Case:

A month old infant manifested severe hyperammonemia. This child had low levels of blood urea, elevated levels of serum transaminase activity, and a generalized hyperaminoacidemia and aminoaciduria. In addition, the blood levels of citrulline, arginosuccinate, and argine were low. Biopsy of liver tissue revealed that the level of mitochondrial carbamoyl phosphate synthetase was about 15-25% that of normal. The levels of all other enzymes in the urea cycle were normal.

The urea cycle disorders (UCD) results from the defects in the metabolism of the extra nitrogen produced by the breakdown of protein and other nitrogen-containing molecules. Severe deficiency or total absence of activity of any of the fuirst four enzymes (CPSI, OTC, ASS, ASL) in the urea cycle or the cofactor producer (NAGS) results in the accumulation and other precursor metabolites during the first few day of life. Infants with urea cycle disorder often appears normal at birth, but rapidly develops cerebral edema and the related signs of lethargy, anorexia, hyperventilation or hypoventilation, hypothermia; seizures and coma. In mild urea cycle disorders, ammonia accumulation may be triggered by illness or stress at most times, which results to multiple mild elevations of plasma ammonia accumulation. (Roth)

Why are blood levels of ammonia high in this child?

Hyperammonemia is often the sign of underlying metabolic disturbance. Normally, excess dietary and waste nitrogen, which remain after protein synthesis, are converted to urea through the urea cycle. Urea is produced in the liver where the urea cycle converts 80% of the excreted nitrogen. The five enzymes that make up the urea cycle, shown in figure 1, are regulated long term by the quantity of protein in the diet. Disorders of any urea cycle enzymes lead to accumulation of ammonia and it precursors (thus the high ammonia level of the infant). This can lead to encephalophaty and death devastating neurologic sequelae if not treated promptly. (Joseph)

Once hyperammonemia is established, further laboratory testing is needed to determine the specific diagnosis. Repeat plasma ammonia levels would be obtained frequently during the acute stage. A reminder that proper processing of the blood is of utmost importance so that levels are truly reflective. Barsotti mentions that the steps to obtain an accurate level includes a.) Ideally, patient must fast for at least 6 hours before drawing the sample, b.) Ammonia free heparin should be used; heparin because it has shown its capability to reduce red blood cell ammonia production, c.) a chilled heparinized vacuum tube that is immediately placed in ice, d.) the plasma should be separated within 15 minutes if allowed to sit, blood and ammonia concentration will spontaneously increase secondary to ongoing production of ammonia from red blood cells and the deamination of amino acids especially glutamine.  (Joseph)

Figure 1. The Urea Cycle (Joseph)

Why are the blood levels of citrulline, arginosuccinate, arginine, and urea low in this child?
Citrulline

Citrulline is the resultant product of the condensation reaction that occurs during normal function of the ornithine transcarbamylase reaction. Under normal circumstances, citrulline is condensed with aspartic acid to form argininosuccinic acid (ASA), which is a reaction mediated by the argininosuccinic acid synthase enzyme. Participation of aspartate in the reaction fixes a second waste nitrogen atom into the reaction product, ASA; the first waste nitrogen molecule derives from free ammonia in the carbamyl phosphate synthetase (CPS) reaction. Deficiency of ASA synthase leads to accumulation of citrulline, or citrullinemia. Citrulline can be metabolized outside of the liver, and ASA synthase is normally expressed in the brain, kidney, and skin fibroblasts. (Roth)

In citrullinemia, the genetic defect is expressed in all of these tissues. The body is unable to circumvent the defect by conversion of citrulline to arginine, as it can under normal circumstances. As mentioned, by reaction of citrulline with aspartic acid, a second waste nitrogen molecule is incorporated into the urea cycle; however, this reaction is impaired and results in reduction of the overall capacity of the urea cycle to dispose of ammonia by 50%. Accordingly, affected individuals have a propensity for developing hyperammonemia. (Roth)

Arginosuccinate

Argininosuccinate (ASA) lyase deficiency results in defective cleavage of ASA, which leads to accumulation in cells and excessive excretion in urine. In virtually all respects, this disorder shares the characteristics of each of the family of urea cycle defects. This deficiency’s most important characteristic is its propensity to cause hyperammonemia in the affected individual. (Roth)

The hepatic urea cycle is the major route for waste nitrogen disposal; generation of nitrogen is chiefly from protein and amino acid metabolism. Low-level synthesis of certain cycle intermediates in extrahepatic tissues makes a small contribution to waste nitrogen disposal. A portion of the cycle is mitochondrial in nature; mitochondrial dysfunction may impair urea production and result in hyperammonemia. Overall, activity of the cycle is regulated by the rate of synthesis of N-acetylglutamate, the enzyme activator that initiates incorporation of ammonia into the cycle. (Roth)

The rate-limiting step is carbamyl phosphate synthetase (CPS) disposal of waste nitrogen. In cases of genetic deficiency of an additional enzyme beyond CPS in the cycle, however, the deficient enzyme becomes rate limiting. This is the situation in argininosuccinic aciduria, despite the fact that formation of this substance ensures incorporation of the 2 waste nitrogen molecules normally found in urea. Although failure to release the arginine limits the cycle rate and slows hepatic regeneration of the distal intermediates of the cycle, this is unlikely to entirely explain the clinical findings, because ASA is excreted by the kidney at a rate practically equivalent to the glomerular filtration rate (GFR). (Roth)

Whether ASA itself may cause a degree of toxicity due to hepatocellular accumulation is unknown; such an effect could help explain hyperammonemia development in affected individuals. In any case, the rapid clearance of ASA in urine has given the name to the disease, although elevations of ASA can be found in plasma. Hyperammonemia in this disease manifests with the typical findings and have all of the attendant consequences seen in the other diseases of this category. (Roth)

Arginine

Arginase deficiency is thought to be the least common of the urea cycle disorders. This entity also manifests itself in a fashion somewhat different from the other members of the group. Two separate isozymes of the enzyme arginase exist. Type I is found in the liver and contributes the vast majority of hepatic arginase activity, while type II is inducible and found in extrahepatic tissues. The disease is caused by deficiency of arginase type I in the liver. (Roth)

The hepatic urea cycle is the major route for waste nitrogen disposal, which is generated chiefly from protein and amino acid metabolism. Low-level synthesis of certain cycle intermediates in extrahepatic tissues makes a small contribution to waste nitrogen disposal as well. A portion of the cycle takes place in mitochondria; mitochondrial dysfunction may impair urea production and result in Hyperammonemia. Overall, activity of the cycle is regulated by the rate of synthesis of N-acetylglutamate, the enzyme activator that initiates incorporation of ammonia into the cycle. (Roth)

The reaction normally mediated by arginase is the terminal step in the urea cycle, which liberates urea with regeneration of ornithine (Image 1). Consequently, as in argininosuccinic aciduria, both waste nitrogen molecules normally eliminated by the urea cycle are incorporated into the arginine substrate molecule in the reaction. (Roth)

The severe hyperammonemia observed in other urea cycle defects is rarely observed in arginase deficiency for at least 2 identifiable reasons. The first reason is that formed arginine, which contains 2 waste nitrogen molecules, can be released from the hepatocyte and excreted in urine. The second reason may be attributable to the inducibility of the type II isozyme in peripheral tissues, which can attack the arginine released by the hepatocyte and produce urea and ornithine. The ornithine returns to the liver for use in the urea cycle, while the urea is excreted. A 4-fold increase in renal type II arginase has been demonstrated in an affected patient. (Roth)

Table 2 Urea Cycle Disorders (Joseph)

Disorder                                        Deficient enzyme                                   Inheritance pattern

Carbamoyl phosphate synthetase deficiency

Carbamoyl phosphate synthetase

Autosomal recessive

Ornithine transcarbamylase deficiency

Ornithine transcarbamylase

X- linked

Citrullinemia
Arginosuccinate synthetase

Autosomal recessive

Argininosuccinic aciduria

Arginosuccinate lyase

Autosomal recessive

Argininemia

Arginase

Autosomal recessive

A colleague suggests that part of the treatment for this child is to administer arginine. Do you agree or disagree? State your reasons.

The next goal is to remove the excess nitrogen in the infant’s body. Hemodialysis is the only means of rapidly removing ammonia from the body. Peritoneal dialysis does not remove ammonia quickly enough to minimize brain injury. While preparing for hemodialysis or extracorporeal membrane oxygenation, ammonia scavenging therapy should be initiated, even prior to the diagnosis of a specific metabolic etiology, if it is felt that alteration of the central nervous status is secondary to the high ammonia level. Loading dose of L-arginine-HCL (600 mg/kg per dose), sodium benzoate (250 mg/kg per dose) and sodium phenylacetate (250 mg/kg per dose) all in 25 to 35 mg/kg 10% dextrose solution given over 90 minutes have been recommended for emergency treatment of infants with hyperammonemia and the sustained infusion of these three medications is recommended over the next 24 hours. (Joseph)

Table 3 Treatment of Intercurrent Episodes of Hyperammonemia in Patients with Urea Cycle Disorders (Joseph)

Disorder
Initial infusion

Administer over 90 min
Maintenance infusion

Daily dose given as

24 – hr infusion

Carbamyl phosphate

synthetase deficiency or

ornithine transcarbamylase deficiency
Sodium benzoate, 250 mg/ kg

Sodium phenylacetate, 250 mg/ kg

10% arginine HCl, 2 ml/ kg

Sodium phenylacetate, 250 mg/ kg

Sodium benzoate, 250 mg/ kg

10% arginine HCl, 2 ml/ kg

Citrullinemia

Sodium benzoate, 250 mg/ kg

Sodium phenylacetate, 250 mg/ kg

10% arginine HCl, 6 ml/ kg
Sodium benzoate, 250 mg/ kg

Sodium phenylacetate, 250 mg/ kg

10% arginine HCl, 6 ml/ kg

Arginosuccinic aciduria
10% arginine HCl, 6 ml/ kg
10 % arginine HCl, 6 ml/ kg

Your colleague also suggests that you should restrict the dietary intact of pyridoxine and ?-keto acids of essential amino acids. Do you agree or disagree? State your reasons.

The true mechanism of neurotoxicity in hyperammonemia is not yet fully determined. Irrespective of the underlying cause, the clinical picture is relatively constant. This implies that the pathophysiologic mechanism, focusing on the CNS, is common to all individuals with hyperammonemia. (Roth)

The normal process of removing the amino group present on all amino acids produces ammonia. The a-amino group is a catabolic key that protects amino acids from oxidative breakdown. Removing the a-amino group is essential for producing energy from any amino acid. (Roth)

Vitamin B6 (pyridoxine) is converted into pyridoxal phosphate, the coenzyme for transamination, decarboxylation, deamination, racemization, and aldol-like condensations of amino acids. In the presence of the enzyme, the coenzyme forms a Schiff base with the amino acid. There are several water-soluble B vitamins that are coenzymes or form parts of coenzymes or prosthetic groups (TABLE 1). Pyridoxal phosphate is a coenzyme of reactions involving all amino acids. Folic acid and cobalamine are coenzymes for reactions, some of which involve amino acids (viz., glycine, serine, and methionine) L-Glutamic acid, being specifically permeable to the inner mitochondrial membrane, passes to the cytoplasm. There, other nonessential (dispensable) amino acids can be formed by transamination. Because

transamination is reversible, the nitrogen of other amino acids can collect in L-glutamic acid. L-Glutamic acid can then return to the matrix of the mitochondria. There it can undergo transamination in the presence of oxaloacetate and mitochondrial GOT to form Laspartic acid. Alternatively, it can be deaminated by mitochondrial glutamic dehydrogenase (GDH) (Gupta)

However, before we discuss deamination further, let us repeat that transaminases are found both in the cytoplasm and in the mitochondria of eukaryotic cells, that the enzymes in each region have characteristic properties, and that they are responsible for synthesis of nonessential amino acids, provided a source of nitrogen is available.

Most nitrogen gets removed from amino acids via L-glutamic acid. Specifically, ammonia is removed by oxidative deamination of L- glutamic acid, a reaction catalyzed by glutamic dehydrogenase. This reaction is reversible so nitrogen can also enter amino acid synthesis by reductive amination of a -ketoglutarate. Thus, L-glutamic acid is the first amino acid to be labeled after 15 NH4+ is administered. However, this reaction is of much less importance in the total nitrogen pool than is transamination. It also specifically requires NADPH for reversal. (Gupta)

What would be your recommendations regarding protein intact for this child? State your reasons.

Protein should be withheld for a short period of time. Without protein, the body goes into negative nitrogen balance, which increases the need for nitrogen excretion and worsens hyperammonemia.

Parenteral nutrition is needed to prevent protein catabolism. Use glucose and intravenous fat emulsions, without protein, to maximize calories. Once the ammonia level is ;100 µM/1 and the patient is table, oral or nosagastric feeds may be initiated using a protein-free poweder. Essential amino acid supplementation may be needed, along with L- citrulline or L- arginine to “prime” the urea cycle. (Joseph)

You also run a battery test for determining the concentration of different amino acids in the blood and urine. You assay for Ala, Ser, pro, Glu, Lys, Gln, Asp, Val, and lie. Which ones would you expect to see elevated? State your reasons.

The biologic requirement for tight regulation is satisfied because the capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation in the periphery and transfer into the blood. Hyperammonemia never results from endogenous production in a state of health. (Roth)

An elevated blood ammonia level, although it may be secondary, must never be ignored. Moreover, since the normal ureagenic capacity of the liver is so great in relation to physiologic load, such a finding points directly to an impairment of the urea cycle in the liver. (Roth)

The CNS is most sensitive to the toxic effects of ammonia. Many metabolic derangements occur as a consequence of high ammonia levels, including alteration of the metabolism of important compounds, such as pyruvate, lactate, glycogen, and glucose. High ammonia levels also induce changes in N-methyl D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptors and causes downregulation in astroglial glutamate transporter molecules. (Roth)

As ammonia exceeds normal concentration, an increased disturbance of neurotransmission and synthesis of both GABA and glutamine occurs in the CNS. A correlation between arterial ammonia concentration and brain glutamine content in humans has been described. Moreover, brain content of glutamine is correlated with intracranial pressure. In vitro data also suggest that direct glutamine application to astrocytes in culture causes free radical production and induces the membrane permeability transition phenomenon, which leads to ionic gradient dissipation and consequent mitochondrial dysfunction. However, the true mechanism for neurotoxicity of ammonia is not yet completely defined. The pathophysiology of hyperammonemia is that of a CNS toxin that causes irritability, somnolence, vomiting, cerebral edema, and coma that leads to death. (Roth)

Reference:

Gupta, R. “Biochemistry and Molecular Biology: Nitrogen Metabolism.” SIU School of Medicine  (1999).

Joseph, M. M.D ; Hageman, J. M.D. “Perinatal/Neonatal Casebook: Neonatal Transport: A 3-Day-Old Neonate with Hypothermia, Respiratory Distress, Lethargy and Poor Feeding.” Journal of Perinatology 22 (2002): 506-09.

Roth, K. S. “Hyperammonemia”.  New York, 2006.  (July 5, 2007):  eMedicine. May 1 2007. ;http://www.emedicine.com/PED/topic1057.htm;.