Homogeneous assay for measuring low-density lipoprotein cholesterol in serum with triblock copolymer and alpha-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, alpha-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 approximately 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, alpha-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and VLDL. A combination of POE-POP with alpha-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.
Sugiuchi
H
H
Department of Central Laboratory, Kumamoto University Hospital, Japan. sugiuchi@gpo.kumamoto-u.ac.jp
Irie
T
T
Uji
Y
Y
Ueno
T
T
Chaen
T
T
Uekama
K
K
Okabe
H
H
D001331:Automation / Q000379:methods; D002784:Cholesterol / Q000097:blood; D002789:Cholesterol Oxidase; D008078:Cholesterol, LDL / Q000097:blood*; D002850:Chromatography, Gel / Q000379:methods; D003505:Cyclodextrins; D006801:Humans; D006949:Hyperlipidemias / Q000097:blood; D007202:Indicators and Reagents; D008075:Lipoproteins, HDL / Q000097:blood / Q000302:isolation & purification; D008077:Lipoproteins, LDL / Q000097:blood / Q000302:isolation & purification; D008079:Lipoproteins, VLDL / Q000097:blood / Q000302:isolation & purification; D010743:Phospholipids / Q000097:blood; D011060:Poloxalene; D011549:Pseudomonas / Q000201:enzymology; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D002787:Sterol Esterase; D013501:Surface-Active Agents; D014280:Triglycerides / Q000097:blood; D047391:alpha-Cyclodextrins
D008078:Cholesterol, LDL; D003505:Cyclodextrins; D007202:Indicators and Reagents; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D010743:Phospholipids; D013501:Surface-Active Agents; D014280:Triglycerides; D047391:alpha-Cyclodextrins; D011060:Poloxalene; D002784:Cholesterol; D002789:Cholesterol Oxidase; D002787:Sterol Esterase; C032613:alpha-cyclodextrin