Aging Aging

204 McEneny and Young5. Size-exclusion chromatography for desalting crude LDL using the PD10 column.Crude LDL is contaminated with KBr. The sample also contains small moleculessuch as urate that can alter its susceptibility to oxidation. Desalting is performedas follows: 0.5 mL of crude LDL is added to the prewashed PD10 columns; this isallowed to enter the gel bed. Two milliliters of PBS is then added and also allowedto enter the gel bed. Both these eluants are discarded. The LDL is then obtainedby adding a second 2-mL aliquot of PBS and this eluant is collected into a 10-mLtube. The LDL sample is now termed “desalted” (PD10) LDL and is placed onice until the sample is ready to be oxidized after protein determination. (Aftercollection of the LDL sample start preparing the columns for reuse by washingwith PBS before sealing).Note: Subjecting LDL to column chromatography renders it less stable. CrudeLDL can be stored at 4°C for up to 2.5 h with little deterioration of lag time.However, PD10-treated LDL must be used as rapidly as possible. It is thereforeimportant to prepare all stages post PD10 treatment in advance while ultracentrifugationis being performed (e.g., quartz cells and protein assay).6. Protein determination and standardization of PD10 LDL: LDL protein is readagainst the prepared BSA standard curve. The dilution of LDL is taken intoaccount by multiplying the figure obtained by a factor of 15, as 100 µL of PD10LDL is added to 1100 µL of water plus 300 µL of Bio-Rad dye reagent, giving afinal volume of 1500 µL and thus a dilution of 15. The LDL is then standardizedto 50 µg/mL of protein with PBS, taking into account the volume of copper solutionadded. The final volume of each cuvet is 1000 µL.The volume of LDL (Y) and PBS (Z) required can be obtained using thefollowing formula:Y µL of LDL = 50/X µg/mL LDL × 1000Z = 1000 µL –(Y µL of LDL + 50 µL Cu 2+ )where X is the concentration of protein determined from the standard curve × 15,Y is the volume of LDL required in the cell to give a final concentration of50 µg/mL when the volume is made up to 1000 µL, and Z is the volume of PBSrequired to dilute the LDL to the required concentration (see Note 5).Dilution of the LDL sample is performed in the quartz cuvet. The copper chloridesolution is added after LDL and PBS. (The action of the pipet is used to mixthe copper solution with the LDL/PBS mixture by expelling and drawing thecopper solution back and forth several times. In our experience this procedure ismore effective than cell inversion). The samples are then placed into the spectrophotometerand data collection initiated.7. Oxidation of the sample is followed in the thermostatically controlled spectrophotometer.Change in absorbance is followed at 234 nm and recorded every2 min. The change in absorbance is recorded on a Nimbus PC-386 utilizing theEnzyme Kinetic Data System software package. This automated system removesthe need for lengthy manual recording.8. Oxidation profile: A typical profile is shown in Fig. 2A, and has two distinct phases: