Mitochondria Isolated from Rat Brown Adipose Tissue and BY J. D. LEVER, M~D, AND J. B. CHAPPELL, PH.D

Mitochondrial fractions, relatively free from contsmination by other cytopla-~mic structures, have been isolated by differential centrifugation from homogenates of brown adipose tissue from starved rats. It was possible in such fractions to distinguish two types of mitochondria in this tissue. Type I mitochondria, when morphologically intact, are limited by a bilaminar membrane and show regular parallel cristae. In isolated fractions, a proportion of these mitochondria are swollen, vacuolation occurring within the cristae between their limiting membranes. Type II mitochondria are distinguished from the more numerous type I bodies by the opaque appearance of their matrix. They are limited by a membrane which is in part single, and in part double. They show a few, but crisply outlined internal membranes. Vacuolation of this type of mitochondrion has not been observed. Vacuolation comparable to that in brown fat mitochondria was also observed between the two ]aminae of the enclosing membrane and within the cristae of liver mitochondria. Liver The cells of the brown fat tissue of starved rats are almost free of lipide droplets and are tightly packed with mitochondria (1). Extremely regular internal cristae and obvious bilaminar limiting membranes are apparent in electron micrographs of these mitochondria. Since the endoplasmic reticulum in brown adipose tissue occupies a small part of the cell volume and is relatively inconspicuous, it was considered, therefore, that this tissue would lend itself to the isolation of relatively pure mitochondrial fractions, free from microsomal contamination. This paper describes the appearance of the mitochondria isolated by differential centrifugation from brown fat tissue and makes comparison with corresponding studies on liver preparations. In the present study two types of brown fat mitochondria have been observed in vitro. Materials and Methods The material for this investigation was removed, under ether anesthesia, from the subcutaneous interscapular mass of brown fat of albino rats (1) starved for 7 to 10 days; and from the normal rat liver. For the study of the whole tissue small pieces were cut from the main mass, which was used for the isolation of mitochondria, and immediately fixed in Dalton's (2) buffered saline---1 per cent osmic acid solution for 1 to 2 hours at 5°C. After brief washing, dehydration in ethanol, and embedding in methacrylate, thin sections (silver and gold colored) were cut with a Porter-Blum microtome. Mitochondria were isolated from the adipose tissue by homogenization in 0.44 ~-sucmse and differential centrifugation, employing a method which has been elaborated by Chappell and Greville (3) for rat liver mitochondria. The once-washed particles were sedimented by centrifugation to form a thin pellet, which was then fixed and examined in the same way as was the intact tissue. A Siemens electron microscope was used. OBSERVATIONS AND DISCUSSION Brown Fa~ Mitoch~ndria: One of the most satisfactory results of the present study has been the reproducible production of mitochondrial fractions relatively free from microsomal and other detectable contamination (Fig. 4). However two varieties of mitochondria