Aging Aging

200 McEneny and Youngoxidized, there is a second plateau period, which may be followed by a slightdecrease in absorption due to decomposition of lipid hydroperoxides.Because this technique involves isolation of LDL from aqueous phase chainbreaking antioxidants such as urate and ascorbate, the susceptibility of LDL tooxidation is influenced mainly by its composition and size (5). Increased antioxidantcontent, particularly of α-tocopherol and ubiquinol, favors increasedresistance to oxidation. The fatty acid composition is also important, with anincreased percentage content of monounsaturated fatty acids protecting againstoxidation and an increased content of polyunsaturated fatty acids decreasingresistance to oxidation. An increase in the cholesteryl ester to cholesterol ratioalso results in increased susceptibility to oxidation, as does a preponderance ofsmall dense LDL particles.Increased susceptibility of LDL to oxidation is a feature of many of thedegenerative diseases associated with aging. In healthy elderly men lag timesare reduced in comparison with younger subjects (6). Increased LDL oxidationis also a feature of established coronary artery disease, peripheral vascular disease,diabetes mellitus, and stroke (7–10). Oxidation of LDL may contribute toimpaired vascular endothelial reactivity in old age (11), and hence contributeto the development of both myocardial ischemia and cerebrovascular disease.Various interventions have been shown to improve the resistance of LDL tooxidation, including antioxidant supplements and change to a diet rich inmonounsaturated fatty acids (12,13). However, as yet there are no prospectivestudies showing that a reduced lag time is an independent risk factor for thedevelopment of vascular disease.The assay as originally described by Esterbauer involved a prolonged ultracentrifugationstep (up to 24 h) to isolate LDL from EDTA plasma, followed bydialysis to remove potassium bromide, EDTA, and small chain breaking antioxidantssuch as urate and ascorbate. In total, the procedure required approx48 h, and artifactual oxidation and loss of endogenous antioxidants from LDLwas therefore a problem (14). The modified procedure as described here has anumber of significant advantages. First, a rapid ultracentrifugation protocol usinga bench top ultracentrifuge allows the isolation of LDL in 1 h. Second, LDL ispurified by size-exclusion chromatography on a Sephadex column, obviatingthe need for prolonged dialysis. Third, the measurement of the lag time prior tothe onset of oxidation and the calculation of rate of propagation has been automated,removing any possibility of subjective bias in determining results.2. MaterialsMetal ion contamination in buffers and solutions used in the extraction andpurification of LDL can initiate oxidation prematurely. To minimize their presenceall solutions are made using Millipore quality water.