Dynamic reaction in a homogeneous HDL-cholesterol assay visualized by electron microscopy.
BACKGROUND
Measurement of HDL-cholesterol (HDL-C) by homogeneous assays with automated analyzers is replacing precipitation methods. However, in this reaction-type assay, interactions between the reagents and lipoproteins remain unknown.


METHODS
Electron microscopy was used to investigate the reactions in a homogeneous HDL-C assay. Negative staining with 10 g/L uranyl acetate was performed for lipoprotein visualization by electron microscopy. Observations of the interactions between lipoproteins and the reagents of a polyanion-polymer/detergent assay were achieved by cooling the reaction mixture in ice water. This treatment also allowed observation of the time course of the reaction.


RESULTS
In the first-reagent reaction (polyanion-polymer), every lipoprotein aggregated almost completely. In the second-reagent reaction (enzymes and detergent), only HDL in the lipoprotein aggregates was selectively resolved and reacted enzymatically. Reagent 1 contains two important substances: polyanion and synthetic polymer. Using x-ray microanalysis, we confirmed that aggregation of lipoproteins in the first reaction occurred through interaction with the phosphotungstate of the polyanion.


CONCLUSIONS
Electron microscopy morphologically revealed the dynamic reaction in a homogeneous HDL-C assay.
Kondo
A
A
Department of Laboratory Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu City, 431-3192 Japan. akikondo@hama-med.ac.jp
Muranaka
Y
Y
Ohta
I
I
Kanno
T
T
D001322:Autoanalysis; D008076:Cholesterol, HDL / Q000032:analysis* / Q000097:blood; D015243:Cholesterol, VLDL / Q000032:analysis / Q000097:blood; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008854:Microscopy, Electron; D011933:Reagent Kits, Diagnostic; D013194:Staining and Labeling; D014965:X-Rays
D008076:Cholesterol, HDL; D015243:Cholesterol, VLDL; D011933:Reagent Kits, Diagnostic