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Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial effects on citrin deficiency by increasing the carbohydrate tolerance of Ctrn/mGPD-KO mice, as observed through increased food intake and maintenance of body weight., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.

The expression of carbamoyl phosphate synthetase I (CPS) gene is suppressed in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice at weaning and under starvation at adult age. To clarify the suppression mechanism, we produced CPSL transgenic JVS mice carrying a transgene composed of the chloramphenicol acetyltransferase (CAT) gene with the upstream region (-12 kb to +138) of the rat CPS gene and CPSE transgenic JVS mice carrying a transgene composed of the luciferase gene with minimal promoter (299 bp from -161 to +138) and enhancer (469 bp around -6.3 kb) fragments of the rat gene. The expression of the CAT gene as well as the endogenous CPS was suppressed in CPSL transgenic JVS mice, but luciferase gene expression was not suppressed in CPSE transgenic JVS mice. We isolated the 5'-upstream region of the mouse CPS gene and identified an activator protein-1 (AP-1) site downstream of the minimum enhancer region of both rat and mouse CPS genes. In conjunction with the 313-bp mouse promoter region, the 714-bp mouse enhancer fragment conferred a cell-type-dependent hormone responsiveness. In rat primary cultured hepatocytes, the addition of oleic acid suppressed reporter gene expression induced by dexamethasone in the construct containing the enhancer fragment of 714 bp with the AP-1 site, but not in its AP-1 site mutants or in 519 bp without the AP-1 site. These results strongly suggest that direct protein-protein interaction between AP-1 and glucocorticoid receptor is not involved in the suppression of the CPS gene in JVS mice and that the AP-1 element is the cis-element which is responsible for the suppression., (Copyright 1999 Academic Press.)

Sparse fur with abnormal skin and hair (spf-ash) mice are deficient in ornithine carbamoyltransferase (OCT) activity, but their OCT protein is kinetically normal. We administered ammonium chloride to spf-ash mice, in order to analyze ammonia metabolism and to find a rationale for the therapy of OCT deficiency. Ammonia concentration in the liver of spf-ash mice increased to a level much higher than in the control. Ammonium chloride injection caused an increase in ornithine (Orn) 5 min after injection and an increase in the sum of Orn, citrulline (Cit) and arginine (Arg) for at least 15 min in the liver of control mice, but no increase in Orn, Cit and Arg in the liver of spf-ash mice. Treatment of spf-ash mice with Arg 5-20 min prior to the injection of ammonium chloride kept the hepatic ammonia concentration at a level comparable to that without the load. A significant reciprocal relationship between ammonia and Orn concentrations in the liver of spf-ash mice 5 min after an ammonium chloride load with or without Arg strongly suggests that ammonia disposal is dependent on the supply of Orn. In spf-ash mice loaded with tryptone as a nitrogen source, Arg supplementation showed a dramatic decrease in urinary orotic acid excretion in a dose-dependent manner. Similar effects were observed with Cit and Orn at the same dose, and a long-lasting effect with an ornithine aminotransferase inactivator, 5-(fluoromethyl)ornithine, at a much lower dose. The rate of urea formation in liver perfused with ammonium chloride was lower in spf-ash mice than in controls, but with the addition of Orn to the medium it increased to a similar level in control and spf-ash mice. These results indicate that OCT is not saturated with Orn in vivo under physiological conditions and that the administration or enrichment of the urea cycle intermediate amino acids enhances the OCT reaction so that the ammonia metabolism of OCT-deficient spf-ash mice is at least partially normalized.

Juvenile visceral steatosis (JVS) mice have been reported to have systemic carnitine deficiency, and the carnitine concentration in the liver of JVS mice was markedly lower than that of controls (11.6 +/- 2.6 versus 393.5 +/- 56.4 nmol/g of wet liver). To evaluate the role of carnitine in mitochondrial beta-oxidation in liver, we examined the effects of carnitine on ketogenesis in perfused liver from control and JVS mice. In control mice, ketogenesis was increased by the infusion of 0.3 mM oleate, but not by L-carnitine. In contrast, although ketogenesis in JVS mice was not increased by the infusion of oleate, it was increased 2.5-fold by the addition of 1000 microM L-carnitine. Addition of 50, 100, and 200 microM L-carnitine increased ketogenesis in a dose-dependent manner. The infusion of 0.3 mM octanoate or butyrate increased ketogenesis in a carnitine-independent fashion in both control and JVS mice. These findings suggest that endogenous long chain fatty acids from accumulated triglycerides may be used as substrates in the presence of carnitine in JVS mice. The relationship between ketogenesis and free carnitine concentration was examined in livers from JVS mice. Ketogenesis increased as free carnitine levels increased until concentrations exceeded about 100 nmol/g of wet liver (340 microM). The free carnitine concentration required for half-maximal ketone body production in liver of JVS mice was 45 microM (13 nmol/g of wet liver), which corresponds to a K(m) value of carnitine palmitoyltransferase I. We conclude that carnitine is a rate-limiting factor for beta-oxidation in liver only when the carnitine level in liver is very low.

Systemic carnitine-deficient juvenile visceral steatosis (JVS) mice exhibit decreased expression of some liver-selective genes including those for the urea cycle enzymes during the infantile period. At 25 days, carbamoylphosphate synthetase (CPS) mRNA level was remarkably low in the liver of JVS mice, and the HNF-4 and C/EBP-alpha mRNA contents were also reduced. HNF-3 alpha and C/EBP-beta mRNAs were slightly higher in the liver of JVS mice, and HNF-1 mRNA remained normal. These results, together with the developmental changes of these transcription factor mRNA levels, suggest that HNF-4 and C/EBP-alpha are involved in the suppression of CPS expression. If JVS mice survived the crisis at 4-5 weeks, their body weight caught up with that of control mice around 7 weeks. The steady-state levels of CPS and argininosuccinate synthetase (ASS) mRNAs in the liver of JVS mice were normalized by no later than 8 weeks. Starvation for 48 h caused an increase of about twofold in CPS and ASS mRNA levels in the liver of control mice, while the same treatment failed to increase their levels in the liver of JVS mice. The starvation similarly caused increases in HNF-4 and C/EBP-beta mRNA levels in the liver of both control and JVS mice, but the increases were significantly less in JVS mice than in control mice. Thus, the lack of induction of CPS and ASS mRNAs during development and under starvation in JVS mice correlated with the lower induction of HNF-4 and C/EBP-alpha mRNAs, and of HNF-4 and C/ EBP-beta mRNAs, respectively. Furthermore, all these changes seemed to correlate with the presence of fatty liver and the high serum free fatty acid levels, suggesting that disturbance of fatty acid metabolism affects nitrogen metabolism at least in part via altered gene expression of transcription factors such as HNF-4, C/EBP-alpha, and C/EBP-beta.

In order to test the possibility that free fatty acids are the mediator of the abnormal expression of urea cycle enzyme genes in carnitine-deficient juvenile visceral steatosis (JVS) mice, the effects of fatty acids on urea cycle enzyme, carbamoylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), mRNA levels were examined in rat primary cultured hepatocytes. Addition of a synthetic glucocorticoid hormone, dexamethasone, caused increases in CPS and ASS mRNAs. Further addition of oleic acid suppressed the induction of CPS and ASS mRNAs by dexamethasone. In contrast, the phosphoenolpyruvate carboxykinase (PEPCK) mRNA level induced by dexamethasone was enhanced in the presence of oleic acid. The effects were reversed on further addition of carnitine. The mRNA levels of these enzymes induced by dibutyryl cAMP were not affected by the addition of oleic acid. A study of the specificity of fatty acids revealed that long-chain fatty acids of more than 16 carbons chain length had a suppressive effect on the CPS mRNA level induced by dexamethasone and that the presence of double bonds enhanced the effect. The changes in gene expression of CPS, ASS and PEPCK caused by the fatty acids in the cultured hepatocytes were very similar to those observed in the liver of JVS mice. The AP-1 DNA binding activity in the presence of dexamethasone was slightly enhanced by the addition of oleic acid. These results suggest that the long-chain fatty acids not metabolized in JVS mice are mediators of the abnormal gene expression in the liver which results in hyperammonemia.

To study liver functions of chicken, we examined the primary culture of chicken hepatocytes, and found an easy method of long-term culture with free atmosphere exchange. Chicken hepatocytes were obtained by collagenase perfusion and cultured at 37 degrees C as a monolayer without substratum in serum-free L-15 medium (pH 7.8) with free atmosphere exchange. The amounts of albumin and transferrin in medium were assayed by ELISA. The culture of chicken hepatocytes was maintained in the serum-free L15-medium )pH 7.) and 37 degrees C with free atmosphere exchange for 20 days. The amount of albumin secreted in the medium decreased to low levels early in culture; however, this was followed by marked increase from day 9 to day 17 of culture. The amount of transferrin was constant until day 6, then it too increased with further culture. We reported an easy method for the simple monolayer culture of chicken hepatocytes in serum-free L12 medium (pH 7.8) with free atmosphere exchange over an extended period. Expression of liver-specific functions, viz. albumin and transferrin synthesis, was observed after 1 week of culture.

A quantitative study of the effect of carnitine deficiency on expression of glycolytic and gluconeogenic enzymes was performed using juvenile visceral steatosis mice which are systemically deficient in carnitine. The amounts of glucokinase and L-type pyruvate kinase mRNA were reduced in homozygotes, compared to heterozygotes and normal controls at 2 and 8 weeks. Liver-type phosphofructokinase, however, did not differ significantly. The abundance of fructose 1,6-bisphosphatase mRNA was unchanged at 2 and 8 weeks. The level of phosphoenolpyruvate carboxykinase mRNA was increased slightly at 2 weeks, but not at 8 weeks. A part of these changes could not be explained by the plasma glucose or insulin level. Carnitine administration restored the mRNA of these enzymes to normal levels. These results suggest that carnitine deficiency affects the expression of these liver enzymes.

We conducted a quantitative study of the effect of carnitine deficiency on the mRNA level of carnitine palmitoyltransferase II in the liver, muscle and heart of mice with juvenile visceral steatosis, a strain that is systematically deficient in carnitine. The amount of carnitine palmitoyltransferase II mRNA was increased in liver and muscle of homozygotes, as compared with heterozygotes and normal controls, at 2, 4, and 8 wk of age. The mRNA levels of this enzyme were normalized after carnitine administration. The mRNA level of carnitine palmitoyltransferase II in the heart was increased only at 8 wk, and was not affected by carnitine administration. These results suggest that carnitine displays some effect on the mRNA level of the carnitine palmitoyltransferase II gene in liver and muscle, probably through fatty acid metabolic change.

Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia. Type II citrullinemia is found in most patients with adult-onset citrullinemia in Japan, and ASS is deficient specifically in the liver. Previous studies have shown that the decrease of hepatic ASS activity is caused by a decrease in enzyme protein with normal kinetic properties and that there are no apparent abnormalities in the amount, translational activity, and nucleotide sequence of hepatic ASS mRNA. Recent results of homozygosity testing indicate that the primary defect of type II citrullinemia is not within the ASS gene locus. In this present work, to understand the pathogenesis and pathophysiology of type II citrullinemia, we have characterized the alterations of gene expression in the liver of type II patients using the recently developed mRNA differential display method. Some cDNA bands expressed differently in type II citrullinemia patients and control were selected, cloned, and sequenced. Nucleotide sequence analysis and homology searching revealed an interesting clone which has 99% homology with the human pancreatic secretory trypsin inhibitor (hPSTI). Northern blot and RT-PCR analyses showed that the expression of hPSTI mRNA increased significantly in the liver of all type II patients tested. Furthermore, the concentration of hPSTI protein was found to be higher in the liver of type II citrullinemia than in control. These results suggest that hPSTI may be related to the primary defect of type II citrullinemia and may be useful as a diagnostic marker, although the detailed mechanism of the high expression of hPSTI mRNA in type II liver is not yet known.

Carnitine-deficient jvs mice expressed reduced levels of a group of genes which are preferentially expressed in the liver, including urea cycle enzyme genes (Biochim. Biophys. Acta 1138, 167-171, 1992). The expression of alpha-fetoprotein and aldolase A was elevated, indicating that the liver of jvs mice is undifferentiated or dedifferentiated (FEBS Lett. 311, 63-66, 1992). Studies of the hormone signal transduction pathway showed that serum cortisol and plasma glucagon levels of jvs mice were 2 and 3 times higher, respectively, than those of normal mice, and that the hormone binding activity of glucocorticoid receptor (GR) in the cytosol of jvs liver was 50% of normal mice, which reflected the amount of receptor protein in the cytosol. On the other hand, GR protein accumulated in the nuclear fraction in jvs mice. Exogenously administrated dexamethasone induced carbamoyl phosphate synthetase (CPS) and tyrosine aminotransferase (TAT) mRNAs in jvs mice, indicating that CPS and TAT genes in jvs mice are responsive to induction by glucocorticoid and cAMP. Analysis of transacting factors by gel retardation assay revealed that HNF-1, COUP-TF and SP-1 were detected at almost the same level in the hepatic nuclear fraction of jvs mice as in normal littermates, and C/EBP and CREB were a little higher in jvs mice, suggesting that these factors are probably not targets of jvs mutation causing abnormal gene expression in the liver. On the other hand, AP-1 binding activity was much higher in jvs mice from an early age, preceding the abnormal expression of urea cycle enzyme, and carnitine administration normalized AP-1 binding activity. We suggest that elevated AP-1 binding induced by carnitine deficiency is closely connected with the abnormal gene expression in the liver.

We determined the mRNA levels of c-jun and c-fos in the liver of C3H-H-2 degrees jvs mice. Both were higher in jvs mice than in normal mice. The level of c-jun mRNA increased gradually after birth, but in the control mice there was almost no change. In addition, alpha-fetoprotein and aldolase A mRNA levels were also higher than in normal littermates. These results suggest that the pattern of the gene expression in jvs mice partly resembles the one that occurs in undifferentiated hepatocytes and/or hepatocellular carcinoma.

Previous studies in our laboratories have revealed that juvenile visceral steatosis mice show suppressed transcription of urea cycle enzyme genes during development and are systemically deficient in carnitine. It has not yet been explained, however, how this carnitine deficiency relates to the abnormal gene expression. We investigated the effect of carnitine on abnormal gene expression, growth retardation, and fatty liver. Carnitine administration relieved the suppression of the developmental induction of two urea cycle enzymes examined, carbamoyl-phosphate synthetase and argininosuccinate synthase, and kept the activities of enzymes normal. However, carnitine did not reduce accumulated lipid in the liver to the normal level. These results suggest that carnitine deficiency plays an important role in the abnormal expression of urea cycle enzyme genes and that the abnormal expression of the genes is not directly caused by lipid accumulation in the liver.

Hyperammonemia, abnormalities in plasma amino acids and abnormalities of standard liver functions were corrected by orthotopic liver transplantation in a 14-day-old boy with carbamyl phosphate synthetase-I deficiency and in a 35-yr-old man with argininosuccinic acid synthetase deficiency. The first patient had high plasma glutamine levels and no measurable citrulline, whereas citrulline values were markedly increased in Patient 2. Enzyme analysis of the original livers showed undetectable activity of carbamyl phosphate synthetase-I in Patient 1 and argininosuccinic acid synthetase in Patient 2. Both patients were comatose before surgery. Intellectual recovery of patient 1 has been slightly retarded because of a brain abscess caused by Aspergillus infection after surgery. Both patients are well at 34 and 40 mo, respectively, after surgery. Our experience has shown that orthotopic liver transplantation corrects the life-threatening metabolic abnormalities caused by deficiencies in the urea cycle enzymes carbamyl phosphate synthetase-I and argininosuccinic acid synthetase. Seven other patients--six with ornithine transcarbamylase deficiency and another with carbamyl phosphate synthetase-I deficiency--are known to have been treated elsewhere with liver transplantation 1 1/2 yr or longer ago. Four of these seven recipients also are well, with follow-ups of 1 1/2 to 5 yr. Thus liver transplantation corrects the metabolic abnormalities of three of the six urea cycle enzyme deficiencies, and presumably would correct all.

We analyzed carnitine profiles in C3H-H-2 degrees strain of mouse associated with fatty liver, hyperammonemia and hypoglycemia (Koizumi et al., 1988). Carnitine levels in serum, liver and muscle of mouse with fatty liver were markedly decreased in comparison with those of control mouse (littermates without fatty liver). This is a useful animal model to analyze the role of carnitine in lipid, amino acid and carbohydrate metabolism.

Argininosuccinate synthetase is an enzyme which has been found to be a specific marker for liver damage. In patients with acute hepatitis, the concentration in serum increases at the onset of the disease, but later decreases more quickly, so that the time required for normalization is shorter than that of alanine aminotransferase. This is probably caused by rapid clearance of argininosuccinate synthetase from the serum. Rapid clearance was demonstrated in experimental animals given purified enzymes intravenously. Argininosuccinate synthetase disappeared from the serum with a half life of about 15 min, while the half lives of alanine aminotransferase and aspartate aminotransferase were 4 and 5 h, respectively, under the same conditions.

We determined the activities of urea cycle enzymes in the liver of C3H-H-2 degree-jsv mice. The activities of all urea cycle enzymes decreased in the latter period of lactation. The activities of carbamylphosphate synthetase and ornithine transcarbamylase in some affected mice were undetectable. On the other hand, the activities of enzymes other than urea cycle enzymes were normal. We consider that the decrease in the urea cycle enzymes is caused by an abnormality in the mechanism of gene expression.

A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.35 kilobase pairs in length. In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed. The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.

The authors analyzed the heterogeneous distribution of hepatic argininosuccinate synthetase of type II citrullinemia in reference to its specificity and clinical implications. The low content of the enzyme in the liver of type II citrullinemic patients is associated with two kinds of the enzyme distribution that can be visualized by means of an immunohistochemical method (Saheki and colleagues. Biomed Res 1983;4:235-238). Among the 25 cases of type II citrullinemia examined, 11 exhibited homogeneous distribution of the enzyme, as in the control livers. On the other hand, 14 presented the clustered distribution, in which the hepatocytes stained positively with antisera to argininosuccinate synthetase formed a cluster among the poorly stained cells. No clustered distribution of the enzyme was present in the liver of control patients either with or without liver diseases. No clustered distribution of arginase and aldolase B was observed even in the liver of type II citrillinemic patients. These results suggest that clustered distribution is specific to argininosuccinate synthetase in the liver of type II citrullinemic patients. From considerations concerning the heterogeneous distribution of the enzyme and certain clinical parameters as well, the authors suggest that the clustered type in type II citrullinemia has a less favorable prognosis with regard to fatality.

Capacities for urea synthesis and amino acid patterns in the perfused livers isolated from rats fed low and high-protein diets were compared. Urea formation with amjonium chlorode as the nitrogen source in perfused livers isolated from rats fed on a 70% casein diet was rapid and the efficiency of conversion of ammonia to urea was 97.9%. However, that in livers isolated from rats fed on a 5% casein diet was much slower and the efficiency of conversion of ammonia to urea was only 36.1%. The ratios of the rate of urea formation from ammonium chloride to activity of ornithine transcarbamylase [EC 2.1.3.3.] in the perfused livers of rats fed on 5 and 70% casein diets were calculated. The ratio of the former condition was much lower than that of the latter. The ratios reached nearly the same level by the addition of ornithine and N-acetylglutamate, the addition of which to the perfusate caused marked elevation of the ratios in both cases. In the perfused livers from rats fed on a 5% casein diet a considerable portion of the ammonia added to the perfusate was fixed into an amino ro an amide group of amino acids such as alamin, aspartate, and glutamine. On the other hand, in the perfused livers from rats fed on a 70% casein diet most of the ammonia added was converted to urea. The regulation of urea synthesis and the relation between anabolism and catabolism of amino acids in rat livers subjected to different dietary conditions were compared.

The enzyme defects in two cases of argininosuccinic aciduria were examined at the molecular level by enzymatic and immunological methods. No argininosuccinate lyase activity was detected in the liver or erythrocytes of either patient nor in the kidney or brain of one of the patients even in the presence of high concentrations of the substrate. The titration curve of antiserum to human argininosuccinate lyase with the liver extract from a control subject was not affected by the addition of the liver extracts from one of the patients. Double immunodiffusion analysis revealed a single precipitin line between the purified antiserum and the liver extract from a control, but no precipitin lines between the antisera and the liver extracts from the two patients. These results indicate a complete or almost complete defect of an immunologically cross-reactive material in the liver of the patients.

Lipoamide dehydrogenase (LADase) was purified to homogeneity from rat liver mitochondria, and the intracellular distribution and biosynthesis of the LADase were investigated with antibody prepared against the purified enzyme. 1) LADase activity was mostly found in mitochondria; the activity in cytosol was about one-tenth of that in mitochondria. 2) LADase in the crude mitochondrial and cytosolic extracts and the purified LADase were immunologically identical as judged from the Ouchterlony double diffusion test. These LADases were indistinguishable from each other on immunochemical titration; i.e., the amount of LADase precipitated by a fixed amount of the anti-LADase antibody was the same for all the preparations. However, cytosolic LADase activity was inhibited by the antibody more strongly than mitochondrial LADase activity. 3) Two min after intravenous injection of [35S]methionine, more radioactivity was incorporated into cytosolic LADase than into the mitochondrial enzyme in the liver. This result suggests that localization of LADase in the cytosolic fraction is not an artifact due to leakage from mitochondria during homogenization of rat liver. 4) LADase was synthesized predominantly on free ribosomes, which indicates that LADase is synthesized on cytoplasmic ribosomes and translocated into mitochondria just as other mitochondrial proteins are. 5) After cell-free protein synthesis with post-mitochondrial supernatant, radioactivity immunoprecipitated with anti-LADase antibody was detected as a major peak with the same molecular weight as the purified LADase.

Antigen contents of ornithine transcarbamylase (OTC) protein in the liver of 5 patients with OTC deficiency and mutant mice were studied by ELISA. OTC activities in the liver tissues of 2 patients were 13.1% and 5.2% of the controls, respectively. The 2 patients' antigen contents of OTC protein were decreased in parallel with the enzyme activities. OTC antigen contents were not detected in the liver tissues of remaining 3 patients who had the complete type of OTC deficiency. Enzyme activities, kinetic properties and antigen contents of the OTC deficient mutant mice were the same as the results reported previously (Briand et al. 1982). The ELISA method for the assay of OTC antigen contents used in this study is more sensitive as compared with the radial immunodiffusion technique.

Human argininosuccinate synthetase (ASS) activity was found to be inactivated by alpha-dicarbonyls such as 1,2-cyclohexanedione and phenylglyoxal in accordance with pseudo first-order kinetics. The enzyme was almost completely protected from this inactivation by Mg-ATP and partially by its analogues. The strongest protective effect against inactivation was found with Mg-ATP, followed by Mg-ADP, AMP, adenosine and Mg-inorganic pyrophosphate. These results suggest the importance of arginine residue(s) for Mg-ATP binding. We determined the amino acid sequence of the peptide with the highest specific radioactivity derived from ASS which had been labeled with [14C]phenylglyoxal and then cleaved by cyanogen bromide treatment. The sequence obtained, PEFYNRFKGRNDLM, corresponds to residues 148-161 of the amino acid sequence deduced from the cDNA nucleotide sequence determined by Bock et al. [Nucleic Acids Res 11:6505-6512, 1983], and has a high homology with the sequences of ATP-binding sites proposed for several ATP-requiring enzymes.

One cDNA clone for lipoate acetyltransferase, a component enzyme of pyruvate dehydrogenase complex, was isolated from a rat liver cDNA library prepared in the phage expression vector lambda gt11 using immunological screening with affinity purified anti-lipoate acetyltransferase antibody. It was identified tha cDNA insert in this clone codes for lipoate acetyltransferase by immunoblotting of lysogen carrying the isolated clone. Lipoate acetyltransferase antigenic polypeptide in fusion protein was about 11,000 daltons, agreeing with the size of cDNA insert to be 300 base pairs.

Argininosuccinate lyase [EC 4.3.2.1] is an enzyme of the urea cycle in the liver of ureotelic animals. The enzymes of the urea cycle, including argininosuccinate lyase, are regulated developmentally and in response to dietary and hormonal changes, in a coordinated manner. The nucleotide sequence of rat argininosuccinate lyase cDNA, which was isolated previously (Amaya, Y., Kawamoto, S., Oda, T., Kuzumi, T., Saheki, T., Kimula, S., & Mori, M. (1986) Biochem. Int. 13, 433-438), was determined. The cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,549), a 5'-untranslated sequence of 150 bp, and a 3'-untranslated sequence of 41 bp. The amino acid composition of rat liver argininosuccinate lyase predicted from the cDNA sequence is in close agreement with that determined on the purified enzyme. The predicted amino acid sequences of the human and yeast enzymes along the entire sequences (94 and 39%, respectively), except for a region of 66 residues of the human enzyme near the COOH terminus. However, the sequence of this region of the human enzyme predicted from another reading frame of the human enzyme cDNA is homologous with the corresponding sequences of the rat and yeast enzymes. Therefore, the human sequence should be re-examined. Lysine-51, the putative binding site for argininosuccinate, and the flanking sequences are highly conserved among the rat, steer, human, and yeast enzymes.

Pyruvate dehydrogenase complex was purified from rat heart. A new component(mol.wt; 52,000) was found in the purified complex in addition to well known three component enzymes. This component(referred to as component X) was acetylated with [2-14C] pyruvate in the absence of CoA as well as lipoate acetyltransferase. The anti-lipoate acetyltransferase antibody reacted with component X and lipoate acetyltransferase, suggesting that component X shows homology with lipoate acetyltransferase in protein structure. cDNA for lipoate acetyltransferase was isolated from rat liver cDNA library in lambda gt 11. cDNA for lipoate acetyltransferase recognized two kinds of mRNAs of 3.5 Kb and 2.5 Kb.

Properties of argininosuccinate synthetases (ASS), including immunological properties and heat stability, from young and old rats were quite similar. However the degradation rates of ASS from young and old rats were found to be different as shown in the experiment using the double labeling technique. Three forms of this enzyme from young and old rat livers were separated by DEAE-Sephadex A-50 column chromatography, and they were called ASS 1, 2 and 3 in order of elution. The degradation rate of ASS 1 from young and old rat livers was very similar, but the rate of degradation of ASS 2 form old rat livers was greater than that from young rat livers.

We have developed a sensitive enzyme immunoassay (EIA) for the quantification of argininosuccinate synthetase (ASS) in the range of 0.05-1 ng/tube using a Fab'-peroxidase conjugate prepared with maleimide compound devised by Ishikawa et al. Amounts of hepatic ASS in control subjects were determined by this method with the purified enzyme as a standard and their specific activities were calculated to be 1.3-1.4 mumol product/min (U)/mg of ASS, which was quite similar to that of the purified enzyme. Amounts of ASS in the liver of patients with three types of citrullinemia, type I, II and III according to our arbitrary classification, were determined by the EIA method. The following results were obtained: hepatic ASS from a patient diagnosed as type I citrullinemia because of its abnormally large Km values for citrulline and aspartate was calculated to have a specific activity of 0.18 U/mg of ASS, indicating that the enzyme is a variant; specific activities of ASS in the hepatic extract of type II citrullinemic patients were 1.2-1.3 U/mg of ASS, confirming the finding obtained by single radial immunodiffusion method that the decrease of the activity in type II is caused by a decrease in the total amount of ASS protein; a very small amount of immune cross-reactive material was detected in the liver of a patient diagnosed as type III citrullinemia based on findings of undetectable ASS activity in the liver and cultured skin fibroblast.

Two siblings with hyperornithinemia, hyperammonemia, and homocitrullinuria are reported. The clinical picture included protein intolerance, mental retardation, seizures, and stuporous episodes. One patient had cerebellar ataxia, myoclonus, convulsive seizure, and muscular weakness in both legs. Isolated liver mitochondria in the patient revealed that ornithine transport and citrulline synthesis were decreased, but urea cycle enzymes and ornithine aminotransferase were normal. Ornithine metabolism was decreased in cultured skin fibroblasts.

A cDNA expression library constructed from poly(A)+ RNA of rat liver was screened immunologically using an antibody against argininosuccinate lyase (EC 4.3.2.1), a urea cycle enzyme, of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.5 kilobase pairs in length. In the bacterial clone, a specific protein of Mr = about 25,000 was expressed. The argininosuccinate lyase mRNA of about 2.1 kilobases long was detected in the liver and in a lesser amount in the kidney and spleen, but not in the small intestine and heart of the rats.

Urea synthesis was studied using the isolated liver perfusion with ammonium cholride and glutamine as nitrogen sources. The rate of urea formation increases with ammonium cholorde concentration up to 5mM, and the rate remained constant in the range between 5 and 20mM of ammonium chloride as the substrate. The concentration of ammonia in the medium to support the half-maximum velocity of urea formation was 0.7mM. The rate of urea formation was stimulated by the addition of 2.5mM ornithine, and the greater part of the ornithine which was taken up into the liver was accumulated as citrulline in the presence of ammonia. A considerable accelerating effect of N-acetylglutamate on the synthetic rate was observed, but a rather high concentration of N-acetylglutamate was required in order to obtain the maximum effect possibly, because its permeability into liver cells may be limited. A marked additive effect on the rate of urea formation was observed with the combined addition of ornithine and N-acetylglutamate. The metabolic conversion of glutamine nitrogen to urea in the perfused rat liver and the effect of several compounds which stimulated urea synthesis with ammonia were further examined. The process of conversion of glutamine nitrogen to urea might be composed of the following three steps. In the first lag phase, a small amount of glutamine was removed from the medium. In the second stage, the glutamine level decreased rapidly and ammonia was accumulated in the perfusate. The third stage was a period in which glutamine concentration remained at a constant low level, and the accumulated ammonia was rapidly conversed to urea. The rate of urea formation in this third stage was found to be much higher than that with ammonia as the substrate. The maximum rate of glutamine removal was obtained at pH 7.7 of the perfusate and at a concentration of 10mM glutamine. Urea formation with glutamine was also stimulated by the addition of ornithine, malate, or N-acetylglutamate, which had accelerating effects on the urea synthesis with ammonia. This stimulation was due to an effective conversion of ammonia to urea, but no change in the rate of removal glutamine was obtained.

Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.

The translation activity of mRNA coding for argininosuccinate synthetase in total RNA extracted from the liver of three patients with quantitative-type citrullinemia was determined using a cell-free translation system. In two patients, the hepatic content of the enzyme was about 20% of the control value, whereas translatable mRNA level for the enzyme was similar to or slightly lower than those of control livers. In the third patient, the enzyme content was about 50% of the control value, and mRNA activity for the enzyme was low normal. These results indicate that at least in the first two patients, the decrease in the enzyme protein is due either to increased degradation of the enzyme or to decreased translation in the patient's liver.