![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
-Linolenic Acid Canola Oil in Lymph Fistula Sprague-Dawley Rats1 ,2
Department of Pathology, University of Cincinnati Medical Center, Cincinnati, OH 45267 and * Strategic-Discovery R&D, Ross Products Division, Abbott Laboratories, Columbus, OH 43219
3To whom correspondence should be addressed. E-mail: tsopp{at}email.uc.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
-linolenic acid [GLA, 18:3(n-6)] in the seed oil has been developed in our laboratories. This study compares the intestinal absorption and lymphatic transport of this newly developed high GLA content canola oil (HGCO) with traditional GLA-rich borage oil (BO) using a lymph fistula rat model. To assess the extent that 1 mL of GLA in the supplemented oil was absorbed and transported, the fatty acid compositions of triglycerides in mesenteric lymph were compared over a 24-h collection period. The digestion, uptake and lymphatic transport of HGCO and the normal physiologic changes associated with fat absorption (e.g., lymph flow and an increase in lymphatic endogenous lipids outputs, triglycerides, cholesterol and phospholipids) were similar in the HGCO-and the BO-fed rats. The original differences in -linolenic acid content in HGCO and BO were preserved in the fatty acid composition of the rats’ lymph lipid. We conclude that the HGCO derived from the genetically modified canola plant is absorbed and transported into lymph similarly to BO.
KEY WORDS: • -linolenic acid • intestinal lipid absorption • genetically modified canola oil • lymphatic lipids • rats
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
-linolenic acid [GLA,4
18:3(n-6)] has been shown to significantly reduce the formation of atherosclerotic lesions in apolipoprotein E knockout mice (1
) and attenuate the clinical symptoms associated with chronic inflammatory disorders such as rheumatoid arthritis (2
). Severe ventricular fibrillation induced in rats by ligation of coronary arteries was significantly reduced by supplementation of GLA in the diet (3
). These and other beneficial effects of GLA have prompted the demand for GLA-rich oils. At present, commercially available GLA-rich oils, such as borage, evening primrose and black currant seed, are generally expensive; thus, their widespread use in disease management has been limited. Efforts to find efficient and economical sources of GLA have increased. Utilizing transgenic technology and the recombinant expression of 
12- or 6-desaturases in the canola plant, our laboratories have developed a new canola strain capable of producing GLA at a level >30% of the fatty acids in the oil of the seed (4
,5
). In vitro hydrolysis of GLA by pancreatic lipase differed among different GLA-rich oils (6
). In this study, we compared the intestinal absorption and lymphatic transport of this newly developed high GLA canola oil (HGCO) with a traditional GLA-rich borage oil (BO) in lymph fistula rats. Comparing the fatty acid composition of dietary oil and chylomicron triglyceride in intestinal lymph after absorption indicates to what extent a specific fatty acid is absorbed by the intestine (7
). We therefore compared the fatty acid compositions of triglycerides in intestinal lymph of rats fed HGCO and BO. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The animal procedures used in this study were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati in accordance with guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Conscious, male adult Sprague-Dawley (Harlan, Indianapolis, IN) lymph fistula rats, weighing 300–350 g, were used to study the intestinal absorption and lymphatic transport of lipids. The rats were fed Purina Rat Chow (Harlan Teklad LM485, Madison, WI) and housed in a room with constant temperature (30°C) and humidity (50%) and a 12-h light:dark cycle before experimentation. After overnight food deprivation, the rats were anesthetized with halothane and laparotomy was performed. The intestinal lymph duct was cannulated with a clear vinyl tubing (o.d., 0.8 mm) according to the method of Bollman et al. (8
). A second soft silicone tubing (o.d., 1.6 mm) was installed in the stomach through the fundus and secured with a purse-string suture. The rats were infused via the gastric tube with a 50 g/L saline/glucose solution containing 145 mmol/L NaCl, 4 mmol/L KCl and 0.28 mol/L glucose at a rate of 3 mL/h immediately after surgery and throughout the experimental period. The rats recovered overnight in restraining cages before lipid infusion.
Experimental plan.
After overnight recovery, the rats (n = 9/group) were randomly assigned to receive either 1 mL of HGCO or 1 mL of BO (Bioriginal Food and Science, Saskatoon, Canada). The fatty acid compositions of the two oils analyzed by gas chromatography are shown in Table 1
. Generally, HGCO contains more GLA and oleic acid [18:1(n-9)], but less linoleic acid [18:2(n-6)] than BO.
|
Lymph was collected into precooled conical graduated centrifuge tubes for 2 h before oil infusion to provide reference for the fasting lymphatic lipid output. After oil infusion, lymph samples were collected at different intervals: 0–2 h, 2–4 h and hourly between 4 and 8 h. Lymph collected between 8 and 24 h after lipid infusion was also pooled. After the volume of the collected lymph was recorded, the samples were centrifuged for 15 min at 700 x g at room temperature to remove blood cells. Lymph lipids were extracted using the method of Blankenhorn and Ahrens (9
), and aliquots of the extracts were taken for determination of triglycerides (10
), phospholipids (11
,12
) and cholesterol (13
). To examine the fatty acid composition of the lymph triglycerides, equal aliquots of the lipid extracts collected during the 0- to 2-h and 2- to 4-h periods were pooled and labeled as the 0- to 4-h lymph lipid extract. Similarly, the extracts from the 4- to 5-h, 5- to 6-h, 6- to 7-h, and 7- to 8-h periods were pooled and labeled as the 4- to 8-h lymph lipid extract. The lymph lipid extracts (fasting, 0–4 h, 4–8 h and 8–24 h) were then fractionated into different lipid fractions by TLC (14
).
Analysis of fatty acid composition.
Fatty acid methyl esters (FAME) of the purified lymph triglyceride fraction were prepared using 1 mol/L sodium methoxide in methanol/benzene (60:10, v/v) (14
,15
). The FAME were then analyzed using a 5890A Hewlett-Packard gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a 1.83 m column of 10% SP-2300 on 80/100 Supelcoport (Supelco, Bellefonte, PA).
Statistical methods.
All values are expressed as means ± SEM A one-way, repeated-measures ANOVA was used to determine whether differences existed among groups for each interval of lipid infusion for each dependent variable. If a main effect of group or time was significant, Tukey’s Honestly Significant Difference (HSD) test was carried out to determine where the difference occurred. When a significant interaction was present, a one-way, repeated-measures ANOVA was conducted for each group, and a one-way ANOVA was conducted at each time. Means were then subjected to Tukey’s HSD test to determine where the differences occurred. Differences with P < 5% were considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
As shown in Figure 1A
, the rate of the fasting lymph flow ranged between 2.2 and 2.3 mL/h in both groups of rats. The flow rate increased markedly from lipid feeding and reached the maximum level (between 2.8 and 3.3 mL/h) during the 2- to 4-h collection period. The lymph flow then fell to the fasting level during the 8- to 24-h collection period. There were no significant differences in flow rates between the HGCO and BO groups at any time point of lymph collection.
|
B. In both groups, the mean triglyceride output in the fasting lymph was 
7 µmol/h. After oil feeding, the triglyceride output increased rapidly and reached the maximum level (ranging from 30 to 40 µmol/h) during the 4- to 8-h collection period. Thereafter, the level of lymph triglyceride output decreased. The lymph triglyceride output was 20 µmol/h during the 8- to 24-h collection period after feeding. There were no significant differences in lymphatic triglyceride output between the two groups at any time point of measurement.
The fasting lymphatic cholesterol output was 1.2 µmol/h in the BO rats and 1.5 µmol/h in the HGCO rats, rates that did not differ (Fig. 1
C). Lymph cholesterol output increased significantly after feeding 1 mL of either HGCO or BO by gavage. It reached the maximum level (ranging between 3 and 4 µmol/h) during the 4- to 8-h collection period and then declined to the fasting level during the 8- to 24-h collection period. There were no significant differences in lymphatic cholesterol outputs between the two groups during any of the collection periods.
Similar to lymphatic triglycerides, the mean outputs of lymphatic phospholipids collected after fasting and during different time intervals after feeding of either HGCO or BO were not different at any time point (data not shown).
Lymphatic triglyceride fatty acid composition.
To compare the bioavailability of GLA in the HGCO- and BO-fed rats, the fatty acid composition of triglycerides in lymph collected during the fasting, 0- to 4-h, 4- to 8-h and 8- to 24-h collection periods was examined. In the fasting lymph samples, the triglycerides contained the following five main fatty acids: 16:0, 18:0, 18:1(n-9), 18:2(n-6) and 20:4(n-6). The proportions of 16:0, 18:0 and 20:4(n-6) in the lymphatic triglycerides decreased after feeding the HGCO or the BO, but the proportions of 18:1(n-9), 18:2(n-6) and 18:3(n-6) increased significantly. Patterns of change were similar for 16:0, 18:0 and 20:4(n-6), i.e., the fatty acid levels decreased rapidly to a minimum during the 4- to 8-h collection period after oil feeding and returned to the fasting level during the 8- to 24-h collection period. The changes were not significantly different between groups. Conversely, oil feeding significantly increased the proportions of 18:1(n-9), 18:2(n-6) and 18:3(n-6) in lymph triglycerides to a different extent in the groups. The levels of 18:1(n-9) (Figure 2
, panel A), 18:2(n-6) (panel B) and 18:3(n-6) (panel C) did not differ between groups during food deprivation and the lymphatic lipid levels of all three fatty acids increased significantly after the gastric feeding of both oils. However, the levels of 18:1(n-9) and 18:3(n-6) were significantly higher and the levels of 18:2(n-6) lower in the HGCO rats than in the BO rats during the 0- to 4-h and 4- to 8-h collection periods. The extent to which these three fatty acids increased was modulated by their amounts in the oils (Table 1)
. Thus, the differences in the lymphatic triglyceride 18:1(n-9), 18:2(n-6) and 18:3(n-6) contents (g/100 g total fatty acids) were due to the different contents of these three fatty acids in the two oils.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
This study compared the intestinal absorption and lymphatic transport of HGCO and BO in lymph fistula rats. The results show similar lymphatic triglyceride, cholesterol and phospholipid outputs, thereby demonstrating that HGCO and BO are metabolized similarly by the small intestine. Shortly after gavage feeding, the lymph lipid outputs increased by more than fourfold. This finding was consistent with the report that lipid absorption enhances the output of lymphatic lipids (16
). An increase in lymphatic cholesterol output after oil feeding was observed. This increase was likely derived endogenously from bile, villous enterocyte shedding and de novo cholesterol synthesis by the small intestine (16
–18
). Lipid absorption also increased the phospholipid output into lymph (data not shown). This was expected because phospholipids are required for the formation and secretion of chylomicrons for lipid transportation in circulation (19
–21
).
Animal and human studies show that in the normal digestion and absorption of dietary lipids, the fatty acid composition of triglycerides in lymph chylomicrons resembles the lipid consumed (15
,22
–24
). Results from this study comparing the fatty acid composition of lymph triglycerides in rats fed either HGCO or BO support these findings. Data presented in Figure 1
clearly suggest that intestinal absorption and lymphatic transport were similar in both groups. Differences in the levels of 18:1(n-9), 18:2(n-6) and 18:3(n-6) were directly related to the amount of these fatty acids in the dietary oil consumed.
This study has provided for the first time the following important observations: 1) the intestinal absorption and lymphatic transport of HGCO are similar to those of BO; 2) the normal physiologic changes associated with fat absorption (e.g., lymph flow and the increase in lymphatic endogenous lipid outputs, cholesterol and phospholipid) appear to be similar between HGCO and BO; 3) the differences between HGCO and BO in oleic acid, linoleic acid and -linolenic acid content are preserved in the fatty acid composition of lymph triglycerides. We therefore conclude that the HGCO derived from the genetically modified canola plant is digested, absorbed and transported into lymph similarly to BO.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
-linolenic acid canola oil in lymph fistula rats. Vol. 11, #5, 511.).
2 Supported in part by Ross Products Division and DK-56910, 54504 and 56863 from the National Institutes of Health.
4 Abbreviations used: BO, traditional GLA-rich borage oil; FAME, fatty acid methyl esters; GLA 18:3(n-6), -linolenic acid; HGCO, high GLA content canola oil; HSD, honestly significant difference.
Manuscript received 3 August 2001. Revision accepted 15 October 2001.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Fan, Y. Y., Ramos, K. S. & Chapkin, R. S. (2000) Modulation of atherogenesis by dietary gamma-linolenic acid. Adv. Exp. Med. Biol. 469:485-491.
2.
Barham, J. B., Edens, M. B., Fonteh, A. N., Johnson, M. M., Easter, L. & Chilton, F. H. (2000) Addition of eicosapentaenoic acid to -linolenic acid–supplemented diets prevents serum arachidonic acid accumulation in humans. J. Nutr. 130:1925-1931.
3. Charnock, J. S. (2000) Gamma-linolenic acid provides additional protection against ventricular fibrillation in aged rats fed linoleic acid rich diets. Prostaglandins Leukot. Essent. Fatty Acids 62:129-134.[Medline]
4.
Huang, Y.-S., Chaudhary, S., Thurmond, J. M., Bobik, G., Jr., Yuan, L., Chan, G. M., Kirchner, S., Mukerji, P. & Knutzon, D. (1999) Cloning of 12- and 6-desaturases from Mortierella alpina and recombinant production of -linolenic acid in Saccromyces cerevisiae. Lipids 34:649-659.[Medline]
5.
Liu, J.-W., Huang, Y.-S., DeMichele, S. J., Bergana, M., Bobik, E., Jr, Hastilow, C., Chuang, L.-T., Mukerji, P. & Knutzon, D. (2001) Characterization of oil exhibiting high level of -linolenic acid from a genetically transformed canola strain. J. Am. Oil Chem. Soc. 70A:489-493.
6.
Huang, Y.-S., Lin, X., Redden, P. R. & Horrobin, D. F. (1995) In vitro hydrolysis of natural and synthetic -linolenic acid-containing triacylglycerols by pancreatic lipase. J. Am. Oil Chem. Soc. 72:625-631.
7. Tso, P. (1985) Gastrointestinal digestion and absorption of lipids. Adv. Lipid Res. 21:143-186.[Medline]
8. Bollman, J. L., Cain, J. C. & Gindlay, J. H. (1949) Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the rat. J. Lab. Clin. Med. 33:1349-1352.
9.
Blackenhorn, D. H. & Ahrens, E. H. (1955) Extraction, isolation, and identification of hydrolytic products of triglyceride digestion in man. J. Biol. Chem. 212:69-81.
10. Biggs, H. G., Erikson, J. M. & Moorehead, W. R. (1975) Manual colorimetric assay of triglycerides in serum. Clin. Chem. 21:437-441.[Medline]
11. Glass, R. L. (1971) Alcoholysis, saponification, and the preparation of fatty acid methyl esters. Lipids 6:919-925.
12. Parker, F. & Peterson, N. F. (1965) Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin-layer chromatograms. J. Lipid Res. 6:455-460.[Abstract]
13. Rudel, L. L. & Morris, M. D. (1973) Determination of cholesterol using O-phthaladehyde. J. Lipid Res. 14:364-366.[Abstract]
14. Tso, P. & Balint, J. A. (1986) Formation and transportation of chylomicrons by enterocytes to the lymphatics. Am. J. Physiol. 250:G715-G726.
15. Blomstrand, R. & Dahlbäck, O. (1960) The fatty acid composition of human thoracic duct lymph lipids. J. Clin. Investig. 39:1185-1191.[Medline]
16. Simmonds, W. J. (1972) Fat absorption and chylomicrons formation. Nelson, G. J. eds. Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism 1972:705-743 Wiley-Interscience New York, NY. .
17. Spady, D. K. & Dietschy, J. M. (1983) Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster, and rat. J. Lipid Res. 24:303-315.[Abstract]
18.
Stange, E. F. & Dietschy, J. M. (1983) Cholesterol synthesis and low density lipoprotein uptake are regulated independently in rat small intestinal epithelium. Proc. Natl. Acad. Sci. U.S.A. 80:5739-5743.
19. Tso, P., Balint, J. A. & Simmonds, W. J. (1977) Role of biliary lecithin in lymphatic transport of fat. Gastroenterology 73:1362-1367.[Medline]
20. Tso, P., Lam, J. & Simmonds, W. J. (1978) The importance of the lysophosphatidylcholine and choline moiety of bile phosphatidylcholine in lymphatic transport of fat. Biochim. Biophys. Acta 30:364-372.
21. Mansbach, C. M., II (1977) The origin of chylomicrons phosphatidylcholine in the rat. J. Clin. Investig. 60:411-420.
22. Kayden, H. J., Karmen, A. & Dumont, A. (1963) Alterations in the fatty acid composition of human lymph and serum lipoproteins by single feeding. J. Clin. Investig. 42:1373-1381.
23. Wood, P., Imaichi, K., Knowles, J., Michaels, G. & Kinsell, L. (1964) The lipid composition of human plasma chylomicrons. J. Lipid Res. 5:225-231.[Abstract]
24. Zilversmit, D. B. (1965) The composition and structure of lymph chylomicrons in dog, rat, and man. J. Clin. Investig. 44:1610-1622.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
