BLOOD ANALYSIS 295 



method it is possible to make a complete analysis for acetone and 

 diacetic acid and hydroxybutyric acid in from 2-5 c.c. of blood. 



Procedure. Two to 5 c.c. of blood drawn from a superficial arm vein by means 

 of a sterile syringe are run into a small weighed flask containing 50 c.c. of 0.5 

 per cent potassium oxalate solution. The flask is reweighed. The diluted blood 

 is run into 100 c.c. of boiling water acidified with i c.c. of glacial acetic acid 1 

 contained in an 800 c.c. Kjeldahl distilling flask and the procedure is then carried 

 out as described in the Marriott-Scott-Wilson methods for (a) acetone and diac- 

 etic acid and (b) /3-hydroxybutyric acid as outlined on page 284. The precipitate 

 in the mercury reagent is however estimated nephelemometrically. In this case 

 the distillate which should measure 75-100 c.c. is allowed to stand half an hour, 

 then transferred to a graduated cylinder and diluted until an opalescence that 

 can be conveniently read is obtained. The turbidity occasioned by 0.05 mg. of 

 acetone diluted to 100 c.c. is a convenient strength for this purpose, although 

 considerably larger or smaller amounts give good results. With heavy opales- 

 cence it is desirable after diluting to a certain volume, say 250 c.c. to remove 

 an aliquot portion with a pipette and dilute this appropriately. A solution con- 

 taming a known amount of acetone 2 is distilled into an excess of reagent 3 and 

 this distillate which is to be used as the standard is diluted as above. Read in the 

 nephelometer against this standard, taking the readings as quickly as possible 

 after filling the tubes as the suspension settles slowly. 



If the unknown suspension is diluted so as to be not more than 20 per cent 

 different from the standard and if comparisons are made in considerable depths 

 of solution (50-60 mm.) no corrections are necessary. If the two agree within 

 10 per cent accurate comparison may be made at less depths. If divergences are 

 greater and accurate results are desired, Kober's correction equation must be 

 used (see discussion of nephelometer p. 294). 



2. Fat. N ephelometric Method of Bloor. 4 Principle. The protein is precipi- 

 tated with alcohol and ether and the fatty acid in the extract determined nephelo- 

 metrically after saponification. 



Procedure. Extraction. About 2 c.c. of blood are drawn from the vein with a 

 graduated syringe and run at once with stirring into a weighed graduated flask 

 containing about 40 volumes of a mixture of 3 parts alcohol and i part ether. 

 After again weighing to find the weight of blood added, the solution is raised to 

 boiling in a water-bath, cooled under the tap, made to volume with alcohol-ether 

 mixture, mixed and filtered. The filtrate is water clear and almost colorless. 



Determination. From 5-20 c.c. of the extract (containing about 2 mg. of fat) 

 are measured with a pipette into a small beaker and saponified by evaporating 

 nearly but not quite to dryness with 2 c.c. of N/i sodium ethylate. The residue is 

 heated just to boiling after the addition of 5 c.c. of alcohol-ether, and 50 c.c. of 

 distilled water are added. 



A similar solution of the standard is prepared by adding 5 c.c. of the standard 



1 Commercial varieties of acetic acid frequently contain substances which behave 'like 

 acetone. Blank determinations should always be made and corrections made accordingly. 



2 A convenient stock solution contains about 0.03 mg. acetone per c.c. The strength 

 of such a solution is determined by titration of 200 c.c. by the Messinger-Huppert method 

 (see Chapter XXVI). 



3 The solution cannot be added directly to the reagent as a lower result is obtained than 

 when distilled. 



4 Bloor: Jour. Biol. Chem., 17, 377, 1914; 23, 31',, 1915. 



