VII. STANDAEDIZATION OF ACTIVITY 633 



concentration in the range from to about 0.2 7 of calcium pantothenate 

 per 10 ml. The pure vitamin and samples which supply it at several levels 

 within this range are added to individual tubes containing 5 ml. of the 

 double-strength medium. Each tube is then diluted to 10 ml., capped, auto- 

 claved, cooled, and inoculated. Response of the test organism is customarily 

 determined by acid titration after 72 hours of incubation at 30 to 37°, or 

 turbidimetric estimations of growth can be made as early as 24 hours. 

 /3-Alanine does not replace pantothenic acid; fatty acids may interfere 

 slightly, and should be removed by filtration of the samples at pH 4.5 

 before assay. 



Lactobacillus casei was used in many of the earlier procedures,*^ but it was 

 more troublesome than L. ardbinosus because of its more complex nutrition 

 and because fatty acids interfered much more markedly with its response 

 to pantothenic acid. With present knowledge, both disadvantages could be 

 overcome, and further work might lead to a simpler assay procedure with 

 this test organism, inasmuch as it is one of the few investigated for which 

 pantethine (LBF) and pantothenic acid have equimolar activity .^^ 



Early yeast assay methods for pantothenic acid were relatively non- 

 specific when applied to natural materials because of the presence of toxic 

 materials, because /3-alanine could be used in place of pantothenic acid, 

 and because other components of the sample stimulated growth in the 

 simple media originally used.**^ None of these objections apply to a more 

 recently developed procedure utilizing Saccharomyces carlshergensis.^''- '^° 

 For suitably equipped laboratories, this method should be found equally 

 as suitable as that utilizing L. arabinosus. 



VII. Standardization of Activity 



GEORGE M. BRIGGS and FLOYD S. DAFT 



Now that the crystalline calcium salt of pantothenic acid is readily 

 available, the older "units" are obsolete. Since the calcium salt is usually 

 used in biological studies, it is sometimes convenient to know that 1.087 g. 

 of calcium pantothenate equals 1.0 g. of pantothenic acid. Pure pantothenic 

 acid is not used as a standard, since it is an unstable viscous oil and ex- 

 tremely hygroscopic. The sodium salt is also hygroscopic, whereas calcium 

 pantothenate in the dry form is reasonably stable to air and light. 



A unit of coenzyme A has been defined as the amount which will activate 

 the in vitro system of Kaplan and Lipmann to half the maxiinum activity.^ 

 It contains approximately 0.7 7 of bound pantothenic acid. 



" L. Atkin, W. L. Williams, A. S. Schultz, and C. N. Frey, Ind. Eng. Chem. Anal. 



Ed. 16, 67 (1944). 

 ' N. O. Kaplan and F. Lipmann, /. Biol. Chem. 174, 37 fl948). 



