Metabolism of inorganic sulphate in the isolated perfused rat liver. Effect of sulphate concentration on the rate of sulphation by phenol sulphotransferase

1. The metabolism of inorganic [³⁵S]sulphate (Na₂³⁵SO₄) was studied in the isolated perfused rat liver at three initial concentrations of inorganic sulphate in the perfusion medium (0, 0.65 and 1.30mm), in relation to sulphation and glucuronidation of a phenolic drug, harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole). 2. [³⁵S]Sulphate rapidly equilibrated with endogenous sulphate in the liver. It was excreted in bile and reached, at the lowest concentration in the perfusion medium, concentrations in bile that were much higher than those in the perfusion medium; at the higher sulphate concentrations, these concentrations were equal. The physiological concentration of inorganic sulphate in the liver, available for sulphation of drugs, is similar to the plasma concentration. 3. At zero initial inorganic sulphate in the perfusion medium, the rate of sulphation was very low and harmol was mainly glucuronidated. At 0.65mm-sulphate glucuronidation was much decreased and considerable sulphation took place, indicating efficient competition of conjugation by sulphation. At 1.30mm-sulphate the sulphation increased still further. 4. The results suggest that an important factor in sulphation is the relatively high K_m of synthesis of adenosine 3′-phosphate 5′-sulphatophosphate (the co-substrate of sulphation) for inorganic sulphate, which is of the order of the plasma concentration of inorganic sulphate. The steady-state adenosine 3′-phosphate 5′-sulphatophosphate concentration may determine the rate of sulphate conjugation of drugs in the rat in vivo.     

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.     

Gaseous persufflation with carbon monoxide during ischemia protects the isolated liver and enhances energetic recovery

Background

The benefit of carbon monoxide as applied by controlled, continuous gaseous persufflation during liver preservation on postischemic graft recovery was investigated in an isolated rat liver model.

Methods

Livers from male Wistar rats were retrieved 30 min after cardiac arrest of the donor and subjected to 18 h of cold storage. Some grafts were subjected to gaseous persufflation with carbon monoxide (CO, dissolved in nitrogen) during static cold storage at a concentration of 50 ppm or 250 ppm. Graft viability was assessed thereafter upon warm reperfusion in vitro.

Results

CO-persufflation significantly reduced cellular enzyme loss (maximal at 50 ppm) and functional recovery (bile production and energy charge) upon reperfusion by about 50%. The effect was associated with a reduction of free radical-induced lipid peroxidation, lower vascular perfusion resistance, and improved mitochondrial ultrastructure.

Conclusion

Viability of cold stored liver grafts can be notably augmented by gaseous ex vivo application of low dose CO to the isolated organ.     

Metabolic effects of trifluoperazine in the liver and the influence of calcium

The effects of trifluoperazine on hepatic cell metabolism were investigated using isolated perfused rat liver. The following effects of trifluoperazine were found: (1) trifluoperazine inhibited oxygen uptake, the site of action being the mitochondria. Half-maximal inhibition occurred at concentrations around 50 μM; with 100 μM trifluoperazine the effect was already maximal. When Ca²⁺ was withdrawn from the perfusion medium and the intracellular Ca²⁺ pools were exhausted, the inhibitory action on respiration was no longer observable. The rein-troduction of Ca²⁺ restored inhibition. (2) Glycogenolysis and glycolysis were not significantly affected during the infusion of trifluoperazine. After stopping trifluoperazine infusion, however, glycogenolysis (glucose release) experienced a transitory stimulation. (3) Gluconeogenesis from lactate as the carbon source was inhibited by trifluoperazine. This inhibition was approximately proportional to the inhibition of oxygen uptake. Withdrawal of Ca²⁺ diminished, but it did not eliminate, inhibition of gluconeogenesis. (4) Ketogenesis was also inhibited in parallel with the inhibition of oxygen uptake. Withdrawal of Ca²⁺ from the perfusion fluid also abolished this action. (5) The effects of trifluoperazine were reverted very slowly when its infusion was stopped. The recovery of oxygen uptake at 50 min after cessation of the infusion was only 30%. Uptake of the substance was very fast. Absence of Ca²⁺ did not affect uptake. It was concluded that inhibition of mitochondrial energy metabolism is one of the most prominent effects of trifluoperazine in the liver. The fact that this inhibition depends on Ca²⁺ is unique.     

Tissue-specific Short Chain Fatty Acid Metabolism and Slow Metabolic Recovery after Ischemia from Hyperpolarized NMR in Vivo

Mechanistic details of mammalian metabolism in vivo and dynamic metabolic changes in intact organisms are difficult to monitor because of the lack of spatial, chemical, or temporal resolution when applying traditional analytical tools. These limitations can be addressed by sensitivity enhancement technology for fast in vivo NMR assays of enzymatic fluxes in tissues of interest. We apply this methodology to characterize organ-specific short chain fatty acid metabolism and the changes of carnitine and coenzyme A pools in ischemia reperfusion. This is achieved by assaying acetyl-CoA synthetase and acetyl-carnitine transferase catalyzed transformations in vivo. The fast and predominant flux of acetate and propionate signal into acyl-carnitine pools shows the efficient buffering of free CoA levels. Sizeable acetyl-carnitine formation from exogenous acetate is even found in liver, where acetyl-CoA synthetase and acetyl-carnitine transferase activities have been assumed sequestered in different compartments. In vivo assays of altered acetate metabolism were applied to characterize pathological changes of acetate metabolism upon ischemia. Coenzyme pools in ischemic skeletal muscle are reduced in vivo even 1 h after disturbing muscle perfusion. Impaired mitochondrial metabolism and slow restoration of free CoA are corroborated by assays employing fumarate to show persistently reduced tricarboxylic acid (TCA) cycle activity upon ischemia. In the same animal model, anaerobic metabolism of pyruvate and tissue perfusion normalize faster than mitochondrial bioenergetics.     

In vivo assessment of 6-deoxy-6-[18F]fluoro-d-galactose as a PET tracer for studying galactose metabolism

The potential of 6-deoxy-6-[¹⁸F]fluoro-d-galactose (6-[¹⁸F]FdGal) as an in vivo tracer for studying galactose metabolism in tumors and liver was investigated. High uptake and rapid clearance of the radioactivity were observed in many organs of mice after i.v. injection of the tracer. d-Galactose loading did not affect liver uptake. Three experimental tumors showed a slightly higher uptake than other tissues, and rat brain tumor was clearly visualized by autoradiography. However, the radioactivity in tumors decreased rapidly. In the liver, a significant amount of the tracer was found in a galactonate form, while this oxidation was a minor metabolic pathway in the tumors. In both tumor and liver tissues, small amounts of the tracer were incorporated into macromolecular glycoconjugate via phosphate and uridylate forms as intermediate precursors. These results indicate that 6-[¹⁸F]FdGal is not suitable for studying galactose metabolism in vivo because of the low affinity of the tracer for the metabolism.     

Investigation of styrene in the liver perfusion/cell culture system. No indication of styrene-7,8-oxide as the principal mutagenic metabolite produced by the intact rat liver

Mutagenic effect of styrene and styrene-7,8-oxide was studied with the isolated perfused rat liver as metabolizing system and Chinese hamster V79 cells as genetic target cells. Styrene-7,8-oxide which is mutagenic per se was rapidly metabolized by the perfused rat liver. Thus no mutagenic effect was detected neither in the perfusion medium nor in the bile. However when styrene was added to the perfusion system, an increase in V79 mutants was observed regardless of where in the circulating perfusion medium the V79 cells were placed: the same effect was obtained with V79 cells close to the liver as well as at a distance from the liver. No mutagenic effect was observed in the bile. Simultaneous analysis of the styrene-7,8-oxide concentration in the perfusion medium, suggest that this metabolite is not the cause of the mutagenic effect observed during perfusion with styrene.The effect of the two test compounds on some liver functions was also studied. Both styrene and styrene-7,8-oxide changed the bile flow without affecting bile acid secretion: styrene caused a reduction in bile flow as compared to control perfusions and styrene-7,8-oxide increased the bile flow. Styrene, but not styrene-7,8-oxide, reduced gluconeogenesis from lactate. Styrene had no effect on the liver's capacity to incorporate amino acids into plasma proteins, whereas styrene-7,8-oxide reduced the amino acid incorporation. The microsomal cytochrome P-450 content was not affected by the two test compounds. No alteration in microsomal N- and C-oxygenation of N, N-dimethylaniline (DMA) was observed with styrene-7,8-oxide or the lower styrene dose used (240 μmol), whereas the higher styrene concentration (480 μmol) reduced N-oxygenation and thus also the total DMA metabolism.It is suggested that the results on styrene and styrene-7,8-oxide found here using the liver perfusion/cell culture system mimic the metabolism expected to be found in the intact animal, thus indicating that styrene-7,8-oxide is not the principal mutagenic metabolite of styrene in vivo.     

Influence of ethanol on the liver metabolism of fed and starved rats

1. The influence of ethanol on the metabolism of livers from fed and starved rats has been studied in liver-perfusion experiments. Results have been obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in livers from starved rats than in livers from fed rats. Ethanol had no effect on the oxygen consumption of either type of liver. After the addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely, the change being faster in livers from starved rats. 3. With livers from fed rats glucose was released from the liver into the perfusion medium. This release was slightly greater when ethanol was present. With livers from starved rats no release of glucose was observed, and when ethanol was added a marked uptake of glucose from the medium was found. A simultaneous release of glycolytic end products, lactate and pyruvate, into the medium occurred. 4. Acetate was the main metabolite accumulating in the perfusion medium when ethanol was oxidized. With livers from starved rats a slightly increased formation of ketone bodies was found when ethanol was present. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 10 to 87 with livers from fed rats and from 20 to 171 with livers from starved rats when the livers were perfused with ethanol in the medium. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 0·8 to 7·6 with livers from fed rats and from 1·0 to 9·5 with livers from starved rats when ethanol was added to the medium. 6. The effects of ethanol are discussed and related to changes in the redox state of the liver that produce new conditions for some metabolic pathways.     

Adenosine transport in liver before and after organ preservation.

The effect of 24-h cold storage of liver on nucleoside transport was investigated. Nucleoside transport was estimated under conditions when both known types of nucleoside transport, facilitated diffusion and Na+/nucleoside cotransport, were active and when one of these transport mechanisms was inhibited. The rate of adenosine transport was not decreased after long-term cold storage of the liver. Inhibition of one of the transport systems decreased the rate of adenosine uptake before and after preservation of the liver to about the same extent. The adenosine transport rate was maintained during long-term (100 min) liver perfusion ex vivo. Slight activation of energy-dependent transport in the beginning of reperfusion and the slower recovery of this transport after the second transition from Na+-free to Na+-containing perfusion are not regarded as physiologically important because they were observed after changing the ionic content of the extracellular medium. We conclude that the nucleoside transport systems in liver are quite well preserved after 24-h cold storage of the organ.     

Mathematical Model of Leukemic-Cell Circulation in the Mouse

L1210 leukemic cells injected in vivo are eliminated from the blood and disintegrated in organs such as the lungs and liver. We present a compartmental model which reproduces one type of in vivo experiment, based on the so-called perfusion curves. Although the data are not complete and some are only approximated, modeling gives a consistent picture of the process.     

Bivascular liver perfusion in the anterograde and retrograde modes: Zonation of the response to inhibitors of oxidative phosphorylation

The action of cyanide (500 μM ), 2,4-dinitrophenol (50 μM ) and atractyloside (100 μM ) on glycogen catabolism and oxygen uptake was investigated in the bivascularly perfused liver of fed rats. Cyanide, 2,4-dinitrophenol and atractyloside were infused at identical rates into the hepatic artery in either the anterograde or retrograde perfusion. The accessible aqueous cell spaces were determined by means of the multiple-indicator dilution technique. Glucose release, oxygen uptake and glycolysis were measured as metabolic parameters. Oxygen uptake changes per unit cell space caused by atractyloside (inhibition) and 2,4-dinitrophenol (stimulation) were equal in the retrograde perfusion (periportal cells) and the anterograde perfusion (space enriched in perivenous cells); the decreases caused by cyanide were higher in the retrograde perfusion. Glucose release from periportal cells was not increased upon inhibition of oxidative phosphorylation, a phenomenon which was independent of the mechanism of action of the inhibitor. There were nearly identical changes in glycolysis in the periportal and perivenous cells. It was concluded that: (1) oxygen concentration in the perfused rat liver, if maintained above 100 μM , had little influence on the zonation of the respiratory activity; (2) in spite of the lower activities of the key enzymes of glycolysis in the periportal hepatocytes, as assayed under standard conditions, these cells were as effective as the perivenous ones in generating ATP in the cytosol when oxidative phosphorylation was impaired; (3) the key enzymes of glycogenolysis and glycolysis in periportal and perivenous cells responded differently to changes in the energy charge.     

Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt^−/− mice than in Pemt^+/+ mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt^−/− mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.     

Effect of hyperoxia and hypercapnia on tissue oxygen and perfusion response in the normal liver and kidney

Objective

Inhalation of air with altered levels of oxygen and carbon dioxide to manipulate tissue oxygenation and perfusion has both therapeutic and diagnostic value. These physiological responses can be measured non-invasively with magnetic resonance (MR) relaxation times. However, interpreting MR measurements is not straight-forward in extra-cranial organs where gas challenge studies have only begun to emerge. Inconsistent results have been reported on MR, likely because different organs respond differently. The objective of this study was to elucidate organ-specific physiological responses to gas challenge underlying MR measurements by investigating oxygenation and perfusion changes in the normal liver and kidney cortex.

Materials and Methods

Gas challenges (100% O₂, 10% CO₂, and carbogen [90% O₂+10% CO₂]) interleaved with room air was delivered to rabbits to investigate their effect on tissue oxygenation and perfusion. Real-time fiber-optic measurements of absolute oxygen and relative blood flow were made in the liver and kidney cortex.

Results

Only the liver demonstrated a vasodilatory response to CO₂. Perfusion changes to other gases were minimal in both organs. Tissue oxygenation measurements showed the liver responding only when CO₂ was present and the kidney only when O₂ was present.

Conclusion

This study reveals distinct physiological response mechanisms to gas challenge in the liver and kidney. The detailed characterization of organ-specific responses is critical to improving our understanding and interpretation of MR measurements in various body organs, and will help broaden the application of MR for non-invasive studies of gas challenges.     

In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats

Organ decellularization is emerging as a promising regenerative medicine approach as it is able to provide an acellular, three-dimensional biological scaffold material that can be seeded with living cells for organ reengineering. However this application is currently limited to donor-derived decellularized organs for reengineering in vitro and no study has been conducted for re-engineering the decellularized organ in vivo. We developed a novel technique of a single liver lobe decellularization in vivo in live animals. Using a surgical method to generate a by-pass circulation through the portal vein and infra-hepatic vena cava with a perfusion chamber system, we decellularized the single liver lobe and recellularized it with allogenic primary hepatocytes. Our results showed that the decellularization process in vivo can preserve the vascular structural network and functional characteristics of the native liver lobe. It allows for efficient recellularization of the decellularized liver lobe matrix with allogenic primary hepatocytes. Upon the re-establishment of blood circulation, the recellularized liver lobe is able to gain the function and the allogenic hepatocytes are able to secret albumin. Our findings provide a proof of principle for the in vivo reengineering of liver.     

Effect of Oxygenation on Xylose Fermentation by Pichia stipitis

The effect of oxygen limitation on xylose fermentation by Pichia stipitis (CBS 6054) was investigated in continuous culture. The maximum specific ethanol productivity (0.20 g of ethanol g dry weight⁻¹ h⁻¹) and ethanol yield (0.48 g/g) was reached at an oxygen transfer rate below 1 mmol/liter per h. In the studied range of oxygenation, the xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were constant as well as the ratio between the NADPH and NADH activities of xylose reductase. No xylitol production was found. The pyruvate decarboxylase (EC 4.1.1.1) activity increased and the malate dehydrogenase (EC 1.1.1.37) activity decreased with decreasing oxygenation. With decreasing oxygenation, the intracellular intermediary metabolites sedoheptulose 7-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, and malate accumulated slightly while pyruvate decreased. The ratio of the xylose uptake rate under aerobic conditions, in contrast to that under anaerobic assay conditions, increased with increasing oxygenation in the culture. The results are discussed in relation to the energy level in the cell, the redox balance, and the mitochondrial function.     

Nonalcoholic fatty liver disease impairs the cytochrome P-450-dependent metabolism of α-tocopherol (vitamin E)

This study aims to investigate in in vivo and in vitro models of nonalcoholic fatty liver disease (NAFLD) the enzymatic metabolism of α-tocopherol (vitamin E) and its relationship to vitamin E-responsive genes with key role in the lipid metabolism and detoxification of the liver. The experimental models included mice fed a high-fat diet combined or not with fructose (HFD+F) and HepG2 human hepatocarcinoma cells treated with the lipogenic agents palmitate, oleate or fructose. CYP4F2 protein, a cytochrome P-450 isoform with proposed α-tocopherol ω-hydroxylase activity, decreased in HFD and even more in HFD+F mice liver; this finding was associated with increased hepatic levels of α-tocopherol and decreased formation of the corresponding long-chain metabolites α-13-hydroxy and α-13-carboxy chromanols. A decreased expression was also observed for PPAR-γ and SREBP-1 proteins, two vitamin E-responsive genes with key role in lipid metabolism and CYP4F2 gene regulation. A transient activation of CYP4F2 gene followed by a repression response was observed in HepG2 cells during the exposure to increasing levels of the lipogenic and cytotoxic agent palmitic acid; such gene repression effect was further exacerbated by the co-treatment with oleic acid and α-tocopherol and was also observed for PPAR-γ and the SREBP isoforms 1 and 2. Such gene response was associated with increased uptake and ω-hydroxylation of α-tocopherol, which suggests a minor role of CYP4F2 in the enzymatic metabolism of vitamin E in HepG2 cells. In conclusion, the liver metabolism and gene response of α-tocopherol are impaired in experimental NAFLD.     

Arginine supplementation and exposure time affects polyamine and glucose metabolism in primary liver cells isolated from Atlantic salmon

Arginine has been demonstrated to enhance glucose and lipid oxidation in mammals through activation of polyamine turnover. We aimed to investigate how arginine affects energy utilization through polyamine metabolism and whether this effect is time dependent. Primary liver cells were isolated from Atlantic salmon (2.2 kg body weight) fed diets containing 25.5 (low arginine, LA) or 36.1 (high arginine, HA) g arginine/kg dry matter for 12 weeks, to investigate the effect of long-term arginine supplementation. The cells were cultured for 24 h in L-15 medium to which either alpha-difluoromethylornithine (DFMO) or N ¹,N ¹¹-diethylnorspermine (DENSPM) was added. Analysis of the medium by nuclear magnetic resonance revealed significant differences between the two dietary groups as well as between cells exposed to DFMO and DENSPM, with decreased glucose, fumarate and lactate concentrations in media of the HA cells. Liver cells from fish fed the HA diet had higher spermidine/spermine-N1-acetyltransferase protein abundance and lower adenosine triphosphate concentration as compared to the LA-fed fish, while gene expression was not affected by either diet or treatment. Primary liver cells isolated from salmon fed a commercial diet and cultured in L-15 media with or without arginine supplementation (1.82 or 3.63 mM) for 48 h, representing short-term effect of arginine supplementation, showed differential expression of genes for apoptosis and polyamine synthesis due to arginine supplementation or inhibition by DFMO. Overall, arginine concentration and exposure time affected energy metabolism and gene regulation more than inhibition or activation of key enzymes of polyamine metabolism, suggesting a polyamine-independent influence of arginine on cellular energy metabolism and survival.     

Metabolism of [3H]leupeptin by rat liver

Tritium-labeled leupeptin was used to study how this tripeptide proteinase inhibitor interacts with the liver, including the mechanism of its transport into the cell, its subcellular distribution after uptake, and its metabolism once in the tissue. Experiments were done in situ and in a perfused liver. At low concentrations (1 to 10 μm) the uptake of radioactive inhibitor was competed by chemically reduced leupeptin. At high concentrations at least up to 400 μm the uptake was directly proportional to the external concentration of tripeptide. Entry into the tissue essentially stopped at low temperature (<21 °C). [³H]Leupeptin initially was located in the soluble fraction of the liver homogenate and by 15 to 30 min became concentrated in the lysosome-rich fraction. During 2 h of perfusion almost 50% of [³H]leupeptin that had entered the liver was secreted intact into the bile. In addition, a portion of the leupeptin that remained in the liver was degraded during this time period.