286 PHYSIOLOGICAL CHEMISTRY 



for cholesterol obtained. The color is compared with a standard in ; 

 colorimeter. 



Procedure. With an accurate pipette transfer 2 c.c. of whole blood or serun 

 to a 100 c.c. Erlenmeyer flask, and add 20 c.c. of a 25 per cent potassium hydroxidi 

 solution. Heat on the water-bath for two hours shaking frequently and adding i 

 little water if necessary to keep from going to dryness. Pour the undilutei 

 mixture into a separatory funnel and add 25-30 c.c. of chloroform. Shak 

 vigorously for five minutes and separate. Shake out with four more portions o 

 20 c.c. each of chloroform. The combined chloroform extracts are turbid an< 

 of a green or brown color and are clarified by shaking with 5-10 grams of anhy 

 drous sodium sulphate and filtering. Dilute the nitrate to 100 c.c. with chloroform 

 Transfer 5 c.c. of this extract to a small glass-stoppered bottle of about 10 c.c 

 capacity, add 2 c.c. of acetic anhydride and o.i c.c. of concentrated sulphuric acii 

 and shake. Place in a water-bath at 32-35C. and keep in the dark 15 minutes 

 A green color is developed. At the same time a series of standards are prepare* 

 and treated in the same manner. The color of the test is compared with tha 

 of the most similar standard in a colorimeter (see Fig. 153, p. 486) and its colo 

 strength determined. 



Five standards are kept, these being prepared by dissolving in 100 c.c. por 

 tions of chloroform: (i) 3.2; (2) 4.8; (3) 6.4; (4) 8.0; (5) 9.6 mg., respectively o 

 pure cholesterol. In preparing the standards for comparison 5 c.c. portions o 

 each of the above solutions are taken, placed in small glass -stoppered bottle 

 and treated as were the unknowns. Each standard then represents a concen 

 tration of : 160 mg. ; 240 mg. ; 320 mg. ; 400 mg. ; and 480 mg. respectively o 

 cholesterol in 100 c.c. of the original blood or serum. 



10. Chlorides. Method of McLean and Van Slyke. 1 The determination re 

 quires two steps: (i) removal of proteins and (2) titration of chlorides. 



Coagulation of Proteins. Removal of the proteins may be accomplished in tw 

 ways, by coagulation or by ignition. Results are identical by both methods, bu 

 coagulation is the simpler. For coagulation 2 c.c. of oxalated plasma (i c.c. ma; 

 be used if material is limited) are drawn into a 2 c.c. pipette which has been cali 

 brated to contain 2 0.005 c - c - From the pipette the plasma is run into a 20 c.c 

 stoppered volumetric flask which contains 10 c.c. of a 10 per cent magnesium sul 

 phate solution. The pipette is rinsed twice by drawing up into it the solution iron 

 the flask. Two drops of 50 per cent acetic acid are added, the flask is filled to th 

 mark with water, the contents are mixed by inverting the flask, and heated in ; 

 bath to 100 for ten minutes. By keeping the stopper loosely in place evaporatioi 

 is prevented, and when cool the contents return to their original volume. Tei 

 minutes on the steam bath are sufficient to coagulate the albumin and to distribut 

 the chlorides evenly between the fluid and precipitated albumin. The flask is thei 

 allowed to cool, and the contents are poured upon about 0.3 gram of blood char 

 coal 2 in a small beaker, and mixed. After a few minutes the liquid is filterec 

 through a dry folded filter, and a water-clear filtrate obtained. 



Titration of the Protein-free Filtrate. In brief, the chlorides are precipitated ii 

 the presence of nitric acid by standard silver nitrate solution, the silver chloride i 

 removed by nitration, and the excess silver titrated with standard potassiun 



1 McLean and Van Slyke: Jour. Biol. Chem., 21, 361, 1915. 



2 Merck's "Blood Charcoal Reagent," purified by acid and freefr om chloride, is used 

 No other form of charcoal has been found to be of service. 



