Carbamyl Phosphate Synthetase Activity Research Articles

Effects of fishmeal replacement with eight protein sources on growth performance, blood biochemistry and stress resistance in Opsariichthys bidens

Nine experimental diets were formulated to study the effects of fishmeal replacement on Opsariichthys bidens. Weight gain rate, protein efficiency rate and apparent digestibility coefficient of fish in fishmeal (FM), maggot meal (MM) and soybean meal (SM) groups were significantly the highest. There was no significant difference in intestinal amylase activity between FM and SM groups. Principal component analysis showed that SM group and wild fish (WF) individual had similar essential amino acid compositions in muscle. The lowest value of serum malondialdehyde and the highest value of total antioxidant capacity were found in SM group, but fish in MM group did the opposite. Fish in MM group had the higher whole-body lipid, serum triglycerides, glucose, complement C3, C4 and immunoglobulin contents and lysozyme activity. After ammonia exposure, cumulative mortality of fish in FM and SM groups was significantly the lowest. Liver carbamyl phosphate synthetase activity of fish in FM group was significantly the highest, but the highest values of glutamine synthetase and glutamate dehydrogenase were found in SM group. This study suggests that fishmeal replacement with soybean meal is acceptable, and muscle amino acid compositions of fish in SM group were more similar to wild individual.

Gene delivery corrects N-acetylglutamate synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-acetylglutamate synthase

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags−/−) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags−/− mice, established the dose of the vector needed to rescue Nags−/− mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags−/− mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags−/− mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

N-carbamylglutamate enhancement of ureagenesis leads to discovery of a novel deleterious mutation in a newly defined enhancer of the NAGS gene and to effective therapy

N-acetylglutamate synthase (NAGS) catalyzes the conversion of glutamate and acetyl-CoA to NAG, the essential allosteric activator of carbamyl phosphate synthetase I, the first urea cycle enzyme in mammals. A 17-year-old female with recurrent hyperammonemia attacks, the cause of which remained undiagnosed for 8 years in spite of multiple molecular and biochemical investigations, showed markedly enhanced ureagenesis (measured by isotope incorporation) in response to N-carbamylglutamate (NCG). This led to sequencing of the regulatory regions of the NAGS gene and identification of a deleterious single-base substitution in the upstream enhancer. The homozygous mutation (c.-3064C>A), affecting a highly conserved nucleotide within the hepatic nuclear factor 1 (HNF-1) binding site, was not found in single nucleotide polymorphism databases and in a screen of 1,086 alleles from a diverse population. Functional assays demonstrated that this mutation decreases transcription and binding of HNF-1 to the NAGS gene, while a consensus HNF-1 binding sequence enhances binding to HNF-1 and increases transcription. Oral daily NCG therapy restored ureagenesis in this patient, normalizing her biochemical markers, and allowing discontinuation of alternate pathway therapy and normalization of her diet with no recurrence of hyperammonemia. Inc.

先天性高アンモニア血症の1例Carbamyl phosphate synthetase低下を伴えるOrnithine transcarbamylase欠損症

先天性高アンモニア血症の1例Carbamyl phosphate synthetase低下を伴えるOrnithine transcarbamylase欠損症

Favourable long-term outcome after immediate treatment of neonatal hyperammonemia due to N-acetylglutamate synthase deficiency

N-Acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder, which may present in the neonatal period with severe hyperammonemia and marked neurological impairment. We report on a Turkish family with a patient who died due to hyperammonemia in the neonatal period. Reduced activity of NAGS and carbamyl phosphate synthetase were found at autopsy. A second child who developed hyperammonemia on the second day of life was immediately treated with arginine hydrochloride, sodium benzoate and protein restriction. After NAGS deficiency was suspected by enzyme analysis, sodium benzoate was replaced by N-carbamylglutamate (NCG). A third child who developed slight hyperammonemia on the third day of life was treated with NCG before enzyme analysis confirmed reduced NAGS activity. Neither of the patients developed hyperammonemia in the following years. After the human NAGS gene was identified, mutation analysis revealed that the older sibling on NCG therapy was homozygous for a 971G>A (W324X) mutation. The parents and the younger sibling were heterozygous. Therapy was continued in the older sibling until now without any adverse effects and favourable neurodevelopment outcome. In the younger sibling, therapy was stopped without any deterioration of urea cycle function. NAGS deficiency can be successfully treated with NCG and arginine hydrochloride with favourable outcome. Molecular diagnostic rather than enzyme analysis should be used in patients with suspected NAGS deficiency.

Carglumic acid: an additional therapy in the treatment of organic acidurias with hyperammonemia?

BackgroundHyperammonemia in patients with methylmalonic aciduria (MMA) and propionic aciduria (PA) is caused by accumulation of propionyl-CoA which decreases the synthesis of N-acetyl-glutamate, the natural activator of carbamyl phosphate synthetase 1. A treatment approach with carglumic acid, the structural analogue of N-acetyl-glutamate, has been proposed to decrease high ammonia levels encountered in MMA and PA crises.Case presentationWe described two patients (one with MMA and one with PA) with hyperammonemia at diagnosis. Carglumic acid, when associated with standard treatment of organic acidurias, may be helpful in normalizing the ammonia level.ConclusionEven though the usual treatment which decreases toxic metabolites remains the standard, carglumic acid could be helpful in lowering plasma ammonia levels over 400 micromol/L more rapidly.

A novel bifunctional N-acetylglutamate synthase-kinase from Xanthomonas campestris that is closely related to mammalian N-acetylglutamate synthase

BackgroundIn microorganisms and plants, the first two reactions of arginine biosynthesis are catalyzed by N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK). In mammals, NAGS produces an essential activator of carbamylphosphate synthetase I, the first enzyme of the urea cycle, and no functional NAGK homolog has been found. Unlike the other urea cycle enzymes, whose bacterial counterparts could be readily identified by their sequence conservation with arginine biosynthetic enzymes, mammalian NAGS gene was very divergent, making it the last urea cycle gene to be discovered. Limited sequence similarity between E. coli NAGS and fungal NAGK suggests that bacterial and eukaryotic NAGS, and fungal NAGK arose from the fusion of genes encoding an ancestral NAGK (argB) and an acetyltransferase. However, mammalian NAGS no longer retains any NAGK catalytic activity.ResultsWe identified a novel bifunctional N-acetylglutamate synthase and kinase (NAGS-K) in the Xanthomonadales order of gamma-proteobacteria that appears to resemble this postulated primordial fusion protein. Phylogenetic analysis indicated that xanthomonad NAGS-K is more closely related to mammalian NAGS than to other bacterial NAGS. We cloned the NAGS-K gene from Xanthomonas campestis, and characterized the recombinant NAGS-K protein. Mammalian NAGS and its bacterial homolog have similar affinities for substrates acetyl coenzyme A and glutamate as well as for their allosteric regulator arginine.ConclusionThe close phylogenetic relationship and similar biochemical properties of xanthomonad NAGS-K and mammalian NAGS suggest that we have identified a close relative to the bacterial antecedent of mammalian NAGS and that the enzyme from X. campestris could become a good model for mammalian NAGS in structural, biochemical and biophysical studies.

Identification of novel mutations of the human N-acetylglutamate synthase gene and their functional investigation by expression studies

The mitochondrial enzyme N-acetylglutamate synthase (NAGS) produces N-acetylglutamate serving as an allosteric activator of carbamylphosphate synthetase 1, the first enzyme of the urea cycle. Autosomal recessively inherited NAGS deficiency (NAGSD) leads to severe neonatal or late-onset hyperammonemia. To date few patients have been described and the gene involved was described only recently. In this study, another three families affected by NAGSD were analyzed for NAGS gene mutations resulting in the identification of three novel missense mutations (C200R [c.598T > C], S410P [c.1228T > C], A518T [c.1552G > A]). In order to investigate the effects of these three and two additional previously published missense mutations on enzyme activity, the mutated proteins were overexpressed in a bacterial expression system using the NAGS deficient E. coli strain NK5992. All mutated proteins showed a severe decrease in enzyme activity providing evidence for the disease-causing nature of the mutations. In addition, we expressed the full-length NAGS wild type protein including the mitochondrial leading sequence, the mature protein as well as a highly conserved core protein. NAGS activity was detected in all three recombinant proteins but varied regarding activity levels and response to stimulation by l-arginine. In conclusion, overexpression of wild type and mutated NAGS proteins in E. coli provides a suitable tool for functional analysis of NAGS deficiency.

Continuous Renal Replacement Therapy in the Initial Management of Neonatal Hyperammonemia Due to Urea Cycle Defects

After completing this article, readers should be able to: 1. Describe the metabolic acidosis that may be seen in urea cycle defects. 2. Delineate the most important factor in determining neurologic outcome in urea cycle defects. 3. Explain the factors that affect the blood ammonia concentration in continuous renal replacement therapy (CRRT). 4. Delineate the most difficult issue in pursuing CRRT in neonates. 5. Describe the morbidities associated with CRRT. MP was born at 39 weeks' estimated gestational age by vaginal delivery to a 35-year-old gravida 3 para 2 woman who had an uncomplicated pregnancy. The 3- and 6-year-old female siblings of the patient were healthy. Apgar scores were 8 at 1 minute and 9 at 5 minutes. The newborn was discharged to home after a 2-day stay in the well baby nursery during which time she appeared to be alert and was breastfeeding appropriately. She presented to the emergency department less than 1 day later after her parents noted increasing lethargy and very poor feeding at home. She was admitted to the neonatal intensive care unit (NICU). Results of a complete blood count with differential count and C-reactive protein evaluation were unremarkable, but the patient progressed to worsening lethargy, respiratory failure, and abdominal distension requiring intubation within hours of admission. The anterior fontanelle was soft on admission physical examination. Further testing revealed a base deficit of 6.1 on arterial blood gas, serum ammonia level of 810 mcmol/L (1,134 mcg/dL), serum lactate level of 5.6 mmol/L (5.6 mEq/L), and serum pyruvate level of 0.06 mmol/L. Results of liver function tests were abnormal, and the infant had a prothrombin time of 35.9 seconds, a partial thromboplastin time of 54.3 seconds, and a fibrinogen concentration of 1.75 g/L (175 mg/dL). Genetic and nephrology consultations were requested immediately. Additional metabolic studies were obtained, including measurement of plasma amino …

Glutamate, at the Interface between Amino Acid and Carbohydrate Metabolism

The liver is the major site of gluconeogenesis, the major organ of amino acid catabolism and the only organ with a complete urea cycle. These metabolic capabilities are related, and these relationships are best exemplified by an examination of the disposal of the daily protein load. Adults, ingesting a typical Western diet, will consume approximately 100 g protein/d; the great bulk of this is metabolized by the liver. Although textbooks suggest that these amino acids are oxidized in the liver, total oxidation cannot occur within the confines of hepatic oxygen uptake and ATP homeostasis. Rather, most amino acids are oxidized only partially in the liver, with the bulk of their carbon skeleton being converted to glucose. The nitrogen is converted to urea and, to a lesser extent, to glutamine. The integration of the urea cycle with gluconeogenesis ensures that the bulk of the reducing power (NADH) required in the cytosol for gluconeogenesis can be provided by ancillary reactions of the urea cycle. Glutamate is at the center of these metabolic events for three reasons. First, through the well-described transdeamination system involving aminotransferases and glutamate dehydrogenase, glutamate plays a key catalytic role in the removal of alpha-amino nitrogen from amino acids. Second, the "glutamate family" of amino acids (arginine, ornithine, proline, histidine and glutamine) require the conversion of these amino acids to glutamate for their metabolic disposal. Third, glutamate serves as substrate for the synthesis of N-acetylglutamate, an essential allosteric activator of carbamyl phosphate synthetase I, a key regulatory enzyme in the urea cycle.

Types of carbamyl phosphate synthetases and subcellular localization of urea cycle and related enzymes in air-breathing walking catfish,Clarias batrachus

The types of carbamyl phosphate synthetase (CPS), the subcellular localization of urea cycle enzymes and glutamine synthetase (GS) in liver and kidney (potential ureogenic tissues) and also the possible involvement of some extra-hepatic tissues for urea synthesis via the urea cycle were studied in an Indian ureogenic amphibious air-breathing walking catfish, Clarias batrachus. Mitochondrial CPS III [glutamine- and N-acetyl-L-glutamate (NAG)-dependent] and cytosolic CPS II (glutamine-dependent) activities were found to be present in liver, analogous to that described for two other teleosts that have CPS III activity. The same activities and subcellular localization were found in kidney. Unexpectedly, a CPS I-like (ammonia- and NAG-dependent) activity was found to be present at levels higher than the CPS III activity in the mitochondrial fraction of both liver and kidney analogous to another Indian amphibious air-breathing teleost fish, Heteropneustes fossilis. The two other urea cycle enzymes, ornithine transcarbamylase (OTC) and arginase (ARG), and GS were found to be localized primarily in the mitochondria, and argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) in the cytosol analogous to H. Fossilis and elasmobranchs. Significant levels of urea cycle enzymes along with GS activity could also be detected in muscle, intestine and brain tissues, except ASL in muscle and ASS in brain. Our results also indicated that probably all three different types of CPS activities are also expressed in all three extra-hepatic tissues. The urea cycle–related CPS III found in invertebrates and fish is considered to be the evolutionary precursor of the urea cycle–related CPS I in ureotelic mammalian and amphibian species. Whether or not the CPS I-like activity reported here (1) is due to the presence of a separate CPS I gene in addition to a CPS III gene or (2) represents an adapted CPS III activity in C. batrachus, these results suggest that the presence of both CPS I-like and CPS III activities along with the presence of urea cycle enzymes in extra-hepatic tissues may play an important physiological adaptive role for survival of these walking catfish in higher ambient ammonia or living in air or while burrowing inside mud during habitat drying. J. Exp. Zool. 283:121–130, 1999. © 1999 Wiley-Liss, Inc.

Allosteric regulation and substrate channeling in multifunctional pyrimidine biosynthetic complexes: analysis of isolated domains and yeast-mammalian chimeric proteins

The initial steps of pyrimidine biosynthesis in yeast and mammals are catalyzed by large multifunctional proteins of similar size, sequence and domain structure, but appreciable functional differences. The mammalian protein, CAD, has carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities. The yeast protein, ura2, catalyzes the first two reactions and has a domain, called pDHO, which is homologous to mammalian DHOase, but is inactive. In CAD, only CPSase is regulated, whereas both CPSase and ATCase in the yeast protein are inhibited by UTP. These functional differences were explored by constructing a series of mammalian yeast chimeras. The isolated ATCase domain is catalytically active, but is not regulated. The inclusion of the yeast sequences homologous to the mammalian regulatory domain (B3) and the intervening pDHO domain did not confer regulation. Chimeric proteins in which the homologous regions of the mammalian protein were replaced by the corresponding domains of ura2 exhibited full catalytic activity, as well regulation of the CPSase, but not the ATCase, activities. The yeast B3 subdomain confers UTP sensitivity on the mammalian CPSase, suggesting that it is the locus of CPSase regulation in ura2. Taken together, these results indicate that there are regulatory site(s) in ura2. Channeling is impaired in all the chimeric complexes and completely abolished in the chimera in which the pDHO domain of yeast is replaced by the mammalian DHO domain.

In vivo Mutational Analysis of Highly Conserved Amino Acid Residues of the Small Subunit Cpa1p of the Carbamylphosphate Synthetase ofSaccharomyces cerevisiae

The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site-directed mutagenesis to change all residues to aspartic acid. Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo. The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate. These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site. Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS. C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mM). The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mM, suggesting that this substitution is critical to NH3-dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme.

Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains.

Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.

Dietary Calorie Restriction in Mice Induces Carbamyl Phosphate Synthetase I Gene Transcription Tissue Specifically

Dietary calorie restriction (CR) delays age-related physiologic changes, increases maximum life span, and reduces cancer incidence. Here, we present the novel finding that chronic reduction of dietary calories by 50% without changing the intake of dietary protein induced the activity of mouse hepatic carbamyl phosphate synthetase I (CpsI) 5-fold. In liver, CpsI protein, mRNA, and gene transcription were each stimulated by approximately 3-fold. Thus, CR increased both the rate of gene transcription and the specific activity of the enzyme. Short-term feeding studies demonstrated that higher cpsI expression was due to CR and not consumption of more dietary protein. Intestinal CpsI activity was stimulated 2-fold, while its mRNA level did not change, suggesting enzyme activity or translation efficiency was stimulated. CpsI catalyzes the conversion of metabolic ammonia to carbamyl phosphate, the rate-limiting step in urea biosynthesis. cpsI induction suggests there is a shift in the metabolism of calorie-restricted animals toward protein catabolism. CpsI induction likely facilitates metabolic detoxification of ammonia, a strong neurotoxin. Enhanced protein turnover and metabolic detoxification may extend life span. Physiologic similarities between calorie-restricted and hibernating animals suggest the effects of CR may be part of a spectrum of adaptive responses that include hibernation.

De novo pyrimidine nucleotide biosynthesis in synchronized Chinese hamster ovary cells.

Cellular requirements for pyrimidine nucleotides are fulfilled through the de novo or salvage biosynthetic pathways. In the de novo biosynthetic pathway, UMP, the first major end-product of the pathway, is synthesized from bicarbonate, glutamine, ATP, aspartate and phosphoribosyl pyrophosphate catalyzed by six major enzymes: carbamyl phosphate synthetase, aspartate transcarbamylase, dihydroorotase, didhydroorotate dehydrogenase, orotate phosphoribosyl transferase and orotidylate decarboxylase1. Mitchell and Hoogenraad2 studied de novo pyrimidine biosynthesis in synchronized rat hepatoma and mouse embryo fibroblast (3T3) cells. They reported that the carbamyl phosphate synthetase and aspartate transcarbamylase enzyme activities and the incorporation of [14 C] bicarbonate into acid soluble pyrimidine nucleotides and RNA were high during the S phase of the cell cycle and low during the G2 and M phases.

Expressing enzymatic domains of hamster CAD in CAD-deficient Chinese hamster ovary cells.

The first three enzymes of de novo pyrimidine biosynthesis, carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase), are domains of a single Polypeptide (called CAD) in species from Dictyostelium to humans. One approach to demonstrate the independence of the enzymatic domains of CAD has been to express portions of the hamster CAD cDNA in E. coli defective in synthesizing pyrimidines de novo. For example, the CAD sequence encoding the ATCase domain cloned into a bacterial expression vector was transformed into ATCase-deficient E. coli (Maley and Davidson, 1988). The transformants were able to grow in the absence of uracil and contained hamster protein with ATCase activity. This system is proving to be a powerful strategy for studying the structure and evolution of the enzymatic domains of CAD (e.g. Musmanno et al., 1991; Zimmermann and Evans, 1993; Williams et al., 1993). However, in order to study the significance of CAD structure to pyrimidine metabolism in mammals, it may be helpful to express the enzymatic domains of CAD separately in a hamster cell line deficient in CAD. This was first achieved with the successful expression of the CPSase domain in a CAD-deficient hamster cell line (Musmanno et al., 1992). In this report the ATCase and DHOase domains are expressed as separate proteins in a CAD-deficient cell line, and the application of this approach in generating a carbamyl phosphate synthetase activity unresponsive to feedback inhibition by UTP is described.

Ｃａｒｂａｍｙｌｐｈｏｓｐｈａｔｅ ｓｙｎｔｈｅｔａｓｅ活性の低下を伴い多彩な併発症を示した成人型シトルリン血症の１例

Carbamylphosphate synthetase (CPS)活性の低下を伴い多彩な併発症を示した成人型シトルリン血症を経験したので報告する.症例は31歳女性で,全身倦怠感を主訴に当院受診.ネフローゼ症候群,高脂血症の診断で入院した.入院中に異常行動,意識障害を認め,検索の結果著明な高アンモニア血症,高シトルリン血症,さらに原発性甲状腺機能低下症が認められた.肝生検組織を用い尿素サイクル酵素活性を測定したところ,argininosuccinate synthetase (ASS)活性は正常対照の9.2%に,CPS活性は同じく25%に低下していた.一方ornithine transcarbamylase (OTC)活性及び,argininosuccinate lyase (ASL)活性は正常範囲内であった.組織学的に脂肪肝の像を認めたが,尿素サイクルの酵素活性を低下させるまでの肝硬変の所見はなく,同時に測定したFructose-1,6-diphosphate (FDP), Fructose-1-phosphate (F1P) aldolase活性及びその比は正常であった.以上のことから本症例はCPS活性低下を伴ったシトルリン血症と考えられた.本症例で認められた多彩な病態と,この複合型尿素サイクル酵素活性低下との関連については現時点では不明であり,今後の検討を要した.

Use of Placental Cord Serum in Cultures of Human Fetal Hepatocytes: Its Assessment with Carbamyl Phosphate Synthetase and Argininosuccinate Lyase as Marker Enzymes.

Isolated human fetal hepatocytes could be successfully cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with placental cord serum, which is a rich source of various growth factors. These cultured hepatocytes were found to maintain their viability, morphological integrity, and detoxifying capacity. The expression of two urea-cycle enzymes, i.e., carbamyl phosphate synthetase (CPS) and argininosuccinate lyase (ASL), was studied in isolated and cultured hepatocytes from human fetuses of different gestational age classified as Group I: 17-20 weeks, Group II: 21-24 weeks, Group III: 25-28 weeks, Group IV: 29-32 weeks, and Group V: 33-36 weeks. We found that on culture the activities of the two enzymes increased for a period of 4-6 days. Hepatocytes of Group I and Group II showed maximum expression of CPS by the 6th day of culture, whereas those of Groups III, IV, and V showed theirs by the 5th day of culture. In the case of ASL the maximum levels were seen by the 4th day of culture, after which the activities of both enzymes decreased. CPS activity in cultured hepatocytes was about 1.4-4.7 times higher whereas that of ASL was about 1.2-1.6 times higher, when compared with that of freshly isolated cells.This study indicates that isolated hepatocytes undergo reversible changes and that on culture for 4-6 days, liver cell functions recover with enhanced enzyme activities. Cultured human fetal hepatocytes of Group III gestation would appear to be more suitable for transplantation than isolated hepatocytes.

Changes in Liver Concentration of N-Acetylglutamate and Ornithine are Involved in Regulating Urea Synthesis in Rats Treated with Thyroid Hormone

The purpose of this study was to elucidate the mechanism by which thyroid hormone alters urea synthesis. A set of three experiments was investigated in three groups of rats given 6-propyl-2-thiouracil (a thyroid inhibitor) without triiodothyronine treatment, treated with 6-propyl-2-thiouracil plus triiodothyronine or neither 6-propyl-2-thiouracil nor triiodothyronine (control). We attempted to determine whether the concentration of ornithine and N-acetylglutamate regulated urea synthesis and whether activities of two ornithine-catabolizing enzymes accounted for changes in ornithine concentration. Urinary excretion of urea and the liver concentration of N-acetylglutamate and ornithine in rats given 6-propyl-2-thiouracil plus triiodothyronine were significantly lower than in rats given 6-propyl-2-thiouracil alone. The liver concentration of N-acetylglutamate was correlated to urea excretion (r = 0.911, P < 0.001). The activities of carbamylphosphate synthetase, ornithine aminotransferase and ornithine decarboxylase in liver of the group treated with 6-propyl-2-thiouracil alone were significantly lower than those of the 6-propyl-2-thiouracil plus triiodothyronine-treated group. The results suggest that a higher liver concentration of N-acetylglutamate and ornithine in the hypothyroid (6-propyl-2-thiouracil only) rats is likely to stimulate urea synthesis. The thyroid hormone-induced increase in activities of ornithine catabolizing enzymes may be primarily responsible for changes in ornithine concentration.