V. ESTIMATION 409 



yields practically a straight line.^ This relation, although possibly only 

 empirical, proves to be applicable to data from different laboratories, using 

 different procedures for estimating prothrombin and different methods of 

 administering supplements (Fig. 4). 



Stamler et al.,-'' employing this relation to compare calculated prothrom- 

 bin levels with observed levels, have stated that the agreement is not "very 

 exact." Very exact results are rarely obtained in biological assays; never- 

 theless the data plotted by the direct method of Almquist and Klose^ ap- 

 pear to be quite consistent (Fig. 4, curve 3). Quick and Stefanini^^ have 

 objected to the use of an extract of chicken muscle instead of chicken brain 

 as a clotting agent for prothrombin determination. Whether or not this 

 criticism is important, the assay method operates well with either prepara- 

 tion (Fig. 4, curves 1 and 2). Of course the linear relation no longer holds 

 after an optimal prothrombin level or time has been reached. 



In any assay it is desirable to establish some such line by means of at 

 least two groups fed different levels of a standard preparation, such as 

 2-methyl-l ,4-naphthoquinone. It is then possible to interpolate and express 

 the potency of an assayed supplement in terms of the reference standard. ^^ 

 Reproducibility of results by this method has been adequately demon- 

 strated. 



Another method is to measure whole blood-clotting time to the nearest 

 minute and then to compare the percentages of the chicks in which the 

 clotting time is reduced to 10 minutes or less;^- or the quantity of material 

 which will cure 50 % of the group by the above criteria may be estimated 

 from several dose levels.^^ This is called the CD50 and is compared with the 

 CD 50 of some standard substance which was employed in the same assay. 

 Improved methods have been suggested from time to time. The reader is 

 referred to papers by Almquist,^i Quick and Stefanini,^i Stamler et al.,^"^ 

 and Dam ei al.^ 



6. Standards of Potency 



It has been suggested^^ that the highly potent compound, 2-methyl-l ,4- 

 naphthoquinone, can be employed as a standard of activity and that 1 

 unit be defined as the antihemorrhagic activity of 1 7 of this compound. 

 The name menadione has been adopted as a non-proprietary term for this 



31 H. J. Almquist, Biol. Symposia 12, 508 (1947). 



32 S. Ansbacher, /. Nutrition 17, 303 (1939). 



"S. A. Thayer, D. W. MacCorquodale, R. W. McKee, and E. A. Doisy, J. Bi'' 



Chem. 123, cxx (1938). 

 3* H. Dam, I. Kruse, and E. S0ndergaard, cited b.y H. Dam, Ann. Rev. Biochern. 20, 



265 (1951). 

 " S. A. Thayer, S. B. Binkley, D. W. MacCorquodale, E. A. Doisy, A. D. Emmett, 



R. A. Brown, and O. D. Bird, /. Am. Chem. Soc. 61, 2563 (1939). 



