The objectives of this study were to identify the subcellular fraction responsible for fatty acid synthesis in rabbit aorta and to determine the effect of cholesterol feeding on the system. A method for homogenization of aorta is described which permitted the isolation of subcellular components of aorta, including mitochondria that were morphologically and functionally intact. Mitochondria were identified as the major site of fatty acid synthesis in this tissue. Cofactor requirements and products showed that the synthetic system operates by chain elongation. Mitochondria from atherosclerotic aortas incorporated acetate into fatty acids faster than did mitochondria from control aortas. It is concluded that cholesterol feeding leads to alterations of aortic mitochondrial function and accelerates the fatty acid elongation pathway.

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