Ascorbic Acid Oxidase in Determining Vitamin C in Lens and Aqueous Humor

The vitamin C content of the eye has been the subject of a great deal of speculation by investigators. In the normal lens and aqueous, there is an appreciable quantity of the vitamin. In the cataractous lens, however, this quantity is markedly decreased. 1 , 2 At first, biological methods were used for determination of vitamin C. Later, it was estimated by titration with Tillman's reagent, 2, 6-dichlorophenolindophenol. It was accepted that reduction of the dye at pH 2 was entirely due to vitamin C. However, so far as the lens is concerned, this has been challenged by Fujita, Iwatake, and Miyata, 3 who, by use of Folin's tungstate reagent, have found that only 79% of the reduction of indophenol depends upon vitamin C. Tauber, Kleiner, and Mishkind 4 recently found in the Hubbard squash an oxidase enzyme which is specific for vitamin C. Use of this, then, should furnish direct evidence as to whether the substance in the lens and aqueous, which reduces the dye, is totally vitamin C. We first undertook to substantiate the work of Tauber, et al. The results of our experiments on the action of the oxidase at various pH values are given in Table I. As seen from our data, the optimum pH range of the enzyme is 5.5–6.0,—which confirms the work of the previous investigators. To determine vitamin C in the lens, it was extracted twice with 5% metaphosphoric acid to precipitate the proteins, centrifuged, washed, and made to a total volume of 50 cc. A one cc. aliquot of this was titrated with the dye. Another one cc. aliquot was incubated for 30 minutes at pH 5.5 with one cc. of the enzyme extract. The solution was then brought to pH 2 with sulphuric acid and titrated.