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The distribution pattern of rat liver parenchymal cells of different ploidy classes has been investigated with isolated liver cell preparations on four different time points of the day. The percentage distribution of different ploidy populations was found to fluctuate along the activity-rest cycle of the animals. The mononuclear tetraploid (MT) cell population showed the strongest time-dependent distribution pattern with a 10% variation around the 24 h mean, the variations in the binuclear diploid (BD) cells being somewhat less obvious, but clearly present. The mononuclear diploid cells and the binuclear tetraploid cells occurred in a virtually constant percentage. An inverse correlationship was noted between MT and BD cells, the increase in the number of MT cells being accompanied by a decrease in the number of BD cells. This interdependency in the presence of the two cell types can be related to their relationship in the process of formation.

A recently developed method for the (quantitative) demonstration of glucose-6-phosphate dehydrogenase activity in individual cells with the use of a polyacrylamide carrier has been extended for other enzyme cytochemical techniques. Isolated hepatocytes have been incorporated in the matrix of a thin transparent polyacrylamide gel prior to incubation in a cytochemical medium. The techniques which have been applied are the synthetizing reaction technique for glycogen phosphorylase, the indigogenic method for nonspecific esterase, the metal salt method for glucose-6-phosphatase, the post-azo-coupling technique for acid phosphatase, and the tetrazolium salt technique for succinate and lactate dehydrogenase activities. In all cases a few major problems which occur in the cytochemistry on single cells seem to be solved. The morphology is very well preserved, the final reaction product seems to be precipitated at the expected site of enzyme activity and the coloured end-product is highly specific for the enzyme activity to be studied, as has been demonstrated well with control experiments. The conclusion is reached, therefore, that this relatively simple device can be used routinely for the optimalization of enzyme cytochemistry of single cells.

The time-dependent variation of glucose-6-phosphate dehydrogenase (G6PDH) activity during 24 h has been investigated by cytophotometric analysis in cytochemically stained, isolated hepatocytes of different ploidy classes from adult male rats. It was found that the amplitude of the variation in the total amount of parenchyma was 22%, with a maximum in the middle of the resting phase of the animals (13%). This small variation could not be detected subjectively in cryostat sections of liver that had been stained histochemically; nor could a metabolic zonation of the liver parenchyma be observed except the intermediate zone, which stained more strongly for G6PDH activity at 13% and 22%. The quantitative study on the isolated hepatocytes, however, revealed a relatively strong variation of 48% during the day in enzyme activity of the mononuclear diploid cells and no significant variation in the binuclear tetraploid cells. The cells of the in-between ploidy classes showed an amplitude of variation of 38% (binuclear diploid cells) and 24% (mononuclear tetraploid cells). All cells showed a maximum activity at 13% and a minimum activity during the night. It is concluded that this ploidy-dependent variation might play a role in the generation of pentoses for RNA synthesis. The findings indicate that for metabolic studies the heterogeneity of the liver parenchyma should be taken into account with respect to the different ploidy classes, as well as the well-known metabolic zonation of the acinus.