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Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein
Published on: October 12, 2017
Development and Clinical Application of an Enzyme-Linked Immunosorbent Assay for Oxidized High-Density Lipoprotein
Takeshi Okada1,2, Mizuki Sumida3, Tohru Ohama1,4
1Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine.
Insights
A new assay measures oxidized high-density lipoprotein (Ox-HDL), revealing higher levels in dyslipidemic patients. Probucol treatment was associated with reduced Ox-HDL levels, suggesting a potential therapeutic target.
Area of Science:
- Cardiovascular Science
- Biochemistry
- Clinical Chemistry
Background:
- High-density lipoprotein (HDL) possesses anti-atherogenic properties.
- Dysfunctional HDL, often observed in atherosclerotic patients, may result from component oxidation.
- Investigating oxidized HDL (Ox-HDL) has been hindered by a lack of reliable assay systems.
Purpose of the Study:
- To develop and validate a novel sandwich enzyme-linked immunosorbent assay (ELISA) for quantifying oxidized HDL (Ox-HDL).
- To assess serum Ox-HDL levels in healthy individuals and patients with dyslipidemia.
- To explore the relationship between Ox-HDL levels and specific dyslipidemias and the effect of probucol treatment.
Main Methods:
- A sandwich ELISA was developed using antibodies specific to oxidized phosphatidylcholine in HDL phospholipids (HDL-PL).
- Forced oxidation defined the unit of measurement: 1 U/L Ox-HDL equals 1 mg/L oxidized HDL-PL.
- Serum Ox-HDL levels were measured in 94 healthy subjects and 177 dyslipidemic patients.
Main Results:
- The developed Ox-HDL ELISA demonstrated good assay precision (CVs 12.5-13.5%).
- Serum Ox-HDL levels were elevated in dyslipidemic patients (50.4±13.3 U/L) compared to healthy subjects (28.5±5.0 U/L).
- Patients with familial hypercholesterolemia treated with probucol showed significantly lower Ox-HDL and Ox-HDL/HDL-PL ratios compared to untreated patients.
Conclusions:
- A novel and reliable sandwich ELISA for Ox-HDL has been established, offering a potential tool for assessing HDL functionality.
- Elevated Ox-HDL levels are associated with dyslipidemia, particularly hyperalphalipoproteinemia.
- Probucol treatment appears to reduce Ox-HDL levels, warranting further investigation into its clinical implications for cardiovascular health.
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.
