512 NIACIN 



The conversion of 6-phosphogluconic acid to CO2 and pentose phosphate 

 is also TPN-dependent and can be used as an assay for either 6-phospho- 

 gluconate or TPN, as shown by Horecker and Smyrniotis.^'^- 



6-Phosphogluconate + TPN ;^ TPNH2 + CO2 + pentose phosphate 



These enzymatic methods have largely replaced the older manometric 

 or methylene blue reduction procedures. i^-- is^-ioi 



The spectrophotometric methods referred to above are possible only 

 with purified or semipurified enzyme systems. Chemical and microbiolog- 

 ical methods are available for use in crude tissue extracts (p. 537). However, 

 Feigelson and associates'^- have developed a spectrophotometric method 

 which measures both DPN and TPN and can be used in crude tissue ex- 

 tracts. The tissue is rapidly frozen, homogenized in 2 % trichloroacetic 

 acid with H2O2, to oxidize and stabilize the coenzymes, and centrifuged. 

 The coenzymes in the supernatant are absorbed on charcoal (Nuchar C). 

 After washing with trichloroacetic acid, the coenzymes are eluted with 

 pyridine. The coenzyme concentration is determined by comparing the 

 absorption at 340 m/x of oxidized and hydrosulfite-reduced samples, fol- 

 lowing the method of Gutcho and Stewart.'^'' The concentration of DPN- 

 TPN was calculated on the basis of dilution factors and the fact that 100 

 7 of reduced coenzymes per millihter in a 1-cm. cell has an optical density 

 of 0.840, according to Gutcho and Stewart'^^ and Schlenk.- The correctness 

 of the 0.840 factor just cited seems open to ciuestion. From recent data 

 on the extinction coefficient of DPN-TPN (6.22 X 10^) and with a 3-ml. 

 cell and a 1-cm. light path, this factor should be about 0.939 if only DPN 

 were present. By virtue of its somewhat higher molecular weight, TPN, if 

 present, would reduce this factor sHghtly. Since the method of Feigelson 

 et al. does not distinguish TPN, it would be difficult to determine how much 

 the factor should be reduced, especially when working with a variety of 

 animal tissues. In view of these considerations, it seems possible that the 

 recent intensive studies of the Wisconsin group on DPN and TPN in ani- 

 mal tissue contain a small systematic error, which would, however, not 

 affect the vahdity of the changes in tissue concentration which they ob- 

 served under various conditions. 



1" K. Myrback, Hoppe-Seijler's Z. phijsiol. Chem. 177, 158 (1928). 



188 H. von Euler, Ergeb. Physiol. 38, 1 (1936). 



189 K. Myrback, Ergeb. Enzymforsch. 2, 139 (1933). 



19" A. E. Axelrod and C. A. Elvehjem, /. Biol. Chem. 131, 77 (1939). 



191 P. S. Krishnan, Science 105, 295 (1947). 



192 P. Feigelson, J. N. Williams, Jr., and C. A. Elvehjem, J. Biol. Chem. 185, 741 

 (1950). 



193 S. Gutcho and E. D. Stewart. Anal. Chem. 20, 1185 (1948). 



