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Problems with the measurement of apolipoproteins AI and AII
1Department of Pathology, University of Louisville, KY.
Insights
Accurate measurement of high-density lipoprotein (HDL) apolipoproteins is crucial for assessing coronary artery disease (CAD) risk. Pretreatment with guanidine effectively enhances apolipoprotein AI and AII assays for improved clinical use.
Area of Science:
- Cardiovascular Research
- Biochemistry
- Clinical Diagnostics
Background:
- Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels are linked to coronary artery disease (CAD), but are insensitive risk markers.
- Subcomponents of HDL, particularly apolipoproteins (APOs), may offer more sensitive CAD risk indicators.
- Accurate measurement of APOs is challenging due to lipid interference obscuring immunoreactivity.
Purpose of the Study:
- To investigate methods for improving the accurate measurement of HDL apolipoproteins, specifically APO AI and APO AII.
- To evaluate the effectiveness of denaturing agents, such as guanidine, in enhancing apolipoprotein immunoreactivity for assays.
Main Methods:
- Utilized guanidine as a denaturing agent to probe HDL particles and assess apolipoprotein immunoreactivity.
- Investigated the effect of varying guanidine concentrations on APO AI and APO AII exposure in an immunoassay system.
- Compared the immunoreactivity of APO AII to APO AI under different treatment conditions.
Main Results:
- APO AII immunoreactivity increased up to 50% with 4M guanidine treatment when diluted to <50 mM in the assay.
- High guanidine concentrations previously inhibited antibody-antigen reactions, masking APO AII exposure.
- Current apolipoprotein assays may not optimally expose antigenic sites, but guanidine pretreatment shows promise.
Conclusions:
- Pretreatment with guanidine is a potentially simple and effective method to optimize APO AI and APO AII assays.
- Further research is needed to determine which apolipoprotein assays are adequately designed for optimal antigenic site exposure.
- Optimized apolipoprotein assays could lead to more sensitive detection of CAD risk.
Abstract:
There is considerable evidence demonstrating that increased levels of low density lipoprotein (LDL) cholesterol and decreased levels of high density lipoprotein (HDL) cholesterol are associated with coronary artery disease (CAD). Yet, these lipoprotein markers are insensitive for identifying risk of CAD. Measurement of subcomponents of HDL may offer more sensitive markers. Investigators have focused on protein components (apolipoproteins) of HDL as a potentially important marker. Unfortunately, because much of the immunoreactivity of apolipoproteins is hidden as a result of an association with lipids, it is difficult to measure them accurately. Detergents and other denaturing agents have been used to expose immunoreactivity. Poor correlations among different methods suggest that some detergents presently used may not be adequate for effective measurement of apolipoprotein (APO) AI. Studies using denaturing agents to probe HDL particles indicate that APO AII immunoreactivity is more resistant to exposure than that of APO AI. Data presented here indicate that the immunoreactivity of APO AII can be increased up to 50 percent by treatment with 4M guanidine provided the concentration of guanidine is diluted to less than 50 mM in the assay system. Previous studies failed to notice this effect because high levels of guanidine inhibited the antibody-antigen reaction in the immunoassay, making it appear that APO AII had not been exposed. It is concluded that with our present state of knowledge, it is unclear which, if any apolipoprotein assays, are adequately designed to achieve optimal exposure of antigenic sites, but that pretreatment with guanidine may be a simple, effective way to optimize APO AI and APO AII assays for clinical purposes.
