Homogeneous assay for measuring low-density lipoprotein cholesterol in serum with triblock copolymer and α-cyclodextrin sulfate

We have developed a fully automated method for measuring LDL-cholesterol (LDL-C) in human serum without the need for prior separation, using a nonionic surfactant, polyoxyethylene–polyoxypropylene block copolyether (POE-POP), and a sodium salt of sulfated cyclic maltohexaose, α-cyclodextrin sulfate. Of the surfactants tested, POE-POP with a higher molecular mass of the POP block and a greater hydrophobicity reduced the reactivity of cholesterol in lipoprotein fractions; the reactivity in descending order was LDL ≫ VLDL > chylomicron ≈ HDL. Gel filtration chromatographic studies revealed that POE-POP removed lipids selectively from the LDL fraction and allowed them to participate in the cholesterol esterase–cholesterol oxidase coupling reaction system. By contrast, α-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and VLDL. A combination of POE-POP with α-cyclodextrin sulfate provided the required selectivity for the determination of LDL-C in serum in the presence of magnesium ions and a small amount of dextran sulfate without precipitating lipoprotein aggregates. There was a good correlation between the results of LDL-C assayed by the proposed method and the beta-quantification reference method involving 161 sera with triglyceride concentrations ranging from 0.3 to 22.6 mmol/L.