Bile acid profiles in analbuminemia rats

Bile acid profiles in serum, urine and bile of Nagase analbuminemia rats (NAR) and Sprague-Dawley rats (SDR) were examined. Serum bile acid levels in NAR (2.02 + 0.51 micrograms/ml, n = 15, M +/- S.E.) were markedly decreased as compared with those in SDR (20.86 +/- 3.72 micrograms/ml, n = 10). The unbound fraction of acids in serum examined by equilibrium dialysis was about ten times higher in NAR than in SDR. In the profiles of urinary and biliary bile acids in NAR and SDR, as big differences as seen for serum bile acids were not observed. Low bile acid levels in serum of NAR may reflect low bile acid binding capacity of NAR serum because the absence of albumin was thought to be one of the major causes of low bile acid levels in serum of NAR.     

Subcutaneous administration of HMG-CoA reductase inhibitors in hyperlipidaemic and normal rats.

Recent reports demonstrate a hypocholesterolaemic effect of daily subcutaneous injections of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in different rat models of hyperlipidaemia. However, this effect is not seen after oral administration of HMG-CoA reductase inhibitors in rats. We found that oral administration of the HMG-CoA reductase inhibitor Simvastatin also had no effect on plasma cholesterol in severely hyperlipidaemic Nagase analbuminaemic rats (NAR). Simvastatin (an apolar compound dissolved in propylene glycol) was infused continuously for 28 days into the subcutis of control Sprague-Dawley rats (SDR) and NAR using an implanted osmotic pump. All doses which were effective in reducing cholesterol in the NAR (reductions up to approximately 60%), reduced apolipoprotein AI but not apolipoprotein B and caused a severe inflammatory reaction in the dermis. Similar toxicity was observed in the SDR. Subcutaneous administration of the vehicle (propylene glycol) did not cause this reaction and did not affect plasma lipids. Administration of Lovastatin in osmotic pumps resulted in a similar inflammatory reaction. Incorporation of Simvastatin into liposomes did not diminish the toxic effect. On the other hand, infusion of Pravastatin (a polar HMG-CoA reductase inhibitor dissolved in isotonic saline) caused no changes in the dermis and had no effect on plasma lipids in NAR or SDR. Liver microsomes prepared from the Pravastatin-treated rats demonstrated a 3- to 4-fold increase in HMG-CoA reductase activity as compared to untreated rats, confirming uptake of the drug. We conclude that continuous subcutaneous administration of the HMG-CoA reductase inhibitors Simvastatin, Lovastatin and Pravastatin for 28 days may not reduce plasma cholesterol in rats by a mechanism which is related to inhibition of HMG-CoA reductase activity in the liver. The decrease of plasma cholesterol effected by subcutaneous infusion of Simvastatin or Lovastatin in NAR coincides with, and may be related to inflammatory changes caused by administering these compounds into the dermis.     

Comparison of plasma levels of lipid hydroperoxides and antioxidants in hyperlipidemic Nagase analbuminemic rats, Sprague-Dawley rats, and humans.

The levels of lipid hydroperoxides and antioxidants in plasma samples from Nagase analbuminemic rats (NAR) and control Sprague-Dawley rats (SDR) were measured in comparison with those from normal human subjects. Cholesteryl ester hydroperoxide (CE-OOH) was detected, but phosphatidylcholine hydroperoxide was not. The levels of CE-OOH and the ratios of CE-OOH/CE were found to increase significantly in the order of human < SDR < NAR, suggesting that oxidative stress increases in the same order. NAR have a significantly lower level of ascorbate and lower ratio of ubiquinol/ubiquinone concentrations than SDR. This also suggests that NAR are subject to more oxidative stress than SDR, since ascorbate and ubiquinol are the most effective plasma antioxidants against oxygen radicals.     

Transport and distribution of copper injected into an albumin-deficient (analbuminemic) rat

Copper (Cu) concentrations in blood, liver, kidney, spleen and pancreas of an albumin-deficient (Nagase analbuminemic) rat (NAR) were compared with those of a control (Sprague-Dawley) rat (SDR). Cu concentrations were significantly higher in the blood and significantly lower in the liver of the NAR strain than those of the SDR strain in female control (saline-injected) groups at 8 weeks old. Female NAR and SDR 8-week-old rats were injected i.p. with Cu at a single dose of 2.0 mg/kg body wt and killed 18 hr later. Concentrations of Cu and other essential elements in the blood, liver, kidney, spleen and pancreas were determined simultaneously by inductively coupled plasma-atomic emission spectrometry. Cu concentration in the liver was significantly lower in the NAR than in the SDR strain suggesting a role for albumin as a carrier protein of free Cu ions in the blood. The effects of Cu loading on other essential elements (Zn, Fe, Ca, Mg, P) were also compared between the NAR and SDR strains.     

Abnormal renal response to twenty-four-hour dehydration and fasting in Nagase analbuminemic rats.

We assessed renal function in fasting adult Nagase analbuminemic rats (NAR). Sodium output in male and female NAR was 68% and 46%, respectively, of the output of age- and sex-matched normal Sprague-Dawley (SD) rats. Potassium excretion was significantly greater in female NAR but there was no difference between male NAR and SD rats. The renal clearances of urea and creatinine were reduced in NAR with corresponding increases in plasma concentrations; however, the urea and creatinine concentrations were not different in plasma samples taken from normally fed and hydrated SD and NAR rats. Exchangeable body sodium and sodium space was significantly larger in normally fed and hydrated NAR than in SD but there were no differences in plasma sodium concentrations or plasma volumes. Although plasma concentrations of albumin in NAR were only about 0.07% of the concentration in SD rats, the renal clearance of albumin in NAR was threefold greater. Kidney weights in NAR were 10 to 16% less than in SD rats but liver weights were 22 to 42% greater. Clearly, renal function was markedly abnormal in Nagase rats during a 24-hour fast.     

Reduced activity of androgen biosynthesis in the testes of rats with analbuminemia

Steroid metabolism in Nagase Analbuminemia Rats (NAR), a mutant strain established from Sprague-Dawley rats, was studied. NAR are characterized by lack of serum albumin and hyperlipidemia. Total testosterone concentration in the serum of NAR was lower than that of normal rats, while the serum free testosterone, LH and FSH concentrations were similar. The half lives of 14C-labeled testosterone administered intravenously in NAR and normal Sprague-Dawley (SD) rats were 4.4 and 4.1 min, respectively. The plasma clearance rates of testosterone in NAR and normal rats were 34.7 and 39.1 ml/min per kg body weight. On Sephadex G-100 chromatography, a mixture of [3H]testosterone and normal rat serum gave two protein peaks eluted in the void volume and the albumin fraction, and the radioactivity was eluted all in the albumin fraction. In contrast, a mixture of [3H]testosterone and NAR serum gave a single protein peak eluted in the void volume and the radioactivity was mainly eluted with this protein peak. The association constants of testosterone to NAR and normal rat sera were 1.25 and 2.24 X 10(4) M-1. Enzyme activities related to the synthesis of testosterone by the testicular microsomal fractions of NAR and normal rats were examined. The activities of 3 beta-hydroxysteroid dehydrogenase, 5-ene-4-ene isomerase, 17 alpha-hydroxylase, C-17-C-20 lyase and 17 beta-hydroxysteroid dehydrogenase were lower in NAR than in normal rats. The activity for synthesis of testosterone from pregnenolone by the testicular microsomal fraction of NAR was about 40% of that of normal rats. These findings indicate that the low serum concentration of testosterone in NAR is mainly attributable to decreased biosynthesis of testosterone in the testes.     

Age-related changes in plasma proteins of analbuminemic rats

A mutant strain, Nagase analbuminemia rats (NAR), was established from Sprague-Dawley rats. Age-related changes in plasma proteins of NAR were investigated to obtain information of their abnormalities of protein metabolism. The total protein concentration in the serum of NAR of various ages was almost the same as that of normal rats of the same age. The albumin level of NAR was less than 0.05 mg/ml at all ages examined. The concentrations of serum alpha 1-antitrypsin, alpha-X protein, alpha 2-macroglobulin, transferrin, ceruloplasmin, IgG, IgA and IgM were higher in NAR than in normal rats except for the perinatal stage, but alpha 1-acid glycoprotein level in NAR was normal. The serum transferrin and ceruloplasmin levels were especially high in female adult NAR. The plasma fibrinogen concentration was also increased in NAR. These findings indicate that the normal total serum protein level of NAR was maintained by increase in the globulin concentration.     

Anemia and potassium permeability of red blood cells in analbuminemic rats

A mutant strain, Nagase analbuminemia rats (NAR), was established from Sprague-Dawley rats. Hematological evaluations were made on NAR of 4 to 52 weeks of age. NAR had an abnormally low number of red blood cells (RBC), a low hematocrit, a reduced hemoglobin concentration and an increased number of reticulocytes. Their plasma electrolyte level was normal. Osmotic fragility of RBC was slightly increased in the rats. Thus NAR shows a slight anemia. The in vitro experiments on RBC were performed. The incubation of blood showed a hemolytic tendency and elevated potassium efflux in the blood of NAR. In addition, an increased efflux of potassium was found in the RBC of NAR, when the RBC was washed with phosphate buffered saline and then was incubated with the saline containing CaCl2. This potassium efflux was prevented in the presence of rat albumin. These findings suggest that the deficiency of serum albumin may increase the permeability of potassium in erythrocyte membrane in NAR.     

Hyperlipoproteinemia in Nagase analbuminemic rats: effects of pravastatin on plasma (apo)lipoproteins and lecithin:cholesterol acyltransferase activity.

The present study demonstrates very high levels of plasma lipids and high density lipoprotein (HDL) apolipoproteins (apoA-I and apoE) in female Nagase analbuminemic rats (NAR) fed a semi-synthetic diet in order to further increase the hyperlipidemia present in this strain. Plasma apoB-containing lipoproteins (very low, intermediate, and low density lipoproteins) were also elevated in NAR. Plasma cholesterol was mainly present in lipoprotein particles with a density between 1.02 and 1.12 g/ml. Separation of lipoprotein classes by gel filtration showed that the major cholesterol-carrying lipoprotein fractions in NAR plasma are apoE-rich HDL and apoA-I-rich HDL. The high HDL levels in NAR are explained, at least partly, by the two- to threefold elevated activity of plasma lecithin:cholesterol acyltransferase (LCAT). The lysophosphatidylcholine generated in the LCAT reaction, as well as plasma free fatty acids, are bound to lipoproteins in NAR plasma. A study was carried out to determine whether the elevated LDL and aopoE-rich HDL levels could be corrected by administration of the HMG-CoA reductase inhibitor pravastatin (at a dose of 1 mg/kg per day). Pravastatin treatment results in a 43% decrease in plasma triglycerides in NAR, but not in Sprague-Dawley (SDR) rats, and had no significant effect on plasma total cholesterol, phospholipids apolipoproteins A-I, A-IV, B, or E, as well as on plasma LCAT activity levels in NAR or SDR.     

A high level of transferrin mRNA in the liver of analbuminemic rats

By means of immunological screening, a cDNA clone bearing the mRNA sequence for rat transferrin was isolated from a cDNA library of rat liver mRNA. The amounts of transferrin mRNA in livers of analbuminemic rats (NAR, Nagase analbuminemia rats) and normal rats were determined by RNA blot hybridization using a cloned transferrin cDNA probe. The level of transferrin mRNA in the NAR liver was about 1.7 times that in the normal rat liver. These findings suggest that the enhanced synthesis of transferrin in the NAR liver resulted from an increase in the transferrin mRNA level.     

Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Nagase analbuminemic rats (NAR), a strain which bears a mutation that determines the impossibility of the normal splicing of the albumin mRNA were used as recipients. The transplanted FH immediately migrated to the liver via portal vein, and anchored there. To assess the functional state of the transplanted cells, one month after transplantation, the expression of the albumin gene was studied in the liver of the recipients.     

Albumin is not an irreplaceable carrier for amphipathic mediators of thermoregulatory responses to LPS: compensatory role of alpha1-acid glycoprotein

In view of the potential involvement of peripherally synthesized, circulating amphipathic mediators [such as platelet-activating factor (PAF) and prostaglandin E(2)] in the systemic inflammatory response to lipopolysaccharide (LPS), we hypothesized that transport of amphipaths by albumin is essential for conveying peripheral inflammatory signals to the brain. Our first specific aim was to test this hypothesis by studying LPS-induced fever and hypothermia in Nagase analbuminemic rats (NAR). NAR from two different colonies and normalbuminemic Sprague-Dawley rats were preimplanted with jugular catheters, and their febrile responses to a mild dose of LPS (10 microg/kg i.v.) at thermoneutrality and hypothermic responses to a high dose of LPS (500 microg/kg i.v.) in the cold were studied. NAR of both colonies developed normal febrile and hypothermic responses, thus suggesting that transport of amphipathic mediators by albumin is not indispensable for LPS signaling. Although alternative carrier proteins [such as alpha(1)-acid glycoprotein (AGP)] are known to assume transport functions of albumin in NAR, it is unknown whether inflammatory mediators are capable of inducing their actions when bound to alternative carriers. To test whether PAF, the most potent amphipathic pyrogen, causes fever when administered in an AGP-bound form was our second aim. Sprague-Dawley rats were preimplanted with jugular catheters, and their thermal responses to infusion of a 1:1 [PAF-AGP] complex (40 nmol/kg i.v.), AGP (40 nmol/kg i.v.), or various doses of free (aggregated) PAF were studied. The complex, but neither free PAF nor AGP, caused a high ( approximately 1.5 degrees C) fever with a short (< 10 min) latency. This is the first demonstration of a pyrogenic activity of AGP-bound PAF. We conclude that, in the absence of albumin, AGP and possibly other carriers participate in immune-to-brain signaling by binding and transporting amphipathic inflammatory mediators.     

Hepato-renal transport of phenolsulfonphthalein in analbuminemic rats: albumin is essential for hepatic compensatory elimination of a nephrophilic ligand in animals with renal dysfunction

To investigate a possible function of plasma albumin in partitioning organic anions into bile and urine, phenolsulfonphthalein (PSP) was administered intravenously and its in vivo fate was studied in normal and analbuminemic mutant rats (NAR). No significant change in the rate of PSP disappearance was observed in bilaterally nephrectomized normal rats. However, biliary excretion of the injected dye increased remarkably in nephrectomized normal rats. Intravenously injected PSP disappeared very rapidly from the circulation of NAR. Thus, the plasma clearance and distribution volume of PSP were significantly larger in NAR than in normal rats. Bilateral nephrectomy also failed to decrease the plasma clearance and distribution volume of the dye in NAR. In striking contrast to the experiments in normal rats, bilateral nephrectomy did not increase the biliary secretion of PSP in NAR. When PSP bound to equimolar albumin was injected into bilaterally nephrectomized NAR, the biliary excretion of PSP increased significantly with concomitant decrease in both plasma clearance and distribution volume of the dye. These results indicate that, in cases of renal transport dysfunction, albumin plays a critical role in hepatic compensatory excretion of PSP, a nephrophilic organic anion, whose molecular weight (MW 354) is close to the threshold value for partitioning a ligand to the eliminatory routes in liver and kidney of a rodent.     

Role of plasma albumin in renal elimination of a mercapturic acid. Analyses in normal and mutant analbuminemic rats

Biosynthesis of N-acetylcysteine S-conjugates of xenobiotics (mercapturic acids) occurs via interorgan metabolism and the renal transtubular transport system plays an important role in elimination of the final metabolites from the organism. To assess the behavior of a mercapturic acid in the circulation, plasma clearance of radioactive S-benzyl-N-acetylcysteine and its interaction with plasma proteins were studied in normal and mutant analbuminemic rats (NAR). Intravenously injected S-benzyl-N-acetylcysteine rapidly disappeared from the circulation both in NAR and normal animals. However, its plasma clearance was significantly higher in NAR (45.7 ml kg-1 min-1) than in normal rats (25.2 ml kg-1 min-1). Ultrafiltration analysis revealed that 18.4% and 80.1% of the mercapturate bound to plasma protein(s) from NAR and normal rats, respectively, at 50 microM ligand concentration. The mercapturic acid bound to plasma albumin with an association constant of 2.24 X 10(5) M-1 and the number of binding sites was 1.18/mol albumin. The binding was competitively inhibited by probenecid and L-tryptophan. Concomitant administration of this mercapturic acid with equimolar amounts of albumin resulted in a marked decrease in the plasma clearance (26.2 ml kg-1 min-1) and an increase in the urinary secretion of this ligand in NAR. 30 min after injection of the mercapturic acid (10 mumol/kg body weight), 27.3% and 60.4% of the injected dose was recovered from urine and kidneys of NAR and normal rats respectively. About 41% of the dose was recovered in NAR urine when the ligand was injected bound to an equimolar amount of albumin. These results suggested that albumin is important for the renal accumulation and urinary elimination of the circulating mercapturic acid.     

Expression and distribution of the prolactin receptor in normal rat liver and in experimental liver cirrhosis.

Recent results have suggested a role for prolactin (PRL) as a regeneration factor in the liver. In order to investigate the involvement of prolactin in the pathogenesis of liver cirrhosis, we studied the expression of the prolactin receptor (PRLR) and PRL during the development of cirrhosis in an animal model. 30 male rats were exposed to CCl4 by inhalation. Phenobarbitone was added to the drinking water to accelerate the formation of toxic metabolites by enzyme induction. Two control groups of 30 animals each were treated with phenobarbitone only or received no treatment. 10 animals of each group were sacrificed 35, 55, and 70 days after initiation of treatment. Liver tissue was subjected to histological examination, which demonstrated fibrosis of different grades and cirrhosis in the CCl4-treated rats. Expression of PRLR mRNA was investigated by mRNA extraction, RT-PCR and computer-supported densitometric evaluation. Compared to control liver, PRLR mRNA was expressed at a higher level in fibrotic and cirrhotic liver specimens. In normal tissue, immunohistochemical staining showed a high concentration of PRLR around the central vein and in the epithelium of the bile ducts. This pattern of distribution was lost in fibrosis and cirrhosis. An accumulation of PRLR was demonstrated within the damaged cells. Neither PRL nor PRL mRNA was detectable in normal, fibrotic, or cirrhotic liver. We conclude that PRLR is distributed in normal rat liver in a typical pattern which is lost with increasing fibrosis. PRL is not produced by rat liver, indicating that PRL does not act through autocrine or paracrine mechanisms.     

Iron overload enhances the development of experimental liver cirrhosis in mice

The role of iron in initiating liver fibrosis in iron overload diseases is not clearly established. Partly, this is due to the lack of suitable animal models that can produce the full liver pathology seen in genetic hemochromatosis. Recent advances in this field have demonstrated that iron may be interacting with other potential liver-damaging agents. The aim of this study was to investigate if feeding with carbonyl iron (CI) facilitates the development of carbon tetrachloride (CCl4)-induced liver fibrosis in the mouse. Mice were given a diet containing 3% CI and treated with CCl4 intraperitoneally twice weekly and 5% alcohol added to the drinking water for 12 weeks. Hepatic iron content increased 15- and 22-fold in animals receiving CI and CI + CCl4. At histological examination, iron-laden hepatocytes were found in CI treated animals, whereas these were absent in animals not exposed to CI. Mice receiving iron-enriched diet alone showed a mild fibrosis. Conversely, a marked collagen deposition was observed in CCl4 and CI + CCl4 groups. In particular, in this latter group, there was evidence of liver cirrhosis. Biochemical evaluation of collagen content substantiated histologic analysis. These results demonstrate that the addition of iron facilitates the development of cirrhosis in animals exposed to subtoxic doses of CCl4. This model may be useful in exploring the pathogenesis of liver cirrhosis. Moreover, its use in genetically altered mouse strains might provide new insight on the role of iron in fibrosis.     

Establishment and characteristics of five analbuminemic inbred strains of rats

Five analbuminemic inbred strains of rats (AD/1, AD/2, AD/3, AD/4, AD/5) were established from Nagase analbuminemic rats (NAR). They showed no genetic differences in coat color, biochemical marker gene loci and skin grafting test. Their serum levels of total cholesterol, phospholipids, triglycerides, and beta-lipoproteins were compared with normal inbred strains (L) derived from Sprague-Dawley rats. Their plasma apoproteins were also examined. All inbred strains of analbuminemic rats showed hyperlipidemia progressing with age although there were slight variations in their lipid and apoprotein levels. These analbuminemic inbred strains of rats may be multigenic models of lipid metabolism abnormality.     

The role of albumin in the hepatic transport of bilirubin: studies in mutant analbuminemic rats

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.     

Iron and copper metabolism in analbuminaemic rats fed a high-iron diet

The metabolism of iron and copper in male Nagase analbuminaemic (NA) and Sprague Dawley (SD) rats was compared. Relative liver weight was higher and spleen weight significantly lower in NA than SD rats. In NA rats, red blood cell count, haemoglobin and haematocrit were lower, whereas plasma transferrin, total iron-binding capacity and mean corpuscular haemoglobin were higher when compared with SD rats. Iron concentrations in plasma, liver, kidneys and heart were higher, and those in the spleen and tibia were lower, in NA rats. The iron concentrations in liver and spleen were positively correlated with the amount of brown pigment as observed histopathologically. Bile flow as well as biliary iron and copper excretion were higher in NA than SD rats. Copper concentrations in liver, kidneys and plasma were higher in NA rats. Plasma levels of ceruloplasmin were about two-fold higher in NA rats. The feeding of a high-iron diet reduced kidney copper concentrations in both strains of rats, which was associated with a decrease in the absorption and biliary excretion of copper.     

Copper metabolism in analbuminaemic rats fed a high-copper diet

Copper metabolism in male Nagase analbuminaemic (NA) rats was compared with that in male Sprague Dawley (SD) rats fed purified diets containing either 5 or 100 mg Cu/kg diet. Dietary copper loading increased hepatic and kidney copper concentrations in both strains to the same extent, but baseline values were higher in the NA rats. There was no strain difference in true and apparent copper absorption nor in faecal endogenous and urinary copper excretion. NA rats had higher levels of radioactivity in kidneys at 2 hr after intraperitoneal administration of ⁶⁴Cu. As based on the distribution of added ⁶⁴Cu, about 70% of plasma copper appeared to be in the non-protein compartment in the NA rats, whereas in SD rats, it was only about 1%. It is concluded that the NA rats are able to maintain a relatively normal metabolism of copper, even after dietary copper challenge. In the NA rats, zinc concentrations in kidneys, liver and urinary zinc excretion were elevated when compared with SD rats. The high-copper diet did not affect tissue zinc concentrations and apparent zinc absorption in both strains of rats.