ETIOLOGY OF BERIBERI. 43 



The extract of rice polishings considered in Table II was, 

 therefore, submitted to chemical treatment in order to divide 

 the amid substances into classes and to enable us to test each 

 class by means of experiments on fowls. The following is the 

 process which was employed. 



In order to remove noncoaffulable proteids and other colloidal substances, 

 the extract obtained from 3 kilograms of rice polishings was treated with 

 an excess of basic lead acetate, the precipitate was removed by filtration, 

 washed with water, and redissolved in 40 cubic centimeters of 10 per cent 

 sodium hydroxide solution. The lead in this solution was precipitated with 

 sulphuretted hydroj?en and filtered. The filtrate was rendered slightly 

 acid vdth hydrochloric acid (whereupon the substances originally pre- 

 cipitated by the basic lead acetate were once more thrown down), was 

 allowed to stand over night in order that the excess of sulphuretted 

 hydrogen might be dissipated, and the following day was rendered slightly 

 alkaline for the purpose of again dissolving the precipitate. The solution 

 was then diluted to 3 liters so that each cubic centimeter of solution 

 represented the substances precipitated by basic lead acetate from the 

 extract of 1 gram of polishings. Ten cubic centimeters of this solution 

 were given to fowls daily, and it was denominated Extract Number 24. 



The filtrate remaining after the lead acetate precipitate had been 

 removed was treated with a slight excess of sulphuric acid to remove the 

 lead. The lead sulphate thus formed was filtered. A saturated solution 

 of phospho-tungstic acid was added to the filtrate until precipitation was 

 complete. (About 10 grams of phospho-tungstic acid were required.) The 

 rather bulky, orange-yellow precipitate was removed and mixed with 

 freshly slaked lime and barium hydroxide. The lime unites with the 

 phospho-tungstic acid leaving in solution free bases of the histon group, 

 including arginin. The excess of lime and baryta was removed by pre- 

 cipitation with carbon dioxide and the solution was neutralized with 

 hydrochloric acid, a few drops of sulphuric acid being added to precipitate 

 any barium salt remaining. The filtrate was diluted to 3 liters. The 

 solution thus prepared represented the free bases of the histon group, 

 chiefly arginin and histidin, extracted from the rice polishings, each cubic 

 centimeter being equivalent to 1 gram of polishings. Ten cubic centi- 

 meters were given daily to fowls, and it was denominated Extract Num- 

 ber 25. 



The filtrate remaining after the separation of the phospho-tungstic 

 acid precipitate contained amino-acids and other substances. In order to 

 remove the excess of phospho-tungstic acid, barium hydroxide was added to 

 slight excess and barium phospho-tungstate filtered off. Sulphuric acid 

 was added until the solution was slightly acid and the barium sulphate 

 removed. The filtrate, containing amino-acids and amino-acid amids, was 

 likewise diluted to 3 liters and fed to fowls as Extract Number 26. 



Experiment 10.^ — Four fowls were fed on polished rice plus 

 a daily dose of 10 cubic centimeters of Extract Number 24, 



' Experiments 1 to 9, inclusive, are recorded in the two previous articles 

 referred to under references (1) and (2). 



