Lipoprotein fractionation in deuterium oxide gradients: a procedure for evaluation of antioxidant binding and susceptibility to oxidation.

Hemin binding and oxidation of lipoproteins in serum: mechanisms and effect on the interaction of LDL with human macrophages

“…Once serum was obtained it was made 0.1 mg/ml in Na 2 -EDTA. LDL was prepared by density gradient or differential centrifugation using D 2 O buffers, containing 0.1 mg/ml Na 2 -EDTA, to raise the serum density to 1.019 and 1.063 g/ ml as described (32). This procedure allows fractionation of VLDL and LDL and its direct use in oxidation experiments without need for dialysis.…”

“…When HDL was prepared, the density of serum was raised by addition of solid KBr. Density gradient centrifugations to separate VLDL, LDL, HDL, and lipoprotein-free serum were performed as described (32)(33)(34). For the cell culture experiments LDL was labeled with 125 I.…”

“…The unbound radioactive iodine was eliminated by gel filtration (6). The methods for protein and cholesterol measurements were described previously (32).…”

“…Electrophoresis of LDL and serum lipoproteins was performed in a Bio-Rad 192 cell (Hercules, CA) in the "submarine mode" as described (32). Increasing concentrations of hemin and protoporphyrin IX (0, 10, 20, 40, 80, and 100 mol/L) final concentrations were added to 2-ml serum samples diluted to 20% with PBS that contained 10 mol/L BHT and 0.2 mg/ml EDTA.…”

“…After incubation for 30 min at room temperature, 10-l aliquots were used for electrophoresis. The remaining diluted serum was immediately fractionated using differential centrifugation in D 2 O solutions to isolate LDL in a TLA 120.2 rotor of a Beckman tabletop ultracentrifuge (Fullerton, CA) (32). Aliquots (10 l) of the floated LDL containing 0.5 mg protein/ml were used for electrophoresis.…”