Abstract
Determination of the circulating levels of plasma lipoproteins HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease. More recently it has become apparent that the LDL subclass pattern is a further important diagnostic parameter. The reference method for separation of plasma lipoproteins is ultracentrifugation. However, current methods often involve prolonged centrifugation steps and use high salt concentrations, which can modify the lipoprotein structure and must be removed before further analysis. To overcome these problems we have now investigated the use of rapid self-generating gradients of iodixanol for separation and analysis of plasma lipoproteins. A protocol is presented in which HDL, LDL, and VLDL, characterized by electron microscopy and agarose gel electophoresis, separate in three bands in a 2.5 h centrifugation step. Recoveries of cholesterol and TG from the gradients were close to 100%. The distribution profiles of cholesterol and TG in the gradient were used to calculate the concentrations of individual lipoprotein classes. The values correlated with those obtained using commercial kits for HDL and LDL cholesterol. The position of the LDL peak in the gradient and its shape varied between plasma samples and was indicative of the density of the predominant LDL class. The novel protocol offers a rapid, reproducible and accurate single-step centrifugation method for the determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of LDL subclass pattern.—Sawle, A., M. K. Higgins, M. P. Olivant, and J. A. Higgins. A rapid single-step centrifugation method for determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of predominant LDL subclass.
Highlights
Determination of the circulating levels of plasma lipoproteins HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease
One of the most prevalent lipid/ lipoprotein patterns associated with risk of coronary artery disease is the atherogenic lipoprotein phenotype (ALP), which is characterized by moderately raised plasma TG, low levels of HDL-C, elevated total and LDL-C, and small, dense LDL particles [10, 11]
We have previously reported a novel method for separation of plasma lipoproteins using self-generating iodixanol gradients [15]
Summary
Determination of the circulating levels of plasma lipoproteins HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease. Current methods often involve prolonged centrifugation steps and use high salt concentrations, which can modify the lipoprotein structure and must be removed before further analysis. To overcome these problems we have investigated the use of rapid self-generating gradients of iodixanol for separation and analysis of plasma lipoproteins. We have modified the gradient conditions to improve the separation of lipoprotein classes, especially in individuals with dyslipidaemias, to reduce the amount of plasma needed for assay, and to resolve LDL subclasses This procedure provides a plasma lipoprotein cholesterol and TG profile in which total HDL, LDL, and VLDL can be directly compared in the same sample and in which the predominate LDL subclass can be determined
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