VII. KSIIMATIOX 251 



2. \\ vv ( Ikow I'll 'l'i;s'r 



Assay proccduics which \\i\vv ulili/.cd I he ^lowih ivspoiisc have heoii 

 inoic siifcesst'ul. In spite ot its j>;r(>aler lack ol" specilicily, it is preferred 

 today for the biological assay of vitamin Hg. 



In the first biological assay procedures by the ral growth method, the 

 water-soluble \'itamins, other than those that were available in crystalline 

 form, were supplied in the diet by liver extracts, rice polishing concen- 

 trates, or other natural products. ""^^ Clarke and Lechycka"*^ replaced the 

 natural products in the diets with pure vitamins, except for biotin, and 

 developed a basal ration which could be used successfully to assay for 

 vitamin Be . The vitamins were fed in solution as separate supplements and 

 given three times each week during both the depletion and assay periods. 



Sarma et al.*^' *^ have modified and improved the method of Conger and 

 Elvehjem; theirs is now the biological procedure that is being used most 

 extensively for the liioassay of vitamin Be . They sought to develop a basal 

 ration which would permit the minimum growth in the absence of vitamin 

 Be and the maximum growth with optimal amounts of vitamin Be . Several 

 carbohydrates (sucrose, dextrin, glucose) and proteins (casein, blood fibrin, 

 egg an)umin) were tried in various combinations to meet this criterion. 

 The diet finally selected for the bioassay of vitamin Be had the following 

 composition: sucrose 75 g., blood fibrin 18 g., salts IV 4 g., corn oil 3 g., 

 thiamine 0.2 mg., riboflavin 0.3 mg., nicotinic acid 2.5 mg., calcium pan- 

 tothenate 2 mg., 2-meth34-l ,4-naphthoquinone 1 mg., inositol 10 mg.; 

 choline chloride 100 mg., and biotin 0.01 mg., per 100 g. of diet. Halibut 

 liver oil, diluted 1 :2 with corn oil, was fed at a level of 2 drops per week, 

 with a-tocopherol included at 0.5 mg. per drop. 



Male rats were placed on this depletion diet for two weeks after which 

 they were divided evenly with respect to weight into groups of three and 

 placed on diets containing different amounts of pyridoxine, ranging from 

 to 150 7 per 100 g. of ration in increments of 25 7. They were continued 

 on their respective diets for four weeks. The weight gain per week was 

 approximately linear up to 75 7 of pyridoxine per 100 g. of diet. Twice 

 this amount was required for maximum weight gains of over 30 g. per week. 



The biological activity of pyridoxine, pyridoxal, and pyridoxamine was 

 determined for three methods of administration. The components of the 

 \itamin Be complex were equally active when fed by medicine dropper as 



'' C. E. Edgar, M. M. El Sadr, and T. F. Macrae, Biochem. J. 32, 22()0 (1938). 



" T. W. Conger and C. A. Elvehjem, /. Biol. Chem. 138, 555 (1941). 



'« L. M. Henderson, H. A. Waisman, and C. A. Elvehjem, J. Nutrition 21, 589 (1941). 



" M. F. Clarke and M. Lechycka, /. Nutrition 25, 571 (1943). 



« P. S. Sarma, E. E. Snell, and C. A. Elvehjem, J. Biol. Chem. 165, 55 (1946). 



" P. S. Sarma, E. ]<]. Snell, and C. A. Elvehjem, ./. Nutrition 33, 121 (1947). 



