♦ V. ESTIMATION 403 



method as yet devised is free from some theoretical objections; nevertheless, 

 there are several good procedures which may yield results as consistent and 

 reproducible as can generally be expected from a bioassay with animals. 



The early development of knowledge on vitamin K was accompanied 

 by the usual mistakes and errors in bioassays which have not been uncom- 

 mon with other vitamins. More specifically, the sources of error included 

 the following. 



1. The test animals were so few that individual variability was a large 

 factor in the accuracy of the data. 



2. The assay time was too short (in some cases only a few hours), there- 

 by placing more emphasis upon speed of absorption than upon intrinsic 

 potency in an equilibrated animal. 



3. The dose level was too high or too low, placing the response outside 

 the most sensitive range. 



4. The vitamin was lost or destroyed during assay, as by volatility or 

 oxidation when dispersed over the large surface of a ground diet, or the 

 sharply increased sensitivity to light in purified preparations of the vita- 

 min.^ 



5. The test animals were exposed to the dust of green plants containing 

 vitamin K or other antihemorrhagic materials (cases are known in which 

 alfalfa dust and the manufacture of 2-methyl-l , 4-naphthoquinone nearby 

 were sufficient to vitiate an assay). 



6. Owing to bacterial contamination and synthesis of the vitamin, some 

 ingredient of the diet may unexpectedly contain vitamin K. Also, cop- 

 rophagy may be a significant source of the vitamin. 



1. Animals 



The young chick is the most readily depleted animal and has been used 

 almost exclusively. The chick presents a further advantage in that the 

 platelets do not furnish thromboplastin which might affect the clotting 

 power of the blood sample.- 



Chicks from good stock fed practical breeder rations are most desirable. 

 Although a carryover of the vitamin from the parent hen to the chick may 

 be demonstrated,^' ^'^ this does not seriously interfere with depletion of the 

 chick. Rapid chick growth is most desirable since it leads to earlier deple- 

 tion of the chick in respect to vitamin K. 



Attempts to use the rat for the study of vitamin K have not been suc- 



1 D. W. MacCorquodale, S. B. Binkley, R. W. McKee, S. A. Thayer, and E. A. 

 Doisy, Proc. Soc. Exptl. Biol. Med. 40, 482 (1939). 



2 H. Dam, Acta Physiol. Scaiid. 12, 189 (1946). 



3 H. J. Almquist and E. L. R. Stokstad, J. Nutrition 12, 329 (1936). 



3" W. W. Cravens, S. B. Randle, C. A. Elvehjem, and J. G. Halpin, Poultry Sci. 20, 

 313 (1941). 



