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Background: Vadadustat, a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, is a substance which carries a lifetime ban in both horse racing and equestrian competition. A comprehensive metabolic study of vadadustat in horses has not been previously reported., Objective: Metabolism and elimination profiles of vadadustat in equine plasma and urine were studied for the purpose of doping control., Methods: A nasoesophageal administration of vadadustat (3 g/day for 3 days) was conducted on three thoroughbred mares. Potential metabolites were comprehensively detected by differential analysis of full-scan mass spectral data obtained from both in vitro studies with liver homogenates and post-administration samples using liquid chromatography high-resolution mass spectrometry. The identities of metabolites were further substantiated by product ion scans. Quantification methods were developed and validated for the establishment of the excretion profiles of the total vadadustat (free and conjugates) in plasma and urine., Results: A total of 23 in vivo and 14 in vitro metabolites (12 in common) were identified after comprehensive analysis. We found that vadadustat was mainly excreted into urine as the parent drug together with some minor conjugated metabolites. The elimination profiles of total vadadustat in post-administration plasma and urine were successfully established by using quantification methods equipped with alkaline hydrolysis for cleavage of conjugates such as methylated vadadustat, vadadustat glucuronide, and vadadustat glucoside., Conclusion: Based on our study, for effective control of the misuse or abuse of vadadustat in horses, total vadadustat could successfully be detected for up to two weeks after administration in plasma and urine., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In this 12-week, randomized, double-blind, placebo-controlled trial, the efficacy and safety of transglucosidase (TGD) were compared with placebo in patients with type 2 diabetes mellitus (T2DM). At 12 weeks, TGD 300 mg/day and TGD 900 mg/day significantly reduced HbA1c (0.18 and 0.21%) and insulin concentration (19.4 and 25.0 pmol/l), respectively, vs. placebo. TGD 300 mg/day and TGD 900 mg/day also significantly reduced low-density lipoprotein cholesterol (0.22 and 0.17 mmol/l, respectively). TGD 900 mg/day significantly reduced triglyceride by 0.24 mmol/l and diastolic blood pressure by 8 mmHg. Placebo was associated with a significant increase from baseline in body mass index, alanine aminotransferase and aspartate aminotransferase (0.17 kg/m(2) , 3 and 2 U/l, respectively), whereas TGD was not. TGD 300 mg/day significantly increased high-molecular-weight adiponectin by 0.6 µg/ml. Adverse events did not differ significantly between the groups. TGD resulted in lowering of HbA1c and blood insulin level and improvements in metabolic and cardiovascular risk factors in T2DM., (© 2011 Blackwell Publishing Ltd.)

An adult swan goose (Anser cygnoides) kept in a zoological garden had gross hepatic enlargement with extensive ill-defined white foci. Microscopically, the hepatic lesions were composed of a mixture of adipocytes and myeloid cells. The goose was also affected with systemic amyloidosis and there were areas of osseous metaplasia associated with deposition of amyloid within the liver.

Furazolidone (FZ), one of the nitrofuran fungicides, is used as a veterinary medicine in the Middle and Far Eastern countries. In this study, FZ (125 mg/kg) was administered orally to Wistar rats for 3 days. Results of the Ames test using the S-9 fraction of rats treated with FZ showed a significant increase in the number of revertant colonies. Western blot analysis of hepatic CYP isozymes induced by FZ, revealed a remarkable induction of CYP1A1 apoprotein, but CYP1A2 and CYP2E1 apoproteins were not altered. In addition, the expression of CYP1A1 mRNA level in rats treated with FZ by RT-PCR was significantly enhanced by FZ treatment. We concluded that FZ is apparently mutagenic and induces transcription of the CYP1A1 isozyme, which metabolically activates numerous promutagens, in hepatocytes.

This study undertook the analysis of tissue cadmium (Cd) distribution using in-air micro-particle-induced X-ray emission (PIXE) and the examination of the involvement of metal ions in parenteral Cd toxicity. A mouse was injected intraperitoneally with 3 mg/kg body weight of CdCl2 thrice weekly. After 27 wk, the liver and kidney were excised and fixed in 10% formalin solution for 4 h and then embedded in paraffin. Thin paraffin sections were used to analyze trace elements with in-air micro-PIXE and to examine metallothionein protein and histological changes. Cd distribution was determined by micro-PIXE in the liver and renal cortex of the Cd-exposed mouse, and the net Cd count was higher in the liver than in the renal cortex. The net iron (Fe) count was higher in the liver of the Cd-exposed mouse compared to the control, and an opposite tendency was observed in the renal cortex. Wide cellular Cd distribution was demonstrated in the liver and renal cortex of the chronic Cd-exposed mouse compared to the control. Metallothionein staining was increased by chronic exposure to Cd both in the liver and kidney, and nephrotoxicity was more apparent than hepatotoxicity. The modification of tissue Fe and calcium distribution by an intraperitoneal injection of Cd might be involved in Cd-induced toxicity.

Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SER. var. thunbergii MAKINO, suppressed D-galactosamine-induced liver injury by 85.2% when added to the diet at 1% and fed to rats for fifteen days. The hepatoprotective effect is more potent than that of a milk thistle extract and turmeric powder. Some fractionated extracts showed hepatoprotective activity in the D-galactosamine-induced in vitro liver injury model.

Isolated hepatocytes are known to maintain their physiological functions for over a week when cultured on Matrigel, artificially reconstituted from basement membrane components. Although this culture technique has been frequently used in research on hepatocyte functions, there has been a limitation on its application for small scale experiments due to some technical problems. By using micro-culture plates with 96 round-bottom wells, we succeeded in coating the wells uniformly with Matrigel. When the cultured hepatocytes were treated with either 10 mM, 15 mM, or 20 mM of acetaminophen or 1 mM, 10 mM, or 20 mM of D-galactosamine, the viability of the hepatocytes became 91.1%, 75.3%, 64.7%, and 79.0%, 43.8%, 26.2% of the non-treated control at 48 hours, respectively. Fractionated extracts of Glycyrrhiza glabra L. and Schisandra chinensis Baillon inhibited the action of acetaminophen or D-galactosamine in this model. From these results, we concluded that the microculture system presented here is capable of maintaining the in vivo characteristics of hepatocytes and is suitable for the screening of hepatoprotective substances.

To simplify liver support using an ex vivo perfused liver, an isolated pig liver was perfused with arterial blood from the recipient pig while monitoring the metabolic capacity of the ex vivo perfused liver. It was possible to perfuse the isolated liver for more than 24 h using arterial blood from a pig with ischemic liver failure. The viability of the isolated liver during support from the liver failure pig was well maintained as evidenced by the high adenylate energy charge (0.815) and a constant ketone body ratio (KBR) of over 1.0 sampled from the hepatic vein. Oxygen consumption (mean, 29.0 microl/min/g of liver) and bile production (mean, 24.2 microl/h/g of liver) were significantly higher in the isolated liver connected to the liver failure pig than in the organ connected to the pig without liver failure (15.5 microl/min/g and 7.3 microl/h/g, respectively). These findings suggest that this liver support system has sufficient metabolic capacity to support a failed liver. Further studies may provide the experimental basis necessary for the clinical application of this device in treatment of patients with acute liver failure.

Temperature changes and their distribution induced by 13.56-MHz radiofrequency (RF) heating of agar phantom and porcine and rabbit liver were investigated. It was possible to produce selective local heating of approximately 50 degrees C in the RF field of 2 x 2 x 2 cm(3) of the pig or rabbit liver. Coagulation necrosis after heating became pronounced and the margin between the coagulated lesion and normal tissue became clearer with time. Within 1 week after RF heating at 50 degrees C for 20 min, the coagulated area was replaced selectively and totally by necrotic tissue.

The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + beta-hydroxybutyrate), and the ketone body ratio (KBR: acetoacetate/beta-hydroxybutyrate) in the medium in order to assess the suitability of this system as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental study of hypoxic injury and revascularization in vitro.

Using computed tomography (CT), measurements of whole liver volume have been used for the assessment of pre-operative functional reserve in cirrhotics. However, measurements of hepatocyte volume, which exclude stromal fibrous tissue, are considered to more directly reflect hepatic functional reserve. We investigated the relationship between total hepatocyte volume and each of the parameters of conventional liver function. Indocyanine green (ICG) tests and blood analyses for the assessment of liver function were performed prior to surgery in cirrhotic patients with liver tumours. Pre-operative liver volume was determined by integrating images of each liver area obtained by CT. Liver area was measured by an image processing program that traced the profile of the liver image while excluding the tumorous area. Sections of normal tissue stained by the haematoxylin-eosin method, were obtained from the resected liver. Using these sections, a hepatocyte area: whole tissue area ratio was calculated using the image processing program, by tracing the profiles of the hepatocyte nodules. The total volume of hepatocytes was then calculated by multiplying the liver volume by this ratio. The hepatocyte volume per unit bodyweight was significantly correlated with ICG tests and with many other parameters of normal liver function. However, the liver volume per unit bodyweight was correlated only with the plasma ICG disappearance rate and with the blood platelet count. These observations suggest that the functional reserve of the cirrhotic liver is assessed more precisely by hepatocyte volume than by liver volume.

The effects of inflammatory cytokines (interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha) on energy metabolism were studied in primary cultured rat hepatocytes. Adenine nucleotide (ATP, ADP, and AMP) content, lactate production, the ketone body ratio (acetoacetate/beta-hydroxybutyrate) reflecting the liver mitochondrial redox state (NAD+/NADH), and nitric oxide formation were measured. Insulin increased ATP content in hepatocytes and had a maximal effect after 8-12 h of culture. Both interleukin-1beta and interleukin-6, but not tumor necrosis factor-alpha, significantly inhibited the ATP increase time- and dose-dependently. Interleukin-1beta and interleukin-6 also stimulated lactate production. During the same period, interleukin-1beta but not interleukin-6 decreased the ketone body ratio. Furthermore, interleukin-1beta markedly stimulated nitric oxide formation in hepatocytes, and this increase was blocked by NG-monomethyl-L-arginine (a nitric oxide synthase inhibitor) and by interleukin-1 receptor antagonist. NG-monomethyl-L-arginine reversed inhibition of the ATP increase, decrease in the ketone body ratio, and increase in lactate production, which were induced by interleukin-1beta. Interleukin-1 receptor antagonist completely abolished all of the effects induced by interleukin-1beta. These results demonstrated that interleukin-1beta and interleukin-6 affect the insulin-induced energy metabolism in rat hepatocytes by different mechanisms. Specifically, interleukin-1beta inhibits ATP synthesis by causing the mitochondrial dysfunction, a process which may be mediated by nitric oxide.

A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908 +/- 0.008 n = 9, human: 0.918 +/- 0.014 n = 6, mean +/- SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.

Among phenylbutazone (PZ) and its related compounds, suxibuzone (SUX) caused the most extensive decrease in pyruvate kinase (PK) activity with lower toxicity. Therefore, we studied the effect of SUX on rat hepatocarcinogenesis to confirm our assumption that an agent which causes a prolonged decrease in PK activity in rat liver promotes hepatocarcinogenesis. For initiation rats were fed a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) for 4 weeks. At the end of 53 weeks of the experiment the incidences of liver tumors were 14.3 and 70.0% in the rats fed basal diet and in the rats fed 0.5% SUX diet, respectively, after the initiation. No tumors were observed in rats fed the SUX diet without the initiation. The result shows that SUX promotes hepatocarcinogenesis and supports the above assumption.

Two forms (Peak A and Peak B) of thymidine kinase [EC 2.7.1.75] from regenerating rat liver cytosol were resolved and partially purified by Deae-cellulose chromatography. Both fractions were identical with respect to their substrate requirement, pH optima, metal requirements, and molecular weight, as judged by their sedimentation in sucrose density gradient centrifugation. Peak B differed from Peak A in heat sensitivity, inhibition by dCTP and Km for thymidine and ATP. Peak B enzyme was the only enzyme found in normal adult liver and Peak A enzyme was the form increasing predominantly in regenerating liver.

A probably promoter-specific decrease of L-type pyruvate kinase (L-PK) in Wistar rat liver is described. The possibility of utilizing the decrease in L-PK activity for screening of hepatic promoters is discussed. A significantly decreased level of activity of L-PK was observed during continuous feedings of the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene [(3'-MeDAB) CAS: 55-80-1], which initiates and promotes hepatocarcinogenesis, and of the known hepatic promoters phenobarbital [(PB) CAS: 50-06-6] and dichlorodiphenyltrichloroethane (CAS: 50-29-3) for at least 4 weeks. In contrast, if it occurred, the decrease in L-PK activity by the nonpromoting agents amobarbital (CAS: 57-43-2) and diphenylhydantoin. (CAS: 57-41-0) was temporary and almost overcome by the 4th week. The depression of L-PK activity caused by PB was reversible, was inversely correlated with PB concentration in the diet, and seemed to be organ-specific. Although hepatic promoters lowered L-PK activity in this study, data are so limited that a much more extensive study is necessary before a general conclusion can be drawn. In contrast to L-PK activity, the activity of K-type pyruvate kinase (K-PK) was induced by injections of the carcinogen diethylnitrosamine (CAS: 55-18-5) or the hepatotoxin CCl4 (CAS: 56-23-5) or by the feeding of 3'-MeDAB. However, feeding of PB or 2-methyl-4-dimethylaminoazobenzene (CAS: 54-88-6), which initiates but does not promote hepatocarcinogenesis, did not increase K-PK activity.

Pharmacological doses of niacin and its analogues were given intraperitoneally to rats with and without coadministration of a hepatocarcinogenic dose of diethylnitrosamine (DEN), and their effects on the induction of ornithine decarboxylase (ODC, EC 4.1.1.17) activity in the rat liver were studied. The induction of ODC activity by DEN was inhibited by 74.3, 85.5, 94.6, 97.6, 72.6 and 55.2% by nicotinamide, nicotinic acid, 3-hydroxymethylpyridine, beta-picoline, pyridine-3-aldehyde and ethylnicotinate respectively. When given alone, these analogues did not induce ODC activity. All these compounds are known to have a niacin effect. DEN-induced ODC activity was also inhibited by 84.0, 93.3, 52.8 and 75.9% by 6-aminonicotinamide, picolinic acid, pyridine-3-sulfonic acid and thionicotinamide, respectively, but, peculiarly, they induced ODC activity by their administration alone. These niacin analogues are known to have anti-niacin effects. Tryptophan, N'-methylnicotinamide and isonicotinic acid hydrazide did not affect the DEN-induced ODC activity but could induce ODC by themselves. Tryptophan belongs to the former group and isonicotinic acid hydrazide to the latter group. The reason for these discrepancies is discussed.