Aging Aging

202 McEneny and Young16. Disposable cups, 20 mL (Sarstedt, cat. no. 73.1056).17. Nusonics ultrasonic cleaner (Quayl Dental, Worthing, Sussex).18. Copper chloride solution (40 µmol/L, BDH cat. no. 10088). This solution isstored at 4°C and is made in sufficient quantities for use in a series of experiments(see Note 3).19. Saline solution (0.9% NaCl: BDH cat. no. 10241AP).20. Decon-90 (4% solution; Decon Laboratories, E. Sussex).21. HCl (0.5 M made from 37%, Janssen cat. no. 12.463.47).22. Thermostatically controlled spectrophotometer (37°C, Hitachi U-2000-1, cat. no.HIT/121-0032), containing automatic six-cell positioner (Hitachi cat. no. HIT/121-0304) linked to a PC with software package for automatic handling of data(Hitachi, Enzyme Kinetic Data System).23. Microsoft Excel software package for data computation utilizing a specially writtenmacroprogram (15) (a copy of this macro is available from the authors).3. MethodsSeparation of the lipoprotein species is achieved by flotation nonequilibriumultracentrifugation. The method described is very rapid and requiresless than 2 h total preparation time (including ultracentrifugation) prior tooxidation.1. To a Beckman 3-mL ultracentrifuge tube containing 0.4451 g of KBr is added0.9 mL of heparinized plasma. This plasma is added from a pipet connected tofine-bore tubing (approx 2.5 cm in length and a diameter small enough to enterthe neck of the ultracentrifuge tube). The ultracentrifugation tube is temporarilysealed with parafilm, allowing gentle inversion to dissolve the salt. This solutionhas a resultant density of 1.30 g/mL.2. To this density-adjusted plasma, NaCl (d = 1.006 g/mL) is added, using the tubingand 21G needle connected to the peristaltic pump. The needle requires shapingto enable it to rest on the inside of the ultracentrifuge tube. Allow the solutionto gently run down the side onto the top of the density adjusted plasma (ensure nomixing or distortion of the layers occurs during this procedure).3. The ultracentrifugation tubes are sealed using the Beckman Tube Topper Sealer.They are gently placed in the Beckman rotor together with the Beckman spacers.Ultracentrifugation is performed using the following settings: 4°C, 100,000 rpm(541,000g) for 1 h. An acceleration and deceleration setting of 6 is chosen; totalultracentrifugation run time is 1 h and 6 min. The acceleration and decelerationparameters prevent disturbance of the density gradient created during ultracentrifugation.Note: During ultracentrifugation the following steps are performed.a. Preparation of columns: one column per sample is prepared (at 4°C) by washingthrough with 25 mL of PBS prior to use. These columns are designed toretain a reservoir of buffer within the gel bed. They do not run dry and may beleft unattended for short periods of time.