256 PYRIDOXINE AND RELATED COMPOUNDS 



that have different activities for different test organisms, and occur in 

 different ratios in different foodstuff's; (2) the bound forms of these vita- 

 mins are inactive for many microorganisms and show unusual hydrolytic 

 behavior, in that they are more rapidly hydrolyzed by low acid concentra- 

 tions than by high; (3) these facts were not known in earlier studies dealing 

 with chemical and biological estimation of vitamin Be , and have been 

 insufficiently considered in many later studies. 



Saccharomyces carlshergensis and Neurospora sitophila respond equally to 

 each of the three forms of vitamin Be and, when used with a satisfactory 

 hydrolytic procedure, should give reliable estimates for the vitamin Be 

 content of a sample. Values obtained with these organisms and by rat assay 

 are listed as "preferred values" in the tables below. The hydrolytic proce- 

 dure used for liberating the vitamin is indicated by a letter (a-m) in paren- 

 theses; of these procedures, a and h appear most satisfactory at present. 



Figures obtained by rat assay, although listed with the "preferred 

 values," are nonetheless subject to considerable error, since on some but 

 not all rations pyridoxal and pyridoxamine are somewhat less active than 

 pyridoxine in supporting growth. 



The various hydrolytic procedures used for liberating vitamin Be are 

 listed in summary form below: 



Extractant Procedure 



(a) 0.055 N H2SO4 , 180 ml./g. finely di- Autoclave at 15 lb. (120°) for 1 hr. 

 vided sample 



(b) 1 N HCl, 40 ml./l-5 g. Autoclave at 15 lb. for 1 hr. 



(c) 2 iV H2SO4 , 1:200 dilution Autoclave at 15 lb. for 30 min. 



(d) 1 N H2SO4 , 50 ml./g. Autoclave at 15 lb. for 30 min. 



(e) 20 mg. of papain and of takadiastase Incubate at 37° for 24 hr. 

 per gram of sample 



(f) 2 N HCl, 10 ml./g. Autoclave at 20 lb. for 5 hr. 



(g) NaOH pretreatment of sample followed by HCl hydrolysis as in (b) 

 (h) 0.055 N HCl, 180 ml./g. Autoclave at 20 lb. for 5 hr. 

 (i) 0.1 N H2SO4 at 80° for 30 min.; pepsin digestion at 38° for 24 hr. 



(j) No hydrolysis; unknown finely divided, shaken with adsorbent prior to chemical 



assay 

 (k) 0.04 N H2SO4 , Autoclave at 15 lb. for 1 hr., then 70 ml. papain-takadiastase 



as in (e) 

 (1) 4 N HCl 100° for 1 hr. 



(m) 2% CH3COOH 100° for 2 min. 



Table II gives values for the vitamin Be content of several foodstuffs that 

 have been assayed repeatedly by different procedures; an idea of the mag- 

 nitude of the variation observed (due to variations both in procedure and 

 in the samples themselves) may be gained from it. 



In Table III, the vitamin Be content of a variety of foods, together with 

 the assay organism and extraction procedure, is summarized. In all cases, 



