VII. ESTIMATION 243 



liinii\ai>' tissiu' ili^vstioii is usually rciiuii'cd tor liic ([uaut itat ivo extraction 

 ot' the a(ii\(> material ])i'i()i' to physical, (•iiciiiical, and niiciohiolo.iiical as- 

 say.' Meliiick and coworkers- liave tested \arious methods tor liheiatiufi; 

 houiul xitamin Br and ha\e found tlie followinji; hyiholytic procedures to 

 l)e satisfactory: (1) auto;'la\inji; \ho materials at 1") |h. j)ressure in 0.05o 

 .V IIjSO^ for 00 miiuites; (2) autoclavin};- at lo Ih. pressure in 2 .V TI.;S()4 for 

 .SO minutes; (3) suspenchn^ the materials in 1 .V IICI at 100° for (K) minutes. 

 Tiiere aic modifications of this type of iiydiolytic procechu'e wliicfi lia\(! 

 been successfully used; tliey all employ an acid hych'olysis, since it has been 

 demonstrated that the three members of the vitamin Be complex are stable 

 in acid solution^ and unstable in neutral or alkaline solution. Pepsin and 

 papain diiiestions in acid media have also been utilized to liberate bound 

 vitamin Be. 



A. PHYSICAL METHODS 



HENRY SHERMAN 



1. Spectrophotometric 



Pyridoxine exhibits a typical ultraviolet absorption spectrum which 

 changes markedlj^ with variations in hydrogen ion concentration.'*' ^ 

 Melnick ct al.- have shown that pyridoxal and pyridoxamine have similar 

 absorption curves and behave similarly with changes in pH. They have 

 used these facts as the basis of their spectrophotometric method for the 

 assay of \itamin Be. "The ultraviolet absorption curves show no maxima 

 common to all three compounds. However, if readings are taken at 325 

 m/i of solutions at pH 6.75, close estimates of the total amount of the com- 

 pounds present may be obtained, despite variations in their relative con- 

 centrations. At that wave length there is an absorption maximum common 

 to pyridoxine and pyridoxamine, and while pj^ridoxal absorbs approxi- 

 mately 20 per cent more light at its 315 m^ maximum, at 325 mn all three 

 compounds absorb to the same extent. The .^l^m. values at 325 m/u in 

 pure solutions at pH 6.75 all approximate 440 ±1.5 percent expressed in 

 terms of the free bases. Spectrophotometric analyses, of course, cannot be 

 applied unless the test solutions are free from irrelevant light-absorbing 

 materials, or unless blank solutions containing none of these vitamin Be 

 factors are a\'ailable for evaluating the interference." This spectrophoto- 



> L. .\tkin, \. S. Schultz. W. L. Williams, and C. N. Yroy. Ind. Enij. Chcm. Aunl. 



Ed. 15. 141 (1943). 

 «D. Mehiick, M. Hochhorn, H, W. Iliinos, atui H. L. Osor, ./. Biol. Chcm. 160. 1 



(1945). 

 ' E. Cunningham and E. E. Snell, ./. Biol. ('hem. 158, 491 (1945). 



.1. C. Keresztesy and J. R. Stevens, J. Am. Chem. Sor. 60, 1267 (1938). 

 ' E. T. Stiller, J. C. Keresztesy, and J. R. Stevens, J. Am. Chem. Soc. 61, 1237 (1939). 



