Bile secretion in hemoglobin-free perfused rat liver

Hemoglobin-free perfused rat liver was demonstrated to be a suitable experimental model in studying bile secretion. Bile flow slowly decreased to more than 3 h of perfusion. Despite differences in metabolic states, the bile flow was the same in the recirculating as in the nonrecirculating mode of perfusion. Sulfobromophthalein stimulated bile flow at high rates of infusion. In bile, the ratio conjugated to unconjugated sulfobromophthalein also increased with sulfobromophthalein infusion rate. The access of [14C]insulin, [14C] sucrose, and inorganic [32P] phosphate from perfusate into bile was restricted. Bile flow, secretion of taurocholate and sulfobromophthalein, and bile pressure are compared with values from anesthetized animals and from isolated livers perfused with medium containing erythrocytes.     

Impaired sensitivity to insulin of rat livers perfused with blood of diminished haematocrit.

1. In livers from fed rats perfused with homologous whole blood of a haematocrit value of 37%, insulin decreased the perfusate concentrations of glucose and amino acids, production of ketone bodies (3-hydroxybutyrate + acetoacetate) and increased bile flow. 2. Perfusion with blood diluted with buffer to a haematocrit value of 17% decreased hepatic O2 consumption by 40-50%. Perfusate concentrations of glucose and lactate, the rate of ketogenesis and the ratios [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] were all increased. 3. In livers perfused with blood of diminished haematocrit, effects of insulin on perfusate glucose an amino acids, ketogenesis and bile flow were abolished.     

Effect of dimethylsulphoxide on the functional characteristics of isolated perfused rat liver

Exposure of rat liver, perfused with 7% BSA in Krebs-Ringer bicarbonate buffer, to 1.4 m Me₂SO at 35 °C had no effect on the release of potassium from the livers, but the rate of urea synthesis fell from 0.6 to 0.1 μmol/min. Bile production also decreased and the total amount collected during perfusion was only half that produced by controls. After perfusion for 4 hr at 35 °C control livers and those exposed to Me₂SO started to release GOT into the perfusate but livers exposed to the cryoprotective compound released the enzyme at a faster rate.Exposure of livers to Me₂SO at 5 °C resulted in potassium being released at a slower rate (0.98 μmol/min) than from cooled controls (1.19 μmol/min) and urea synthesis was decreased from 0.8 to 0.2 μmol/min. Bile production also declined but, because bile flow normally ceases during hypothermia, the effect on this aspect of liver function was probably less than was found at 35 °C. Release of GOT from livers exposed to Me₂SO at 5 °C was quite different from that observed at 35 °C; the enzyme appeared in the perfusate after about 8 hr and it was present in much lower concentration than was found with appropriately cooled controls which started to release the enzyme after 6 hr.Thus, exposure of rat liver to Me₂SO at 5 °C appears to be slightly less damaging than exposure at 35 °C and it may even have a beneficial effect on some aspects of liver function in vitro.     

Flow dependence of first-order uptake of substances by heterogeneous perfused organs

A commonly accepted model of steady first-order uptake of substances by a perfused organ consisting of capillaries acting in parallel assumes that the capillaries are functionally identical. The model predicts the constancy of the quantity F 1n (c_i/c_o), where F is the rate of flow of perfusate through the organ, and c₁ and c_o are the inlet (arterial) and outlet (venous) concentrations, respectively, of the substance being taken up. The failure of that constancy under variations of F at constant values of physiological control data, as observed in liver and in muscle, is explained here in its general features in terms of a narrow statistical distribution of the relevant properties of individual capillaries. The coefficient of variation ? of that distribution is a measure of the functional heterogeneity of the organ. A detailed quantitative interpretation of published data on the uptake of colloidal CrP0₄ by the Kupffer cells of perfused isolated rat livers is carried through. The resulting value of e is found to be smaller than the upper limit for the corresponding value of e pertaining to the elimination of galactose by the hepatocytes of isolated perfused rat livers, estimated earlier by different experimental and theoretical methods. Transformation of bolus flow into Poiseuille flow by the removal of erythrocytes from the perfusate is shown to increase significantly the value of ?.     

Criteria for Viability Assessment of Discarded Human Donor Livers during Ex Vivo Normothermic Machine Perfusion

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37°C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.     

Regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase. In vitro inhibition by a protein present in bile.

The activity of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-limiting enzyme of cholesterol biosynthesis, is inhibited in vitro by factors present in both rat and bovine bile. The inhibitory factor from bovine bile has been purified to near homogeneity and is a high molecular weight lipoprotein with a density (p = 1.024) and lipid composition similar to serum β-lipoprotein. Analysis of the interaction of the enzyme and inhibitor demonstrate that the observed inactivation/inhibition is a function of lipoprotein concentration, microsomal protein concentration and duration of interaction. The observed inhibition is apparently irreversible and while neither substrate alone protects the enzyme, both substrates decrease the rate of inactivation several fold.     

Protein synthesis in the perfused rat liver

1. When the rat livers are perfused under the conditions of these experiments with rat blood diluted with saline, the livers remain capable of removing colloidal chromic phosphate normally for 4 hours or more; that is, the reticuloendothelial system continues to function normally. 2. Good rates of bile flow continue, generally for 4 hours. 3. The livers incorporate radioactivity from the amino acids methionine, lysine, and histidine at rapid rates for 1 or 2 hours. Thereafter the rates fall. 4. The specific activity of the free lysine and histidine in the perfusate falls rapidly during the experiments (to 25 or 35 per cent of its original value at 10 minutes). 5. The fall in rate of incorporation of radioactivity is attributable to the fall in amino acid specific activity. 6. Addition of a complete amino acid mixture to the perfusate does not appear to have any stimulatory effect on incorporation of radioactivity from labelled amino acids. 7. With lysine, on the assumption that incorporation is due to new protein formation, there is a rate of synthesis equivalent to 230 mg. of plasma protein per 100 gm. of rat per day. This result is in agreement with turnover data obtained from rats in vivo. 8. The results emphasize once again the importance of precursor specific activity in the interpretation of metabolic experiments with labelled amino acids.     

Abnormal secretion of proteins into bile from colchicine-treated isolated perfused rat livers.

The microtubule poison, colchicine, caused an abnormal output of a variety of proteins into rat bile. After 3 h of exposure to the drug, livers were isolated and perfused with media of defined protein composition. There was no essential change in permeability of the hepatobiliary system to proteins (e.g. bovine serum albumin) entering bile from the perfusion fluid. The rat (serum) albumin and fibrinogen that were secreted into bile from colchicine-treated livers were probably derived from the hepatocytes. Disruption of the microtubular system reduces the secretion of proteins at the sinusoidal face of the hepatocyte and results in an accumulation of secretory vesicles in the cytoplasm. It is suggested that under these conditions some of the vesicles discharge their contents into the bile canaliculus.     

[14C]Formate and [8-14C]Adenine incorporation into purine nucleotides of ischaemic rat liver after re-establishment of blood flow

• 1.1. After restoration of blood flow the incorporation rate of [¹⁴C]formate and [8-¹⁴C]adenine into purine nucleotides of ischaemic rat liver was investigated.
• 2.2. The incorporation rate of [¹⁴C]formate into adenine nucleotides was nearly 5-fold greater in post-ischaemic livers than in controls at 120 min point following blood restoration.
• 3.3. No difference in the rate of [8-¹⁴C]adenine incorporation into adenine nucleotides of post-ischaemic and control rat liver lobes was found.
• 4.4. After re-establishment of blood circulation a predominant contribution of the de novo biosynthetic pathway in the recovery of purine nucleotides of the ischaemic rat liver is emphasized.

     

Effect of HDL1 infusion on biliary secretion in perfused rat liver

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in thein vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.     

Biliary protein output by isolated perfused rat livers. Effects of bile salts.

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.     

Placental Transfer of Lactate,Glucose and 2-deoxyglucose in Control and Diabetic Wistar Rats

Placental transfer of lactate, glucose and 2-deoxyglucose was examined employing the in situ perfused placenta. Control and streptozotocin induced diabetic Wistar rats were infused with [U¹⁴C]-glucose and [³H]-2-deoxyglucose (2DG). The fetal side of the placenta was perfuseci with a cell free medium and glucose uptake was calculated in the adjacent fetuses. Despite the 5-fold higher maternal plasma glucose concentration in the diabetic dams the calculated fetal glucose metabolic index was not significantly different between the 2 groups. Placental blood flow was reduced in the diabetic animals compared with controls but reduction of transfer of [U¹⁴C]-glucose and [³H]-2-deoxyglucose and endogenously derived [¹⁴C]-Lactate to the fetal compartment, could not be accounted for by reduced placental blood flow alone. There was no significant net production or uptake of lactate into the perfusion medium that had perfused the fetal side of the placenta in either group. The plasma lactate levels in the fetuses adjacent to the perfused placenta were found to be higher than in the maternal plasma and significantly higher in the fetuses of the diabetic group compared with control group. In this model the in situ perfused placenta does not secrete significant quantities of lactate into the fetal compartment in either the control or diabetic group.     

Machine perfusion at 20°C reduces preservation damage to livers from non-heart beating donors

We previously reported that machine perfusion (MP) performed at 20 °C enhanced the preservation of steatotic rat livers. Here, we tested whether rat livers retrieved 30 min after cardiac arrest (NHBDs) were better protected by MP at 20 °C than with cold storage. We compared the recovery of livers from NHBDs with organs obtained from heart beating donors (HBDs) preserved by cold storage. MP technique: livers were perfused for 6 h with UW-G modified at 20 °C. Cold storage: livers were perfused in situ and preserved with UW solution at 4 °C for 6 h. Both MP and cold storage preserved livers were reperfused with Krebs-Heinselet buffer (2 h at 37 °C). AST and LDH release and mitochondrial glutamate dehydrogenase (GDH) levels were evaluated. Parameters assessed included: bile production and biliary enzymes; tissue ATP; reduced and oxidized glutathione (GSH/GSSG); protein–SH group concentration. Livers preserved by MP at 20 °C showed significantly lower hepatic damage at the end of reperfusion compared with cold storage. GDH release was significantly reduced and bile production, ATP levels, GSH/GSSG and protein–SH groups were higher in livers preserved by MP at 20 °C than with cold storage. The best preserved morphology and high glycogen content was obtained with livers submitted to MP at 20 °C. Liver recovery using MP at 20 °C was comparable to recovery with HBDs. MP at 20 °C improves cell survival and gives a better-quality of preservation for livers obtained from NHBDs and may provide a new method for the successful utilization of marginal livers.     

Effects of ethynyloestradiol on the metabolism of [1-14C]-oleate by perfused livers and hepatocytes from female rats

Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.     

Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony-forming cells

Background aimsA hierarchy of endothelial colony-forming cells (ECFC) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. ECFC has recently become an attractive target for new vascular regenerative therapies; however, in vitro expansion of ECFC typically depends on the presence of fetal bovine serum (FBS) or fetal calf serum (FCS) in the culture medium, which is not appropriate for its therapeutic application.MethodsTo identify optimal conditions for in vitro expansion of ECFC, the effects of human endothelial serum-free medium (SFM) supplemented with six pro-angiogenic cytokines and human umbilical cord blood plasma (HCP) were investigated. The in vitro morphology, proliferation, surface antigen expression and in vivo vessel-forming ability were utilized for examining the effects of medium on ECFC.ResultsThis novel formulation of endothelial cell culture medium allows us, for the first time, to isolate and expand human ECFC efficiently in vitro with a low concentration of HCP (1.5%) and without bovine serum additives. In this serum-reduced medium (SRM), human ECFC colony yields remained quantitatively similar to those cultured in a high concentration (10%) of bovine serum-supplemented medium. SRM-cultured ECFC displayed a robust clonal proliferative ability in vitro and human vessel-forming capacity in vivo.ConclusionsThe present study provides a novel method for the expansion of human ECFC in vitro and will help to advance approaches for using the cells in human therapeutic trials.     

Inhibition of mixed-function oxidation of p-nitroanisole in perfused rat liver by 2,4-dinitrophenol

The effect of dinitrophenol (52 μm), an uncoupler of oxidative phosphorylation, on p-nitroanisole O-demethylation in the perfused rat liver was examined. Dinitrophenol inhibited p-nitroanisole metabolism 70% in perfused livers from fasted, phenobarbital-treated rats, and 30% in livers from normal rats, but had no effect on this reaction in isolated microsomes. Rates of p-nitroanisole O-demethylation in livers from fed, phenobarbitaltreated rats were not inhibited by dinitrophenol unless the pentose phosphate shunt was first inhibited by 6-aminonicotinamide pretreatment. Dinitrophenol diminished cellular concentrations of ATP and NADPH 30 and 50%, respectively. Since mixed-function oxidation requires NADPH, these data are consistent with the hypothesis that dinitrophenol interrupts the synthesis and/or transfer of reducing equivalents from the mitochondria into the extramitochondrial space by interfering with energy-dependent NADPH synthesis and substrate shuttle mechanisms.In addition, dinitrophenol diminished conjugation reactions 57 and 89% in all metabolic states studied, most likely because it decreased UDP-glucose levels considerably (40 to 60%).     

Effects of monensin on vesicular transport pathways in the perfused rat liver

In the rat hepatocyte, the internalization and degradation of asialoglycoproteins and the secretion of plasma and biliary proteins require specific intracellular sorting of vesicles. To aid in the biochemical characterization of these different vesicular pathways, we examined the effects of the ionophore monensin on the uptake and degradation of 125I-asialoorosomucoid (ASOR) and on the secretion of plasma and biliary proteins by the in situ perfused rat liver. In control livers, 77% of injected 125I-ASOR was extracted on first pass; 93% of the extracted radioactivity was released back into the circulation (totally degraded and some intact ASOR was found); and approximately 2% was recovered in the bile, some of which was intact. Monensin treatment decreased first pass uptake of 125I-ASOR to 57% and abruptly blocked the release of radioactivity into the perfusate and the bile. When hepatic proteins were biosynthetically labeled with 3H-leucine, monensin treatment dramatically reduced and delayed the secretion of newly synthesized proteins into both the perfusate and the bile. In contrast with control livers, in which secretion of protein into the perfusate preceded secretion of protein into the bile, TCA-precipitable 3H-protein appeared in bile about 20 min before TCA-precipitable 3H-protein appeared in the perfusate in monensin-treated livers. Thus, monensin treatment in the perfused liver blocked the degradation of asialoglycoproteins and inhibited the secretion of plasma proteins but had less effect on biliary protein secretion. These data document physiologic effects of monensin in an intact organ and suggest that biochemical distinctions between different vesicular pathways exist in the rat hepatocyte.     

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 mm of oleate (fatty acid/albumin molar ratio $\text{7}\text{1}$). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.     

Effect of fatty acids on physical properties of microsomes from isolated perfused rat liver

A computer-centered spectrofluorimeter was used to examine the physicochemical properties of hepatic microsomes and microsomal lipids obtained from isolated rat livers perfused with medium containing palmitate or oleate. The fatty acid composition and degree of unsaturation of the liver microsomal lipids reflected that the fatty acid present in the perfusate. The absorption corrected fluorescence, relative fluorescence efficiency, polarization, and fluorescence anisotropy of several fluorescent probe molecules were measured to determine if their different microenvironments may be altered by the type of fatty acid infused. The probe molecules β-parinaric acid and 1.6-diphenyl-1,3,5-hexatriene had higher values for each of these parameters when incorporated into microsomes obtained from livers perfused with a medium containing palmitate than with oleate. The same parameters measured for cholesta-5,7,9(11)-trien-3β-ol and N-phenyl-1-naphthylamine were not altered. These differences appeared to be primarily due to alterations in microviscosity of the probe microenvironments since the rotational correlation time of 1,6-diphenyl-1,3,5-hexatriene was 25% lower in the microsomes from livers perfused with oleate as compared to livers perfused with palmitate. Thermal discontinuities in Arrhenius plots were noted in the intact microsomes but not in the isolated microsomal lipids with the fluorescence probe molecule β-parinaric acid. Break points occurred at 10°C and 26°C for microsomes from livers perfused with palmitate and at 12°C and 17°C for microsomes from livers perfused with oleate containing medium. These results suggest that the physicochemical properties of liver microsomes were determined in part by the fatty acid in the perfusate.     

In vivo relevance of Mrp2-mediated biliary excretion of the Amanita mushroom toxin demethylphalloin

To determine which efflux carriers are involved in hepatic phalloidin elimination, hepatobiliary [³H]-demethylphalloin (DMP) excretion was studied in normal Wistar rats and in Mrp2 deficient TR(−) Wistar rats as well as in normal wild-type FVB mice, Mdr1a,b(−/−) knockout mice, and Bcrp1(−/−) knockout mice by in situ bile duct/gallbladder cannulation. A subtoxic dose of 0.03 mg DMP/kg b.w. was used, which did not induce cholestasis in any tested animal. Excretion of DMP into bile was not altered in Mdr1a,b(−/−) mice or in Bcrp1(−/−) mice compared with wild-type FVB mice. Whereas 17.6% of the applied dose was excreted into bile of normal Wistar rats, hepatobiliary excretion decreased to 7.9% in TR(−) rats within 2 h after intravenous application. This decrease was not due to reduced cellular DMP uptake, as shown by normal expression of Oatp1b2 in livers of TR(−) rats and functional DMP uptake into isolated TR(−) rat hepatocytes. Tissue concentrations of phalloidin were also not altered in any of the transgenic mice. Interestingly, the decrease of biliary DMP excretion in the TR(−) rats was not followed by any increase of phalloidin accumulation in the liver but yielded a compensatory excretion of the toxin into urine, indicating that hepatocytes of TR(−) rats expelled phalloidin back into blood circulation.