ESTIMATION OF FAT. 71 



that the extiact contains phosphorus and possibly also nitrogen, 

 which indicates that lipoids are being dissolved, but it is impossible 

 to draw any line of distinction. In some cases lipoids will be 

 extracted from the first. Moreover, although these bodies are 

 sometimes themselves insoluble in ether, they invariably dissolve 

 when the ether contains fats or other lipoid material. 



It is obviously of importance that these lipoid bodies should 

 not be regarded simply as impurities, for they have an invaluable 

 function in metabolism, and in judging of the nutritive value of 

 foodstuffs their importance should be recognised. 



With regard to the separation of lipoids from true fats, no 

 satisfactory method has been yet suggested. The best procedure 

 is that of Zuelzer'^. The fatty extract is dissolved in the smallest 

 quantity of ether, and acetone is added in excess, the acetone-ether 

 solution then contains the fat, and the lipoid material is precipi- 

 tated. 



The writer has found that with Zuelzer's method as much as 

 25 per cent, of lipoid may remain with the fat. Many lipoids are 

 undoubtedly soluble in acetone. 



The Determination of Fatty Acids. — For the reasons given above, 

 most workers prefer to estimate not the fat but the fatty acid. 

 It is quite unnecessary to extract the fat for this purpose — on the 

 contrary, such a procedure is obviously inaccurate. The material 

 is mixed with 40 per cent. KOH and saponified directly. This 

 method was first used by Liebermann * and has since been 

 modified by Kumagawa and Suto.'^ Hartley,^ Mottram,i° and 

 others. Their method is briefly as follows : — The tissue is saponified 

 for varying times with 20-30 per cent, alcoholic potash or dissolved 

 by adding KOH and then saponified by adding alcohol and boiling 

 as before. The alcohol is removed, the soap and unsaponified 

 material dissolved in water, HCl is then added and the liberated 

 fatty acids and unsaponifiable material dissolved with ether. 

 Mottram's treatment is then as follows :■ — The ether solution of 

 fatty acid and unsaponifiable material obtained above is mixed 

 with an approximately equal volume of dilute NaOH and the 

 soap solution is separated in this way from the ether solution, the 

 operation being repeated till all the fatty acid is removed. The 

 ether solution then contains only unsaponifiable material. In this 

 way one avoids the emulsions which so interfere with such separa- 

 tions. 



But to carry out the method as described by Mottram, one 

 employs only small amounts of material ; the method is not appli- 

 cable for obtaining large quantities of fatty acid. 



The writer 11 has set himself the problem of determining the 

 quantities of various fatty acids in certain tissues, and it became of 

 importance to find a method by which it would be possible to 

 combine accuracy with the obtaining of a sufficient amount of 

 fatty acids to permit of their individual recognition. It is always 

 desirable to employ a method which is applicable to large as well 



