Development of a homogeneous assay for measurement of small dense LDL cholesterol.
BACKGROUND
Plasma concentrations of small dense (sd)-LDL are associated with the prevalence of cardiovascular events. However, the special equipment and long assay times required for sd-LDL measurement have hindered its clinical application. Herein, we report development of a simple homogeneous assay for sd-LDL-cholesterol (C) adaptable to autoanalyzers.


METHODS
We identified suitable surfactants and phospholipases by screening for those selective for the sd-LDL fraction (d 1.044-1.063 kg/L) and for the dissociation of other lipoproteins, including large buoyant LDL (lb-LDL). Principal characteristics of this assay were compared with ultracentrifugal isolation of LDL subfractions and with our previous heparin-magnesium precipitation assay for sd-LDL. We measured sd-LDL-C concentrations in 460 healthy, normolipidemic individuals.


RESULTS
We used a polyoxyethylene benzylphenyl ether derivative to dissociate triglyceride-rich lipoproteins and HDLs, whereas sphingomyelinase proved most effective for dissociation of lb-LDL from LDL owing to the higher sphingomyelin content in the lb-LDL subfractions. A polyoxyethylene styrenephenyl ether derivative protected sd-LDL against the dissociative actions of sphingomyelinase and cholesterol oxidase/esterase during an initial incubation step. Next, polyoxyethylene alkyl ether dissociated sd-LDL-C and the cholesterol released from sd-LDL were subsequently measured by using cholesterol oxidase/esterase. The homogeneous method correlated excellently with ultracentrifugation for sd-LDL-C (y = 0.99x-0.09, R(2) = 0.91, n = 60) and exhibited within-run precision CVs <1.1%. The distribution of sd-LDL-C was skewed, and the central 95% of sd-LDL-C concentrations ranged from 0.24 to 0.88 mmol/L (9.4-34.0 mg/dL).


CONCLUSIONS
The homogeneous assay allows reproducible measurement of sd-LDL-C within 10 min and appears promising in further investigations of the clinical significance of sd-LDL-C.
Reagent R&D Department, Denka Seiken Co., Ltd., Tokyo, Japan. y_ito@denka-seiken.co.jp
Yasuki
Y
Ito
Miki
M
Fujimura
Motoko
M
Ohta
Tsutomu
T
Hirano
D002789:Cholesterol Oxidase / Q000737:chemistry; D008078:Cholesterol, LDL / Q000097:blood*; D004987:Ethers; D006801:Humans; D011092:Polyethylene Glycols; D012016:Reference Values; D012044:Regression Analysis; D015203:Reproducibility of Results; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry; D002787:Sterol Esterase / Q000737:chemistry; D013501:Surface-Active Agents
D008078:Cholesterol, LDL; D004987:Ethers; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D002789:Cholesterol Oxidase; D002787:Sterol Esterase; D013108:Sphingomyelin Phosphodiesterase