Ultracytochemical Detections of Some Lipids in Tissues by Enzymic Digestive Methods

Abstract

Some purified esterases for lipids, e. g. mould lipase, snake venom phospholipase A², cholesterol esterase from microorganism, were applied for the analysis of smaller fat granules in ultrastructure. The enzymes were purchased from Böhringer-Mannheim Co., and another mould lipase was donated from Drs. Tsujisaka and Iwai of Osaka Municipal Technical Research Institute. Some organs containing various amounts of lipids in rats, e. g. early and recovery stages of the Ethionine-induced fatty liver in females, the liver of fasting females, the Gentamicininjured renal cortex, the lactating mammary gland and the adrenal cortex of resting males, were perfused with a heparin-containing saline for 2 min and subsequently with 1/2 Karnovsky fixative for 3 min. Both tissue blocks, sized 0.5-1cm³, from the perfused organs and the atheromatous focus of human aorta were incubated in cold Karnovsky fixative for 1-2 hr, washed in cold cacodylate buffer with sucrose overnight, cut to 40μ-thick sections by Vibratom. The free-floating sections were treated in the following digestion media, respectively, at 37°C for 1/22 hr, while the medium was usually shaken rhythmically on the machine. The digestion medium was composed of 4m1 of 0.05 Tris buffer,1 ml of 2% calcium chloride and an enzyme. The enzyme was added as follows; 0.075 ml of lipase of Drs. Tsujisaka and Iwai at pH 7.2 for triglycerides detection,0.5 ml of snake venom phospholipase A2 of Böhringer at pH 7.5 for phospholipidsdetection, or 0.21 ml of cholesterol esterase of Böhringer at pH 7.6 for cholesterol esters detection. Duplicate control sections were treated in the reaction medium without enzyme. After treatment the sections were washed in cold distilled water, immersed in 0.1% lead nitrate for 15 min, washed again thoroughly in distilled water, post fixed in cold osmium buffer for 1.5 hr, dehydrated in cold graded ethanols according to Idelman's procedure, and finally embedded in epoxy resin. The ultrathin sections with or without metal staining were observed under an electron microscope. Derived fatty acids after the digestion with a specific enzyme were detected as lead soaps which had not been seen in the duplicate control section. These reaction products should indicate the location of the substrate lipid. Derived fatty acids could be also observed by means of Holczinger's method for fatty acids under a light microscope, when both pretreatments of removal of fatty acids and calcium in tissues should be done.
Many co arse reaction products for triglycerides could be seen on newly appearing and disappearing fine lipid materials within the hepatic cells. Those for phospholipids revealed in some cytosegresomes of renal cortex, milk fat globule membranes, and biomembrane system of the examined cells, More trials are requested for ultrastructural detections of cholesterol esters, but the reaction products after cholesterol esterase digestion could be seen in adrenal cortical cells and macrophages in atheromatous foci. Both utility in histochemical analysis for the mixed phase of lipid spheres and limitation depending on the size of lipid particles were discussed on the ultracytochemical observations by this enzymic digestive method.