Inborn Errors Of Urea Synthesis

Group 1 Section A2
Bicera, Genesis Bido, Amabel Diane Bigay, Donna Karen Bilang, Janine Bondoc, Alvin Rey Bravo, Vincent Martin

I.) Urea Cycle

Urea Cycle 1. Site since ammonia is toxic to CNS even in traces liver rapidly removes ammonia from circulation and converts it to a non-toxic water soluble urea. Hence site of urea synthesis is liver. 2. The reactions leading to formation of urea from ammonia are proposed by Krebs and Henseleit. Hence, urea cycle is also called as Krebs-Henseleit cycle. 3. Formation of urea from ammonia in urea cycle occurs in five reactions. However the first reaction is not a part of the cycle but for the continuation of the cycle which consist of remaining four reactions product of the first reaction is essential. Further, the intermediates of the four reactions are aminoacids. However no codons exist for them. 4. Synthesis of urea from ammonia is a energy dependent process. 5. Enzymes of urea cycle are present in mitochondria and cytosol. 6. First two reactions of urea formation occurs in mitochondria and remaining reactions

occur in cytosol. REACTION SEQUENCE OF UREA FORMATION For the synthesis of urea only one ammonia molecule is used as such. Aspartate serves as donor of another molecule of ammonia. HCO3 serves as source of CO2 required for urea formation. Formation of Carbamoyl Phosphate 1. First reaction leading to urea formation is condensation of ammonia and HCO3 At the expense of two high-energy bonds to form carbamoyl phosphate. The reaction is catalyzed by mitochondrial carbamoyl phosphate synthetase-I. The enzyme requires N-acetyl glutamate and Mg2+. N-acetyl glutamate is synthesized from acetyl-CoA and glutamate in the liver. 2ATP molecules are hydrolyzed to 2 ADP and 2Pi in the first reaction. Of the 2Pi one Pi is consumed in the reaction. Since the product of the reaction carbamoyl phosphate is high energy compound its formation thermodynamically pulls subsequent reactions of urea cycle towards urea formation. Reactions of Urea Cycle 2. Now the first reaction of urea cycle is catalyzed by ornithine transcarbamoylase. It condenses carbamoyl phosphate and ornithine to form citrulline. This enzyme is present in mitochondria. Since the subsequent reactions of urea cycle occurs in cytosol, citrulline formed eners cytosol through specific transporter present in inner mitochondrial membrane. 3. Arginino succinate synthetase present in cytosol catalyzes second reaction of urea cycle. It condenses citrulline and aspartate at the expense of two high energy bonds to form argininosuccinate. One high energy bond is consumed by the hydrolys is of ATP to AMP and PPi. Further hydrolysis of PPi to 2Pi by pyrophosphatase leads to consumption of another high energy bond. 4. In the third reaction of urea cycle argininosuccinate is cleaved by argininosuccinase to arginine and fumarate. 5. Regeneration of ornithine and formation of urea from arginine is the final reaction of urea cycle. This reaction is catalyzed by arginase. The ornithine so formed enters mitochondria through specific transporter present in inner mitochondrial membrane to start reactions of urea cycle once again. Overall equation for urea formation

Fate of Urea Urea has no physiological function. Hence it is transported to kidneys where it is excreted in urine. It is major end product of protein catabolism in humans. About 10-25 gm of urea is excreted in urine per day which makes up to 80-90% of total nitrogen excreted per day. However, blood also contains some urea.

Blood Urea Normal blood urea level is 16-36 mg/100 ml. Fate of Fumarate Fumarate is recycled in urea cycle by converting it to aspartate. First fumarate is converted to malate by fumarase which is present in cytosol. Malic enzyme generates pyruvate from malate in the next reaction. Next pyruvate is converted to oxalo acetate by pyruvate carboxylase. Oxalocetate also can be formed directly from malate by the action of malate dehydrogenase. Finally aspartate is generated from oxaloacetate by transamination in which -ketoglutarate. Thus operation of this system cause net supply of one ammonia molecule to urea synthesis. Regulation of Urea Formation The urea cycle operates only to eliminate excess ammonia. The excess comes mainly from ingested amino acids not used promptly for the synthesis of proteins; only a few nitrogenous compounds comprising a small percentage of nitrogen turnover, such as purines, pyrimidines, creatine and nicotinic acid, are not degraded to ammonia. The general nutritional status does not influence the degradation of excess amino acids; whether the carbon skeletons from amino acids are used immediately to produce ATP or are stored as fat and glycogen, the amino groups are released as ammonia, which must be eliminated. In nitrogen balance the amount of urea produced represents metabolic wear and tear, a poorly understood combination of cell death and regeneration and protein turnover with partial, but never complete, reuse of amino acids. These metabolic processes persist even during protein deficiency in a high-fat diet, high-carbohydrate diet so that urea production never ceases. The rate, however, is precisely regulated to the need to eliminate potentially toxic ammonia. 1.) Regulation by substrate availability the higher the rate of ammonia production, the higher the urea formation 2.) Allosteric activation of carbamoyl phospatase I (CPS I) by N-acetylglutamate (N-AGA). N-AGA is formed specifically to activate CPS I; it has no other known function. Formation of urea is regulated by activity of carbamoyl phosphate synthetase-I. This enzyme catalyzes committed step in urea synthesis. N-acetylglutamate regulates this enzyme activity. It is an allosteric activator. 3.) Induction/repression of the synthesis of urea cycle enzymes the induction of urea cycle enzymes occurs in response to conditions which require increased protein metabolism, such as high protein diet or prolonged fasting. In both of these physiological states, as amino acid carbon is converted to glucose, amino acid nitrogen is converted to urea. High protein in take leads to more N-acetylglutamate formation. Thus high protein in take influences urea formation. In starvation also urea synthesis is more mostly due to increased protein breakdown.

II.) Inborn errors associated with deficiency of each enzyme of the urea cycle (Urea Cycle Disorders)
Urea cycle disorders are genetic disorders caused by a deficiency of one of the enzymes in the urea cycle which is responsible for removing ammonia from the blood circulation. Urea cycle disorders or defects are usually characterized by hyperammonemia, encephalopathy, and respiratory alkalosis and can lead to coma in severe cases. The diagnosis requires measurement of plasma ammonia, plasma amino acids, and urine orotic acid, useful for differentiating ornithine transcarbamylase deficiency from carbamyl phosphate synthase-1 and N-acetylglutamate synthase deficiency. Deficiencies of carbamoyl phosphate synthetase I, ornithine transcarbamoylase, argininosuccinate synthetase, and argininosuccinate lyase or argininosuccinase, can lead to the accumulation of precursors of urea, usually ammonia and the amino acid glutamine. There is also a decrease in the level of the amino acid arginine in the blood (except for arginemia) due to the failure of the cycle to synthesize the said amino acid. Ammonia intoxication is most severe when the metabolic block occurs at reactions 1 or 2, for if citrulline can be synthesize, some ammonia has already been removed by being covalently linked to an organic metabolite. Clinical symptoms common to all urea cycle disorders include vomiting, avoidance of highprotein foods, intermittent ataxia, irritability, lethargy, and severe mental retardation. The most dramatic clinical presentation occurs in full-term infants who initially appear normal, then exhibit progressive lethargy, hypothermia, and apnea due to high plasma ammonia levels. Acute liver failure can also be observed in adults. Females with ornithine transcarbamoylase deficiency or milder forms of other urea cycle defects can present at time of childbirth due to the combination of involuntary fasting and stress that favor catabolism. The clinical features and treatment of all five disorders are similar. They are all transmitted as autosomal recessive traits, with the exception of ornithine transcarbamoylase deficiency, which is X-linked. Hepatocytes of females with ornithine transcarbamylase deficiency express either the normal or the mutant allele due to random Xinactivation and may be unable to remove excess ammonia if mutant cells are predominant The usual therapy for these inborn errors includes limiting protein intake to lessen the potential build up of ammonia in the body. Lactulose or Levulose are also administered to promote the excretion of ammonia in the feces. Antibiotics are also given to patients to kill the ammonia producing bacteria that are normally seen in the intestines. Excess ammonia can also be removed from the body by using compounds that covalently binds to amino acids and produces nitrogen containing molecules that are excreted in the urine. An example of the this compounds are benzoate that binds to glycine and forms hippuric acid. Another example is phenylacetate that binds to glutamine and produces phenylacetylglutamine. Replacement of the missing intermediates from the cycle is also done. The most advisable intermediate that is given is glutamic acid. The following are the inborn errors of metabolism related to the urea cyle:

A.) Hyperammonemia Type I

In this condition the enzyme deficient is Carbamoyl Phosphate Synthetase 1. This condition is an autosomal recessive disorder that causes ammonia to accumulate in the blood. Carbamoyl phosphate synthetase I deficiency often becomes evident in the first few days of life. An infant with this condition may be lethargic or unwilling to eat, and have a poorly controlled

breathing rate or body temperature. Some infants with this disorder may experience seizures or unusual body movements like intermittent ataxia, or go into a coma in severe cases. Complications of carbamoyl phosphate synthetase I deficiency may also include developmental delay and mental retardation. In some affected individuals, signs and symptoms of carbamoyl phosphate synthetase I deficiency may be less severe, and may not appear until later in life. These symptoms correlate with an ammonia level of 100-150 mol/L, which is 2-3 times higher than the reference range.

B.) Hypermmonemia Type II

In this condition the Enzyme deficient is Ornithine Transcarbamoylase. Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder. It is an x-linked recessive disorder caused by a number of different mutations in the OTC gene. In this condition a mutant enzyme protein impairs the reaction that leads to condensation of carbamoyl phosphate and ornithine to form citrulline. This impairment leads to reduced ammonia incorporation, which, in turn, causes symptomatic hyperammonemia, hence it is called hyperammonemia type II. Ornithine transcarbamoylase deficiency often becomes evident in the first few days of life, however it can present at middle age. Classical symptoms of this condition is similar to the symptoms usually observed in all inborn errors of the urea cycle like lethargy, vomiting, avoidance of high-protein foods, intermittent ataxia, irritability and severe mental retardation. In severe cases it can also lead to coma. In some affected individuals, signs and symptoms of ornithine transcarbamoylase deficiency may be less severe, and may not appear until later in life. Some female carriers become symptomatic later in life in times of metabolic stress. This can happen as a result of anorexia, starvation, malnutrition, pregnancy or even as a result of gastric bypass surgery. Like the other urea cycle disorders this condition also exhibits a high level of ammonia in the blood and can be accompanied by build up of orotic acid in the blood which leads to orotic aciduria, because carbamoyl phosphate can enter the pyrimidine synthesis and be converted to orotic acid. Ornithine build up in the body is also evident due to the failure of the cycle to incorporate it with carbamoyl phosphate.

C.) Citrullinemia
In this condition the enzyme deficient is Argininosuccinate Synthetase. This is an autosomal recessive urea cycle disorder that causes ammonia and other toxic substances to accumulate in the blood. There are two types of citrullinemia which differs from each other based on the signs and symptoms, onset of the disorder and the genes that is mutated. The type I is more common and presents a mutation in the ASS (argininosuccinate synthetase) gene which makes the enzyme argininosuccinate synthetase. This condition usually becomes evident in the first few days of life and presents symptoms that are similar to other urea cycle disorder. Affected infants typically appear normal at birth, but as ammonia builds up in the body they develop a lack of energy (lethargy), poor feeding, vomiting, seizures, and loss of consciousness. These medical problems can be life-threatening in many cases. A milder form of type I citrullinemia is less common in childhood or adulthood. Some people with gene mutations that cause type I citrullinemia never experience signs and symptoms of the disorder. Type II citrullinemia usually appear during adulthood and affects the nervous system mainly. Mutations in the SLC25A13 gene are responsible for type II citrullinemia. This gene makes a protein called citrin, which normally shuttles certain molecules in and out of mitochondria. These molecules are essential for the urea cycle and are also involved in making proteins and nucleotides.

Mutations in the SLC25A13 mutation typically prevent the production of any functional citrin, which inhibits the urea cycle and disrupts the production of proteins and nucleotides. Like the other urea cycle disorders this condition also exhibits a high level of ammonia in the blood and an elevated level of citrulline in the blood and citrullinuria is also evident in some cases.

D.) Argininosuccinate Aciduria

In this condition the deficient enzyme is Argininosuccinase or Argininosuccinate Lyase. Argininosuccinate lyase is a cytosolic enzyme which catalyzes the fourth reaction in the cycle and the first degradative step, that is, the breakdown of argininosuccinic acid to arginine and fumarate. Deficiency of this enzyme results in an accumulation of argininosuccinic acid in tissues, and excretion of argininosuccinic acid in urine leading to the condition argininosuccinic aciduria (ASA). ASA is an autosomal recessive disorder and is the second most common urea cycle disorder. In addition to the accumulation of argininosuccinic acid, argininosuccinate lyase deficiency results in decreased synthesis of arginine, a feature common to all urea cycle disorder except argininemia. Argininosuccinic aciduria may become evident in the first few days of life because of high blood ammonia, or later in life presenting with "sparse" or "brittle" hair, developmental delay, and tremors. An infant with argininosuccinic aciduria may seem lethargic or be unwilling to eat, have poorly-controlled breathing rate or body temperature, experience seizures or unusual body movements, or go into a coma. Complications from argininosuccinic aciduria may include developmental delay and mental retardation. Progressive liver damage, skin lesions, and brittle hair may also be seen. Immediate treatment and lifelong diet management may prevent many of these complications. Occasionally, an individual may inherit a mild form of the disorder in which ammonia accumulates in the bloodstream only during periods of illness or other stress.

E.) Argininemia or Arginemia

In this condition the deficient enzyme is Arginase. Arginase is the final enzyme in the urea cycle that catalyzes the breakdown of arginine to ornithine and urea, which is the major metabolite carrying waste nitrogen destined for urinary excretion. Patients with arginase deficiency have elevated levels arginine in the blood, a contrast with the other urea cycle disorders. A mutation in the ARG1 gene causes argininemia and the deficient arginase gene is located on chromosome 6. This disorder most often follows an autosomal recessive inheritance pattern. Argininemia usually becomes evident by about the age of 3. It most often appears as stiffness, especially in the leg area, caused by abnormal tensing of the muscles or spasticity. Other symptoms may include slower than normal growth, developmental delay, mental retardation, seizures, and ataxia. Occasionally, high protein meals or stress caused by illness or periods without food like in fasting may cause ammonia to accumulate more quickly in the blood. This rapid increase in ammonia may lead to episodes of irritability, refusal to eat, and vomiting. In some affected individuals, signs and symptoms of argininemia may be less severe, and may not appear until later in life.

F.) N-Acetylglutamate synthetase deficiency

Based on the name of the disorder the deficient enzyme is N-Acetylglutamate Synthetase. This enzyme is responsible for the production of N-Acetylglutamate (N-AGA) which

is the natural activator of the enzyme carbamoyl phosphate synthetase I (one of the rate limiting enzymes in the urea cycle and responsible for the production of carbamoyl phosphate). The chromosome found to be carrying the gene encoding for N-acetylglutamate synthetase is chromosome 17q in humans. This disorder like the other urea cycle disorder (except for hyperammonemia type II) is also inherited in an autosomal recessive inheritance pattern. The symptoms are visible within the first week of life and if not detected and diagnosed correctly immediately consequences are fatal. Clinical signs and symptoms of this condition occur when ammonia fails to fix into carbamoyl phosphate effectively, thus disabling the urea cycle. This leads to accumulation of alanine and glutamine (transamination products of pyruvate and glutamate, respectively) and, finally, of ammonia. Although there is currently no cure, treatment includes injections of structurally similar compound, N-Carbamoyl-L-glutamate, an analogue of N-Acetyl Glutamate. This analogue likewise activates carbamoyl phosphate synthetase I. This treatment mitigates the intensity of the disorder. If symptoms are detected early enough and the patient is injected with this compound, levels of severe mental retardation can be slightly lessened, but brain damage is irreversible. Early symptoms include lethargy, vomiting, and in some cases deep coma.

G.) Ornithine Transporter Deficiency

This condition results from mutation of the ORNT1 gene that encodes the mitochondrial membrane ornithine transporter and the mode of inheritance is autosomal recessive. The failure to import cytosolic ornithine into the mitochondrial matrix renders the urea cycle inoperable and subsequently leads to the HHH syndrome (hyperornithinemia, hyperammonemia, and homocitrullinuria). Growth and developmental delays, learning disabilities (especially speech delay), periodic confusion and ataxia are typical presenting symptoms. Clotting factors VII and X may be deficient in patients with hyperornithinemiahyperammonemia-homocitrullinemia syndrome leading to mild coagulopathy. Episodic lethargy and vomiting may also be evident in patients with this condition. Patients with hyperornithinemia-hyperammonemia-homocitrullinemia syndrome have abnormally high ornithine levels despite normal ornithine transcarbamoylase function, because the urea cycle cannot continue without ornithine inside the mitochondria, therefore ammonia disposal is also slow and blood ammonia levels rises. Ornithine transcarbamoylase within the mitochondrial matrix may convert lysine to homocitrulline in the absence of ornithine, causing high blood levels of homocitrulline and homocitrullinuria. However, this theory is controversial because some studies have shown no correlation between lysine supplementation and homocitrulline levels; moreover, the role of the lysine transcarbamoylase that lies outside the inner mitochondrial membrane is still not known.

III.) Explain the following manifestations in the different cases that are discussed below CASE 1
Patient: Male infant born after a full-term pregnancy and spontaneous vaginal delivery.

First 36 hours well following birth but later became lethargic and irritable and had hyperactive deep tendon reflexes. He was afebrile. blood culture rule out suspected sepsis. Antibiotics were then administered. Serum electrolytes were within normal limits except for a slightly decreased HCO3- concentration. The arterial blood pH was 7.50 (normal 7.35-7.45) and the PCO2 was 25 torr (normal, 35-45 torr); the blood urea nitrogen (BUN) level was 2 mg/Dl (normal, 5-20 mg/Dl. hyperventilating, chest x-ray was done and showed normal results. Over the ensuing 24 hours lethargy progressed to stupor then coma, thus requiring mechanical ventilation Three days later infant was transferred to a newborn intensive care unit where the sepsis workup (including a lumbar puncture) was repeated. The head circumference was 2 cm greater than at birth. Day 5 plasma ammonium level 1800 mol/L (normal <35 mol/L). Magnetic resonance imaging of the head revealed cerebral edema. While awaiting the results of plasma amino acid analysis and urinary orotic acid (orotate) analysis hemodialysis was started. After 4 hours plasma ammonium had decreased to 150 mol/L. Despite control of his hyperammonemia, the baby remained in deep coma with no evidence of spontaneous respiration or neurological function. Life support systems were discontinued after consultation with the family Day 7 infant died Plasma amino acid values Glutamine level of 1500 mol/L (normal 550-650), no detectable Citrulline (normal, 10-30 mol/L) Arginine level of 20 mol/L (normal 40-80 mol/L). Urinary orotate was 100-fold greater than normal. Postmortem analysis of hepatic ornithine transcarbamylase (OTC) activity revealed it to be <2% of normal. A review of the family history, showed the two of the mothers brother had died in coma within the first week of life. Encephalitis (inflammation of the brain) had been given as the cause of death.

Although this neonatal onset case is frequently, but erroneously, cited as the characteristic of OTC deficiency, it has become apparent over the past several years that OTC deficiency, the most common of the urea cyle disorders, occurs most often in older patients.

CASE 2
Patient: 9 months old boy Before 9 months of age, this boys medical history was unrevealing and his development normal at 9 months of age, At 9 months of age following antibiotic treatment of otitis media, he was admitted to the hospital with a chief complaint of irritability, vomiting and lethargy. Over a period of 24 hours, he rapidly progressed to coma and was unresponsive except to painful stimuli & was afebrile Diagnosis of Reye Syndrome Transferred to The John Hopkins Hospital. Laboratory studies on admission revealed plasma ammonium level of 607 mol/L Plasma glutamine level of 1188 mol/L Plasma citrulline level of 6 mol/ Urinary orotate excretion of 2669 mol/L creatinine (normal, < 2 mol/L). Emergency treatment Intravenously administered sodium benzoate, phenylacetate, and arginine hydrochloride, after which his plasma ammonium value decreased to 153 mol/L and 50 mol. One and 12 hours later and remained normal thereafter. Computerized tomography (CT) scan of head revealed severe cerebral edema. Coma persisted for 4 days Liver biopsy revealed 5.7% of normal OTC activity and normal carbamyl phosphate synthetase activity.

Case 3
Patient: 5-year old girl Uncomplicated as were her first 3 years of life 3 years of age abrupt onset of episodes of grogginess approximately every 4 to 6 months, often associated with combative behavior and biting but not associated with any discrete observable illness. During one of these episodes, she was admitted to a regional medical center, Plasma ammonium level noted to be increased but this finding was disregarded because ammonium levels were said to be problematical. Hospitalized for 5 days during that episode and recovered after a course of intravenous fluids.

 Three weeks before admission to The John Hopkins Hospital, the patient had been in and out of grogginess. Sent home from school because of excessive sleepiness. During this episode, she had the previously observed aggressive outbursts that included awakening her parents by biting. Admitted to the local hospital Lethargic, agitated and ataxic, Afebrile Plasma ammonium level was 193 mol/L Arterial blood gases pH 7.45, Pco2 = 30 torr Within 48 hours unresponsive to all but painful stimuli and require mechanical ventilation Transferred to The John Hopkins Hospital. On arrival, deeply comatose, unresponsive and on a ventilator Plasma ammonia levels 314 mol/L Plasma glutamine levels 1321 mol/L Urine orotate was five-fold greater than normal CT Scan cerebral edema Promptly treated intravenous benzoate/phenylacetate and arginine 24 hours after admission Plasma ammonia concentration 34 mmol/L Diagnosis: OTC deficiency Over the ensuing week she gradually improved, became ambulatory and communicative, but it became apparent that she was cortically blind. Three weeks after admission, vision improved and was discharged Most recent school evaluation suggests that she has changed from a bright interactive 5-year old girl to a withdrawn, confused, inattentive child.

CASE 4
Patient: 30-year old white man admitted to a local hospital for lethargy, confusion and forgetfulness. For the next 5 days Continued to be disoriented with vomiting, loss of memory, ataxia and slurred speech. Diagnosis of encephalopathy of unknown etiology was made Physical examination was normal with the exception of the neurologic exam, afebrile Unable to perform simple arithmetic tasks and had poor short- and long-term memory Asterixis noted Initial laboratory tests including liver function tests, were within normal limits. Day 5 plasma ammonia level 104 mol/L rising to 194 mol/L the following day Gradually imporved and was discharged several days later with a diagnosis of Reye syndrome Positive allopurinol test Diagnosis: OTC deficiency Within his pedigree there are 6 other late-onset OTC-deficient males

DIAGNOSIS The first case exhibits the clinical course common to all neonatal-onset urea cycle disorders. The following are important points: 1) The infant is almost always full term 2) The clinical symptoms usually start 24 hours after birth because it takes time for ammonium to accumulate (during pregnancy the placenta clears the ammonium from the blood of the fetus and the maternal liver detoxifies it) 3) Respiratory alkalosis (evidence of overbreathing caused by hyperammonemia) 4) High plasma ammonium concentration 5) Glutamine, a storage site for ammonium, accumulates 6) There is an absence (or nearly so) of plasma citrulline (the product of OTC reaction) 7) Coma can occur 8) Orotic aciduria (a pyrimidine) occurs as a consequence of accumulation of carbamyl phosphate (the substrate for ornitine transcarbamylase), which promotes biosynthesis. The history of male deaths in the maternal line strongly supports the diagnosis of OTC deficiency, an X-linked disorder. Three cases of late-onset OTC deficiency (the most commonly occurring urea cycle disease) are included to emphasize the great phenotypic and genetic variability of this inborn error of metabolism. Thus, it may be assumed that because OTC deficiency is an X-linked disease, the variability in males is a function o a mutation at the OTC locus that permits the synthesis of some OTC activity. Variability in females is best explained by the phenomenon of random X-chromosome inactivation. When a large proportion of a females active X-chromosome has a mutation at its locus, the female then has a limited ability to synthesize OTC. Hence, she will have restricted ability to synthesize urea and will be vulnerable to hyperammonemic encephalopathy.

Manifestations of Inborn Errors of Urea Synthesis

A.) Hyperammonemia
Ammonia toxicity Since ammonia is toxic to central nervous system particularly to glial cells blood ammonia level must be within normal range. If blood ammonia level raises due to any reason symptoms of ammonia intoxication appears. They are slurred speech, blurred vision and tremors. Coma and death can occur in severe cases. Mechanism of ammonia toxicity Mechanism of toxic effect of ammonia on brain is not clearly understood. However ammonia can cause brain toxicity by three ways.

1. The entry of ammonia into brain leads to formation of glutamate by the reversal of glutamate ehydrogenase reaction. This depletes available -keto glutarate in the brain. As a result citric acid cycle operation is impaired and ATP production diminishes. This leads to brain cell dysfunction. 2. Since the brain is rich in glutamine synthetase the ammonia which enters brain is used for glutamine synthesis. This leads to depletion of cellular ATP and cell dysfunction. 3. Since glutamate is considered as neurotransmitter the toxic effect of ammonia may be due to over stimulation of nerve cells by glutamate formed from ammonia and -keto glutarate by the action of glutamate dehydrogenase. Causes for ammonia toxicity 1. If hepatic function is impaired plasma ammonia rises to toxic level. Liver function can impair in cases of poisoning due to carbon tetra chloride, heavy metals and viral infections. 2. If collateral communication is developed between portal vein and systematic blood plasma ammonia rises to toxic level. In cirrhosis collateral communication develops between portal vein and systematic blood. 3. Consumption of protein rich diet after gastro intestinal hemorrhage can cause ammonia toxicity.

B.) Orotic Aciduria

A pyrimidine occurs as a consequence of accumulation of carbamoyl phosphate (the substrate for ornithine transcarbamylase), which promotes pyrimidine biosynthesis. The history of male deaths in the maternal line strongly supports the diagnosis of OTC deficiency, an X-linked disorder. It arises secondary to blockage of the urea cycle, particularly in ornithine transcarbamylase deficiency (or OTC deficiency). You can distinguish this increase in orotic acid secondary to OTC deficiency from hereditary orotic aciduria by looking at blood ammonia levels and the BUN. In OTC deficiency, because the urea cycle backs up, you will see hyperammonemia and a decreased BUN.

C.) Hyperglutaminemia
Hyperglutaminemia manifests as a psychological problem, this is due to a buildup of glutamate and ammonia, since the body can break down glutamine into ammonia and glutamate. Glutamine proliferates in the CNS, and its interstitial and cerebrospinal fluid concentrations are at least one order of magnitude higher than of any other amino acid. Glutamine transport from blood to the brain is insufficient to meet the demand of the brain tissues for this amino acid. This demand is met by intracerebral Glutamine synthesis from glutamate, a reaction carried out by Glutamine Synthetase, an enzyme residing in astrocytes. A major proportion of astroglia-derived Glutamine is shuttled to neurons where it is degraded by phosphate-activated glutaminase giving rise to the excitatory neurotransmitter amino acid glutamate. Glutamate released from neurons is taken up by astrocytes, and reconverted to glutamine. Glutamine effluxes from astrocytes, its neuronal uptake and enters to the

blood via the cerebral capillary endothelial cells are mediated by different amino acid carriers. The glutamine, specific carriers also largely contribute to glutamine efflux from the brain to the vascular bed. Excessive accumulation of glutamine in brain cells may be deleterious to brain function. The brain relies on glutamine synthesis for the removal of excess ammonia and the temporary storage of nitrogen. As this process is predominantly localized in the astrocytes, acute hyperammonemia causes increased intracellular production of glutamine from glutamate and ammonia via glutamine synthetase, which leads to astrocyte swelling and dysfunction. Excess Gln is released into the extracellular space, and directly alters the local astrocyte-neuronal synaptic environment. A consequent imbalance of excitatory versus inhibitory neurotransmission occurs because of increased glutamate production in conjunction with decreased synaptic uptake of glutamate. In hyperammonemia, excess glutamine leads to cerebral edema, which largely results from its interference with mitochondrial function.

D.) Low levels of Citrulline

Citrulline plasma concentrations are likely to be low during a hyperammonemic decompensation. Citrulline is a precursor of arginine. Arginine is a contributing member of the various amino acids found in the urea cycle, which is responsible for detoxifying ammonia. Ammonia is a byproduct of bacterial metabolism in the intestinal tract and must be detoxified in the liver. If the citrulline level is low, arginine will not be synthesized, this will result in accumulation of ammonia.

E.) Altered level of consciousness

Altered level of consciousness is evident when there is already hyperammonemia. It would lead to accumulation of ammonia and glutamine in the brain. This will now increase the level of glutamine; excessive accumulation of Gln in brain cells may be deleterious to the brain function.

IV.) The rationale behind the use of benzoate and phenylacetate

The administration of Na benzoate and Na phenylacetate to humans activates the biosynthesis of two nitrogen-containing compounds, hippurate and phenylacetylglutamine. The net effect of these reactions is to stimulate alternative routes for nitrogen dispossal. Hippurate is the product of detoxification of sodium benzoate by elimination as a conjugate with glycine, provides a pathway for the disposal of waste nitrogen. Phenylacetate supports the production of phenylacetylglutamine. Because of its repulsive odor, phenylacetate is supplied as phenylbutyrate. Phenylbutyrate is a prodrug that is rapidly converted in vivo to phenylacetate, which combines with glutamine to form phenylacetylglutamine. The phenylacetylglutamine, containing two atoms of nitrogen, is excreted in the urine, therefore assisting in clearance of nitrogenous waste.