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Urea Cycle Enzymopathies

R. Butterworth

1 2 2.1 2.2 2.3 3 4 4.1 4.2 4.3 5 5.1 5.2 5.3 5.4 5.5

Experimental Urea Cycle Enzymopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Neurotransmitter Changes in OTC Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 PeripheralType (Mitochondrial) Benzodiazepine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 The Cholinergic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Serotonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 Pathogenesis of Neuronal Cell Death in Congenital OTC Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Brain Energy Deficit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 NMDA ReceptorMediated Excitotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Current Therapy in Congenital OTC Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Sodium Benzoate, Phenylacetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 LCarnitine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 AcetylLCarnitine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Liver Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Springer-Verlag Berlin Heidelberg 2007

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Abstract: Inborn errors of urea cycle enzymes and transporters related to the urea cycle have been described. Ornithine transcarbamylase (OTC) deciency, an X-linked genetic disorder is well characterised from the molecular genetic standpoint and over 100 mutations have been described. Clinical symptomatology varies according to the residual enzyme activity and may include seizures, mental retardation and cerebral palsy. Neuropathologic evaluation reveals brain atrophy, ventricular dilatation and delayed myelination. A well characterised animal model of OTC deciency, the sparse fur (spf ) mouse has been employed to study the neurochemistry of the disorder; abnormalities of the glutamatergic, cholinergic and serotoninergic neurotransmitter systems have been identied. Studies on the pathogenesis of the neuronal cell death in congenital OTC deciency suggest that cerebral energy compromise and NMDA receptormediated excitotoxicity are implicated. Current therapy in congenital urea cycle disorders involves the reduction of circulating ammonia (using agents such as sodium benzoate and phenylacetate), L-carnitine administration to improve cellular energy metabolism and liver transplantation. Clinical trials using gene therapy are currently under evaluation. List of Abbreviations: ATP, adenosine triphosphate; KGOH, -ketoglutarate dehydrogenase; CP, candate-puramen; CPSase, carbamyl phosphate synthetase; CSF, cerebrospinal uid; CAT, choline actyltransferase; CoA, coenzyme A; CT, computed tomographic; FC, frontal cortex; GABA, -aminobutyric acid; GP, globus pallidus; 5HIAA, 5-hydroxyindoleacetic acid; MAO, monoamine oxidase; NMDA, N-methyl-Daspartate; NOS, nitric oxide synthase; OTC, ornithine transcarbamylase; OLT, orthotopic liver transplantation; PC, parietal cortex; 5HT, serotonin; spf, sparse fur The major pathway for ammonia detoxication in mammalian systems depends on its conversion to urea in the liver by a series of enzymatic reactions shown schematically in > Figure 111. Although a complete urea cycle is only expressed in the liver, other tissues including the brain may express some of the constituent enzymes (> Table 111). Inborn errors of six constituent enzymes and two transporters related to the urea cycle have been described (Brusilow and Horwich, 1995). The prevalence of these disorders in the USA is approximately 1 in 8,000. All congenital disorders of the urea cycle are inherited as autosomal
. Figure 111 Schematic representation of the urea cycle showing intra and extramitochondrial localization of constituent enzymes, their substrate cofactors, and the ornithine transporter

Urea cycle enzymopathies . Table 111 Constituent enzymes of the urea cyclea Enzyme Argininosuccinate synthetase EC 6.3.4.5 Arginosuccinate lyase EC 4.3.2.1 Arginase EC 3.5.3.1 Nacetylglutamate synthetase EC 2.3.11 Carbamyl phosphate synthetase EC 6.3.4.16 Ornithine transcarbamylase EC 2.1.3.3
a

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Compartment Cytoplasm Cytoplasm Cytoplasm Mitochondrial matrix Mitochondrial matrix Mitochondrial matrix

Activity (mmol/h/g) 90 220 86,600 0.301.49 279 6,600

Tissue distribution Liver, kidney, brain (trace) Liver, kidney Liver, kidney, brain (trace) Liver, intestine Liver, intestine Liver, intestine

Enzyme activity is expressed as micromoles per hour per gram wet weight for human liver

recessive traits with the exception of ornithine transcarbamylase (OTC) deciency, which is Xlinked. OTC deciency is the best studied of the urea cycle disorders from the point of view of molecular genetics, pathophysiology, and treatment. OTC is a mitochondrial enzyme that catalyzes the conversion of ornithine and carbamyl phosphate to citrulline (> Figure 111). More than 100 mutations have been described in families with OTC deciency where large deletions are encountered in 8% of patients, and small deletions/insertions in a further 10%. The remaining mutations are single-base substitutions (Tuchman et al., 1996). Many hemizygous males die (presumably) from severe ammonia intoxication during the neonatal period. Heterozygous females manifest a variable clinical course (Brusilow and Horwich, 1995). A recent study in a 2yearold female patient revealed OTC deciency presenting with strokelike episodes (Christodoulou et al., 1993). Depending on the residual enzyme activity and the degree of hyperammonemia, infants and children with congenital OTC deciency present with a spectrum of clinical symptoms, including failure to thrive, lethargy, and hypotonia. Seizures are not uncommon and a large percentage of survivors go on to manifest severe developmental disabilities, including mental retardation and cerebral palsy (Msall et al., 1984). Neuropathology in congenital OTC deciency consists of atrophy, cystic degeneration, ventricular dilatation, delayed myelination, and Alzheimer type II astrocytosis (Harding et al., 1984; Dolman et al., 1988; Harper and Butterworth, 1997). Cerebral edema sufcient to lead to intracranial hypertension and brain herniation has been described in congenital OTC deciency (Kendall et al., 1983). Computed tomographic (CT) abnormalities were reported in the brain of a 2yearold patient with a residual liver OTC activity of 7% (Takayanagi et al., 1984). Such abnormalities included diffuse hypointensities in frontal and parietal lobes. Followup CT scanning showed bilateral ventricular dilatation and cerebral atrophy. It has repeatedly been suggested that such neuropathologic damage in congenital OTC deciency may be acquired in utero (Harding et al., 1984; Filloux et al., 1986). Deciency of carbamyl phosphate synthetase1 (CPSase1) results (in addition to hyperammonemia) in increases of plasma glutamine and alanine with low to absent citrulline and arginine. CPSase1 deciency results in high mortality; survivors have a high prevalence of mental retardation and suffer frequent hyperammonemic crises during intercurrent illness or catabolic stress. Citrullinemia results from arginosuccinate synthase deciency.

Experimental Urea Cycle Enzymopathies

Experimental animal models of congenital urea cycle disorders have been described; some are spontaneous mutants while others have appeared with the advent of gene targeting paradigms. By far the most widely

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used and best characterized model of a congenital urea cycle disorder is the sparsefur (spf ) mouse with a defect in OTC. The Xlinked spf mutation arose spontaneously in the progeny of an irradiated male mouse, at Oakridge Laboratories, USA, maintained on various genetic backgrounds (Demars et al., 1976; Qureshi, 1992). Young spf/Y males are characterized by their small size, relatively sparse fur, and wrinkled skin traits that are visible as early as 7 days postnatally. The spf mouse harbors a structural mutation in the OTC molecule consisting of a single point substitution mutation of a cytosine to adenine within the coding region of OTC cDNA, which results in a change of amino acid 117 from a histidine to an arginine residue (Veres et al., 1987). This mutation affects the pH dependence of the enzyme and limits the enzymes capacity to form citrulline from carbamyl phosphate and ornithine. Residual OTC activities in the livers of spf/Y mice are in the 1015% range (Qureshi, 1992). Brain ammonia concentrations are increased in the spf mutant compared to littermate controls (> Table 112) (Batshaw et al., 1988; Ratnakumari et al., 1992) and,
. Table 112 Hepatic activities of OTC, brain ammonia, and amino acid concentrations in spf (OTC-decient) mice Hepatic OTC activity (mmol/min/mg) Brain Ammonia (mmol/g) Brain Amino acids Ornithine (nmol/g) Citrulline (nmol/g) Arginine (nmol/g) Glutamine (mmol/g) Glutamate (mmol/g)
*p < 0.01 compared to control values

Control 64.2 4.0 1.41 0.05 11.7 3.1 12.0 0.3 128.0 3.1 3.93 0.7 8.78 1.0

OTC decient 6.3 0.8* 2.85 0.16* 6.9 0.7* 8.0 0.1* 78.0 7.5* 10.11 1.3* 5.91 0.8*

since glutamine synthesis is the major route for ammonia removal by brain, glutamine concentrations are also increased (Batshaw et al., 1988; Ratnakumari et al., 1994a). Increased brain glutamine concentrations have also been reported using magnetic resonance spectroscopy in children with OTC deciency (Connelly et al., 1993).

Neurotransmitter Changes in OTC Deciency

2.1 Amino Acids

Glutamate concentrations in cerebral cortex of spf mice are signicantly reduced (> Table 112) (Ratnakumari et al., 1994a), whereas concentrations of aspartate are increased in several brain regions of these animals. GABA concentrations, although unchanged in cerebral cortex of spf mice, are signicantly increased in striatum, hippocampus, and midbrain, and it was suggested that these increases reect increased ux through the GABA shunt pathway (Ratnakumari et al., 1994a). However, activities of the GABA synthetic enzyme, glutamic acid decarboxylase, are unchanged (Ratnakumari et al., 1993a). Glutamate (NMDA)-binding sites were evaluated using the selective ligand [3H]MK801 in the brains of spf mice. [3H]MK801 sites were reduced in density by up to 70% in several brain regions (Ratnakumari et al., 1995b) (> Figure 112). Whether these changes result from a loss of neurons expressing NMDA receptors or are the consequence of downregulation as a result of increased exposure to endogenous ligands (such as glutamate or quinolinate) is unclear, but it was suggested that the loss of NMDA receptor sites in congenital OTC deciency could represent an adaptive mechanism of protection against further excitotoxic injury (Ratnakumari et al., 1995b). More recently it has been suggested that NMDA sites may also be expressed by

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. Figure 112 Regional distribution of [3H]-MK801binding sites in the brains of control () and OTCdecient ( ) mice. Values represent mean S.E. of data from ve animals per group. Values signicantly differ from control indicated by *p < 0.01 by Students ttest. Abbreviations: FC, frontal cortex; PC, parietal cortex; CP, caudateputamen; GP, globus pallidus; CA1, CA2, CA3: regions of hippocampus

astrocytes suggesting that the loss of [3H]MK801 sites could also be the consequence of astrocytic changes in OTC deciency.

2.2 PeripheralType (Mitochondrial) Benzodiazepine Receptors

Peripheraltype benzodiazepine receptors (PTBRs) are localized on the outer mitochondrial membrane of peripheral tissues and brain where they are predominantly astrocytic in localization. Using [3H] PK11195, a radioligand with high selectivity for the PTBR, these sites were assessed in brains and peripheral tissues of spf mice. Densities of [3H]PK11195 binding sites were signicantly increased in brain, kidney, liver, and testes of OTCdecient animals (Raghavendra Rao et al., 1993), and it was suggested that these increases were the result of exposure to increased ammonia concentrations. Increased expression of PTBRs in brain could also be the result of reactive astrocytosis following neuronal cell loss in congenital OTC deciency since reactive astrocytes are known to express increased quantities of PTBRs. However, this explanation is unlikely since PTBR increases were particularly apparent in the brainstem, a brain region which shows relatively little loss of neurons in spf mice brains. More likely, increased densities of PTBRs reect astrocytic changes resulting from exposure to ammonia. Putative roles for the PTBR in brain include oxidative metabolism (Anholt, 1986) and in the synthesis of neurosteroids, some of which have positive allosteric modulatory properties at GABAA receptors (Simmonds, 1991). Activation of PTBRs in the brain in OTC deciency, therefore, could indirectly result in increased GABAergic neurotransmission.

2.3 The Cholinergic System

Ratnakumari et al. (1994b, 1995a, 1996a) performed a series of studies of the cholinergic system in OTC deciency. Activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were measured in the brains of spf/Y mice and CDl/Y controls. CAT activities were found to be reduced in frontal cortex, striatum, hippocampus, thalamus, and brainstem of spf mice (> Figure 113), and CATpositive neurons were reduced in number in cerebral cortex, septal area, and diagonal band of these animals (Ratnakumari

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. Figure 113 Choline acetyltransferase activities in the brain regions of control () and congenitally hyperammonemic ( ) spf/Y mice. FC, frontal cortex; CS, striatum; HC, hippocampus. Each value represents mean S.E. of triplicate determinations in ve animals per group. Signicant differences between data from spf/Y and control are indicated by *p < 0.05, **p < 0.01 by unpaired Students ttest

et al., 1994b). These ndings are consistent with a loss of forebrain cholinergic neurons in OTC deciency. In a followup study conducted at various times during postnatal development, CAT activities were found to be reduced as early as 35 days postnatally in the brainstem of spf mice and highafnity [3H]choline uptake by cerebral cortical synaptosomes from spf mice was signicantly reduced as early as 21 days postnatally (Ratnakumari et al., 1995a). Subsequent studies of muscarinic cholinergic M1- and M2-binding sites in the brains of spf mice using quantitative receptor autoradiography revealed up to 54% increased binding sites for the M1 receptor ligand [3H]pirenzepine and a concomitant loss of binding sites for the M2 receptor ligand [3H]AFDX384 (Ratnakumari et al., 1996a). Since the M1 sites are predominantly postsynaptic and the M2 sites presynaptic in localization, signicant reductions of M2 sites and concomitant increases of M1 sites are, in the light of the previous nding of a loss of the cholinergic nerve terminal enzyme CAT, conrmative of a selective loss of cholinergic neurons in this model of congenital OTC deciency. The increase in binding-site densities for the postsynaptic M1 ligand [3H]pirenzepine in brain regions of spf mice could be the consequence of receptor upregulation following loss of presynaptic neurons, which is similar to ndings in autopsied brain tissues from patients with Alzheimers disease (Arauja et al., 1988). Basal forebrain cholinergic neuronal loss could readily explain the severe cognitive dysfunction characteristic of congenital OTC deciency (Gropman and Batshaw, 2004).

Serotonin

Increased brain concentrations of the serotonin metabolite 5hydroxyindoleacetic acid (5HIAA) have been reported in the brains of spf mice (Bachmann and Colombo, 1984) and in the cerebrospinal uid (CSF) of children with congenital hyperammonemia (Hyman et al., 1988). Concentrations of tryptophan, the amino acid precursor of serotonin (5HT), are increased in the blood and brain tissues of spf mice (Chaouloff et al., 1985; Inoue et al., 1989), and increases in the 5HIAA/5HT ratio, indicative of increased ux through the 5HT pathway, were also reported. One possible explanation for the increased 5HT metabolism in the brains of spf mice is provided by the subsequent reporting of increased activity of the 5HTmetabolizing enzyme monoamine oxidaseA (MAO-A) (Raghavendra Rao et al., 1994). Receptorbinding studies revealed a signicant loss of binding sites for the 5HT2 receptor ligand [3H]ketanserin and a concomitant increase in binding sites for the 5HT1A site ligand [3H]8OHDPAT (Robinson et al., 1992). Neurobehavioral assessment of spf mice revealed abnormalities in 5HTrelated behaviors in these animals.

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Pathogenesis of Neuronal Cell Death in Congenital OTC Deciency

4.1 Brain Energy Decit

In vitro studies demonstrate that millimolar concentrations of ammonia inhibit aketoglutarate dehydrogenase (aKGDH) in both brain and heart preparations (Lai and Cooper, 1986). Brain tissue from spf mice contains signicantly increased concentrations of alanine and lactate (Ratnakumari et al., 1994a) consistent with diminished pyruvate oxidation in the brains of these animals, a phenomenon which could result from an inhibitory effect of ammonia on aKGDH. If aKGDH inhibition is sufciently prolonged and severe, a cerebral energy decit could result. Consistent with such a possibility, direct measurements reveal signicant losses of ATP in the brains of spf mice (Ratnakumari et al., 1992).

4.2 Nitric Oxide

Both plasma and brain concentrations of Larginine are reduced in human and experimental OTC deciency (Ratnakumari et al., 1996b). In the spf mouse, brain concentrations of several guanidino compounds including Larginine were found to be signicantly decreased (Ratnakumari et al., 1996b). Nitric oxide is synthesized by nitric oxide synthase (NOS) from Larginine via oxidation of the guanidino nitrogen. Not surprisingly, Larginine deciency in the spf mouse resulted in decreased brain activities of NOS, and it was suggested that decreased synthesis of nitric oxide could be of pathophysiologic importance in relation to the central nervous system dysfunction characteristic of congenital OTC deciency. Supplementary Larginine forms part of the treatment regimen currently used in congenital OTC deciency; its benecial effects could conceivably result not only from priming of the urea cycle but also from normalization of nitric oxide synthesis.

4.3 NMDA ReceptorMediated Excitotoxicity

Tryptophan may be oxidized to quinolinic acid, an excitotoxic compound which causes neuronal cell death via its action on NMDA receptors (Schwarcz et al., 1983). Quinolinic acid concentrations are increased by up to tenfold in CSF of severely affected children with congenital OTC deciency (Batshaw et al., 1993) and studies in the spf mouse reveal twofold increases of quinolinic acid in several brain regions (Robinson et al., 1992, 1995a) including striatum where neuropathologic evaluation showed a loss of medium spiny neurons and increases in microglia (Robinson et al., 1995a); similar neuropathologic patterns of cellular changes were previously reported following direct infusions of quinolinic acid into the brains of normal mice (Bakker and Foster, 1991). The loss of NMDA sites reported in the brains of spf mice (Ratnakumari et al., 1995b) could reect downregulation of these sites following chronic exposure to quinolinic acid and in this way represent a protective mechanism against further NMDA receptormediated excitotoxicity and neuronal cell death. These ndings suggest the potential for the eventual use of NMDA receptor antagonists for the prevention of neuronal loss in congenital OTC deciency.

Current Therapy in Congenital OTC Deciency

5.1 Sodium Benzoate, Phenylacetate

In 1979, Brusilow and coworkers proposed that the conjugation of sodium benzoate with glycine to form hippurate could represent an alternative pathway for the excretion of waste nitrogen in children with congenital urea cycle deciencies. Subsequent studies in these disorders demonstrated a lowering effect on ammonia with concomitantly improved neurological outcome and survival. Sodium benzoate prescribed in combination with Larginine and sodium phenylacetate remains the treatment of choice for congenital OTC

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deciency (Brusilow and Horwich, 1995). With this therapy, survival rates for patients with OTC deciency have increased to >50%. However, more than 75% of children with neonatalonset OTC deciency still suffer severe brain damage resulting in seizures, mental retardation, and developmental disabilities (Msall et al., 1984). In studies in spf mice, sodium benzoate was found to be effective in lowering brain ammonia and glutamine concentrations (Ratnakumari et al., 1993b). However, high doses of sodium benzoate led to a rebound in brain ammonia and to a signicant reduction in acetyl-CoA and ATP concentrations (Ratnakumari et al., 1993b). Similar changes had previously been reported in the livers of rats following sodium benzoate treatment (Kalbag and Palekar, 1988; Michalak and Qureshi, 1995a). In an analogous fashion to sodium benzoate, sodium phenylacetate removes ammonia by conjugating with glutamine to form phenylacetylglutamine (Brusilow et al., 1979).

5.2 LCarnitine
administration signicantly attenuates ammonia neurotoxicity (Matsuoka et al., 1991), an effect which is mediated by maintenance of cerebral energy metabolites (Matsuoka and Igisu, 1993). It was suggested that the potentiation of ammonia toxicity at higher doses of sodium benzoate (see above) could be prevented by the administration of Lcarnitine (OConnor et al., 1986). A subsequent study demonstrated a doserelated benecial effect of Lcarnitine on liver (Michalak and Qureshi, 1995b) and brain (Ratnakumari et al., 1993c) acetyl-CoA and on ATP decits caused by sodium benzoate in spf mice. On the basis of these ndings, it was suggested that Lcarnitine supplementation could be benecial during benzoate therapy in congenital OTC deciency in humans.
LCarnitine

5.3 AcetylLCarnitine
AcetylLcarnitine is a physiological substance found in many tissues including brain where it appears to act as a carrier for acetyl groups across the inner mitochondrial membrane (Tucek, 1985) and, by virtue of its proposed role in cholinergic neurotransmission, has been suggested to be benecial in the treatment of cognitive disorders (Bassi et al., 1988). In view of the cholinergic decit described in OTC deciency (Ratnakumari et al., 1994b, 1995a), acetylLcarnitine was administered to pregnant spf rats and its effects on cholinergic parameters in the brains of offspring were assessed (Ratnakumari et al., 1995a). AcetylL carnitine treatment resulted in a partial correction of the developmental prole for the cholinergic nerve terminal enzyme CAT, suggesting a potential therapeutic role for this compound in the prevention of cholinergic neuronal loss in OTC deciency.

5.4 Liver Transplantation

Liver transplantation continues to gain acceptance as an effective treatment of urea cycle enzymopathies. To date, it has been used to treat cases of OTC deciency, arginosuccinic aciduria, and citrullinemia. Recent improvements in organ procurement, surgical techniques, and immunosuppression have signicantly improved morbidity and mortality. Several cases of OTC deciency have been successfully treated by orthotopic liver transplantation (OLT). In all cases, OLT led to correction of hyperammonemia and plasma amino acid proles. Survival rates are currently >80% and most survivors have a normal mental status posttransplantation (Todo et al., 1992; Hasegawa et al., 1995; McBride et al., 2004). Living donor (partial) liver transplants have successfully been used in cases of citrullinemia (Takenaka et al., 2000) and OTC deciency (Nagasaka et al., 2001). Initial studies using isolated hepatocyte transplantation in a patient with OTC deciency resulted in metabolic improvement and temporary relief of hyperammonemia prior to full liver transplantation (Horslen et al., 2003).

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5.5 Gene Therapy

Clinical trials of gene therapy have been considered for patients with congenital urea cycle disorders. On the basis of studies in the spf mouse, a twostep process was suggested (Robinson et al., 1995b) consisting of both in vivo and ex vivo gene therapy. Step one would address the treatment of neonatal hyperammonemic coma using in vivo gene therapy and once stabilized, longterm correction would be attempted by ex vivo therapy using recombinant retroviruses. Modications of adenoviral vectors to decrease immunogenicity could then potentially permit longterm correction (Morsy and Caskey, 1994; Robinson et al., 1995b). A series of adenoviral vectors containing mouse OTC cDNA used in a study in the spf mouse revealed that recombinant adenoviruses deleted in El and with a temperaturesensitive mutation in E2 successfully corrected the OTC deciency and subsequent metabolic abnormalities for several months (Ye et al., 1996). Adenoviral vectors have been explored as a treatment for citrullinemia in two models of arginosuccinate synthase deciency namely a naturally occurring bovine model and a murine model created by molecular mutagenesis (Patejunas et al., 1998). Mice treated with adenoviral vectors expressing arginosuccinate synthase lived longer than the untreated animals, but the treatment was suboptimal. In the bovine model, reports of either no improvement (Patejunas et al., 1998) or signicant clinical improvement and normalization of plasma glutamine (Lee et al., 1999) have been reported using a different adenoviral vector. Studies so far in human OTC deciency have not been encouraging with both lethal complications and adverse effects including ulike episodes and thrombocytopenia being reported (Raper et al., 2002).

Acknowledgments
Studies from the authors research unit were funded by The Canadian Institute of Health Research and the Canadian Liver Foundation.

References
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