2Q2 



PHYSIOLOGICAL CHEMISTRY 



electric hot plate, the chloroform made up to some suitable volume, such as 

 20 c.c., filtered if necessary, and colorimetric estimation carried out as follows : 

 5 c.c. of the chloroform extract are pipetted into a dry test tube, and 2 c.c. of 

 acetic anhydride and o.i c.c. of concentrated sulphuric acid (best with o.i c.c. 

 pipette) are added. After thorough mixing, the solution is placed in the dark 

 for exactly 10 minutes 1 to allow the color to develop, and then compared with a 

 standardized 0.005 per cent aqueous solution of naphthol green B in a Bock- 

 Benedict or Kober colorimeter. If the Duboscq colori- 

 meter is used, it is necessary that the cups should be 

 remounted in plaster of Paris instead of balsam. 



With a good grade of acetic anhydride, it has been 

 found that when an 0.005 per cent solution of naphthol 

 green B is used as a standard and set a 15.5 mm. on 

 the Duboscq or Kober instrument, 0.4 mg. of cholesterol 

 in 5 c.c. of chloroform treated with 2 c.c. of acetic anhy- 

 dride and o.i c.c. of concentrated sulphuric acid will read 

 15 mm. The color curves for both the cholesterol and 

 naphthol green B appear to fall in a straight line so that 

 readings somewhat above or below the standard are 

 accurate. 



Calculation. If a cholesterol standard containing 

 0.4 mg. to 5 c.c., or naphthol green B standard of 

 equivalent strength, are employed, the following formula 

 may be used for the calculation : 



FIG. 93. EXTRACTION 

 APPARATUS FOR CHOLES- 

 TEROL DETERMINATION. 



(From Myers: "Prac- 

 tical Chemical Analysis 

 of Blood," C. V. 

 Mosby Co., St. Louis, 

 1921). 



~ X 0.0004 X X 100 = Cholesterol content of 



K 5 



blood in per cent, 



for 100 c.c. 



in which S stands for the depth of standard in mm., 

 R for the reading of the unknown, 0.0004 the equiva- 

 lent amount of cholesterol in 5 c.c. of chloroform, D 

 the dilution of the chloroform extract from the i c.c. of 

 blood, 5 the dilution of the standard and 100 the factor 

 For example : 



- X 0.0004 X - X 100 = o.i 60 per cent. 

 15 5 



Interpretation. Normal blood serum contains from 0.15 to o.i 8 

 per cent of cholesterol and whole blood about 0.14 to 0.17 per cent. 

 Values of from 0.170 to 0.350 have been noted in chronic and acute 

 nephritis. In diabetes, values of 0.150 to 0.300, and in lipemia ex- 

 tremely high values (up to 3.6 per cent) are found. An increase 

 is also found in pregnancy. In -cholelithiasis high values are fre- 

 quently obtained. In pernicious anemia values as low as 0.07 per 

 cent are noted. Cholesterol is increased by a high lipoid diet and 

 decreased by a diet low in cholesterol. 



1 In order to get the proper temperature for color development in warm weather it is 

 advisable either to keep the reagents in a cool place or to insert the tubes in water during 

 the development of the color. 



