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AIM: Analysis of long-term outcomes of carriage of hepatitis B virus surface antigen (HBsAg). MATERIAL AND METHODS: Complete clinical, device, serological and partial morphological examinations of patients with latent form of HBV infection were performed 16-22 years after the infection with HBV. RESULTS: Progression to chronic viral hepatitis (morphologically--hepatitis B) with a trend to annular hepatic tissue rearrangement was stated in all the examinees. Despite the development of chronic hepatitis, serological profile and PCR indicated HBV elimination. CONCLUSION: All patients with latent HBV infection need regular observation because in the absence of symptoms worsening of clinico-biochemical and morphological indices takes place. Therefore, these patients should receive specific antiviral therapy.

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Glycogen content was determined in hepatocytes of different lobule zones of the normal human liver (23 patients without any liver pathology) and the liver of patients with chronic viral B hepatitis (30 patients) and chronic alcohol hepatitis (28 patients). All the patients were males and aged between 17-50 years. Quantitative analysis of the glycogen content in hepatocytes of portal and central lobule zones was carried out in sections of the human liver (material of functional biopsies) stained with PAS-reaction. The measurements were carried out using an image analyser 'Magiscan' which allows combined cytophotometric analysis of a substance in cells and determination of the cell localization in tissue. The results showed significant differences of the glycogen content in different lobule zones in the normal liver and in the liver in chronic viral and alcohol hepatitis. Ratios of glycogen content in hepatocytes of the portal and the central zones of liver lobule were 1.128 +/- 0.004 and 1.061 +/- 0.003 in normal human liver, and liver of patients with chronic viral hepatitis respectively, i.e. the glycogen content in hepatocytes of the portal lobule zone was much higher than in the central lobule zone in the normal liver and in the liver of patients with chronic viral B hepatitis. The ratio in patients with chronic alcohol hepatitis was less than 1.0 (0.930 +/- 0.003), i.e. a significantly higher glycogen content was found in hepatocytes of the central liver lobule zone. Possible mechanisms of this phenomenon are discussed. Thus, the pattern of the glycogen content in hepatocytes of different lobule zones can be used as an indicator of etiology of chronic hepatitis.

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Investigation of glycogen function in hepatocytes of different liver lobule zones is particularly important in understanding glycogen metabolism in humans and animals in norm and pathology. The present study was done to investigate glycogen contents in hepatocytes of different lobule zones of human liver in norm, and in patients with chronic hepatitis of viral or alcohol etiology. Quantitative analysis of glycogen content in hepatocytes of portal and central lobule zones was conducted on slices of human liver (the material of series live punctional biopsies) stained using a quantitative variant of PAS-reaction (Kudryavtseva et al., 1970, 1974). The measurements were done by image analyzer <>, which allows to make jointly cytophotometric analysis of substance in cells and definition of cell localization in tissue. The results showed clear differences of glycogen contents in different lobule zones in normal liver and in liver during chronic viral and alcohol hepatitis. Glycogen contents in hepatocytes of portal lobule zone were significantly higher than in the central lobule zone in patients with chronic viral hepatitis. Opposite data were obtained in patients with chronic alcohol hepatitis. Significantly higher glycogen contents were found in hepatocytes of the central liver lobule zone. Possible mechanisms of such a phenomenon are discussed . Thus, if glycogen contents in hepatocytes may be taken as an indicator of liver chronic damage degree (as has been shown elsewhere: Kudryavtseva, 1987; Kudryavtseva et al., 1988) the pattern of distribution of hepatocytes with different glycogen content in the liver lobule can be used as an indicator of etiology of chronic hepatitis. The obtained data seem to be important and actual, particularly for diagnostic of subclinical and symptomless forms of these diseases. Further investigation is required to find out reasons and mechanisms of this phenomenon.

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Reversibility of hepatocyte functional activity is shown by cytofluorometric and microbiochemical methods in human and rat liver during postcirrhosis rehabilitation. Contents of the total glycogen and its fractions in liver cells were defined on smears of isolated hepatocytes obtained from the live puncture liver biopsies. A double increase of glycogen level is shown, in average, in hepatocytes during experimental liver cirrhosis in rats. At the same time, a relative content of the hard soluble fraction of glycogen increases by 5-8 times. The glycogen level falls to reach the norm already within one month after shutting off the pathogenic influence. However, in some animals after 6 months this level becomes even lower than the normal one. Again, the ratio between the hard soluble fraction and light one remains. In men with cirrhosis glycogenosis of hepatocytes can be expressed in greater degree (increase by 4-5 times): it depends on the illness heaviness. Further changes in glycogen content depend on the pathological process development. Under experimental cirrhosis the activity of glucose-6-phosphatase decreases by 4.3 times. Within one month after stopping the pathogenic influence the activity of this enzyme increases by 3-4 times, but later, in 6 months, it decreases to reach 55-65% of the norm. No actual changes were observed in the activities of other enzymes.

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Levels of hepatocyte ploidy were investigated in patients with chronic viral hepatitis, alcohol hepatitis, and liver cirrhosis. It was shown that in spite of some decrease in the share of mononucleate diploid hepatocytes these cells equally make the main part of hepatocyte population (about 80% on the average) both during chronic liver diseases and in the norm. In chronic hepatitis and cirrhosis processes of polyploidization in the human liver, unlike those in livers of laboratory animals (rats and mice), are accomplished with much lower rates and characterized by accumulation of binucleate diploid cells. These processes differ from those occurring under ageing. Reparative regeneration of human liver under chronic hepatitis and cirrhosis is accomplished on the whole by proliferation of mononucleate diploid hepatocytes but processes of polyploidization in the liver of some patients can play a significant role in reparative liver growth.

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By cytofluorometric method, a study was made of the total glycogen and its two fractions in liver parenchymal cells both in the donors (20 men) and in patients with cirrhosis of different etiology (39 men). The examination was performed on preparations--smears of isolated hepatocytes, obtained from the live functional liver biopsies. The quantitative analysis has shown an increase in the total glycogen content in hepatocytes of patients with cirrhosis by 3 times compared to the norm, and this increase is independent on the etiology of liver cirrhosis. To study the mechanism of the discovered glycogenosis, the activity of key enzymes of glycogenolyses was determined. It was shown that glucose-6-phosphatase and glycogen-phosphorylase activity in the liver with cirrhosis was lower than in the norm. The most considerable changes were shown in hepatocytes of patients with liver cirrhosis in fractional glycogen composition and, even more significant, in the content of a hard soluble fraction. The hard soluble fraction portion was higher in hepatocytes of the patients with liver cirrhosis of alcohol etiology. The quantitative analysis of glycogen fraction contents in liver cells may be the best marker in the differential diagnosis of symptomless elapsing liver cirrhosis.

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By cytofluorometry employing the cytofluorometric PAS reaction, a study was made of the total glycogen and of its two fractions in liver parenchymal cells, both in the norm and in patients with chronic alcoholism (alcoholic steatosis, chronic alcoholic hepatitis, and mixed forms of alcoholic-viral hepatitis, viral hepatitis with steatosis and also viral hepatitis). The examination was performed on preparations-smears of isolated hepatocytes, obtained from the live puncture liver biopsies. The quantitative analysis has shown the increase in the total glycogen content in hepatocytes of patients with alcoholic hepatitis in comparison with the norm and with chronic viral hepatitis. The transition from a reverse stage--alcoholic steatosis--to alcoholic hepatitis was accompanied by a sharp increase in the total glycogen content and by an obvious change in the ratio of glycogen fractions, towards the hard soluble fraction in liver cells. The quantitative analysis of glycogen fractions in liver cells of patients with chronic alcoholic disease may be an appreciated marker of differential diagnostics of different stages and forms of alcoholic liver disease.

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A cytofluorometric study was made of total glycogen and two glycogen fractions--the one easily soluble (ES) and the other hard soluble (HS) in isolated liver cells (needle aspiration biopsy) of patients in the norm and with chronic viral hepatitis (CVH) and chronic alcoholic hepatitis (CAH). The amount of LS in hepatocytes of patients with CAH was lower than that in patients with the norm or with CVH. This distinction was shown already at the beginning of chronic disease, and then, in spite of a considerable increase in the total glycogen content in hepatocytes, with progression of the disease did not change. The quantitative analysis of glycogen fraction contents in liver cells may be an additional differential diagnostic marker for the etiological distinction of chronic hepatitis.

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Dry weight of hepatocytes of 44 patients both normal and suffering from chronic hepatitis was measured interferometrically. The hepatocyte dry weight of the normal liver was about 431 pg. With the progression of the disease, this weight increased up to 640 pg. The dry weight concentration in this case did not change. The dry weight of hepatocytes increases proportionally to the polyploidy degree both in the norm and at chronic hepatitis. It was shown that cell hypertrophy not associated with cell proliferation and polyploidy is of great importance for the reparative liver growth. In patients with strong liver disease, it was increased by 30% in comparison with the normal condition.

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DNA contents in mono- and binucleated hepatocytes have been measured using cytofluorimetry on slides of isolated human liver cells. In all, 28 patients, with the mean age of 33.7 years (ranging from 17 to 50 years), were examined, including both healthy (normal) ones and those with chronical hepatitis. The normal livers were shown to include approximately 88% of mononucleated diploid hepatocytes, and 9% of binucleated hepatocytes with diploid nuclei, whereas mononucleated tetraploid cells, binucleated cells with tetraploid nuclei and mononucleated octaploid cells made all together only a bit slightly more than 3%. The patients with hepatitis displayed markedly increased numbers of binucleated hepatocytes with diploid nuclei; some of these having more than 40% of such cells. The number of mononucleated tetraploid hepatocytes, binucleated hepatocytes with tetraploid nuclei and of mononucleated octaploid hepatocytes was seen to increase very slightly not reflecting the degree of the liver disease. It has been proposed that the normal and the reparative growth of human liver is accomplished due mainly to mitotic divisions of mononucleated diploid hepatocytes; the role of polyploidy in these processes being insignificant.

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Using cytofluorimetry, glycogen amount was measured in hepatic parenchymal cells on stained slides made of biopsied liver tissue taken from patients with chronic hepatitis. The glycogen content has been shown to increase progressively with the progression of the disease (by almost 3 times); the population of hepatocytes becoming more and more heterogenous as to their glycogen level. Simultaneously some typical morphological changes of hepatocytes were also found: increased dencondensation and vacuolization of the cytoplasm, a loss of cytoplasmic and nuclear boundaries. It is suggested that the glycogen content and morphological features of hepatocytes may be indicative as criteria of the hepatic disease dignosis.

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