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.     

Transhepatic transport of taurocholic acid in normal and mutant analbuminemic rats

To elucidate a possible function of plasma albumin in vectorial transport of various cholephilic organic anions, such as bile acids, plasma clearance and transhepatic transport of radioactive taurocholate were studied in vivo in normal and mutant analbuminemic rats. Intravenous administration of taurocholate was followed by its rapid disappearance from the circulation in both animal groups. However, plasma clearance of taurocholate was significantly larger in analbuminemic (68.3 ml/min per kg of body weight) than in normal rats (29.8 ml/min per kg of body weight) at a dose of 8 mumol/kg of body weight. The increased plasma clearance in analbuminemic rats was accompanied by a more prompt biliary secretion of the ligand than occurred in normal animals; 79 and 42% of the injected dose was recovered in analbuminemic and normal rat bile, respectively, within 10 min after administration. Ultrafiltration analysis revealed that the binding of taurocholate to serum protein(s) was significantly lower in analbuminemic rats as compared with that in normal rat serum; 24 and 76% of taurocholate bound to protein fractions of analbuminemic and normal rat serum, respectively, at 0.5-mM ligand concentration. Binding of taurocholate to cytosolic proteins of normal and analbuminemic liver were similar; 23 and 28% of taurocholate bound to protein fractions from analbuminemic and normal rat, respectively, at 10 mg protein/ml and 20-microM ligand concentration. These results indicate that plasma albumin does not play a role in directing circulating taurocholate to the liver and that transhepatic transport of the bile acid increases with the increase in concentration of unbound ligand in the circulation.     

Hypertriacylglycerolemia and adipose tissue lipoprotein lipase activity in the Nagase analbuminemic rat

In Nagase analbuminemic rats, serum triacylglycerol levels were significantly elevated. This abnormality was accompanied by decreased adipose tissue fat stores, and both were more marked in female than in male rats. Parametrial adipose tissue lipoprotein lipase activity was determined in normally fed female rats. When expressed per mg protein, the activity in analbuminemic rats was only 35% of that in control rats. The activity in analbuminemic rats, however, could be increased as in control rats by refeeding starved animals with a fat-free and carbohydrate-rich diet, and the peak values recorded were the same with the two groups. Treatment of animals with streptozotocin lowered adipose tissue lipoprotein lipase activity in both groups to similar levels. These results suggest that hypertriacylglycerolemia associated with analbuminemia may be caused, at least in part, by altered hormonal control of adipose tissue lipoprotein lipase activity.     

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.     

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.     

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.     

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.     

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.     

Characteristics and significance of albumin-positive hepatocytes in analbuminemic rats

Analbuminemic rats (NAR) are a mutant strain in which splicing of the albumin mRNA is blocked due to a seven-base-pair deletion in an intron of the albumin gene. NAR liver contains a few hepatocytes that react with anti-rat albumin antibody (Alb+ hepatocytes), and these cells increase in number during aging and on treatment with hepatocarcinogens. To characterize these Alb+ hepatocytes, we examine their albumin mRNA, the biochemical specificity of their albumin, and its intracellular distribution. Signals of albumin mRNA were observed in a few hepatocytes by in situ hybridization. Moreover, a small amount of cytoplasmic albumin mRNA was detected by RNA blot analysis in the liver of aged NAR and NAR treated with 3'-methyl-4-diaminoazobenzene (DAB). Immunoelectron microscopic examination revealed the cisternae of the rough and smooth endoplasmic reticula, Golgi complexes, and secretory vesicles of the Alb+ hepatocyte of NAR being filled with material that reacted with anti-rat albumin antibody. These facts suggested that albumin was gradually synthesized in Alb+ hepatocytes but that its secretion was disturbed. The albumin-like proteins of NAR were shown by Western blot analysis to consist of three species of 68 kDa, 50 kDa, and 25 kDa proteins. The 50 kDa albumin was thought to be formed by exon-skipping splicing of the albumin mRNA precursor, which was recently reported by Shalaby and Shafritz (Proc. Natl. Acad. Sci. USA 87, 2652-2656 (1990)). The 25 kDa protein was suspected to be formed by fragmentation of the 50 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute phase response of plasma proteins in analbuminemic rats

In healthy Nagase analbuminemic rats (NAR), the highest degree of relative increase in serum protein concentration was found for alpha-2-macroglobulin, the most prominent acute phase protein in rats. Its levels were about 30- and 60-fold higher in males and females, respectively, than those in the control Sprague-Dawley (SD) rats. In terms of absolute concentration, however, alpha-1-inhibitor 3 (also called alpha-X-protein or murinoglobulin) showed the most conspicuous change, its levels being higher by about 7 mg/ml than those in SD. When the acute phase reaction was induced by subcutaneous injection of turpentine, the levels of alpha-1- and alpha-2-macroglobulins, alpha-1-cysteine proteinase inhibitor, alpha-1-antiproteinase, and alpha-1-inhibitor 3 in NAR changed in essentially the same way as in SD: alpha-1-inhibitor 3 decreased markedly while the rest increased further. These results suggest that mechanisms responsible for the elevation of serum globulins in healthy NAR are not directly related to those involved in the acute phase response. On the other hand, the antithrombin III levels in healthy NAR were about twice the control values and changed little during the inflammation. In contrast, this protein in SD doubled during the acute phase, its maximal levels being close to those in healthy or inflamed NAR. This suggests that the antithrombin III level in healthy NAR is regulated by a mechanism similar to that in SD maximally reacting to the acute phase stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of liver cirrhosis induction by thioacetamide in Nagase analbuminaemic rats

Repeated administration of thioacetamide (TA), either intraperitoneally or in drinking water, produced liver cirrhosis in normal Sprague-Dawley rats (SDR) with significant histological alterations similar to those observed in human cirrhosis. In the present study, we evaluated the ability of TA to induce liver cirrhosis in mutant Nagase analbuminaemic SDR. Thioacetamide was administered either intraperitoneally up to 4 months or in drinking water up to 6 months to normal and to Nagase analbuminaemic SDR. Nagase analbuminaemic rats (NAR) were also administered TA in drinking water up to 10 months. Liver cirrhosis development was determined by macroscopic and microscopic analysis. In contrast to normal SDR, no histological characteristics of cirrhosis could be observed in NAR submitted to a 4 or 6 months treatment with TA. Such failure to induce cirrhosis in Nagase rats was confirmed even after prolonged TA administration in drinking water for up to 10 months. In contrast, fibrosis and cholangiolar proliferation occurred in the 10-month TA-treated analbuminaemic rats, suggesting that the mechanisms involved in cirrhosis induction are different from those involved in fibrosis development and carcinogenesis. It is unlikely that the protective effect against TA-induced cirrhosis observed in analbuminaemic rats is related to the absence of albumin in this rat strain, since a co-administration of TA with albumin in analbuminaemic rats did not restore the potential for TA to induce cirrhosis in this rat strain. In conclusion, the fact that induction of cirrhosis by TA is prevented in the inherently hyperlipidaemic and hypercholesterolaemic analbuminaemic rats could be considered for potential application in the treatment of clinical cirrhosis.     

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.     

Establishment and characteristics of three analbuminemic congenic strains of rats

We have established three analbuminemic congenic strains of rats (ACI-alb, F344-alb, and SHR-alb) by repeated backcrossing with a progeny test or intercrossing. Some coat color and biochemical marker genes of each congenic strain agreed with those of the background inbred strain of rats, except for the alb gene locus. These established congenic strains were maintained by cross-intercrossing. Body weights, organ weights and serum lipid concentrations of each strain were measured up to 30 weeks of age. Body weights of ACI-alb congenic strains (alb/alb and alb/+) were similar to those of the original ACI(+/+) strain, but those of F344-alb and SHR-alb were heavier in the order of +/+, alb/+ and alb/alb. The liver and adrenal weights of all strains were higher in the order of alb/alb, alb/+ and +/+. Serum lipid concentrations were also higher in the same order. These three analbuminemic congenic strains originating from different inbred strains should be useful in studies of carcinogenesis and genetically modified mechanisms of albumin functions.     

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.     

Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats

A plasmid (palb3) was constructed containing the structural gene for human serum albumin driven by mouse albumin enhancer-rat albumin promoter elements. Using an asialoglycoprotein-polycation conjugate consisting of asialoorosomucoid coupled to poly-L-lysine, a soluble DNA complex was formed that was capable of targeting specifically to hepatocytes via asialoglycoprotein receptors present on these cells. Groups of Nagase analbuminemic rats were injected with complexed DNA or controls, followed by two-thirds partial hepatectomy to stimulate hepatocyte replication. Using a cDNA probe for the human albumin structural gene, hybridizable sequences were detected in analbuminemic rats treated with complex as determined by Southern blot analysis. Two weeks post-injection, the targeted DNA was found to exist primarily in plasmid form with an average copy number of 1000/diploid cell. Human albumin mRNA was detected by dot-blot hybridization with a specific oligonucleotide cDNA probe and confirmed by RNase protection assay using a vector-specific probe. Circulating human albumin was detected in the serum of palb3-treated Nagase analbuminemic rats by Western blots using an antibody specific for human serum albumin. A time course demonstrated that circulating human albumin was not detectable 24 h after injection, but became measurable at a level of 0.05 micrograms/ml within 48 h and increased in concentration to a maximum of 34 micrograms/ml by 2 weeks post-injection. This level of expression remained stable through 4 weeks after injection and partial hepatectomy.     

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.     

Pharmacokinetics of theanine enantiomers in rats

Theanine, first discovered in tea, is a chiral nonproteinic amino acid that has been reported to have cardiovascular, neurological, and oncological effects. It is being considered as a therapeutic/medicinal agent and additive in consumer products. The present study evaluated the pharmacokinetics of D-theanine, L-theanine, and D,L-theanine in plasma and urine using LC-ESI/MS in rats after oral (p.o.) and intraperitoneal (i.p.) administration. Oral administration data indicated that gut absorption of d-theanine was far less than that of L-theanine. However, after i.p. administration, plasma theanine concentrations of L- and D-theanine were similar. This indicated that D- and L-theanine may exhibit a competitive effect with respect to intestinal absorption. Regardless of the route of administration, p.o. or i.p., the presence of the other enantiomer always decreased theanine plasma concentrations, indicating D,L-theanine competition with respect to urinary reabsorption. Data on urinary concentrations of D-theanine suggested that the D-isomer may be eliminated with minimal metabolism. L-Theanine appeared to be preferentially reabsorbed and metabolized by the kidney while D-theanine was preferentially excreted. Clearly, the bioequivalencies of D,L-theanine and its enantiomers were found to be quite different from one another. Consequently, the efficacy of commercial theanine products containing D-theanine, L-theanine, or D,L-theanine may be quite different.     

Elevation of serum albumin concentration in analbuminemic rats by administration of 3'-methyl-4-dimethylaminoazobenzene

Albumin-like protein was detected in the serum of analbuminemic rats which have a mutation affecting albumin mRNA processing and genetically lack serum albumin. This protein was identified as rat serum albumin on the basis of molecular weight, immunological property and digestion patterns with proteases. The serum albumin level in analbuminemic rats was 5 μg/ml 4 weeks after birth and increased slightly during aging. By continuous administration of a hepatocarcinogen, 3′-methyl-4-dimethylaminoazobenzene, the level of serum albumin in the mutant rats increased over that of untreated rats and reached a maximum of 32 μg/ml at the 15th week of the carcinogen administration, whereas that of untreated rats was 16 μg/ml at the same time.