VII. ESTIMATION 537 



tinamide using a fluorometer equipped with the same filters as for thio- 

 chrome. As Httle as 0.3 7 of the compound per milHHter of dikited urine 

 can be detected easily. This usually permits a sufficient dilution to eliminate 

 interference from highly pigmented urines and other substances. Urines 

 giving high blank values can be decolorized with charcoal and acetic acid. 



4. 6-Pyridone of N^-Methylnicotinamide 



This compound^^ can be assayed directly by means of its intense ultra- 

 violet absorption which has maxima at 260 and 290 m/i. As little as 1 7 

 per milliliter can be assayed in this way. Rosen et al.^^ found the spectro- 

 photometric technique somewhat inadequate in that the values were too 

 high largely because of inability to obtain a suitable blank. They devised 

 a fluorometric method which gave satisfactory results. This method, al- 

 though somewhat laborious, is useful and does not detect N^-methylnico- 

 tinamide. 



5. Trigonelline (N^-Methyl Nicotinic Acid Betaine) 



In spite of the close structural similarity of this substance to N^-methyl- 

 nicotinamide, it is now well established that it is not a metabolic product 

 of nicotinic acid in mammals. Several methods have been devised to measure 

 it in urine. ^^' ^^- ^^'^ 



6. DPN and TPN (Coenzymes I and II) 



Enzymatic spectrophotometric methods have been described elsewhere 

 (p. 509). Total pyridine nucleotides in red blood cells may be determined 

 by means of a fluorometric procedure^^ based on the same principle as 

 Huff, Perlzweig, and Tilden's fluorometric determination of N^-methyl- 

 nicotinamide in urine.^- The blood is made protein-free with trichloroacetic 

 acid. Acetone condensation is then carried out in the presence of alkali, 

 yielding a fluorescent derivative which can be quantitated fluorometrically. 

 The procedure yields results very similar to those obtained by Hemophilus 

 parainfluenza assay. 



Essentially the same procedure can be applied to assay the contents of 

 coenzymes I and II in the tissues." In this instance the tissue to be assayed 

 must be immersed promptly in 2 % nicotinamide solution to inhibit break- 



" W. E. Knox and W. I. Grossman, /. Biol. Chem. 168, 121 (1947). 



3" F. Rosen, W. A. Perlzweig, and I. G. Leder, J. Biol. Chem. 179, 157 (1949). 



35 S. W. Fox, E. W. McNeil, and H. Field, Jr., J. Biol. Chem. 147, 645 (1943). 



"" H. P. Sarett, W. A. Perlzweig, and E. D. Levy, /. Biol. Chem. 135, 483 (1940). 



38 N. Levitas, J. Robinson, F. Rosen, J. W. Huff, and W. A. Perlzweig, J. Biol. Chem. 



167, 169 (1947). 

 " J. Robinson, N. Levitas, F. Rosen, and W. A. Perlzweig, J. Biol. Chem. 170, 653 



(1947). 



