172 VITAMIN D GROUP 



age groups. But the work did inspire further attempts to demonstrate the 

 synthesis of vitamin D under controlled conditions. 



gjljgisoa ]jept young catfish in darkened aquaria for six months on a diet 

 of veal muscle, which contained no detectable amount of vitamin D. At 

 the end of this period the fish had doubled in weight, and their visceral oil 

 deposits had increased. The visceral oil of these fish contained at least as 

 much vitamin D per gram as the oil of fish assayed at the beginning. Al- 

 though this experiment was not conclusive, it strongly suggested that syn- 

 thesis had occurred. Hess et al."^ tried to demonstrate synthesis by allowing 

 cod livers to digest with added ergosterol, but the findings were negative. 

 In another experiment, ^^^ captive codfish were given ergosterol by mouth, 

 and also intramuscularly, but there was no indication that any of it was 

 converted to vitamin D. 



Fish feed extensively on mollusks, such as whelk, ^^^ and thereby ingest 

 quantities of various provitamins D, yet the chief sterol of fish is cholesterol 

 containing only the traces of provitamin which are characteristic of the 

 higher animals. To associate the ingestion of much provitamin D with the 

 accumulation of much vitamin D implies a synthesis, for which there is 

 considerable evidence but as yet no proof. An up-to-date version of the 

 experiments of Hess and Bills would comprise the digesting of 7-dehydro- 

 cholesterol with the intestinal tissue or with the enzyme-rich pyloric cecum 

 of species known to be good accumulators of vitamin D, such as the tunas. 



2. Isolation from Natural Sources 



Isolation of vitamin D is a task beset with difficulties. In the first place, 

 the amount of vitamin D present, even in such rich sources as fish oils, is 

 small. Assuming that the vitamin D of cod liver oil has the same potency 

 as vitamin D3, it can be figured that a ton of oil contains only 2 or 3 g. of 

 vitamin. But this vitamin, according to the evidence of molecular distilla- 

 tions,^^^ exists in several forms. Furthermore, when taken out of its protect- 

 ing esters and oily vehicle, it is unstable. It is thus understandable that 

 vitamin D has only once been obtained in the crystalline state from a 

 natural source. 



The crystalline preparation was isolated from tuna liver oil by Brockman 

 and Busse.^^^ The procedure, in brief, consisted in the following steps: (1) 

 saponification and separation of the unsaponifiable fraction of the oil, (2) 

 partial separation of vitamin D from vitamin A by partitioning between 



132 A. F. Hess, C. E. Bills, M. Weinstock, and M. Imboden, Proc. Soc. Expil. Biol. 



Med. 29, 1227 (1932). 

 13' A. H. Cooke, in Cambridge Natural History, Vol. 3, p. 59. Macmillan, London, 



1913. 

 134 H. Brockmann, Hoppe-Seyler's Z. physiol. Chevi. 241, 104 (1936); H. Brockmann 



and A. Busse, ibid. 249, 176 (1937). 



