V. ESTIMATION 251 



(1) Hydrogen Sulfide. TillmansS'- 9» and other workers"- •'»• »'. 92 showed 

 that, whereas 2,6-dichlorophenohndophcnol did not measure this oxidized 

 form, sample extracts could l)e reduced with hydrogen sulfide, which was 

 removed before titration of the total ascorbic acid. Several investigators 

 48.71.86, 93 have shoAvn, however, that this method cannot be applied to 

 some types of sample, owing to the possibility of production by the hy- 

 drogen sulfide of substances, other than ascorbic acid, which may react 

 ^vith the dye. 



(2) 2,4-Dinilrophenylhydrazme. Subsequently, Roe and Kuether^'* de- 

 veloped a method which, they claimed, measured dehydroascorbic acid 

 as well as reduced ascorbic acid in blood and urine. Oxidation is effected by 

 Norit, and then the oxidation products are condensed with 2,4-dinitro- 

 phenylhydrazine. The osazone so produced is soluble in sulfuric acid, 

 gi\-ing a red color which can be measured photometrically, the intensity 

 being proportional to the quantity of ascorbic acid originally present, 

 together with any dehydroascorbic acid. Acetic acid was subsequently sug- 

 gested as a substitute for sulfuric acid,^^ but it is less effective in inhibiting 

 interfering substances. ^^ Roe and Oesterling adapted the Roe and Kuether 

 method for the determination of dehydroascorbic acid in the presence of 

 ascorbic acid in plant tissue.*^ Oxidation with Norit was omitted, and 

 thiourea was added to stabilize the reduced form. Substances other than 

 dehydroascorbic acid, such as reductones and the biologically inactive 

 2 , 3-diketo-L-gulonic acid,^^ also react with 2,4-dinitrophenylhydrazine, 

 and the method has been found not specific for aerated orange juice'^ and 

 stored solutions of dehydroascorbic acid and reduced ascorbic acid.^^ Guild 

 et al?^ have made the interesting suggestion that the Roe and Kuether 

 method may successfully be applied to oxalic acid slurries of garden fresh 

 fruits or vegetables which have had to be stored or transported before 

 assay, since decomposition products of the original dehydroascorbic acid 



85 J. H. Roe and M. J. Oesterling, /. Biol. Chem. 152, 511 (1944). 



86 IM. C. Miller, Food Research 12, 343 (1947). 



" F. C. Lamb, L. D. Lewis, and D. G. White, Food Tcchnol. 5, 269 (1951). 



88 T. J. McMillan and E. N. Todhunter, Science 103, 196 (1946). 



89 J. Tillmans, P. Hirsch, and F. Siebert, Z. Untersuch. Lebensm. 63, 21 (1932). 

 9" J. Tillmans, P. Hirsch, and H. Dick, Z. Untersuch. Lebensm. 63, 267 (1932). 

 »' A. Emmerie, Biochem. J. 28, 268 (19.34). 



92 M. Van Eekelen and A. I^mmerie, Biochem. J. 30, 25 (1936). 



"W. W. Woessner, C. \. Elvehjem, and H. A. Schuette, Nutrition 20, 327 (1940). 



" J. H. Roe and C. A. Kuether, J. Biol. Chem. 147, 399 (1943). 



»6 R. A. Boloniey and A. R. Kemmerer, J. Biol. Chem. 165, 377 (1946). 



9«M. B. Mills and J. H. Roe, /. Biol. Chem. 170, 159 (1947). 



9" J. R. Penney and S. S. Zilva, Biochem. J. 37, 403 (1943). 



98 IVL Pijoan and H. J. Gerjovitch, Science 103, 202 (1946). 



