SILVER NITRATE TEST FOR ASCORBIC ACID 11 



The Silver Nitrate-Acetic Acid Test for Ascorbic Acid 



When tissue is treated by silver nitrate dissolved in acetic 

 acid, it is commonly found that deposits of silver are formed 

 due to reduction of the silver nitrate. It has been claimed that 

 reduction of silver under these conditions is a specific test for 

 ascorbic acid and also that the localization of the silver de- 

 posits is a close guide to the localization of ascorbic acid. How- 

 ever, owing to the diffusion factors involved, these conclusions 

 can hardly be true; indeed this system is an excellent example 

 of the complications which are bound to ensue when such chemi- 

 cal studies are attempted on small molecules. Molecules of low 

 molecular weight, such as ascorbic acid, silver nitrate, and 

 probably also the initial reaction product of ascorbic acid with 

 silver nitrate, are highly diffusible. It is thus easy to say much 

 in theory, but it is impossible to prove the precise localization 

 of ascorbic acid by this method. As the silver nitrate-acetic 

 acid mixture diffuses into cells a mixing zone will be established 

 somewhere close to the cell wall in which ascorbic acid and 

 silver nitrate will react. As the reaction proceeds fresh silver 

 nitrate and fresh ascorbic acid will be recruited into the mixing 

 zone until the ascorbic acid supply is exhausted. This inter- 

 action in the mixing zone is inevitable with two such highly 

 diffusible substances. Consequently, if . ascorbic acid exists in 

 cells as such, it can only be demonstrated in the mixing zone, 

 which is an artefact of fixation. In fact, the silver which is 

 formed by reduction in this procedure is not found in the mix- 

 ing zone but attached to mitochondria, etc. It is thus clear 

 that either one of three things must be true. The reaction may 

 be truly occurring at the surface of the mitochondria; but if 

 this is true, it is not with free ascorbic acid that the reaction 

 is occurring but a substance bound to the mitochondria so as to 

 render it non-diffusible. An alternative explanation is that the 

 technique does involve a reaction of silver with free ascorbic 

 acid but that the results of the reaction appear adsorbed on 

 surfaces which have a high affinity for the reduced silver and 

 which bear no relationship to the distribution of ascorbic acid 

 in the living cell. A second alternative is that diffusible ascor- 

 bic acid is released from the interior of the mitochondria by fix- 



