TURNOVER OF PHOSPHATIDES 353 



of BoLLMAN et aJ. are calculated both according to their and our 

 method, almost identical figures being obtained in l)oth cases. 



The method of calculating the experimental results can thus not 

 be responsible for the difference in the renewal percentage obtained 

 by different workers and we have thus to consider other explanations. 



As shown in the present communication, the ratio of the specific 

 activities of the orthophosphate P and phosphatide P of the liver varies 

 with the age of the rat, and these variations can be assumed to be at 

 least partly responsible for the discrepancies mentioned al)Ove<^\ 



EXPERIMENTAL 



To each of more than 100 rats of known age, kept on normal diet, 0.1 ml physiol. 

 NaCl solution containing 32p of 2 /(curie activity and a negligible ^ip content 

 was administered by subcutaneous injection. Two hours later, batches of 4 or 

 irioro rats were pooled, the animals were killed by decapitation, bled, and the 

 isolated organs frozen with sohd COg. An aliquot of the liver, spleen and kidney 

 samples was used to determine the specific activity of the inorganic P, another 

 for the total P, while a third was used for the determination of the specific activity 

 of phosphatide P. In the case of the Uver, the labile P of ATP was investigated 

 as well. All organs were cut into small pieces and average fractions were obtained. 

 To measure the inorganic P values, fractions (0.2 — 0.3 gm) were extracted with 

 cold CCI3COOH. The total P was obtained by wet ashing of about 0.2 gm fresh 

 tissue, while a few gm were used for the extraction of phosphatides. 



Before extracting phosphatides the tissue was treated with 200 ml acetone 

 for 15 minutes. The filtrate was dried in a COg atmosphere and the residue extracted 

 with ether. The acetone treated tissue was extracted by grinding it in a mortar 

 twice with 150 ml ether and once with 1 : 3 ether-alcohol mixture for 15 minutes. 

 The residue was then extracted for 8 hours in a Soxhlet flask with 150 ml boiling 

 ether-alcohol mixture (1 :3). All ether and alcohol fractions were united and diied 

 in a CO2 atmosphere. To free the phosphatides from traces of inorganic P and 

 other P compounds the residue was dissolved in 300 ml ether and shaken with 

 450 ml 0.1 n HCl -|- 0.01 n NaCl solution in a separating funnel. This procedure 

 was repeated four times, as suggested by Hahn and Tyren (1945). 



The ethereal solution was evaporated in a Kjeldahl flask and then ashed by 

 a mixture of HgSO. and HNO3. An idiquot of this solution was used for colorimet- 

 ric determination and another precipitated as magnesium ammonium phosphate 

 and its radioactivity determined. 



To determine the specific activity of the labile P of ATP about 8 gm of liver 

 tissue were extracted with 3 volumes cold CCI3COOH solution. The cooled filtrate 

 was neutrahzed to phenolphtalein with cooling by adding solid Ba(0II)2. 



The precipitate containing adenosine triphosphate, adenosine diphosphate, 

 orthophosphate, and some other minor fractions of organic P compounds was 

 washed with a little Ba(0n)2 and neutralized with CClgCOOII. Subsequently 

 it was dissolved in 15 ml n HNO3. To the solution, as suggested by Sacks and 

 Altschuler (1942), NH^NOg was added until a concentration of 5 per cent was 



(1) That the spec, activity of the liver P of mice declines from 3.7 to l.G when 

 the age increases from H to 24 weeks was observed by Falkenheim (1943). 



23 Jievesy 



