Evaluation of an immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma

Abstract

Substantial evidence indicates that triglyceride-rich lipoprotein remnants are atherogenic. Additional research has, however, been limited by available methods for separation and quantification of remnants. We have evaluated an immunoseparation assay developed to measure cholesterol in remnant-like particles (RLP-C). This method uses monoclonal antibodies to human apolipoproteins B-100 and A-I to remove most of the apolipoprotein B-100-containing lipoproteins (namely LDL and nascent VLDL) and apolipoprotein A-I-containing lipoproteins (namely chylomicrons and HDL), leaving behind a fraction of triglyceride-rich lipoproteins, including chylomicron and VLDL remnants, both of which are enriched in apolipoprotein E. Cholesterol in the unbound fraction is measured with a sensitive enzymatic assay. The RLP-C concentration was highly correlated with total triglyceride-rich lipoproteins (sum of VLDL-cholesterol and IDL-cholesterol) separated by ultracentrifugation and by polyacrylamide gel electrophoresis (r = 0.86 and 0.76, respectively). The within-run and run-to-run imprecision (CV) of the assay was ∼6% and 10%, respectively. The assay was not affected by hemoglobin up to 5000 mg/L (500 mg/dL), bilirubin up to 342 mmol/L (20 mg/dL), glucose up to 67 mmol/L (1200 mg/dL), or ascorbic acid up to 170 mmol/L (3.0 mg/dL). In 726 subjects (men, n = 364; women, n = 362) in the US, the 75th percentiles of RLP-C concentration were 0.17 mmol/L (6.6 mg/dL) and 0.23 mmol/L (8.8 mg/dL) in sera obtained after overnight fasting or randomly, respectively. A group of 151 patients from nine US centers and one Canadian center with coronary artery atherosclerosis established by angiography had higher median RLP-C concentrations than 302 gender- and age-matched controls (P <0.05). We conclude that the RLP-C assay compares favorably to ultracentrifugation and electrophoresis and provides a convenient and economical approach to measure triglyceride-rich lipoprotein remnants in routine clinical laboratories.

Remnant Lipoproteins Are Residual Risk Factor for Future Cardiovascular Events in Patients With Stable Coronary Artery Disease and On-Statin Low-Density Lipoprotein Cholesterol Levels <70 mg/dL

“…We also performed category-free net reclassification improvement (NRI) and integrated discrimination improvement (IDI) to analyze the degree to which the addition of a simple and reliable technique for measuring remnant-like lipoprotein particle cholesterol (RLP-C) using an immunoseparation method has been developed. 16,17 We have previously shown that RLP-C levels are an important predictor of secondary CVD events in patients with hypertriglyceridemia. [18][19][20][21] The present study prospectively examined whether RLP-C levels predict the risk of future CVD events (CVEs) in patients with CAD who had achieved LDL-C levels <70 mg/dL during statin therapy.…”

“…RLP-C levels were measured using an immunoseparation assay as previously described. [16][17][18][19][20][21] Intra-and interassay coefficients of variation of RLP-C levels were 2.3% and 3.6%, respectively. Left ventricular ejection fraction (LVEF) was measured by echocardiography using the Teicholz formula.…”

Lipoprotein risk factors for cardiovascular disease in patients with type 2 diabetes mellitus treated with oral antihyperglycaemic agents

“…Plasma glucose concentration was measured as previously described [12], as were plasma TG and cholesterol concentrations [13]. Chylomicron and very low‐density lipoprotein remnant particles were isolated by an immunoseparation technique as described [14,15], and LDL particle diameter measured via gradient gel electrophoresis [16].…”

Characteristic comparison of triglyceride-rich remnant lipoprotein measurement between a new homogenous assay (RemL-C) and a conventional immunoseparation method (RLP-C)

“…Two clinically available methods to determine cholesterol levels of remnant lipoproteins have ever been developed, but these assay procedures are basically different. First, remnant-like particle-cholesterol (RLP-C), an immunoaffinity separation method (RLP-C assay; Otsuka, Japan) was developed, and this assay isolates remnant-like particles (RLPs) from human serum using an immunoaffinity gel containing two different immobilized monoclonal antibodies to human apolipoproteins A-1 and B-100 [ 9 , 10 ]. Many clinical studies have demonstrated that RLP-C is a risk factor for cardiovascular disease, and serum RLP-C levels are higher in patients with coronary artery disease, diabetes, and metabolic syndrome than in healthy subjects [ 2 , 4 , 11 ].…”

“…Cholesterol values of HDL, LDL, IDL, VLDL and chylomicron measured by this HPLC method are correlated to those estimated by the ultracentrifugation method [ 14 ], and therefore this HPLC method may be employed as a substitute of the ultracentrifugation method. RLP-C correlated well with VLDL-cholesterol but poorly with IDL-cholesterol, measured by the HPLC method [ 14 ] similarly as RLP-C was correlated to VLDL-cholesterol but not to IDL-cholesterol, measured by the ultracentrifugation method [ 10 ]. However, the associations of RemL-C with VLDL and IDL have never been examined quantitatively by the HPLC method, although the RemL-C values contain VLDL remnant (IDL) cholesterol concentrations qualitatively estimated by the gel filtration method and polyacrylamide gel electrophoresis analysis in the previous report [ 12 ].…”