101
Biochimica et Biophysics Acta, 1044 (1990) 101-110 Elsevier
BBALIP
53389
Metabolism of [ 3H]arachidonic acid- and [14C]linoleic acid-labelled chylomicrons in essential fatty acid-deficient rats Lena Hjelte I, Birgitta Strandvik
’
and Ake Nilsson 2
’ Department of Pediatrics and Clinical Research Center, Karolinska Institutei, Huddinge Hospital, Stockholm and ’ Department of Medicine, Hospital of Lund, Lund (Sweden)
(Revised
Key words:
Arachidonic
(Received 15 August 1989) manuscript received 30 November
acid; Chylomicron
metabolism;
Essential
1989)
fatty acid deficiency;
Linoleic
acid; (Rat)
Mesenteric duct chylomicrons labelled with [ 3H]arachidonic (20 : 4) and [ “C]linoleic (18: 2) acid were injected intravenously into essential fatty acid-deficient (EFAD) and control rats. The rats were killed after 5, 10, 20 and 240 min, and serum and different organs were analysed for radioactivity of different lipid classes. 3H and 14C in the triacylglycerol (TG) and phosphatidylethanolamine (PE) fractions were cleared from blood faster in the EFAD than in the control rats. The incorporation of [ “C]18: 2 into liver and heart phospholipids was increased compared to control rats at all time intervals, the increase in the incorporation of [ 3H]20 : 4 being less pronounced. Furthermore, the total incorporation of [ r4C]18: 2 was increased in the heart of the EFAD group. The increased incorporation into ~~phatidylcholine (PC) and particularly into PE was observed already at S-20 min, whereas a marked increase in the C radioactivity of cardiolipin occurred between 20 and 240 min. The 3H and r4C radioactivity per g white adipose tissue was lower in the EFAD group than in the controls. After S-20 min there were no differences between the groups in the lipid radioactivity of the stomach and the small and the large intestine. In the upper small intestine, the 3H radioactivity in both groups and the 14C radioactivity in the EFAD group increased markedly between 20 and 240 min. The study demonstrates an increased plasma clearance and an efficient hepatic uptake and initial incorporation into PC and PE of chylomicron [ 14C]18: 2 and [ 3H]20: 4 in EFAD rats. In these rats a marked selective transfer of [ “C]18: 2 to cardiolipin from other tissue lipids occurred with time. In addition, the study demonstrates a preferential transfer of injected [ ‘H]20: 4, and in EFAD rats also of [ 14C]18: 2, into lipids of the upper small intestine, possibly by secretion of [ 3H]20 : 4 and [ 14C]18 : 2 in bile phospholipids.
Introduction During the development of essential fatty acid (EFA) deficiency, the preservation of the fatty acid pattern varies between different organs and between individual phospholipid classes [l-3]. The mechanisms by which this is achieved have not been clarified. Obviously the
EFA, essential fatty acid; EFAD, essential fatty acid-deficient; BHT, butylated hydroxytoluene; DG, diacylglycerol; CE, cholesteryl ester; CL, cardiolipin; FFA, free fatty acids; HL, hepatic lipase; LPC, lysophosphatidylcholine; LPL, lipoprotein hpase; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; TG, triacylglycerol; TLC, thin-layer chromatography; 18 : 2, linoleic acid; 20 : 4, arachidonic acid. Correspondence: L. Hjelte, Hospital, S-141 86 Huddinge, 0005-2760/90/$03.50
Department Sweden.
of
Pediatrics,
0 1990 Elsevier Science Publishers
Huddinge
B.V. (Biomedical
fatty acid specificity in the pathways of biosynthesis and degradation of phospholipids may be important, but also the regulation of the transport processes by which polyunsaturated fatty acids reach different organs. In an earlier study, we fed [‘4C]linoleic (18 : 2) and [ 3H]aracbidonic (20 : 4) acids to EFA-deficient (EFAD) rats and to control rats and observed an increase in the incorporation of the labelled fatty acids into phospholipids of several organs, particularly into the phosphatidylethanolamine (PE) fraction. The increase in the retention of [i4C]18 : 2 in phosphatidylcholine (PC), PE and cardiolipin (CL) of the small intestine, liver and heart was particularly pronounced [25]. Earlier studies [4,5] have shown that the pattern of incorporation of dietary 20 : 4 into chylomicrons as well as the action of lipoprotein lipase (LPL) on arachidonic acid ester bonds in the chylomicrons might differ from that of the predominant 16-18 carbon fatty acids. Division)
102 Arachidonic acid was overrepresented in chylomicron phospholipids, particularly in PE which is a preferred substrate for the hepatic lipase (HL) [6]. Furthermore, the 20: 4 esters of chylomicron triacylglycerols (TG) exhibited a relative resistance to LPL and accumulated in remaining remnant TG and in 1,2-X-diacylglycerols (DG) 151. This DG and remaining remnant [ ‘HI20 : 4 TG could act as substrates for HL. In vivo experiments also have shown a higher uptake of chylomicron [ ‘HI20 : 4 than of [14C]18 : 2 in the liver in contrast to adipose tissue [7]. These findings combined with the recent observation, that the level of HL is increased in EFAD rats (Nilsson, A., Hjelte, L., Nilsson-Ehle, P. and Strandvik, B., submitted for publication) suggested that the HL may have a selective role in the uptake of polyunsaturated fatty acids by the liver. The tissue uptake of the polyenoic fatty acids might then be regulated in a different manner than the tissue uptake of the predominant 16-18 carbon fatty acids of the chylomicrons which is mediated by LPL. The purpose of this study was to examine whether differences seen in the incorporation patterns of dietary [14C]18 : 2 and [3H]20: 4 in different organs might be due to differences in the metabolism of the chylomicrons between the EFAD and control rats. To elucidate this question we injected linoleate-rich chylomicrons labelled with [r4C]18 : 2 and [ 3H]20 : 4 intravenously into EFAD rats and into controls and followed the plasma clearance and uptake by different organs during 20 min. We also studied the redistribution of radioactive fatty acids that had occurred after 4 h.
Material and Methods
Animals and diets Pregnant white Sprague-Dawley rats were obtained from ALAB AB (Stockholm, Sweden). 10 days prior to delivery and during lactation, one group of the pregnant animals as well as their off-spring (EFAD rats) were fed a semi-synthetic diet deficient in EFA. Another group of pregnant rats and their off-spring (controls) received the same diet except that hydrogenated lard was replaced by soybean lipid. The composition of the diets is given in Table I. The rats had free access to tap water and their respective diets until they were killed. Male rats, 14 weeks old, were used for the experiments. The EFAD rats showed retarded growth and scaliness of hindlegs and tail, characteristic symptoms of EFA deficiency. The mean weight of these rats was 227 g (range 180-268 g) and that of the control rats 300 g (range 250-350 g). The average triene/tetraene ratio (20 : 3 (n - 9)/20 : 4(n - 6)), used as biochemical criterion to define EFA deficiency (2 0.4 [S]), was 30 (range 15-44) in serum phospholipids of the EFAD rats.
TABLE
I
Casein (vitamin-free) Potato starch Glucose Cellulosa fluor Salt mixture’ Vitamins h Hydrogenated lard Soy bean oil
EFAD (wt.%)
Control (wt.%)
20.0 54.0 10.0 4.0 4.0 1.0 7.0 _
20.0 54.0 10.0 4.0 4.0
1.o 1.3 5.1
a Salt mixture containing Ca 0.55%. P 0.31%, K 0.44%. Mg 0.04%. Cl 0.48%. Na 0.28%, Fe 292 ppm, Mn 68 ppm. Cu 10 ppm, Zn 7 ppm. Co 0.4 ppm, J 2 ppm, MO 0.18 ppm, As 0.23 ppm, B 0.17 ppm, Se 0.1 ppm. h Vitamin mixture containing Vit A 11.9 [E/g, Vit D, 1.5 IE/g. Vit B, 4 pg/g, Vit B, 12.4 pg/g, Vit $ 5 pg/g. calcium pantotenate 45% 10 pg/g, niacin 40 pg/g, Vit B,, 0.04 pg/g, Vit K, 1.5 pg/g, biotin 1% 0.3 pg/g, Vit C 480 pg/g, inositol 30 pg/g, Vit E-500 mg/g 42 pg/g, choline chloride 50% 1000 pg/g, p-aminobenzoic acid (PABA) 250 pg/g, folinic acid 0.5 pg/g.
[5,6,9,11,12,14,15-“HI20 : 4 (83.8 Ci/mmol) and [li4C]18 : 2 (52.6 mCi/mmol) were purchased from New England Nuclear Corporation (Boston, MA). Unlabelled 20 : 4 and 18 : 2 were obtained from Nu-ChekPrep (Elysian, MN). Intralipid@ was purchased from Kabi-Vitrum AB (Stockholm, Sweden). This lipid emulsion contains 200 mg soybean TG and 12 mg phospholipid per ml. Mesenteric duct cannulations and stomach fistula operations were performed after an overnight fast and the rats were treated post-operatively as described earlier 141. 50 PCi of [3H]20 : 4 and 50 PCi of [14C]18 : 2 were blown to dryness with nitrogen together with 1 mg of egg-PC, which was dispersed in 2 ml 0.9% NaCl and added to 2 ml of 20% Intralipid. 2 ml of the mixture with labelled fatty acids were fed as a single dose through the stomach fistula at least 20 h after operation and lymph was collected for 8 h on ice. Na,EDTA was added and after filtration chylomicrons were isolated by vacuum ultracentrifugation at 25 000 rpm for 2 h at 4” C in a Beckman SW 41 Swingout bucket rotor. The top layer was collected and redispersed in 0.9% NaCl. TG of the chylomicrons was determined by an enzymatic kit method (Boehringer-Mannheim, F.R.G.). In an earlier study [4], the fatty acid composition of TG, PC and PE of chyle prepared under identical conditions was determined. The TG contained 36.0 + 1.7%, 18 : 2, and 0.80 k 0.08%, 20: 4 (mean + S.E.. n = 3). Two of these chyle preparations were subjected to transmethylation and the fatty acid methyl esters were separated according to the number of double bonds by argentation thin-layer chromatography [9]. 95% of the ‘H and
103 97% of the i4C migrated as 20 : 4 and 18 : 2, respectively. Less than 2% of the 14C migrated as 20: 4, indicating that no major conversion of [14C]18 : 2 to [14C]20 : 4 had occurred in the intestine, before the incorporation into the chylomicrons. The chylomicrons (0.3 ml, containing 1.8 mg TG, per 100 g body wt.) were injected intravenously into the jugular vein under light ether anaesthesia. The radioactivity of the chylomicrons was 4.68. lo5 dpm 3H and 5.81. lo5 dpm 14C per 0.3 ml. After time intervals indicated in the tables and the figures, the rats were again anaesthetized with diethyl ether and killed by aortic puncture. After being rapidly rinsed with saline the intestine was divided in an upper and a lower half and put into chloroform/methanol (1 : 1, v/v) containing 0.005% (w/v) butylated hydroxytoluene (BHT). From other tissues lipids were also extracted with chloroform/methanol (1 : 1, v/v) containing 0.005% BHT. After removing the precipitate by centrifugation, the solvent proportions were adjusted to chloroform/ methanol/water (2 : 1 : 1, v/v) by adding chloroform and 0.1 M KH,PO,. The lower phases were blown to dryness with nitrogen and redissolved in a small volume of chloroform. Aliquots were then taken for thin-layer chromatography (TLC). Non-polar lipids were separated by TLC on silica gel G plates, which were developed in petroleum ether/ diethyl ether/ acetic acid (80 : 20 : 2, v/v) and phospholipids were separated on Merck silica gel 60 plates which were developed in chloroform/ methanol/ acetic acid/ water (100 : 80 : 12 : 1.2, v/v). The spots were identified by staining with iodine and scraped into counting vials. 1 ml methanol/ water (1 : 1, v/v) and 10 ml Instagel/ toluene (1 : 1, v/v) were added and the radioactivity of the samples was determined in a Packard TriCarb 460 CD liquid scintillation system, using the computerized automatic external standard for quench correction.
TABLE
II
Disrrihubon of llpid radioactivity in injected chylomicrons Percent distribution of ‘H and 14C among different lipid classes in chylomicrons used for the injection experiments. Values are means of duplicate analyses. DG, diacylglycerol; FFA, free fatty acids; TG. triacylglycerol; CE, cholesteryl ester; PC, phosphatidylcholine; PE, phosphatidylethanolamine. 1,2-DG
1.3-DG
FFA
TG
CE
PC
PE
‘H
1.3
1.6
3.9
70.5
0.3
14.9
2.8
14 C
0.7
1.4
4.2
88.6
0.4
3.6
0.3
chylomicrons. Also, when the clearance of [3H]TG and [14C]TG was compared, the [14C]TG disappeared faster than the [3H]TG in both animal groups. In the EFAD rats the disappearance of both 3H and 14C radioactivity from serum and the disappearance of labelled PE were faster than in the controls, although the differences were statistically significant only for some of the time
Statistical analysis
Student’s t-test was used for statistical analysis. Variations were indicated by standard errors of the mean (_t S.E.).
loo-
Results Clearance
of ‘H- and “C-labelled
chylomicrons from blood
As previously demonstrated [4], the [3H]20 : 4 and [14C]18 : 2 were not equally distributed between chylomicron phospholipids and TG (Table II). The mean 3H/‘4C ratio of chylomicron TG was 0.8 compared to 4.1 in the PC and 9.3 in the PE fraction. As in an earlier study in normal rats [7], the disappearance of 14C from serum was faster than that of 3H (Fig. 1). The chylomicron TG and PE were cleared from blood faster than PC which partly reflects that a larger proportion of the ‘H than of the 14C was in PC in the injected
25. 0 5
10
20
Fig. 1. Relative recovery of different lipid classes in serum. Rats were injected intravenously with 300 ~1 (per 100 g body wt.) freshly prepared chylomicrons labelled with [ 3H]20 : 4 and [14C]18 : 2. Values are given in percentage of administered radioactivity in respect.ive lipid class and represent means f S.E. of five animals. [ 3H]20 : 4 to the left, [14C]18 : 2 to the right. From above: A, total amount; B, triacylglycerol (TG); C, phosphatidylethanolamine (PE); and D, phosphatidylcholine (PC). n, control rats; 0, essential fatty acid-deficient rats. * P i 0.05, * * P < 0.01, * * * P i 0.001.
III
of lipid radioactivity
14 c
3H
Polar lipids
46.1 f 7.2
25.2 f 1.3
20
240
71.5 f 2.2
45.8 f 1.5
20
240
3.0
9.9f
32.4f
5
10
1.1
26.5 + 3.8
48.5 Yk4.4
5
C
10
(fin)
Time
*P
Biochimica et Biophysics Acta, 1044 (1990) 101-110 Elsevier
BBALIP
53389
Metabolism of [ 3H]arachidonic acid- and [14C]linoleic acid-labelled chylomicrons in essential fatty acid-deficient rats Lena Hjelte I, Birgitta Strandvik
’
and Ake Nilsson 2
’ Department of Pediatrics and Clinical Research Center, Karolinska Institutei, Huddinge Hospital, Stockholm and ’ Department of Medicine, Hospital of Lund, Lund (Sweden)
(Revised
Key words:
Arachidonic
(Received 15 August 1989) manuscript received 30 November
acid; Chylomicron
metabolism;
Essential
1989)
fatty acid deficiency;
Linoleic
acid; (Rat)
Mesenteric duct chylomicrons labelled with [ 3H]arachidonic (20 : 4) and [ “C]linoleic (18: 2) acid were injected intravenously into essential fatty acid-deficient (EFAD) and control rats. The rats were killed after 5, 10, 20 and 240 min, and serum and different organs were analysed for radioactivity of different lipid classes. 3H and 14C in the triacylglycerol (TG) and phosphatidylethanolamine (PE) fractions were cleared from blood faster in the EFAD than in the control rats. The incorporation of [ “C]18: 2 into liver and heart phospholipids was increased compared to control rats at all time intervals, the increase in the incorporation of [ 3H]20 : 4 being less pronounced. Furthermore, the total incorporation of [ r4C]18: 2 was increased in the heart of the EFAD group. The increased incorporation into ~~phatidylcholine (PC) and particularly into PE was observed already at S-20 min, whereas a marked increase in the C radioactivity of cardiolipin occurred between 20 and 240 min. The 3H and r4C radioactivity per g white adipose tissue was lower in the EFAD group than in the controls. After S-20 min there were no differences between the groups in the lipid radioactivity of the stomach and the small and the large intestine. In the upper small intestine, the 3H radioactivity in both groups and the 14C radioactivity in the EFAD group increased markedly between 20 and 240 min. The study demonstrates an increased plasma clearance and an efficient hepatic uptake and initial incorporation into PC and PE of chylomicron [ 14C]18: 2 and [ 3H]20: 4 in EFAD rats. In these rats a marked selective transfer of [ “C]18: 2 to cardiolipin from other tissue lipids occurred with time. In addition, the study demonstrates a preferential transfer of injected [ ‘H]20: 4, and in EFAD rats also of [ 14C]18: 2, into lipids of the upper small intestine, possibly by secretion of [ 3H]20 : 4 and [ 14C]18 : 2 in bile phospholipids.
Introduction During the development of essential fatty acid (EFA) deficiency, the preservation of the fatty acid pattern varies between different organs and between individual phospholipid classes [l-3]. The mechanisms by which this is achieved have not been clarified. Obviously the
EFA, essential fatty acid; EFAD, essential fatty acid-deficient; BHT, butylated hydroxytoluene; DG, diacylglycerol; CE, cholesteryl ester; CL, cardiolipin; FFA, free fatty acids; HL, hepatic lipase; LPC, lysophosphatidylcholine; LPL, lipoprotein hpase; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; TG, triacylglycerol; TLC, thin-layer chromatography; 18 : 2, linoleic acid; 20 : 4, arachidonic acid. Correspondence: L. Hjelte, Hospital, S-141 86 Huddinge, 0005-2760/90/$03.50
Department Sweden.
of
Pediatrics,
0 1990 Elsevier Science Publishers
Huddinge
B.V. (Biomedical
fatty acid specificity in the pathways of biosynthesis and degradation of phospholipids may be important, but also the regulation of the transport processes by which polyunsaturated fatty acids reach different organs. In an earlier study, we fed [‘4C]linoleic (18 : 2) and [ 3H]aracbidonic (20 : 4) acids to EFA-deficient (EFAD) rats and to control rats and observed an increase in the incorporation of the labelled fatty acids into phospholipids of several organs, particularly into the phosphatidylethanolamine (PE) fraction. The increase in the retention of [i4C]18 : 2 in phosphatidylcholine (PC), PE and cardiolipin (CL) of the small intestine, liver and heart was particularly pronounced [25]. Earlier studies [4,5] have shown that the pattern of incorporation of dietary 20 : 4 into chylomicrons as well as the action of lipoprotein lipase (LPL) on arachidonic acid ester bonds in the chylomicrons might differ from that of the predominant 16-18 carbon fatty acids. Division)
102 Arachidonic acid was overrepresented in chylomicron phospholipids, particularly in PE which is a preferred substrate for the hepatic lipase (HL) [6]. Furthermore, the 20: 4 esters of chylomicron triacylglycerols (TG) exhibited a relative resistance to LPL and accumulated in remaining remnant TG and in 1,2-X-diacylglycerols (DG) 151. This DG and remaining remnant [ ‘HI20 : 4 TG could act as substrates for HL. In vivo experiments also have shown a higher uptake of chylomicron [ ‘HI20 : 4 than of [14C]18 : 2 in the liver in contrast to adipose tissue [7]. These findings combined with the recent observation, that the level of HL is increased in EFAD rats (Nilsson, A., Hjelte, L., Nilsson-Ehle, P. and Strandvik, B., submitted for publication) suggested that the HL may have a selective role in the uptake of polyunsaturated fatty acids by the liver. The tissue uptake of the polyenoic fatty acids might then be regulated in a different manner than the tissue uptake of the predominant 16-18 carbon fatty acids of the chylomicrons which is mediated by LPL. The purpose of this study was to examine whether differences seen in the incorporation patterns of dietary [14C]18 : 2 and [3H]20: 4 in different organs might be due to differences in the metabolism of the chylomicrons between the EFAD and control rats. To elucidate this question we injected linoleate-rich chylomicrons labelled with [r4C]18 : 2 and [ 3H]20 : 4 intravenously into EFAD rats and into controls and followed the plasma clearance and uptake by different organs during 20 min. We also studied the redistribution of radioactive fatty acids that had occurred after 4 h.
Material and Methods
Animals and diets Pregnant white Sprague-Dawley rats were obtained from ALAB AB (Stockholm, Sweden). 10 days prior to delivery and during lactation, one group of the pregnant animals as well as their off-spring (EFAD rats) were fed a semi-synthetic diet deficient in EFA. Another group of pregnant rats and their off-spring (controls) received the same diet except that hydrogenated lard was replaced by soybean lipid. The composition of the diets is given in Table I. The rats had free access to tap water and their respective diets until they were killed. Male rats, 14 weeks old, were used for the experiments. The EFAD rats showed retarded growth and scaliness of hindlegs and tail, characteristic symptoms of EFA deficiency. The mean weight of these rats was 227 g (range 180-268 g) and that of the control rats 300 g (range 250-350 g). The average triene/tetraene ratio (20 : 3 (n - 9)/20 : 4(n - 6)), used as biochemical criterion to define EFA deficiency (2 0.4 [S]), was 30 (range 15-44) in serum phospholipids of the EFAD rats.
TABLE
I
Casein (vitamin-free) Potato starch Glucose Cellulosa fluor Salt mixture’ Vitamins h Hydrogenated lard Soy bean oil
EFAD (wt.%)
Control (wt.%)
20.0 54.0 10.0 4.0 4.0 1.0 7.0 _
20.0 54.0 10.0 4.0 4.0
1.o 1.3 5.1
a Salt mixture containing Ca 0.55%. P 0.31%, K 0.44%. Mg 0.04%. Cl 0.48%. Na 0.28%, Fe 292 ppm, Mn 68 ppm. Cu 10 ppm, Zn 7 ppm. Co 0.4 ppm, J 2 ppm, MO 0.18 ppm, As 0.23 ppm, B 0.17 ppm, Se 0.1 ppm. h Vitamin mixture containing Vit A 11.9 [E/g, Vit D, 1.5 IE/g. Vit B, 4 pg/g, Vit B, 12.4 pg/g, Vit $ 5 pg/g. calcium pantotenate 45% 10 pg/g, niacin 40 pg/g, Vit B,, 0.04 pg/g, Vit K, 1.5 pg/g, biotin 1% 0.3 pg/g, Vit C 480 pg/g, inositol 30 pg/g, Vit E-500 mg/g 42 pg/g, choline chloride 50% 1000 pg/g, p-aminobenzoic acid (PABA) 250 pg/g, folinic acid 0.5 pg/g.
[5,6,9,11,12,14,15-“HI20 : 4 (83.8 Ci/mmol) and [li4C]18 : 2 (52.6 mCi/mmol) were purchased from New England Nuclear Corporation (Boston, MA). Unlabelled 20 : 4 and 18 : 2 were obtained from Nu-ChekPrep (Elysian, MN). Intralipid@ was purchased from Kabi-Vitrum AB (Stockholm, Sweden). This lipid emulsion contains 200 mg soybean TG and 12 mg phospholipid per ml. Mesenteric duct cannulations and stomach fistula operations were performed after an overnight fast and the rats were treated post-operatively as described earlier 141. 50 PCi of [3H]20 : 4 and 50 PCi of [14C]18 : 2 were blown to dryness with nitrogen together with 1 mg of egg-PC, which was dispersed in 2 ml 0.9% NaCl and added to 2 ml of 20% Intralipid. 2 ml of the mixture with labelled fatty acids were fed as a single dose through the stomach fistula at least 20 h after operation and lymph was collected for 8 h on ice. Na,EDTA was added and after filtration chylomicrons were isolated by vacuum ultracentrifugation at 25 000 rpm for 2 h at 4” C in a Beckman SW 41 Swingout bucket rotor. The top layer was collected and redispersed in 0.9% NaCl. TG of the chylomicrons was determined by an enzymatic kit method (Boehringer-Mannheim, F.R.G.). In an earlier study [4], the fatty acid composition of TG, PC and PE of chyle prepared under identical conditions was determined. The TG contained 36.0 + 1.7%, 18 : 2, and 0.80 k 0.08%, 20: 4 (mean + S.E.. n = 3). Two of these chyle preparations were subjected to transmethylation and the fatty acid methyl esters were separated according to the number of double bonds by argentation thin-layer chromatography [9]. 95% of the ‘H and
103 97% of the i4C migrated as 20 : 4 and 18 : 2, respectively. Less than 2% of the 14C migrated as 20: 4, indicating that no major conversion of [14C]18 : 2 to [14C]20 : 4 had occurred in the intestine, before the incorporation into the chylomicrons. The chylomicrons (0.3 ml, containing 1.8 mg TG, per 100 g body wt.) were injected intravenously into the jugular vein under light ether anaesthesia. The radioactivity of the chylomicrons was 4.68. lo5 dpm 3H and 5.81. lo5 dpm 14C per 0.3 ml. After time intervals indicated in the tables and the figures, the rats were again anaesthetized with diethyl ether and killed by aortic puncture. After being rapidly rinsed with saline the intestine was divided in an upper and a lower half and put into chloroform/methanol (1 : 1, v/v) containing 0.005% (w/v) butylated hydroxytoluene (BHT). From other tissues lipids were also extracted with chloroform/methanol (1 : 1, v/v) containing 0.005% BHT. After removing the precipitate by centrifugation, the solvent proportions were adjusted to chloroform/ methanol/water (2 : 1 : 1, v/v) by adding chloroform and 0.1 M KH,PO,. The lower phases were blown to dryness with nitrogen and redissolved in a small volume of chloroform. Aliquots were then taken for thin-layer chromatography (TLC). Non-polar lipids were separated by TLC on silica gel G plates, which were developed in petroleum ether/ diethyl ether/ acetic acid (80 : 20 : 2, v/v) and phospholipids were separated on Merck silica gel 60 plates which were developed in chloroform/ methanol/ acetic acid/ water (100 : 80 : 12 : 1.2, v/v). The spots were identified by staining with iodine and scraped into counting vials. 1 ml methanol/ water (1 : 1, v/v) and 10 ml Instagel/ toluene (1 : 1, v/v) were added and the radioactivity of the samples was determined in a Packard TriCarb 460 CD liquid scintillation system, using the computerized automatic external standard for quench correction.
TABLE
II
Disrrihubon of llpid radioactivity in injected chylomicrons Percent distribution of ‘H and 14C among different lipid classes in chylomicrons used for the injection experiments. Values are means of duplicate analyses. DG, diacylglycerol; FFA, free fatty acids; TG. triacylglycerol; CE, cholesteryl ester; PC, phosphatidylcholine; PE, phosphatidylethanolamine. 1,2-DG
1.3-DG
FFA
TG
CE
PC
PE
‘H
1.3
1.6
3.9
70.5
0.3
14.9
2.8
14 C
0.7
1.4
4.2
88.6
0.4
3.6
0.3
chylomicrons. Also, when the clearance of [3H]TG and [14C]TG was compared, the [14C]TG disappeared faster than the [3H]TG in both animal groups. In the EFAD rats the disappearance of both 3H and 14C radioactivity from serum and the disappearance of labelled PE were faster than in the controls, although the differences were statistically significant only for some of the time
Statistical analysis
Student’s t-test was used for statistical analysis. Variations were indicated by standard errors of the mean (_t S.E.).
loo-
Results Clearance
of ‘H- and “C-labelled
chylomicrons from blood
As previously demonstrated [4], the [3H]20 : 4 and [14C]18 : 2 were not equally distributed between chylomicron phospholipids and TG (Table II). The mean 3H/‘4C ratio of chylomicron TG was 0.8 compared to 4.1 in the PC and 9.3 in the PE fraction. As in an earlier study in normal rats [7], the disappearance of 14C from serum was faster than that of 3H (Fig. 1). The chylomicron TG and PE were cleared from blood faster than PC which partly reflects that a larger proportion of the ‘H than of the 14C was in PC in the injected
25. 0 5
10
20
Fig. 1. Relative recovery of different lipid classes in serum. Rats were injected intravenously with 300 ~1 (per 100 g body wt.) freshly prepared chylomicrons labelled with [ 3H]20 : 4 and [14C]18 : 2. Values are given in percentage of administered radioactivity in respect.ive lipid class and represent means f S.E. of five animals. [ 3H]20 : 4 to the left, [14C]18 : 2 to the right. From above: A, total amount; B, triacylglycerol (TG); C, phosphatidylethanolamine (PE); and D, phosphatidylcholine (PC). n, control rats; 0, essential fatty acid-deficient rats. * P i 0.05, * * P < 0.01, * * * P i 0.001.
III
of lipid radioactivity
14 c
3H
Polar lipids
46.1 f 7.2
25.2 f 1.3
20
240
71.5 f 2.2
45.8 f 1.5
20
240
3.0
9.9f
32.4f
5
10
1.1
26.5 + 3.8
48.5 Yk4.4
5
C
10
(fin)
Time
*P
