908 ADVENTURES IN RADIOISOTOPE RESEARCH 



days in the labile portions of the epiphysis, the rest being found in the 

 stable portion. The estimation of such magnitudes encounters great 

 difficulties in view of the very complicated way in which the labeUing of 

 the bone tissue takes place. The degree of renewal of the mineral consti- 

 tuents of the different parts of the skeleton which takes place within 

 a time interval can, however, be determined in the way described on 

 p. 204. The degree of renewal in these experiments means the percentage 

 of bone tissue newly formed once or several times since the start of 

 the experiment. 



RATE OF RENEWAL OF THE FISH SKELETON 



We investigated the rate of renewal of the skeleton of sticklebacks (Gasterosteus 

 ■actileatus). These fish, weighing 1 — 3 gm have a lifetime of about one year and 

 can be expected not to grow any longer when one year old. Their small size has 

 the advantage that the use of a large sea-water volume and, thus, an unduly 

 large amounts of labelled phosphate, can be avoided. Our experiments, in which 

 the sticklebacks were kept for up to six weeks in labelled sea-water, were carried 

 out with radio-phosphorus having an initial activity of 0.05 millicurie, which 

 was found to be ample to give an active skeleton. 



The small size of the fish facilitates, furthermore, their ashmg, which is to be 

 carried out when we want to determme the total phosphorus content or the total 

 32P content of the fish. Wet ashing was carried out by heating with 1 ml of cone, 

 sulphuric acid containing some nitric acid and, in the last phase of the experiment, 

 some hydrogen peroxide. 24 sticklebacks were kept in 3 htres of sea- water to 

 which 10 ml of a radioactive solution containing 0.05 millicurie and 3.4 mgm of 

 sodium phosphate (pH — 7.6) were added. After the lapse of 6 weeks, this acti- 

 vity declined to Vs of its initial value. The water was daily renewed, as was its 

 labelled phosphate content. 



The sticklebacks were investigated at different intervals. After killing the fish 

 and washing it very carefully with sea-water, the liver was taken, and the "free" 

 phosphate was extracted from it with cold 5 per cent trichloracetic acid. The solu- 

 tion was then brought up to 25 ml. While the "free" phosphate content of 20 ml 

 was precipitated as magnesium ammonium phosphate, the activity of which was 

 measured, the residual part used in a colorimetric determination of the free P 

 content of the extract. The activity measurements are much simplified when 

 the samples have about the same weight. To obtain such samples, we added to 

 the above mentioned 20 ml so much sodium phosphate that the precipitate obtained 

 weighed 60 mgm. As mentioned above, the determination of the rate of renewal 

 is hased upon the comparison of the activity of 1 mgm of free plasma P and 1 mgm 

 of mineral skeleton P. It is, however, extremely difficult to secure blood from 

 fish weighing a couple of grams and, therefore, we replaced the determination 

 of the activity of the plasma P by a determination of the activity of the free liver P. 

 In view of the great ease with which phosphate ions penetrate the liver cells and 

 vice versa, the activitv level of the free phosphate P of the liver differs not much 

 from the activitv level of the free phosphate of the plasma. The writer is much 

 indebted to Mr." Tryggve Gustavson for his very effective help m removing 

 the hvers, weighing 45 — 70 mgm. 



To remove the organic constituents of the skeleton, we treated the bones with 

 boiling glycol containing 6 gm KOH per 100 ml for several hours, until the bones 



