Research in Veterinary Science 1992, 53, 172-178
Effects of oral sunflower oil and olive oil on serum and cutaneous fatty acid concentrations in dogs K. L. CAMPBELL, G. P. DORN, Department of Veterinary Clinical Medicine, College of
Veterinary Medicine, University of Illinois, 1008 W Hazelwood Drive, Urbana, Illinois 61801, usa
The effects of dietary fatty acids on serum and cutaneous fatty acids of healthy dogs were evaluated under controlled conditions. Beagle puppies (n = 12) were fed a standard diet supplemented with sunflower oil (group A), olive oil (group B) or no supplementation (group C) for 12 weeks. There were no significant differences in food intake or growth rates between the three groups. Dogs in group A had significant increases (P < 0"05) in serum 18:2n6 (linoleic acid) and 20:3n6 (dihomogamma-linolenic acid), and cutaneous 18:2n6 with significant decreases in serum 20:4n6 (arachidonic acid) and cutaneous 18:1n9 (oleic acid) and 18:3n3 (alpha-linolenic acid). Dogs in group B had significant increases in serum 18:1n9, 20:3n6 and cutaneous 18:1n9 with decreases in serum 20:4n6, 22:4n6, 22:5n3 and 22:5n6, and cutaneous 18:2n6, 18:3n3 and 20:4n6. There were no significant changes in serum or cutaneous fatty acids for the dogs in group C. This study demonstrates that fatty acid supplements can be used to alter the serum and cutaneous fatty acid compositions of dogs.
THE importance of dietary fats for the health of the skin of dogs was recognised by Hansen and Wiese (1943) over 45 years ago. Functions of fatty acids include the maintenance of cell membrane integrity and fluidity, maintenance of cell membrane permeability and serving as substrates for the production of eicosanoids (Prottey 1976, 1977). Eicosanoids, which include prostaglandins, thromboxanes, hydroxyeicosatetraenoic acids and leukotrienes, have numerous functions including regulation of D N A synthesis in keratinocytes and serving as mediators of inflammation (Goetzl 1981, Samuelsson 1981). Puppies fed diets deficient in essential fatty acids (I~FAs) develop a seborrhoea characterised by
scaliness and greasiness of the skin and haircoat (Hansen and Wiese 1943). EVA-deficient puppies have lower serum iodine numbers (implying fewer unsaturated double bonds in serum fatty acids) than puppies fed control diets (Hansen and Wiese 1943). This suggests a relationship can exist between dietary fat, serum fatty acid composition, and the development of seborrhoea in dogs. Alterations in fatty acid concentration profiles are seen in a variety of diseases (see discussion) in association with clinical signs of skin disease. The effectiveness of dietary fatty acid supplementation in correcting disease-associated alterations in fatty acid concentration profiles is the subject of current investigations. In the past, veterinary dermatologists advocated the addition of equal parts of vegetable and animal fats to the diet of dogs with seborrhoea (Kunkel et al 1980, Muller et al 1989). More recently, fatty acids have been proposed as 'magic bullets' in the treatment of a variety of disorders on the hypothesis that they can be used to influence the amounts and types of eicosanoids formed in the body (Brenner 1980, Kunkel et al 1980). To date, no controlled studies of the effects of dietary fats on serum and cutaneous fatty acid concentrations in dogs have been reported. The purpose of this study was to evaluate the effects of dietary fatty acids on serum and cutaneous fatty acids of healthy dogs under controlled conditions. Materials and methods
Dogs Twelve eight-week-old, sexually intact male beagles were individually housed indoors in steel cages. They had access to water ad libitum. The dogs had been weaned at six weeks and were
172
Fatty acid concentrations in dogs consuming a dry kibble diet (Wayne Puppy Food; Wayne Pet Foods, Indiana).
Experimental design Dogs were randomly assigned to three groups (A, B, C) of four dogs each. Dogs in group A were fed a standardised dry kibbled diet (Piasa Pet Products, Rolla, Missouri, USA. Fatty acid composition is 16:0 = 24 per cent, 16:1 = 6 per cent; 18:1n9 = 44 per cent, 18:2n6 = 24 per cent, 18:3n3 = 2 per cent, 20:4n6 = 0.2 per cent, 22:4n6 = 0.5 per cent) supplemented with 10 per cent (by diet weight) sunflower oil (Sunlite Wesson; Beatrice/Hunt Wesson. 18:1n9 = 11 per cent 18:2n6 = 78 per cent), dogs in group B were fed the standardised diet supplemented with 10 per cent olive oil (Pompeian. 18:1n9 -- 75 per cent, 18:2n6 --- 7 per cent), and dogs in group C were fed the standardised diet with no supplementation. Fresh diet was provided twice daily; the dogs were allowed to eat all they wanted during a 30 minute period. After 30 minutes, the food was removed and the amount of food consumed was recorded (in grams). The dogs were weighed once weekly during the 12 week trial.
Serum lipid analysis Serum was obtained following a 16 hour fast on days 0, 21, 42, 63 and 94. One millilitre of serum was added to 20 ml of chloroform: methanol (2:1, v/v) containing 0-005 per cent butylated hydroxytoluene, and the mixture was homogenised (Polytron homogeniser) for two minutes. The homogenate was centrifuged (500 g, 10 n~inutes, 4°C), and the chloroform layer was transferred to another tube and dried under nitrogen. The extracted lipids were methylated using four per cent sulphuric acid in methanol. The fatty acids were separated from dimethyl acetals and cholesterol dienes by sequential extractions after alkalinisation and acidification. The fatty acids were then remethylated using 4 per cent sulphuric acid in methanol (Johnston 1971). The fatty acid methyl esters were analysed (Rowland 1974) by use of gas liquid chromatography (Nu Chek Prep) on a 0.53 ~tm diameter, 30 m long column. The column conditions were: initial temperature, 220°C; hold four minutes; increase to 250°C at 2°C min-1; hold 20 minutes. The methyl esters were identified by comparison
173
with standards. An integrator (Model 4290, Varian) was used to determine the area of each fatty acid peak. Fatty acids evaluated in this study were 18:1n9 (oleic acid), 18:2n6 (linoleic acid), 18:3n6 (y-linolenic acid), 18:3n3 (~-linolenic acid), 20:3n6 (dihomo-y-linolenic acid), 20:4n6 (arachidonic acid), 20:5n3 (eicosapentaenoic acid), 22:4n6 (adrenic acid), 22:5n3 (docosapentaenoic acid) and 22:6n3 (docosahexaneoic acid). For each fatty acid, the areas were multiplied by the relative response factor of the detector and the results were expressed as milligrams of individual fatty acid 100 mg 1 of total fatty acids (weight percentage) (Rowland 1974). Serum cholesterol and triglyceride concentrations (Boehringer/Mannheim Diagnostics) were determined by use of enzymatic assays.
Cutaneous fatty acid analysis Skin biopsies were obtained on days 0, 21, 42, 63 and 94 of the study. Lignocaine hydrochloride (2 per cent) was administered subcutaneously in an area on the lateral aspect of the thorax and a 6 mm biopsy punch (Key Pharmaceuticals) was used to obtain the skin sample. The subcutaneous fat was removed by dissection with metzenbaum scissors from the overlying dennis and epidermis. The skin was then placed in chloroform:methanol (2:1) containing 0.005 per cent butylated hydroxytoluene and homogenised using a tissue homogeniser (Polytron; Brinkman). The solution was filtered, and the fatty acids were extracted, methylated and quantified using the same methods as used for serum.
Diet fatty acid analysis Diets were homogenised, lipids were extracted by refluxing with ether in a soxhlet extraction apparatus (Fisher Scientific) with an allihn condenser (Fisher Scientific). The extracted fatty acids were methylated and quantified using the same methods as used for serum.
Statistical analysis Analysis of variance (SAS Institute 1985) was used to evaluate serum and cutaneous fatty acid concentrations of each group of dogs at days 0, 21, 42, 63 and 94 (time as the test variable) and to evaluate serum and cutaneous fatty acid concentrations of the three groups of dogs on day
174
K. L. Campbell, G. P. Dorn
94 (group as the test variable). The level of significance was chosen at P
Effects of oral sunflower oil and olive oil on serum and cutaneous fatty acid concentrations in dogs K. L. CAMPBELL, G. P. DORN, Department of Veterinary Clinical Medicine, College of
Veterinary Medicine, University of Illinois, 1008 W Hazelwood Drive, Urbana, Illinois 61801, usa
The effects of dietary fatty acids on serum and cutaneous fatty acids of healthy dogs were evaluated under controlled conditions. Beagle puppies (n = 12) were fed a standard diet supplemented with sunflower oil (group A), olive oil (group B) or no supplementation (group C) for 12 weeks. There were no significant differences in food intake or growth rates between the three groups. Dogs in group A had significant increases (P < 0"05) in serum 18:2n6 (linoleic acid) and 20:3n6 (dihomogamma-linolenic acid), and cutaneous 18:2n6 with significant decreases in serum 20:4n6 (arachidonic acid) and cutaneous 18:1n9 (oleic acid) and 18:3n3 (alpha-linolenic acid). Dogs in group B had significant increases in serum 18:1n9, 20:3n6 and cutaneous 18:1n9 with decreases in serum 20:4n6, 22:4n6, 22:5n3 and 22:5n6, and cutaneous 18:2n6, 18:3n3 and 20:4n6. There were no significant changes in serum or cutaneous fatty acids for the dogs in group C. This study demonstrates that fatty acid supplements can be used to alter the serum and cutaneous fatty acid compositions of dogs.
THE importance of dietary fats for the health of the skin of dogs was recognised by Hansen and Wiese (1943) over 45 years ago. Functions of fatty acids include the maintenance of cell membrane integrity and fluidity, maintenance of cell membrane permeability and serving as substrates for the production of eicosanoids (Prottey 1976, 1977). Eicosanoids, which include prostaglandins, thromboxanes, hydroxyeicosatetraenoic acids and leukotrienes, have numerous functions including regulation of D N A synthesis in keratinocytes and serving as mediators of inflammation (Goetzl 1981, Samuelsson 1981). Puppies fed diets deficient in essential fatty acids (I~FAs) develop a seborrhoea characterised by
scaliness and greasiness of the skin and haircoat (Hansen and Wiese 1943). EVA-deficient puppies have lower serum iodine numbers (implying fewer unsaturated double bonds in serum fatty acids) than puppies fed control diets (Hansen and Wiese 1943). This suggests a relationship can exist between dietary fat, serum fatty acid composition, and the development of seborrhoea in dogs. Alterations in fatty acid concentration profiles are seen in a variety of diseases (see discussion) in association with clinical signs of skin disease. The effectiveness of dietary fatty acid supplementation in correcting disease-associated alterations in fatty acid concentration profiles is the subject of current investigations. In the past, veterinary dermatologists advocated the addition of equal parts of vegetable and animal fats to the diet of dogs with seborrhoea (Kunkel et al 1980, Muller et al 1989). More recently, fatty acids have been proposed as 'magic bullets' in the treatment of a variety of disorders on the hypothesis that they can be used to influence the amounts and types of eicosanoids formed in the body (Brenner 1980, Kunkel et al 1980). To date, no controlled studies of the effects of dietary fats on serum and cutaneous fatty acid concentrations in dogs have been reported. The purpose of this study was to evaluate the effects of dietary fatty acids on serum and cutaneous fatty acids of healthy dogs under controlled conditions. Materials and methods
Dogs Twelve eight-week-old, sexually intact male beagles were individually housed indoors in steel cages. They had access to water ad libitum. The dogs had been weaned at six weeks and were
172
Fatty acid concentrations in dogs consuming a dry kibble diet (Wayne Puppy Food; Wayne Pet Foods, Indiana).
Experimental design Dogs were randomly assigned to three groups (A, B, C) of four dogs each. Dogs in group A were fed a standardised dry kibbled diet (Piasa Pet Products, Rolla, Missouri, USA. Fatty acid composition is 16:0 = 24 per cent, 16:1 = 6 per cent; 18:1n9 = 44 per cent, 18:2n6 = 24 per cent, 18:3n3 = 2 per cent, 20:4n6 = 0.2 per cent, 22:4n6 = 0.5 per cent) supplemented with 10 per cent (by diet weight) sunflower oil (Sunlite Wesson; Beatrice/Hunt Wesson. 18:1n9 = 11 per cent 18:2n6 = 78 per cent), dogs in group B were fed the standardised diet supplemented with 10 per cent olive oil (Pompeian. 18:1n9 -- 75 per cent, 18:2n6 --- 7 per cent), and dogs in group C were fed the standardised diet with no supplementation. Fresh diet was provided twice daily; the dogs were allowed to eat all they wanted during a 30 minute period. After 30 minutes, the food was removed and the amount of food consumed was recorded (in grams). The dogs were weighed once weekly during the 12 week trial.
Serum lipid analysis Serum was obtained following a 16 hour fast on days 0, 21, 42, 63 and 94. One millilitre of serum was added to 20 ml of chloroform: methanol (2:1, v/v) containing 0-005 per cent butylated hydroxytoluene, and the mixture was homogenised (Polytron homogeniser) for two minutes. The homogenate was centrifuged (500 g, 10 n~inutes, 4°C), and the chloroform layer was transferred to another tube and dried under nitrogen. The extracted lipids were methylated using four per cent sulphuric acid in methanol. The fatty acids were separated from dimethyl acetals and cholesterol dienes by sequential extractions after alkalinisation and acidification. The fatty acids were then remethylated using 4 per cent sulphuric acid in methanol (Johnston 1971). The fatty acid methyl esters were analysed (Rowland 1974) by use of gas liquid chromatography (Nu Chek Prep) on a 0.53 ~tm diameter, 30 m long column. The column conditions were: initial temperature, 220°C; hold four minutes; increase to 250°C at 2°C min-1; hold 20 minutes. The methyl esters were identified by comparison
173
with standards. An integrator (Model 4290, Varian) was used to determine the area of each fatty acid peak. Fatty acids evaluated in this study were 18:1n9 (oleic acid), 18:2n6 (linoleic acid), 18:3n6 (y-linolenic acid), 18:3n3 (~-linolenic acid), 20:3n6 (dihomo-y-linolenic acid), 20:4n6 (arachidonic acid), 20:5n3 (eicosapentaenoic acid), 22:4n6 (adrenic acid), 22:5n3 (docosapentaenoic acid) and 22:6n3 (docosahexaneoic acid). For each fatty acid, the areas were multiplied by the relative response factor of the detector and the results were expressed as milligrams of individual fatty acid 100 mg 1 of total fatty acids (weight percentage) (Rowland 1974). Serum cholesterol and triglyceride concentrations (Boehringer/Mannheim Diagnostics) were determined by use of enzymatic assays.
Cutaneous fatty acid analysis Skin biopsies were obtained on days 0, 21, 42, 63 and 94 of the study. Lignocaine hydrochloride (2 per cent) was administered subcutaneously in an area on the lateral aspect of the thorax and a 6 mm biopsy punch (Key Pharmaceuticals) was used to obtain the skin sample. The subcutaneous fat was removed by dissection with metzenbaum scissors from the overlying dennis and epidermis. The skin was then placed in chloroform:methanol (2:1) containing 0.005 per cent butylated hydroxytoluene and homogenised using a tissue homogeniser (Polytron; Brinkman). The solution was filtered, and the fatty acids were extracted, methylated and quantified using the same methods as used for serum.
Diet fatty acid analysis Diets were homogenised, lipids were extracted by refluxing with ether in a soxhlet extraction apparatus (Fisher Scientific) with an allihn condenser (Fisher Scientific). The extracted fatty acids were methylated and quantified using the same methods as used for serum.
Statistical analysis Analysis of variance (SAS Institute 1985) was used to evaluate serum and cutaneous fatty acid concentrations of each group of dogs at days 0, 21, 42, 63 and 94 (time as the test variable) and to evaluate serum and cutaneous fatty acid concentrations of the three groups of dogs on day
174
K. L. Campbell, G. P. Dorn
94 (group as the test variable). The level of significance was chosen at P
