F. A. Csonka 3 403 
Methods. 
ip 
The saponification of the alcohol-ether extract was preferred 
to the direct saponification of whole blood because of the incon- 
venience of working with such large quantities, and the large 
volumes, required to saponify in alcoholic KOH medium in a 
concentration sufficient to be sure that the esters of cholesterol 
are also split. 
Blood was taken from the vein before breakfast.!° A few crystals of 
potassium oxalate were added to prevent clotting. 75 to 125 cc. of blood 
were measured with a pipette and run slowly into 250 to 400 cc. of 95 per 
cent alcohol with constant stirring. After the protein matter had settled 
it was filtered through a Buchner funnel by suction. The blood proteins 
were put into a large extraction shell and extracted by absolute alcohol 
for 24 hours and again by petroleum ether for 24 hours in a Soxhlet appa- 
ratus. The filtrate from the blood protein as well as the absolute alcohol 
extract was evaporated on the water bath to dryness. It was redissolved 
in the petroleum ether extract, filtered through cotton, measured, and 
about an eighth of it was used for the determination of the fat and lipoid 
content. From the remainder the petroleum ether was distilled off, the 
residue saponified, and the fatty acid determined according to the method 
of Gephart and Csonka.?! 
The first step in the separation of the unsaturated from the 
saturated fatty acids was to convert them into.their lead soaps. 
These were extracted by ether, according to the method of Var- 
rentrapp, which dissolves the lead soaps of the unsaturated fatty 
acids. This method is not strictly quantitative, but it is the best 
at present devised for the separation of unsaturated from satu- 
rated fatty acid in general.” 
10 T wish to thank Drs. C. C. Hartman and W. T. Mitchell, Jr., for col- 
lecting the blood samples used in this work. 
11 Gephart, F. C., and Csonka, F. A., J. Biol. Chem., 1914, xix, 521. 
12To demonstrate how this method worked in my hands I give one of 
the preliminary analyses made of Merck’s oleic acid labeled as highly 
purified. (A), 0.1700 gm. of oleic acid gave 0.1490 gm. of unsaturated 
fatty acids; yield 87.6 per cent. (B), 0.1080 gm. of oleic acid gave 
0.0950 gm. of unsaturated fatty acids; yield 87.9 per cent. A third sample, 
where 0.1635 gm. of oleic acid was saponified first, gave 0.1558 gm. of 
unsaturated fatty acids; yield 95.3 per cent. 
