96 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 



Materials and Methods 



Litters of Wistar strain rats were di\ided into two "roups. The individuals 

 of one group received 750 r of x-irradiation to the head only (the pituitary 

 was irradiated as well as the brain ) at 2 days of age and were kept for vary- 

 ing lengths of time before sacrifice. Individuals of the other group served as 

 litter-mate controls. 



At sacrifice (by decapitation), the brains of all rats were separated into 

 three precisely resolvable parts: brain stem, cerebellum, and cortex. Each 

 sample was frozen and stored in a freezer. On thawing, the total wet weights 

 of the organs were determined, as were total water contents, total solids, 

 total nitrogens, total lipids, and total phospholipids. Selected samples were 

 analyzed for wet volumes of nuclei, mitochondria, and microsomal fractions; 

 total lipids were extracted and their fatty acid profiles obtained by gas 

 chromatography. 



Water content was determined by disintegrating the thawed tissue in a 

 piston-type disintegrator, adding acetone to a weighed sample of the wet 

 mash, evaporating thrice under a stream of nitrogen at room temperature 

 (Sperry, 1955), and then drying in an Abderhalden apparatus over boiling 

 water for 3 hours in the presence of PO5. Water loss was measured gravi- 

 metrically. Total lipid was extracted from the dried solids by addition of 

 10 ml of a 2:1 chloroform-methanol mixture. The extract was pvnified by 

 Sperry's modification of the procedure of Folch-Pi rf al. ( Spen7, 1955), the 

 total lipid being measured gravimetrically. Total phospholipids were deter- 

 mined gravimetrically by extracting the total lipids with 15.0 ml of acetone 

 to which one drop of saturated MgClj had been added. Nitrogen analyses 

 on the solid residue remaining after lipid extraction was done by the micro- 

 Kjeldahl technique. 



Subcellular components were isolated by differential centrifugation, and 

 measurements of wet volumes of the fractions were carried out as detailed in 

 a previous publication ( Schjeide, d al., 1960). 



Gas chromatography of the fatty acids derived from nuclei and mito- 

 chondria^ was performed on the Model 10 Barber-Coleman apparatus, using 

 a 50-in. column packed with commercially obtained ethylene glycol suc- 

 cinate at 170°C. The detector was of the argon-ionization type. A 3 /aI 

 aliquot of 2.5% in petroleum ether solution was injected into the column 

 for each analysis. 



' The methyl esters of these fatty acids were prepared by solubilizing the total lipid 

 in 1 ml of dried benzene, placing this in a 15-ml glass-stoppered centrifuge tube to 

 which was added 2 ml of 4% anhydrous methanolic HCl, refluxing 4 hours over 

 methanol, extracting the pentane-soluble lipids in 10 ml of pentane, washing thrice 

 with distilled H2O, shaking with magnesium sulfate (to remove residual H:;0), and 

 finally absorbing away the nonesterified lipids on powdered alumina. 



