FURTHER CONTRIBUTIONS TO THE MICRO DETERMINATION OF CHOLESTEROL

抄録

For a long time the gravimetric digitonin method has been regarded as a most reliable one for the determination of cholesterol, but it has not been generally adopted because it needed large amounts of cholesterol and expensive digitonin for single determination, and the procedures were complicated and time consuming. Recent workers have therefore attempted to devise a substitute for the gravimetry. As the gravimetry needs relatively a large amount of the material, however it may be modified, a method consisted of different principle was searched (1931). The oxidative micro method was intended to satisfy the above need which enabled one to determine less than 1mg. of cholesterol with simple procedures. The gasometric micro determination (1934) was next published by which much less cholesterol was determined. These two methods agreed in oxidizing cholesterol digitonide with chromic acid, but the determination of lipids including cholesterol by oxidative principles was not free from discussion that the oxidation of the contaminated materials would alter the final result (1933). Meanwhile, the colorimetric procedure which was thought less adequate than the digitonin method because of the well known disadvantage was revived by Schoenheimer and Sperry who applied the color reaction to cholesterol digitonide. The two familiar principles of cholesterol determination, digitonin and Liebermann-Burchard reaction, were thus combined in one method. The idea was not entirely new, but no one had ventured to realize it, as digitonin was known to give color-yellowish brown-in Lieberniann-Burchard reaction. The above authors took the advantage of the photometer through which the color given by digitonin was filtered.
In the method of this communication cholesterol was precipitated as digitonide and freed from contaminated substances in the lipid extract by washing after that digitonin of digitonide was removed by simple technique, leaving only cholesterol for the colorimetry. Such a treatment would undoubtedly be more desirable than to apply the color reaction to digitonide directly, and the objections raised to the old colorimetry of cholesterol would be eliminated.
The saponification of bound cholesterol, subsequent formation of digitonide and its separation were critically studied, and as the result the whole procedures of cholesterol determination were significantly simplified.
The writer wishes to acknowledge his indebtedness to Professor S. Kakiuchi of Tokio Imperial University for helpful criticism of this work.