BLOOD ANALYSIS 275 



proteins are removed from the blood by precipitation with hot dilute 

 acetic acid in the following manner: 



(a) Folin-Denis Procedure. The blood is drawn into small, wide-mouthed 

 bottles previously weighed and containing a small amount (about o.i gram) of 

 finely powdered potassium oxalate. From the subsequent weight of each bottle 

 is obtained the weight of the blood. Five times this weight of N/ioo acetic acid 

 solution 1 is transferred to an ordinary 1000 c.c. flask and heated to boiling. The 

 oxalated blood is then poured into this boiling acetic acid solution, stirring con- 

 stantly, and the heating is continued until the solution has again begun to boil. 

 The mixture is filtered while still hot. The coagulated material on the filter paper 

 is transferred back into the flask (by means of a small spoon or a spatula), about 

 200 c.c. of boiling water 2 are poured over it and it is allowed to stand for five 

 minutes. This mixture is then filtered through the same filter as was used for the 

 first filtration. The filtrate in the receiving flask should be very nearly as clear 

 as water, and will be found to be so if the original blood was promptly shaken 

 with the oxalate, so that no clotting has taken place. 



If clotting has occurred, the coagulation and washing of the blood is a little 

 more complicated. The clot leads to so much bumping in the boiling acetic acid 

 solution that it is not practical or safe to try to heat the mixture to boiling. 

 The filtration is, therefore, made earlier. The partially coagulated clot is then 

 broken up with a glass rod, transferred to a mortar and there ground into a paste 

 in the presence of hot water. This suspension is then poured on the filter. The 

 protein material on the filter is then washed, as before, with about 200 c.c. of hot 

 water. In this case the combined filtrates are, however, never colorless but more 

 or less reddish. On being heated to boiling a second small coagulum will be 

 obtained and the filtrate will then be practically as clear as water. 



The combined filtrate and washings, containing the uric acid and other solu- 

 ble materials, is further acidified by the addition of 5 c.c. of 50 per cent acetic 

 acid and is evaporated, over a free flame in a suitable dish, 3 to a very small 

 volume (about 3 c.c.). The liquid is then poured into an ordinary centrifuge tube 

 and the dish washed with two successive portions of o.i per cent lithium car- 

 bonate solution, using about 2 c.c. for each rinsing. Any solid material adhering 

 to the sides of the dish is removed by rubbing with a rubber-tipped stirring rod. 

 This solid material can be removed by centrifuging and pouring the superna- 

 tant liquid into another tube, washing the sediment with lithium carbonate solu- 

 tion (Smith). 



The liquid in the centrifuge tube, which at this stage should not be 

 more than 10 c.c. in volume, is then treated as in the method for urine. 



(b) Benedict Procedure. Benedict 1 has suggested that the pre- 

 cipitation of proteins by the method of Folin and Denis is incomplete, 

 and proposes the use of colloidal iron for the completion of precipitation 



1 Prepared by diluting 0.6 c.c. of glacial acetic acid to i liter. 



2 For this washing, water is used rather than N/ioo acetic acid, because if the latter is 

 used the coagulum will give off more or less of the blood pigment and the nitrates are less 

 clear. 



3 Deep (half globular) dishes 10 cm. in diameter and having a capacity of 250 c.c. are 

 very good for this purpose. While free flames are the most convenient for concentrating 

 the uric acid solutions, care must, of course, be taken not to char the contents toward the 

 end of the operation. Unless the solution can be watched carefully at this stage, it is safer 

 to finish the concentration on the water-bath. 



4 Benedict:/. Biol. Chem., 20, 629, 1915. 



