Vitamin D Assay
Vitamin D is determined most successfully at the present by animal assay. Rats or chicks are employed, and the standardized procedures yield excellent and reproducible results. The rat assay method is used for vitamin products intended for human consumption. Chicks do ot respond to vitamin D2 as they do to D3 on a rat unit basis. In the assay of vitamin D .roducts for the poultry industry it is necessary to use chicks as the test animal. In this ,rocedure the vitamin product is fed to groups of chicks at various levels with a standardized 'et, and the level of bone ash after a certain number of days is the criterion used to establish ilie vitamin level in the test material. The rat, as indicated elsewhere, is unique as regards the development of rickets.
No extreme caution is required to deprive a ration of vitamin D for the production of rickets in this species. It is only necessary to arrange the calcium-phosphorus ratio of the diet to about 4 or 5 to 1. Of the various rachitic rations developed, the Steenbock 2965 ration is employed most generally. This is composed of wheat gluten, yellow corn, calcium carbonate, and salt. Weanling rats restricted to water and this diet for 18 to 21 dpys develop a rather reproducible rachitic condition. Small additions of test material are given daily for the following five days (in one procedure) or eight days to groups of the standard rachitic rats.
One group receives a known amount of vitamin daily (USP Reference cod liver oil). After a further day or two without supplement, the animals are sacrificed. The rachitic metaphysis of the distal ends of the longitudinally sectioned radius and ulna are examined for new bone growth brought about by the vitamin administered. This is readily seen on gross examination after treating the bones with AgN03 solution and then reducing the silver phosphate by light. Black areas develop wherever bone mineral exists. At the provisional zone of calcification a rather straight continuous line of new bone is seen when the proper amount of vitamin D was administered, which led to the name "line test" for such an assay.
The extent of new calcification compared with that found in the animals receiving the known amount of vitamin D is an index of the vitamin intake and thus the content of the test material. Details of the method may be found in recent volumes of the U.S. Pharmacopoeia. The official assay method of the United Stales Pharmacopeia XVII involves the reaction of vitamin D with antimony trichloride (SbCI3). The method is that of Wilkie and co-workers. Vitamin A and cholesterol do not interfere when present in reasonable quantities. The reaction is satisfactory only with concentrated preparations of the vitamin.
