Southwestern. Medical. Center,. 5323 Harry ..... S. Rankin. SM. Role of oxidized low density lipoprotein in atherogenesis. Prog. Lipid. Res. 1992;31:127-43. 5.
CLIN. CHEM. 41/11,
Effect Anne
1628-1632
of Aging Schmuck,’
According
#{149}Lipids
J. Fuller,
of Low-Density
Sridevi
Treatment
Devaraj,
Education
Program,
elderly subjects. The LDL oxidation showed the two
groups
Panel
and
National risk factor for heart disease. To assess the relation between age and LDL oxidizability, we studied copper-mediated LDL oxidation in 13 healthy elderly subjects (>59 years) and 13 sex-matched healthy young controls (28 g (1 oz.)/day, lowering medication,
diabetes,
hypertension,
tion in the last 6 weeks before elderly individuals who responded flier, 13 fulfilled all the criteria study. All subjects gave informed donation. Weight to calculate
and body
angina
myocardial infarction or peripheral thyroid dysfunction, alcohol intake smoking, hormonal treatment, lipidor vitamin or iron supplementa-
height mass
were index
entry. From the 30 to the advertised to be entered in the consent before blood
determined (BMI).
for After
all an
subjects overnight
fast, 60 mL of blood was drawn into EDTA tubes (1 g/L) for LDL isolation. Glucose, creatinine, and lipid profile were determined on EDTA-plasma by standard laboratory techniques. Vitamins were measured in heparinized plasma. Samples for plasma ascorbate were deproteinized with 10% metaphosphoric acid, centrifuged, and the supernate was purged with nitrogen and stored at -18 #{176}C before analysis. Ascorbate concentrations were determined spectrophotometrically after derivatization with 2,4-dinitrophenylhydrazine (16). Plasma samples for a-tocopherol and (3-carotene determination were purged with nitrogen and frozen at -80 #{176}C until analysis. The measurements were done by reversed-phase HPLC after extraction with hexane
(17). Both plasma a-tocopherol and (3-carotene were standardized to total plasma lipids Plasma and LDL fatty acids were determined by gas-liquid chromatography after extraction and transmethylation as previously reported (19). An internal standard ofCl7:0 (NuChek Prep, Elysian, MN) was added to all samples. Total PUFA was calculated as the sum of 18:2, 18:3, and 20:4 fatty acids. LDL (d 1.019-1.063 kgfL) was isolated by preparative ultracentrifugation in NaCIINaBr solutions containing 1 gIL EDTA as previously described (20). The isolated LDL was dialyzed at 4 #{176}C against 10 L of 150 mmolIL NaC1, 1 mmolfL EDTA, pH 7.4, and then filtered and stored at 4 #{176}C under nitrogen. Protein was measured by the method of Lowry et al. standardized with bovine serum albumin. Oxidation studies were performed within 48 h of LDL isolation. LDL oxidation was undertaken after an overnight dialysis against 1 L of phosphate-buffered saline (PBS; 10 mmolIL phosphate, 150 mmolIL NaC1), pH 7.4, at 4 #{176}C. LDL (100 mg of protein per liter) was oxidized with 5 Mmol/L copper in PBS at 37 #{176}C (22). The oxidation was monitored over an 8-h period. Each time point was set up in duplicate. At 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, and 8 h, oxidation was stopped by refrigeration and the addition of 200 Mm0IJL EDTA and 40 MmolJL butylated hydroxytoluene. Three indices of oxidation were used to monitor LDL oxidation. The amount of conjugated dienes formed was determined by measuring the absorbance of the LDL at 234 nm The data were expressed as the increase in conjugated dienes over baseline (M234). The lipid hydroperoxide content was determined by a modification of the method of El-Saadani et al. (24). To 200 ML of supernate, 800 ML of cholesterol oxidaseiodide reagent was added. The tubes were vortexmixed, covered, and kept at room temperature for 1 h in the dark. The absorbance was read at 365 nm against a reagent blank. To obtain an index of protein modification, we measured the fluorescence of apolipoprotein B (apo B) by spectrofluorometry (excitation 360 nm, emission 430 nm) against a PBS blank (25). The kinetics of LDL oxidation were determined as previously described (26). The rate of LDL oxidation was determined from the propagation phase of the time-course curve by using a spline function. The lag phase was obtained by drawing a tangent to the slope of the propagation phase and extrapolating it to the horizontal axis. The lag time constitutes the interval from zero time to the intersection point. Analyses were performed with BMDP#{174}programs 3D and 2V (BMDP Statistical Software, Los Angeles, CA). The repeated-measures ANOVA was used to assess differences between groups for the kinetics of LDL (18).
(21),
tween age and oxidation statistical significance was
kinetics. P
