Dietary (n-3) fatty acids alter plasma fatty acids and leukotriene B synthesis by stimulated neutrophils from healthy geriatric Beagles.

Abstract

The study objective was to determine the effect of feeding food enriched in (n-3) fatty acids (FA) on plasma FA profiles and leukotriene B (LTB) synthesis by stimulated peripheral blood neutrophils from dogs. For 36 weeks, two groups of dogs (n = 5) were fed food that contained either a low ratio of (n-6)-(n-3) FA (1.31:1; fish oil-enriched food) or a high ratio of (n - 6)-(n-3) FA (40.6:1; corn oil-enriched food). Consumption of food enriched in fish oil resulted in higher plasma concentrations of eicosapentaenoic acid and docosahexaenoic acid and lower concentrations of arachidonic acid. Neutrophils from dogs fed fish oil-enriched food produced 7.6-fold more LTB(5)(P = 0.002), and the ratio of LTB(5)-LTB(4) concentrations was 8.3-fold higher (P < 0.001) compared with dogs fed corn oil-enriched food. Dietary FA can modulate leukotriene production by neutrophils in dogs, and suggests that foods enriched in (n-3) FA from fish oil may have value in the treatment of canine inflammatory diseases.

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Dietary (n-3) fatty acids alter plasma fatty acids and leukotriene B synthesis by stimulated
neutrophils from healthy geriatric Beagles

By: J.A. Hall*, L.R. Henry, S. Jha†, M.M. Skinner‡, D.E. Jewell§, and R.C. Wander**

Hall, J.A., Henry, L.R., Jha, S., Skinner, M.M., Jewell, D.E., and Wander, R.C. 2005. Dietary
(n-3) fatty acids alter plasma fatty acids and leukotriene B synthesis by stimulated
neutrophils from healthy geriatric Beagles. Prostaglandins, Leukotrienes, Essential Fatty
Acids. Nov 73(5): 335-341.

Made available courtesy of Elsevier:
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Abstract:
The study objective was to determine the effect of feeding food enriched in (n-3) fatty
acids (FA) on plasma FA profiles and leukotriene B (LTB) synthesis by stimulated
peripheral blood neutrophils from dogs. For 36 weeks, two groups of dogs (n = 5) were fed
food that contained either a low ratio of (n-6)–(n-3) FA (1.31:1; fish oil-enriched food) or a
high ratio of (n-6)–(n-3) FA (40.6:1; corn oil-enriched food). Consumption of food
enriched in fish oil resulted in higher plasma concentrations of eicosapentaenoic acid and
docosahexaenoic acid and lower concentrations of arachidonic acid. Neutrophils from
dogs fed fish oil-enriched food produced 7.6-fold more LTB5 (P = 0.002), and the ratio of
LTB5–LTB4 concentrations was 8.3-fold higher (P<0.001) compared with dogs fed corn oil-
enriched food. Dietary FA can modulate leukotriene production by neutrophils in dogs,
and suggests that foods enriched in (n-3) FA from fish oil may have value in the
treatment of canine inflammatory diseases.

Article:
1. Introduction
A variety of diets and fatty acid (FA) supplements rich in (n-3) fatty acids (FAs) from fish
oil are currently marketed for use in dogs. We have previously shown that healthy, geriatric
Beagles fed foods enriched in (n-3) FAs from fish oil for 12 weeks have altered plasma FA
profiles [1], similar to what has been reported in humans, horses, and laboratory animals [2–5].
In addition, a ratio of dietary (n-6)–(n-3) FAs of 1.4:1 depressed cell- mediated immunity,

* Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Dryden Hall 206,
Corvallis, OR 97331, USA
† Department of Animal Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR 97331,
USA
‡ Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Dryden Hall 206,
Corvallis, OR 97331, USA
§ Science and Technology Center, Hill’s Pet Nutrition Inc, 1035 NE 43rd Street, Topeka, KS 66617-1587, USA
** Department of Nutrition, Human Nutrition Research Laboratory, School of Human Environmental Sciences, The
University of North Carolina at Greensboro, Greensboro, NC 27402-6170, USA
Supported in part by a grant from Hill’s Pet Nutrition, Inc., P.O. Box 1658, Topeka, KS 66601-1658, USA.

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based on results of a delayed-type hypersensitivity (DTH) skin test, and decreased pros-
taglandin E2 (PGE2) production by stimulated mononuclear cells [1]. Altering the ratio
of (n-6)–(n-3) FAs also affected T-cell subpopulations in aged dogs, in that after
immunization with a novel protein these dogs had decreased numbers of CD4+
lymphocytes and decreased CD4+-to-CD8+ ratios [6]. Furthermore, we have shown
that there is an interaction between dietary vitamin E and (n-3) FAs such that the effects
of an optimum amount of dietary vitamin E concentration on the DTH response are
blunted by dietary (n-3) FAs [7]. This study is a continuation of these investigations in
aged Beagles, whereby we report the effect of consumption of a diet enriched in (n-3) FAs
for 36 weeks on plasma FA profiles and on leukotriene (LT) B4 and LTB5 production
by peripheral blood neutrophils.

The type of LT that cells produce, and consequently, communication between cells of the
immune system, can potentially be modulated through dietary supplementation of (n-3)
FAs [2,8–10]. When immune cells are activated by a chemical or physical insult, FAs
are mobilized from cell membrane phospholipids and metabolized into eicosanoids
[11]. Leukotrienes are eicosanoids with chemotactic properties; they are also involved
in regulation of inflammatory and immune processes. Arachidonic acid [AA; 20:4(n-6)]
is a 20 carbon (n-6) polyunsaturated FA that is incorporated into cell membrane
phospholipids. In neutrophil cell membranes, AA is metabolized by 5-lipoxygenase to
yield LTA4, which is converted to LTB4 by LTA4-hydrolase. Production of LTB4
by stimulated peripheral blood neutrophils reflects the plasma concentration of AA, the
FA from which LTB4 is derived [3]. Leukotrienes of the 4-series regulate inflammatory
cytokine production [8]. The types of leukotrienes produced from eicosapentaenoic
acid [EPA; 20:5 (n-3)], a 20 carbon (n-3) polyunsaturated FA, are less biologically
potent than analogues synthesized from AA [8]. For example, LTB5 which is
produced by metabolism of EPA, is 10-fold less potent as a neutrophil
chemoattractant than LTB4 [12,13].

The goal of this investigation was to determine the effect of feeding different amounts of (n-3)
FAs on plasma FA profiles and leukotriene production by peripheral blood neutrophils from
dogs. To achieve this goal, we measured LTB4 and LTB5 production by calcium-ionophore
stimulated peripheral blood neutrophils of dogs fed either corn oil- or fish oil- enriched foods.

2. Materials and methods

2.1. Animals
Ten healthy, female, geriatric (7- to 10-year-old) Beagles that weighed between 7.6 and 13.1 kg
were selected for this study (Hill’s Pet Nutrition Inc., Topeka, KS, USA).

All dogs had been previously vaccinated against canine distemper, parvovirus, and
rabies according to standard protocols, and none had chronic systemic disease, as
determined on the basis of results of physical examination, complete blood count (CBC)
determination, serum biochemical analyses, urinalysis, and fecal examination for parasites.
Dogs were housed in pairs of two in indoor runs and fed once daily in the morning. Dogs
experienced enrichment through interactions with each other and with the caretakers. Four

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additional healthy Beagles used in the teaching program at the College of Veterinary
Medicine, Oregon State University, Corvallis, OR, USA were also included in the study.
The experimental protocol was reviewed and approved by the Oregon State University
Animal Care and Use Committee in accordance with principles outlined by the
National Institutes of Health [14].

2.2. Foods
For 90 days before the study, the experimental dogs consumed a commercial food (Science
Diet Canine Maintenance, Hill’s Pet Nutrition Inc.) without enhancement of (n-6) and
(n-3) FAs. The ratio of (n-6)– (n-3) FAs was 18:1 (Table 1). The source of (n-3) FAs in this
food was 90% plant-derived from a-linolenic acid (soybean oil). The healthy Beagles used in
the teaching program also consumed the same baseline food (Science Diet Canine
Maintenance, Hill’s Pet Nutrition Inc.) as the experimental dogs, before the latter were
switched to their respective experimental foods.

The experimental foods were prepared by a commercial company (Hill’s Pet Nutrition Inc.).
Experimental foods varied in the amount of (n-3) FAs they contained. The low (n-3) FA
food contained a minimal amount of (n-3) FAs (0.5g [n-3] FAs/kg of food, wet-weight
basis) and, thus, had a high ratio of (n-6)–(n-3) FAs (40.6:1). The high (n-3) FA food
contained a larger amount of (n3) FAs from fish oil (5.9 g [n-3] FAs/kg of food, wet- weight
basis) and had a low ratio of (n-6)–(n-3) FAs (1.31:1). The amount of 18:3(n-3) FA (a-
linolenic acid) was low in both foods and roughly equivalent. Both foods contained 101
mg/kg of all-rac-a-tocopherol acetate. The basal food ingredients (by weight) included
54.8% water, 20.3% turkey, 15.0% corn, 4.5% pork liver, 2.0% soy meal, 1.0% beet pulp
and 0.4% vitamin and mineral premixes. Rice hulls were used as the carrier for the vitamin
premix, which contained 25 mg/kg cholecalciferol, 7500 mg/kg nictotinic acid, 5000 mg/kg
calcium D-pantothenate, 21,800mg/kg thiamine mono- nitrate, 1250 mg/kg riboflavin, 2430
mg/kg pyridoxine hydrochloride, 250 mg/kg folic acid, 50 mg/kg biotin and 50mg/kg vitamin
B-12. Calcium carbonate was used as the carrier for the mineral mix, which contained 80
g/kg zinc as zinc oxide, 6.0 g/kg manganese as manganese oxide, 280 g/kg iodine as
calcium iodate, 1.0 g/kg cobalt as cobalt carbonate, 180 mg/kg selenium as selenium
selenite, and 2.5 g/kg copper as copper chloride. The remaining 2% of the food was provided
as added oil. The source of oil for the (n-3) enriched food was Menhaden fish oil (Menhaden
fish oil, Zapata Protein, Reedville, VA, USA), whereas the source for the (n-6) enriched food
was corn oil (Mazola corn oil, Mazola, Englewood Cliffs, NJ, USA). Foods were analyzed
at a commercial laboratory (Woodson-Tenent Laboratories, Des Moines, IA, USA).
Nutrient composition (by weight) was 77.4% moisture, 5.8% protein, 4.5% fat, 1.3% ash and
0.7% crude fiber and 10.3% carbohydrate. FA composition of the two experimental foods
was determined (Table 1).

2.3. Study design
Dogs were ranked on the basis of body weight and assigned to two groups of five dogs
each, such that body

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Table 1
Composition of selected fatty acids in foods fed to geriatric Beagles

Fatty acid Baseline food without
enhancement of (n-6) and (n-3)
fatty acid content (g/kg of food)
Corn oil-enriched food with
low (n-3) fatty acid content
(g/kg of food)
0.2

7.5

2.5

10.2

1.1

14.5

15.8

19.9

0.5

<0.1

0.4

<0.1

<0.1

<0.1

<0.1

20.8

20.3

0.5

40.6:1

Fish oil-enriched food with
high (n-3) fatty acid content
(g/kg of food)



10.9

2.5

9.0

11.8

7.2

0.5

0.6

0.5

0.3

1.9

0.4

2.2

13.3

7.7

5.9

1.31:1

14:0 0.2






































1.4
7.1
2.3






































16:0
18:0
Sum of SFAt
16:1(n-7)
18:1(n-9)c
Sum of MUFA$
18:2(n-6)
18:3(n-3)
18:4(n-3)
20:4(n-6)
20:4(n-3)
20:5(n-3)
22:5(n-3)
22:6(n-3)
Sum of PUFA§
Sum of (n-6) fatty acids
Sum of (n-3) fatty acids
Ratio (n-6)–(n-3)
tSum of the saturated fatty acids (SFA) was determined as follows: 8:0+ 10:0+ 11:0+ 12:0+ 14:0+ 15:0+ 16:0+ 17:0+ 18:0 +20:0 +22:0 +24:0.
6.9
3.3
10.4
0.9
12.4
13.6
7.2
0.3
<0.1
0.1
<0.1
<0.1
<0.1
<0.1
8.0
7.5
0.4
18.0:1
$Sum of the monounsaturated fatty acids (MUFA) was determined as follows: 14:1 + 15:1 + 16:1(n-7) + 17:1 + 18:1(n-9)c+
18:1(n-7)+ 18:1 (n-9)t+ 20:1(n-9) + 22:1(n-9) + 24:1.
§Sum of the polyunsaturated fatty acids (PUFA) was determined as follows: 18:2(n-6)+18:3(n-6)+18:3(n-3)+18:4(n-
3)+20:2(n-6)+20:3 (n-6) + 20:3(n-3) + 20:4(n-6) + 20:4(n-3) + 20:5(n-3) + 21:5(n-3) + 22:2(n-6) + 22:4(n-6) + 22:5(n-6) + 22:5(n-3) +
22:6(n-3).
weights were evenly distributed between the groups. Body weights were measured before
the study began and weekly during the study. Food intake was quantitated and adjusted
such that dogs did not gain or lose weight. Blood samples were collected after the
experimental dogs had consumed their respective foods for 36 weeks. Blood samples were
collected from the healthy Beagles used in the teaching program after they had consumed
the baseline food for 4 weeks. Blood samples were collected before the daily meal
offering into evacuated tubes containing EDTA (final concentration, 1.5 g/L), and plasma
was harvested.

2.4. Plasma FAs
FA content of plasma samples was determined by use of gas chromatography, as previously
described [15], using heptadecanoic acid as the internal standard. FA concentration was
expressed as g/100g of FAs.

2.5. Leukotriene B4 and B5 quantification
Neutrophils were isolated and purified as previously described [16] from blood collected at
36 weeks for the experimental dogs and at 4 weeks for the healthy Beagles used in the
teaching program. Aliquots of 1 x 106 neutrophils were transferred to test tubes and the
volume adjusted to 495 µL with Hanks balanced salt solution containing 0.8 mmol/L

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CaCl2. Neutrophils were then stimulated with 5µL of calcium ionophore A23187 (Sigma
Chemical Co., St. Louis, MO, USA) in 0.2% dimethyl sulfoxide (Syntex, West Des Moines,
IA, USA) such that the final concentration of A23187 was 10 mmol/L, while
unstimulated neutrophils received 5 µL of 0.2% dimethyl sulfoxide without calcium
ionophore. All tubes were incubated for 5 min at 37'C as previously described [16].
Supernatants were stored at -70'C until subsequent LTB4 and LTB5 measurements were
made. LTB4 and LTB5 were extracted, separated, and quantified as previously described
[16]. Standard calibration curves for LTB4 and LTB5 were made by adding 100 ng of
PGB3 as an internal standard to samples containing 0.47–40 ng of LTB4 and LTB5
(Sigma). PGB3 was chosen as the internal standard because it was widely separated from
LTB4 present in actual samples during HPLC separation, whereas PGB2 coeluted with LTB5
and PGB1 coeluted with LTB4 [17]. Concentrations of leukotrienes in test samples were
calculated with reference to the standard curves. Final LTB4 and LTB5 concentrations
were reported as nanograms of LTB4 and LTB5 per 1 x 106 cells.

2.6. Statistical analysis
Data are reported as means ±SEM. Using the Kolmogorov–Smirnov test, data that
were found to be normally distributed were analyzed using analysis of variance. On the
basis of the Homogeneity of Variance test, data that were homoscedastic were analyzed
using analysis of variance followed by post hoc separation of the means using the Tukey–
Kramer Multiple-Comparison test. When the assumptions of normality and equal variance
were suspect, data were analyzed by the nonparametric Kruskal–Wallis Test, followed
by post hoc separation of the means using the Kruskal–Wallis Z-Test. Correlations were
evaluated with the Pearson correlation test. Overall significance was set at P<0.05.
Statistical analyses were performed using the Number Cruncher Statistical System (NCSS)
(Kaysville, UT, USA), version 2004 (www.ncss.com).

3. Results

3.1. Animals and foods
All dogs readily consumed their respective FA- enriched foods. At the beginning of
the study dogs consuming the high (n-3) FA food weighed 10.0±0.7 kg (mean±SEM). At
the end of the study these dogs weighed 10.8±0.7kg. At the beginning of the study the
dogs consuming the low (n-3) FA food weighed 10.5 ± 0.8 kg (mean ± SEM). At the
end of the study these dogs weighed 12.5±0.9 kg. Body weights were not significantly
different between groups, although dogs consuming the low (n-3) FA food weighed
significantly more after 36 weeks compared to baseline (P = 0.02).

3.2. Plasma FA profiles
The FA composition of plasma at the beginning of the study was similar for experimental dogs
regardless of the projected dietary intervention (Table 2). The healthy Beagles used in the
teaching program differed from the experimental dogs in total saturated fatty acids
(SFA)(P<0.001), arachidonic acid (P<0.001), total polyunsaturated fatty acids (PUFAs) (P =
0.01), and total (n-6) FAs (P = 0.01). Plasma from these dogs had approximately 18% more
SFA (primarily palmitic acid and to a lesser extent myristic acid) than experimental dogs,