72 PROCEEDINGS OF SECTION B. 



as small quantities of material. For this reason it may not perhaps 

 be out of place to describe an improved method by which this end 

 may be achieved. 



The material under investigation was highly rich in lipoid and 

 practically devoid of free fat. Saponification thus required at 

 least ten hours and it was customary to extend this time to twenty 

 hours. Saponification is complete when the ether extract of un- 

 saponilied material does not contain phosphorus. With ordinary 

 animal or vegetable tissues such a lengthy saponification would not 

 be required. The remainder of the procedure would, however, be 

 the same in all cases. 



Details of the improved method for the determination of fatty 

 acids and cholesterol in animal or vegetable tissues : — 



1. A suitable quantity of the material (50-100 gms.) is 



dissolved by adding KOH or NaOH with or without a 

 small quantity of water. When quite fluid (this state 

 may be accelerated by placing in steam bath or steri- 

 liser for half an hour), alcohol is added to make the 

 solution of KOH about 20 per cent, and the saponifica- 

 tion continued with reflux condenser. The alcohol is 

 then partly removed by distillation. 



2. The saponified mass is now poured into a porcelain dish 



and mixed with NaHCOs. The mass is thoroughly 

 dried and may then be powdered and transferred to a 

 bottle when it can be repeatedly extracted with 

 anhydrous ether. To render the ether perfectly anhydrous 

 the addition of freshly burned Na2C03 is recommended. 



3. The ether extracts are united and evaporated. The 



extract from animal tissue should be quite white and 

 consists of cholesterol. 



4. The powder now free from cholesterol is dissolved in hot 



water, acid is added in excess and the fatty acids col- 

 lected on filter paper and dissolved in ether, or they 

 may be dissolved directly from the soap solution by 

 adding first ether and then hydrochloric acid. An 

 aliquot portion of the ether solution is evaporated to 

 determine the percentage of total fatty acids. 



References : 



(1) I'flug, Arch. 65, s. 90, 1896. 



(2) Zeitschr. j. Btol., xxxv., s. 555, 1897. 



(3) Biochem. Zeit., Bd. ix., s. 212. 



(4) Cbl. inn. Med., 33. 



(5) Pflug, .Arch. 95, s. 107. 



(6) " Handbuch der Biochemie " (Oppenheimer), 1907. 



(7) H. S. Zeit. Physl. Chem. 27, s. 259, 1899. 



(8) Pflug, Arch. 72, s. 360. 



(9) Journal oj Phys. vol. xxxviii., p. 353, 1909. 



(10) Ibid., vol. xl., 1910. 



(11) Proc. Linn. Soc. New South Wales, vol. xxxv., part i, 1910. 



