VITAMIN C 217 



fraction precipitated at pH 5.4 to 7.2 which contained almost the entire 

 amount of the vitamin C in the original juice, while the fraction pre- 

 cipitated by neutral lead acetate at pH 5.4, although totally inactive in 

 antiscorbutic properties, showed an even higher reducing capacity than 

 the active fraction. On adding phenolindophenol to decitrated lemon 

 juice until the indicator was no longer reduced and testing the treated 

 solution immediately for vitamin C no appreciable loss in antiscorbutic 

 activity was observed. These findings were thought to furnish additional 

 proof that there is no direct connection between the reducing and anti- 

 scorbutic activity, or at least that the antiscorbutic factor is of a lower 

 reducing intensity than the reducing agency. Attention was then directed 

 to the possible function of the reducing agency as a stabihzing agency 

 for the antiscorbutic factor. This possibility seems to be borne out by 

 the following observations : 



Decitrated lemon juice purified by precipitation with alcohol was 

 tested for both reducing and antiscorbutic properties after storage for 

 24 hours and for one week. The deterioration of the two properties 

 was more or less of the same order, although there was possibly a 

 greater loss of reducing than of antiscorbutic properties. Decitrated 

 lemon juice adjusted to pH 7 and heated for 1 hour in a steam auto- 

 clave under a pressure of one atmosphere showed little loss in either 

 antiscorbutic or reducing properties when tested immediately, but almost 

 complete loss in both after storage in the cold for a week. In com- 

 menting upon these observations Zilva stated: "It would almost appear 

 as if the stability of the antiscorbutic factor depends on a chain of 

 reactions which are kept in equilibrium in the living cell and that 

 on damaging the cell the equilibrium is disturbed and the individual 

 links are progressively damaged with the ultimate inactivation of the 

 vitamin. The presence of some impurities would, according to this 

 view, be a necessary condition for establishing the antiscorbutic potency, 

 since the removal of such stabilizing substances would inactivate the 

 vitamin before the preparation could be tested." 



Zilva (1928) soon presented further evidence favoring the 

 hypothesis that in the chemical fractionation of vitamin C a reducing 

 substance is removed which in its natural medium exerts a protective 

 action on the vitamin. 



After the reducing substance in decitrated lemon juice had been 

 neutralized with phenolindophenol and the solution adjusted imme- 

 diately to pH 7 the antiscorbutic activity of the juice disappeared within 

 24 hours. Concentrates, prepared as previously described, lost their anti- 

 scorbutic property much more rapidly than did decitrated lemon juice 



