血清リポ蛋白の三次元形態の検討

Abstract

Listen

Translate article

Recent trends in biological science indicate that there is close correlation between biological function and shape of macromolecules. We consider this also as relevant in the study of serum lipoproteins. However, the morphological aspects of lipoprotein metabolism have not been widely studied, because of the technical difficulties and instrumental limitations.We approached this field by employing scanning electron microscopy (SEM), and explored in this study the methodology for SEM of serum lipoproteins. Using Chylomicron and VLDL as lipoprotein model, we checked the following five points: i) effect of isolation procedures of lipoproteins, ii) suitable fixation, iii) influence of carbon and gold coating, iv) comparison for configurations between transmission electron microscopy (TEM) and SEM, and v) variance due to different serum lipid levels.Both lipoproteins isolated by ultracentrifugation at 9, 500g for 45 minutes for chylomicron and at 82, 000g for 16 hours for VLDL and those isolated by gel-filtration with Bio-Gel A-150m had no deterious effects, while lipoproteins gained by precipitation with heparin-managenese showed the surface fused with several particles. There was no difference in size and shape of SEM between the single fixation with 1% OsO4 for 16 hours and the double fixation with 3% glutaraldehyde for 30 minutes plus 1% OsO4, 16 hours. However, there was a great difference in shape of lipoproteins when we compared TEM of the lipoproteins negatively stained with 3% phosphotungstic acid for 60 seconds and SEM of the lipoproteins fixed with 1% OsO4 for 16 hours. The former had occasionally collapsed, polygonal particles with the surface shrinked and uneven, while the latter revealed the spheres with the surface plump and smooth.From these results, we conclude that SEM gives a better undistorted, three dimensional forms of lipoproteins than TEM of negatively stained lipoproteins. The methods for the SEM of serum lipoproteins in the final form are, thus, as follows; chylomicron (d<0.9) and VLDL (d<1.006) were isolated by ultracentrifugation, and washed with physiological saline solution three times, then fixed with 1% OsO4 for 16 hours. The fixed lipoproteins were washed with distilled water more than 10 times, and mounted on a specimen holder to coat with carbon and gold, then viewed under SEM at 10, 000-30, 000x magnifications.A preliminary application of this method revealed that VLDL from normolipidemic subjects had an average diameter of 561±197 Å, while those from hypertriglyceridemic patients had a greater diameter ranging from 1995±1355Å to 3040±922Å. SEM of serum lipoprotein will be used as a useful tool for the morphological studies on serum lipoproteins.