Development of a homogeneous assay for measurement of high-density lipoprotein-subclass cholesterol.
BACKGROUND
Several studies have suggested that measurement of high-density lipoprotein (HDL) 2 and HDL3 subfractions might be more useful for evaluating coronary risk than total HDL-cholesterol (C). However, methods of measuring HDL2 and HDL3 are quite laborious for general clinical use. Development of a quick and easy method of measuring HDL subfractions has been long-awaited.


METHODS
Triglyceride (TG) rich lipoproteins (TRLs), low-density lipoprotein (LDL), HDL2, and HDL3 were used for screening of surfactants and enzymes to react selectively with HDL3-C and to decompose other lipoproteins.


RESULTS
In order to develop HDL3-C homogeneous assay, polyoxyethylene styrenated phenyl ether derivative, for which the hydrophilic lipophilic balance (HLB) value is 13.6, was adopted as the most effective and specific surfactant for selection of HDL3 from HDL. Sphingomyelinase (SMase) reacted with TRLs and LDL preferentially, and decomposed them. HDL2-C was estimated by subtracting measured HDL3-C from total HDL-C, directly measured by homogeneous method. The homogeneous assay exhibited excellent correlations with the results of HDL3-C and HDL2-C measured by standard ultracentrifugation (R(2)=0.848 and 0.982, respectively).


CONCLUSIONS
We established a rapid and effective, fully-automated assay for the measurement of HDL3-C. Furthermore, the subtraction of HDL3-C from total HDL-C allows concurrent determination of HDL2-C.
Reagent R&D department, Denka Seiken Co., Ltd., 1359-1, Kagamida, Kigoshi, Gosen-shi, Niigata 959-1695, Japan. Electronic address: y_ito@denka-seiken.co.jp.
Yasuki
Y
Ito
Noriyuki
N
Satoh
Takayoshi
T
Ishii
Junko
J
Kumakura
Tsutomu
T
Hirano
D000328:Adult; D000368:Aged; D057205:Automation, Laboratory; D005260:Female; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood* / Q000145:classification*; D008297:Male; D008875:Middle Aged; D055815:Young Adult
D008075:Lipoproteins, HDL
4-AA; 4-Aminoantipyrine; BES; DS; HDL subclasses; HDL3: HDL3-C; HLB; Hep–Mn; N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, dehydrate; SMase; Surfactant; TG rich lipoproteins; TOOS; TRLs; dextran sulfate; heparin–manganese; hydrophilic lipophilic balance; sphingomyelinase