158 METHODS FOR THE ANALYSIS OF BLOOD 



represents the number of milligrams of cholesterol in 100 c.c. of 

 whole blood, blood serum or corpuscles. 



CALCIUM IN BLOOD 



Method of Lyman l 



Principle. Blood is freed from protein by trichloracetic acid. 

 Calcium is precipitated as the oxalate, dissolved in HC1, and 

 treated with a solution of ammonium stearate. The resulting 

 cloudiness due to formation of calcium soap is compared in a 

 nephelometer with a standard solution of calcium soap. 



Procedure. To draw the blood, a paraffined pipette attached 

 to a hollow needle by a piece of rubber tubing is employed. The 

 potassium oxalate is naturally omitted. Run 5 c.c. of blood into 

 a small flask containing 15 c.c. of trichloracetic acid, 6.5 per cent, 

 while agitating the flask. Mix and let stand for a few minutes. 

 Filter through a folded calcium-free filter paper. Pipette 10 c.c. 

 of the filtrate into an Erlenmeyer flask of about 50 c.c. capacity. 

 Add one drop of methyl orange, 0.1 per cent. Add 2 N ammonium 

 hydrate drop by drop until just yellow. Add nitric acid, 0.05 N, 

 dropwise until pink, and then 1 c.c. more. Add 1 c.c. of oxalic 

 acid, 4 per cent. Add 1 c.c. of sodium acetate, 20 per cent, drop- 

 wise. Cool under the water tap until a faint cloud appears. Shake 

 ten minutes or stand over night at room temperature, as con- 

 venient. Rinse the stopper with a few drops of ammonium 

 oxalate, 0.5 per cent. Pour into a centrifuge tube and centrifuge. 

 Pipette off supernatant liquor. Rinse the flask with 5 c.c. of 

 ammonium oxalate, 0.5 per cent, pour into centrifuge tube, stir, 

 rinsing with rod with 0.5 per cen ammonium oxalate, and again 

 centrifuge. Pipette off supernatant liquor. Dissolve precipitate 

 in 5 c.c. of 0.1 N nitric acid by means of stirring, and pour into 

 original flask. Agitate a moment to dissolve any precipitate 

 adhering to the walls. Rinse the rod and centrifuge tube with 

 5 c.c. of water, and pour into the flask. 



Into another flask of about 100 c.c. capacity, pipette 20' c.c. 



of the standard calcium oxalate solution. Pipette 50 c.c. and 25 



c.c. respectively of the ammonium stearate reagent into two clean 



dry beakers. Pour the standard solution into the 50 c.c. f 



1 Lyman: Jour. Biol. Chem., 1917, 29, 169. 



