Hepatic accumulation of lysosomes and defective transcytotic vesicular pathways in cirrhotic rat liver.

To investigate the potential role of lysosomes in cirrhosis, we analyzed the activity of lysosomal enzymes in rats exposed long-term to phenobarbital and carbon tetrachloride. The activity of lysosomal enzymes was markedly increased in the homogenate of cirrhotic livers (e.g., arylsulfatase 9 +/- S.D.2 vs. 16 +/- 6 nmoles.min-1.mg-1 in control rats and cirrhotic rats, respectively; p less than 0.001). The corresponding plasma levels were also increased (7 +/- 1 vs. 12 +/- 3 nmoles.min-1.mg-1; p less than 0.01), whereas biliary excretion was diminished (16 +/- 7 vs. 7 +/- 2 pmol.min-1.gm liver-1; p less than 0.05) in cirrhotic rats. Stereological quantification of lysosomes visualized cytochemically revealed an increase of pericanalicular lysosomes averaging 1.5 +/- 0.4 around a canaliculus in controls and 3.7 +/- 1.0 in cirrhotic rats (p less than 0.01). Because this suggested a defect in the transcellular vesicular pathway, we investigated the biliary excretion of horseradish peroxidase and epidermal growth factor in perfused livers. Bile flow and total horseradish peroxidase excretion were similar in control rats and cirrhotic rats. However, the early peak of biliary horseradish peroxidase excretion--usually taken as evidence of paracellular transport--was increased in cirrhotic rats (13 +/- 7 vs. 57 +/- 22%; p less than 0.01), whereas the second peak--reflecting the transcellular vesicular pathway(s)--was markedly reduced (87 +/- 7 vs. 43 +/- 22%; p less than 0.001). A similar reduction in the biliary excretion of intact epidermal growth factor and of its degradation products was found. These results demonstrate an increased number of lysosomes in hepatocytes of cirrhotic livers; this appears to be the result of accumulation rather than proliferation, in view of the reduced transcellular vesicular movement of different markers into bile.     

Taurocholate transport by liver plasma membrane vesicles is not altered in cirrhotic rats

Recently, we have shown that taurocholate transport is impaired in hepatocytes isolated from CCl4 cirrhotic rats. Na+,K+-ATPase activity depends on the lipid composition of the surrounding membrane. Therefore, we performed this study in order to detect differences in plasma membrane composition and membrane functions between livers of CCl4 cirrhotic (n = 17) and of control rats (n = 15). After biochemical characterization of the animals we isolated basolateral and canalicular membrane vesicles and determined membrane enzyme activities, transport functions and lipid composition. We found no differences in the isolation characteristics of the plasma membranes between the two groups. The lipid composition of the membrane fractions was not altered, except for a lower cholesterol content in the canalicular membranes of the cirrhotic group (200 +/- 15 vs. 246 +/- 18 micrograms/mg protein, P less than 0.05). Taurocholate transport into basolateral membrane vesicles and marker enzyme activities of the membrane fractions were also equal in control and cirrhotic animals. We conclude that the plasma membrane composition and membrane enzyme/transport activities have returned to normal in CCl4 cirrhotic rats 14 days after cessation of exposure to CCl4. Thus, a disturbed transport system is not the cause for the observed decreased taurocholate transport into hepatocytes from cirrhotic rats. Even a cirrhotic liver has a high potential for recovery after acute CCl4 intoxication.     

Decreased hepatic glycogen content and accelerated response to starvation in rats with carbon tetrachloride-induced cirrhosis

Glucose homeostasis and fatty acid metabolism are abnormal in patients with cirrhosis. To assess the metabolic response to starvation in an animal model of cirrhosis, glycogen and fuel metabolism were characterized in rats with CCl4-induced cirrhosis studied 2 wk after 10 weekly doses of CCl4. Plasma concentrations of glucose and beta-hydroxybutyrate were not different between fed CCl4-treated and control rats, but plasma nonesterified fatty acid concentrations were higher in cirrhotic animals (0.25 +/- 0.01 vs. 0.39 +/- 0.04 mmol/L; p less than 0.05). After 12 hr of starvation, the plasma nonesterified fatty acid concentration had reached 0.58 +/- 0.04 mmol/L in CCl4-treated rats, compared with 0.38 +/- 0.04 mmol/L in control rats (p less than 0.05). The redistribution of the hepatic carnitine pool toward acylcarnitines, which is characteristic of starvation, was complete after fasting for 12 hr in the CCl4-treated rats, compared with the 24 hr required in control rats. In fed cirrhotic rats, liver glycogen content per gram liver was decreased by 64% compared with control rats (30.0 +/- 5.1 vs. 10.8 +/- 1.1 mg/gm liver wet wt; p less than 0.05). After 12-hr fasting, hepatic glycogen content had fallen to 14.3 +/- 3.9 and 4.8 +/- 0.4 mg/gm liver wet wt (p less than 0.05) in control and cirrhotic animals, respectively. To further characterize the status of glycogen metabolism in cirrhotic livers, activities of glycogen synthase and glycogen phosphorylase were determined. Hepatic active and total glycogen phosphorylase activities normalized to hepatocellular content were unaffected by CCl4 treatment, whereas total glycogen synthase activity was increased by 45%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urea cycle enzyme activities are normal and inducible by a high-protein diet in CCl4 cirrhosis of rats

We produced moderately severe, inactive micronodular cirrhosis in rats using CCl4 and measured the urea cycle enzyme activities in liver after feeding a 15% casein diet for 1 week and again after a 60% casein diet for 1 week. There was no deficiency of any of the five urea cycle enzymes in cirrhotic livers of rats pair-fed the 15% casein diet. Argininosuccinate synthetase and carbamyl phosphate synthetase activities were lower than in non-pair-fed controls by some baselines. All five enzymes in cirrhotic livers were induced 1.5- to 3-fold by the high-protein diet expressed as units per 100 gm of rat. The level of carbamyl phosphate synthetase activity was lower in the livers of rats pair-fed the 60% casein diet than in control livers based on wet weight, collagen-free protein and DNA, but the activities were equal expressed as units per 100 gm of rat. This example of CCl4-induced cirrhosis in the rat does not serve as a good model for human cirrhosis, in which the urea cycle enzymes are reported to be decreased in activity.     

Lipid peroxidation and extracellular matrix in normal and cirrhotic rat livers following 70% hepatectomy

BACKGROUND/AIMS: The aim was to measure the deposition of collagens and proteoglycans and the underlying mechanism leading to lipid peroxidation due to oxidative stress in partially hepatectomized normal and cirrhotic rats. METHODOLOGY: Four groups of adult Wistar rats were used comprising normal livers, regenerated normal livers, cirrhotic livers, and regenerated cirrhotic livers. Cirrhosis was induced by intragastric administration of carbon tetrachloride and phenobarbital in the drinking water of the rats. Hydroxyproline, as a constituent of collagens, uronic acid, as a constituent of proteoglycans, and malondealdehyde, an end-product of lipid peroxides, were measured in normal and cirrhotic rats, and following partial hepatectomy. RESULTS: Hydroxyproline, uronic acid and malondealdehyde levels were 234.2 +/- 41.2, 11.82 +/- 1.92, 46.3 +/- 5.8 and 211.8 +/- 43.6, 9.16 +/- 1.41, 48.5 +/- 7.5 for normal and regenerated normal livers respectively. The values after partial hepatectomy in cirrhotic and regenerated cirrhotic livers were 396.9 +/- 48.5, 17.96 +/- 1.62, 144.5 +/- 25.1 and 309.6 +/- 43.2, 13.35 +/- 1.72, 229.9 +/- 24.4, respectively. When the cirrhotic liver group was compared with the normal liver group, the levels of hydroxyproline, uronic acid and malondealdehyde were significantly higher (p < 0.001). Uronic acid levels of regenerated normal and regenerated cirrhotic livers and hydroxyproline level of regenerated cirrhotic liver were significantly less than those of their non-regenerated states (p < 0.01). Although the malondealdehyde levels of normal and regenerated normal livers did not differ significantly (p > 0.05), the malondealdehyde levels of regenerated cirrhotic liver was significantly higher than cirrhotic liver (p < 0.01). The histopathological examination with light microscopy did not reveal any obvious difference between the groups other than between normal and cirrhotic. CONCLUSIONS: Cirrhotic livers revealed a significantly higher amount of extracellular matrix constituents and lipid peroxidation than normal livers. Although partial hepatectomy in cirrhotic livers caused decreases in the tissue levels of collagens and proteoglycans, it did not actually lower the ongoing oxidative stress, known as physiological lipid peroxidation, in normal and cirrhotic livers following partial hepatectomy.     

Altered transmembrane ionic flux in hepatocytes isolated from cirrhotic rats

BACKGROUND/AIMS: Although cirrhosis is known to be associated with many hepatocyte abnormalities, there is no well-established model to study the cellular drug uptake process independent of hemodynamic effects. The purpose of the present study was to test the following hypothesis: hepatocytes isolated from cirrhotic animals may be used as a model to study the cellular abnormalities associated with cirrhosis. Our hypothesis was tested by comparing the membrane potential (PD) of hepatocytes in anesthetized healthy and cirrhotic animals, and the PD and [3H]palmitic acid clearance rate of hepatocytes isolated from healthy and cirrhotic animals. METHODS: Mild to moderate cirrhosis was induced in female Sprague-Dawley rats by CCl4 administration. PD was recorded in anesthetized animals using intracellular microelectrodes. Hepatocytes from those livers were subsequently isolated by collagenase perfusion for further determinations of PD and [3H]palmitic acid uptake. RESULTS: The mean (+/-SEM) hepatocyte PD from intact rat livers was 38+/-1 mV (control) and -32+/-1 mV (cirrhosis; n=6/group, p<0.01). The PD (mean+/-SEM) in isolated hepatocytes was -21+/-1 mV (control) and -15+/-1 mV (cirrhosis, n=13/group, p<0.01). The clearance rate of [3H]palmitic acid was lower in hepatocytes isolated from cirrhotic animals (26%) than in those isolated from healthy control animals (p<0.01). CONCLUSION: The results of this study indicate that hepatocytes isolated from cirrhotic animals may be used to study the cellular abnormalities associated with cirrhosis.     

Noninvasive measurement of liver regeneration with positron emission tomography and [2-11C]thymidine

The feasibility of liver regeneration determination with [2-11C]thymidine and positron emission tomography was investigated in partially hepatectomized rats. Serial tomographic scans were performed over a 120-minute period after injection of [2-11C]thymidine together with tritium-labeled thymidine. Within 10 minutes after injection, positron emission tomography scans showed a twofold higher hepatic uptake in regenerating than in nonregenerating livers. Time-activity curves over the liver area indicated that the maximal uptake was followed by a faster decrease of 11C radioactivity in controls than in regenerating animals, so that total 11C activity remaining in the liver at 120 minutes accounted for 68% of maximum in regenerating and only 38% in controls. Tissue distribution studies performed at 120 minutes showed that total 11C radioactivity, expressed in percent injected dose per gram, was six times higher in regenerating livers than in controls (0.62% +/- 0.07% in regenerating livers and 0.10% +/- 0.03% in nonregenerating livers; P less than 0.001) and correlated with 3H radioactivity measured in the nuclear fraction (r = 0.92; P less than 0.001). When the hepatic uptake was expressed in percent of dose per organ, the difference between both groups increased (2.31% +/- 0.23% in regenerating livers and 0.29% +/- 0.02% in nonregenerating livers; P less than 0.001) because of higher weight of regenerating livers than of nonregenerating livers (3.83 +/- 0.11 g in regenerating livers and 2.96 +/- 0.16 g in nonregenerating livers; P less than 0.001). In other organs examined, no difference in 11C radioactivity was found between the two groups of rats. These results indicated the potential usefulness of [2-11C]thymidine and positron emission tomography for noninvasive measurement of liver regeneration.     

Primary cirrhotic hepatocytes resist TGFbeta-induced apoptosis through a ROS-dependent mechanism

BACKGROUND/AIMS: The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis. METHODS: Hepatocytes were isolated from normal and CCl(4)-treated mice and treated with TGFbeta, TNFalpha, and UV-C, known apoptotic agents. RESULTS: Cirrhotic hepatocytes were less sensitive to TGFbeta- (45+/-5 vs. 15+/-3%; P<0.003), TNFalpha- (59+/-21 vs. 21+/-8%; P=0.02), and UV-C-induced (31+/-4 vs. 17+/-4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFbeta-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFbeta treatment. CONCLUSIONS: These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFbeta-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.     

Increased blood flow through the portal system in cirrhotic rats

Portal venous pressure is the result of the interplay between portal venous blood flow and the vascular resistance offered to that flow. Whether portal hypertension is maintained only by an increased portal venous resistance or also by an increased blood flow within the portal venous system is still open to speculation. To resolve these differences, splanchnic and systemic hemodynamics were evaluated in cirrhotic rats, induced by CCl4. Blood flow and portal-systemic shunting were measured by radioactive microsphere techniques. All cirrhotic rats had portal hypertension (portal venous pressure 13.5 +/- 1.1 vs. 9.0 +/- 0.5 mmHg, in normal control rats; p less than 0.01), but portal-systemic shunting in cirrhosis (31% +/- 13% vs. 0.2% +/- 0.02%; p less than 0.05) was variable, ranging from 1% to 97%. Portal venous inflow, the total blood flow within the portal system, was increased in cirrhotic rats (5.75 +/- 0.04 vs. 4.52 +/- 0.36 ml/min per 100 g; p less than 0.05). Total splanchnic arterial resistance was reduced in cirrhotic rats (3.3 +/- 0.2 vs. 5.8 +/- 0.5 dyn X s X cm-5 X 10(5); p less than 0.01). Portal venous resistance, however, was not abnormally elevated in cirrhotic rats (4.6 +/- 0.5 vs. 4.7 +/- 0.5 dyn X s X cm-5 X 10(4), p = NS). Splanchnic hemodynamics in cirrhotic rats demonstrate that portal hypertension is maintained, at least in part, by a hyperdynamic portal venous inflow. The hemodynamic data in cirrhotic rats provided evidence that supports the role of an increased portal blood flow in portal hypertension and gives a quantitative definition of splanchnic hemodynamics in intrahepatic portal hypertension.     

Expression of growth hormone receptor and its mRNA in hepatic cirrhosis

AIM: To investigate the expression of growth hormone receptor (GHR) and mRNA of GHR in cirrhotic livers of rats with the intension to find the basis for application of recombinant human growth hormone (rhGH) to patients with liver cirrhosis.METHODS: Hepatic cirrhosis was induced in SpragueDawley rats by administration of thioacetamide intraperitoneally for 9-12 weeks. Collagenase Ⅳ was perfused in situ for isolation of hepatocytes. The expression of GHR and its mRNA in cirrhotic livers was studied with radio-ligand binding assay, RT-PCR and digital image analysis.RESULTS: One class of specific growth hormone-binding site, GHR, was detected in hepatocytes and hepatic tissue of cirrhotic livers. The binding capacity of GHR (RT, fmol/mg protein) in rat cirrhotic liver tissue (30.8±1.9) was significantly lower than that in normal control (74.9±3.9) at the time point of the ninth week after initiation of induction of cirrhosis (n=10, P＜0.05), and it decreased gradually along with the accumulation of collagen in the process of formation and development of liver cirrhosis (P＜0.05). The number of binding sites (×10 4/cell) of GHR on rat cirrhotic hepatocytes (0.86±0.16) was significantly lower than that (1.28±0.24)in control (n= 10, P＜0.05). The binding affinity of GHR among liver tissue, hepatocytes of various groups had no significant difference (P＞0.05). The expression of GHR mRNA (riOD,pixel) in rat cirrhotic hepatic tissues (23.3±3.1) was also significantly lower than that (29.3±3.4) in normal control (n=10, P＜0.05).CONCLUSION: The growth hormone receptor was expressed in a reduced level in liver tissue of cirrhotic rats,and lesser expression of growth hormone receptors was found in a later stage of cirrhosis. The reduced expression of growth hormone receptor was partly due to its decreased expression on cirrhotic hepatocytes and the reduced expression of its mRNA in cirrhotic liver tissue.     

Relationship between hepatic mitochondrial functions in vivo and in vitro in rats with carbon tetrachloride-induced liver cirrhosis

BACKGROUND/AIMS: The metabolic capacity of liver mitochondria is impaired in rats with carbon tetrachloride (CCl4)-induced cirrhosis. These studies were performed to find out whether benzoate and/or palmitate are suitable substrates for assessing hepatic mitochondrial function in vivo. METHODS: In vivo metabolism of benzoate and 1-14C-palmitate was assessed by monitoring urinary excretion of hippurate and exhalation of 14CO2, respectively, in cirrhotic and control rats (n=8 for each group). Isolation of liver mitochondria, and in vitro benzoate and palmitate metabolism were performed by methods published previously. The hepatic content of mitochondria was assessed by stereological analysis of the volume of hepatocytes and by biochemical determination, using the activity of citrate synthase. RESULTS: Renal excretion of hippurate following i.p. administration of benzoate was reduced in cirrhotic rats (64+/-15 vs. 85+/-14% of administered dose over 24 h), and showed a linear correlation with hippurate formation by isolated mitochondria. The activities of benzoyl-CoA synthase and benzoyl-CoA:glycine N-acyltransferase were reduced by approximately 60%, and the coenzyme A content by 50% in hepatic mitochondria from cirrhotic rats, explaining impaired hippurate formation. Peak exhalation of 14CO2 after i.p. administration of 1-14C-palmitate was reduced by 44% and the area under the 14CO2 exhalation-time curve by 34% in cirrhotic rats. Peak 14CO2 exhalation revealed a linear correlation with oxidative metabolism of palmitoylcarnitine in isolated mitochondria. Both in vivo benzoate and palmitate metabolism showed a linear correlation with the volume fraction of hepatocytes. The mitochondrial protein content was reduced in cirrhotic rats per g liver and per liver but equal to control rats per volume of hepatocytes. CONCLUSIONS: In vivo metabolism of both palmitate and benzoate reflects hepatic mitochondrial function in rats with CCl4-induced cirrhosis. Hepatic mitochondrial function is impaired in rats with CCl4-induced cirrhosis due to both reduced mitochondrial volume per liver and impaired metabolism of the remaining mitochondria. In contrast to rats with secondary biliary cirrhosis, rats with CCl4-induced cirrhosis showed no hepatic mitochondrial proliferation to counteract reduced mitochondrial function.     

Significance of hepatic arterial responsiveness for adequate tissue oxygenation upon portal vein occlusion in cirrhotic livers

We investigated sinusoidal blood flow and hepatic tissue oxygenation during portal vein occlusion in cirrhotic rat livers to examine the effect of cirrhosis on the properties of hepatic microvascular blood flow regulation. After 8 weeks of CCl4/phenobarbital sodium treatment to induce cirrhosis Sprague-Dawley rats were prepared surgically to allow assessment of portal venous and hepatic arterial inflow using miniaturized flow probes with simultaneous analysis of hepatic microcirculation and tissue oxygenation by fluorescence microscopy and polarographic oxymetry. Age-matched noncirrhotic animals served as controls. Upon portal vein occlusion in cirrhotic livers (flow reduction to < 20%), hepatic arterial blood flow increased 1.5-fold (61 +/- 8 ml/min per 100 g liver) of baseline (40 +/- 7 ml/min per 100 g liver), reflecting an appropriate hepatic arterial buffer response (HABR), similarly as seen in control livers. The net result was a reduction in total liver inflow from 90 +/- 12 to 72 +/- 11 ml/min per 100 g liver, which was associated with a significant decrease in both sinusoidal red blood cell velocity and volumetric blood flow to approx. 71% and 76% of baseline values. However, portal vein occlusion did not cause a deterioration in hepatic tissue pO2 (11 +/- 3 vs. 10 +/- 3 mmHg at baseline). Sinusoidal diameters were found unchanged, disproving a major role of the sinusoidal tone in the regulation of HABR. Microvascular response of cirrhotic livers did not generally differ from that in noncirrhotic livers upon portal inflow restriction. We conclude that HABR in cirrhotic livers operates sufficiently to meet the liver tissue oxygen demand, most probably by an increased relative contribution of arterial perfusion of hepatic sinusoids.     

Enhanced vasoconstrictor response of the isolated perfused cirrhotic rat liver to humoral vasoconstrictor substances found in portal venous blood

Humoral vasoconstrictor factors in portal venous blood have an important influence on hepatic vascular tone. The aim of this study was to determine whether there is altered reactivity of the intrahepatic portal vascular bed of cirrhotic livers to such factors. Isolated perfused rat liver preparations (IPRLP) obtained from rats with carbon tetrachloride-induced cirrhosis and from normal controls were treated with small aliquots of fresh, heparinized venous blood (4% vol/vol) added to a synthetic perfusate composed of 2.5% bovine serum albumin in Krebs-Henseleit buffer. Compared with blood from the inferior vena cava, portal venous blood produced a greater increase in perfusion resistance of normal IPRLP (2.8 +/- 0.7 vs 15 +/- 3%, P less than 0.05). There was no significant difference in the response of normal IPRLP to portal venous blood obtained from cirrhotic animals compared with portal blood from normal controls (10 +/- 4 vs 15 +/- 3%). However, cirrhotic IPRLP were significantly (P less than 0.05) more responsive to portal venous blood than were control livers, regardless of whether the blood was obtained from control (28 +/- 6%) or cirrhotic (24 +/- 6%) rats. The response of both control and cirrhotic IPRLP to portal blood could be partially inhibited by the alpha-adrenoceptor antagonist phentolamine (5 x 10(-6) mol/L) and cirrhotic IPRLP were more responsive than controls to exogenous noradrenaline (518 +/- 27 vs 363 +/- 21%, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic Alcohol Treatment Results in Disturbed Vitamin D Metabolism and Skeletal Abnormalities in Rats

The effect of chronic alcohol consumption on the skeleton was investigated in rats. The treated group received ethanol administered as 38% of caloric intake in a liquid diet (Sustacal) for 10 months. The control rats were pair weighted to the ethanol-treated animals throughout the study; the growth curves of the two groups were the same. The controls were given the same liquid diet except that dextrin:maltose (3:1) was substituted isocalorically for ethanol. Ethanol-treated rats did not differ from the pair-weighted controls in mean serum calcium, phosphorous, or creatinine. In contrast, serum magnesium was reduced (p less than 0.02) in alcohol-treated rats. Ethanol treatment also resulted in changes in the serum concentrations of vitamin D metabolites; serum 25-hydroxyvitamin D3 was increased (p less than 0.001), while serum 1,25-dihydroxyvitamin D3 was decreased (p less than 0.01). Tibial length was reduced in ethanol-treated rats (p less than 0.05) but there was no change in femoral length. Medullary area was increased in tibial diaphyses from alcohol-treated rats compared to weight matched control animals (p less than 0.01), indicating a net increase in resorption. The cross-sectional area of the tibial diaphysis of ethanol-treated rats was the same as the matched controls. Trabecular bone was decreased in the tibial metaphysis of ethanol-treated rats compared to the matched controls (p less than 0.05) indicating a net loss of trabecular bone. Ethanol treatment did not have an effect on the organic weight of the femur but the ash weight was reduced (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of the impaired diuretic and natriuretic responses to a sustained and moderate saline infusion in rats with experimental cirrhosis

Kidney function and tubular handling of water and sodium by superficial nephrons, packed cell volume, total plasma proteins and albumin distribution space were studied in control and cirrhotic rats before and after a moderate and sustained saline infusion (3% body weight per 30 min + reposition of urinary losses). Tubular fluid samples were obtained from late proximal, early distal and late distal convolutions of superficial nephrons using micropuncture. Protein distribution was assessed by intravenous injection of 0.5 muCi of (125I)-albumin. In basal conditions, both groups of rats showed similar glomerular filtration rate and renal plasma flow, but cirrhotic animals had lower sodium excretion (fractional excretion of sodium = 0.04 +/- 0.01% vs. 0.22 +/- 0.02%, p less than 0.05) and urinary volume (4.31 +/- 0.41 vs. 7.57 +/- 0.53 microliter per min; p less than 0.05). After saline infusion, total plasma proteins decreased more in cirrhotic than in control rats (-18.5 +/- 2.7 vs. -12.9 +/- 2.2%, p less than 0.05). The opposite was observed for albumin distribution space (34.5 +/- 6.1 vs. 22.1 +/- 3.5%, p less than 0.05). Fractional sodium excretion increased to 2.98 +/- 0.15% in control rats but only to 0.61 +/- 0.080% in cirrhotic rats. The ratio single nephron glomerular filtration rate/glomerular filtration rate increased from 19.6 +/- 0.7 to 21.2 +/- 1.0 (X10(-6), p less than 0.005) in control animals but did not change in cirrhotic rats. These animals were unable to decrease adequately fractional fluid reabsorption in the proximal tubule and the loop of Henle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intact hepatocyte theory of impaired drug metabolism in experimental cirrhosis in the rat.

The elimination of propranolol by perfused livers of rats made cirrhotic by chronic carbon tetrachloride inhalation during phenobarbital treatment has been compared with control animals receiving only phenobarbital. Cirrhosis reduced propranolol clearance at a constant flow of 20 ml/min from 1.43 +/- 0.08 to 1.12 +/- 0.08 ml/min/g liver (P less than 0.025). In addition, an increase in intrahepatic shunting of 15-micron microspheres from 0.41 +/- 0.01 to 9.4 +/- 4.1% was found in cirrhotic livers (P less than 0.05). Finally, in cirrhotic livers, reducing blood flow did not produce the normal rise in hepatic extraction ratio, which actually fell from 0.873 +/- 0.021 at 20 ml/min to 0.836 +/- 0.025 at 15 ml/min and 0.823 +/- 0.026 at 10 ml/min. At each flow the observed extraction was significantly lower than that predicted to result from a reduced enzyme activity alone, consistent with the development of functionally significant intrahepatic shunts. An operational model is proposed that explains impaired drug metabolism in cirrhosis on the basis of the development of intrahepatic shunts which perfuse nonfunctioning tissue, while the remaining blood flow is exposed to a reduced mass of hepatocytes with an apparently normal amount of drug metabolizing enzyme (the intact hepatocyte theory).     

Canalicular bile flow and bile salt secretion are maintained in rats with liver cirrhosis. Further evidence for the intact cell hypothesis

Different aspects of biliary physiology were studied in rat model of liver cirrhosis induced by CCl4/phenobarbitone. We measured bile flow, bile salt secretion, biliary secretion pressure and bile-to-plasma ratios of inert solutes under basal conditions and during infusion of taurocholate (0.4 and 0.8 mumol.min-1.100 g body wt.-1) in 11 cirrhotic and 10 control male Sprague-Dawley rats. Bile flow and biliary bile salt secretion did not differ between the two groups. Analyzing the relationship between bile salt secretion and bile flow, however, we found an increased slope (P less than 0.02) in the cirrhotic animals, suggesting a higher apparent osmotic activity of the bile salts secreted. Maximal biliary secretion pressure was maintained in cirrhotic animals (22.5 +/- 2.5 vs. 25.0 +/- 2.9 cmH2O) in the absence of exogenous bile salt. During taurocholate infusion it decreased to a lesser extent (P less than 0.001) in cirrhotic animals (13.5 +/- 3.4 vs. 19.3 +/- 3.8 cmH2O). Bile-to-plasma ratios of [3H]sucrose and [14C]ferrocyanide were markedly increased in cirrhotic rats. Biliary [14C]erythritol clearance was equal to bile flow in both groups. In the cirrhotic group, the [3H]sucrose bile/plasma ratio was positively correlated with spleen weight (r = 0.744, P less than 0.01), serum concentration of alkaline phosphatase (r = 0.583, P less than 0.05) and basal maximum biliary secretion pressure (r = 0.801, P less than 0.001). We conclude that chronic portal hypertension is associated with increased permeability of the blood/bile barrier. Nevertheless, bile flow and bile salt secretion are maintained in cirrhotic rats, giving support to the intact cell hypothesis for this important hepatocellular function.     

Vitamin D2 is as effective as vitamin D3 in maintaining circulating concentrations of 25-hydroxyvitamin D

CONTEXT: Two reports suggested that vitamin D2 is less effective than vitamin D3 in maintaining vitamin D status. OBJECTIVE: Our objective was to determine whether vitamin D2 was less effective than vitamin D3 in maintaining serum 25-hydroxyvitamin D levels or increased the catabolism of 25-hydroxyvitamin D3. SUBJECTS AND DESIGN: This was a randomized, placebo-controlled, double-blinded study of healthy adults ages 18-84 yr who received placebo, 1000 IU vitamin D3, 1000 IU vitamin D2, or 500 IU vitamin D2 plus 500 IU vitamin D3 daily for 11 wk at the end of the winter. RESULTS: Sixty percent of the healthy adults were vitamin D deficient at the start of the study. The circulating levels of 25-hydroxyvitamin D (mean+/-sd) increased to the same extent in the groups that received 1000 IU daily as vitamin D2 (baseline 16.9+/-10.5 ng/ml; 11 wk 26.8+/-9.6 ng/ml), vitamin D3 (baseline 19.6+/-11.1 ng/ml; 11 wk 28.9+/-11.0 ng/ml), or a combination of 500 IU vitamin D2 and 500 IU vitamin D3 (baseline 20.2+/-10.4 ng/ml; 11 wk 28.4+/-7.7 ng/ml). The 25-hydroxyvitamin D3 levels did not change in the group that received 1000 IU vitamin D2 daily. The 1000 IU dose of vitamin D2 or vitamin D3 did not raise 25-hydroxyvitamin D levels in vitamin D-deficient subjects above 30 ng/ml. CONCLUSION: A 1000 IU dose of vitamin D2 daily was as effective as 1000 IU vitamin D3 in maintaining serum 25-hydroxyvitamin D levels and did not negatively influence serum 25-hydroxyvitamin D3 levels. Therefore, vitamin D2 is equally as effective as vitamin D3 in maintaining 25-hydroxyvitamin D status.     

Aminopyrine N-demethylation by rats with liver cirrhosis. Evidence for the intact cell hypothesis. A morphometric-functional study

The intact cell hypothesis states that a reduced number of intrinsically normal hepatocytes, together with hemodynamic alterations, explains decreased drug metabolism in cirrhosis. We explored this hypothesis by comparing results of the aminopyrine breath test with in vitro measurements of aminopyrine N-demethylation and morphometrically determined liver cell volume in a rat model of cirrhosis. Aminopyrine N-demethylation in vivo (ABT-k) was 0.98 +/- 0.10/h (mean +/- SD) in controls. The cirrhotic rats were separated into those with normal (NCR) and those with abnormal ABT-k (PCR). Microsomal aminopyrine N-demethylase averaged 2.08 +/- 0.77 and 2.09 +/- 0.54 mumol/min in controls and NCRs, respectively; it was reduced to 1.00 +/- 0.81 mumol/min (p less than 0.02) in PCRs. Morphometrically determined hepatocellular volume was 18.8 +/- 2.8, 17.1 +/- 1.9, and 11.6 +/- 6.1 ml in controls, NCRs, and PCRs, respectively, PCRs being lower than controls (p less than 0.01) and NCRs (p less than 0.05). When N-demethylase and cytochrome P450 were related to hepatocellular volume (in milliliters), no significant difference between the three groups was apparent. We conclude that reduced aminopyrine N-demethylation in progressed cirrhosis is mainly due to a loss of liver cell volume. The function per liver cell volume remains constant, however, thus favoring the intact cell hypothesis for the handling of slowly metabolized compounds such as aminopyrine.     

Effect of volume expansion on hemodynamics, capillary permeability and renal function in conscious, cirrhotic rats

General and splanchnic hemodynamics (radioactive microspheres), renal function, spontaneous and histamine-mediated vasopermeability and albumin distribution space were studied in conscious control and nonascitic cirrhotic rats, before and after a moderate and sustained saline infusion (3% of body weight per 30 min + repletion of urinary losses). In basal conditions, cirrhotic rats showed lower fractional sodium excretion than did control rats (0.09 +/- 0.01 vs. 0.15 +/- 0.01%, p less than 0.005). In addition, cirrhotic animals showed higher cardiac output (161.4 +/- 12.8 ml per min) and lower total peripheral resistance (0.63 +/- 0.05 mm Hg . min per ml) and mean arterial pressure (102.9 +/- 3.9 mm Hg) than did control rats (cardiac output: 89.0 +/- 7.6; total peripheral resistance: 1.31 +/- 0.11; mean arterial pressure: 117.5 +/- 5.11; p less than 0.005). No differences in portal-systemic shunt rate or vasopermeability between both groups were observed. After saline infusion, fractional sodium excretion increased to 4.31 +/- 0.16% in controls but only 2.11 +/- 0.02% in cirrhotic animals. In this group, cardiac output decreased by 49.6 +/- 5.1% whereas mean arterial pressure and total peripheral resistance increased by 7.1 +/- 0.6 and 112 +/- 10%, respectively. In control rats, no significant hemodynamic changes were observed. Blood gases did not change after expansion in any group. Saline infusion induced an increase in histamine-mediated vasopermeability in cirrhotic rats but not in control rats. Also albumin distribution space increased more in cirrhotic than in control animals. Heart weight was higher in cirrhotic rats.(ABSTRACT TRUNCATED AT 250 WORDS)