BLOOD ANALYSIS 293 



(6) (Method of Bloor). Principle. The method consists in the application of 

 the Autenrieth-Funk procedure 1 to the alcohol-ether extract of blood or serum 

 prepared as for the determination of fat (see nephelometric methods). 



Procedure. Measure 10 c.c. of the extract into a small beaker, and evaporate 

 just to dryness on the water-bath or electric stove. (Any heating after dryness is 

 reached produces a brownish color, which makes the determination difficult or 

 impossible.) 



Extract the cholesterol from the dry residue by boiling out 3 or 4 times with 

 small portions (2-3 c.c.) of chloroform and decanting.. Evaporate the combined 

 extracts to a little less than 5 c.c., transfer to a 10 c.c. graduated cylinder, and 

 make the volume up to 5 c.c. A little turbidity does not matter, since it disappears 

 on adding the reagents. Measure 5 c.c. of a standard cholesterol solution in chloro- 

 form, containing 0.5 mg. of cholesterol into a similar 10 c.c. graduate. Add to 

 each 2 c.c. of acetic anhydride and o.i c.c. of concentrated H 2 SO4. Mix the solu- 

 tions by inverting two or three times, and set the cylinders in the dark for 15 

 minutes; then transfer the solutions to the colorimeter cups, and compare as usual, 

 setting the standard at 15 mm. 2 



Interpretation. See above. 



4. Calcium. Method of Halverson and Bergeim. 3 Principle. The method 

 depends upon, (i) the removal of protein by means of sodium picrate and heat, 



(2) the precipitation of calcium from the protein free material as the oxalate, and 



(3) titration of the calcium oxalate with very dilute standard potassium per- 

 manganate solution. 



Procedure. A. Removal of Protein. Whole blood is preserved with powdered 

 sodium citrate to make approximately 1.5 per cqnt. An additional i per cent 

 of citrate should be added to plasma if this is not to be analyzed at once. Direc- 

 tions given below are for serum or plasma. Twice the quantity of whole blood 

 should be employed and reagents increased proportionately. 



Pipette 5 c.c. of serum or plasma into a 50 c.c. volumetric flask containing 

 exactly 20 c.c. of distilled water. Rinse by once drawing the solution up into 

 the pipette. While rotating the flask add from a pipette 5 c.c. (i c.c. per c.c. of 

 plasma or serum) of a 4 per cent solution of sodium picrate. 4 In the same manner 

 add slowly 5 c.c. of hydrochloric acid (1:2). Heat in a boiling water-bath with 

 occasional rotation for 15 minutes. Cool to a little below room temperature in 

 cold water. Pour onto a folded calcium-free filter paper and allow to drain well. 



B. Precipitation of Calcium. Measure an aliquot (usually 25 c.c.) of the filtrate 

 into a 50 c.c. Erlenmeyer flask (Pyrex). Neutralize cautiously with concentrated 

 ammonium hydrate added drop by drop from a burette, using one or two drops of 



1 Bloor: Jour. Biol. Chem., 24, 227, 1916; 29-, 437, 1917. 

 Autenrieth and Funk: Munch, med. Wochnschr., 69, .1243, 1913. 



2 The cement of the colorimeter cups must, of course, not be soluble in chloroform. 

 Plaster-of-Paris has been found satisfactory, or even ordinary glue, if the cups are not 

 used for any other purpose. 



3 Halverson and Bergeim: Jour. Biol. Chem., 32, 159, 1917. For colorimetric method 

 see Marriott and Rowland: Jour. Biol. Chem., 32, 233, 1917 and for nephelometric method 

 seeLyman: Jour. Biol. Chem., 29, 169, 1917; see also the microtitration method of Kramer 

 and Rowland: Jour. Biol. Chem. 43, 35, 1920. 



4 Sodium Picrate Solution, 4 Per Cent. To 40 gm. of dry purified picric acid add a 

 little^ calcium-free water and 10 gm. of highest purity anhydrous sodium carbonate 

 (calcium-free) dissolved in 50 c.c. of water. Dilute to i liter. Shake until the picric acid 

 is completely dissolved. Add concentrated hydrochloric acid until a slight permanent 

 precipitate of picric acid forms. Filter through highest grade filter paper. 



