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Effects of a "tall oil" -derived phytosterol mixture on the development of atherosclerotic lesions in apo E-deficient mice

University of British Columbia

Background: The effects of a phytosterol mixture (FCP-3PI) on the plasma cholesterol concentrations and development of atherogenic lesions have been evaluated in apo E-knockout (apo E-KO) mice using a number of biochemical and histological methods. In addition, the systemic effects and the tolerance to FCP-3PI have also been tested in this animal model. FCP-3PI, which is composed of S-sitosterol (69%), campesterol (15%) and sitostanol (16%), was extracted from "tall oil" soap, a by-product of the pulp and paper industry. The degree of purity of the final product was approximately 95% as assessed by gas chromatography. Objectives:The objectives of this thesis were: 1) to determine the effects of dietary supplementation with FCP-3PI on plasma lipid concentrations in apo E-KO mice; 2) to evaluate the cholesterol-lowering properties of FCP-3PI in wild-type normolipidemic mice; 3) to evaluate the effects of FCP-3PI on the quality and extent of atherosclerotic lesions in apo E-KO mice; 4) to compare and contrast cholesterol-lowering and anti-atherogenic effects of FCP-3PI to those of probucol, a well-known lipid lowering agent with antioxidant properties; 5) to test systemic effects of FCP-3PI following its parenteral administration in apo E-KO mice; and finally, to investigate the tolerance of apo E-KO mice to FCP-3PI and its safety when administered over the long-term. Results: Atherosclerosis progression experiments revealed that addition of 2% (w/w) FCP-3PI to a typical "Western" diet resulted in a significant reduction in average total plasma cholesterol concentrations in the treated animals compared to controls. This was accompanied by a significant (p<0.0001) reduction in the average lesion area in the treated animals. This reduced lesion area in the aortic sinuses was accompanied by a substantial reduction in all lesional components, reflecting a delay in the progression of atheromatous changes. The lesion size was strongly correlated with the average plasma total cholesterol concentrations (r=0.69). Cholesterol lowering effects of FCP-3PI were tested in a number of male CD1 mice in the presence or absence of additional dietary cholesterol. FCP-3PI did not significantly reduce plasma total cholesterol in these normolipidemic mice. The lack of cholesterol-lowering effects of FCP-3PI may be due to limited cholesterol absorption in normolipidemic mice. The next experiment was carried out to investigate possible mechanisms of FCP-3PI effects on cholesterol metabolism and atherosclerotic lesion development, and then to compare FCP-3PI effects to those of probucol in apo E-deficient mice. The cholesterol-lowering and anti-atherogenic activities of FCP- 3PI were accompanied by significant alterations in several other features which may be directly/indirectly involved in atherogenesis. Thus, FCP-3PI treatment caused a significant increase in the activity of hepatic HMG-CoA reductase and to a lesser extent in the activity of hepatic cholesterol 7 -hydroxylase. These changes were associated with a significant decrease in hepatic cholesterol content and a 50% increase in fecal cholesterol excretion compared to controls. Hepatic lipase activity was also significantly reduced by FCP-3PI treatment. In addition, FCP-3PI caused a 20% decrease in plasma fibrinogen concentrations. Unlike FCP-3PI, probucol caused a marked decrease in plasma total, VLDL-, LDL-, and HDL-cholesterol concentrations which was associated with a significant increase in atherosclerotic lesion size in the aortic roots of the mice. Probucol also caused a significant increase in plasma fibrinogen concentration compared to controls. These changes were associated with a significant increase in plasma antioxidant enzyme activities. In addition, probucol, unlike FCP-3PI, reduced hepatic LDL receptor binding to two-thirds of that in controls. The activity of both hepatic HMG-CoA reductase and cholesterol 7 hydroxylase was increased in the probucol-treated animals compared to controls. The atherosclerosis regression study revealed no evidence for regression of pre-established atherosclerotic lesions in the aortic roots of the mice fed regular mouse chow supplemented with 2% (w/w) FCP-3PI for 25 weeks. Intraperitoneal injection of FCP-3PI into apo E-KO mice resulted in a significant reduction in the activity of hepatic HMG-CoA reductase, a nonsignificant reduction in plasma total cholesterol concentration and a significant increase in the activity of plasma antioxidant enzymes as compared to controls. Finally, parallel to the first experiment, tolerance of orally administered FCP-3PI was evaluated in apo E-deficient mice. Histological examination revealed no abnormality in tissues examined except for a certain degree of testicular atrophy. FCP-3PI treatment prevented the development of cutaneous xanthomatosis. Urinalysis and hematological data were comparable between the control and FCP-3PI-treated animals except for a significant decrease in platelet count in the treated group. The erythrocytes of the treated mice showed a decreased susceptibility to hypotonic lysis in vitro. Conclusions: We have demonstrated that FCP-3PI has cholesterollowering and anti-atherogenic effects in apo E-KO mice. Moreover, we have demonstrated, for the first time, the effectiveness of FCP-3PI in preventing cutaneous xanthomatosis and retarding the development of atherosclerotic lesions in this animal model. Our data suggest that these effects of FCP-3PI may be mediated through a decrease in plasma VLDL-cholesterol, an increase in fecal cholesterol excretion (most likely due to decreasing cholesterol reabsorption and increasing biliary cholesterol excretion), a decrease in hepatic lipase activity and decrease in plasma fibrinogen concentrations. The lack of toxicity and its abundance in nature along with its low cost make potential use of FCP-3PI in prevention and treatment of human hypercholesterolemia attractive.

Thesis/Dissertation

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2009-06-02

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http://hdl.handle.net/2429/8597

Pathology

University of British Columbia

UBCV

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