APPLICATION OF SCHIFF'S REAGENTS WITH VARIOUS SPECTRAL CHARACTERISTICS TO DETERMINE THE CONTENT OF LABILE AND STABLE
GLYCOGEN FRACTIONS IN ISOLATED HEPATOCYTES
N. N. Bezborodkina, E. I. Kirshina, E. V. Mushinskaya, B. N. Kudryavtsev
Institute of Cytology RAS, St. Petersburg;
e-mail: cellpath@mail.cytspb.rssi.ru
The dynamics of the total glycogen accumulation, its labile and stable fraction contents were studied in hepatocytes after administration of 30 % glucose to rats that
had been starving for 48 h. In the study, the original method based on the use of Schiff's reagents with various spectral characteristics
(auramine-SO2 - Au-SO2 - and ethidium bromide-SO2 - EtBr-SO2) was applied allowing detection and quantitative
determination of the labile and stable glycogen fractions in the same hepatocytes to be achieved. Staining of the preparation by Au-SO2 during 40 min revealed
the labile glycogen fraction (green fluorescence, λmax = 526 nm). The following staining of the same sample by EtBr-SO2 during 50 min
revealed stable glycogen fractions (red fluorescence, λmax = 620 nm) in the cells. The measurement of fluorescence intensity in the corresponding
spectrum permits the quantity of each glycogen fractions in isolated liver cells to be determined. It has been shown that as the gly-cogen accumulates in the hepatocytes
its labile fraction content increases. The correlation coefficient (r) between the labile fraction content and the total glycogen content in hepatocytes obtained from the
liver of starving rats was 0.95, and in hepatocytes isolated 20 and 30 min after the beginning of glucose refeeding to starving rats it was 0.95 and 0.98, respectively. 90 and
120 min after administration of glucose to starving rats, when the hepatocyte population becomes non-synchronous with respect to synthesis and degradation of glycogen
in cells, the dependence of the labile fraction content upon the quantity of the total glycogen becomes less expressed (r = 0.71 and 0.82, respectively). Thus the
application of Schiff's reagents with various spectral characteristics enables us to obtain valuable information about glycogen structure in cells and about the processes
of its synthesis and degradation in the cell population.
Key words: glycogen fractions, hepatocytes, auramine, ethydium bromide, Schiff's reagent
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