190 0 1992 Elsevier
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Biochimiw et Biophysicu Acril, I 124 ( 1992) 1OO- 194 Publishers B.V. All rights reserved !KlOS-27~0/92/$05.00
53843
Intestinal absorption of fish oil in rats previously adapted to diets containing fish oil or corn oil
(Revised
Key words:
(Received 5 June 1991) manuscript received 30 September
Fish oil; Fatty acid, YI- 3: Icosapentaenoic
1991)
acid: Docosahexaenoic
acid; Intestinal
absorption
A study was conducted to evaluate whether the composition of previous dietary fat affects the absorption and composition of lymph obtained after a meal of fish oil. Adult male Sprague-Dawley rats were fed diets containing either corn oil or fish oil (MaxEPA) for 2 weeks. They were then given intraduodenaIiy a bolus of an emulsion of 0.5 ml of fish oii plus 0.5 ml of 20 mM sodium taurocholate. Intestinal lymph was collected from a cannula in the main intestinal lymph trunk for various times after oil administration. Rats previously fed fish oil absorbed a greater proportion of the test dose of fish oil than those fed corn oil. There was an effect of previous diet on the fatty acid composition of the lymph. Rats fed fish oil had a higher percentage of eicosapentaenoic and docosahexaenoic acids in the lymph lipids than those fed corn oil while those fed corn oil had a higher percentage of linoleic acid. These results rule out decreased intestinal absorption as a mechanism for the hypotriacylglycerolemic effect of dietary fish oils. They also indicate a significant contribution of endogenous lipids to the fatty acids in lymph.
Introduction One of the major effects of dietary fish oils is the reduction in circulating triacylglycerols which has been attributed to the effects of the long chain 12- 3 fatty acids [I]. It has been shown that dietary fish oils reduce the synthesis and secretion of hepatic VLDL [l-31 and it is likely that this is the major factor in their hypotriacylglycerolmic effect. There is also evidence of increased oxidation of the long chain n - 3 fatty acids, both in the liver [2] and in muscle (Ref. 4, Levy and Herzberg, unpublished data). There have been reports that the long chain ?Z- 3 fatty acids in triacylglycerols are resistant to hydrolysis by pancreatic lipase ISI although this has been refuted [6,7]. We have recently shown that there is no difference between the intestinal absorption of fish oil and olive oit in rats [7]. Harris et al. [8] reported that humans previously fed a diet rich in fish oil had a smaller area under their fat
Correspondence: G.R. Flerzberg Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland A 1B 3X9, Canada.
tolerance curves than those previously fed vegetable oil. They suggested that this might be due to an adaptation of intestinal absorption such that fat absorption was reduced by a fish oil diet. It has also been shown that endogenous lipids make a significant contribution to intestinal lymph lipids [S-11]. Thus, it might be expected that a fish oil diet would alter the composition of endogenous lymph lipid. Such an effect has not previously been examined in fish oil-fed rats. Because our previous study was done in chow fed rats, we felt it necessary to reexamine fat absorption in rats that had been previously fed either a fish or vegetable oil containing diet. Materials
and Methods
Male, Sprague-Dawley rats (250-300 g) were obtained from Canadian Hybrid Farms (Halls Harbour, N.S.). They were maintained on diets containing either 10% corn oil or 10% MaxEPA (R.P. Scherer, Windsor Ontario). The diets had the following composition (g/kg): glucose-600; casein-200; fat-loo; cellulose-50; AIN mineral mix-35; AIN vitamin mix-lo; methionine3, choline chloride-2, TBHQ-0.02. Diets were prepared
191 TABLE I Fatty acid composition
of the dietary fats (weight %)
Fatty acid
Corn oil
MaxEPA
14:o 16:O 16: l(n -7) 18:0 18: l(n -9) 18:2(n -6) 18:3(n -3) 18:4(n -3) 20:l(n-9) 20:4(n -6) 20:5(n-3) 22:l(n-11) 22:.5(n -3) 22:6(n -3)
1.0 9.9 0.2 2.5 27.9 53.0 0.9
5.1 14.3 6.9 4.5 13.3 3.0 0.7 4.5 1.8 0.9 15.7 1.3 2.3 11.6
0.2 -
and stored so as to minimize oxidation (121. Two dietary fats were used, either 100 g of corn oil/kg or 100 g of MaxEPA/kg of diet. The dietary fats had the composition given in Table I. The composition of the MaxEPA used for duodenal infusions was the same as that of the dietary MaxEPA given in Table I. Rats were fed the experimental diets for two weeks before the absorption studies we;e conducted. Rats were fitted with exteriorized cannulas in the duodenum and the the main intestinal lymphatic trunk as previously described [7]. Following surgery, the rats were immobilized in Bollman restraint cages and placed in a warm, dark room. The rats had free access to drinking water. Lymph flow was promoted by continuous infusion of saline through the duodenal cannula for the duration of the experiment (2.5 ml/h). Following an overnight recovery, basal lymph flow rates and collections were made for analysis of endogenous fatty acid composition of the lymph triacylglycerols. Coagulation of lymph was prevented by the addition of sodium citrate to tared containers. Each rat was given intraduodenally a 1.0 ml bolus dose containing 0.5 ml of MaxEPA oil plus 0.5 ml of 20 mM sodium taurocholate and the saline infusion was continued. The oil and sodium taurocholate were sonicated just before infusion to ensure homogeneity. Lymph was cotlected and pooled at 60 min intervals for 6 h and then pooled for the remaining 18 h. Basal lymph was collected for 30 min prior to the intraduodenal introduction of the fish oil. BasaI TAG output and fatty acid composition are shown as the zero time values in the figures and tables. A 100 ~1 sample was taken from each sample, diluted with 900 ~1 of saline and assayed for triacylglycerol using a Hitachi 70.5 Automatic Analyzer [13]. Only the 0, 1, 2 and 3 h lymph samples were analyzed for fatty acid composition. Samples of lymph were mixed with 19 volumes of CHCl,/CH,OH (2: 1, v/v) and shaken with 3.8 volumes of 0.9% saline f14].
The lower phases containing the lipid were collected. Samples of the diet were treated similarly. Solvents contained 0.25 g/l of recrystallized hydroquinone as antioxidant. Portions of the lipid extracts were transmethylated in acidified methanol as previously described [15]. Methyl heptadecanoate was used as an internal standard. The methyl esters were analyzed using a 30 m X 0.25 mm column coated with SP 2330 (Supelco, Bellefonte, PA) in a Perkin-Elmer 8310 gas chromatograph (Norwalk, CT). Oven temperature was 18O”C, and the injection port and flame ionization detector temperatures were 230°C. Quantitation was carried out with the data-handling and control unit of the instrument. The triacylglycerol recovery and the fatty acid composition data were analyzed by two way ANOVA (factors were time and diet). The percent composition data were arc-sin transformed before the analysis was performed [161. Results
The triacylglycerol output during the first 6 h of collection is shown in Fig. 1. The change in output with time is the same for both diets. The dietary treatment did not alter the time at which maximal output was reached (2 h) which was the same as in our previous study of chow-fed rats [71. However, at each time, except t = 0, the TAG output was greater in rats prefed MaxEPA. This is shown in Table II where the cumulative tria~lglycerol recovery al 6 and 24 h is illustrated. There was SignificantIy greater recovery of triacylglycerol in the lymph in animals prefed the fish oil diet (P < 0.02) in both the first 6 hours and the
Fig. 1. Lymph triacylglycerol output (pmol/h) after intraduodenal administration of MaxEPA. Rats had previously been fed diets containing corn oil (n = 7) or MaxEPA (n = 6) for 2 weeks. Values are means + S.E.
25
22:6
20
o0
60
120
180
60
0
120
180
25
20: 1 acids were constant throughout the collection period (P > 0.05 for the effect of time). 18 : 0, 18 : 2 and 20: 4 all declined during the collection while 14 : 0, 18 : 4, 20 : 5, 22 : 5 and 22 : 6 all increased (P < (LOOS). Only four of the fatty acids were significantly (P < 0.001) affected by diet, linoleic, 20 : 1, eicosapentaenoic and docosahexaenoic. These results are illustrated in Fig. 2. For animals prefed the corn oil diet, 1X:2, the predominant fatty acid in the diet, was higher throughout the collection period. Similarly, 20: 5 and 22: 6 which are absent from corn oil but make up about 20% of the fish oil, were higher in the lymph of fish oil fed animals through~~ut the collection.
2O:l
20 i
0 0
60
Time Fig. 2. Fatty
120
0
180
(mint
acid c~~rnp~)siti~n (weight%)
tr~jd~lt~den~~i~ldminisfr~~ti(~n of MaxEPA. fed diets containing weeks.
Values
corn oil (CO,
are means+S.E.
there was a significant 70: I. 1O:S. 22:h)
60
120
Time
(min)
of lymph lipids after Rats had previously
N = 7) or MaxEPA Only
180
those
fatty
in-
been
01 = h) for 2
acids for which
effect of diet on lymph composition
(18:2,
are shown. (3, corn oil fed rats; 0. MaxEPA
Discussion
fed
rats.
The observation that the TAG output into lymph was greater in fish oil-fed animals (Fig. 1 and Table II) suggests that the lowering of the fat tolerance curve in fish oil fed subjects reported by Harris et al. [S] was not due to a decreased ability to absorb fish oil. Recently, Hall et al. [17] reported that there was decreased
24 h collection period. The lymph fatty acid composition over the first 3 h of collection is presented in Table III. For simplicity, only those fatty acids which made up at least 1% of the total lymph fatty acids at at least one time are presented. Palmitic and oleic and entire
TABLE
III
Eiltry trciri c~ompositio~tof lynlph lipid af times after ir?fd~rodtwd ir!firsim yf MaxEPA Rats were fed diets containing composition
was determined.
acids present at greater Fatty acid
either
corn oil or MaxEPA
Time after MaxEPA
Corn oil
Ih:O 16:
I
or seven (corn oil) individual
as described
in the text and the lipid fatty acid
experiments
k S.D. For clarity. only those fatty
than I% at at least one time are presented. infusion (min) 60
0
14:o
for 2 weeks. Lymph was collected
Results are means of six (MaxEPA)
MaxEPA
2.2+_ 15 _ 1X.2* 2.4 2.2 * 0.4
lX:ll
Il.hi
IX: !
13.7f3.1
1.1
IX:’
ih.8 + 2.x
IX:4
0.2 f 0.6
20: I
I .8 i 3.0
I20
Corn oil 4.2+ 2.9 17.5 i_ 2.‘)
I 6.h+ 0.7 6.4 * 2. 13.Yi2.0
MaxEPA 4.9 2 2.5 1X.6&3.0 6.4i_ 1.Y 6.h& I.5
I x0
Corn oil 6.4 I35 IY.4& I.8 x.2*
1.2
i.3iO.h
MaxEPA
--
6.5 & 3.‘) 10.9*2.1 7.7 i 2.X 5.7 * I. 1 14.4 * 2.4
Corn oil 5.7 2 2.0 19.9 * 0.9 7.0 * i .fi
MaxEPA
5.x * 2.4 l7.hi
1.5
5.9 + 1. I
5.3 * 0.8
4.8 + 0.5
IS.1 f I.9
14.1 t I .i
14.7 & 1.2
11.2 * 2.0
9.8 _L2.2
7.hi_2.2
7.4 + 2.3
s.3*
7.0 +0.x
4.3i
I.6
3.0 + 0.7
‘t.l&
I.3
3.7 * 0.6
3.1 * I.6
1.72
3.x*
I.1
I .Y IO.8
0.8+
I.0
2.0 + 0.5
I .o ?r 1.2
2.0 * 0.6
1.3
1.4+ I.1
I.4
4.6 * 0.9
4.3 i 2.3
12.0 -* 2.5
12.0 f 1.3
15.xi- I.0
1.0
0.0 _t 0.0
0.3 * 0.8
0.6 + I .o
1.3 + 0.9
I.3 & I .2
2.2 & I .o
8.4 * 2.3
x.3 * 0.Y
20:4
8.3 i 2.7
6.7 * 2.2
6.5 + 4.0
3.7*0.7
20:5
Y.o*
I.3
Y.Y _I 2.3
13.x + I .4
IO.1 i2.8
22:5
0.5 * 1.3
0.2 i 0.4
0.4 * 0.6
1.2*
22:s
1.4k2.2
I.65
2.2+
1.3
22:6
7.5+1.x
fIl.9+ 1.4
8.2 + 3.6
7.9 + 2.8
1.5
1.5
3.7*
l.7_+ 1.3 11.7*
1.h
193 secretion of triacylglycerol by CaCo-2 cells incubated with fish oil fatty acids. They suggested that this supported a role for reduced enterocyte triacylglycerol secretion in the reduction of postprandial triacylglycerolemia in humans consuming fish oil diets. Yang et al. [lS] have shown that the intestinal absorption of the methyl esters of fish oil fatty acids is limited due to the limited capacity of the glycerol phospate pathway for reesterification. The glycerol phosphate pathway is the dominant pathway for reesterification in the absence of monoacylglycerol. In their experiments, Hall et al. [I71 used free fatty acids without monoacylglycerol. Thus the limited capacity of the gly~erolphosphate pathway may explain the reduced tria~ylglycerol secretion they observed. Interestingly, Trotter and Starch [19] have reported that CaCo-2 cells do not have the monoacylglycerol pathway and rely almost completely on the glycerolphosphate pathway for fatty acid esterification. This suggests that CaCo-2 cells may not be a good model for the study of the absorption of dietary fish oils. In any case, our results in vivo demonstrate that there is no reduction in the absorption of fish oil in animais previousIy fed a fish oii containing diet. TAG output into lymph collected prior to the intraduodenal introduction of MaxEPA was the same on both diets (P > 0.5). This suggests that the effect of prior diet on the absorption of MaxEPA is not due to a greater supply of endogenous lipid in fish oil-fed animals. However, it is possible that there is a greater increase in endogenous lipid during exogenous lipid absorption in animals prefed fish oil [lO,ll]. The changes in lymph fatty acid composition are consistent with our previous report [71 that the composition of lymph during fish oil absorption reflected the fatty acid composition of the fish oil. This is particularly apparent for the long chain IZ- 3 fatty acids 20 : 5 and 22 : 6. There was no evidence of significant elongation, desaturation or chain shortening of the a - 3 fatty acids although evidence has been presented that the intestinal epithelium has the enzymatic capacity for such reactions. This can be seen by examining Fig. 2. It can be seen that once the increased output of TAG is established, the composition of 20: 5 and 22: 6 are constant from 1 to 3 h. This is also true for 22 : 5 (Table III). If there had been significant elongation/ desaturation of 20 : 5, there should have been a decline in its content over time and an increase in 22 : 5 and/or 22: 6. Thus we conclude that there is no significant elongation/desaturation of the long chain n - 3 fatty acids during intestinal absorption. Interestingly, there was no EPA in the lymph of corn oil fed animals at the beginning of the collection but there was a significant amount of docosahexaenoic acid at that time. This is consistent with a biliary phospholipid source of the DHA. Lands et al. [20] found significant amounts of DHA but no EPA in the
plasma and liver phospholipids of corn oil fed rats although both fatty acids were absent from the diet. The higher 20: 1 in corn oil compared to fish oil fed rats is paradoxical since there is more 20: 1 in MaxEPA than corn oil. The observation suggests either increased 18: 1 elongation in corn oil fed rats or increased utilization of 20: 1 in fish oil fed rats. We have no data supporting either possibility. The higher 18:2 in the corn oil fed rats and higher 20: 5 and 22: 6 in fish oil fed rats observed before infusion of the test dose of fish oil continued during absorption which suggests an endogenous source for these fatty acids. It has previously been shown that endogenous lipid makes a significant contribution to the lipid in intestinal lymph. Baxter [9] estimated that 50% of the endogenous contribution came from bile lipid. Other endogenous sources could not be identified although circulating free fatty acid made little if any contribution. Karmen et al. [lo] showed that the contribution of endogenous lipids increased during lipid absorption. This has been confirmed by Bergstedt et al. [II] who found an increased output of fatty acids other than oleate in rats given only triolein intraduodenally. They also found that oleic acid only accounted for 74% and stearic acid only 49% of lymph lipid fatty acids in rats intraduodenally infused with triolein or tristearin respectively. This clearly points to a significant (25.50%) contribution of endogenous lipid to lymph lipid. Tso et al. [21] have previously shown that the fatty acid composition of lymph in the absence of triacylglycerol absorption reflects the composition of bile phospholipid. We believe that the differences we see in the pre-infusion lymph composition of 18:2, 20: 1, 20 : 5 and 22 : 6 reflect differences in bile phospholipid fatty acid composition. We have previously found that the long chain n - 3 fatty acids 20: 5 and 22: 6 are enriched in hepatic triacylglycerols of fish oil fed rats, suggesting an increased availability of these fatty acids in the liver (Sheppard and Herzberg, unpublished data). Such an increased supply could also be available for bile phospholipid synthesis. We could not find any data on the bile phospholipid composition of fish oil fed rats and it is presently under investigation in our laboratory. Our finding of an influence of prior diet on the fatty acid composition of lymph Iipids suggests that this may be useful for obtaining further insight into the quantitative contribution of endogenous lipids to the lipids of lymph. There is essentially no 20: 5 in the fasting lymph of rats previously fed a corn oil diet but this fatty acid makes up about 8% in fish oil fed rats. Corn oil has no 20: 5. If we prefed rats fish oil and then measured lymph lipid composition and quantified the lipids after a dose of corn oil all the 20: 5 must have come from endogenous sources. By quantitating bile iipid and determining its fatty acid composition we
194
could determine what fraction of 20 : 5 and, by extension to the other fatty acids in bile, what fraction of the lymph lipids came from bile. If this did not account for all the 20:5, then plasma must have provided the rest. By determining plasma fatty acid composition we could then calculate the contribution of plasma lipids to lymph. In this manner the contributions of bile, plasma and dietary lipid to lymph lipid could be estimated without the complications introduced through the use of labelled lipids. In conclusion, we have shown that prefeeding rats fish oil increases the absorption of a test dose of fish oil. This rules out reduced absorption as a contributor to the hypolipidemic effect of dietary fish oils. In addition we have shown that dietary corn and fish oil affect the fatty acid composition of lymph in characteristic ways. This observation has suggested an experimental approach to examine the contributions of endogenous lipids to lymph. Acknowledgements
This work was supported by the Medical Research Council of Canada. We thank R.P. Scherer (Canada) for the gift of MaxEPA oil. References 1 Harris, W.S. (1989) J. Lipid Res. 30, 785807. 2 Wong, S.H., Nestel, P.J., Trimble, R.P., Storer, G.B., Illman, R.J. and Topping, D.L. (1984) Biochim. Biophys. Acta 792, 103-109.
3 Herzberg, G.R. and Rogerson, M. (1988) J. Nun. 118, 1061-1~67. 4 Herzberg, G.R. (1991) Can. 3. Physiol. Pharmacol., in press. 5 Bottino, N.R., Vandenburg, CA. and Reiser, R. (1967) Lipids 2, 489-493. 6 Brockerhofff, H. and Hoyle, R.J. (1963) Arch. Biochem. Biophys. 102, 452-455. 7 Chernenko, G.A., Barrowman, J.A., Kean, K.T., Herzberg, G.R. and Keough, K.M.W. (1989) Biochim. Biophys. Acta 1004,95-102. 8 Harris, W.S., Connor, W.E., Alam, N. and Illingworth, D.R. (1988) J. Lipid Res. 29, 1451-1460. 9 Baxter, J.N. (1966) J. Lipid Res. 7, 158-166. IO Karmen, A., Whyte, M. and Goodman, D.S. (S963) J. Lipid Res. 4, 312-321. 11 Bergstedt, S.E., Hayashi, H., Kritchevsky, D. and Tso, P. (1990) Am. J. Physiol. 259, G386-G393. 12 Fritsche, K.L. and Johnston. P.V. (1988) J. Nun. 118, 425-426. 13 Wahlfeld, A.W. (1974) in Methods of Enzymatic Analysis, 2nd English Edn. (Bergmeyer, H.U., ed.), pp. 1831-1835, Verlag Chemie, Weinheim and Academic Press, New York. 14 Folch, J., Lees. M. and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. 15 Keough, K.M.W. and Kariel, N. (1987) Biochim. Biophys. Acta 902, 11-18. 16 Zar, J.H. (1984) Biostatistical Analysis, 2nd Edn. pp. 239-241, Prentice Hall Canada, Toronto. 17 Hall, J.A., Connor, W.E.. Anderson, G.J. and Newcomb. K.C. (19911 FASEB J. 5, AIROI. 18 Yang, L.-Y.. Kuksis. A. and Myher, J.J. (1990) B&hem. Cell Biol. 68, 480-491. 19 Trotter. P.J. and Starch, J. t19Yl) FASEB J. 5. A1640. 20 Lands. W.E.M., Morris, A. and Libelt, B. (1990) Lipids 9,505-516. 21 Tso, P., Baiint, J.A. and Simmonds, W.J. (1977) Gastroenterology 12, 1362-1367.
BBALIP
Science
Biochimiw et Biophysicu Acril, I 124 ( 1992) 1OO- 194 Publishers B.V. All rights reserved !KlOS-27~0/92/$05.00
53843
Intestinal absorption of fish oil in rats previously adapted to diets containing fish oil or corn oil
(Revised
Key words:
(Received 5 June 1991) manuscript received 30 September
Fish oil; Fatty acid, YI- 3: Icosapentaenoic
1991)
acid: Docosahexaenoic
acid; Intestinal
absorption
A study was conducted to evaluate whether the composition of previous dietary fat affects the absorption and composition of lymph obtained after a meal of fish oil. Adult male Sprague-Dawley rats were fed diets containing either corn oil or fish oil (MaxEPA) for 2 weeks. They were then given intraduodenaIiy a bolus of an emulsion of 0.5 ml of fish oii plus 0.5 ml of 20 mM sodium taurocholate. Intestinal lymph was collected from a cannula in the main intestinal lymph trunk for various times after oil administration. Rats previously fed fish oil absorbed a greater proportion of the test dose of fish oil than those fed corn oil. There was an effect of previous diet on the fatty acid composition of the lymph. Rats fed fish oil had a higher percentage of eicosapentaenoic and docosahexaenoic acids in the lymph lipids than those fed corn oil while those fed corn oil had a higher percentage of linoleic acid. These results rule out decreased intestinal absorption as a mechanism for the hypotriacylglycerolemic effect of dietary fish oils. They also indicate a significant contribution of endogenous lipids to the fatty acids in lymph.
Introduction One of the major effects of dietary fish oils is the reduction in circulating triacylglycerols which has been attributed to the effects of the long chain 12- 3 fatty acids [I]. It has been shown that dietary fish oils reduce the synthesis and secretion of hepatic VLDL [l-31 and it is likely that this is the major factor in their hypotriacylglycerolmic effect. There is also evidence of increased oxidation of the long chain n - 3 fatty acids, both in the liver [2] and in muscle (Ref. 4, Levy and Herzberg, unpublished data). There have been reports that the long chain ?Z- 3 fatty acids in triacylglycerols are resistant to hydrolysis by pancreatic lipase ISI although this has been refuted [6,7]. We have recently shown that there is no difference between the intestinal absorption of fish oil and olive oit in rats [7]. Harris et al. [8] reported that humans previously fed a diet rich in fish oil had a smaller area under their fat
Correspondence: G.R. Flerzberg Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland A 1B 3X9, Canada.
tolerance curves than those previously fed vegetable oil. They suggested that this might be due to an adaptation of intestinal absorption such that fat absorption was reduced by a fish oil diet. It has also been shown that endogenous lipids make a significant contribution to intestinal lymph lipids [S-11]. Thus, it might be expected that a fish oil diet would alter the composition of endogenous lymph lipid. Such an effect has not previously been examined in fish oil-fed rats. Because our previous study was done in chow fed rats, we felt it necessary to reexamine fat absorption in rats that had been previously fed either a fish or vegetable oil containing diet. Materials
and Methods
Male, Sprague-Dawley rats (250-300 g) were obtained from Canadian Hybrid Farms (Halls Harbour, N.S.). They were maintained on diets containing either 10% corn oil or 10% MaxEPA (R.P. Scherer, Windsor Ontario). The diets had the following composition (g/kg): glucose-600; casein-200; fat-loo; cellulose-50; AIN mineral mix-35; AIN vitamin mix-lo; methionine3, choline chloride-2, TBHQ-0.02. Diets were prepared
191 TABLE I Fatty acid composition
of the dietary fats (weight %)
Fatty acid
Corn oil
MaxEPA
14:o 16:O 16: l(n -7) 18:0 18: l(n -9) 18:2(n -6) 18:3(n -3) 18:4(n -3) 20:l(n-9) 20:4(n -6) 20:5(n-3) 22:l(n-11) 22:.5(n -3) 22:6(n -3)
1.0 9.9 0.2 2.5 27.9 53.0 0.9
5.1 14.3 6.9 4.5 13.3 3.0 0.7 4.5 1.8 0.9 15.7 1.3 2.3 11.6
0.2 -
and stored so as to minimize oxidation (121. Two dietary fats were used, either 100 g of corn oil/kg or 100 g of MaxEPA/kg of diet. The dietary fats had the composition given in Table I. The composition of the MaxEPA used for duodenal infusions was the same as that of the dietary MaxEPA given in Table I. Rats were fed the experimental diets for two weeks before the absorption studies we;e conducted. Rats were fitted with exteriorized cannulas in the duodenum and the the main intestinal lymphatic trunk as previously described [7]. Following surgery, the rats were immobilized in Bollman restraint cages and placed in a warm, dark room. The rats had free access to drinking water. Lymph flow was promoted by continuous infusion of saline through the duodenal cannula for the duration of the experiment (2.5 ml/h). Following an overnight recovery, basal lymph flow rates and collections were made for analysis of endogenous fatty acid composition of the lymph triacylglycerols. Coagulation of lymph was prevented by the addition of sodium citrate to tared containers. Each rat was given intraduodenally a 1.0 ml bolus dose containing 0.5 ml of MaxEPA oil plus 0.5 ml of 20 mM sodium taurocholate and the saline infusion was continued. The oil and sodium taurocholate were sonicated just before infusion to ensure homogeneity. Lymph was cotlected and pooled at 60 min intervals for 6 h and then pooled for the remaining 18 h. Basal lymph was collected for 30 min prior to the intraduodenal introduction of the fish oil. BasaI TAG output and fatty acid composition are shown as the zero time values in the figures and tables. A 100 ~1 sample was taken from each sample, diluted with 900 ~1 of saline and assayed for triacylglycerol using a Hitachi 70.5 Automatic Analyzer [13]. Only the 0, 1, 2 and 3 h lymph samples were analyzed for fatty acid composition. Samples of lymph were mixed with 19 volumes of CHCl,/CH,OH (2: 1, v/v) and shaken with 3.8 volumes of 0.9% saline f14].
The lower phases containing the lipid were collected. Samples of the diet were treated similarly. Solvents contained 0.25 g/l of recrystallized hydroquinone as antioxidant. Portions of the lipid extracts were transmethylated in acidified methanol as previously described [15]. Methyl heptadecanoate was used as an internal standard. The methyl esters were analyzed using a 30 m X 0.25 mm column coated with SP 2330 (Supelco, Bellefonte, PA) in a Perkin-Elmer 8310 gas chromatograph (Norwalk, CT). Oven temperature was 18O”C, and the injection port and flame ionization detector temperatures were 230°C. Quantitation was carried out with the data-handling and control unit of the instrument. The triacylglycerol recovery and the fatty acid composition data were analyzed by two way ANOVA (factors were time and diet). The percent composition data were arc-sin transformed before the analysis was performed [161. Results
The triacylglycerol output during the first 6 h of collection is shown in Fig. 1. The change in output with time is the same for both diets. The dietary treatment did not alter the time at which maximal output was reached (2 h) which was the same as in our previous study of chow-fed rats [71. However, at each time, except t = 0, the TAG output was greater in rats prefed MaxEPA. This is shown in Table II where the cumulative tria~lglycerol recovery al 6 and 24 h is illustrated. There was SignificantIy greater recovery of triacylglycerol in the lymph in animals prefed the fish oil diet (P < 0.02) in both the first 6 hours and the
Fig. 1. Lymph triacylglycerol output (pmol/h) after intraduodenal administration of MaxEPA. Rats had previously been fed diets containing corn oil (n = 7) or MaxEPA (n = 6) for 2 weeks. Values are means + S.E.
25
22:6
20
o0
60
120
180
60
0
120
180
25
20: 1 acids were constant throughout the collection period (P > 0.05 for the effect of time). 18 : 0, 18 : 2 and 20: 4 all declined during the collection while 14 : 0, 18 : 4, 20 : 5, 22 : 5 and 22 : 6 all increased (P < (LOOS). Only four of the fatty acids were significantly (P < 0.001) affected by diet, linoleic, 20 : 1, eicosapentaenoic and docosahexaenoic. These results are illustrated in Fig. 2. For animals prefed the corn oil diet, 1X:2, the predominant fatty acid in the diet, was higher throughout the collection period. Similarly, 20: 5 and 22: 6 which are absent from corn oil but make up about 20% of the fish oil, were higher in the lymph of fish oil fed animals through~~ut the collection.
2O:l
20 i
0 0
60
Time Fig. 2. Fatty
120
0
180
(mint
acid c~~rnp~)siti~n (weight%)
tr~jd~lt~den~~i~ldminisfr~~ti(~n of MaxEPA. fed diets containing weeks.
Values
corn oil (CO,
are means+S.E.
there was a significant 70: I. 1O:S. 22:h)
60
120
Time
(min)
of lymph lipids after Rats had previously
N = 7) or MaxEPA Only
180
those
fatty
in-
been
01 = h) for 2
acids for which
effect of diet on lymph composition
(18:2,
are shown. (3, corn oil fed rats; 0. MaxEPA
Discussion
fed
rats.
The observation that the TAG output into lymph was greater in fish oil-fed animals (Fig. 1 and Table II) suggests that the lowering of the fat tolerance curve in fish oil fed subjects reported by Harris et al. [S] was not due to a decreased ability to absorb fish oil. Recently, Hall et al. [17] reported that there was decreased
24 h collection period. The lymph fatty acid composition over the first 3 h of collection is presented in Table III. For simplicity, only those fatty acids which made up at least 1% of the total lymph fatty acids at at least one time are presented. Palmitic and oleic and entire
TABLE
III
Eiltry trciri c~ompositio~tof lynlph lipid af times after ir?fd~rodtwd ir!firsim yf MaxEPA Rats were fed diets containing composition
was determined.
acids present at greater Fatty acid
either
corn oil or MaxEPA
Time after MaxEPA
Corn oil
Ih:O 16:
I
or seven (corn oil) individual
as described
in the text and the lipid fatty acid
experiments
k S.D. For clarity. only those fatty
than I% at at least one time are presented. infusion (min) 60
0
14:o
for 2 weeks. Lymph was collected
Results are means of six (MaxEPA)
MaxEPA
2.2+_ 15 _ 1X.2* 2.4 2.2 * 0.4
lX:ll
Il.hi
IX: !
13.7f3.1
1.1
IX:’
ih.8 + 2.x
IX:4
0.2 f 0.6
20: I
I .8 i 3.0
I20
Corn oil 4.2+ 2.9 17.5 i_ 2.‘)
I 6.h+ 0.7 6.4 * 2. 13.Yi2.0
MaxEPA 4.9 2 2.5 1X.6&3.0 6.4i_ 1.Y 6.h& I.5
I x0
Corn oil 6.4 I35 IY.4& I.8 x.2*
1.2
i.3iO.h
MaxEPA
--
6.5 & 3.‘) 10.9*2.1 7.7 i 2.X 5.7 * I. 1 14.4 * 2.4
Corn oil 5.7 2 2.0 19.9 * 0.9 7.0 * i .fi
MaxEPA
5.x * 2.4 l7.hi
1.5
5.9 + 1. I
5.3 * 0.8
4.8 + 0.5
IS.1 f I.9
14.1 t I .i
14.7 & 1.2
11.2 * 2.0
9.8 _L2.2
7.hi_2.2
7.4 + 2.3
s.3*
7.0 +0.x
4.3i
I.6
3.0 + 0.7
‘t.l&
I.3
3.7 * 0.6
3.1 * I.6
1.72
3.x*
I.1
I .Y IO.8
0.8+
I.0
2.0 + 0.5
I .o ?r 1.2
2.0 * 0.6
1.3
1.4+ I.1
I.4
4.6 * 0.9
4.3 i 2.3
12.0 -* 2.5
12.0 f 1.3
15.xi- I.0
1.0
0.0 _t 0.0
0.3 * 0.8
0.6 + I .o
1.3 + 0.9
I.3 & I .2
2.2 & I .o
8.4 * 2.3
x.3 * 0.Y
20:4
8.3 i 2.7
6.7 * 2.2
6.5 + 4.0
3.7*0.7
20:5
Y.o*
I.3
Y.Y _I 2.3
13.x + I .4
IO.1 i2.8
22:5
0.5 * 1.3
0.2 i 0.4
0.4 * 0.6
1.2*
22:s
1.4k2.2
I.65
2.2+
1.3
22:6
7.5+1.x
fIl.9+ 1.4
8.2 + 3.6
7.9 + 2.8
1.5
1.5
3.7*
l.7_+ 1.3 11.7*
1.h
193 secretion of triacylglycerol by CaCo-2 cells incubated with fish oil fatty acids. They suggested that this supported a role for reduced enterocyte triacylglycerol secretion in the reduction of postprandial triacylglycerolemia in humans consuming fish oil diets. Yang et al. [lS] have shown that the intestinal absorption of the methyl esters of fish oil fatty acids is limited due to the limited capacity of the glycerol phospate pathway for reesterification. The glycerol phosphate pathway is the dominant pathway for reesterification in the absence of monoacylglycerol. In their experiments, Hall et al. [I71 used free fatty acids without monoacylglycerol. Thus the limited capacity of the gly~erolphosphate pathway may explain the reduced tria~ylglycerol secretion they observed. Interestingly, Trotter and Starch [19] have reported that CaCo-2 cells do not have the monoacylglycerol pathway and rely almost completely on the glycerolphosphate pathway for fatty acid esterification. This suggests that CaCo-2 cells may not be a good model for the study of the absorption of dietary fish oils. In any case, our results in vivo demonstrate that there is no reduction in the absorption of fish oil in animais previousIy fed a fish oii containing diet. TAG output into lymph collected prior to the intraduodenal introduction of MaxEPA was the same on both diets (P > 0.5). This suggests that the effect of prior diet on the absorption of MaxEPA is not due to a greater supply of endogenous lipid in fish oil-fed animals. However, it is possible that there is a greater increase in endogenous lipid during exogenous lipid absorption in animals prefed fish oil [lO,ll]. The changes in lymph fatty acid composition are consistent with our previous report [71 that the composition of lymph during fish oil absorption reflected the fatty acid composition of the fish oil. This is particularly apparent for the long chain IZ- 3 fatty acids 20 : 5 and 22 : 6. There was no evidence of significant elongation, desaturation or chain shortening of the a - 3 fatty acids although evidence has been presented that the intestinal epithelium has the enzymatic capacity for such reactions. This can be seen by examining Fig. 2. It can be seen that once the increased output of TAG is established, the composition of 20: 5 and 22: 6 are constant from 1 to 3 h. This is also true for 22 : 5 (Table III). If there had been significant elongation/ desaturation of 20 : 5, there should have been a decline in its content over time and an increase in 22 : 5 and/or 22: 6. Thus we conclude that there is no significant elongation/desaturation of the long chain n - 3 fatty acids during intestinal absorption. Interestingly, there was no EPA in the lymph of corn oil fed animals at the beginning of the collection but there was a significant amount of docosahexaenoic acid at that time. This is consistent with a biliary phospholipid source of the DHA. Lands et al. [20] found significant amounts of DHA but no EPA in the
plasma and liver phospholipids of corn oil fed rats although both fatty acids were absent from the diet. The higher 20: 1 in corn oil compared to fish oil fed rats is paradoxical since there is more 20: 1 in MaxEPA than corn oil. The observation suggests either increased 18: 1 elongation in corn oil fed rats or increased utilization of 20: 1 in fish oil fed rats. We have no data supporting either possibility. The higher 18:2 in the corn oil fed rats and higher 20: 5 and 22: 6 in fish oil fed rats observed before infusion of the test dose of fish oil continued during absorption which suggests an endogenous source for these fatty acids. It has previously been shown that endogenous lipid makes a significant contribution to the lipid in intestinal lymph. Baxter [9] estimated that 50% of the endogenous contribution came from bile lipid. Other endogenous sources could not be identified although circulating free fatty acid made little if any contribution. Karmen et al. [lo] showed that the contribution of endogenous lipids increased during lipid absorption. This has been confirmed by Bergstedt et al. [II] who found an increased output of fatty acids other than oleate in rats given only triolein intraduodenally. They also found that oleic acid only accounted for 74% and stearic acid only 49% of lymph lipid fatty acids in rats intraduodenally infused with triolein or tristearin respectively. This clearly points to a significant (25.50%) contribution of endogenous lipid to lymph lipid. Tso et al. [21] have previously shown that the fatty acid composition of lymph in the absence of triacylglycerol absorption reflects the composition of bile phospholipid. We believe that the differences we see in the pre-infusion lymph composition of 18:2, 20: 1, 20 : 5 and 22 : 6 reflect differences in bile phospholipid fatty acid composition. We have previously found that the long chain n - 3 fatty acids 20: 5 and 22: 6 are enriched in hepatic triacylglycerols of fish oil fed rats, suggesting an increased availability of these fatty acids in the liver (Sheppard and Herzberg, unpublished data). Such an increased supply could also be available for bile phospholipid synthesis. We could not find any data on the bile phospholipid composition of fish oil fed rats and it is presently under investigation in our laboratory. Our finding of an influence of prior diet on the fatty acid composition of lymph Iipids suggests that this may be useful for obtaining further insight into the quantitative contribution of endogenous lipids to the lipids of lymph. There is essentially no 20: 5 in the fasting lymph of rats previously fed a corn oil diet but this fatty acid makes up about 8% in fish oil fed rats. Corn oil has no 20: 5. If we prefed rats fish oil and then measured lymph lipid composition and quantified the lipids after a dose of corn oil all the 20: 5 must have come from endogenous sources. By quantitating bile iipid and determining its fatty acid composition we
194
could determine what fraction of 20 : 5 and, by extension to the other fatty acids in bile, what fraction of the lymph lipids came from bile. If this did not account for all the 20:5, then plasma must have provided the rest. By determining plasma fatty acid composition we could then calculate the contribution of plasma lipids to lymph. In this manner the contributions of bile, plasma and dietary lipid to lymph lipid could be estimated without the complications introduced through the use of labelled lipids. In conclusion, we have shown that prefeeding rats fish oil increases the absorption of a test dose of fish oil. This rules out reduced absorption as a contributor to the hypolipidemic effect of dietary fish oils. In addition we have shown that dietary corn and fish oil affect the fatty acid composition of lymph in characteristic ways. This observation has suggested an experimental approach to examine the contributions of endogenous lipids to lymph. Acknowledgements
This work was supported by the Medical Research Council of Canada. We thank R.P. Scherer (Canada) for the gift of MaxEPA oil. References 1 Harris, W.S. (1989) J. Lipid Res. 30, 785807. 2 Wong, S.H., Nestel, P.J., Trimble, R.P., Storer, G.B., Illman, R.J. and Topping, D.L. (1984) Biochim. Biophys. Acta 792, 103-109.
3 Herzberg, G.R. and Rogerson, M. (1988) J. Nun. 118, 1061-1~67. 4 Herzberg, G.R. (1991) Can. 3. Physiol. Pharmacol., in press. 5 Bottino, N.R., Vandenburg, CA. and Reiser, R. (1967) Lipids 2, 489-493. 6 Brockerhofff, H. and Hoyle, R.J. (1963) Arch. Biochem. Biophys. 102, 452-455. 7 Chernenko, G.A., Barrowman, J.A., Kean, K.T., Herzberg, G.R. and Keough, K.M.W. (1989) Biochim. Biophys. Acta 1004,95-102. 8 Harris, W.S., Connor, W.E., Alam, N. and Illingworth, D.R. (1988) J. Lipid Res. 29, 1451-1460. 9 Baxter, J.N. (1966) J. Lipid Res. 7, 158-166. IO Karmen, A., Whyte, M. and Goodman, D.S. (S963) J. Lipid Res. 4, 312-321. 11 Bergstedt, S.E., Hayashi, H., Kritchevsky, D. and Tso, P. (1990) Am. J. Physiol. 259, G386-G393. 12 Fritsche, K.L. and Johnston. P.V. (1988) J. Nun. 118, 425-426. 13 Wahlfeld, A.W. (1974) in Methods of Enzymatic Analysis, 2nd English Edn. (Bergmeyer, H.U., ed.), pp. 1831-1835, Verlag Chemie, Weinheim and Academic Press, New York. 14 Folch, J., Lees. M. and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. 15 Keough, K.M.W. and Kariel, N. (1987) Biochim. Biophys. Acta 902, 11-18. 16 Zar, J.H. (1984) Biostatistical Analysis, 2nd Edn. pp. 239-241, Prentice Hall Canada, Toronto. 17 Hall, J.A., Connor, W.E.. Anderson, G.J. and Newcomb. K.C. (19911 FASEB J. 5, AIROI. 18 Yang, L.-Y.. Kuksis. A. and Myher, J.J. (1990) B&hem. Cell Biol. 68, 480-491. 19 Trotter. P.J. and Starch, J. t19Yl) FASEB J. 5. A1640. 20 Lands. W.E.M., Morris, A. and Libelt, B. (1990) Lipids 9,505-516. 21 Tso, P., Baiint, J.A. and Simmonds, W.J. (1977) Gastroenterology 12, 1362-1367.
