VITAMIN D 315 



the long bones entirely free from visible calcium deposits. Fifteen rats 

 were placed on such a diet until it was evident that they could live but 

 a short time, at which time there was added to the diet of eight of the 

 test animals 1 or 2 per cent of cod-liver oil. After 2 to 8 days all the 

 rats were killed, and the bones examined. Seven of the 8 animals re- 

 ceiving cod-liver oil showed upon histological examination of the bones, 

 a line of freshly laid down calcium, while none of the control animals 

 showed deposits of calcium. (McCollum, Simmonds. Parsons. Shipley 

 and Park, 1921.) Out of this experience and other work which soon 

 followed was developed a test for calcium-depositing (antirachitic) sub- 

 stances. This "line test" as originally described may be briefly sum- 

 marized as follows : The rickets-producing diet is fed to rats 55 to 60 

 grams in weight (weight seemed to be more important than age) for 

 28 to 40 days. By the time the rats are ready for the test they have a 

 tottering gait and their hind quarters waver in walking. By this time 

 the metaphyses of the bones are usually free from calcium salts. Then 

 the test food is fed for a designated time. During the test, records of 

 food consumption must be kept, because starv-ation causes a deposition 

 of calcium salts in the epiphyseal cartilage, probably due to release of 

 phosphorus of disintegrating tissue. At the end of the experimental 

 period the proximal end of the tibia is examined for evidences of rickets. 

 The bones are split with a scalpel, immersed in 1 per cent silver nitrate, 

 exposed to light, washed in distilled water, and then studied for signs 

 of deposition of calcium. This treatment stains the newly deposited 

 calcium black. Under the binocular microscope it appears like a cross 

 section of blackened honey-comb (the matrix, not the cartilage cell, is 

 calcified). In a "negative line test" there is no calcification of the 

 epiphyseal cartilage and little if any in the metaphysis. In a "positive 

 line test" there is a broad linear deposit of calcium salts on the meta- 

 physeal side of the epiphyseal cartilage. 



For a very detailed description of this technique and for a recent 

 discussion of factors involved in determining A-itamin D quantitatively, 

 by means of it. the reader is referred to an article by Bills, Honeywell 

 and MacXair (1928). 



A scale showing line tests of varying degrees of healing has been 

 published by Bills. This is useful in classifying results obtained by this 

 rapid method of diagnosis. 



For preparing animals for this line test McCollum, Simmonds. Par- 

 sons, Shipley and Park (1921) and McCollum, Simmonds, Shipley and 

 Park (1922) recommended first the following diet, which has been used 

 extensively : 



