610 PANTOTHENIC ACID 



carried out by charcoal adsorption as described by Gregory et al."- ^^ The 

 prepurified material was then coprecipitated with reduced glutathione 

 by the Hopkins reagent, cuprous oxide. The glutathione was removed by 

 resin chromatography. In this manner, in a rather good yield, 75 % pure 

 CoA was obtained. 



A very good preparation of CoA, averaging 360 units per milligram (about 

 75 to 80% pure) is now commercially available from the Pabst Labor- 

 atories, 1037 McKinley Avenue, Milwaukee 3, Wisconsin. 



D. SOME NUTRITIONAL ASPECTS OF THE CoA PROBLEM 



1. Assay of Pantothenic Acid Based on the Methodology 

 OP Pantothenic Acid Liberation from CoA 



The difficulties encountered with the liberation of pantothenic acid 

 from its coenzyme, which have been described above, eventually resulted 

 in the elaboration of the dual enzyme treatment. ^^ Since, as shown in Table 

 II, fresh tissues contain almost solely CoA and very little free pantothenic 

 acid, it is obvious that the methodology for liberation of pantothenic acid 

 from the coenzyme should be generally applicable for microbiological 

 determination of pantothenic acid in natural sources, including foodstuffs. 

 With the treatment by intestinal phosphatase and liver enzyme, the first 

 fully satisfactory method for a complete Uberation of pantothenic acid 

 from natural sources was developed as an accidental result of our studies 

 on CoA. The data in Table III may serve as an example of the effective- 

 ness of the method for reaching all bound pantothenic acid. 



The importance of this method for the evaluation of true pantothenic 

 acid content in food is amplified by the work of Neilands and Strong, ^^ 

 who compared the old clarase-papain procedure with the phosphatase-liver 

 enzyme method (cf. Table III). They found that the new method yielded 

 in some cases up to four times as much pantothenic acid as the old one. A 

 somewhat troublesome high blank in the liver enzyme has recently been 

 ehminated by Novelli and Schmetz^* through Dowex treatment of the liver 

 enzyme solution. Recent work in our laboratory by Levintow indicates 

 that pig kidney may even be a better and more convenient source of the 

 second enzyme than bird liver. 



Earlier discrepancies of pantothenic acid values obtained with the chick 

 and microbiological assays^* have now found their explanation in the dif- 

 ference in availability of CoA-bound pantothenic acid. Hegsted and Lip- 



62 J. B. Neilands and F. M. Strong, Arch. Biochem. 19, 287 (1948). 

 " G. D. Novelli and F. J. Schmetz, Jr., /. Biol. Chem. 192, 181 (1951). 

 " T. H. Jukes, Biol. Symposia 12, 253 (1947). 



