In vitro LDL oxidation studies
A volume of 3 ml plasma was centrifuged at d = 1.006
kg/l in an ultracentrifuge (Sorvall Ultra 80) at 40,000 rpm
using a T-865 rotor at 14jC for 10 h [18]. After ultracentrifugation
floating VLDL and chylomicrons were removed
and LDL was separated by precipitation from the solution
[19]. Forty microliters of 4% phosphotungstic acid in 1 M
NaOH was added, stirred and 10 Al of 2 M MgCl26H20
was added and centrifuged at 1500 g for 30 min at 4jC.
The supernatant was discarded and the precipitated LDL
was redissolved in 0.4 ml 0.5 M Na2CO3, kept in ice
overnight and dialyzed against three changes of PBS for
12 h.
Isolated LDL (200 Ag protein/ml) was preincubated with
50 Ag PF from the respective oils for 30 min. LDL oxidation
was initiated by adding 1.7 mM copper sulphate to the
reaction mixture [20]. The thiobarbituric acid reactive substance
(TBARS) was determined after 6 h by the procedure
described by Wills [21].