404 VITAMIN K GROUP 



cessful quantitatively except when the bile is diverted away from the gut 

 by ligation of the bile duct, thus interfering with absorption.^- ^ Bile- 

 fistula dogs have been used in studies of vitamin K and blood prothrom- 

 bin level.® Dogs in which the bile was diverted to the kidneys also became 

 vitamin K deficient and were used for estimajting the requirement of this 

 species.^ Individually standardized rabbits were employed in the investiga- 

 tions of the hemorrhagic sweet-clover disease which also produces prothrom- 

 bin variations in the blood. ^ 



2. Diet 



The formulation of a successful diet involves the problem of avoiding 

 ingredients which may contain vitamin K, at the same time providing all 

 other nutrients for substantially normal maintenance and growth of the 

 animal during the period of the assay. For the chick assay, the basal test 

 ration^ given in Table I, or minor variations of it, has been widely used. 



The ration is principally ground polished rice, chosen because this type 

 of cereal is free of vitamin K or any extraneous material that may contain 

 the vitamin. As a protein supplement to the polished rice, the most satis- 

 factory single product is sardine fish meal that has been extracted contin- 

 uously with ethyl ether for 24 hours or more to remove small amounts of 

 vitamin K. The fish meal also provides most of the mineral supplements in 

 the form of bone and trace elements. Instead of the 17.5 parts of fish meal 

 a mixture of 12.0 parts of ether-extracted casein, 2.5 parts of ether-ex- 

 tracted gelatin, and 3.0 parts of bone ash may be used. 



Dried brewer's yeast is a convenient source of the water-soluble vitamins 

 required by the chicks. The yeast should be extracted with ethyl ether to 

 remove possible traces of vitamin K. The full amount specified is needed 

 to supplement the ration adec^uately in the well-known and less well-known 

 vitamins and dietary factors of the water-soluble class required by chicks. 



Cod liver oil U.S. P. is recommended as a source of vitamins A and D. 

 Other types of animal feeding oils have been found to contain significant 

 amounts of vitamin K and should not be used in vitamin K assay. Puri- 

 fied /3-carotene and synthetic vitamins A and D may also be used in an 

 oil such as refined cottonseed oil with an antioxidant, such as hydroquinone. 



4 J. E. Flynn and E. D. Warner, Proc. Soc. Exptl. Biol. Med. 43, 190 (1940). 



5 J. D. Greaves and C; L. A. Schmidt, Proc. Soc. Exptl. Biol. Med. 37, 43 (1937). 



6 H. P. Smith, E. D. Warner, K. M. Brinkhous, and W. H. Seegers, /. Exptl. Med. 

 67, 911 (1938). 



' A. J. Quick and G. E. Collentine, Ain. J. Physiol. 164, 716 (1951) ; /. Lab. Clin. Med. 

 36, 976 (1950). 



8 R. S. Overman, M. A. Stahmann, W. R. Sullivan, C. F. Huebner, H. A. Campbell, 

 and K. P. Link, J. Biol. Chem. 142, 941 (1942). 



9 H. J. Almquist and A. A. Klose, Biochem. J. 33, 1055 (1939). 



I 



