 3.344-1).3
~h.5 + 1.2 83.2 .t 1.5 88.3± 1.0
13.2± 1.5 6.4 ~-115
a Values show for each fatty acid its percentage of total triacylglycerol and p h o s p h o l i p i d fatty acid ma~', (molC/~).
h Values show for each fatty acid its distribution in mol% between triacylglycerol and phospholipid.
9.9+- 1.0
262
r ~ 10'
o,oj oo
30'
.L
°"1,o~>--~ 60'
10'
..~]
30"
60"
~,
exhibiting the slowest d i s a p p e a r a n c e rate o f ' a l l fatty acids. 2 0 : 4 and 2 0 : 5 also a c c u m u l a t e d in diacylglycerol and monoacylglycerol to a larger extent than the other fatty acids. The 2 0 : 3 , 2 2 : 5 and 2 2 : 6 esters exhibited an i n t e r m e d i a r y lipolysis pattern. The c h a n g e s d u r i n g incubation in the content of different fatty acids in the phospholioid fraction were s small that they could not account for any i m p o r t a n t portion of the F A being formed.
Pattern of lipolysis with lipoprotebt lipase attd hepatic lipase using radioactive "hy.',omic:ons
10' ~0" 60 10" 30" 60" Fig. I. Fatty acid patterns in lipolysis products from fish oil chylomicram, hydrolyzed with lipoprotcin lipase. The added chylomicrons contained 34.2 mg triacylglyccrol. The activity of the lipoprotcin lipi~s.2 preparation, measured at pH 8.5, 25 °C [81 was 0.36 v.mol free fatty acid/vl pet min and the added volume was 25 V,I. Values indicate for each fatty acid the distribution of percent among nonpolar llpids. Valu,,'sarc means of three incubations with the same chyle. S.E.M. is not indicated everywhere to make the figures less complicated. The symbols indicate: c,. 20:4; • 20:5; t2.22:5; U. 22:6; o, 2(1: 3: and O. 14-18 carbon filtty acids. Siguificances of differences between the individual 20-22 carbon fatty acids as compared to the 14-18 carbon fatty acids as a group were estimated by Student's t-test, and arc indicated * P
