Rat liver responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia

Hepatic responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia was investigated. For this purpose, livers were perfused with a saturating concentration of 2 mM glycerol, 5 mM L-alanine or 5 mM L-glutamine as gluconeogenic substrates. All experiments were performed 1 h after an ip injection of saline (CN group) or 1 IU/kg of insulin (IN group). The IN group showed higher (P<0.05) hepatic glucose production from glycerol, L-alanine and L-glutamine and higher (P<0.05) production of L-lactate, pyruvate and urea from L-alanine and L-glutamine. In addition, ip injection of 100 mg/kg glycerol, L-alanine and L-glutamine promoted glucose recovery. The results indicate that the hepatic capacity to produce glucose from gluconeogenic precursors was increased during insulin-induced hypoglycemia.

Reaction of diphenyl diselenide with hydrogen peroxide and inhibition of delta-aminolevulinate dehydratase from rat liver and cucumber leaves

The interaction of the product of H2O2 and (PhSe)2 with delta-aminolevulinate dehydratase (delta-ALA-D) from mammals and plants was investigated. (PhSe)2 inhibited rat hepatic delta-ALA-D with an IC50 of 10 µM but not the enzyme from cucumber leaves. The reaction of (PhSe)2 with H2O2 for 1 h increased the inhibitory potency of the original compound and the IC50 for animal delta-ALA-D inhibition was decreased from 10 to 2 µM. delta-ALA-D from cucumber leaves was also inhibited by the products of reaction of (PhSe)2 with H2O2 with an IC50 of 4 µM. The major product of reaction of (PhSe)2 with H2O2 was identified as seleninic acid and produced an intermediate with a lambdamax at 265 nm after reaction with t-BuSH. These results suggest that the interaction of (PhSe)2 with mammal delta-ALA-D requires the presence of cysteinyl residues in close proximity. Two cysteine residues in spatial proximity have been recently described for the mammalian enzyme. Analysis of the primary structure of plant delta-ALA-D did not reveal an analogous site. In contrast to (PhSe)2, seleninic acid, as a result of the higher electrophilic nature of its selenium atom, may react with additional cysteinyl residue(s) in mammalian delta-ALA-D and also with cysteinyl residues from cucumber leaves located at a site distinct from that found at the B and A sites in mammals. Although the interaction of organochalcogens with H2O2 may have some antioxidant properties, the formation of seleninic acid as a product of this reaction may increase the toxicity of organic chalcogens such as (PhSe)2.

Responsiveness of glycogen breakdown to cyclic AMP in perfused liver from rats with insulin-induced hypoglycemia

The responsiveness of glycogen breakdown to cAMP was investigated in isolated perfused liver from male Wistar fed rats (200-220 g) with insulin-induced hypoglycemia. The activation of glycogenolysis by 3 µM cAMP was decreased (P<0.05) in livers from rats with hypoglycemia induced by the administration of insulin or during the direct infusion of insulin into the isolated liver. The direct effect of insulin on glycogen catabolism promoted by 3 µM cAMP occurred as early as 3 min after starting insulin infusion. In contrast, the cAMP agonists resistant to phosphodiesterases, 8Br-cAMP and 6MB-cAMP, used at the same concentration as cAMP, i.e., 3 µM, did not modify the effect of insulin. The data suggest that the decreased hepatic responsiveness of glycogen breakdown during insulin-induced hypoglycemia is a direct effect of insulin decreasing the intracellular levels of cAMP.

Induction of liver monooxygenases by annatto and bixin in female rats

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and ß-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (ß-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin) and bixin (95% pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.

Effect of lung resection and sham surgery on the frequency of infection in alloxan-diabetic rats

The present study was carried out in order to determine the effect of lung resection on the frequency of infections in alloxan-diabetic rats. Adult female Wistar rats were injected with alloxan (40 mg/kg, iv) to induce diabetes mellitus (group D; N = 45) or with vehicle (1.0 ml/kg, iv) to be used as controls (group C; N = 45). Thirty-six days after receiving alloxan both groups were randomly divided into three subgroups: no operation (NO; N = 15), sham operation (SO; N = 15), and left pneumonectomy (PE; N = 15). The rats were sacrificed 36 days after surgery and their lungs were examined microscopically and macroscopically. The occurrence of thoracic wall infection, thoracic wall abscess, lung abscess and pleural empyema was similar in groups D and C. In contrast, the overall infection rate was higher (P<0.05) in the diabetic rats (SO-D and PE-D subgroups, but not in the NO-D subgroup). Considering that the overall infection rate was similar in the SO-D and PE-D subgroups, we suggest that surgery but not pneumonectomy was related to the higher prevalence of infection in diabetic rats.

Isolation of a ß-galactoside-binding lectin from cat liver

A lectin from cat liver has been identified and purified by affinity chromatography on asialofetuin-Sepharose. One hundred micrograms of lectin was obtained from one cat liver with a purification factor of 1561. The lectin agglutinates trypsin-treated rabbit and cow erythrocytes. Hemagglutination was inhibited only by saccharides containing ß-galactosyl residues, of which the 1-amine-1-deoxy-ß-D-galactose was the most potent one by inhibiting hemagglutination at a concentration of 12.5 mM, followed by melibiose, trehalose and galactose. The lectin has a subunit molecular mass of 14.4 kDa determined by SDS-PAGE under reducing conditions and a pI of 4.85. Compared with the composition of lectins from calf heart and porcine heart, cat liver lectin contains approximately the same amount of cysteine, half the amount of glycine, twice as much arginine and threonine, and three times the amounts of tyrosine and methionine. Cat liver lectin contains four cysteine residues per subunit, all of them in the reduced form. Their lack of reactivity towards thiol-reactive supports suggests they are not exposed on the lectin surface. The protein apparently has a blocked N-terminus. The purified lectin was stable for up to 20 months stored at +4ºC in buffer supplemented with 4 mM ß-mercaptoethanol. Results indicated that this lectin belongs to the family of soluble ß-galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues.

A randomized double-blind study of the short-time treatment of obese patients with nonalcoholic fatty liver disease with ursodeoxycholic acid

In order to determine the effect of ursodeoxycholic acid on nonalcoholic fatty liver disease, 30 patients with body mass indices higher than 25, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) or gamma-glutamyltransferase (gamma-GT) at least more than 1.5 times the upper limit of normality, and hepatic steatosis demonstrated by ultrasonography were randomized into two groups of 15 patients to receive placebo or 10 mg kg-1 day-1 ursodeoxycholic acid for three months. Abdominal computed tomography was performed to quantify hepatic fat content, which was significantly correlated with histological grading of steatosis (r s = -0.83, P < 0.01). Patient body mass index remained stable for both groups throughout the study, but a significant reduction in mean (± SEM) serum levels of ALT, AST and gamma-GT was observed only in the treated group (ALT = 81.2 ± 9.7, 44.8 ± 7.7, 48.1 ± 7.7 and 52.2 ± 6.3 IU/l at the beginning and after the first, second and third months, respectively, N = 14, P < 0.05). For the placebo group ALT values were 66.4 ± 9.8, 54.5 ± 7, 60 ± 7.6 and 43.7 ± 5 IU/l, respectively. No alterations in hepatic lipid content were observed in these patients by computed tomography examination (50.2 ± 4.2 Hounsfield units (HU) at the beginning versus 51.1 ± 4.1 HU at the third month). These results show that ursodeoxycholic acid is able to reduce serum levels of hepatic enzymes in patients with nonalcoholic fatty liver disease, but this effect is not related to modifications in liver fat content.

Immunological basis of septal fibrosis of the liver in Capillaria hepatica-infected rats

Rats infected with the helminth Capillaria hepatica regularly develop septal fibrosis of the liver similar to that induced by repeated ip injections of pig serum. Fibrosis starts when the focal parasitic lesions begin to show signs of resorption, thus suggesting an immunologically mediated pathogenesis of this fibrosis. To explore this possibility, the development of C. hepatica-related hepatic fibrosis was observed in rats exposed to worm antigens from the first neonatal day onward. Wistar rats (150 g) were either injected ip with an extract of C. hepatica eggs (protein concentration: 1 mg/ml) or received immature eggs by gavage from the first neonatal day until adult life and were then infected with 500 embryonated eggs. Changes were monitored on the basis of serum levels of anti-worm antibodies and hepatic histopathology. Rats submitted to immunological oral tolerance markedly suppressed C. hepatica-related serum antibodies and septal fibrosis of the liver when infected with the helminth later on. Tolerance trials with ip injections of worm antigens gave essentially negative results. The partial suppression of septal fibrosis of the liver after the induction of immunological tolerance to C. hepatica antigens in rats indicates an immunological basis for the fibrosis and emphasizes the importance of immunological factors in the pathogenesis of hepatic fibrosis.

Noninvasive classification of liver disease in asymptomatic and oligosymptomatic male alcoholics

The predominant type of liver alteration in asymptomatic or oligosymptomatic chronic male alcoholics (N = 169) admitted to a psychiatric hospital for detoxification was classified by two independent methods: liver palpation and multiple quadratic discriminant analysis (QDA), the latter applied to two parameters reported by the patient (duration of alcoholism and daily amount ingested) and to the data obtained from eight biochemical blood determinations (total bilirubin, alkaline phosphatase, glycemia, potassium, aspartate aminotransferase, albumin, globulin, and sodium). All 11 soft and sensitive, and 13 firm and sensitive livers formed fully concordant groups as determined by QDA. Among the 22 soft and not sensitive livers, 95% were concordant by QDA grouping. Concordance rates were low (55%) in the 73 firm and not sensitive livers, and intermediate (76%) in the 50 not palpable livers. Prediction of the liver palpation characteristics by QDA was 95% correct for the firm and not sensitive livers and moderate for the other groups. On a preliminary basis, the variables considered to be most informative by QDA were the two anamnestic data and bilirubin levels, followed by alkaline phosphatase, glycemia and potassium, and then by aspartate aminotransferase and albumin. We conclude that, when biopsies would be too costly or potentially injurious to the patients to varying extents, clinical data could be considered valid to guide patient care, at least in the three groups (soft, not sensitive; soft, sensitive; firm, sensitive livers) in which the two noninvasive procedures were highly concordant in the present study.

DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells

The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared by each fixation procedure was submitted to DNA extraction with the Wizard® genomic DNA purification kit for DNA quantification and five of the preparations were amplified by multiplex PCR (azoospermia factor genes). The amount of DNA extracted varied from 20 to 8,640 µg, with significant differences between fixation methods. Scrape preparation fixed in 95% ethanol provided larger amount of extracted DNA. However, the mean for all groups was higher than the quantity needed for PCR (50 ng) or Southern blot (500 ng). There were no qualitative differences among the different material and fixatives. The same results were also obtained for glass slides stored at room temperature for 6, 12, 18 and 24 months. We conclude that touch and scrape preparations fixed in 95% ethanol are a good source of DNA and present fewer limitations than cell culture, tissue paraffin embedding or freezing that require sterile material, culture medium, laboratory equipment and trained technicians. In addition, they are more practical and less labor intensive and can be obtained and stored for a long time at low cost.

Effect of hyperbaric oxygenation on the regeneration of the liver after partial hepatectomy in rats

The aim of the present study was to assess the influence of hyperbaric oxygenation (HBO) on rat liver regeneration before and after partial hepatectomy. Rats were sacrificed 54 h after 15% hepatectomy, liver and body weights were measured, and serum alanine transaminase (ALT) and aspartate transaminase (AST) activity and albumin levels were determined. The lipid peroxide level, as indicated by malondialdehyde production in the remnant liver was measured, and liver sections were analyzed by light microscopy. Five groups of 10 rats in each group were studied. The preHBO and pre-hyperbaric pressure (preHB) groups were treated before partial hepatectomy with 100% O2 and 21% O2, respectively, at 202,650 pascals, daily for 3 days (45 min/day). The control group was not treated before partial hepatectomy and recovered under normal ambient conditions after the procedure. Groups postHBO and postHB were treated after partial hepatectomy with HBO and HB, respectively, three times (45 min/day). The preHBO group presented a significant increase in the initiation of the regeneration process of the liver 54 h postoperatively. The liver/body weight ratio was 0.0618 ± 0.0084 in the preHBO compared to 0.0517 ± 0016 g/g in the control animals (P = 0.016). In addition, the preHBO group showed significant better liver function (evaluated by the lowest serum ALT and AST activities, P = 0.002 and P = 0.008, respectively) and showed a significant decrease in serum albumin levels compared to control (P < 0.001). Liver lipid peroxide concentration was lowest in the preHBO group (P < 0.001 vs control and postHBO group) and light microscopy revealed that the composition of liver lobules in the preHBO group was the closest to normal histological features. These results suggest that HBO pretreatment was beneficial for rat liver regeneration after partial hepatectomy.

Effect of bullfrog (Rana catesbeiana) oil administered by gavage on the fatty acid composition and oxidative stress of mouse liver

The aim of the present study was to investigate the effects of daily intragastric administration of bullfrog oil (oleic, linoleic and palmitoleic acid-rich oil), corresponding to 0.4% of body weight for four weeks, on fatty acid composition and oxidative stress (lipid peroxidation and catalase activity) in mouse liver. The activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT), biomarkers of tissue injury, were determined in liver homogenates and serum. The proportions of 18:2n-6, 20:4n-6, 20:5n-3, and 22:6n-3 (polyunsaturated fatty acids, from 37 to 60%) in the total fatty acid content were increased in the liver of the bullfrog oil-treated group (P < 0.05) compared to control. At the same time, a significant decrease in the relative abundance of 14:0, 16:0, and 18:0 (saturated fatty acids, from 49 to 25%) was observed. The hepatic content of thiobarbituric acid reactive substances (TBARS) was increased from 2.3 ± 0.2 to 12.3 ± 0.3 nmol TBA-MDA/mg protein and catalase activity was increased from 840 ± 32 to 1110 ± 45 µmol reduced H2O2 min-1 mg protein-1 in the treated group. Bullfrog oil administration increased AST and ALP activities in the liver (from 234.10 ± 0.12 to 342.84 ± 0.13 and 9.38 ± 0.60 to 20.06 ± 0.27 U/g, respectively) and in serum (from 95.41 ± 6.13 to 120.32 ± 3.15 and 234.75 ± 11.5 to 254.41 ± 2.73 U/l, respectively), suggesting that this treatment induced tissue damage. ALT activity was increased from 287.28 ± 0.29 to 315.98 ± 0.34 U/g in the liver but remained unchanged in serum, whereas the GGT activity was not affected by bullfrog oil treatment. Therefore, despite the interesting modulation of fatty acids by bullfrog oil, a possible therapeutic use requires care since some adverse effects were observed in liver.

Diagnosis, staging and treatment of hepatocellular carcinoma

Hepatocellular carcinomas are aggressive tumors with a high dissemination power. An early diagnosis of these tumors is of great importance in order to offer the possibility of curative treatment. For an early diagnosis, abdominal ultrasound and serum alpha-fetoprotein determinations at 6-month intervals are suggested for all patients with cirrhosis of the liver, since this disease is considered to be the main risk factor for the development of the neoplasia. Helicoidal computed tomography, magnetic resonance and/or hepatic arteriography are suggested for diagnostic confirmation and tumor staging. The need to obtain a fragment of the focal lesion for cytology and/or histology for a diagnosis of hepatocellular carcinoma depends on the inability of imaging methods to diagnose the lesion. Several classifications are currently available for tumor staging in order to determine patient prognosis. All take into consideration not only the stage of the tumor but also the degree of hepatocellular dysfunction, which is known to be the main factor related to patient survival. Classifications, however, fail to correlate treatment with prognosis and cannot suggest the ideal treatment for each tumor stage. The Barcelona Classification (BCLC) attempts to correlate tumor stage with treatment but requires prospective studies for validation. For single tumors smaller than 5 cm or up to three nodules smaller than 3 cm, surgical resection, liver transplantation and percutaneous treatment may offer good anti-tumoral results, as well as improved patient survival. Embolization or chemoembolization are therapeutic alternatives for patients who do not benefit from curative therapies.

Plasma hydroxy-metronidazole/ metronidazole ratio in hepatitis C virus-induced liver disease

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 ± 0.0044 vs 0.0742 ± 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 ± 0.0032 vs 0.0438 ± 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 ± 0.0026 (mild chronic hepatitis) and vs 0.0478 ± 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

Serum laminin, type IV collagen and hyaluronan as fibrosis markers in non-alcoholic fatty liver disease

Hepatic fibrosis in patients with non-alcoholic fatty liver disease is associated with progression of the disease. In the present study, we analyzed the discriminative ability of serum laminin, type IV collagen and hyaluronan levels to predict the presence of fibrosis in these patients. In this preliminary report, we studied 30 overweight patients divided into two groups according to the absence (group I, N = 19) or presence (group II, N = 11) of fibrosis in a liver biopsy. Triglycerides, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltranspeptidade, hyaluronan (noncompetitive fluoroassay), type IV collagen, and laminin (ELISA) were determined. Group II presented significantly higher mean laminin, hyaluronan, type IV collagen, and aspartate aminotransferase values, which were due to the correlation between these parameters and the stage of fibrosis in the biopsy (Spearman's correlation coefficient, rS = 0.65, 0.62, 0.53, and 0.49, respectively). Analysis of the ROC curve showed that laminin values >282 ng/ml were those with the best diagnostic performance, with 87% accuracy. Association of laminin with type IV collagen showed improvement in the positive predictive value (100%), but with reduction in diagnostic sensitivity (64%). When compared with the criteria of Ratziu et al. [Gastroenterology (2000) 118: 1117-1123] for the diagnosis of septal fibrosis, laminin values presented a better diagnostic accuracy (83 vs 70%). Determination of extracellular matrix components in serum, especially of laminin, may identify patients with non-alcoholic fatty liver disease and fibrosis and these components may be used as indicators for liver biopsy in these patients.