218 VITAMIN D GROUP 



for the separation of vitamin A from vitamin D, are not, in Green's opinion, 

 good ones, for considerable losses of vitamin D may be encountered. 



C. BIOLOGICAL METHODS 



The author has elsewhere-^ presented a chapter on the use of the rat in 

 the estimation of vitamin D, and Waddell and Kennedy ^^ have published 

 a companion chapter on the use of the chick for this purpose. New workers 

 in the field of bioassay are advised first of all to absorb the wisdom in 

 Coward's^* "Biological Standardization of the Vitamins." The practical, 

 theoretical, and statistical aspects of this subject are also covered by 

 Gyorgy,^ and in particularly fine detail. 



The official method of estimating vitamin D in medicinal oils is described 

 in U.S. Pharmacopeia (14th revision). It is essentially a 7-day curative 

 technique, observations being made by the line test on rats previously 

 rendered ricketic. The ofl^icial procedure for estimating vitamin D in milk 

 is given in the A.O.A.C. "Methods of Analysis."^* It is similar to the U.S.P. 

 method but requires the feeding of skim milk powder to the reference rats 

 in amounts equivalent to the milk given the test animals, thus to counter- 

 balance the interfering action of milk per se. The A.O.A.C. method for 

 estimating vitamin D intended for poultry use is a 21 -day prophylactic 

 technique with baby chicks, observations being made on the percentage of 

 ash in the tibiae. These are all pass-or-fail tests, useful in quality control, 

 but not otherwise very informative as to the amount of vitamin present. 

 To gain quantitative information, one must either repeat the assays at 

 numerous levels or, better, interpret the findings by means of response 

 curves. 



To illustrate the use of response curves, two are shown in Fig. 17. Let us 

 consider the cod liver oil curve, bearing in mind that it portrays the typical, 

 or ideal, response of chicks to graded doses of vitamin D under an established 

 set of conditions. (With a different basal diet, or with chicks of different age 

 or different breed, the curve would be different.) Under the prescribed con- 

 ditions, 10 I.U. of cod liver oil vitamin D will produce, on the average, a 

 bone ash of 43 %. Now suppose that an assay is conducted with two groups 

 of chicks. The reference group is given 10 I.U. of vitamin D per 100 g. of 

 diet in the form of a reference cod liver oil (the curve was made when cod 



22 C. E. Bills, Biol. Symposia 12, 409 (1947). 



23 J. Waddell and G. H. Kennedy, Biol. Symposia 12, 435 (1947). 



2'* K. H. Coward, The Biological Standardization of the Vitamins, 2nd ed. Williams 

 and Wilkins, Baltimore, 1947. 



25 Methods of Analysis, 7th ed. Association of Official Agricultural Chemists, Wash- 

 ington, 1950. 



