By Michael Sanfilippo (M.S.)
The remnant lipoprotein-cholesterol (RLP-C) assay is a quantitative immunoseparation assay performed on human serum or plasma, for use in cardiovascular risk assessment and disease management. The RLP-C assay is recently FDA-cleared as an aid in the assessment of coronary artery disease, in addition to its initial clearance as an aid in the diagnosis of type III familial dyslipidemia. Further studies show that elevated RLP-C is an independent factor in detecting and predicting cardiac events in individuals with normal cholesterol and triglyceride levels. The assay can also be used to monitor the efficacy of treatment therapies.
Physiology
Plasma triglycerides are contained predominantly in two major lipoprotein classes: very low density lipoprotein (VLDL-primarily Apo B-100) and chylomicrons (CM-primarily Apo B-48). Remnant lipoproteins (RLPs) are produced from chylomicrons (intestine) and VLDL (liver) by the action of lipoprotein lipase (LPL). There is a certain amount of apolipoprotein exchange among the different lipoprotein classes during the formation of smaller and more dense particles, RLPs. The remnants are particularly rich in Apolipoprotein E which is critical for removal of atherogenic remnants from the circulation by receptor mechanisms. The Apo E serves as a ligand for the uptake of RLP by vascular wall cells as part of the scavenger receptor process. Substantial evidence indicates that these remnants are atherogenic. The newly formed RLP particles are depleted of triglycerides, phospholipids, and Apo A and C and are enriched in cholesteryl esters and Apo E – which are believed to be more atherogenic than the larger triglyceride rich lipoproteins. Pro-atherogenic properties of RLP particles are: the ability to inhibit endothelium-dependent vasorelaxation; their uptake up by macrophages without modifications; and their role in enhanced platelet aggregation.
Clinical Observance
The relationship between certain lipoprotein metabolism disorders and the risk of coronary heart disease (CHD) is well established. Several well-recognized lipoproteins, such as LDL, HDL, Lp(a) and remnant lipoproteins, contribute independently or synergistically to the development of CHD. The RLP-Cholesterol Assay provides a means to measure one of these lipoproteins, namely remnant lipoproteins (RLP-C). Depending upon the patient and the assay’s use, the results are interpreted in slightly different ways.
To aid in the assessment of CHD risk, the RLP-Cholesterol Assay is used in addition to clinical evaluation, patient risk assessment and other lipoprotein tests, such as LDL and HDL for individuals who have triglyceride concentrations <800 mg/dL. To diagnose familial type III hyperlipoproteinemia, the test results are used in combination with total serum triglyceride (TG) and total cholesterol in individuals who have total cholesterol concentrations >200 mg/dL and triglyceride concentrations between 200 and 800 mg/dL.
During the past 20 years, evidence has accumulated to suggest that remnant lipoprotein particles are the atherogenic component of triglyceride rich lipoproteins. These particles remain in circulation for extended periods of time post prandially – even in individuals with normal fasting triglyceride levels.
In the U.S., increased RLP-C levels were found in normolipidemic patients with angiographically defined coronary artery disease. This and numerous other studies suggest that RLP-C analysis may aid in detecting and predicting cardiac events in individuals with elevated RLP-C and normal cholesterol and triglyceride levels.
Methodology
Remnant lipoproteins are extremely heterogeneous in size, hydrated density, chemical composition and charge. As a result of this, RLP measurement has been challenging.
A novel immunoseparation assay has been developed for measuring RLP-cholesterol concentrations based on both the lipoprotein content and immunospecificity. This method provides a quantitative measure of remnant status and is easily adapted to the clinical laboratory.
The intra-assay and inter-assay C.V.’s of the assay were demonstrated to be 5% and 10%, respectively.
The RLP-cholesterol assay uses two monoclonal antibodies to isolate remnant lipoproteins. The first one (JI-H) is raised against human Apo B-100. It recognizes an epitope near the Apo B-51 region of the Apo B-100 molecule and removes LDL, Lp(a) and nascent VLDL. The second one (H-12) is raised against human Apo A-I, and removes HDL. The monoclonal antibodies are conjugated to sepharose-4B beads and separate the bound lipoproteins from the remnant lipoproteins that remain in the unbound faction. Cholesterol in the unbound fraction is then quantified by an enzymatic assay.
The unbound fraction contains essentially chylomicron remnants and VLDL remnants which are enriched in cholesteryl esters and Apo B. Remnant lipoproteins isolated by the RLP-Cholesterol Assay have b or slow pre-b mobility on AGE, whereas the VLDL recognized by the JI-H monoclonal antibody have pre-b mobility.
Current and Future Utility
In May 2001, the National Institute of Health published its new standard for both risk categories and healthy levels of various lipids. This report, the Adult Treatment Panel (ATP-III), created new risk categories, recognizing the need to identify life-habit risk factors as well as including emerging lipid tests alongside the traditional panel of cholesterol and triglycerides.
RLP’s appear to contribute to CHD risk, and their measurement can be a factor in clinical decisions on the type and intensity of risk reduction therapies needed to manage an individual’s disease. This is significant since at least half of the patients observed to have myocardial infarction in an emergency department had normal cholesterol and/or triglyceride levels which would have given no warning of an impending coronary event. Studies have shown that elevated RLP levels predict coronary events in CHD patients independently of traditional risk factors.
Significant correlation of RLP-C to carotid intimal medial thickness has been demonstrated in healthy 50 year old men. Elevated remnant lipoproteins have also been found in survivors of myocardial infarction and in individuals with significant coronary atherosclerosis. Two prospective studies demonstrated that elevated remnant lipoproteins were predictors of progression of coronary atherosclerosis and coronary events. In one of these studies, remnant lipoproteins significantly contributed to the progression of mild/moderate coronary lesions whereas LDL significantly contributed to the progression of severe lesions, suggesting that remnant lipoproteins and LDL play independent roles in the development of CHD.
RLP-C has been documented to be modulated by lipid-lowering therapy. This is important in order to effectively assess the efficacy of treatments and care plans, which can include statins, antioxidants and fibrates. Gemfibrozil was shown to reduce median RLP-C levels by 34% and was associated with a decrease in vein-graft stenosis.
The new emerging markers will play a major role in prediction and assessment of CHD, especially in those patients demonstrating normal conventional lipid profiles. The American Medical Association has also recognized the importance of new technologies by establishing the new CPT Category III codes for emerging technologies. RLP-C has been recognized as one of these emerging markers and has been assigned CPT code 0026T.
The clinical benefits of the assay are:
- Diagnosis Type II Hyperproteinemia
- Aid in the risk assessment of CVD
- Predictor of clinical events associated with CHD
- Monitor the efficacy of therapy
Michael Sanfilippo, M.S., is the technical product manager at Polymedco in Cortlandt Manor, New York.
For more information, select Reader Service Number 260, or contact Polymedco, Inc. at 914-739-5400 or www.cholesterol-tests.com.
