VII. ESTIMATION 539 



purposes, quite apart from their pathogenic character.^* Some nicotinic 

 acid-requiring yeasts utihze trigoneUine,''^ and special procedures to correct 

 for this substance, which has no activity for animals, are necessary when 

 they are used. A wide variety of lactic acid bacteria require nicotinic acid; 

 furthermore, the most widespread of the active forms of the vitamin (nico- 

 tinic acid, nicotinamide, coenzymes I and II) are equally active on the 

 molar basis. The method of Snell and Wright,^^ which uses Lactobacillus 

 arabinosus as the assay organism, has been widely studied and has proved 

 highly successful both in its original form and in any of several minor modi- 

 fications. 



The assay procedure of choice is the modification adopted after wide 

 collaborative study by the U S. Pharmacopeia'*'' and the American Associ- 

 ation of Agricultural Chemists. The method has been treated in detail 

 with respect to procedure, specificity, and reliability in several readily 

 available treatises.^"* ■ ^^"^^ Grow^th of L. arabinosus in the niacin-free basal 

 medium used increases with the niacin concentration in the range from to 

 about 0.40 7 per 10 ml. of medium. Pure nicotinic acid and samples to 

 supply the vitamin at several levels within this range are added to indi- 

 vidual tubes containing 5 ml. of the double-strength basal medium. Each 

 tube is then diluted to 10 ml., capped, autoclaved, cooled, and inoculated. 

 Response of the test organism is usually determined by acid titration after 

 72 hours incubation at 30 to 37°; under favorable conditions, turbidimetric 

 estimations of growth can be made as early as 24 hours. The niacin con- 

 tent of the sample is then determined by interpolation of the response 

 obtained with known levels of the samples onto the standard curve obtained 

 by plotting the responses to known levels of nicotinic acid. The procedure 

 is usually considered accurate to within ±10%, which compares very 

 favorably with the best of the chemical procedures. 



The extraction of nicotinic acid from natural materials preparatory to 

 assay offers little difficulty because of the looseness with which the vitamin 

 is bound, and because of the stability of nicotinic acid. Autoclaving of the 

 finely divided sample at 15 lb. pressure for 15 minutes with 0.1 A^ sulfuric 

 acid is the preferred procedure.'*'^ • ^^ Few interfering materials are known. 

 In contrast to animals, the test organism is unable to utilize tryptophan or 

 the intermediates between tryptophan and niacin for growth in the absence 



" E. E. Snell, Biol. Symposia 12, 183 (1947). 



^5 W. L. Williams, J. Biol. Chem. 166, 397 (1946). 



« E. E. Snell and L. D. Wright, J. Biol. Chem. 139, 675 (1941). 



" U. S. Pharmacopeia, 14th revision, p. 737, 1950. 



« E. E. Snell in Vitamin Methods, Vol. I, pp. 360-370. Academic Press, New York, 



1950. 

 ^' Association of Vitamin Chemists, Methods of Vitamin Assay, 2nd ed., p. 245. In- 



terscience Publishers, New York, 1951. 



