Development and Clinical Application of an Enzyme-Linked Immunosorbent Assay for Oxidized High-Density Lipoprotein

Aims: HDL particles have various anti-atherogenic functions, whereas HDL from atherosclerotic patients was demonstrated to be dysfunctional. One possible mechanism for the formation of dysfunctional HDL is the oxidation of its components. However, oxidized HDLs (Ox-HDLs) remain to be well investigated due to lack of reliable assay systems. Methods: We have developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) for Ox-HDL by using the FOH1a/DLH3 antibody, which can specifically recognize oxidized phosphatidylcholine, a major component of HDL phospholipid (HDL-PL). We defined forced oxidation of 1 mg/L HDL-PL as 1 U/L Ox-HDL. We assessed serum Ox-HDL levels of normolipidemic healthy subjects (n = 94) and dyslipidemic patients (n = 177). Results: The coefficients of variation of within-run and between-run assays were 12.5% and 13.5%. In healthy subjects, serum Ox-HDL levels were 28.5 +/- 5.0 (mean +/- SD) U/L. As Ox-HDL levels were moderately correlated with HDL-PL (r= 0.59), we also evaluated the Ox-HDL/HDL-PL ratio, which represents the proportion of oxidized phospholipids in HDL particles. In dyslipidemic patients, Ox-HDL levels were highly variable and ranged from 7.2 to 62.1U/L, and were extremely high (50.4 +/- 13.3U/L) especially in patients with hyperalphalipoproteinemia due to cholesteryl ester transfer protein deficiency. Regarding patients with familial hypercholesterolemia, those treated with probucol, which is a potent anti-oxidative and anti-hyperlipidemic drug, showed significantly lower Ox-HDL (16.2 +/- 5.8 vs. 30.2 +/- 5.4, p<0.001) and Ox-HDL/HDL-PL ratios (0.200 +/- 0.035 vs. 0.229 +/- 0.031, p= 0.015) than those without probucol. Conclusion: We have established a novel sandwich ELISA for Ox-HDL, which might be a useful and easy strategy to evaluate HDL functionality, although the comparison study between this Ox-HDL ELISA and the assay of HDL cholesterol efflux capacity remains to be done. Our results indicated that probucol treatment may be associated with lower Ox-HDL levels.

Automated summary

A novel sandwich ELISA for Ox-HDL was developed to evaluate HDL functionality, with healthy subjects having levels of 28.5 ± 5.0 U/L and dyslipidemic patients showing higher levels, especially in patients with hyperalphalipoproteinemia due to cholesteryl ester transfer protein deficiency. Probucol treatment was associated with significantly lower Ox-HDL levels in patients with familial hypercholesterolemia.