806 ADVENTURES IN RADIOISOTOPE RESEARCH 



carbon fragments find their way into cholesterol, steroid hormons, 

 hemin and fatty acids on one side and into citric acid, glutamic acid, 

 proline, arginine on the other side. The suppression of any of these paths 

 may lead to an increased i*C incorporation in the metabolic products 

 involved in the remaining paths. 



EXPERIMENTAL 



The X-ray tube applied, was run with 180 kW and 7 milliamp. The distance 

 between the anticathode and the surface of the paperbox in which the mice were 

 placed was 40 cm. 



Adult mice were irradiated for 3 min. with a dose of 295 r per min. The mice 

 had access to food aU through the experiment. They were injected intraperitoneaUy 

 or, in some cases, subcutaneously mostly with 0.1 ml of physiological sodium 

 chloride solution containing in the carboxyl group labelled sodium acetate of an 

 activity of 2 to 6 microcuries. In some experiments, injection with acetate took 

 place immediately after irradiation, in others up to several hours later. At least 

 10 mice were irradiated in each experiment. The mice were killed by decapitation. 

 In most experiments blood plasma, hver, kidneys, intestinal mucosa, muscles 

 and brain, is some cases femur, lungs and spleen were secured as weU. The intestines 

 were carefuUy rinsed before securing the mucosa. An ahquot of the organs was 

 dried and the activity of the samples of the pooled organs of irradiated mice and 

 that of the controls compared. From another aliquot of the organs the total fats 

 were obtained by extracting the ground organs with a boiling mixture of ether- 

 alcohol (1 : 3) for 3 hr. The filtrate obtained was evaporated in vacuo and the 

 residue extracted with petroleum-ether. The evaporation of the petroleum-ether 

 gave the total fats. In our later experiments, these were purified with coUoidal 

 iron foUowing the procedure of Folch and Van Slyke as described below. 



In order to obtain the phosphatides, the total fats were dissolved in petroleum- 

 ether and precipitated with twice the amount of cold acetone and 5 drops of an 

 alcoholic solution of 4.5% MgClj, precipitation being completed by standing for 

 2 hr in the refrigeiator. The phosphatides were centrifuged. The precipitate dis- 

 solved in petroleum-ether was reprecipitated in the same way. After centrifugation 

 the precipitate dissolved in petroleum-ether was washed with distihed water. 

 Evaporation of the petroleum-ether gave the phosphatides. 



The supernatants from the precipitation of the phosphatides were combined and 

 evaporated. The residue was saponified by boihng for 8 hr with 10 cc. of an aqueous 

 solution of 40% KOH and 20 cc. alcohol. After saponification the solution was 

 extracted several times with petroleum-ether. To the petroleum-ether solution 

 after being brought to a small volume twice its volume of a 0.5% solution of digi- 

 tonin in 80% alcohol was added. The quantity of digit onin was about 5 times the 

 amoimt of cholesterol expected. The precipitated cholesterol was first washed 

 with 80% alcohol to ehminate the excess of digitonin, then with a mixture of 

 acetone-ether (1 : 2) and finally with ether; it was dried at 37°. ^^^ 



When investigating the effect of irradiation on cholesterol formation, we com- 

 pared the activity of cholesterol digitonin samples. The presence of digitonin 

 reduced the activity of cholesterol to about to 1/4 of its value, conversion of cho- 



(i^ P. A. SvEBE, I. L. Chaikoff and W. G. Dauben, J. Biol. Chem. 176, 829 

 (1948). 



