Simpler technique for measuring oxidative susceptibility by heparin affinity column isolation of lipoproteins

Variations in the in vitro oxidative susceptibility or resistance of lipoproteins have been used to test the effect of ingested antioxidants and may prove to be a marker for coronary artery disease. Here we describe a simple technique for isolating and oxidizing beta-lipoproteins that may have utility in the clinical laboratory. Electrophoretic profiles showed that beta-lipoproteins were separated from alpha-lipoproteins and essentially from most other serum proteins using heparin affinity columns. Lipoproteins were normalized in the reaction mixture by measuring apo B in the beta-lipoprotein eluate using an automated apo B method, which gave good recoveries of 106-112%. Copper mediated oxidation was monitored by measurement of conjugated dienes formation at 234 nm for 300 min. When the reaction mixture included beta-lipoprotein eluate containing 0.03 g/L of apoB and 5 micromol/L copper sulfate, conditions were effective for obtaining complete oxidation while allowing reproducible measurements, with between day coefficients of variation of 2.35%, 14.6%, and 10.5% for lag, propagation and plateau phases, respectively. Beta-lipoproteins isolated from serum were more susceptible to oxidation than beta-lipoproteins from plasma apparently due to inhibition of oxidation by fibrinogen which co-eluted with beta-lipoprotein from plasma. For this reason, we recommend using serum preserved with EDTA for this assay.