ASCORBIC ACID 245 



13. After 10 min., promptly measure the color (it slowly fades 

 and becomes turbid) spectrophotometrically at 535 m/x. From to 

 1.5 milligram per cent allantoin there is a linear relationship between 

 absorption and concentration. 



14. Run the standard soln. in the same manner and at the same 

 time as the unknown. 



ASCORBIC ACID* 



The method of Roe and Kuether (1943) for assay of ascorbic acid 

 was adapted by Lowry, Lopez, and Bessey ( 1945) to determinations 

 on amounts of blood serum down to 10 fd. Measurements in the 

 range 0.3 to 1.4 milligram per cent have been made with a standard 

 deviation, in single determinations, of the order of 0.03 milligram 

 per cent. Ascorbic acid is converted to dehydroascorbic acid, the 

 latter is treated with 2,4-dinitrophenylhydrazine, and the osazone 

 formed is made to yield a colored dehydration product through the 

 action of sulfuric acid. Pijoan and Gerjovich ( 1946) pointed out that, 

 while this method is reliable for use on blood, its application to 

 tissues must be made with caution in regard to oxidation products of 

 ascorbic acid. This follows since the phenylhydrazine reaction is not 

 specific for dehydroascorbic acid but can react with structures, such 

 as diketogulonic acid, which bear no antiscorbutic properties. Before 

 applying the procedures to tissues, it would be desirable, and perhaps 

 necessary, to ascertain in advance whether interfering substances 

 were present in the tissue. A titrimetric method, employing dichloro- 

 phenol indophenol, which measures ascorbic acid directly and not 

 dehydroascorbic acid, is given on page 300. 



Lowry, I^opez, and Bessey Method for Ascorbic Acid 



SPECIAL REAGENTS 



Osazone Reagent. Prepare a soln. of 2% dinitrophenylhydrazine 

 and 0.25% thiourea in 9 N sulfuric acid; centrifuge or filter 

 through sintered glass if a precipitate develops. Store in a re- 

 frigerator and discard after 1 month. 



65% Sulfuric Acid. Add 70 ml. of the concentrated acid to 30 ml. 

 water. 



* See Bibliography Appendix, Refs. 33, 34, and 43. 



