Effect of chronic renal failure with metabolic acidosis on alanine metabolism in isolated liver cells.

BACKGROUND & AIMS: Decreased ureagenesis and gluconeogenesis from alanine have been reported during chronic renal failure in rat. This study addressed the respective roles of plasma-membrane transport and intracellular metabolism in these abnormalities of alanine pathways. METHODS: In hepatocytes isolated from uremic and control rats, we investigated: (1) the influence of uremia on gluconeogenesis and ureagenesis during incubations with alanine; (2) the kinetics of alanine plasma-membrane transport; (3) the relationships between intracellular alanine concentrations and its metabolism. Plasma-membrane alanine transport was assessed after addition of alanine (2 mM) by measuring its intracellular accumulation from 0 to 10 min, in the presence of a transaminase inhibitor. Alanine metabolism was studied in perifused hepatocytes by measuring intracellular alanine concentration together with urea, glucose and lactate production in the presence of increasing concentrations of alanine (0-8 mM). RESULTS: Uremic rats showed decreased plasma bicarbonate. Uremia induced (P<0.05) a decrease in both gluconeogenesis (36%) and ureagenesis (22%). Alanine plasma-membrane transport decreased by 20% during uremia. During perifusions, uremia induced a 30-40% decrease in urea, glucose, and lactate production without modifying intracellular alanine concentration. CONCLUSIONS: In uremic rats with acidosis, hepatocyte alanine utilization was impaired at both plasma-membrane transport and intracellular transamination steps.

Chronic renal failure-induced changes in serum and urine bile acid profiles.

Although the kidney is believed to play a minor role in bile acid (BA) excretion, chronic renal failure (CRF) has been reported to be accompanied by alterations in the BA balance. The aim of the present work was to evaluate the changes in BA serum concentrations and renal excretion in patients with different stage of CRF or after kidney transplantation and to elucidate whether these might play a role in the development of pruritus, a common symptom in this disease. This study was carried out on 125 patients. None of them had a history or signs of hepatobiliary malfunction. They belonged either to a control group (N = 31) or to one of the three following CRF groups: patients maintained only on a low-protein diet (diet group, N = 23); the same, together with periodic sessions of hemodialysis (dialysis group, N = 42); and patients who had undergone a kidney transplant more than 1 and less than 2 years before (transplanted group, N = 29). Serum and urine BA concentrations were assayed by gas chromatography-mass spectrometry. Pruritus was quantified by means of a questionnaire answered at the time of sample collection. A marked hypercholanemia together with a reduction in BA output into urine and profound alterations in the profiles of these compounds in both serum and urine in patients with CRF were observed. The levels of total BAs in serum, but not the proportions of molecular species, were corrected by hemodialysis. By contrast, kidney transplant reversed BA serum patterns to normality but, due to immunosuppressive therapy with cyclosporin A, total serum BA concentrations were consistently elevated in this group. Pruritus was more frequent in patients with impaired kidney function and hypercholanemia, although no significant correlation between the degree of this symptom and the magnitude of the serum concentrations of total or individual BAs were found. By contrast, in spite of hypercholanemia, once renal function had been restored by kidney transplantation, none of the patients suffered from pruritus. These results suggest that the kidney plays an important role in determining the serum BA pool size and composition and that hypercholanemia may be a contributing factor, but not the only one, determining the development of pruritus in patients with CRF.

Changes in the pattern of bile acids in the nuclei of rat liver cells during hepatocarcinogenesis.

Bile acids reach the nuclei of hepatocytes, where they may play an important role in controlling gene expression by binding to nuclear receptors. In previous studies, changes in the amounts of the different molecular species of bile acids in the hepatocyte nucleus during rat liver regeneration have been reported. The aim of the present work was to investigate whether this also occurs during rat hepatocarcinogenesis. Liver cell nuclei were isolated after homogenization of livers from healthy adult rats (controls) and from rats at different time points during chemically induced hepatocarcinogenesis, corresponding to the stages of foci (12 weeks), hepatoma (20 weeks) and carcinoma (32 weeks). Bile samples from the cannulated common bile duct were collected for 1h from different sets of animals undergoing hepatocarcinogenesis. Bile acids in bile, liver homogenates and isolated nuclei were measured by GC-MS. Because the yield of nuclei isolated changed during the course of hepatocarcinogenesis (control, 20.1%; 12 weeks, 23.6%; 20 weeks, 7.8%; 32 weeks, 5.1%), amounts of bile acids in nuclei were corrected for the amount of DNA in each preparation. During hepatocarcinogenesis, bile acid concentrations in liver homogenates were reduced to approximately half the values obtained in control livers, while the levels of bile acids in both isolated nuclei and bile were not decreased. Hepatocarcinogenesis induced changes in the composition of bile acid pools. These were manifest as an increase in the proportion of cholic acid and a decrease in that of ursodeoxycholic acid in both bile and liver. These modifications differed from the changes seen in the nuclear bile acid pool, where a decrease in the proportion of cholic acid together with an increase in that of ursodeoxycholic acid were the major changes observed during hepatocarcinogenesis. With regard to the 'flat' bile acids (allo-cholic acid plus Delta(5)- or Delta(4)-unsaturated bile acids), a marked hepatocarcinogenesis-induced increase in the output of these species in bile was found. However, these bile acids were only found in liver homogenates at the hepatoma stage, whereas they were not detected in isolated nuclei at any stage of hepatocarcinogenesis. In summary, these results support the existence of a bile acid pool in hepatocyte nuclei whose composition differs from that of the extranuclear bile acid pool. Moreover, they indicate that, during hepatocarcinogenesis, the composition of the nuclear pool undergoes important alterations.

Cytokine-mediated inhibition of ketogenesis is unrelated to nitric oxide or protein synthesis.

Cytokines play an important role in the lipid disturbances commonly associated with sepsis. Ketogenesis is inhibited during sepsis, and tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) have been suggested to mediate this impairment, irrespective of the ketogenic substrate (fatty acid or branched chain ketoacid). However, the underlying mechanism of cytokine action is still unknown. First we investigated the possible role of the induction of nitric oxide (NO) synthesis, using rat hepatocyte monolayers. Hepatocytes were incubated for 6 h, with either alpha -ketoisocaproate (KIC) (1 mM) or oleic acid (0.5 mM) in the presence or absence of TNF alpha (25 microg/L) and IL-6 (15 microg/L). In some experiments, cells were incubated with NO synthase (NOS) inhibitors. The ketone body (beta -hydroxybutyrate and acetoacetate) production and nitrite production were measured in the incubation medium. Our results indicated no involvement of nitric oxide in the inhibitory action of cytokines on ketogenesis. Secondly, we showed that cycloheximide (10(-4)M) did not counteract the cytokine-mediated ketogenesis decrease; hence, the effects of cytokines on ketogenesis are not protein synthesis-dependent. The cytokine-mediated inhibition of ketogenesis is therefore unrelated to either NO production or protein synthesis.

Glucose 6-phosphate hydrolysis is activated by glucagon in a low temperature-sensitive manner.

Glucagon affects liver glucose metabolism mainly by activating glycogen breakdown and by inhibiting pyruvate kinase, whereas a possible effect on glucose-6-phosphatase has also been suggested. Although such a target is of physiological importance for liver glucose production it was never proven. By using a model of liver cells, perifused with dihydroxyacetone, we show here that the acute stimulation of gluconeogenesis by glucagon (10(-7) m) was not related to the significant inhibition of pyruvate kinase but to a dramatic activation of the hydrolysis of glucose 6-phosphate. We failed to find an acute change in glucose-6-phosphatase activity by glucagon, but the increase in glucose 6-phosphate hydrolysis was abolished at 21 degrees C; conversely the effect on pyruvate kinase was not affected by temperature. The activation of glucose 6-phosphate hydrolysis by glucagon was confirmed in vivo, in postabsorptive rats receiving a constant infusion of glucagon, by the combination of a 2-fold increase in hepatic glucose production and a 60% decrease in liver glucose 6-phosphate concentration. Besides the description of a novel effect of glucagon on glucose 6-phosphate hydrolysis by a temperature-sensitive mechanism, this finding could represent an important breakthrough in the understanding of type II diabetes, because glucose 6-phosphate is proposed to be a key molecule in the transcriptional effect of glucose.

Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.

The aim of this work was to investigate the reappearance during liver neoplasia of bile acids (BAs) species, which are unusual in healthy adults, but common in fetuses. Serum and urine samples were collected from patients with hepatocellular carcinoma (HCC; n=27), and for comparative purposes, with liver cirrhosis (n=49), liver metastasis (n=19), chronic viral hepatitis (n=11) and healthy volunteer (control group; n=26) groups. BAs were identified and measured by GC--MS. Hypercholanaemia was found in all groups of patients. In HCC, this was characterized by a marked increase in the chenodeoxycholate/cholate ratio in both serum and urine. Although increased levels of BAs, with hydroxylations at unusual positions, and oxo-BAs were found in HCC, these were not significantly different from those observed in other groups. However, BAs with a flat structure, i.e. Delta(4)-unsaturated- and 5 alpha- or allo-BAs, which were almost absent in healthy subjects, were markedly increased in the serum and urine of HCC patients. They were also detected, although in much lower amounts, in liver metastasis and liver cirrhosis, but not in viral hepatitis. Flat-BAs were better detected in urine than in serum. Urinary Delta(4)-unsaturated-BA output was significantly lower in patients with small tumours (<3 cm) compared with those with higher size tumours. No correlation between flat-BA output into urine and serum alpha-fetoprotein or total BAs was found. These results suggest that Delta(4)- and/or allo-BAs are particularly elevated in patients with HCC, which may be a potentially useful complementary, rather than alternative, marker for early detection of liver neoplasia.

Exogenous Mg-ATP induces a large inhibition of pyruvate kinase in intact rat hepatocytes.

Mg-ATP infusion in vivo has been reported to be beneficial both to organ function and survival rate in various models of shock. Moreover, a large variety of metabolic effects has been shown to occur in several tissues due to purinergic receptor activation. In the present work we studied the effects of exogenous Mg-ATP in rat liver cells perifused with dihydroxyacetone to investigate simultaneously gluconeogenetic and glycolytic pathways. We found a significant effect on oxidative phosphorylation as characterized by a decrease in oxygen consumption rate and in the cellular ATP-to-ADP ratio associated with an increase in lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid and reversible inhibition of both gluconeogenesis and glycolysis. The main effect on gluconeogenesis was located at the level of the fructose cycle, whereas the decrease in glycolysis was due to a strong inhibition of pyruvate kinase. Although pyruvate kinase inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes. This newly described short-term regulation of pyruvate kinase occurs only in the intact cell and may open new potentials for the pharmacological regulation of pyruvate kinase in vivo.

Enhancement of bile acid pool size, synthesis and secretion by epomediol in the rat.

Epomediol is a terpenoid compound that has been reported to reverse 17alpha-ethinylestradiol-induced cholestasis and to have a choleretic effect related to the biliary secretion of epomediol glucuronide. The aim of this study was to investigate the contribution of changes in bile acid metabolism to epomediol-induced effects on bile formation. Twenty-four-hour bile collections were performed in animals that had received intraperitoneal epomediol for five days at 100 mg/kg daily. Epomediol-treated rats had a 24% larger bile acid pool and 28% greater bile acid synthesis than controls when measured by the "washout" technique. There was no change in the fractional turnover rate and the cycling frequency of the pool. Both basal bile flow and bile acid secretion were significantly increased (+42% and +74%, respectively). Linear regression analysis between bile flow and bile acid secretion revealed that both bile acid-dependent fraction and bile acid-independent fraction were significantly increased (+40 and +27, respectively), with no change in the choleretic capacity of bile acids. Cholesterol secretion was increased by 42%, but there were no significant differences in phospholipid secretion. Cholesterol 7alpha-hydroxylase and HMG-CoA reductase activities were significantly higher in epomediol-treated rats (+39% and +97%, respectively). The activities of NADPH-cytochrome c reductase and aniline hydroxylase were also significantly elevated (+26% and +64%, respectively). It is concluded that epomediol treatment expands the bile acid pool through an enhanced bile acid synthesis. Choleresis induced by the drug is partly related to the increase in bile acid secretion.

Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I.

We report here a new mitochondrial regulation occurring only in intact cells. We have investigated the effects of dimethylbiguanide on isolated rat hepatocytes, permeabilized hepatocytes, and isolated liver mitochondria. Addition of dimethylbiguanide decreased oxygen consumption and mitochondrial membrane potential only in intact cells but not in permeabilized hepatocytes or isolated mitochondria. Permeabilized hepatocytes after dimethylbiguanide exposure and mitochondria isolated from dimethylbiguanide pretreated livers or animals were characterized by a significant inhibition of oxygen consumption with complex I substrates (glutamate and malate) but not with complex II (succinate) or complex IV (N,N,N',N'-tetramethyl-1, 4-phenylenediamine dihydrochloride (TMPD)/ascorbate) substrates. Studies using functionally isolated complex I obtained from mitochondria isolated from dimethylbiguanide-pretreated livers or rats further confirmed that dimethylbiguanide action was located on the respiratory chain complex I. The dimethylbiguanide effect was temperature-dependent, oxygen consumption decreasing by 50, 20, and 0% at 37, 25, and 15 degrees C, respectively. This effect was not affected by insulin-signaling pathway inhibitors, nitric oxide precursor or inhibitors, oxygen radical scavengers, ceramide synthesis inhibitors, or chelation of intra- or extracellular Ca(2+). Because it is established that dimethylbiguanide is not metabolized, these results suggest the existence of a new cell-signaling pathway targeted to the respiratory chain complex I with a persistent effect after cessation of the signaling process.

Cholephilic characteristics of a new cytostatic complex of cisplatin with glycocholate (Bamet-R2).

The aim of this work was to investigate both the existence of enterohepatic circulation of cisplatin-cholylglycinate complex, Bamet-R2, and the relevance of biliary versus urinary excretion of this compound. Two experimental models were used: (i) intraluminal perfusion of 'in situ' ileum in anaesthetized rats bearing a biliary catheter that permitted bile sample collection and (ii) conscious rats in which a permanent intraarterial catheter had been implanted to carry out sequential blood sampling after intravenous (i.v.) or intragastric (i.g.) drug administration. Total platinum in serum, bile, ileum, liver, urine and feces was measured by flameless atomic absorption spectroscopy. Serum concentration versus time curves obtained after i.v. administration of 1 micromol Bamet-R2 or cisplatin revealed that the area under the curve was significantly higher for Bamet-R2 than for cisplatin (+48%). Non-ultrafiltrable platinum accounted for 54.8 and 48.4% of serum platinum 168 h after cisplatin and Bamet-R2 i.v. administration, respectively. When the animals received i.g. 1 micromol cisplatin or Bamet-R2, serum concentrations of total platinum were markedly higher (three-fold) after Bamet-R2 than after cisplatin administration. The area under the curve was, also in this case, significantly higher for Bamet-R2 than for cisplatin (+28%). This was in part due to the enhanced intestinal absorption of Bamet-R2, as confirmed in experiments on perfused rat ileum, where a markedly higher amount of the drug was found in ileum tissue and bile after perfusion with media containing Bamet-R2 as compared with experiments where cisplatin instead of Bamet-R2 was added to perfusion media. Moreover, after i.v. administration to conscious rats, excretion of Bamet-R2 by the kidney was three-fold lower than that of cisplatin, while elimination of the former compound into feces was four-fold higher than that of the latter. In summary, these results indicate that in addition to the previously reported cytostatic activity of Bamet-R2, this complex has interesting cholephilic characteristics typical of bile acids, such as low urinary excretion together with enhanced intestinal absorption and biliary secretion, probably endowed by the cholylglycyl moiety included in the Bamet-R2 molecule.

Transport and biotransformation of the new cytostatic complex cis-diammineplatinum(II)-chlorocholylglycinate (Bamet-R2) by the rat liver.

Rat liver uptake and bile output of the cytostatic complex cis-diammineplatinum(II)-chlorocholylglycinate (Bamet-R2) were studied. Up to 100 microM, Bamet-R2 uptake by rat hepatocytes in primary culture followed saturation kinetics (Vmax = 0.65 +/- 0.12 nmol/5 min per mg protein; K(M) = 45.2 +/- 10.7 microM). Bamet-R2 uptake was lower than that of cholylglycinate (CG) but higher than that of cisplatin. Replacement of 116 mM NaCl by 116 mM choline chloride did not significantly reduce Bamet-R2 uptake. Addition of 500 microM CG, cholic acid, estrone sulfate, or ouabain to 50 microM Bamet-R2-containing incubation media inhibited Bamet-R2 uptake. No liver biotransformation of Bamet-R2 occurred, as indicated by HPLC analysis of bile collected from anesthetized rats after intravenous administration of the drug. Bamet-R2 uptake and secretion into bile by isolated rat livers exceeded those of cisplatin but were lower than those of CG. Differences between Bamet-R2 and CG were more marked for bile output than for liver uptake. Thus, higher Bamet-R2 than CG or cisplatin liver content was found. Co-administration of Bamet-R2 and CG revealed that CG induced a slight reduction in Bamet-R2 uptake and a marked inhibition in Bamet-R2 bile output. By contrast, Bamet-R2 had no effect on CG on either liver uptake or bile output. In sum, the present data indicate that Bamet-R2 is efficiently taken up and secreted into bile by the rat liver by mechanisms shared in part by natural bile acids.

In vitro test to determine the effect of cytostatic drugs on co-cultured rat hepatocytes and hepatoma cells.

Using uptake of the fluorescent bile acid derivative cholylglycylamido-fluorescein (FITC-GC) as a measurement of liver cell population size and function, the antiproliferative and toxic effects of the well known cytostatic drug, cisplatin was evaluated on rapidly growing rat hepatoma McA-RH7777 cells and rat hepatocytes in primary culture under non-proliferating conditions. Co-culture set up to mimic the in vivo situation of tumour and extratumoural liver tissue exposed to cytostatic chemotherapy does not markedly affect the survival or the growth dynamics of both cell types. FITC-GC uptake as corrected for DNA and protein contents in the dish was significantly lower in hepatoma cells than in rat hepatocytes throughout the experimental period (96 h). Effect of 0.1-100 microM cisplatin exposure from 24 to 96 h of culture on cell population size, as measured by protein and DNA contents in the culture dishes, were consistent with changes observed in total FITC-GC uptake. Cisplatin concentrations lower than 50 microM did not affect FITC-GC uptake by rat hepatocytes. By contrast, a progressively increasing effect on hepatoma cells as from 2 microM cisplatin was observed. Two phases in the decay of FITC-GC uptake versus cisplatin concentrations were found in co-cultures exposed to this drug. The first segment, between 2 microM and 50 microM, was characterized by a slow decay that matched the response of hepatoma cells to cisplatin exposure. This was considered to be due to the antiproliferative effect of cisplatin. The second segment, with a steeper decay, matched the effect of cisplatin on hepatocytes. This was interpreted as being due to non-specific toxicity. These results suggest that FITC-GC uptake by co-culture of hepatocytes and tumour cells provides a useful experimental model to explore the mechanism of action and the size of beneficial effect window for new drugs in vitro.

Rat liver transport and biotransformation of a cytostatic complex of bis-cholylglycinate and platinum (II).

BACKGROUND/AIMS: Bile acids have previously been used as shuttles for directing organic drugs to the liver. The aim of this study was to investigate liver transport and biotransformation of a new cytostatic bioinorganic complex (Bamet-H2), that was obtained by binding platinum(II) to two cholylglycinate moieties. METHODS: Using rat hepatocytes in primary culture, the kinetics of cholylglycinate, cisplatin and Bamet-H2 uptake were studied. Sodium-dependency of Bamet-H2 uptake was investigated by replacement of 116 mM NaCl by 116 mM choline chloride. Liver biotransformation was investigated by HPLC analysis of bile samples collected from anesthetized rats following intravenous Bamet-H2 administration. Using isolated rat liver preparations, which were perfused with erythrocyte- and albumin-free Krebs-Henseleit solutions for 40 min, measurement of cholylglycinate, cisplatin and Bamet-H2 uptake and bile output was carried out. Interaction between Bamet-H2 and cholylglycinate for liver transport was studied by co-administration of 1 microM Bamet-H2 plus 500 microM cholylglycinate and 1 microM [14C]-cholylglycinate plus 500 microM Bamet-H2. RESULTS: Both cholylglycinate and Bamet-H2 uptake by rat hepatocytes followed saturation kinetics. Comparison between the two compounds indicated that the Vmax (22.2 versus 8.5 nmol.5 min(-1).mg protein(-1)), and Kt (365 versus 171 microM) were higher for Bamet-H2 uptake. The efficiency of Bamet-H2 uptake (Vmax/Kt) was significantly reduced (-35%) in the absence of sodium. Cisplatin uptake by rat hepatocytes was approximately 10-fold lower than that for Bamet-H2 at any dose used. Moreover, this was not saturable up to 400 microM cisplatin. Bamet-H2 was not biotransformed during its intrahepatic residence in anesthetized rats. Bamet-H2 uptake and secretion into bile by isolated rat livers exceeded cisplatin but were less than cholylglycinate. Differences between Bamet-H2 and cholylglycinate were more marked for bile output than for liver uptake. Thus, higher drug liver content was found after perfusion with Bamet-H2 than with cholylglycinate or cisplatin. Co-administration of Bamet-H2 and cholylglycinate revealed the existence of partial cross-inhibition in both liver uptake and bile output. Bamet-H2 induced a more profound alteration on cholylglycinate uptake and bile secretion than cholylglycinate on both process for Bamet-H2. CONCLUSION: These results suggest that in the transfer of Bamet-H2 from the sinusoids to the canaliculi both bile acid and non-bile acid transport systems are involved.

Fetal excretion of the fluorescent bile acid derivative cholylglycylamido-fluorescein (FITC-GC) by the rat placenta-maternal liver tandem.

Bile acid transfer from the fetus to maternal bile was studied using in situ perfused rat placenta on day 21 of gestation and a fluorescent derivative of glycocholate (GC): cholylglycylamido-fluorescein (FITC-GC). Single-pass perfusion of the placenta with 0.25 mumol FITC-GC via the umbilical artery over 5 min was followed by the output of 6 per cent of this amount in maternal bile collected over the ensuing 120 min. This amount was reduced (-35 per cent) by simultaneous administration of 2.5 mumol GC through the jugular vein of the mother. This inhibition was stronger (-73 per cent) when 2.5 mumol GC was co-infused with FITC-GC through the umbilical artery. These results suggested that FITC-GC was, at least in part, transported by bile acid carriers across both the liver and the placenta. Using isolated perfused rat livers obtained from female virgin or 21-day pregnant rats, a slight increase in the residence time of FITC-GC in the liver of pregnant rats was found. However, no change in the ability of the liver to take up FITC-GC was observed. By contrast, when FITC-GC was injected into the left jugular vein of anaesthetized pregnant rats, a delayed plasma disappearance of this compound was seen, which may have been due in part to the existence of a transient and reversible FITC-GC exchange with the placental-fetal compartment. The maximal rate of FITC-GC output into bile after FITC-GC administration (1 mumol/100 g body weight) to pregnant rats was approximately 0.2 mumol/min, while maximal FITC-GC bile output was approximately 1 nmol/min when this compound was given through the umbilical artery (2.5 mumol). Therefore, the rate of FITC-GC output into bile was considered to reflect the rate of transfer across the placenta. Using this approach no saturation but rather a linear regression (slope = 1.1 microliters/min, p < 0.05) was found between placental transfer and placental perfusate concentrations in the 10-1000-mumol/l FITC-GC range. In summary, the in situ perfused rat placenta is a useful model to study the fetal excretion of cholephilic compounds, and transfer across the trophoblast would be the limiting step in the excretion of fetal bile acids by the placenta-maternal liver tandem.

Effect of maternal cholestasis on biliary lipid and bile acid secretion in the infant rat.

Partial and reversible impairment of bile formation has been reported to occur in the offspring of rats undergoing common bile duct ligation during the last third of pregnancy. This situation was defined as latent cholestasis of the neonate and was suggested to be related to the multilamellar bodies partially occupying the canalicular lumen. The current study was undertaken to investigate the presence of alterations in the secretion of biliary lipids in these infant rats. Using both high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analyses, no changes caused by maternal cholestasis were found in either the conjugation pattern, or in the ratio of primary to secondary major bile acids in bile samples collected from 4-week-old and 8-week-old rats. However, a decrease in the proportion of cholate together with an increase in the amount of alpha- and omega-muricholate were found at 4 weeks of age. These changes were different from those observed in the pattern of maternal plasma bile acids, in which beta-, but not alpha-muricholate, concentrations were increased. Moreover, studies performed by labeling the bile acid pool of the cholestatic mother-fetus tandem with [14C]glycocholic acid (GC) at day 16 of pregnancy indicated that only a minor proportion (approximately 10%) of bile acids found in 4-week-old pups was of maternal origin. Changes in the bile acid pool composition were fully reversed by 8 weeks of age. Bile lecithin and cholesterol output were determined by enzymatic techniques, both under basal conditions and during stepwise taurocholate (TC) infusion. At the time when multilamellar bodies were found, i.e., 4 weeks after birth, no change in either nonstimulated or TC-induced cholesterol output was observed. By contrast, both spontaneous and TC-induced lecithin secretion were markedly higher (+200%) in pups of cholestatic mothers as compared with control rats. These differences were abolished at 8 weeks of age. At this time, cholesterol output was significantly lower than that found in younger animals. This reduction was more pronounced in the control than in the cholestatic group. Histological examination of liver samples collected from the cholestatic group at 4 weeks of age revealed the presence of multilamellar bodies not only in the canalicular lumen but also within vesicular structures located in the pericanalicular area or near the Golgi apparatus. Both intracellular and intracanalicular bodies were present before and after TC infusion for 2 hours. These results indicate that maternal cholestasis in rats induces profound alterations in biliary lipids and bile acid secretion in their pups. Because bile acids are important activators of different steps responsible for biliary lipid secretion (intracellular trafficking, releasing into bile, and solubilization), alterations in maternal bile acid pool size and composition may affect the fetal development of biliary lipid secretion mechanisms, which may result in the appearance of multilamellar bodies within bile canaliculi, which in turn may be involved in the reversible latent cholestasis observed in these infants rats.

Effect of bile acids on hepatobiliary transport of cisplatin by perfused rat liver.

The liver and kidney collaborate in the excretion of the cytostatic drug, cis-diamminedichloroplatinum(II) (cisplatin) from the body. Enhancement of this process is envisaged as a way of reducing cisplatin toxicity, thus allowing increases in the doses administered. In this sense, using different compounds, several attempts have been made to enhance cisplatin biliary excretion. In this study, the ability of endogenous compounds belonging to the bile acid family to improve cisplatin excretion by the isolated perfused rat liver was investigated. A highly choleretic bile acid (ursodexoycholic acid) and two others bile acids with marked micelle-forming properties (glycocholic acid and chenodeoxycholic acid) were chosen for study. When these drugs were given at concentrations (1 microM) that did not affect the viability of liver preparations, a correlation between the biliary excretion of platinum and bile acid output was found. This was not due to the incorporation of cisplatin into mixed micelles because no correlation between the biliary output of lecithin or cholesterol and platinum was observed. Moreover, a wash-out effect of bile acids was probably not the cause of bile acid-induced platinum output into bile because no correlation between this and bile flow was found. An enhancement in cisplatin transport processes by the hepatocyte or by direct binding of cisplatin to bile acid monomers or aggregates cannot be ruled out. In spite of the biliary induction of cisplatin output, the net excretion of platinum was reduced under bile acid administration. This was related to lower platinum contents in the liver tissue, probably due to an inhibition of the ability of the hepatocyte to take up and/or retain cisplatin while subject to bile acid infusion. In summary, our results indicate that bile acids reduce the net excretion of cisplatin by the liver even though they induce an enhancement in the transport of this compound from the hepatocyte into bile.

Bile acid secretion during synchronized rat liver regeneration.

One major difficulty in interpreting the changes occurring during liver regeneration is the co-existence of non-activated cells and proliferating hepatocytes at all stages of differentiation. The aim of this study was to investigate bile acid (BA) secretion into bile during normal (NLR) and synchronized (SLR) liver regeneration in rats. Regeneration was synchronized by reversible inhibition of ribonucleotide reductase by 10 h treatment with hydroxyurea (HU) shortly after two-third partial hepatectomy. Total BA output as measured by GC-MS increased immediately after partial hepatectomy. This was followed by a further transient enhancement during the next day in the NLR. HU treatment did not significantly modify total BA output, but after releasing synchronized regeneration a marked reduction was observed. This was followed by a recovery to reach values close to those of NLR on day 7 of the regenerative process in SLR. Amidated BA output as measured by HPLC analysis revealed an early enhancement in the proportion of non-conjugated BAs in bile in NLR. However, the profile of conjugated BAs, which was not affected by HU treatment, matched that of total BAs during the first stage of SLR. By contrast, the increase in BA output observed on day 3 of the regenerative process in this group was accounted for by an enhancement in non-conjugated BA secretion. On day 7 of the regenerative process, the proportion of conjugated BA in bile was restored to approximately 100% in this group. Most BA molecules were conjugated with taurine rather than with glycine in all experimental groups, during both NLR and SLR. GC-MS determinations indicated that the magnitude of the cholic acid predominance in all bile samples was significantly modified during liver regeneration. This was increased early after partial hepatectomy and declined toward control values after few (2-3) days. Enhancement in the cholic acid predominance was due to a reduction in the proportion of all other major BAs, above all ursocholic acid and omega-muricholic acid. By contrast, minor BAs in normal control rat bile such as allo-cholic acid were increased during both NLR and SLR, and remained at detectable levels up to day 7. Changes in the proportion of secreted BA species were similar in SLR and NLR except that the early reduction in the proportion of BAs other than cholic acid was more pronounced in SLR and the quantitative importance of the diversity in BA species was recovered earlier in SLR than in NLR. In summary, these results indicate that profound changes in BA secretion during rat liver regeneration do occur. Most of them are probably related to the existence of retro-differentiation/re-differentiation processes which are speeded up by hydroxyurea-induced synchronization of the wave of hepatocyte proliferation.

Relationship between bile acid transplacental gradients and transport across the fetal-facing plasma membrane of the human trophoblast.

Bile acids and bilirubin are synthesized by the fetal liver very early on during intrauterine life. The main fate of these compounds is to be transferred to the mother. This excretory role of the placenta is primarily determined by the ability of the trophoblast to transport them, which is believed to occur mainly by carrier-mediated processes. The aim of this study was to investigate the role of the cholephilic organic anion exchanger located in the fetal-facing plasma membrane of the human trophoblast in placental "biliary-like" function. No relationship between the magnitude of transplacental gradients for total bile acids and bilirubin was found. However, transport studies, which were carried out by using purified plasma membrane vesicles derived from the fetal-facing pole of the human trophoblast, revealed that [14C]taurocholate transport was affected by both another bile acid (taurochenodeoxycholic acid) and a non-bile acid cholephilic organic anion (bromosulfophthalein). On plotting the ability of different major bile acid species to inhibit radiolabeled taurocholate uptake by these vesicles versus their concentrations in fetal serum or the magnitude of their transplacental gradients, inverse relationships were found. Lower fetal serum concentrations and transplacental gradients were found for bile acid species with higher abilities to affect this transport and presumably to interact with the carrier. By contrast, the magnitude of the transplacental gradient for bile acid species was not correlated with their hydrophobic/hydrophilic balance, as would be expected if diffusion across the lipidic structures of the placental barrier would be the major pathway for the flux of bile acid across this organ.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of hepatobiliary function and placental barrier integrity in pregnant rats by combination of bromosulphophthalein and fluorescein isothiocyanate plasma disappearance tests.

The permeability of the placental barrier to bromosulphophthalein (BSP) is believed to be very low. Whether this property is shared by other cholephilic organic anions, such as fluorescein isothiocyanate (FITC), is not known. When BSP was injected (140 mumol/kg body wt) into the left jugular vein of non-pregnant or pregnant female rats (at the 21st day of pregnancy), a similar and rapid plasma disappearance was observed during the first few minutes; afterwards, a slower disappearance phase was found. This phase was different in these groups, that is, a longer retention of BSP in the maternal bloodstream of pregnant rats was accompanied by a slower BSP output into bile. It was impossible to demonstrate the presence of BSP in fetal blood or the placenta by colorimetric methods. These results are consistent with the modifications occurring in the hepatic handling of BSP during pregnancy together with a marked impermeability of the placenta to the dye, at least in the mother-to-fetus direction. After intravenous FITC (10 mumol/kg body wt) administration to the mother, the compound was rapidly transferred into both the maternal bile and the fetal blood. Thereafter, FITC refluxed back from the fetal-placental compartment to the maternal blood as soon as the maternal liver reduced its plasma concentrations, which were first higher (approximately threefold) and then similar to those found in fetal blood. The reversible retention of FITC by the fetal-placental compartment accounts for a significant delay in both FITC bile output and plasma disappearance as compared with those observed in non-pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of bile acid facilitation of biliary protoporphyrin excretion in rat liver.

The mechanism(s) by which bile acids increase biliary protoporphyrin excretion was characterized using perfused rat livers. We determined 1) relationships between biliary bile acids, phospholipid, and protoporphyrin, using rapid kinetic analyses; 2) protoporphyrin excretion in livers with defective canalicular multispecific organic anion transport; 3) effects of intracellular vesicular transport inhibition with colchicine and monensin; and 4) the role of luminal bile acids, using retrograde intrabiliary taurocholate injections. Biliary protoporphyrin excretion peaked with phospholipid excretion 14-18 min after loading. Protoporphyrin excretion induced by taurocholate was not related to effects on intracellular transport, including colchicine- and monensin-inhibitable vesicular systems. Eisai hyperbilirubinemic rat livers excreted protoporphyrin similarly to controls. Retrograde intrabiliary taurocholate injections increased protoporphyrin output. Collectively, these data suggest that 1) intracellular protoporphyrin transport is mediated by nonvesicular carriers targeted to the canalicular membrane, and 2) bile acid facilitates protoporphyrin translocation into bile in the same manner it effects phospholipid excretion.