- Email: [email protected]
Chylomicron remnants and lipid accumulation in J774 macrophages: influence of cellular oxidation state
Posters 14. Nut&ion The lower serum PON-1 activity on a diet rich in truns fatty acids might have resulted in increased concentrations of lipid peroxides in serum, which may impair endothelial ftmction. This effect could be part of the mechanism by which trans fat increases risk of cardiovascular disease. IP256
CIIYLOMICRON REMNANTS AND LIPID ACCUMULATION IN 5774 MACROPILAGES: OF CELLULAR OXIDATION STATE
and 2.83f0.4 (chol) vs 1.6&0.3 (chol + Cu) (p < 0.05) respectively). These data suggest that dietary copper supplementation reduces atherosclerotic lesion development in the cholesterol-fed rabbit, perhaps by increaseing nitric oxide availability, and is associated with a decrease in both smooth muscle cell content and cellular apoptosis. We thank the British Heart Foundation for support.
INFLUENCE
lp2581 PLANT
K.M. Botham’, M. Napolitano, R. Rivabene, M. Avella’ , E. Bravo. Istituto Superiore di Sanitci, Laboratov of Metabolism and Pathological Biochemistry. Rome, Italy; ‘Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 OTU, UK The accumulation of lipid in macrophages in the artery wall, leading to the formation of foam cells, is an early event in the development of atherosclerosis. The lipid is taken up from plasma lipoproteins, and it has been shown that changes in the cellular oxidant/antioxidant balance can affect this process. The role of low density lipoprotein (LDL) in foam cell formation has been extensively studied, but much less is known about the part played by chylomicron remnants (CR), which carry lipid Tom the diet. In this study, we have investigated the uptake of lipid from CR by the murine macrophage cell line 5774, and how it is influenced by changes in the intracellular redox balance. Chylomicrons radiolabelled in triacylglycerol (TG) with [3H]oleate were collected from rats after cannulation of the thoracic duct, and labelled CR were then prepared in oitro by incubation with lipoprotein lipase. The CR were incubated with J774 macrophages (4 h, l&150 ug remnant cholesterol/ml) after exposure of the cells to CuSO4 (pro-oxidant, 2.5 PM), N-acetylcysteine (NAC, antioxidant, 5 mM) or the same volume of culture medium (control) for 24 h, and the incorporation of [3H]oleate into TG, phospholipid (PL), cholesteryl ester (CE) and free fatty acids (FFA) was determined. ln control cells, label was shown to be incorporated into cellular lipid, and was mainly found in TG (52%) and PL (37%), with only a small amount in CE (8%) and FFA (3%). Afier incubation with CuSO4 or NAC, the glutathione: oxidised glutathione ratio in the cells was lowered (-56%) or raised (+77%), respectively, indicating that the cells has been shifted into pro-oxidising or pro-reducing states. Furthermore, incorporation of label into macrophage TG and PL was increased by 20-30% in CuSO4- as compared with NAC-treated cells. These findings demonstrate that macrophages take up lipid from chylomicron remnants, and that uptake is increased in pro-oxidising conditions. Thus, lipids of dietary origin can contribute to lipid acccumulation in macrophages in foam cell formation, and their role appears to be enhanced by an increase in the intracellular oxidation state. IP257
111
COPPER SUPPLEMENTATION INCREASES NITRIC OXIDE AND DECREASES INTIMAL SMOOTH MUSCLE CONTENT, APOPTOSIS AND ATHEROSCLEROSIS IN CHOLESTEROL-FED RABBITS
D.J. Lamb’, T.Y. Avades’, S. Papachristou’, K. Anw&, G. Kass2, G.L. Reeves’, D. Prentice’, S. Hourani’, G.A.A. Ferns’. ‘Cenhe for Clinical Science and Measurement: ‘School of Biological Sciences, University of Surrey, Guildfoord UK We have shown previously that dietaq copper supplementation in the cholesterol-fed rabbit significantly reduces the formation of atherosclerotic lesions in their aortae. NZW rabbits were fed a 1% cholesterol diet (chol; n = 8) or a 1% cholesterol diet + 0.2% copper acetate (chol + Cu; n = 8) for 13 weeks. Rabbits were killed by anaesthetic overdose, perfused with isotonic PBS and arterial tissues recovered for analysis. Aortic segments were also embedded in paratin wax and sectioned. Copper supplementation significnatly improved carotid artery relaxation to calcium ionophore, but not to sodium nitroprusside, in organ bath experiments compared to arteries from non-supplemented animals. Copper supplementation significantly increased aortic copper content @g/g tissue) 1.8f0.2 (chol) vs 4.0f0.8 (chol + Cu) (p < 0.05). Copper supplementation also significantly reduced the intimal area: medial area ratios of WG stained sections (1.1 f0.2 (chol) vs 0.4&O. 1 (chol + Cu) (p < 0.01)). Copper supplementation significantly reduced the cross-sectional area of lesion staining for smooth muscle (8.OhO.6 (chol) vs 5.7 f 0.6 (chol + Cu) (p < 0.05)), but did not affect the area staining for macrophages (3.3fO.6% (chol) vs 3.8f0.7% (chol + Cu)). There was a significant inverse relationship between aortic copper content and SMC area (R’ = 0.6, p < 0.05). Copper supplementation reduced the number of Tdt-mediated dUTP nick end labelling (TUNBL) positive cells in both the endothelium and intima (1.7f0.8 (chol) vs 0.4& 0.3 (chol + Cu) 0, > 0.05)
STEROL ESTER-ENRICHED SPREAD LOWERS PLASMA TOTAL AND LDL CHOLESTEROL IN CHILDREN WITH FAMILAL HYPERCHOLESTEROLEMIA
A.L. Amundsen’, L. Ose’, E Ntanios2. ‘Lipid Clinic, National Hospital, Oslo, Norway; ’ Unileuel; Vlaardingen, The Netherlands Reduced intake of saturated fat and cholesterol and increased intake of unsaturated fat, are the main dietary advises for the treatment of high blood cholesterol in young children with familial hypercholesterolemia (FH). Recent research do also support a favourable effect of naturally occurring plant sterols on blood cholesterol levels. New technology has made it possible to extract plant sterols from vegetable oils and incorporate them into food. The aim of the present study was to study the effect of 20 grams of plant sterol ester-enriched (SE) spread on serum lipids and lipoproteins as well as safety parameters in children with FH. The study was a randomised, double-blind cross-over study lasting for 2 x 8 weeks. Thirty-eight children with FH consumed 18.2f1.5 grams of SE spread daily corresponding to 1.46f0.12 g of plant sterols. Blood lipids, lipoproteins, blood chemistry, haematology and fat-soluble vitamins were analysed at start and end of the SE and control (C) periods. Plasma concentration of LDL cholesterol decreased by 10.2% (p = 0.003) during the SE period compared with the C period. Total cholesterol was reduced by 7.4% (p = 0.007) and APO-B lipoprotein by 7.4% (p = 0.020). No changes were observed in HDL cholesterol, triglycerides or ApoAI lipoprotein. Serum concentration of lipid adjusted lycopene decreased significantly by 8.1% 0, = 0.0 15) in the SE period, with no changes in other carotenoids, while serum retinol and a-tocopherol concentrations increased by 15.6% (p < 0.001) and 7.1% (p = 0.027), respectively. There was a significant increase (16.8%, p = 0.04) in alanine transaminase (ALT) in the SE period but this may be explained by a significant lower baseline concentration in the plant sterol period compared with the control period. No other effects were observed in clinical blood chemistry or haematology. The children followed a recommended AHA Step-I diet and there was no difference in diet composition between the diet periods. In conclusion, this study shows that in children with FH following a recommended diet, a daily intake of 1.5 grams of plant sterols as plant sterol ester-enriched spread, induces an additional reduction in LDL cholesterol of about 10% without adverse effects.
IP259
DIETARY ASSOCIATES OF SERUM TOTAL CHOLESTEROL IN YOUNG MEN AND WOMEN
V Rosanov, E. Shugaeva, A. Alexandrov, Ch. Pugoeva, A. Olferjev. National Research Centre for Preventive Medicine, Moscow, Russia The objective of the study was to examine the dietary interrelations of serum cholesterol in men and women 35 years old. The sample included 82 men and 94 women born 1964, of them 18 and 12 correspondingly suffered hypercholesterolemia (HC) (serum total cholesterol 2230 mg/dl). Control group consisted of 64 men and 82 women (serum total cholesterol ~200 mgdl). Factual diet was assessed by means of 24 hour recall method and the usage of moulages of dishes with known mass. Daily ration of men with HC as compared with men from control group included less fiber (2.8 and 4.1 g correspondingly, p < 0.05), vegetable oil (0.2 and 2.7 g, p < 0, 05), beta-carotene (0.7 and 2.4 mg, p < 0.001) and vitamin C (31.1 and 68.5 mg, p < O.OOl), but more animal proteins (11.5 and 9.1 g, p < 0,05). There were no differences in cholesterol -&take (523.1 and 512.9 mg, p=O.9). Men with HC consumed less fruit and fruit iuices than men from control group (12.0 and 86.4 g correspondingly, p < OyOl). Women with HC as compared with women from control group consumed less fiber absolutely (1.8 and 3.4 g, p=O.OOl) as well as relatively (1.1 and 2.1 g/1000 ccal, p < 0.05), though there were no significant differences in cholesterol intake (515.3 and 301.5 mg correspondingly, p=O.3). Women with HC consumed less vegetables as compared to women from control group (115.4 and 202.3 g, p c 0,05). It can be concluded that HC is associated not with the intake of cholesterol but with deficit of fiber, vegetable oil and vitamins-antioxidants
72nd EAS Congress
CIIYLOMICRON REMNANTS AND LIPID ACCUMULATION IN 5774 MACROPILAGES: OF CELLULAR OXIDATION STATE
and 2.83f0.4 (chol) vs 1.6&0.3 (chol + Cu) (p < 0.05) respectively). These data suggest that dietary copper supplementation reduces atherosclerotic lesion development in the cholesterol-fed rabbit, perhaps by increaseing nitric oxide availability, and is associated with a decrease in both smooth muscle cell content and cellular apoptosis. We thank the British Heart Foundation for support.
INFLUENCE
lp2581 PLANT
K.M. Botham’, M. Napolitano, R. Rivabene, M. Avella’ , E. Bravo. Istituto Superiore di Sanitci, Laboratov of Metabolism and Pathological Biochemistry. Rome, Italy; ‘Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 OTU, UK The accumulation of lipid in macrophages in the artery wall, leading to the formation of foam cells, is an early event in the development of atherosclerosis. The lipid is taken up from plasma lipoproteins, and it has been shown that changes in the cellular oxidant/antioxidant balance can affect this process. The role of low density lipoprotein (LDL) in foam cell formation has been extensively studied, but much less is known about the part played by chylomicron remnants (CR), which carry lipid Tom the diet. In this study, we have investigated the uptake of lipid from CR by the murine macrophage cell line 5774, and how it is influenced by changes in the intracellular redox balance. Chylomicrons radiolabelled in triacylglycerol (TG) with [3H]oleate were collected from rats after cannulation of the thoracic duct, and labelled CR were then prepared in oitro by incubation with lipoprotein lipase. The CR were incubated with J774 macrophages (4 h, l&150 ug remnant cholesterol/ml) after exposure of the cells to CuSO4 (pro-oxidant, 2.5 PM), N-acetylcysteine (NAC, antioxidant, 5 mM) or the same volume of culture medium (control) for 24 h, and the incorporation of [3H]oleate into TG, phospholipid (PL), cholesteryl ester (CE) and free fatty acids (FFA) was determined. ln control cells, label was shown to be incorporated into cellular lipid, and was mainly found in TG (52%) and PL (37%), with only a small amount in CE (8%) and FFA (3%). Afier incubation with CuSO4 or NAC, the glutathione: oxidised glutathione ratio in the cells was lowered (-56%) or raised (+77%), respectively, indicating that the cells has been shifted into pro-oxidising or pro-reducing states. Furthermore, incorporation of label into macrophage TG and PL was increased by 20-30% in CuSO4- as compared with NAC-treated cells. These findings demonstrate that macrophages take up lipid from chylomicron remnants, and that uptake is increased in pro-oxidising conditions. Thus, lipids of dietary origin can contribute to lipid acccumulation in macrophages in foam cell formation, and their role appears to be enhanced by an increase in the intracellular oxidation state. IP257
111
COPPER SUPPLEMENTATION INCREASES NITRIC OXIDE AND DECREASES INTIMAL SMOOTH MUSCLE CONTENT, APOPTOSIS AND ATHEROSCLEROSIS IN CHOLESTEROL-FED RABBITS
D.J. Lamb’, T.Y. Avades’, S. Papachristou’, K. Anw&, G. Kass2, G.L. Reeves’, D. Prentice’, S. Hourani’, G.A.A. Ferns’. ‘Cenhe for Clinical Science and Measurement: ‘School of Biological Sciences, University of Surrey, Guildfoord UK We have shown previously that dietaq copper supplementation in the cholesterol-fed rabbit significantly reduces the formation of atherosclerotic lesions in their aortae. NZW rabbits were fed a 1% cholesterol diet (chol; n = 8) or a 1% cholesterol diet + 0.2% copper acetate (chol + Cu; n = 8) for 13 weeks. Rabbits were killed by anaesthetic overdose, perfused with isotonic PBS and arterial tissues recovered for analysis. Aortic segments were also embedded in paratin wax and sectioned. Copper supplementation significnatly improved carotid artery relaxation to calcium ionophore, but not to sodium nitroprusside, in organ bath experiments compared to arteries from non-supplemented animals. Copper supplementation significantly increased aortic copper content @g/g tissue) 1.8f0.2 (chol) vs 4.0f0.8 (chol + Cu) (p < 0.05). Copper supplementation also significantly reduced the intimal area: medial area ratios of WG stained sections (1.1 f0.2 (chol) vs 0.4&O. 1 (chol + Cu) (p < 0.01)). Copper supplementation significantly reduced the cross-sectional area of lesion staining for smooth muscle (8.OhO.6 (chol) vs 5.7 f 0.6 (chol + Cu) (p < 0.05)), but did not affect the area staining for macrophages (3.3fO.6% (chol) vs 3.8f0.7% (chol + Cu)). There was a significant inverse relationship between aortic copper content and SMC area (R’ = 0.6, p < 0.05). Copper supplementation reduced the number of Tdt-mediated dUTP nick end labelling (TUNBL) positive cells in both the endothelium and intima (1.7f0.8 (chol) vs 0.4& 0.3 (chol + Cu) 0, > 0.05)
STEROL ESTER-ENRICHED SPREAD LOWERS PLASMA TOTAL AND LDL CHOLESTEROL IN CHILDREN WITH FAMILAL HYPERCHOLESTEROLEMIA
A.L. Amundsen’, L. Ose’, E Ntanios2. ‘Lipid Clinic, National Hospital, Oslo, Norway; ’ Unileuel; Vlaardingen, The Netherlands Reduced intake of saturated fat and cholesterol and increased intake of unsaturated fat, are the main dietary advises for the treatment of high blood cholesterol in young children with familial hypercholesterolemia (FH). Recent research do also support a favourable effect of naturally occurring plant sterols on blood cholesterol levels. New technology has made it possible to extract plant sterols from vegetable oils and incorporate them into food. The aim of the present study was to study the effect of 20 grams of plant sterol ester-enriched (SE) spread on serum lipids and lipoproteins as well as safety parameters in children with FH. The study was a randomised, double-blind cross-over study lasting for 2 x 8 weeks. Thirty-eight children with FH consumed 18.2f1.5 grams of SE spread daily corresponding to 1.46f0.12 g of plant sterols. Blood lipids, lipoproteins, blood chemistry, haematology and fat-soluble vitamins were analysed at start and end of the SE and control (C) periods. Plasma concentration of LDL cholesterol decreased by 10.2% (p = 0.003) during the SE period compared with the C period. Total cholesterol was reduced by 7.4% (p = 0.007) and APO-B lipoprotein by 7.4% (p = 0.020). No changes were observed in HDL cholesterol, triglycerides or ApoAI lipoprotein. Serum concentration of lipid adjusted lycopene decreased significantly by 8.1% 0, = 0.0 15) in the SE period, with no changes in other carotenoids, while serum retinol and a-tocopherol concentrations increased by 15.6% (p < 0.001) and 7.1% (p = 0.027), respectively. There was a significant increase (16.8%, p = 0.04) in alanine transaminase (ALT) in the SE period but this may be explained by a significant lower baseline concentration in the plant sterol period compared with the control period. No other effects were observed in clinical blood chemistry or haematology. The children followed a recommended AHA Step-I diet and there was no difference in diet composition between the diet periods. In conclusion, this study shows that in children with FH following a recommended diet, a daily intake of 1.5 grams of plant sterols as plant sterol ester-enriched spread, induces an additional reduction in LDL cholesterol of about 10% without adverse effects.
IP259
DIETARY ASSOCIATES OF SERUM TOTAL CHOLESTEROL IN YOUNG MEN AND WOMEN
V Rosanov, E. Shugaeva, A. Alexandrov, Ch. Pugoeva, A. Olferjev. National Research Centre for Preventive Medicine, Moscow, Russia The objective of the study was to examine the dietary interrelations of serum cholesterol in men and women 35 years old. The sample included 82 men and 94 women born 1964, of them 18 and 12 correspondingly suffered hypercholesterolemia (HC) (serum total cholesterol 2230 mg/dl). Control group consisted of 64 men and 82 women (serum total cholesterol ~200 mgdl). Factual diet was assessed by means of 24 hour recall method and the usage of moulages of dishes with known mass. Daily ration of men with HC as compared with men from control group included less fiber (2.8 and 4.1 g correspondingly, p < 0.05), vegetable oil (0.2 and 2.7 g, p < 0, 05), beta-carotene (0.7 and 2.4 mg, p < 0.001) and vitamin C (31.1 and 68.5 mg, p < O.OOl), but more animal proteins (11.5 and 9.1 g, p < 0,05). There were no differences in cholesterol -&take (523.1 and 512.9 mg, p=O.9). Men with HC consumed less fruit and fruit iuices than men from control group (12.0 and 86.4 g correspondingly, p < OyOl). Women with HC as compared with women from control group consumed less fiber absolutely (1.8 and 3.4 g, p=O.OOl) as well as relatively (1.1 and 2.1 g/1000 ccal, p < 0.05), though there were no significant differences in cholesterol intake (515.3 and 301.5 mg correspondingly, p=O.3). Women with HC consumed less vegetables as compared to women from control group (115.4 and 202.3 g, p c 0,05). It can be concluded that HC is associated not with the intake of cholesterol but with deficit of fiber, vegetable oil and vitamins-antioxidants
72nd EAS Congress