Aging Aging

206 McEneny and Youngeffect have been utilized. When the propagation phase is complete a plateauof maximum absorbance is reached.9. Data analysis: See Fig. 2B. Determination of lag time using the mathematicalprogram removes subjective error which may occur if determined manually. Thisis calculated using a specially written macroprogram after the results obtained onthe PC are converted into ASCII file format and imported into the spreadsheetprogram Excel. The lag time is calculated as the intercept between the line ofmaximum slope of the propagation phase and the baseline where absorbance wasat time = zero (see Note 6).4. Notes1. Heparinized plasma is used in preference to serum purely for logistic reasons, asserum collection lengthens total preparation time. However, this technique maybe applied equally well to serum samples with no detectable difference in lagtime when compared to heparinized plasma.2. Protein standards and protein estimation: Stock BSA (25 µg/mL) is prepared byplacing 12.5 mg of BSA into a 500-mL beaker. Add approx 300 mL of deionizedwater and gently stir (care is required as vigorous mixing can cause BSA to foamand make it difficult to bring to the correct volume). Add this solution to a 500-mLvolumetric flask and rinse the beaker with remaining water to bring to correctvolume. Aliquot 7 mL of this solution into 10-mL tubes, cap, and freeze at –20°C.This solution is stable for up to 6 mo.Working standard solutions (0, 2.5, 10, 15, 20 µg/mL) are prepared from thestock BSA. These together with the samples are diluted with distilled water to afinal volume of 1.2 mL and prepared in duplicate in the 4-mL tubes. Threehundred microliters of Bio-Rad dye reagent is then added, making a final volume1.5 mL. The standards and samples are then gently inverted and absorbance at595 nm recorded within 5–60 min. The PD10 LDL is made up to 1.2 mL using0.1 mL of sample and 1.1 mL of distilled water. If the protein concentration ofcrude LDL is required use only 10 µL and dilute to 1.2 mL with water (dilutionfactor for crude LDL is 150).3. Copper chloride solution: The working solution of copper chloride is 40 µM. Thissolution is made by serial dilution of a 33.2 mM stock solution.a. Solution A: 33.2 mM — 0.567 g of CuCl 2·2H 2 O made in a 100-mL volumetricflask using deionized water.b. Solution B: 332 µM —1 mL of solution A is added to a 100-mL volumetricflask and the volume is made up using saline solution.c. Solution C: 40 µM working solution — 6.024 mL of solution B is added to a50-mL volumetric flask and the volume made up using either PBS or salinesolution (addition of PBS in the earlier solutions causes precipitation).4. Preparation of quartz cells: New or used cells must undergo the following procedurebefore use.Each cell is rinsed at least 10× with distilled water. They are then placed into aseparate disposable cup containing sufficient 4% Decon–90 to cover them. The