Abstract
Two methods are described for the study of adipose tissue cellularity and metabolism. In the first, 8 M urea was used to liberate osmium tetroxide-fixed adipocytes from the connective tissue matrix. In the smaller-sized cell ranges there was a significant reduction in apparent adipocyte number of rat, pig, and beef adipose tissue with 8 M urea treatment. This was attributed to solubilization of connective tissue debris that was counted as adipocytes in samples isolated without urea. There was no effect on the larger cell-size fractions with 8 M urea treatment. Eight molar urea had no effect on fixed adipocyte retention of radioactivity. The second method entailed the use of hydrogen peroxide to volatilize the black, osmium tetroxide-fatty acid complex of osmium tetroxide-fixed adipocytes, containing radioactivity, resulting in colorless lipid suitable for liquid scintillation counting. This latter technique permits incubation of unfixed adipose tissue slices with a radioactive substrate, followed by fixation with osmium tetroxide and subsequent separation of the adipocytes, by screening, into the desired size ranges. Adipocytes in various size fractions can then be counted, sized, and then decolorized with hydrogen peroxide in order to quantitate the amount of radioactivity within the adipocytes. There was no loss of radioactivity from the fixed cells with hydrogen peroxide treatment.
Highlights
Treatment of fixed adipose tissue slices with 8 M urea to liberate fixed adipocytes had a significant effect upon the respective diameter histograms (Fig. 2)
In all three species there was a marked reduction in the apparent numberof cells in the 20-30 p m range with 8 M urea treatment
Non-Urea connective tissue debris being counted as adipocytes
Summary
Summary Two methods are described for the study of adipose tissue cellularity and metabolism. The second method entailed the use of hydrogen peroxide to volatilize the black, osmium tetroxide-fatty acid complex of osmium tetroxidefixed adipocytes, containing radioactivity, resulting in colorless lipid suitable for liquid scintillation counting This latter technique permits incubation of unfixed adipose tissue slices with a radioactive substrate, followed by fixation with osmium tetroxide and subsequent separation of the adipocytes, by screening, into the desired size ranges. In an effort to study metabolic changes associated with cell size, Bjorntorp and Karlsson [5] first attempted to isolate adipocytes of different sizes Their method involved the incubation of human adipose tissue slices with collagenase to liber-. The fixed cells were fractionated by filtration into different diameter ranges prior to quantitation of radioactivity Neither of these methods is adaptable to a larger number of cells, per sample, because of color quenching due to the presence of osmium tetroxide. The osmium-fixed adipocytes were decolorized with hydrogen peroxide in order to quantitate the amount of radioactivity present and its distribution into glyceride-glycerol and fatty acids
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