Aging Aging

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Susceptibility of LDL to Oxidation 207cups are placed in the sonic bath and sonicated for 5 min. The cells are theninverted (not the cups), while trying to retain as much solution as possible withinthe cells. They are then sonicated again for 5 min, removed, rinsed with distilledwater (10×), and placed in 0.5 M HCl for at least 1 h prior to use/reuse.Before use in the oxidation experiment the acid-soaked cells must be thoroughlycleaned. The cells are rinsed with distilled water (10×), placed into cleancups containing water, and sonicated for 5 min, before again inverting and sonicatingfor a further 5 min. The cells are then allowed to drain on absorbent paperand are now ready for use.The reason for this rigorous cleaning regime is because oxidation of LDL produceslipoperoxides which may adhere to the side of the cells. These seed theoxidation reaction and would artefactually increase the rate of oxidation in thesample being examined. In our hands this rigorous cleaning procedure is essentialif reproducible results are to be obtained.5. A worked example of the formula is shown here. If the concentration of LDLprotein is 157 mg/mL, determined from the standard curve X dilution factor, thevolumes of LDL (Y) and PBS (Z) are as follows:Y = 50/157 × 1000 = 318 µL of LDLAs the LDL is made up to a final volume of 1000 µL the volume of PBS required,taking into account the 50 µL of copper to be added, isZ = 1000 – (318 µL of LDL + 50 µL of Cu) = 632 µL of PBSTherefore to the cell is added 318 µL of LDL + 50 µL of Cu + 632 µL of PBS. Thefinal volume is 1000 µL.6. The lag time is determined by taking the point of maximum slope over an intervalof 20 min and fitting a line by the least squares method. The point of maximumslope was found by repeated computation of the average slope for 11 consecutivepoints moving along 1 point at a time, enabling deviations due to random noisefrom the spectrophotometer to be removed.References1. Witztum, J. L. (1993) Susceptibility of low-density lipoprotein to oxidative modification.Am. J. Med. 94, 347–356.2. Heinecke, J. W. (1997) Mechanisms of oxidative damage of low density lipoproteinin human atherosclerosis. Curr. Opin. Lipidol. 8, 268–274.3. Westhuyzen, J. (1997) The oxidation hypothesis of atherosclerosis: an update. Ann.Clin. Lab. Sci. 27, 1–10.4. Esterbauer, H., Striegl, G., Puhl, H., and Rotheneder, M. (1989) Continuous monitoringof in vitro oxidation of human low-density lipoprotein. Free Radic. Res.Commun. 6(1), 67–75.5. Esterbauer, H., Gebicki, J., Puhl, H., and Jurgens, G. (1992) The role of lipidperoxidationand antioxidants in oxidative modification of LDL. Free Radic. Biol.Med. 13(4), 341–390.
208 McEneny and Young6. Napoli, C., Abete, P., Corso, G., Malorni, A., Postiglione, A., Ambrosio, G.,Cacciatore, F., Rengo, F., and Palumbo, G. (1997) Increased low-density lipoproteinperoxidation in elderly men. Coron. Art. Dis. 8, 129–136.7. Karmansky, I., Shnaider, H., Palant, A., and Greuner, N. (1996) Plasma lipid oxidationand susceptibility of low density lipoproteins to oxidation in male patients withstable coronary artery disease. Clin. Biochem. 29, 573–579.8. Iribarren, C., Folsom, A. R., Jacobs, D. R., Gross, M. D., Belcher, J. D., Eckfeldt,J. H. (1997) Association of serum vitamin levels, LDL susceptibility to oxidation,and autoantibodies against MDA-LDL with carotid atherosclerosis — a case-controlstudy. Arterioscler. Thromb. Vasc. Biol. 17, 1171–1177.9. Yoshida, H., Ishikawa, T., and Nakamura, H. (1997) Vitamin E lipid peroxide ratioand susceptibility of LDL to oxidative modification in non-insulin-dependent diabetesmellitus. Arterioscler. Thromb. Vasc. Biol. 17, 1438–1446.10. Palinski, W. and Horkko, S. (1997) Biological consequences of oxidation in atherosclerosisand other pathologies. Lipoprotein oxidation in brain arteries. Nutr. Metab.Cardiovasc. Dis. 7, 237–247.11. Khder, Y., Bray, L., Boscs, D., Aliot, E., and Zannad, F. (1996) Endothelial, viscoelasticand sympathetic factors contributing to the arterial wall changes duringageing. Cardiol. Elderly 4, 161–165.12. deWaart, F. G., Moser, U., and Kok, F. J. (1997) Vitamin E supplementation in elderlylowers the oxidation rate of linoleic acid in LDL. Atherosclerosis 133, 255–263.13. Parthasarathy, S., Grasse, B. J., Miller, E., Almazan, F., Khoo, J. C., Steinberg, D.,and Witztum, J. L. (1991) Using an oleate-rich diet to reduce susceptibility of lowdensitylipoprotein modification in humans. Am. J. Clin. Nutr. 54, 701–706.14. Scheek, L. M., Wiseman, S. A., Tijburg, L. M., and Vantol, A. (1995) Dialysis ofisolated low density lipoprotein induces a loss of lipophilic antioxidants and increasesthe susceptibility to oxidation in vitro. Atherosclerosis 117, 139–144.15. McDowell, I. F., McEneny, J., Trimble, E. R. (1995) A rapid method for measurementof the susceptibility to oxidation of low-density-lipoprotein. Ann. Clin.Biochem. 32, 167–174.
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Understanding Aging 9the intestine
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Understanding Aging 117. Do long-li
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Understanding Aging 172. Pearl, R.
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Understanding Aging 1945. Perovic,
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24 Cristofalo, Volker, and Allenmen
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60 Pawelection, the VERUM Foundatio
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Telomeres and Replicative Senescenc
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Detection of Molecular Events 715De
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Detection of Molecular Events 772.
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Detection of Molecular Events 79Fig
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86 Kirk and Millerexperiments on un
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88 Kirk and MillerFig. 1. (A) Analy
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98 Engel, Adibzadeh, and PawelecPCR
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Xenobiotic-Metabolizing Enzymes 119
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Assessing Antioxidant Status 1339As
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Assessing Antioxidant Status 135Fig
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Assessing Antioxidant Status 137Tab
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Assessing Antioxidant Status 139add
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Assessing Antioxidant Status 14121.
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Comet Assay of DNA Damage 14310Meas
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Comet Assay of DNA Damage 145contro
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Comet Assay of DNA Damage 1476. Lys
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Page 176 and 177: 190 HouThe HPRT mutational spectrum
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Page 194 and 195: Analysis of Pentosidine 20916Measur
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Page 204 and 205: 222 Miquellipoperoxides and malonal
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Mitochondrial DNA Mutations 261X-10
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Mitochondrial DNA Mutations 26315.
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Mitochondrial DNA Mutations 26520An
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Mitochondrial DNA Mutations 269muta
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Mitochondrial DNA Mutations 2717. P
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Mitochondrial DNA Mutations 2733.2.
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Mitochondrial DNA Mutations 275that
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Mitochondrial DNA Mutations 27711.
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NK Cell Function in Aging 31123Age-
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NK Cell Function in Aging 3199mL of
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Immunogenetics and Life-Span 349to
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Genetically Engineered Mice 36126Th
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Aging Aging

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