§ 7 THE LUTEIDIN PROBLEM 4 i 



in the fishes until some time afterwards, so that any hydro- 

 lysis of the active component of the urine might proceed 

 unhindered before the actual test began. In this experiment 

 he actually found a reduction of the latent period, usually a 

 little less than the time during which the urine had been at 

 22° C, before the fishes had entered the water. Finally, a 

 constant latent period of 2 hours was reached. 



Attempts at extraction of luteidin at room temperature 

 with ether or benzene were without result, because these 

 extracts were invariably inactive; it was found, however, that 

 an extract could be made with butanol, in the cold, which 

 contained all active substances from the urine. To this end 

 the urine is shaken up with butanol three times; the resultant 

 emulsion is disintegrated by centrifugation; the solvent is 

 evaporated in a vacuum, and the residue is put in alcohol for 

 testing purposes. The urine extracted is invariably completely 

 inactive. The latent period of the active extracts is always 

 5 Yz hours, and retains this value until the extract is dissolved 

 in water, for then hydrolysis sets in again with its measur- 

 able rate. 



All this seems to point to the fact that luteidin itself may 

 be an inactive substance, the active component being freed 

 only by hydrolysis. This is not surprising, since it is well 

 known that substances strongly active physiologically are 

 often inactivated by the body through coupling to another 

 substance before being secreted into the urine. Thus, estrone 

 is secreted in the form of inactive estrone-sulphate, and estriol 

 in the form of glucuronidate. 



The identity of the active component of luteidin, how- 

 ever, has not yet been determined. Actually, of all the steroids 

 examined, only allo-pregnanediol, 3-20 produces a curve 

 resembling in any way that of hydrolised luteidin; a latency 

 period of 2 hours, linear growth for 5 Yz hours; the maximum 

 of growth, however, is only about 3,2 A.U., and is attained 

 only with quantities above 225 7. 



Well then; a urine, 2 ml of which already produces 

 maximal growth would have to contain 500 times this 

 quantity, or 1 10 mg allo-pregnanediol per litre, whereas the 



