METHODS FOR STUDY OF DIGESTIBILITY OF LIPIDS 1209 



of the rat experiments reported recently, the determination of lipids has 

 been made on the dried feces. Several objections have been raised to this 

 procedure in comparison with the use of methods in which the wet feces 

 are employed directly. These are: (1) that, during drying, some hydroly- 

 sis of fat may take place, and that unsaturated acids may become oxidized ; 

 (2) that volatile acids might escape during drying; and (3) that the proce- 

 dures in which drying is required are quite time-consuming. 



There are a number of modifications of the technic for lipid determina- 

 tion in dried samples. In the first place, aliquots of the dried feces may 

 be extracted with diethyl ether, as carried out in the U.S.D.A. studies 1 

 (I), or with petroleum ether (II). Another general method involves 

 saponification of the stools with alcoholic potash, extraction of the acidi- 

 fied solution with diethyl ether or petroleum ether, drying of the ethereal 

 solution with anhydrous sodium sulfate or with an adequate drying agent, 

 followed by removal of the solvent (III). It is obvious that, by the use 

 of Methods I or II, only neutral fat, fatty acids, and components of the 

 non-saponifiable fraction are extracted, leaving the soap with the residue. 

 In Method III, all neutral fats, fatty acids, soaps, and the non-saponifiable 

 fraction are extracted; however, the glycerol has previously been re- 

 moved from the tri-, di-, and monoglycerides by the saponification pro- 

 cedure, and one cannot determine definitely how large a proportion of 

 fat was originally present in the form of free fatty acids or soaps. One 

 can correct for the loss of glycerol by the addition of a calculated weight 

 based upon the total fatty acid content. It is of course evident that the 

 corrected value becomes progressively less accurate as the proportion of 

 fatty acid derived from triglyceride is decreased. 



Augur et a/. 39 have modified Method I by a two-stage Soxhlet extrac- 

 tion, so that the soap fraction can be included. The neutral fats, fatty 

 acids, and unsaponifiable extract are first removed, the fecal residue is 

 acidified by the dropwise addition of 50% sulfuric acid, and the acidified 

 mixture is subjected to a second Soxhlet extraction. As indicated earlier, 

 a metabolic soap exists which must be used as a correction factor in the 

 calculation of the digestibility of a fat when this analytical procedure is 

 used. 



The method of Folin and Wentworth 40 likewise includes the soaps in 

 the analysis. The air-dried feces are extracted for twenty hours with 

 diethyl ether containing 10% hydrochloric acid. After removal of the 

 diethyl ether, the extract is again subjected to extraction with petroleum 



39 V. Augur, H. S. Rollman, and H. J. Deuel, Jr., /. Nutrition, S3, 177-186 (1947). 



40 O. Folin and A. H. Wentworth, J. Biol. Chem., 7, 421-426 (1909-1910). 



