ANALYSIS OF TISSUES 60 1 



H. FAT. 



It has been shown by Kumagawa and Suto 1 that the methods of Rosenfeld 

 and of Pfliiger for the estimation of fat in tissues are inaccurate and that accu- 

 rate estimations of fat in tissues can only be obtained by converting the 

 fat into fatty acids, i.e. estimating the fat as fatty acid. This method has 

 been adopted by most workers and they have introduced some slight 

 improvements (Hartley, Mottram). Tamura 2 does not consider that these 

 improvements are necessary. 



A convenient way of effecting the estimation is described by Mottram 3 : 



The organ is minced and thoroughly mixed after removal of the visible 

 fat and connective tissue. Portions of 10-15 gm. are introduced through a 

 funnel into weighed pressure bottles of about 150 c.c. capacity. The bottles 

 are weighed and the same volume of 60 per cent, potassium hydroxide as 

 there are grams of tissue are added. The bottles are closed with rubber 

 stoppers, exhausted with a water pump and heated for 1*5 hours in a boiling 

 water-bath, during which time they are frequently shaken, especially at first. 

 The alkaline solution is transferred to a 250 c.c. separating funnel, the 

 stoppers of which have been moistened with glycerin, and the bottle is rinsed 

 out with warm water. The combined solution and washings are cooled with 

 water and acidified with 20 c.c. of concentrated hydrochloric acid which is added 

 slowly. A copious precipitate is formed. The acid solution is cooled and 

 shaken for 30 seconds with 50 c.c. of purified ether. Separation takes place 

 rapidly and the acid solution is run into a beaker until the precipitate begins 

 to leave the funnel. The ethereal solution is siphoned into a CO 2 flask and 

 the funnel rinsed twice with 10 c.c. of ether, the rinsings being siphoned into 

 the same flask. 



The precipitate which remains is dissolved in a few drops of strong 

 potash and the solution is shaken for 30 seconds with 50 c.c. of ether. Before 

 the emulsion has time to settle the acid solution in the beaker is added and 

 the mixture shaken for 30 seconds. If separation does not take place at once, 

 the funnel may be put in an incubator at 37 for a few minutes. The acid 

 solution, but not the precipitate is run off and the ether siphoned into the 

 CO 2 flask, the funnel being rinsed as before. 



The solution of the precipitate is repeated, but 25 c.c. of ether are used 

 in the extraction which is carried out as before. 



1 Biochem. Zs., 1908, 8, 212. *Ibid., 1913, 51, 463. 



3 J. Physiol., 1910,40, 131. 



