V. ESTIMATION 243 



containing all known nutrients except ascorbic acid and supplemented 

 witli graded amounts of the test sample. One group was kept as negative 

 controls. At the end of 6 to 10 weeks, the degree of protection against 

 scurvy was determined by signs, sur\dval period, and autopsy findings. 

 The amount of test material just sufficient to prevent scurvy was taken as 

 equivalent to one Sherman unit or 0.5 to 0.6 mg. of ascorbic acid. 



Many modified procedures were subsequently suggested, and of these 

 the most frequently used are the preventive dental histology method and 

 the curative growth method. The dental histology method arose from the 

 work of Hojer,^ who described ten stages in histological changes in the 

 teeth during the development of scurvy. These stages were based on the 

 amount of disorganization of the odontoblasts, the width, irregularity 

 and structure of the dentine, and the degree of calcification of the pre- 

 dentine. Key and Elphick* subsequently defined four arbitrary stages of 

 deficiency, and the degree of protection is more usually assessed by ref- 

 erence to these standards. The general procedure is to place standardized 

 guinea pigs for a few days on a basal scurvy-producing diet, supplemented 

 with 15 g. of cabbage daily, until the weight is between 250 and 300 g. The 

 animals are then divided into groups and for a period of 2 weeks are given 

 the basal diet, supplemented with graded levels of the test substance in 

 some groups and of ascorbic acid in others. Another group on the basal 

 ration alone serves as a negative control. The animals are then killed, the 

 lower jaws removed, and sections made of the decalcified incisors. The 

 degree of protection is assessed by microscopic examination of the sections 

 for the histological changes described above, ^ and by comparison of the 

 results of the standard ascorbic acid with the sample doses. 



In the simple curative method, use is made of the fact that, if male guinea 

 pigs are placed on a basal scurvy-producing diet, a decline in weight begins 

 on about the eleventh day.® The animals, each weighing exactly 300 g., 

 are therefore given this diet for 10 days and then, before any appreciable 

 loss in weight has yet occurred, are placed on graded doses of the test 

 sample and of ascorbic acid, a further group of the guuiea pigs being kept 

 as negative controls. Changes of body weight are recorded daily over a 

 further period of 20 to 30 days, and the biological values of the sample 

 are calculated from a comparison of the resultant dose-response curves. 

 The advantage of this over the previous method from the point of view of 

 time and simplicity is self-evident, and, although theoretically the speci- 

 ficity might be regarded as less, this is not found in practice. 



' J. A. Hojer, Brit. J. Exptl. Pathol. 7, 356 (1926). 



* K. M. Key and G. K. Elphick, Biochem. J. 25, 888 (1931). 



' K. H. Coward, Biological Standardization of the Vitamins, 2nd ed., p. 93. Bail- 



li^re, Tindall and Cox, London, 1947. 

 « L. J. Harris and M. Olliver, Biochem. J. 36, 155 (1942). 



