VITAMINS 363 



In general the principles involved in all biological assays are 

 similar. Control groups of animals are feci a so-called chemically 

 purified ration consisting of all known nutrients (protein, fat, carbo- 

 hydrate, mineral salts, and vitamins) necessary for normal health and 

 growth. This control ration contains optimal amounts of all vitamins, 

 including the vitamin to be tested. 



Similar groups of experimental animals receive the same ration as 

 that fed to the control animals, except that the vitamin to be measured 

 is omitted. As a result these latter groups develop typical symptoms 

 of vitamin deficiency which can be prevented or cured by addition of 

 the food material containing the vitamin to be assayetl. Biological 

 assays are of two general types: (1) prophylactic, and (2) cinative. 



Prophylactic tests are made by adding small graduated amounts of 

 the food to be tested to the vitamin-deficient ration at the beginning 

 of the feeding tests. In this way it is possible to find the least amount 

 of food necessary to prevent vitamin-deficiency symptoms and to 

 produce a rate of growth comparable to that obtained with the com- 

 plete control rations containing known amounts of the vitamin. When 

 the curative methed is used, several groups of animals receive the 

 vitamin-deficient ration until they begin to lose weight and develop 

 symptoms of vitamin deficiency. At this point control groups receive 

 a sufficient amount of the pure vitamin to cure the deficiency symp- 

 toms and cause the animals to grow at a required rate. Comparable 

 experimental groups of vitamin-deficient animals receive graduated 

 amounts of the food to be tested until an amount is found which 

 contains sufficient vitamin to bring about growth responses comparable 

 to those obtained with the control animals. 



Reference standards in the form of chemically pure vitamins are 

 used for control studies whenever possible. Crystalline thiamine 

 hydrochloride, riboflavin, and L-ascorbic acid are examples of pure 

 reference standards used in assaying foods for vitamins Bj, Bo, and C, 

 respectively. Crystalline ^S-carotene was the international standard 

 for vitamin A assays, but it has been superseded by crystalline vitamin 

 A acetate. 



Vitamin D is determined biologically by producing vitamin D 

 deficiency (rickets) in rats and finding the minimum amount of food 

 required to initiate recalcification in the bones. This method involves 

 sacrificing the animals in order that the bones can be examined 

 microscopically. When chickens are used for vitamin D assays, the 

 prophylactic method is followed, and the ash content of the bones is 

 considered the criterion of vitamin D potency. Rations of a special 



