252 ASCORBIC ACID 



will also be determined. Experimental data to confirm the validity of this 

 proposal have been put forward by Goldblith and R. S. Harris.^^ 



Roe et al.^^ have developed a method for the simultaneous determina- 

 tion of ascorbic acid, dehydroascorbic acid, and diketogulonic acid in the 

 one sample extract. Stannous chloride is used so that the ascorbic acid in a 

 metaphosphoric acid extract is stabilized and does not react with 2,4- 

 dinitrophenylhydrazine, which is then used to determine the dehydro- 

 ascorbic acid and diketogulonic acid content. The dehydroascorbic acid 

 is then reduced by hydrogen sulfide and the diketogulonic acid content, 

 which is not affected, is determined by the dinitrophenylhydrazine. After 

 removal of hydrogen sulfide from the filtrate, bromine is used to yield a 

 solution containing all the oxidized forms of the ascorbic acid. The value of 

 this method when testing aged and some processed foodstuffs has been 

 emphasized in a subsequent publication.'"" The advantage of substituting 

 thiourea for stannous chloride in the stabilization of ascorbic acid has 

 recently been claimed.^'' Lowry et al. adapted the dinitrophenylhydrazine 

 test for the determination of ascorbic acid in amounts as small as 0.01 

 ml. of serum. '"^ This micro method has been found reliable for urine and 

 blood and has the advantage of allowing ascorbic acid determinations in 

 whole blood^^' ^•'^' ^"^ Attention has, however, been drawn to the necessity 

 of adopting suitable precautions against possible extraction of interfering 

 substances from rubber stoppers.^"* 



(3) Bacteria. A proposed improvement over the hydrogen sulfide method 

 for dehydroascorbic acid, with regard to both specificity and time involved, 

 was suggested by Gunsalus and Hand, who used Escherichia coli to reduce 

 dehydroascorbic acid in fruit juices, milk, and urine, although they found 

 this inapplicable to some vegetable extracts.^"* Stewart and Sharp, how- 

 ever, satisfactorily used a strain of Staphylococcus alhus for a large number of 

 vegetable juices as well as for milk, fruit juices, urine, and blood plasma.'"^ 

 They also reported that E. coli could be used for the quantitative deter- 

 mination of dehydroascorbic acid in vegetable extracts, if the pH of titra- 

 tion were kept above 4.5 after inactivation of the bacteria. A pH of 3.5 or 



89 J. H. Roe, M. B. Mills, M. J. Oesterling, and C. M. Damron, J. Biol. Chem. 174. 



201 (1948). 

 i"" M. B. Mills, C. M. Damron, and J. H. Roe, Anal. Chem. 21, 707 (1949). 

 1" O. H. Lowry, J. A. Lopez, and O. A. Bessey, J. Biol. Chem. 160, 609 (1945). 

 102 B. B. Lloyd, H. M. Sinclair, and G. R. Webster, Proc. Biorhew. J. 49, XVII 



(1945) 

 10^ W. Daul)('nm(>rkl, Acta Pharmacol. Toxicol. 5, 270 (1949). 

 10" R. L. Ck)0(lhuui, R. S. Sealock, N. S. Scrimshaw, and L. C. Clark, Science 109, 



494 (1949). 

 los I. C. Gunsalus and D. B. Hand, J. Biol. Chem. 141, 853 (1941). 

 106 A. P. Stewart and P. F. Sharp, Ind. Eng. Chem. Anal. Ed. 17, 373 (1945). 



