Urea Cycle Defects

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46.1 Clinical Features

and Laboratory Investigations There are five well-documented urea cycle defects (Fig. 46.1):

– Carbamyl phosphate synthetase deficiency (CPSD) – Ornithine transcarbamylase deficiency (OTCD) – Argininosuccinate synthetase deficiency (ASSD),

also called citrullinemia

– Argininosuccinate lyase deficiency (ASLD), also called argininosuccinic aciduria

– Arginase deficiency, also called hyperargininemia These disorders have an autosomal recessive mode of inheritance, with the exception of OTCD, which has an X-linked recessive inheritance. Clinical signs of metabolic derangement may appear at any time from early infancy to adulthood, but peak periods include the neonatal period, change to a diet with high pro- tein content (replacement of milk feeding by a diet with higher protein content, parenteral nutrition), and episodes of infectious diseases. Valproate may al- so induce an episode of metabolic derangement.

In the case of neonatal presentation, a normal ba- by is born after normal pregnancy and delivery. After

one or two days the child becomes lethargic and hy- potonic. Vomiting, seizures, hypothermia, and hyper- ventilation occur. Lethargy increases and coma fol- lows. There are signs of elevated intracranial pressure with a bulging fontanel and increasing head size. In most cases the disease progresses rapidly to death within a few days. Survivors almost always have severe neurological sequelae.

In the case of later onset, the disease is chronic and episodic. Occurrence of symptoms may be related to protein intake, infections, trauma, surgery, or initia- tion of valproate treatment, but not infrequently an episode occurs without any obvious cause. The episodes are characterized by headache, lethargy, irri- tability, agitation, confusion, hallucinations, vomit- ing, hypotonia, ataxia, dysarthria, or coma. The pre- sentation may also be stroke-like with signs of dys- function of one cerebral hemisphere, in particular hemiplegia. In patients with later onset of clinical symptoms the mortality rate is still high, and highest during the initial presenting illness. In addition to the episodic worsenings, there are often chronic clinical signs, which may include learning problems or psy- chomotor retardation of variable severity, behavioral problems, ataxia, seizures, and hepatomegaly. Some patients voluntarily restrict their protein intake. Nor- mal development and neurological function up to adulthood have been reported in rare cases. In ASLD, coarse and friable hair (trichorrhexis nodosa) is a special characteristic.

The clinical features and course of disease are in- distinguishable in CPSD, OTCD, ASSD, and ASLD. It is only in hyperargininemia that the clinical manifesta- tions differ. In the latter disease, the clinical symp- toms are slowly progressive and include growth fail- ure, psychomotor retardation, progressive spastic tetraplegia (the legs being more severely involved than the arms), tremor, ataxia, choreoathetosis, epilepsy, and hyperactivity. In addition, episodes of lethargy, vomiting, and coma may occur. There is some variability in onset and rate of progression of the disease. Life span is usually longer than in the oth- er urea cycle defects.

Females heterozygous for OTCD are usually free of symptoms, but approximately 10% become sympto- matic and have a milder and more variable course of disease than affected males. Symptoms are episodic and include headaches, vomiting, irritability, bizarre behavior, lethargy, ataxia, tremors, seizures, and co-

Urea Cycle Defects

Chapter 46

Fig. 46.1. The urea cycle. CPS, carbamyl phosphate syn- thetase; OTC, ornithine transcarbamylase; ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase

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ma. The presentation may also be stroke-like with recurrent episodes of hemiparesis. A high-protein diet, infection, surgery, and the postpartum state may precipitate attacks. Deterioration following use of val- proate has been described repeatedly.

Laboratory investigations reveal hyperammone- mia in all urea cycle disorders, with the exception of hyperargininemia, in which blood ammonium levels may be normal. Respiratory alkalosis is often present.

Urinary orotic acid is increased because of the shunt- ing of nitrogen waste from the urea cycle. In CPSD and OTCD citrulline is decreased in plasma. In ASSD plasma citrulline is elevated, whereas in ASLD argini- nosuccinate is elevated and citrulline is moderately increased. In these four disorders, plasma levels of glutamine and alanine are frequently raised, whereas arginine and ornithine are decreased. In hyper- argininemia, arginine is elevated in plasma. In urine, elevation of arginine, lysine, cystine, ornithine, cit- rulline, glutamine, and orotic acid is found. A definite diagnosis can be established by enzyme assessment in liver cells. In hyperargininemia and ASLD, enzyme assessment is also possible in erythrocytes; in ASSD and ASLD, enzyme assessment is also possible in fibroblasts. DNA confirmation is possible for all urea cycle defects.

All five urea cycle defects can be diagnosed antena- tally. The techniques available for prenatal diagnosis vary from measurement of abnormal metabolites in amniotic fluid, to DNA analysis in chorionic villi or amniocytes, to measurement of enzyme activity in cultured amniocytes or in utero liver biopsy samples.

Protein loading, alanine loading, and allopurinol challenge to induce orotic aciduria can be used for carrier detection in OTCD. However, a negative test does not rule out the carrier status. DNA techniques can be used for carrier detection if the mutation in the affected patient is known.

46.2 Pathology

Neuropathological findings are variable and depend on the age of the patient and on the relative effects of present and past acute and chronic metabolic de- rangements. Neuropathological findings are similar in the different urea cycle disorders.

Actual high elevations of ammonium levels lead to brain swelling. On light and electron microscopy, as- trocyte swelling is found. Hyperammonemia induces the so-called Alzheimer type II change in astrocytes.

This change consists of an increase in the number and size of astrocytic nuclei, which may become nearly twice their normal size. These nuclei are vesicular with a prominent nuclear membrane and an optical- ly empty nucleoplasm with sparse chromatin parti- cles. The cytoplasm is not discernible with the light

microscope. Alzheimer type II astrocytes are mainly present in cerebral cortex, basal nuclei, cerebellar cor- tex and nuclei, and brain stem nuclei. The presence of Alzheimer type II cells depends on the presence of hyperammonemia. In adequately treated patients with normal ammonia levels Alzheimer type II cells are absent.

In neonates who die in the acute phase of metabol- ic decompensation, diffuse brain swelling is found.

Alzheimer type II astrocytes are present in gray matter structures. There may be signs of acute neu- ronal injury. Myelination is normal for age. In some neonates a status spongiosus of cortex and white matter is observed.

In older infants, children, and adults, atrophy of the brain is often found with widening of the lateral ven- tricles and spaces between the sulci. The atrophy varies from mild to severe. Variable focal and multi- focal corticosubcortical necrosis may be seen with a tendency to microcavitation. Acute lesions are swollen; old lesions are atrophic with presence of ule- gyria. On microscopic examination, cortical findings vary from normal to pseudolaminar neuronal necro- sis to complete depopulation of the cortex and diffuse gliosis. The cortical damage may have a spongiform appearance. Neuronal loss, gliosis, and spongy changes may also be seen in basal nuclei, thalamus, and brain stem. White matter changes are variable.

Myelination may be normal or mildly to severely delayed. Signs of active myelin breakdown may be present, but are not seen in all cases. The white matter may show diffuse gliosis. The white matter changes may be spongiform with presence of myelin splitting and vacuolation. In severe cases, there is diffuse loss of axons and myelin, and the white matter may be largely replaced by gliotic tissue.

In female carriers of OTCD, neuropathological findings are apparently mainly related to chronic hyperammonemia, which leads predominantly to neuronal damage. Variable, sometimes extreme cere- bral atrophy is the predominant finding. The hemi- spheric walls are thin and the lateral ventricles are dilated. The cerebral cortex shows signs of neuronal loss and gliosis. There is also loss of neurons in the basal nuclei and thalamus. Alzheimer type II astro- cytes are present in the cerebral cortex, basal nuclei, dentate nuclei, and brain stem nuclei. The white mat- ter may be rarefied and gliotic with a reduced number of myelinated fibers.

46.3 Pathogenetic Considerations

The urea cycle (Fig. 46.1) serves two purposes: it con-

tains, in part, the biochemical reactions required for

the de novo biosynthesis and degradation of arginine,

and it incorporates the surplus of nitrogen into urea,

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which serves as a waste nitrogen product. The en- zymes involved in urea synthesis are partly located within the mitochondria (carbamyl phosphate syn- thetase, ornithine transcarbamylase), whereas the other enzymes are located within the cytosol (ar- gininosuccinate synthetase, argininosuccinate lyase, arginase). Carbamyl phosphate synthetase requires N-acetylglutamate for full activity. Most urea cycle metabolic capacity resides in the liver. The gene loca- tions of the urea cycle enzymes have been identified and the genes have been characterized. The gene for carbamyl phosphate synthetase is located on chromo- some 2q35; the gene for ornithine transcarbamylase is located on chromosome Xp21.1; the gene for argininosuccinate synthetase is located on chromo- some 9q34; the gene for argininosuccinate lyase is located on chromosome 7cen-q11.2; and the gene for arginase is located on chromosome 6q23.

A urea cycle defect has two consequences: arginine becomes an essential amino acid (except in hyper- argininemia), and nitrogen accumulates in a variety of molecules, in particular ammonia. Ammonia is highly toxic to the brain where it interferes with ener- gy production and normal metabolism of neuro- transmitters. Ammonia influences the glutamine–

glutamate–GABA balance. Glutamate is the most im- portant excitatory neurotransmitter, whereas GABA (g-aminobutyric acid), formed by decarboxylation from glutamate, is the most important inhibitory neu- rotransmitter. In the presynaptic neuron, glutamate is formed from glutamine by glutaminase. After release by the presynaptic neuron, glutamate is taken up by the astrocyte, in which it is processed by glutamine synthetase into glutamine. Glutamine is transported to the presynaptic neuron, where glutaminase cat- alyzes the formation of glutamate available for neuro- transmission. Hyperammonemia has a great impact on this cycle by stimulating synthesis of glutamine from ammonia and glutamate with consumption of ATP. The disturbance of the balance between excitato- ry and inhibitory neurotransmitters may contribute to the cerebral dysfunction. Glutamine synthetase is mainly located in astrocytes, and high plasma levels of ammonia lead to accumulation of glutamine with- in astrocytes. During hyperammonemia, the concen- tration of glutamine in the brain becomes highly ele- vated. It has been proposed that the consequent osmotic effect causes astrocytes to swell, with sub- sequent cerebral edema.

The excitotoxin quinolinic acid has been proposed to explain aspects of neuronal injury. Quinolinic acid accumulates under hyperammonemic conditions and derives from tryptophan metabolism. Under such conditions there is increased transport of tryptophan across the blood–brain barrier. Tryptophan oxidation leads to the formation of quinolinic acid, which acts as an excitotoxin at the N-methyl-

D

-aspartate

(NMDA) receptors. Moderate elevations of CSF levels of quinolinic acid have been found in patients with a urea cycle defect.

Unlike patients with liver failure, in whom ammo- nia is only one of several toxins, ammonia appears to be the only cause of the acute encephalopathy seen in patients with urea cycle defects, except for those with hyperargininemia, in whom an increase in arginine may also play a role. In hyperammonemia due to liver failure, MRI of the brain shows, as expected in generalized disorders, a symmetrical pattern. There are changes in signal intensity in the basal nuclei, in particular due to T

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shortening. In contrast, brain pathology in urea cycle disorders is often character- ized by focal, usually asymmetrical lesions. It remains to be explained why cerebral abnormalities related to hyperammonemia in urea cycle disorders are so dif- ferent from those observed in liver failure, and why the lesions tend to be asymmetrical.

In all urea cycle disorders apart from hyper- argininemia, arginine is deficient unless externally supplied. Chronic arginine deficiency is character- ized by dermatological features with erythematous scaling. A dramatic improvement of this cutaneous eruption occurs with dietary arginine supplementa- tion. High levels of citrulline or argininosuccinate are probably not toxic. The similarity in presentation among the different urea cycle defects is related to hyperammonemia. The variability in clinical severity is probably related to differences in mutations (and differences in levels of residual enzyme activity), dif- ferences in other genetic factors and environmental factors. In females carrying an OTC mutant allele on one chromosome, variability in expression is related to the proportion of hepatocytes in which the normal or mutant allele is active (lyonization).

Hyperargininemia has a clinical picture that is partially different from that of the other urea cycle defects. The progressive spasticity that dominates the clinical picture in hyperargininemia is not a feature of the other urea cycle defects. It has been proposed that arginine and its metabolites, the guanidine com- pounds, are responsible.

46.4 Therapy

Therapeutic strategies in urea cycle defects aim at reduction of protein intake, utilization of alternative pathways of nitrogen excretion, and replacement of deficient nutrients.

Emergency treatment consists of stopping all pro- tein intake, starting high energy intake to prevent catabolic situations with breakdown of endogenous protein, and measures that lead to augmented nitro- gen disposal. Sodium benzoate, sodium phenyl bu- tyrate, or sodium phenyl acetate, supplied orally or

Chapter 46 Urea Cycle Defects 362

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intravenously, can be used as substrates for an alter- nate route of nitrogen disposal. Lactulose binds am- monia in the intestinal tract and can increase ammo- nia disposal in feces. Arginine should be supplement- ed in all urea cycle defects except hyperargininemia.

Hemodialysis can be used in life-threatening situa- tions. Of course, the conditions initiating the deterio- ration (infections, insufficient intake) and complica- tions (seizures) have to be treated too.

Chronic treatment of urea cycle defects consists of a protein-restricted diet, sufficient caloric intake to avoid catabolic situations, and supplementation of absent substances. Protein-restricted diets have to be supplemented with essential amino acid mixtures to avoid deficiencies. In all urea cycle disorders apart from hyperargininemia, arginine cannot be synthe- sized endogenously; it has become an essential amino acid and has to be supplied. If an essential amino acid is lacking, protein breakdown cannot be remedied.

Long-term treatment that restricts intake of high- protein food, including milk and meat, leads to defi- ciency of minerals, trace elements, and vitamins, which also have to be supplemented. Infections and insufficient caloric intake due to anorexia carry the risk of triggering an episode of severe hyperammone- mia. Therefore, such conditions have to be treated vigorously. The use of valproic acid as an antiepilep- tic drug should be avoided.Valproic acid may acceler- ate the appearance of hyperammonemia.

The overall long-term prognosis has improved with treatment, in particular in patients presenting after the neonatal period. However, a high percentage of treated patients are still mentally retarded. The mortality rate in the group with neonatal presenta- tion is still very high, and most (or all) surviving patients are severely handicapped.

Enzyme replacement therapy through liver trans- plantation has been attempted, and it has been shown to be effective in correcting the metabolic defect.

After liver transplantation no further brain damage is to be expected, but significant recovery of prior brain damage does not occur. Therefore, liver transplanta- tion may be an excellent treatment for patients with- out major brain injury. However, in view of the poten- tial morbidity and mortality associated with liver transplantation, the present experience is still too limited to allow a balanced opinion. Genetic correc- tion of the patient’s liver cells is a promising future option.

46.5 Magnetic Resonance Imaging

In urea cycle defects, cerebral abnormalities change depending on the stage of disease. In acute episodes of metabolic derangement, lesions appear, which may improve under treatment but leave traces. Chronic hyperammonemia also has deleterious effects. MRI has the advantage over neuropathological examina- tions of being able to depict the dynamics of cerebral lesions in urea cycle disorders.

In neonates, neuroimaging shows severe brain swelling. On CT diffuse cerebral hypodensity with loss of contrast between cortex and white matter may be seen. MRI shows diffuse cerebral edema (Fig. 46.2) and may demonstrate involvement of the basal gan- glia with a high signal in the caudate nucleus, puta- men, and/or globus pallidus on T

2

-weighted images (Fig. 46.2) and a high signal in the globus pallidus and to a lesser extent the putamen on T

1

-weighted images.

The deep sulci of the insular and perirolandic region may also display T

1

shortening. MR spectroscopy may contribute by showing highly elevated glutamine lev- els. If the patient survives, diffuse brain atrophy fol- lows. In some cases, focal or diffuse gross cystic de- generation of the cerebral hemispheres is seen on fol- low-up. The basal ganglia are often prominently in- volved as well, but the thalamus, brain stem, and cerebellum tend to be relatively spared.

In acute metabolic derangement in older infants and children, small and large areas of abnormal sig- nal intensity are seen in the brain, most often involv- ing both cortex and underlying white matter, giving them an infarct-like aspect (Figs. 46.4 and 46.5).

Sometimes, the signal abnormalities involve only or mainly the cortex (Fig. 46.3). The acute lesions are moderately swollen. Often multiple lesions are pre- sent. The distribution of the lesions is as a rule asym- metrical or even unilateral (Figs. 46.4 and 46.5). In some cases one hemisphere is totally involved. How- ever, in other patients almost the entire brain is affect- ed (Fig. 46.3). A combination of high plasma ammo- nia levels and an MRI picture with one or more large, moderately swollen areas involving cortex and white matter is highly suggestive of a urea cycle defect. This pattern can be found in all urea cycle defects when an episode of acute metabolic derangement is present, including those in female carriers of OTCD (Fig. 46.4) and patients with arginase deficiency (Fig. 46.5). In the chronic stage, swelling resolves and focal areas of atrophy and patchy signal changes of cortex and white matter remain (Fig. 46.6). Sometimes, the le- sions are restricted to the white matter.

In chronic hyperammonemia, defective myelina-

tion and progressive cerebral atrophy are seen. Some

patients have some nonspecific focal white matter

abnormalities.

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Fig. 46.2. MRI series of a baby boy, 6 days old, with ASLD and extreme hyperammonemia. The T1-weighted sagittal image shows generalized edema and tonsillar herniation. The T2- weighted transverse images show generalized edema with, in

particular, severe swelling of the brain stem. Note the signal abnormalities in the putamen and caudate nucleus. MR spec- tra of this patient are shown in Chap. 108

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Fig. 46.3. A 2-year-old girl, a carrier of OTCD, with an acute episode of neurological deterioration and coma. The T2- weighted transverse images show diffuse involvement of the

cerebral cortex and subcortical white matter. Courtesy of Dr. S.

Blaser, Department of Diagnostic Imaging, Hospital for Sick Children, Toronto, Canada

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Fig. 46.4. A 2-year-old girl, a carrier of OTCD, with an acute episode of neurological deterioration and hemiplegia. The T2- weighted images show an extensive area of high signal intensity and swelling in the left frontal and parietal white matter and cortex with blurring of the corticomedullary junction. A

smaller area of abnormal signal in the cortex and subcortical white matter is seen in the right frontal region. Courtesy of Dr.

S. Blaser, Department of Diagnostic Imaging, Hospital for Sick Children, Toronto, Canada

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Fig. 46.5. A boy, 6 years of age, with hyperargininemia.The MR images were made during an episode of acute metabolic decompensation following protein-rich gavage feeding. The T2- weighted transverse images show the signal changes bilater-

ally in the frontal lobes, accentuated on the left side, with involvement of both gray and white matter, blurring the gray–white matter junction. The corpus callosum is not involved, nor are the basal ganglia

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Fig. 46.6. The same boy as in Fig. 46.5, now 2 years later. The acute abnormalities have disappeared. Note the atrophy, most marked in the left frontal area