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)     

Discrimination between periportal and pericentral necrosis of rat liver by determination of glutamine synthetase and other enzyme activities in serum

Periportal or pericentral necrosis of rat liver was produced by injection of allyl-alcohol or bromobenzene, respectively. Activities of predominantly periportal and perivenous enzymes were determined in serum during maximal necrosis. Aspartate aminotransferase, which is more or less homogeneously distributed in the liver acinus, exhibited similar activities in serum after periportal and pericentral injury. Serum activities of the mainly periportal enzymes alanine aminotransferase and fructose 1,6-bisphosphatase were 1.5- to 2-fold higher after periportal as compared to pericentral necrosis. Serum activity of the mainly pericentral glutamate dehydrogenase was 3-fold higher after pericentral than after periportal damage. However, due to individual variations necrosis could not be definitively localized in any case by measurement of these enzyme activities. Better discrimination between periportal and pericentral necrosis was achieved by the serum activity of the exclusively pericentral enzyme glutamine synthetase, which was 8-fold higher after pericentral as compared to periportal necrosis. Conclusive discrimination was obtained by the activity ratio fructose 1,6-bisphosphatase/glutamine synthetase in serum.     

New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.     

Adenylate cyclase activity in microdissected rat liver tissue: periportal to pericentral activity gradient

Adenylate cyclase activity was measured in microdissected samples from lyophilized cryostat sections of rat liver by means of an improved assay. Livers were obtained from adult Sprague-Dawley rats fasted for 22 hr. Adenylate cyclase activities, basal and those elicited by various agents, were determined in dissected samples from periportal and pericentral regions of the classic liver lobule. In all samples, enzyme activity was strongly stimulated by glucagon, cholera toxin, guanosine-5'-O-(3-thiotriphosphate), sodium fluoride and forskolin. The beta-adrenergic agonist isoproterenol produced very weak, if any, enzyme stimulation. Angiotensin II did not inhibit the activity elicited by lithium chloride and GTP at high concentrations, and pertussis toxin did not enhance the GTP-stimulated activity. We observed a periportal-to-pericentral gradient for basal and agent-stimulated activities.     

Extracellular matrix remodeling at the early stages of liver regeneration in the rat

Previous studies have shown that activity of urokinase-type plasminogen activator (u-PA) increases very rapidly (within 1 minute) after partial hepatectomy. In view of the well-recognized roles of u-PA as one of the major initiators of the matrix proteolysis cascade and as an activator of plasminogen and hepatocyte growth factor (HGF), we studied matrix degradation in liver shortly after partial hepatectomy. The activation of plasminogen to plasmin following partial hepatectomy was examined by Western blot analysis, and a small increase in plasmin at approximately 15 minutes followed by a large elevation at approximately 3 to 6 hours after partial hepatectomy was detected. In addition, we found that fibrinogen, the major substrate for plasmin, begins to be degraded at approximately 15 to 30 minutes following partial hepatectomy. Using immunohistochemical staining, we detected that the distribution of fibrinogen in normal liver is localized to the perisinusoidal space surrounding the periportal region. A decreased distribution of fibrinogen in the periportal region was found by 15 minutes and continued through 24 hours following partial hepatectomy. In addition, the distribution of fibronectin in normal liver was localized to the perisinusoidal space surrounding the periportal and the pericentral regions. A strikingly decreased distribution of fibronectin in the periportal region was found at 5 minutes after partial hepatectomy. Furthermore, we observed that the protein levels of laminin, entactin, and fibronectin in an extracellular matrix (ECM)-enriched preparation decreased shortly after partial hepatectomy, and were restored later. No changes were observed with either vitronectin or the integrin chain alpha(v). In contrast to the protein levels of the ECM components, the messenger RNA (mRNA) levels of fibronectin, integrin chain beta1, and integrin chain alpha(v) gradually increased over 18 hours and then decreased thereafter. Taken together, these results suggest that rapid reorganization of selected ECM components are important for hepatocyte proliferation at the early stages of liver regeneration.     

Kinetic parameters of lactate dehydrogenase in liver and gastrocnemius determined by three quantitative histochemical methods

We determined the Michaelis constant (K(m)) and maximal velocity (Vmax) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities (vi) of LDH, corrected for "nothing dehydrogenase," were determined as described in the previous article. The Km and Vmax were calculated from Hanes plots of s/vi on L-lactate concentration (s). The Km values obtained with three assay methods were similar and in the range of 21.1-21.9 mM for pure LDH, 8.62-13.5 mM for LDH in mouse periportal hepatocytes, and 13.3-17.9 mM for LDH in mouse skeletal muscle fibers. The Vmax values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreement but were 53-65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity kcat of LDH is retarded by the high viscosity of PVA media because PVA hardly inhibited the enzyme. The K(m) values of LDH determined histochemically in skeletal muscle fibers and periportal hepatocytes were respectively three to five times and two to three times higher than those determined biochemically. These differences may be due to interactions of LDH with intracellular components.     

Nerve tissue-specific human glutamate dehydrogenase that is thermolabile and highly regulated by ADP

Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue-specific GDH showed only 5% of its maximal activity, compared with approximately 40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05-0.25 mM) induced a concentration-dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550-1,300%) than of the housekeeping enzyme (by 120-150%). Magnesium chloride (1-2 mM) inhibited the nonactivated housekeeping GDH (by 45-64%); this inhibition was reversed almost completely by ADP. In contrast, Mg2+ did not affect the nonstimulated nerve tissue-specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05-0.25 mM). Heat-inactivation experiments revealed that the half-life of the housekeeping and nerve tissue-specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue-specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.     

Glutamate dehydrogenase, alanine aminotransferase, thymidine kinase, and arginase in fetal and adult human and rat liver

In fetal livers of both man and rat thymidine kinase activity was 12 times higher than in the adult, glutamate dehydrogenase and arginase were present at 20-50% of their adult values, whereas alanine aminotransferase activity was only an insignificant fraction of that in the adult. Although the developmental changes for the four enzymes were quantitatively similar in both species, qualitatively there were some significant differences. In adult human liver, glutamate dehydrogenase activity was distributed almost equally between the cytosol and particles; the concentration of only the soluble enzyme increased after birth. In rat liver, glutamate dehydrogenase remained exclusively a particulate enzyme. The soluble hepatic alanine aminotransferase activity rose in both species after birth (from less than 2 U/g to 41-57 U/g, respectively). Thymidine kinase was wholly soluble in the fetal livers; only in adult human liver was additional activity (at least 50% of the total) found in the particles. Arginase isozymes, identical and apparently the same single isozyme in fetal and adult rat liver, show an ontogenetic change in man. A shift from a single form, common to human fetal liver and fetal kidney, to at least two variants in adult human liver, indicates another complexity of the fully differentiated liver in man.     

Morphology of liver stellate cells and liver vitamin A content in 3,4,3',4'-tetrachlorobiphenyl-treated rats

BACKGROUND/AIMS: Because xenobiotics decrease the vitamin A stores localized in the liver stellate cells, we investigated morphological alterations in the liver of rats exposed to 3,4,3',4'-tetrachlorobiphenyl. Special attention was given to the morphology of the liver stellate cells and to their relationship to the liver vitamin A content. METHODS: Six rats received an intraperitoneal injection of 3,4,3',4'-tetrachlorobiphenyl (300 mumol/kg) in soyabean oil. A further six rats received the vehicle alone. After 7 days, all rats were killed and their livers assayed for vitamin A. Liver stellate cells were examined and counted on liver sections, stained with toluidine blue or immunocytochemically for desmin and, for some animals, for alpha-smooth muscle actin. RESULTS: In the livers of 3,4,3',4'-tetrachlorobiphenyl-treated rats, we found spotty and bridging necrosis, with inflammation and accumulation of desmin-positive liver stellate cells. Steatosis and mild portal inflammation were also observed. 3,4,3',4'-Tetrachlorobiphenyl decreased the liver vitamin A content by 38%, whereas morphometric analyses showed a 40% decrease of the number of toluidine blue-detected liver stellate cells and an 11% increase of desmin-detected liver stellate cells, indicating a likely differentiation of liver stellate cells into myofibroblast-like cells. 3,4,3',4'-Tetrachlorobiphenyl treatment did not modify the expression of alpha-smooth muscle actin. Morphological alterations were more pronounced in periportal than in pericentral areas. The liver vitamin A content was positively correlated (r = 0.56, p < 0.005) with the number of toluidine-blue detected liver stellate cells. CONCLUSIONS: 3,4,3',4'-tetrachlorobiphenyl administration results in an accumulation of liver stellate cells that start differentiating into myofibroblast-like cells. The 3,4,3',4'-tetrachlorobiphenyl-induced decrease in liver vitamin A probably results from this differentiation, although other mechanisms cannot be excluded.     

Decreased uptake of 14C-labeled dicarboxylic amino acids in rapidly growing hepatomas

In contrast to the increased uptake of amino acids which has been found in many neoplastic cells, we have observed a decrease in the net uptake of [14C]aspartate and [14C]glutamate in rapidly growing hepatomas relative to rat host liver. When measured 10 min after s.c. injection, the radioactivity from 14C-labeled dicarboxylic amino acids was greater in liver than in all other tissues examined (blood, skeletal, muscle, heart, spleen, lung, and brain) except kidney, where there was an approximately 2-fold greater uptake of aspartate and 10-fold greater uptake of glutamate. Mean uptakes in the rapidly growing Morris hepatomas 7288CTC and 7777 were 19 to 26% of corresponding values for the host livers. Comparison with uptake of 3H2O indicated that these low values were not solely due to differences in circulation. Decreased uptake was not accompanied by equivalent decreases in the concentration of aspartate and glutamate in the tumors. There were small changes in the net uptake of these amino acids in the slowly growing hepatoma 7787 and no significant differences in regenerating liver and hepatoma 5123C, a tumor of intermediate growth rate. The net uptake of [14C]arginine and [14C]lysine in the hepatomas was similar to that in host livers, except for a 250% increase in uptake of [14C]lysine in hepatoma 5123C. A decreased uptake of the magnitude seen with dicarboxylic amino acids in rapidly growing hepatomas has not been observed with other amino acids.     

Pigmented uveal tumours in a transgenic mouse model

Lectins are sensitive probes which bind carbohydrate structures specifically. In this study, we modified the lectin staining procedure for sensitive detection of carbohydrate structures in formalin-fixed, paraffin-embedded sections of normal and heterologous serum-induced fibrotic livers. The liver sections were heated in hot distilled water at 100 degrees C for 10 min (thermo-treatment: TT), and then stained with 24 different lectins. In comparison with the results from sections without TT (nonTT), enhanced and/or alternated staining patterns of 19 lectins were demonstrated in sections with TT, and enhanced staining of Vicia villosa agglutinin seen in Kupffer cells was noted. Interestingly, no positive staining was seen with Dolichos biflorus agglutinin, peanut agglutinin or soybean agglutinin (SBA), which recognize O-linked carbohydrate chains, in Kupffer cells of non-TT sections, but strong positive staining was demonstrated in those of TT sections. SBA-positive staining in the cytoplasm of some scattered hepatocytes located in the periportal and perifibrous zones and central zone of pseudolobules was demonstrated only in the fibrotic liver sections with TT. Such findings indicate the heterogeneity of hepatocytes in the liver with fibrosis. Formalin fixation causes masking of lectin binding sites, especially O-linked carbohydrate chains, and TT may recover such masking reactions. TT improved the staining reactions for many lectins in formalin-fixed, paraffin-embedded liver sections, and new staining patterns appear after TT. Modified TT staining procedures may be useful for the diagnosis and prognosis of liver fibrosis.     

Connexin 32 gap junctions enhance stimulation of glucose output by glucagon and noradrenaline in mouse liver

Gap junctions connect neighboring cells via intercellular channels composed of connexins (Cx). Connexin 32 (Cx32) is the main connexin in hepatocytes. Gap junctions propagate a signal from periportal to perivenous hepatocytes generated by electrical stimulation of sympathetic liver nerves. Therefore, it was the aim of this study to examine the involvement of hepatocellular gap junctions in hormonal regulation. In perfused livers from wild-type mice and Cx32-deficient mice, the stimulation of glucose release by varying noradrenaline and glucagon concentrations was investigated. At saturating hormone concentrations, glucose release was the same in wild-type and Cx32-deficient livers. However, glucose output was significantly smaller in Cx32-deficient than wild-type livers at half-maximally effective hormone concentrations. Because the two hormones circulate at less than half-saturating concentrations and because they are degraded during passage of blood through the liver, they lose efficiency from the periportal to the perivenous zone. In wild-type livers, this decrease in efficiency can be partially compensated by intercellular signal propagation through gap junctions, resulting in higher hormone actions than in Cx32-deficient livers. It is concluded that gap junctions are not only involved in intercellular propagation of nervous, but also of hormonal signals from periportal to perivenous hepatocytes.     

Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes

The metabolic fate of glutamate in astrocytes has been controversial since several studies reported > 80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U-13C] glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 mM [U-13C] glutamate 85% of the 13C metabolized was converted to glutamine. The formation of [1,2,3-13C3] glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2-0.5 mM glutamate, 13C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [13C] lactate was essentially unchanged within the range of 0.2-0.5 mM glutamate, whereas the amount of [13C] aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 mM, suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 mM and astrocytes from both rat and mouse brain are consistent with these findings.     

Expression of adhesion molecules on endothelial cells after contact with knitted Dacron

Glutamine synthetase and carbamoylphosphate synthetase I expression was examined immunohistochemically in livers of spf-ash homozygous and hemizygous mice, in which one of the urea cycle enzymes (ornithine carbamoyltransferase) is deficient and hyperammonemic disorders are obvious. In the mutant adult mouse liver, only hepatocytes lining central veins expressed glutamine synthetase. In contrast, other hepatocytes expressed carbamoylphosphate synthetase I but not glutamine synthetase. This complementary expression pattern is similar to that seen in wild-type mouse liver. In the liver of mutant young mice, which showed severe retarded growth and abnormal hair and skin development, the developmental expression pattern of both enzymes was also similar to that of the corresponding wild-type liver. However, suppression of carbamoylphosphate synthetase I expression in the pericentral hepatocytes occurred later in the mutant than in wild-type liver. These results show that high plasma concentrations of ammonium ions, which are one of the substrates for both the enzymes, do not change their complementary expression. Instead they support the idea that factor(s) associated with central veins rather than humoral factors direct pericentral hepatocytes to express glutamine synthetase and to suppress carbamoylphosphate synthetase I expression.     

Electrophysiological elucidation of pathways of intrinsic horizontal connections in rat visual cortex

In single pass perfused rat liver, rapid osmotic water shifts across the plasma membrane in response to hyperosmolar urea were followed by monitoring liver mass and transient concentrating or diluting effects on Na+ concentration in effluent perfusate. Sudden addition or removal of hyperosmolar urea (200mM, resulting in a step change of the perfusate osmolarity from 305 to 505 mosmol/l) had little effect on liver mass or Na+ activity in the effluent perfusate, suggesting that urea equilibrated at a rate similar to that of water across the liver plasma membrane. When, however, phloretin (0.2mM) was present, sudden addition (removal) of urea (200mM) induced within seconds a marked and transient decrease (increase) of both liver mass and effluent Na+ concentration, suggestive of transient osmotic water shifts out of/into the cells. Although to a lesser extent, comparable effects were induced when urea was added/removed in the presence of the phloretin-related phenol compounds 2,4,6-trihydroxyacetophenone (5mM) and 2,4,5-trihydroxybutyrophenone (5mM). Phloretin-induced inhibition of urea export from livers preloaded with [14C]urea was reversible, and no saturation of urea transport was found at concentrations up to 200mM. In contrast to [14C]urea transport, [3H]water transport across the plasma membrane was not affected by phloretin. The data indicate that urea export across the hepatocyte plasma membrane is almost as fast as water export. The urea transport mechanism is sensitive to phloretin and other phenol compounds, works with high capacity and is distinct from the water-transporting system. The regulation of this putative transport mechanism and its relevance for hepatic nitrogen metabolism remain to be established.     

Essential active-site lysine of brain glutamate dehydrogenase isoproteins

Two soluble forms of bovine brain glutamate dehydrogenase (GDH) isoproteins were inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff's base formation with amino groups of the enzyme. Sodium borohydride reduction of the pyridoxal 5'-phosphate-inactivated GDH isoproteins produced a stable pyridoxyl enzyme derivative that could not be reactivated by dialysis. The pyridoxyl enzyme was studied through fluorescence spectroscopy. No substrates or coenzymes separately gave complete protection against pyridoxal 5'-phosphate. A combination of 10 mM 2-oxoglutarate with 2 mM NADH, however, gave complete protection against the inactivation. Tryptic peptides of the isoproteins, modified with and without protection, resulted in a selective modification of one lysine. In both GDH isoproteins, the sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other GDH species.     

Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were colocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Ultrastructure of periportal and centrilobular hepatocytes in human fatty liver of various aetiology

The ultrastructure of periportal and centrilobular hepatocytes of fatty liver from 2 alcoholic patients, 2 diabetic patients and 2 obese patients is described and compared with that of hepatocytes in 2 patients with normal liver. A striking difference between periportal and centrilobular hepatocytes in all cases of fatty liver is deomonstrated. The periportal hepatocytes contain abnormal mitochondria, while centrilobular hepatocytes have normal mitochondria, but fatty vacuoles, peroxisomes and lysosomes are more abundant. The nature and degree of ultrastructural changes showed no correlation with aetiology of the fatty liver. The normal livers showed no such changes or distinct differences between periportal and centrilobular hepatocytes.