- Email: [email protected]
Plasma lipid peroxidation in hyperlipidemic chickens
Atherosclerosis, 51 (1985) 119-122 Elsevier Scientific Publishers Ireland,
119 Ltd.
ATH 03675
Preliminary
Note
Plasma Lipid Peroxidation in Hyperlipidemic Chickens Terrance
Smith,
’ Takayoshi
Toda
2 and Fred
A. Kummerow
’
’Department of Food Science, University of Illinois, Burnsides Research Laboratory, 1208 West Pennsylvania Avenue, Urbana, IL 61801 (U.S.A.) and’ Department of Pathology, School o/Medicine. Ryukyu University, Okinawa 903 (Japan) (Received 29 January. 1985) (Revised, received 19 March, 1985) (Accepted 26 March, 1985)
Summary Laying and genetically defective non-laying hens were evaluated for plasma lipid and plasma peroxidation levels. The non-layers developed extreme hyperlipidemia as well as greatly increased levels of lipid peroxidation. It was concluded that the concurrent presence of lipid peroxidation products must be considered when evaluating hyperlipidemic causes of atherosclerosis in the chicken. Key words:
Atherosclerosis - Hyperlipidemia - Peroxidation
Introduction In recent years lipid peroxidation and its possible role in heart disease has received increasing attention. The presence of serum peroxidation products in patients with atherosclerosis [1,2] suggests that peroxidative reactions may be involved in the disease. That lipid peroxides might be capable of initiating atherosclerosis is suggested by the occurrence of aortic lesions in rabbits injected with linoleic acid hydroperoxides [3]. Clues to the action of lipid peroxides in causing or contributing to atherosclerosis come from studies done at the cellular level. Endothelial cells in tissue culture are injured and killed by low density lipoprotein which contains peroxidation products [4]. Macrophages ingest LDL by a well-regulated receptor-mediated endocytosis. Lipoperoxide-modified LDL is not recognized by this receptor but is taken up by a non-regulated secondary receptor 0021-9150/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
Ltd.
120
[5]. The resulting unrestrained accumulation of cholesterol ester is thought to cause the conversion of macrophages to foam cells. Since lipid peroxides may be atherogenic it is important to consider the peroxidative state of lipids from animal models used in atherosclerosis research. In this paper we report that hyperlipidemia in restricted ovulator hens is closely paralleled by an increase in plasma lipid peroxides. Materials and Methods Diagnostic kits to determine total serum cholesterol and triglycerides were obtained from Sigma Chemical Co., St. Louis, MO. Thiobarbaturic acid, tetramethylpropane, and phosphotungstic acid were also obtained from Sigma. All solvents were reagent grade or better. Chickens were housed in individual wire cages and fed a commercial chicken mash ad libitum. At 6 months of age the photoperiod was increased to 18 h. Those hens which then laid no eggs were classified as ‘non-layers’, those which laid 5-6 eggs per week ‘layers’ and those which laid l-4 eggs per week were excluded. At 1 year of age blood was drawn from a wing vein using a heparinized syringe and was then cooled on ice. After separating the plasma by centrifugation, the plasma lipid peroxidation (malondialdehyde) level was determined by the method of Yagi [6]. Plasma triglyceride and total plasma cholesterol were determined according to the instructions supplied with the commercial assay kits used. To determine plasma phospholipid content, an aliquot of plasma was extracted by the method of Folch et al. [7] and assayed for phosphorous after perchloric acid digestion [8]. Statistical analysis was done using a one-tailed Student’s t-test. Results The plasma lipid levels of layers and non-layers used in this study are shown in Table 1. Cholesterol, triglyceride and phospholipid plasma levels are significantly elevated in the non-layers and are in agreement with plasma lipid levels previously reported [9]. The concentration of the peroxidation product malondialdehyde was also elevated in the plasma of non-layers. When peroxide levels were expressed on the basis of triglyceride or phospholipid, there was no statistical difference in the extent of plasma peroxidation. Discussion In this laboratory we have used restricted ovulator hens as an animal model for atherosclerosis [lo]. Restricted ovulator hens, when exposed to a prolonged photoperiod, reabsorb the egg consequently developing hyperlipidemia [ll] and then atherosclerosis [12]. According to the currently accepted theory the hypercholesterolemia of these hens is responsible for the development of atherosclerosis. However, there are considerable data suggesting that lipid peroxides are also atherogenic [l-5]. We have demonstrated that not only were the plasma lipid components elevated
1
AND
84*15 64Ok91
b
Cholesterol
Component
LIPIDS
540*340 4800+210 b
CHICKENS
5.6k 1.0 76 + 12.0 b
Plasma (nmol MDA/ml)
AND NON-LAYER
Phospholipid
OF LAYER
1100+ 900 12000+1600 b
Triglyceride
(mg/dl)
PEROXIDATION
a Values represent the mean f standard deviation of at least 3 samples. b Significantly greater than layer, a = 0.05, one-tailed Student’s r-test.
Layer Non-layer
PLASMA
TABLE
4.6 f 3.1 12 k2.0 b
Cholesterol
nmol MDA/mg
’
0.84 & 0.02 0.65 + 0.104~1.4
Triglyceride
component
f 0.4
0.82 _+0.01
Phospholipid
122
in non-laying chickens, but the level of malondialdehyde was also greatly increased (Table 1). Non-layers had 14 times the amount of this peroxidation product per milliliter of plasma compared to layers. This suggested that the extent of peroxidation of the serum lipid was the same in layers and non-layers, but due to hyperlipidemia the total concentration of peroxidation product was greatly increased in non-layers. It is conceivable that hypercholesterolemia and lipid peroxidation play complementary parts in atherogenesis. The presence of lipid peroxidation products might damage the endothelium and deregulate the uptake of LDL by macrophage. At the same time, hypercholesterolemia would provide an ample supply of cholesterol to convert macrophages to foam cells. The precise role of lipid peroxidation and its interaction with hypercholesterolemia remains to be determined. It is clear from the data presented here that future experiments on atherosclerosis using restricted ovulator hens must consider the presence of lipid peroxidation products as well as hypercholesterolemia. References Sato, Y., Hotta, N., Sakamoto, N., Matsuoka, S., Ohishi, N. and Yagi, K. Lipid peroxide level in plasma of diabetic pateints, Biochem. Med., 21 (1979) 104. Goto, Y., Lipid peroxides as a cause of vascular disease. In K. Yagi (Ed.), Lipid Peroxides in Biology and Medicine, Academic Press, 1983, p. 295. Yagi, K., Ohkawa, H., Ohishi, N., Yamashita, M. and Nakashima, T., Lesion of aortic intima caused by intravenous administration of linoleic acid hydroperoxide, J. Appl. B&hem., 3 (1981) 58. Hessler, J.R., Morel, D.W., Lewis, L.J. and Chrisolm, L.W., Lipoprotein oxidation and lipoprotein-induced cytotoxicity, Arteriosclerosis, 3 (1983) 215. Brown, MS. and Goldstein, J.L., Lipoprotein metabolism in the macrophage - Implications for cholesterol in atherosclerosis, Ann. Rev. B&hem., 52 (1983) 223. Yagi, K., A simple fluormetric assay for lipoperoxide in blood plasma, Biochem. Med., 15 (1978) 212. Folch, J., Lees, M. and Sloane-Stanley, G.H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. Eng, L.F. and Noble, E.P., The maturation of rat brain myelin, Lipids, 3 (1968) 157. Cho, B.H.S., Lawson, L.D., Toda, T. and Kummerow, F.A., Oxidation of fatty acid by heart mitochondria of chickens with endogenous hyperhpidemia, Biochem. Med., 31 (1984) 347. I. and Kummerow, F., Animal model of atherosclerosis Experimental 10 Toda, T., Nishimori, atherosclerosis in the chicken animal model, J. Japan Atheroscl. Sot., 11 (1983) 755. 11 Ho, K.J., Lawrence, W.D., Lewis, L.A., Liu, L.B. and Taylor, C.B., Hereditary hyperlipidemia in non-laying chickens, Arch. Path., 98 (1974) 161. 12 Tokuyasu, T., Imai, H., Taura, S., Cho, B.H.S. and Kummerow, F.A., Aortic lesions in non-laying hens with endogenous hypeylipidemia, Arch. Path. Lab. Med., 104 (1980) 41.
119 Ltd.
ATH 03675
Preliminary
Note
Plasma Lipid Peroxidation in Hyperlipidemic Chickens Terrance
Smith,
’ Takayoshi
Toda
2 and Fred
A. Kummerow
’
’Department of Food Science, University of Illinois, Burnsides Research Laboratory, 1208 West Pennsylvania Avenue, Urbana, IL 61801 (U.S.A.) and’ Department of Pathology, School o/Medicine. Ryukyu University, Okinawa 903 (Japan) (Received 29 January. 1985) (Revised, received 19 March, 1985) (Accepted 26 March, 1985)
Summary Laying and genetically defective non-laying hens were evaluated for plasma lipid and plasma peroxidation levels. The non-layers developed extreme hyperlipidemia as well as greatly increased levels of lipid peroxidation. It was concluded that the concurrent presence of lipid peroxidation products must be considered when evaluating hyperlipidemic causes of atherosclerosis in the chicken. Key words:
Atherosclerosis - Hyperlipidemia - Peroxidation
Introduction In recent years lipid peroxidation and its possible role in heart disease has received increasing attention. The presence of serum peroxidation products in patients with atherosclerosis [1,2] suggests that peroxidative reactions may be involved in the disease. That lipid peroxides might be capable of initiating atherosclerosis is suggested by the occurrence of aortic lesions in rabbits injected with linoleic acid hydroperoxides [3]. Clues to the action of lipid peroxides in causing or contributing to atherosclerosis come from studies done at the cellular level. Endothelial cells in tissue culture are injured and killed by low density lipoprotein which contains peroxidation products [4]. Macrophages ingest LDL by a well-regulated receptor-mediated endocytosis. Lipoperoxide-modified LDL is not recognized by this receptor but is taken up by a non-regulated secondary receptor 0021-9150/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
Ltd.
120
[5]. The resulting unrestrained accumulation of cholesterol ester is thought to cause the conversion of macrophages to foam cells. Since lipid peroxides may be atherogenic it is important to consider the peroxidative state of lipids from animal models used in atherosclerosis research. In this paper we report that hyperlipidemia in restricted ovulator hens is closely paralleled by an increase in plasma lipid peroxides. Materials and Methods Diagnostic kits to determine total serum cholesterol and triglycerides were obtained from Sigma Chemical Co., St. Louis, MO. Thiobarbaturic acid, tetramethylpropane, and phosphotungstic acid were also obtained from Sigma. All solvents were reagent grade or better. Chickens were housed in individual wire cages and fed a commercial chicken mash ad libitum. At 6 months of age the photoperiod was increased to 18 h. Those hens which then laid no eggs were classified as ‘non-layers’, those which laid 5-6 eggs per week ‘layers’ and those which laid l-4 eggs per week were excluded. At 1 year of age blood was drawn from a wing vein using a heparinized syringe and was then cooled on ice. After separating the plasma by centrifugation, the plasma lipid peroxidation (malondialdehyde) level was determined by the method of Yagi [6]. Plasma triglyceride and total plasma cholesterol were determined according to the instructions supplied with the commercial assay kits used. To determine plasma phospholipid content, an aliquot of plasma was extracted by the method of Folch et al. [7] and assayed for phosphorous after perchloric acid digestion [8]. Statistical analysis was done using a one-tailed Student’s t-test. Results The plasma lipid levels of layers and non-layers used in this study are shown in Table 1. Cholesterol, triglyceride and phospholipid plasma levels are significantly elevated in the non-layers and are in agreement with plasma lipid levels previously reported [9]. The concentration of the peroxidation product malondialdehyde was also elevated in the plasma of non-layers. When peroxide levels were expressed on the basis of triglyceride or phospholipid, there was no statistical difference in the extent of plasma peroxidation. Discussion In this laboratory we have used restricted ovulator hens as an animal model for atherosclerosis [lo]. Restricted ovulator hens, when exposed to a prolonged photoperiod, reabsorb the egg consequently developing hyperlipidemia [ll] and then atherosclerosis [12]. According to the currently accepted theory the hypercholesterolemia of these hens is responsible for the development of atherosclerosis. However, there are considerable data suggesting that lipid peroxides are also atherogenic [l-5]. We have demonstrated that not only were the plasma lipid components elevated
1
AND
84*15 64Ok91
b
Cholesterol
Component
LIPIDS
540*340 4800+210 b
CHICKENS
5.6k 1.0 76 + 12.0 b
Plasma (nmol MDA/ml)
AND NON-LAYER
Phospholipid
OF LAYER
1100+ 900 12000+1600 b
Triglyceride
(mg/dl)
PEROXIDATION
a Values represent the mean f standard deviation of at least 3 samples. b Significantly greater than layer, a = 0.05, one-tailed Student’s r-test.
Layer Non-layer
PLASMA
TABLE
4.6 f 3.1 12 k2.0 b
Cholesterol
nmol MDA/mg
’
0.84 & 0.02 0.65 + 0.104~1.4
Triglyceride
component
f 0.4
0.82 _+0.01
Phospholipid
122
in non-laying chickens, but the level of malondialdehyde was also greatly increased (Table 1). Non-layers had 14 times the amount of this peroxidation product per milliliter of plasma compared to layers. This suggested that the extent of peroxidation of the serum lipid was the same in layers and non-layers, but due to hyperlipidemia the total concentration of peroxidation product was greatly increased in non-layers. It is conceivable that hypercholesterolemia and lipid peroxidation play complementary parts in atherogenesis. The presence of lipid peroxidation products might damage the endothelium and deregulate the uptake of LDL by macrophage. At the same time, hypercholesterolemia would provide an ample supply of cholesterol to convert macrophages to foam cells. The precise role of lipid peroxidation and its interaction with hypercholesterolemia remains to be determined. It is clear from the data presented here that future experiments on atherosclerosis using restricted ovulator hens must consider the presence of lipid peroxidation products as well as hypercholesterolemia. References Sato, Y., Hotta, N., Sakamoto, N., Matsuoka, S., Ohishi, N. and Yagi, K. Lipid peroxide level in plasma of diabetic pateints, Biochem. Med., 21 (1979) 104. Goto, Y., Lipid peroxides as a cause of vascular disease. In K. Yagi (Ed.), Lipid Peroxides in Biology and Medicine, Academic Press, 1983, p. 295. Yagi, K., Ohkawa, H., Ohishi, N., Yamashita, M. and Nakashima, T., Lesion of aortic intima caused by intravenous administration of linoleic acid hydroperoxide, J. Appl. B&hem., 3 (1981) 58. Hessler, J.R., Morel, D.W., Lewis, L.J. and Chrisolm, L.W., Lipoprotein oxidation and lipoprotein-induced cytotoxicity, Arteriosclerosis, 3 (1983) 215. Brown, MS. and Goldstein, J.L., Lipoprotein metabolism in the macrophage - Implications for cholesterol in atherosclerosis, Ann. Rev. B&hem., 52 (1983) 223. Yagi, K., A simple fluormetric assay for lipoperoxide in blood plasma, Biochem. Med., 15 (1978) 212. Folch, J., Lees, M. and Sloane-Stanley, G.H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. Eng, L.F. and Noble, E.P., The maturation of rat brain myelin, Lipids, 3 (1968) 157. Cho, B.H.S., Lawson, L.D., Toda, T. and Kummerow, F.A., Oxidation of fatty acid by heart mitochondria of chickens with endogenous hyperhpidemia, Biochem. Med., 31 (1984) 347. I. and Kummerow, F., Animal model of atherosclerosis Experimental 10 Toda, T., Nishimori, atherosclerosis in the chicken animal model, J. Japan Atheroscl. Sot., 11 (1983) 755. 11 Ho, K.J., Lawrence, W.D., Lewis, L.A., Liu, L.B. and Taylor, C.B., Hereditary hyperlipidemia in non-laying chickens, Arch. Path., 98 (1974) 161. 12 Tokuyasu, T., Imai, H., Taura, S., Cho, B.H.S. and Kummerow, F.A., Aortic lesions in non-laying hens with endogenous hypeylipidemia, Arch. Path. Lab. Med., 104 (1980) 41.