The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2641S-2644S

Dietary Flaxseed in Dogs Results in Differential Transport and Metabolism of (n-3) Polyunsaturated Fatty Acids^1,2

John E. Bauer³, Brent L. Dunbar, and Karen E. Bigley

Comparative Nutrition Research Laboratory, Texas A&M University, College Station, TX 77843-4474 USA

KEY WORDS: dogs · phospholipids · cholesteryl esters · (n-3) fatty acids · flaxseed

	INTRODUCTION

Introduction References

The essential nature of dietary fatty acids has been recognized since the 1920s. Nonetheless, a complete description of the essential fatty acid requirements of various animal species, even qualitatively, remains to be established. In addition to linoleic acid [LA,⁴ 18:2(n-6)], evidence that (n-3) fatty acids are also essential has been recognized recently in such species as rat, some nonhuman primates, and in humans, especially with respect to nervous system development. The American Institute of Nutrition has recently modified its recommendations for normal laboratory rat diets to contain a fat source such as soybean oil because it contains both 18:2(n-6) and 18:3(n-3) (-linolenic acid, ALA) fatty acids (Reeves et al. 1993). Furthermore, results of animal studies espouse the anti-inflammatory nature of the (n-3) fatty acids and their potential use in the prevention and therapy of chronic diseases (Bauer 1994, Okayuma and Sakai 1991). Most of these studies have been conducted in mice and rats although some reports in horses and dogs have appeared (Bauer 1994, Henry et al. 1990). In some studies, total dietary fatty acid intakes were not completely defined. Thus, specific relationships between dietary fatty acid intake and physiologic responses have not always been clearly established.

The carbon chain length of the (n-3) fatty acids fed may also be important, and evidence suggesting their differential metabolism exists in dogs and rodents; other species differences are likely (Bauer et al. 1997, Okayuma and Sakai 1991). Thus, the extent of such differences in fatty acid desaturation and chain elongation, including newly recognized peroxisomal pathways (Voss et al. 1991), may have specific metabolic consequences. Previous studies indicate that several agents affecting peroxisomal proliferation in mice and rats fail to elicit a similar response in dogs, rhesus monkeys or guinea pigs (Lock et al. 1989); humans also appear to be nonresponsive. In view of these observations, the dog was chosen as an experimental model. This species is not only an important companion animal but canine peroxisomal fatty acid metabolism may also be more similar to that of humans and therefore more representative than rodents in this regard. In this study, the time course of enrichment of the plasma lipid subfraction fatty acids in dogs fed a diet specifically enriched in only 18-carbon (n-6) or (n-3) fatty acids (LA and ALA, respectively) was investigated. Chain elongation and desaturation of the 18-carbon precursors of long-chain polyunsaturated fatty acids were evaluated. The data obtained also confirm and extend our earlier observations regarding aspects of differential metabolism of polyunsaturated fatty acids in the canine species (Bauer et al, 1997).

Materials and methods. 

Flaxseed and sunflower seed.  Whole flax and sunflower seeds were ground and screened at 200 mesh. They were provided by Essential Nutrient Research (ENRECO, Manitowoc, WI). The seeds were ground to control for digestibility differences that might exist due to seed coats. The whole flaxseed contained 52.7% ALA and the sunflower seed contained 68.2% LA. The other major fatty acids [i.e., 16:0, 18:0 and 18:1(n-9)] were distributed nearly equivalently between these two oilseeds. The use of sunflower seed as a control group allowed treatment comparisons such that dietary fatty acids varied predominately between 18-carbon (n-6) and (n-3) fatty acid series.

Animals and diets.  A single lot number of a commercially available canine dry, kibbled diet (Hill's®, Canine Senior) was lightly sprayed with no more than 1% (wt/wt) distilled water in a bakery mixer (Model L800, Hobart Industries, Troy, OH), and the ground seeds were coated on the surface of the kibbled product. The diets were allowed to dry completely and stored indoors in their original bags before being placed in plastic drums fitted with lids until the time of feeding. Portions of these diets were subjected, in triplicate, to total fatty acid extraction, and fatty acid profiles were determined (Table 1).

For the study, 18 adult random gender mixed breed dogs, 10-20 kg in body weight, were divided into two groups of 9 each. Three feeding periods were used so that three dogs from each group (6 dogs per day) were fed the supplemented diets, thus ensuring feeding and sample collections in a timely manner. The dogs are property of Texas A&M University, Veterinary Teaching Hospital and were housed in the Small Animal Clinic. Health examinations are routinely performed on this colony throughout the year. Dogs are individually maintained in kennels according to the American Physiological Society Guidelines for Animal Research and according to guidelines set forth by Texas A&M University Care and Use Committee.

The basal diet contained ~23.8% energy from fat (about 9.7% fat wt/wt as is), and moderate amounts of protein and fiber. This diet contained 32.5% of total fatty acids (wt/wt) and 7.8% energy as LA. By comparison, the supplemented diets contained ~25% energy as fat. The sunflower seed supplemented diet (SUN) contained 37.5% of the total fatty acids as LA and 1.7% as ALA (Table 1). These values corresponded to 9.3% energy as LA and 0.43% energy as ALA. In contrast, the flaxseed supplemented diet (FLX) contained 27.9% of the total fatty acids as LA and 10.1% ALA. In this case, the FLX diet was determined to have 7.3% energy as LA and 2.5% energy as ALA. At 7-9% energy as LA in both diets, it would be expected that tissue levels would be saturated with LA. Thus the proportion of energy from LA was high and nearly constant (1.3-fold difference), whereas the proportion of energy from ALA was about sixfold higher in the FLX vs. the SUN diet. In this way, a comparison between a low (0.43% energy) ALA diet (SUN) and an enriched (2.5% energy) ALA diet (FLX) could be made. It should be noted that both diets contained about three to four times more LA than the typically accepted minimum of ~2% of energy. A minimum amount of dietary ALA has not yet been established for dogs.

All dogs were weighed weekly during the 84-d feeding period. A 2-wk diet acclimation period to the unsupplemented basal diet was used. The dogs were then randomly assigned to either the sunflower or flax group; blood samples were obtained after food was withheld overnight (d 0). Thereafter, the animals were fed their assigned diet and blood samples collected at 4, 7, 14, 28, 56 and 84 d.

Sample analyses.  Serum was freshly harvested from the blood samples on the day of collection. Total lipids were extracted and subfractionated via TLC and analyzed using gas chromatography. The column oven was temperature programmed starting at 180°C, held for 15.5 min, then heated to 210° at 12°C/min and held for 19 min. Other conditions were as reported previously (Bauer et al. 1997).

Statistics.  The data were analyzed with two-factor, repeated measures ANOVA followed by Newman-Keuls multiple comparisons to identify treatment differences. Windows version of SAS/STAT Version 6 was used for the analysis (SAS Institute, Cary, NC) and the levels of significance are as indicated. Fatty acid profiles of serum lipid subfractions taken from dogs on d 0, 4 and 84 are presented.

Results. 

Serum lipids.  Both total (n-6) and (n-3) fatty acids increased over time in all lipid fractions in the SUN and FLX groups. Further inspection of the data revealed that increases in most individual (n-3) fatty acids occurred over time in the FLX but not the SUN group and diet × time interactions were seen. By contrast, increases in the (n-6) fatty acids were observed over time independently of supplement type and without significant diet × time interactions. It is not surprising that an increase of (n-3) fatty acids occurred in the FLX group because the net fatty acid composition of sunflower plus the basal diet is similar to that of the basal diet alone, whereas the flaxseed plus basal composition differs by virtue of its (n-3) fatty acid content (Table 1).

A number of additional differences were observed when the SUN and FLX groups were compared. First, (n-3) fatty acid enrichment of FLX group lipid subfractions occurred early on and was distinctive, depending on the lipid subclass examined. More specifically, in the FLX group, phospholipid (PL) fractions were significantly enriched in 18:3(n-3) and 20:5(n-3) at d 4 and thereafter. At the end of the feeding period, significantly higher PL 18:3(n-3) (fourfold), 20:3(n-3) (fivefold), 20:5(n-3) (threefold), and 22:5(n-3) (twofold) were found along with significantly lower 20:4(n-6), 22:4(n-6) and 22:5(n-6) (Table 2). In the FLX group, it was especially noteworthy that PL-docosahexaenoic acid [DHA;22:6(n-3)] was unchanged during the entire period, indicating some resistance for plasma 22:6(n-3) levels to accumulate in dogs fed 18:3(n-3).

Plasma triacylglycerol (TG) fractions of FLX-fed dogs were also enriched in 18:3(n-3) (threefold), 20:5(n-3) (threefold) and 22:5(n-3) (twofold), but again no change in 22:6(n-3) was found (Table 2). Significant increases over time were seen in 18:2(n-6) and 20:4(n-6). In the SUN group, elevations of 22:4(n-6) [the (n-6) analog of 22:5(n-3)] were seen compared with FLX.

In the cholesteryl ester (CE) fractions, certain (n-3) fatty acids increased early on in the FLX group, whereas others did not (Table 3). Significant time and time × diet effects were seen for 18:3(n-3) and 20:5(n-3) with diet effects of 18:3(n-3) becoming apparent on d 4 (fivefold difference vs. SUN) and fourfold higher 20:5(n-3) on d 84 vs. the SUN group. By contrast, little 22:5(n-3) or 22:6(n-3) was detected over time in CE, with values never exceeding 0.03 weight %.

Discussion.  The results clearly show rapid accumulation of eicosapentaenoic acid (EPA) and certain other (n-3) fatty acids in plasma lipids in the canine model when a diet containing a modest increase of ALA is fed. Similar results have been observed in human studies with a 4-wk feeding period (Mantzioris et al. 1994). The observed changes appeared to achieve a steady state after ~28 d, although EPA continued to increase modestly throughout the 84-d feeding period.

It is especially noteworthy in the FLX group that PL-DHA [22:6(n-3)] was unchanged during the entire period. This finding indicates some resistance for plasma DHA levels to accumulate in dogs fed ALA at the diet level employed in this study. It is known that conversion of 22-carbon precursors to DHA occurs in canine neurologic tissues and is believed to occur in peroxisomes (Alvarez et al. 1994). However, canine hepatic peroxisomes, unlike those of rodents, may not readily proliferate under various conditions, and conversion in canine liver may be subsequently slowed (Lock et al. 1989). A similar finding has recently been reported in cats (Pawlosky et al. 1994). Taken together, these data raise the intriguing possibility that there is limited conversion of ALA to DHA in liver of certain species even when docosapentaenoic acid [DPA; 22:5(n-3)] is produced. This latter fatty acid might then be transported via plasma lipids to important neurologic tissues for subsequent DHA synthesis (Pawlosky et al. 1994).

In an earlier study with dogs, we observed that when fish oils are fed, 22:5(n-3) does not appear in the CE fraction, but that 22:6(n-3) does (Bauer et al. 1997). In this study, using dietary flaxseed, 22:5(n-3) was seen both in plasma PL and TG, but little 22:6(n-3) was found. Again, in spite of this enrichment in the PL fraction, no plasma CE 22:5(n-3) was detected. Taken together, these data suggest the possibility that forward transport of hepatic 22:5(n-3) via plasma PL and TG to tissues is likely, but reverse cholesterol transport of this fatty acid to the liver as CE mediated by the lecithin cholesterol acyltransferase reaction does not occur. The significance of these observations and their similarity to humans remain to be determined. However, we hypothesize that, in dogs, peroxisomal conversion of 22:5(n-3) to 22:6(n-3) is slow and that after hepatic synthesis of 22:5(n-3) from 18:3(n-3), this fatty acid is subsequently transported to important neurologic tissues via plasma PL and TG, especially when dietary sources of 22:6(n-3) are scarce. Because 22:5(n-3) is absent in CE, such transport may be unidirectional. In this way, substrate for DHA synthesis in neurologic tissues may be conserved. Future studies are in progress to investigate these possibilities.

	FOOTNOTES

	LITERATURE CITED

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