Changes in the zonation of lactate dehydrogenase activity in lobules of rat liver after experimentally induced colon carcinoma metastases.

Visualization of lactate dehydrogenase (LDH) activity with Neotetrazolium as final electron acceptor under anaerobic conditions and an incubation medium containing polyvinyl alcohol showed that under normal physiological conditions a zonal distribution of LDH activity is present in the liver lobule of male rats. Periportal hepatocytes contain more LDH activity than pericentral hepatocytes. This difference is due to the role of LDH both in gluconeogenesis (periportal cells) and glycolysis (pericentral cells). In livers containing metastases from colon carcinoma, areas of the parenchyma which are not affected by tumour growth maintain such zonation in the lobule, whereas areas close to metastatic foci show increased activity which is distributed uniformly over the lobule. This change may be explained by a Cori's cycle-like relationship between malignant cells and the surrounding hepatocytes due to glucose consumption and lactate production by the tumour cells. Within the metastatic foci, a zonation of LDH activity was also observed. Malignant cells close to the edge of the tumours contained the lowest activity, whereas activity increased inwards. Cancer cells directly surrounding necrotic areas showed the highest activity. Such patterns are in line with increasing anaerobic glycolysis towards the inner metastatic regions. Anaerobic glycolysis supplies limited amounts of ATP with concomitant lactate production but also large amounts of metabolites for RNA, DNA, lipid and complex carbohydrate synthesis. Lactate that is produced by the metastases induces adaptive changes in surrounding hepatocytes to convert this excess of lactate effectively.

The histochemical G6PDH reaction but not the LDH reaction with neotetrazolium is suitable for the oxygen sensitivity test to detect cancer cells.

We used the oxygen sensitivity of the histochemical reaction to detect glucose-6-phosphate dehydrogenase (G6PDH) activity based on neotetrazolium (NT) reduction to discriminate cancer cells from normal cells. Formazan generation was strongly reduced in normal but not in malignant cells when the incubation was performed in oxygen instead of nitrogen. Competition for reductive equivalents between NT and oxygen via superoxide dismutase (SOD) has been suggested. Since SOD activity is usually decreased in cancer cells, NT reduction would not be hampered in these cells. We tested this hypothesis by demonstrating NAD-dependent lactate dehydrogenase (LDH) activity instead of NADP-dependent G6PDH activity in normal rat liver and colon, in human colon carcinoma, and in experimentally induced metastases of colon carcinoma in rat livers. Reactions for both enzymes were determined cytophotometrically in an atmosphere of pure oxygen or nitrogen. G6PDH acted as described previously, showing distinct activity in cancer cells but strongly reduced activity in normal cells after incubation in oxygen, but this was not the case with LDH because formazan was also generated in normal tissue in oxygen. It appeared that after 5 min of incubation at 37 degrees C the residual activity of G6PDH in an atmosphere of oxygen compared with nitrogen was 0% in normal liver tissue and 15% in normal colon epithelium, whereas in colon carcinoma and in colon carcinoma metastasis in liver it was 48% and 33%, respectively. The residual activity of LDH in oxygen was 30% in normal female rat liver, 75% in normal male rat liver, and 38% in normal colon epithelium, whereas the residual activity in colon carcinoma and metastases in liver was 54% and 24%, respectively. These experiments clearly indicate that the oxygen sensitivity phenomenon is not solely an effect of competition for reducing equivalents between NT and oxygen via SOD, because NADPH generated by G6PDH and NADH generated by LDH have a similar redox potential. Apparently the system is more complex. The role of specifically NADPH-converting cellular systems such as NADPH-cytochrome P450 reductase was excluded because incubations in the presence of exogenous NADPH as substrate for these systems revealed oxygen sensitivity. Involvement of NADPH-dependent lipid peroxidation in the oxygen sensitivity test is discussed.

Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver.

The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) in normal female mouse liver. Quantification of enzyme activity was determined cytophotometrically in periportal (PP), pericentral (PC) and midzonal (MZ) areas. No coloured reaction product was present in PVA media after the incubation period. In contrast, the agarose gels appeared to be highly coloured after incubation. As a consequence, sections incubated with gel media were less intensely stained than those incubated in PVA-containing media. The specific G6PDH reaction (test minus control) yielded approximately 75% less formazan in sections incubated by the agarose gel method than with the PVA method. Further, the amount of formazan deposits attributable to G6PDH activity was highest in the midzonal and pericentral zones of the liver lobule with PVA media, and Kupffer cells could be discriminated easily because of their high G6PDH activity. Significant zonal differences or Kupffer cells could not be observed when agarose gel films were used for the detection of G6PDH activity. The LDH localization patterns appeared to be more uniform after incubation with both methods: no significant differences in specific test minus control reactions were seen between PP, PC and MZ. However, less formazan production (33%) was detected in sections incubated with agarose gels when compared with those incubated with PVA media.(ABSTRACT TRUNCATED AT 250 WORDS)