Comparison of the Effect of Probucol and CS-514 on Plasma Lipids of Hypercholesterolemic Patients
Gen YoshinoTsutomu KazumiMasahide IwaiKohji MatsubaIppei IWATANIToshio KasamaRinzo UenoyamaAkio InuiKoichi YokonoMakoto OtsukiShigeaki Baba
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CS-514, a new inhibitor of endogenous cholesterol biosynthesis, has been shown to reduce plasma cholesterol concentration in both healthy and hypercholesterolemic subjects. In this study we compared the effect of CS-514 and probucol on plasma lipids in hypercholesterolemic subjects. After treatment with probucol, HDL-cholesterol as well as total cholesterol tended to decrease. After a washout period probucol was replaced with comparable doses of CS-514. All the patients showed decreased plasma cholesterol levels after CS-514 treatment. HDL-cholesterol levels increased significantly after CS-514. Plasma triglyceride levels showed no significant change throughout the study period. Thus, the two drugs differed in their effects on HDL-cholesterol.Keywords:
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The tracer [4-14C]cholesterol, incubated at 37° for 1 hr with the subject's own plasma, was administered intravenously to two subjects. The initial rapid decrease of plasma cholesterol specific activity indicated a net disappearance of [4-14C]cholesterol from the bloodstream, presumably due to phagocytosis of the particu-late cholesterol by the reticuloendothelial system. The initially disappeared [4-14C]cholesterol quickly reappeared in the blood. Complete equilibrium between RBC and plasma-free cholesterol was attained within 12-18 hr, whereas the equilibrium between plasma-free and esterified cholesterol required 2 days. Therefore, the com-partmental analysis should be based on the plasma total cholesterol specific activity obtained 2 days after the injection.
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Probucol
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The effect and safety of a new cholesterol- lowering drug, probucol, was investigated in 49 hypercholesterolemia patients in Kyushu multicentral cooperative study (10 hospitals). The patients were allocated to either 750mg or 1000mg per day for 16 weeks. Both doses of probucol lowered serum cholesterol, LDL-cholesterol and HDL-cholesterol and phospholipid significantly without influencing on serum tryglyceride levels. No relationship among two doses of probucol and the degree of cholesterol lowering have been found in this study. And at all doses employed, the drug was well tolerated and no changes attributable to therapy were observed in the laboratory parameters evaluated.
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Probucol
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Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.—Bird, D. A., R. K. Tangirala, J. Fruebis, D. Steinberg, J. L. Witztum, and W. Palinski. Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice. J. Lipid Res. 1998. 39: 1079–1090. Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.—Bird, D. A., R. K. Tangirala, J. Fruebis, D. Steinberg, J. L. Witztum, and W. Palinski. Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice. J. Lipid Res. 1998. 39: 1079–1090. Oxidized lipoproteins may enhance atherosclerosis by a number of mechanisms. These include the recognition of oxidized lipoproteins by macrophage scavenger receptors, their chemotactic and cytotoxic properties, and the modulation of gene expression of vascular cells (reviewed in 1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. Beyond cholesterol. Modifications of low density lipoprotein that increase its atherogenicity.N. Engl. J. Med. 1989; 320: 915-924Google Scholar, 2Steinberg D. Low density lipoprotein oxidation and its pathobiological significance.J. Biol. Chem. 1997; 272: 20963-20966Google Scholar, 3Berliner J.A. Navab M. Fogelman A.M. Frank J.S. Demer L.L. Edwards P.A. Watson A.D. Lusis A.J. Atherosclerosis: Basic mechanisms. oxidation, inflammation, and genetics.Circulation. 1995; 91: 2488-2496Google Scholar). The occurrence of oxidized low density liproprotein (LDL) within atherosclerotic lesions has been amply demonstrated (4Haberland M.E. Fong D. Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits.Science. 1988; 241: 215-218Google Scholar, 5Palinski W. Rosenfeld M.E. Ylä-Herttuala S. Gurtner G.C. Socher S.A. Butler S. Parthasarathy S. Carew T.E. Steinberg D. Witztum J.L. Low density lipoprotein undergoes oxidative modification in vivo.Proc. Natl. Acad. Sci. USA. 1989; 86: 1372-1376Google Scholar, 6Ylä-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Google Scholar, 7Boyd H.C. Gown A.M. Wolfbauer G. Chait A. Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe Heritable Hyperlipidemic rabbit.Am. J. Pathol. 1989; 135: 815-826Google Scholar, 8Rosenfeld M.E. Palinski W. Ylä-Herttuala S. Butler S. Witztum J.L. Distribution of oxidation-specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits.Arteriosclerosis. 1990; 10: 336-349Google Scholar, 9Hammer A. Kager G. Dohr G. Rabl H. Ghassempur I. Jürgens G. Generation, characterization, and histochemical application of monoclonal antibodies selectively recognizing oxidatively modified apoB-containing serum lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 704-713Google Scholar), but to date the most convincing evidence for the atherogenicity of LDL oxidation is the fact that powerful antioxidants, such as probucol, butylated hydroxytoluene, and diphenyl-phenylenediamine (DPPD), significantly reduce atherosclerosis in rabbits, hamsters, and primates (10Kita T. Nagano Y. Yokode M. Ishii K. Kume N. Ooshima A. Yokida H. Kawai C. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia.Proc. Natl. Acad. Sci. USA. 1987; 84: 5928-5931Google Scholar, 11Carew T.E. Schwenke D.C. Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks slowing the progression of atherosclerosis in the WHHL rabbit.Proc. Natl. Acad. Sci. USA. 1987; 84: 7725-7729Google Scholar, 12Björkhem I. Henriksson-Freyschuss A. Breuer O. Diczfalusy U. Berglund L. Henriksson P. The antioxidant butylated hydroxytoluene protects against atherosclerosis.Arterioscler. Thromb. 1991; 11: 15-22Google Scholar, 13Sparrow C.P. Doebber T.W. Olszewski J. Wu M.S. Ventre J. Stevens K.A. Chao Y.S. Low density lipoprotein is protected from oxidation and the progression of atherosclerosis is slowed in cholesterol-fed rabbits by the antioxidant N,N′′-diphenyl-phenylenediamine.J. Clin. Invest. 1992; 89: 1885-1891Google Scholar, 14Parker R.A. Sabrah T. Cap M. Gill B.T. Relation of vascular oxidative stress, α-tocopherol, and hypercholesterolemia to early atherosclerosis in hamsters.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 349-358Google Scholar, 15Sasahara M. Raines E.W. Chait A. Carew T.E. Steinberg D. Wahl P.W. Ross R. Inhibition of hypercholesterolemia-induced atherosclerosis in Macaca nemestrina by probucol. I. Is the extent of atherosclerosis related to resistance of LDL to oxidation?.J. Clin. Invest. 1994; 94: 155-164Google Scholar). The most potent of these compounds, probucol, strongly protects plasma LDL against ex vivo oxidation. However, the mechanisms by which probucol and some other antioxidants reduce atherogenesis have not been established. Probucol also has a variety of cellular effects which may or may not be due to its antioxidant properties (16Chang M.Y. Sasahara M. Chait A. Raines E.W. Ross R. Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol: II. Cellular composition and proliferation.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1631-1640Google Scholar, 17Akeson A.L. Woods C.W. Mosher L.B. Thomas C.E. Jackson R.L. Inhibition of IL-1 beta expression in THP-1 cells by probucol and tocopherol.Atherosclerosis. 1991; 86: 261-270Google Scholar, 18Fruebis J. Gonzales V. Silvestre M. Palinski W. Effect of probucol treatment on gene expression of VCAM-1, MCP-1 and M-CSF in the aortic wall of LDL receptor-deficient rabbits during early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1289-1302Google Scholar). For example, probucol down-regulates baseline gene expression of vascular cell adhesion molecule 1 (VCAM-1) and macrophage colony stimulating factor (M-CSF) in rabbit aortas and prevents the up-regulation of VCAM-1 mRNA and protein (18Fruebis J. Gonzales V. Silvestre M. Palinski W. Effect of probucol treatment on gene expression of VCAM-1, MCP-1 and M-CSF in the aortic wall of LDL receptor-deficient rabbits during early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1289-1302Google Scholar) that occurs during atherogenesis (18Fruebis J. Gonzales V. Silvestre M. Palinski W. Effect of probucol treatment on gene expression of VCAM-1, MCP-1 and M-CSF in the aortic wall of LDL receptor-deficient rabbits during early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1289-1302Google Scholar, 19Li H. Cybulsky M.I. Gimbrone M.A. Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium.Arterioscler. Thromb. 1993; 13: 197-204Google Scholar, 20Cybulski M.I. Gimbrone M.A. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis.Science. 1991; 251: 788-791Google Scholar). Furthermore, it is increasingly recognized that the antiatherogenic effect of antioxidants may not necessarily be due to, or reflected by, the protection of plasma LDL against oxidation. We recently demonstrated in LDL receptor-deficient rabbits that the degree of antioxidant protection of plasma LDL was not a predictor of the anti-atherosclerotic effect of different antioxidants (21Fruebis J. Bird D.A. Pattison J. Palinski W. Extent of antioxidant protection of plasma LDL is not a predictor of the antiatherogenic effect of antioxidants.J. Lipid Res. 1997; 38: 2455-2464Google Scholar). In that study, a combination of antioxidants (vitamin E, a probucol analogue, and a very low dose (0.025%) of probucol) protected plasma LDL to the same extent as 0.091% probucol, yet failed to achieve a significant reduction of atherosclerosis, whereas 0.091% probucol did. Murine models would be particularly attractive for studying atherogenic mechanisms involving oxidative processes because techniques are readily available to delete or overexpress murine genes (22Witztum J.L. Murine models for study of lipoprotein metabolism and atherosclerosis.J. Clin. Invest. 1993; 92: 536-537Google Scholar, 23Breslow J.L. Mouse models of atherosclerosis.Science. 1996; 272: 685-688Google Scholar). Two murine strains are available in which extensive atherosclerosis occurs in the entire aortic tree, either spontaneously or induced by high-cholesterol diets: the apoE-deficient (apoE-/-) mouse (24Plump A.S. Smith J.D. Hayek T. Aalto-Setälä K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.Cell. 1992; 71: 343-353Google Scholar, 25Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Google Scholar) and the LDL receptor-deficient (LDLR-/-) mouse (26Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.J. Clin. Invest. 1993; 92: 883-893Google Scholar). Atherosclerotic lesions in these mice range from fatty streaks to advanced atheromas and show many characteristic features of lesions seen in other animal models and in humans (27Reddick R.L. Zhang S.H. Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression.Arterioscler. Thromb. 1994; 14: 141-147Google Scholar, 28Nakashima Y. Plump A.S. Raynes E.W. Breslow J.L. Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the aortic tree.Arterioscler. Thromb. 1994; 14: 133-140Google Scholar). Support for the involvement of lipoprotein oxidation in the atherogenic process in these murine models consists of the immunocytochemical demonstration of "oxidation-specific" epitopes in their lesions and the presence in their plasma of high titers of autoantibodies against epitopes of OxLDL (29Palinski W. Ord V. Plump A.S. Breslow J.L. Steinberg D. Witztum J.L. ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis: demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde–lysine in serum.Arterioscler. Thromb. 1994; 14: 605-616Google Scholar, 30Palinski W. Tangirala R.K. Miller E. Young S.G. Witztum J.L. Increased autoantibody titers against epitopes of oxidized LDL in LDL receptor-deficient mice with increased atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1569-1576Google Scholar, 31Palinski W. Hörkkö S. Miller E. Steinbrecher U.P. Powell H.C. Curtiss L.K. Witztum J.L. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apoE-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma.J. Clin. Invest. 1996; 98: 800-814Google Scholar, 32Hörkkö S. Miller E. Dudl E. Reaven P.D. Zvaifler N.J. Terkeltaub R. Pierangeli S.S. Curtiss L.K. Branch D.W. Palinski W. Witztum J.L. Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids: recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low-density lipoprotein.J. Clin. Invest. 1996; 98: 815-825Google Scholar). However, to date the atherogenicity of lipoprotein oxidation, and conversely, the antiatherogenic effect of antioxidants, have not been conclusively established in mice. An earlier study from our laboratory suggested that an antioxidant, DPPD, can be effective in murine models. ApoE-/- mice treated with DPPD developed less atherosclerosis (33Tangirala R.K. Casanada F. Witztum J.L. Steinberg D. Palinski W. Effect of the antioxidant N,N′-diphenyl 1,4-phenylenediamine (DPPD) on atherogenesis in apoE-deficient mice.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1625-1630Google Scholar), but the effect was small and DPPD may have toxic side effects (treated animals gained less weight than controls). In contrast, a recent report by Zhang et al. (34Zhang S.H. Reddick R.L. Avdievich E. Surles L.K. Jones R.G. Reynolds J.B. Quarfordt S.H. Maeda N. Paradoxical enhancement of atherosclerosis by probucol treatment in apolipoprotein E-deficient mice.J. Clin. Invest. 1997; 99: 2858-2866Google Scholar) showed that probucol increased atherogenesis in apo E-/- mice. However, the mechanisms influencing atherogenesis in this model may be complex, due to the inability of intimal macrophages to secrete apoE and other consequences of apoE deficiency. Furthermore, to study the effects of probucol unrelated to its modulation of lipid metabolism, it would be desirable to match the cholesterol levels in the treatment and control groups. Probucol has a very powerful hypolipidemic effect in mice, which is difficult, if not impossible, to compensate for in apoE-/- mice, because in control animals extensive hyperlipidemia occurs spontaneously, even on a regular rodent diet. In contrast, extensive hypercholesterolemia and atherosclerosis in LDLR-/- mice is dependent on administration of a high-fat, high-cholesterol diet (26Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.J. Clin. Invest. 1993; 92: 883-893Google Scholar, 30Palinski W. Tangirala R.K. Miller E. Young S.G. Witztum J.L. Increased autoantibody titers against epitopes of oxidized LDL in LDL receptor-deficient mice with increased atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1569-1576Google Scholar, 35Ishibashi S. Goldstein J.L. Brown M.S. Herz J. Burns D.K. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice.J. Clin. Invest. 1994; 93: 1885-1893Google Scholar). This should make it possible to achieve similar plasma cholesterol levels in the treatment and control groups by adjusting the cholesterol content of the respective diets. Therefore, to determine whether murine models are generally suitable to investigate the mechanisms by which probucol and other antioxidants may affect atherogenesis, we carried out a series of intervention studies in LDLR-/- mice. Three consecutive studies (Experiments I – III) were performed using homozygous LDLR-/- mice with a C57BL/6J × 129Sv background from a breeding colony established from animals originally provided by Jackson Laboratories. The design of these experiments is summarized in Table 1.TABLE 1.Experimental protocolExpGroupSexProbucol(% of diet)Cholesterol(% of diet; time-averaged)Matched Overall Cholesterol ExposureMatched Treatment PeriodIControlFnone0.08High probucolF0.51.25yesnoIIControlMnone0.01Low probucol/vitamin Eaa This group received 0.1% vitamin E, in addition to probucol. F, female; M, male; Exp, experiment.M0.0250.83Low probucolM0.0251.12yesnoHigh probucolM0.51.78IIILow cholesterol controlF, Mnone0.01High probucolF, M0.51.25noyesHigh cholesterol controlF, Mnone1.25a a This group received 0.1% vitamin E, in addition to probucol. F, female; M, male; Exp, experiment. Open table in a new tab In Experiment I, 28 female mice were divided into two equal groups, matched for age (3–4 months), body weight, and plasma cholesterol levels. The first group (high-probucol) was treated with 0.5% (w/w) probucol (a generous gift from the Hoechst Marion Roussel Research Institute, Cincinnati, Ohio). This group was initially fed an atherogenic diet containing 21.2% fat and 1.25% cholesterol (TD96121; Harlan Teklad). The second group (control) was also fed a high-fat diet, but one containing a lower amount of cholesterol. None of the diets contained cholate. Because probucol has a very strong lipid-lowering effect in mice, the cholesterol content of the control diet was adjusted throughout the intervention period to achieve plasma cholesterol levels similar to those in the treatment group. In addition, in order to obtain the same overall cholesterol exposure (area under the curve describing cholesterol levels over time) in both groups, the treatment period of the probucol group had to be extended (142 days vs. 112 days in the control group). One control mouse died during blood sampling, reducing the final number of animals in this group to 13. Experiment II followed the same experimental approach. Furthermore, two additional groups were included in Experiment II to test the dose dependency of the effect of probucol and to see whether the effect on atherosclerosis correlated with the degree of antioxidant protection of plasma LDL. Sixty-four male LDLR-/- mice were divided into four groups of 16, matched for age (2–3 months), body weight, and plasma cholesterol levels (Table 1). The first group was again treated with 0.5% probucol (high probucol). The second group did not receive any antioxidant (control). The third group was treated with a low dose (0.025%) of probucol and a high dose (0.1%) of vitamin E (low probucol/vitamin E). The fourth group received only 0.025% probucol (low probucol). As in Experiment I, the dietary cholesterol content was adjusted periodically throughout the study to compensate for the hypocholesterolemic effect of probucol. More specifically, the cholesterol content of the high probucol group was raised from 1.25% to 2% after 52 days and that of the control group was lowered from 0.02% to 0.01% after 24 days. The dietary cholesterol of the low probucol group was lowered from 1.25% to 1% on day 66, and that of the low probucol/vitamin E group was first reduced to 1% (day 42) and then to 0.5% (day 66) and 0.3% (day 94). In order to achieve the same overall cholesterol exposure in all four groups, treatment of the control and low probucol/vitamin E groups was ended after 119 and 118 days, respectively, whereas treatment of the low probucol group was extended to 139 days and that of the high probucol group to 181 days. During routine blood sampling from the retro-orbital plexus 1, 1, 2, and 4 mice died from the control, low probucol/vitamin E, low probucol, and high probucol groups, respectively. Also, 2 mice from the low probucol/vitamin E group died of unknown causes. Experiment III was carried out to compare the effect of probucol in male and female mice and included more than one time point, to test whether the rate of progression of lesion formation was different between sexes. The design of Experiment III was also conceptually different. In Experiments I and II, significantly longer treatment periods of the high probucol groups were necessary to achieve a matched overall cholesterol exposure, because the modulation of the dietary cholesterol by itself had been insufficient to completely compensate for the powerful hypocholesterolemic effect of probucol. In contrast, in Experiment III we assumed that the duration of hypercholesterolemia was more important than an exact match of the cholesterol exposure, and therefore kept the intervention period the same for all groups. In addition, no attempt was made to modulate the cholesterol levels during the intervention phase. The extent of atherosclerosis in mice treated with 0.5% probucol was instead compared to that in two groups of untreated mice fed diets inducing a low or high plasma cholesterol level. In this experiment, 36 female and 24 male mice were divided into three matched groups (age 2–3 months) (Table 1). The first group of 12 females and 8 males (low cholesterol control) was fed a diet containing 21% fat and 0.01% cholesterol. The second group of 12 female and 9 male mice was treated with 0.5% probucol (high probucol) and was fed the high-fat diet containing 1.25% cholesterol. The third group of 12 females and 7 males (high cholesterol control) was fed the high-fat diet with 1.25% cholesterol. After 6 weeks, 6 female mice from each group were killed and analyzed as described below. The remaining mice were treated for 13 weeks. During the intervention phase, 3 mice from the control group and 2 mice from the probucol group died. The size of each group at the end of the study is indicated in Table 2.TABLE 2.Body weights, plasma lipid and antioxidant concentrations in Experiments I–IIIPlasma Concentration at the End of the Intervention PeriodExpGroupSexnDaysBWOverall Cholesterol ExposureTCTGProbucolVitamin Egdays × mg/dl × 103mg/dlμMIControlF1311226.3 ± 1.2111.9± 5.2903 ± 6186 ± 18n.d.n.d.High probucolF1414226.7 ± 1.1103.4 ± 3.7750 ± 76159 ± 24aaP < 0.05270 ± 11n.d.IIControlM1511939.0 ± 2.3127.1 ± 22.71386 ± 109498 ± 88n.d.117 ± 9Low prob/vit EM1311845.3 ± 2.0aaP < 0.05127.2 ± 28.91496 ± 93546 ± 6990 ± 6161 ± 13aaP < 0.05Low probucolM1413942.6 ± 1.6127.4 ± 19.41019 ± 57aaP < 0.05337 ± 38158 ± 10ccP < 0.005 compared to the low dose probucol/vitamin E group.64 ± 4bbP < 0.005 compared to the control group of the same sex.High probucolM1218141.6 ± 3.1130.9 ± 8.3719 ± 46bbP < 0.005 compared to the control group of the same sex.604 ± 130497 ± 47ccP < 0.005 compared to the low dose probucol/vitamin E group.41 ± 4bbP < 0.005 compared to the control group of the same sex.IIILow chol. controlF109121.2 ± 0.757.7 ± 1.8589 ± 38171 ± 60054 ± 14M79133.2 ± 3.8ddP < 0.0565.9 ± 4.5854 ± 123551 ± 102ddP < 0.05083 ± 13High probucolF119124.4 ± 1.1aaP < 0.0556.3 ± 3.5635 ± 43179 ± 49343 ± 2832 ± 4aaP < 0.05M99130.7 ± 2.1ddP < 0.0557.7 ± 1.4650 ± 44255 ± 56aaP < 0.05463 ± 16eeP < 0.005 compared to the female mice of the same group31 ± 2bbP < 0.005 compared to the control group of the same sex.High chol. controlF119123.5 ± 1.492.6 ± 4.7bbP < 0.005 compared to the control group of the same sex.995 ± 101aaP < 0.05202 ± 53059 ± 5M69135.7 ± 1.5ddP < 0.05132.3 ± 10.7bbP < 0.005 compared to the control group of the same sex.,dcP < 0.005 compared to the low dose probucol/vitamin E group.1946 ± 145bbP < 0.005 compared to the control group of the same sex.,edP < 0.05686 ± 101ddP < 0.050116 ± 12eeP < 0.005 compared to the female mice of the same groupIn Experiments I and II, n = number of mice at the end of the intervention period. In Experiment III, n = the total number of mice in each group; 6 female mice were killed after 6 weeks, the rest after 13 weeks. Data represent mean ± SEM. F, female; M, male; Exp, experiment; BW, final body weight; TC, total plasma cholesterol; TG, plasma triglyceride; n.d., not determined.a aP < 0.05b bP < 0.005 compared to the control group of the same sex.c cP < 0.005 compared to the low dose probucol/vitamin E group.d dP < 0.05e eP < 0.005 compared to the female mice of the same group Open table in a new tab In Experiments I and II, n = number of mice at the end of the intervention period. In Experiment III, n = the total number of mice in each group; 6 female mice were killed after 6 weeks, the rest after 13 weeks. Data represent mean ± SEM. F, female; M, male; Exp, experiment; BW, final body weight; TC, total plasma cholesterol; TG, plasma triglyceride; n.d., not determined. Plasma cholesterol and triglyceride levels were determined at 3–4 week intervals, using an automated enzymatic assay (Boehringer Mannheim Diagnostics, Mannheim, Germany) (36Siedel J. Haegele E.O. Ziegenhorn J. Wahlefeld A.W. Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency.Clin. Chem. 1983; 29: 1075-1080Google Scholar). Blood samples for these assays were obtained from the retro-orbital plexus of anesthetized mice and were collected in heparinized tubes. Plasma high density lipoprotein (HDL)-cholesterol levels were determined by precipitating the very low density lipoprotein (VLDL) and LDL with 2 m MgCl2 and 5000 U/ml heparin (37Bachorik P.S. Wood P.D. Albers J.J. Steiner P. Dempsey M. Kuba K. Warnick R. Karlsson L. Plasma high-density lipoprotein cholesterol concentrations determined after removal of other lipoproteins by heparin/manganese precipitation or by ultracentrifugation.Clin. Chem. 1976; 22: 1828-1834Google Scholar) and then measuring the remaining plasma cholesterol as described above. The plasma concentration of probucol was determined either in terminal plasma samples pooled from 3–4 mice (Experiment I) or in individual samples (Experiments II and III), using an HPLC assay previously described (38Fruebis J. Steinberg D. Dresel H.A. Carew T.E. A comparison of the antiatherogenic effects of probucol and of a structural analog of probucol in low density lipoprotein receptor-deficient rabbits.J. Clin. Invest. 1994; 94: 392-398Google Scholar). In brief, plasma samples were extracted with methanol–acetone 3:2 after addition of internal standard, 2-pentanone-bis-(3,5-di-t-butyl-4-hydroxyphenyl) mercaptole. HPLC analysis was performed on a C18-reversed phase column. The samples were eluted with acetonitrile–heptane–0.1 m ammonium acetate 92:6:2 (vol/vol/vol), and probucol was measured by the absorbance at 254 nm. The plasma vitamin E levels were determined using a previously described HPLC method with a C18-reversed phase column (38Fruebis J. Steinberg D. Dresel H.A. Carew T.E. A comparison of the antiatherogenic effects of probucol and of a structural analog of probucol in low density lipoprotein receptor-deficient rabbits.J. Clin. Invest. 1994; 94: 392-398Google Scholar). Briefly, the vitamin E was extracted with heptane and α-tocopherol acetate was added as an internal standard. The mobile phase was acetonitrile–methylene chloride–methanol, 70: 20:10 (vol/vol/vol), and detection was by absorbance at 292 nm. Plasma lipoprotein profiles of control and probucol-treated mice were obtained by FPLC using a 50 cm Sepharose 6B column. A plasma aliquot of 100 μl was injected onto the column, and 250 μl fractions were collected and analyzed for cholesterol and triglyceride content, as described above. LDL (1.019 < d < 1.063 g/ml) was prepared from terminal plasma samples (pooled from 3–4 mice each) by sequential ultracentrifugation (30,000 rpm, 4°C, 14 h, using a
Probucol
Low-density lipoprotein
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Probucol
Low-density lipoprotein
Scavenger
Free radical scavenger
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Probucol
Mode of Action
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42 patients with acute cerebral stroke (average age 70, 83ą9, 95 years) were examined for fluctuations in the level of serum triglyceride, total cholesterol, HDL-cholesterot and LDL-cholesterol within 24 hours after the acute event and on the eight day after the onset of symptoms. Patients did not receive specific hipolipemic drugs during the treatment. On the first day average values of serum triglyceride were 1, 97ą0, 94 ; of total cholesterol 5, 99 ą1, 91 ; of HDL-cholesterol 0, 86ą0, 35 and of LDL-cholesterol 3, 91ą1, 63 mmol/L. On the eight day average values of triglyceride were 2, 26ą2, 31 ; of total cholesterol 4, 59ą1, 49 ; of HDL-cholesterol 1, 2ą0, 54 and of LDL-cholesterol 2, 91ą1, 17 mmol/L. The results revealed a statistically significant (p<0, 05) decrease in total cholesterol and LDL-cholesterol and statistically significaiit increase in HDL-cholesterol, while there was no statistically significant difference between levels of triglyceride. We. conclude that in patients with acute cerebral stroke, triglyceride, total cholesterol and LDL cholesterol is increased, due to stress and increased secretion of catecholamines. On the eight day there is decrease in serum levels of triglyceride, total cholesterol and LDL-cholesterol, while HDL-cholesterol is increased. These changes are probably due to dietary changes after stroke, besides the fact that stress inducing catecholamines overproduction is no long acting.
Stroke
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The nationally-recognized Susquehanna
Chorale will delight audiences of all
ages with a diverse mix of classic and
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performance - and all this while
working at an extremely high
musical level.AÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂA¢AÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂAÂA Experience choral
singing that will take you to new
heights!
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