ASCORBIC ACID AND GLYCOGEN 247 



6. Prepare a standard and blank by adding 4 ml. of the acid- 

 charcoal suspension to 1 ml. aliquots of freshly prepared 1 milligram 

 per cent ascorbic acid soln. and water, respectively. Centrifuge and 

 treat 30 /xl. aliquots in the same fashion as the unknowns. Take care 

 to avoid floating charcoal, which is more of a problem in the absence 

 of serum. Correct both standard and unknown for the blank and cal- 

 culate the result; only the single standard is required since the color 

 is directly proportional to the concentration of ascorbic acid. 



note: Serum may be stored safely for several days in a refrigerator or 

 for several weeks at — 20° after the acid has been added. The supernatant 

 acid extract may be separated and stored safely in a refrigerator for at 

 least several weeks, and presumably at —20° for an indefinite period. The 

 tubes must be well sealed with rubber stoppers to prevent evaporation. 

 When samples are stored in the frozen state it is preferable to separate 

 the supernatant before freezing in order to avoid the troublesome tendency 

 for the charcoal to float as a result of the subsequent necessity for stirring. 

 The rapid loss of ascorbic acid in blood at ordinary temperatures makes it 

 imperative to keep the material cool until it is acidified. 



In the preceding method of Lowry et al. the danger of charring 

 has been minimized by the use of 65% sulfuric acid instead of the 

 85% acid used in the original method of Roe and Keuther; however, 

 it is still necessary to cool the reaction mixture. Bolomey and Kem- 

 merer (1946) suggest the substitution of glacial acetic acid for the 

 sulfuric acid in order to avoid the danger of charring without the 

 necessity of cooling. The color intensity developed with the acetic 

 acid is about half that obtained with the sulfuric acid, which may or 

 may not be important. 



GLYCOGEN 



While colorimetric methods for glycogen have not been developed 

 specifically for histochemical studies, the procedure of Boettiger 

 ( 1946) can be adapted to the micro scale necessary. 



In the method of Boettiger, the glycogen obtained by alcohol pre- 

 cipitation of an alkaline digest of the tissue is dissolved, heated with 

 an acid solution of diphenylamine, and the color which is developed 

 is measured (a filter No. 635 is used with the Evelyn photoelectric 

 colorimeter) . This method enables duplicate determinations on 5-10 

 jug. glycogen, and if reduced to the volumes required in micro- 



