Abstract
Both the hepatic tissues and their microsome fractions of adult female rats, which were sacrificed either after 18-hr fasting or 3-6 hr after an intraperitoneal injection of ethionine, lg/kg, to the 12-hr fasing animals, were observed ultracytochemically with the enzymic digestive methods using purified esterase preparations for lipids. In addition to the prefixed hepatic tissue blocks of the above-mentioned animals, the prefixed pellets of microsome fraction prepared from each hepatic tissue (ca 5g) in Karnovsky's fixative for 1 hr were washed in cacodylate buffer with sucrose overnight and cut to 40 μ-thick sections by vibratome. The thick sections were digested with a mold triglyceride lipase, snake venom phospholipase A2 and bacillus phospholipase C, respectively, at 37°C for 30 min. After digestion, the sections were washed, immersed in 0.1 % lead nitrate for 15 min, washed thoroughly, postfixed in 2% osmium tetroxide, dehydrated and embedded in epon. The ultrathin sections were usually stained doubly by uranium acetate and lead citrate. The reaction products through the enzymic digestive method might indicate the site of substrate with electron microscopy, as compared with the duplicate control section incubated in the reaction medium containing no enzyme preparation.
Membrane-bound liposomes carrying the products through lipase digestion appeared in the liver cells 3 hr after the injection, and they increased in number and size 6 hr after the injection. In surfaces of the developing liposomes the reaction products in either phospholipasedigestive method could be detected. Independent of the development of liposomes, some minute structures carrying the products through phospholipase digestion were observed scatteringly in the cytoplasms of liver cells. They exhibited either round bodies in the lumen of biomembrane system of phospholipase A2-digested materials or comma-shaped features on the biomembrane of phospholipase C-digested materials. Therefore, such minute structures as observed only in the phospholipase-digested materials would be resulted from the digestion of biomembrane phospholipids.
