Direct measurement of high-density lipoprotein cholesterol in serum with polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin.
We have developed an automated method for measuring high-density lipoprotein (HDL)-cholesterol in serum without prior separation, using polyethylene glycol (PEG)-modified enzymes and sulfated alpha-cyclodextrin. When cholesterol esterase and cholesterol oxidase enzymes were modified with PEG, they showed selective catalytic activities towards lipoprotein fractions, with the reactivity increasing in the order: low-density lipoprotein < very-low-density lipoprotein approximately chylomicron < HDL. In the presence of magnesium ions, alpha-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and very-low-density lipoprotein, without the need for precipitation of those lipoprotein fractions. The combination of PEG-modified enzymes with alpha-cyclodextrin sulfate provided selectivity for the determination of HDL-cholesterol in serum in the presence of a small amount of dextran sulfate without the need for precipitation of lipoprotein aggregates. The results of the HDL-cholesterol assayed in serum by this direct method correlated well with those obtained by precipitation-based methods and also that by an ultracentrifugation method.
Sugiuchi
H
H
Department of Laboratory Medicine, Kumamoto University Medical School, Japan.
Uji
Y
Y
Okabe
H
H
Irie
T
T
Uekama
K
K
Kayahara
N
N
Miyauchi
K
K
D000328:Adult; D002789:Cholesterol Oxidase / Q000378:metabolism*; D008076:Cholesterol, HDL / Q000097:blood*; D003505:Cyclodextrins / Q000494:pharmacology*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008297:Male; D008875:Middle Aged; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011786:Quality Control; D012016:Reference Values; D012680:Sensitivity and Specificity; D002787:Sterol Esterase / Q000378:metabolism*
D008076:Cholesterol, HDL; D003505:Cyclodextrins; D007202:Indicators and Reagents; D011092:Polyethylene Glycols; D002789:Cholesterol Oxidase; D002787:Sterol Esterase