§ 7 THE LUTEIDIN PROBLEM 43 



present in 2 ml urine in a quantity of about i o 7. This quite 

 explains the low activity of this fraction. 



After an alcoholic solution had been made of the neutral 

 butanol fraction, we found that, when this had been left un- 

 touched for a week-end at a fairly low temperature, crystals 

 had separated from it, which could be burned without leaving 

 any residue, began to frizzle when heated to 250^ C, and 

 melted, whilst disintegrating, at 255 — 260° C. After back- 

 crystallisation the melting point was 264° C. These pro- 

 perties point in the direction of Na-pregnanediol-glucuronide, 

 which, in the pure state, melts at 268 — 271° C. No signif- 

 icant biological activity was expected of this substance; we 

 therefore first tested the solutions left over. The result was 

 that both the neutral and the acid fraction as well as the layer 

 of water remaining during the operation were tnactivel This 

 result surprised us not a little, for evidently the active element 

 had disappeared from the extract, so active during the initial 

 experiments. The only possibility, to our minds, was that the 

 isolated pregnane-complex, against all expectations, should 

 prove to be active all te same. And indeed, during a pro>- 

 visional test with 3 mg, a growth of 5,5 A.U. was observed, 

 with a latent period of 5 >^ hours. When fresh fishes were 

 put in the same water a few days afterwards, the growth was, 

 if possible, a little greater; the latent period, however, had 

 gone back again to 2 hours. The quantity of material used 

 corresponds to 2 mg pregnanediol, which, according to 

 DUYVENE DE WiT, should produce a growth of 3 A.U., but 

 only after 10 hours, including a latent period of i hour. 



We are therefore faced with the question; what is the 

 reason why this pregnanediol-glucuronide which we have 

 isolated is so much more active than pregnanediol itself, with 

 which we are familiar? It was evident from a few experi- 

 ments that glucuronic acid is completely inactive in the ovi- 

 positor test, and cannot, therefore, be held accountable for 

 the marked increase in activity on the part of pregnanediol. 

 The only way to find the answer to the above question is, 

 therefore, by isolating the pregnane-complex in sufficient 

 quantity and examining it as to its properties, after adequate 



