Katharine H. Coward i^r 



made by Professor Jansen and biological tests carried out by workers in several 

 laboratories showed that its potency was equal to that of the original prepara- 

 tion. W^hen vitamin B^ was synthesised, a large sample of it was adopted as 

 the International Standard of reference and workers in eleven different labo- 

 ratories established the relation between the pure substance and the original 

 standards. Three micrograms of the crystalline material were equivalent in 

 biological activity to lo mg. of the standard, that is, to i International Unit.' 



For vitamin C, in 1931, there -was no known preparation stable enough to 

 use as a standard of reference, and the juice of fresh lemons, thought from its 

 antiscorbutic effect on guinea-pigs not to vary greatly in potency, was adopted. 

 By 1934, however, ascorbic acid had been prepared in a pure state and Prof. 

 A. Szent-Gyorgyi prepared 200 gm. for use as an International Standard of 

 reference. Since then ascorbic acid has been synthesised and the British Phar- 

 macopoeia now accepts chemical and physical tests of purity without biolog- 

 ical evidence of activity.* For vitamin D the original preparation of irradiated 

 ergosterol is still in use. For vitamin E a standard of reference will almost cer- 

 tainly have been adopted by the time this book is in print. It is a preparation 

 f//-a-tocopherol acetate of well-defined chemical and physical properties, shewn 

 to be stable for many months, not only when kept under the best conditions 

 known but also during the varied conditions of actual dosing in the experi- 

 ments carried out by workers in twelve laboratories to determine its activity 

 and the shape of the curve of response given by female rats. Other standards 

 of reference will become necessary as further vitamins are discovered (for 

 example, vitamin K), but it is possible that their chemistry and physics may be 

 so well known by the time their function is recognised that therapeutic prepa- 

 rations, at least, may be determined without biological tests. 



2. Criterion for the Test.— hong before any attempt is made to determine the 

 amount of a vitamin in any substance, a fair idea will have been gained as to 

 its action on some animal suitable for laboratory experiments. Sometimes a 

 vitamin has several functions; for instance, vitamin B^ is necessary for growth 

 (increase in weight in rats), for preventing or curing polyneuritis in pigeons, 

 and for preventing or curing "convulsions" in rats. When increase of weight 

 is the criterion adopted, it is specially important that the basal diet should 

 contain abundance of all substances necessary for growth other than the vita- 

 min in question. This criterion for vitamin B^ has lower limits of error (79- 

 126 per cent for P = 0.95) than the cure of polyneuritis in pigeons Avhose limits 

 of error are 24-417 for P = 0.95. The cure of convulsions in rats produced by 

 a deficiency of vitamin B, and cured by giving that substance has a high degree 

 of accuracy in the hands of Dr. E. M. Nelson.' It must never be assumed that 

 any method has the same degree of accuracy when performed by all workers, 

 nor indeed by any one worker in all his tests.' 



All criteria but increase in weight have the disadvantage of uncertain start- 

 ing-points'and end-points. The onset of xerophthalmia is ill-defined, as also 



