ASSAY METHODS 51 



of the cure, the duration being roughly proportional to the amount of 

 thiamine administered. The same animal can be used over and over, 

 5 to 10 times or more, and if laboratory animals are continually kept in 

 condition for testing, assays can be run with relative speed, and the 

 results may be as accurate as those obtained by the U.S.P. method. 15 



Biological tests for thiamine involving rat growth, 38 pigeon weight 

 maintenance, 39 prevention of bradycardia in rats involving the use of an 

 electrocardiograph, 37 chick growth, 40 and postponement of death from 

 polyneuritis in chicks 41 have all been successfully used; but except for 

 those interested in the particular species involved, these tests are largely 

 of historical interest only, since they are not used at present as thiamine 

 assay methods per se. The "catatorulin" test of Peters and co-workers 42 

 should also be mentioned in this connection. Oxygen consumption by 

 brain tissue from avitaminotic pigeons is low, and is increased by minute 

 amounts of thiamine. The test is sensitive to about 0.2 /xg of thiamine. 



Riboflavin 



Aside from the rat growth and chick growth methods which are basic, 

 but belong in a class by themselves because of cumbersomeness and the 

 time and expense involved, there are two methods that have been widely 

 used and are recommended for riboflavin assay, namely, the fluorometric 

 and the microbiological. 



Fluorometric Method. This method takes advantage of the fact that 

 riboflavin fluoresces strongly when exposed to light of wave length 440 

 to 500 m/x, and the intensity of the fluorescence is proportional to the 

 concentration of riboflavin in the solution examined. It must be performed 

 on solutions or extracts containing riboflavin, and the extraction pro- 

 cedures involve simultaneous hydrolysis of the bound forms (p. 33). If 

 one were dealing with solutions containing riboflavin as the only fluo- 

 rescing substance, the application of the method would be relatively 

 simple. However, in practice, particularly with some types of products, 

 it is necessary to take elaborate precautions to eliminate the effects of 

 other substances. 



One expedient that is always used to eliminate the effect of interfering 

 substances involves the use of a sodium hydrosulfite (dithionite) , 

 NaoSoO.!, which reduces riboflavin quantitatively to its nonfluorescing 

 leuco-form, and leaves unaltered some of the other colored fluorescent 

 substances which may be present. After the fluorometric reading is taken 

 on the final solution, regardless of previous treatments, the riboflavin is 

 destroyed with hydrosulfite and a blank fluorometric reading taken. This 

 reading is subtracted from the riboflavin reading. 



