BIOLOGICAL ESTIMATION 



animals used for testing the unknown sample and the standard are 

 made up of pairs of litter-mates of the same sex and of approximately 

 the same weight, so that as far as possible differences in response due 

 to variations in animals are eliminated. The quantity of aneurine fed 

 is so chosen that the rate of growth will be maximal. It is the normal 

 practice to give the test material at at least three dose levels and the 

 standard at two levels. 



Sherman and Spohn originally recommended a test period of eight 

 weeks, subsequently reduced to four weeks, whilst K. H. Coward ^^ 

 claimed that satisfactory results were obtained after only two weeks 

 and F. W. Schultz and E. M. Knott ^^ believe that results can be 

 obtained in ten days. When the test is complete, a dose-response 

 curve is plotted, the smoothness of which is a check on the reliability 

 of the assay. The vitamin content of the test substance is calculated 

 by reference to this curve. According to Coward the accuracy of the 

 vitamin B^ assay is high for a biological method. The sensitivity is 

 also high, differences between doses of one /xg. being detectable. Care 

 has to be taken to prevent refection ^^ (see page 75). 



A curative method using rats, similar in principle to the original 

 test with pigeons, was proposed by M. I. Smith. ^^ In this method, the 

 rats were fed a basal vitamin B^-free diet until depleted, and the 

 weight gain on the basal diet, to which had been added graded doses 

 of the test solution, was compared with the gain in weight of animals 

 fed on the same basal diet supplemented with known amounts of a 

 standard solution of vitamin B^. Young rats on a vitamin B^-deficient 

 diet were found to develop polyneuritis in fifty to eighty days. Oral 

 administration or injection of aneurine resulted in improvement 

 within three to five hours and definite cure in eighteen to twenty-four 

 hours. After a certain time the effects wore off, symptoms of poly- 

 neuritis recurred and were again alleviated by another dose of the 

 vitamin. Although the curative response was proportional to the 

 dose. Smith did not believe the relationship to be quantitative, and 

 he therefore made his comparisons on the basis of the minimum 

 curative dose. Other workers, although confirming Smith's general 

 conclusions, found considerable variations in the occurrence of poly- 

 neuritis but these, it is suggested, can be reduced by the use of a 

 sufficiently large number of animals. 



Of the four procedures considered by the Committee of Revision 

 of the U.S. Pharmacopoeia, only the rat curative method was recom- 

 mended. The preferred procedure is substantially that of Smith, 

 the main variation being a more adequate diet, which is claimed to 

 give a 100 % incidence of polyneuritis in rats, and repeated production 

 and cure of polyneuritis in the same animal ; as many as ten such 

 periods are said to be possible. 



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