VII. ESTIMATION 253 



calciuni ^luconalc ").() ^., KHoP^^i 1-0 }>;., liver extract. "L" (Wilson and 

 Co.) 2.0 g., wheat germ oil I..") <>;., 100 1) fish liver oil 0.5 g., l-cystine 0.2 

 ti'., choline 0.2 g., inositol 0.1 g., PAllV 30.0 mg., thiamine 2.0 mg., ribo- 

 llavin 2.0 mg., calcium pantothenate 4.0 mg., nicotinic acid 10.0 mg., biotin 

 0.01 mg.. and 2-methyl-l , 1-naphthoquinone 0.4 mg. 



4. Rice Moth L.\uv.\ Test 



Sarma'^'' ^- obser\-ed that the rice moth larva (Corcyra cephalonica St.) 

 letiuired vitamin Be for growth, and that growth was proportional to the 

 amount of A-itamin Be in the diet. An assay procedure based on these ob- 

 serA'ations was dex'eloped. The results obtained with this procedure agreed 

 well with chemical tests. Pseudopyridoxine (pyridoxal and pyridoxamine) 

 was equivalent to pyridoxine in activity. 



1). MICROBIOLOGICAL METHODS 

 ESMOND E. SNELL 



Vitamin Be occurs naturally in three unconjugated and several con- 

 jugated or "bound" forms. The former are pjn'idoxal, pyrido.xamine, and 

 pyridoxine. The latter include pyridoxal phosphate and pyridoxamine 

 phosphate and their combinations with protein, an unidentified conjugate 

 of pyridoxine present in rice bran concentrate, and possibly additional 

 compounds. The bound forms show activity in animal assay, Init not for 

 most microorganisms. Hydrolysis to the unconjugated forms must therefore 

 h<^ carried out preparatory to microbiological assay. ^^ 



Pyridoxal and pyridoxamine phosphates are hydrolyzed more rapidly 

 between pH 1.5 and 2.0 than at lower or higher pH values. Where these 

 compounds are among the most important bound forms of the vitamin, 

 low concentrations of acid are superior to higher concentrations for the 

 lil)eration of vitamin Be ^'^' ^^ This situation exists with many, but not all, 

 natural materials. The procedure of choice for most materials is to heat the 

 finely divided sample with an excess of 0.055 A^ H2SO4 or HCl at 20 lb. 

 pressure in the autoclave for 5 hours. For some materials, higher acid 

 • oncentrations are more effective. Preliminary to extensive study of a 

 ingle material, therefore, the most effective hydrolytic procedure should 

 be determined and used.^'^ 



Pyridoxal, pyridoxamine, and pyridoxine are ecpial in acti\'ity for animals 



■' P. S. tiiinmi, Indian J. Mai. Research 31, 165 (1943). 



-' V. S. Sarma, Indian J. Med. Research 32, 117 (1944). 



' Iv K. Snoll, in \'itainin Methods, Vol. 1, p. 327. .\railemic Press, New YDrk, PI.'jO. 

 • J. C. Ral)iiio\vitz and Iv E. Siioll, Ind. Entj. ('hem. .\nal. Ed. 19, 277 ( l'.ll7i. 



■ S. II. Piil.ii. and .1. Schoincr, ./. Hiol. Chrni. 162, 3S!) (194(1). 



