Changes in the activity of alcohol dehydrogenase isoenzymes during the estrus cycle in the vagina of the rat.

In rodents, the vaginal epithelium undergoes cyclical changes with an alternating pattern of keratinization and mucification. It has been known for decades that vitamin A and its active form retinoic acid are responsible for normal epithelial homeostasis. However, it has not so far been certain which enzymes catalyze the first and rate-limiting step in retinoic acid synthesis. By means of microdissection and ultrathin-layer gel electrophoresis, alcohol dehydrogenase isoenzyme activity was determined quantitatively in the various layers of the vaginal mucous membrane. It was found that, in the rat, only alcohol dehydrogenase 3 and 4 are expressed. Marked cyclical changes of alcohol dehydrogenase 4 activity in the stratum germinativum of the vaginal epithelium strongly support the assumption that this isoenzyme is responsible for retinoic acid synthesis, and that it is essential for the changes accompanying keratinization and mucification.

Spatial distribution of human liver aldehyde dehydrogenase isoenzymes.

To elucidate the pattern of lesions in the liver parenchyma after ethanol ingestion, the quantitative distribution profiles of both the cytosolic and the mitochondrial aldehyde dehydrogenase isoenzyme activities were determined by the use of ultrathin-layer electrophoresis. It was found that in human liver parenchyma, both isoforms of aldehyde dehydrogenase are almost homogeneously represented in the liver acinus. These quantitative data are supported by the results of an improved histochemical technique. Moreover, sex differences were not detected either in activity or in the distribution pattern. Consequently, it can be assumed that it is not the activity of total aldehyde dehydrogenase or its isoforms which is responsible for the higher susceptibility of the perivenous zone to alcohol-dependent damage.

Distribution of alcohol dehydrogenase isoenzymes in the human liver acinus.

By the use of a newly developed technique of ultrathin-layer electrophoresis, class I and class II alcohol dehydrogenase activity could be demonstrated in microdissected samples of the periportal, intermediate, and perivenous zones of the liver acinus in men and women. It could be demonstrated that both classes exhibit low activity in the periportal zone. From there, a rising gradient in the direction of the perivenous end was apparent. This increase, however, was found to be significant only in women. The analysis of class I alcohol dehydrogenase isoenzymes showed that the expression of alpha-, beta-, and gamma-containing isoforms did not differ in relation to the intraacinar position. The constant proportions of the isoenzymes to the maxima and minima of the total alcohol dehydrogenase activity support the view that the adult liver-specific isoenzyme pattern is determined during postnatal development.

Ultrathin-layer zone electrophoresis of alcohol dehydrogenase in partly rehydrated polyacrylamide gels: an alternative to starch gel electrophoresis.

A highly sensitive electrophoretic technique for the separation of alcohol dehydrogenase isoenzymes by zone electrophoresis in partly rehydrated polyacrylamide gels is described. Five hundred microm thin polyacrylamide gels are polymerized under standardized conditions. After polymerization the gels are washed thoroughly with distilled water to remove any unreacted monomers, catalysts or still soluble polymers. The washed gels are then impregnated with 0.5% Tween 20 and dried. Before electrophoresis the dry gels are rehydrated to a thickness of 250 microm, which makes up 50% of the original gel volume. Rehydration is carried out by use of a degassed buffer solution. This method permits the demonstration of the isoenzymes of alcohol-dehydrogenase class I and II in man and allows quantitative determination.

Intra-acinar profiles of cytosolic and mitochondrial malate dehydrogenase isoenzymes in rat liver.

A new method for electrophoretic separation of the isoforms of malate dehydrogenase in microdissected tissue samples was applied to rat liver. The intra-acinar activity profiles of cytosolic (cMDH) and mitochondrial (mMDH) malate dehydrogenase were determined in male and female control animals and in animals fasted for 84 hr. Measurements were carried out on lyophilized liver tissue samples of 50-100 ng from the whole length of the sinusoid. The results showed that both in fed and fasted animals, mMDH activity was almost evenly distributed throughout the acinus in livers of both sexes. cMDH showed higher activity in the periportal area compared with the perivenous area by a factor of approximately 1.35 in all animals studied. Our results favor a slightly higher capacity of the malate-aspartate shuttle in the periportal area in both fed and fasted animals. Furthermore, the distribution pattern of mMDH suggests that this isoenzyme is not a marker for the zonation of the oxidative metabolism in the liver acinus.

Microquantitative determination of lactate dehydrogenase isoenzymes in the myocardium and the conducting system.

A newly developed technique was used for the electrophoretic separation of lactate dehydrogenase (LDH) isoenzymes from lyophilized tissue samples in the nanogram range. In this study portions of 10-200 ng from the myocardium and the conducting system of cattle, sheep, pig and man were microdissected and analysed. In the heart tissues of cattle, sheep and pig, the isoforms LDH1, LDH2 and LDH3 were detected in species-specific varying amounts. In all these animals, the conducting system is marked by high LDH1 activity, which is present at a ratio of about 2:1 compared with the myocardium. The values in man, however, differ from these values, but this might be due to post-mortem changes. The findings are discussed with respect to possible aerobic-anaerobic functions.

Ultrathin-layer microelectrophoretic determination of lactate dehydrogenase isoenzymes in corneal and conjunctival epithelium of the cow.

In order to evaluate the impact of tissue oxygenation on the distribution pattern of lactate dehydrogenase isoenzymes, activities of the isoenzymes were measured in microdissected samples of bovine tissue. A highly sensitive ultrathin-layer electrophoretic technique was used to determine the distribution pattern of lactate dehydrogenase isoenzymes in basal, intermediate and superficial layers of the epithelium of central and peripheral cornea and in the epithelium of the bulbar conjunctiva. Measurements revealed almost homogeneous intraepithelial distribution patterns of lactate dehydrogenase isoenzymes in both tissues. In the cornea the lactate dehydrogenase isoenzymes 4 and 5, which are regarded to be specialized for anaerobic glucose metabolism, were found to predominate. In the well-oxygenated conjunctival epithelium most of the activity could be ascribed to the lactate dehydrogenase isoenzyme 3. In contrast to the isoenzymatic activities, total activity of lactate dehydrogenase was inhomogeneously distributed; maximum activities were found in the basal layer of corneal epithelium and in the intermediate layer of conjunctival epithelium. The results indicate that oxygen supply is relevant rather for the intraepithelial distribution of total enzyme activity than for the expression of lactate dehydrogenase isoenzymes.

Evidence for the colocalization of parvalbumin and glutamate, but not GABA, in the perforant path of the gerbil hippocampal formation: a combined immunocytochemical and microquantitative analysis.

Gerbils (Meriones unguiculatus) are known for their seizure sensitivity, which is dependent on an intact perforant path from the entorhinal cortex to the hippocampus. In contrast with other species, the perforant path in gerbils contains parvalbumin, a cytosolic high-affinity calcium-binding protein. Parvalbumin is known to be present in a subpopulation of GABA-containing neurons and is thought to be responsible for their physiological characteristics of fast spiking activity and lack of spike adaptation. Therefore, the question arose of whether this projection in gerbils is GABAergic or glutamatergic as in other species. In a first approach to this question, the effect of lesioning the origin of the perforant path, the entorhinal cortex, on levels of GABA and glutamate was determined by enzymatic-luminometric assay in single layers of the dentate gyrus of lyophilized brain sections. Parallel sections were cryofixed using an acidified acetone-formaldehyde mixture at -20 degrees C for 48 h, and subsequently stained for parvalbumin immunocytochemistry. Seven days after ablation of the entorhinal cortex, parvalbumin staining was undetectable in the termination zone of the perforant path, the outer two-thirds of the stratum moleculare. In parallel, glutamate content was reduced to 80% of controls (and of the unoperated contralateral side) but unchanged in the inner third of the stratum moleculare and in stratum granulare. GABA content was not significantly altered by the lesion. From these results, we conclude that in the gerbil as in other species, the perforant path contains glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrathin-layer zone electrophoresis of lactate dehydrogenase isoenzymes in microdissected liver samples.

A highly sensitive electrophoretic separation of lactate dehydrogenase isoenzymes in 150-microns ultrathin-layer polyacrylamide gels is described. By means of the incorporation of miniature-sized wells into the gel and by executing all the analytical steps under paraffin oil, this technique allows the exact and sharp separation of the lactate dehydrogenase isoenzymes in nanogram-sized microdissected liver tissue samples. With this method the heterogeneous distribution pattern of lactate dehydrogenase isoenzymes in the liver acinus of various mammals could be demonstrated, and a new interpretation of their functional roles is offered.

The intramucosal distribution of gastric alcohol dehydrogenase and aldehyde dehydrogenase activity in rats.

Using qualitative and microquantitative histo-chemical techniques, alcohol dehydrogenase and aldehyde dehydrogenase activity was studied in the gastric mucosa of male and female rats. Alcohol dehydrogenase was demonstrated by staining reactions with maximum activity in surface and neck cells and with clearly weaker activity also in parietal cells. Aldehyde dehydrogenase could be detected in surface and neck cells, and also to a comparable degree in the parietal cells. Quantitative analyses of microdissected samples yielded high values for alcohol dehydrogenase activity exclusively in the superficial part of the gastric mucosa, whereas low-Km aldehyde dehydrogenase activity showed a decreasing gradient from the surface to the deeper parts of the mucosa. Sex differences could not be confirmed.

Liver architecture.

The development of liver parenchyma starts from entodermal cells which grow out from the gut into the mesenchyma of the septum transversum. In the definitive organ this close association of epithelial cells (hepatocytes) and mesenchyma-derived nonparenchymal cells is maintained. The liver, and with it each hepatocyte, acts in two directions: the vascular poles of the hepatocytes serve in an ingestive sense, while at their biliary poles secretory functions are exerted. Hepatic microvascularization comprises two afferent vessels (arterial and portal terminal branches), the sinusoids and the terminal hepatic venule. Sinusoidal cells surround the capillaries but also have highly specialized functions with regard to filtration, phagocytosis, fat storage and defense. The autonomic innervation plays an important role in the regulation of metabolic functions. Above the cellular level the proper architecture of the liver parenchyma has been the object of controversial discussions for centuries. The concept of the liver lobule, the portal unit, the liver acinus and other structures are presented and discussed. Finally, the liver parenchyma is described as an irregular interdigitating system of regions related to the terminal blood vessels.

Intraacinar profiles of alcohol dehydrogenase and aldehyde dehydrogenase activities in human liver.

The intraacinar activity profiles of alcohol dehydrogenase and the aldehyde dehydrogenases (I, I plus II, and total) were determined, using liver biopsy samples from eight male and eight female patients. Microchemical assays were performed in microdissected tissue samples from the whole length of the sinusoid. Alcohol dehydrogenase activity in men less than 53 years of age showed a maximum in the intermediate zone, whereas in women less than 50 years of age an increase in the gradient toward the perivenous zone was observed. Furthermore, alcohol dehydrogenase activity in the livers of women was significantly higher than in men. After the age of 53 in men and 50 in women, the sex specificity of the distribution profiles was no longer apparent. The intraacinar profiles of aldehyde dehydrogenase isoenzymes showed only minor variations in the different groups; they were not statistically significant. This is also true for low-Michaelis constant (Km) aldehyde dehydrogenase, which is most important for acetaldehyde oxidation in vivo. Thus, of the variations in zonal heterogeneity of ethanol-degrading enzymes, it is mainly the activity of alcohol dehydrogenase that may contribute to the sex- and age-related susceptibility of liver parenchyma.

Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver.

In adult male and female rat liver, the activity of NAD(+)-and NADP(+)-dependent glutamate dehydrogenase (GDH) was microquantitatively measured in tissue samples of 50-150 ng, microdissected continuously along the sinusoidal length. Total activity of GDH with NAD+ as co-factor was found to be higher by a ratio of about 1:2.3 than with NADP+. All intra-acinar enzyme profiles, irrespective of sex, showed an increasing gradient of GDH activity from the periportal beginning to the perivenous end. These findings are at variance with the immunohistochemical localization of GDH in rat liver. The microquantitative GDH profiles with higher perivenous values could indicate a more pronounced glutamine synthesis in Zone 3 of the liver acinus.

Changes in activity and intra-acinar distribution of glucose-6-phosphate dehydrogenase and malic enzyme during pregnancy in rat liver.

Using techniques of microdissection and microassay as well as qualitative histochemistry the activity and intra-acinar distribution of G6PDH and ME were studied on selected days of pregnancy in the rat. Both enzymes show distinct fluctuations during the course of pregnancy in keeping with changes in hepatic lipogenesis. Marked increases in activity are seen as early as the 4th day, while highest values are attained on day 20, with a predominant perivenous induction. On day 22, just before parturition a sharp decrease of both enzyme activities with a flattening of the periportal/perivenous gradient was detected. G6PDH shows proportionally considerably larger increases and more distinct changes in zonation. The perivenous fluctuations in G6PDH activity of late gestation are supposed to be caused primarily by insulin. Although estrogen is known to induce both enzymes, the temporal changes in enzyme activity in pregnancy cannot be related to the action of estrogen alone. The changes in enzyme activity, however, correspond well to those of progesterone, and although no direct action of progesterone on these enzymes has yet been proposed, further work on its effects on enzyme activity and distribution is indicated.

The circadian rhythm of intra-acinar profiles of alcohol dehydrogenase activity in rat liver: a microquantitative study.

Using microquantitative measurements of alcohol dehydrogenase activity in microdissected samples of liver tissue along the sinusoidal length, the intra-acinar distribution profiles were studied in seven groups of female rats at different times during 24 h with a light phase from 6:30 h to 18:30 h. The mean values of alcohol dehydrogenase activity showed a circadian rhythm with a minimum at 13.30 h and a maximum at 17.30 h (p less than 0.001). However, the intra-acinar gradients remained almost unchanged, indicating that increase and decrease in enzyme activity takes place simultaneously in all parts of the liver acinus. This observation, together with data from the literature, suggests that the circadian rhythm of alcohol dehydrogenase activity reflects variations in different liver cell constituents, rather than enzyme protein synthesis or proteolysis.

Nutritional and gonadal effects on the intra-acinar profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver.

Total and low-Km aldehyde dehydrogenase (ALDH) activity was measured in 50-150 ng microdissected liver tissue samples of the entire sinusoidal length. High-Km ALDH activity was calculated by subtracting the low-Km ALDH values from the total ALDH activity. Enzyme activity was measured by a microchemical assay, using the oil-well technique with luminometric determination of NADH. The intra-acinar profiles of high-Km and low-Km ALDH activity could be demonstrated graphically for both male and female rats after 84 h of starvation, and after starvation and refeeding for 6 nights. In addition, the ALDH distribution patterns of juvenile, castrated, and castrated and testosterone-treated rats were determined. It could be demonstrated that starvation, and starvation followed by refeeding, lead to changes in enzyme activity which parallel the loss and regain of liver- and body-weight. The nutritional factors do not essentially alter the normal intra-acinar profiles. In juvenile rats, ALDH is lower by 30% in comparison with the controls, but sex-differences in the distribution profiles are not yet present. Castration has no effect on the amount of enzyme activity but the sex specific distribution profiles are less marked. The main effect of testosterone treatment is an elevation of low-Km ALDH in the perivenous zone. The characteristics of the intra-acinar profiles of high-Km and low-Km ALDH activity are discussed with respect to hepatic acetaldehyde oxidation and alcoholic liver damage.

Microquantitative determination of intra-acinar distribution profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver.

Microquantitative measurements of total and of low-Km aldehyde dehydrogenase (ALDH) activity with millimolar and micromolar concentrations of acetaldehyde and propionaldehyde were carried out on the livers of male and female rats. Lyophilized cryostat sections of liver parenchyma were microdissected along the entire sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ALDH activity was measured in a microbiochemical assay using the oil-well technique with luminometric determination of NADH. On the basis of single measurements, mean values of total, low-Km and high-Km ALDH activity could be calculated and the specific distribution patterns graphically demonstrated. The two substrates acetaldehyde and propionaldehyde yielded similar values of ALDH activity, the intraacinar distribution profiles of which showed characteristic sex differences. In the liver of the male rat high-Km ALDH activity has two flat peaks in the periportal and the perivenous area, while low-Km ALDH activity is almost evenly distributed throughout the acinus. In the livers of female rats, both high-Km and low-Km ALDH activity shows a continuous gradient which decreases from the periportal to the perivenous zone (pp/pv = 1.4:1). It was therefore possible to demonstrate that the maxima of alcohol dehydrogenase activity and of low-Km ALDH activity are localized in opposite parts of the liver acinus of the female rat. This heterotopy should have consequences with respect to hepatotoxicity after alcohol ingestion.