The Urea Cycle in Healthy Individuals
The urea cycle (Fig. 1) has two functions in the human metabolism. It is responsible for the de novo synthesis of arginine and converting waste nitrogen (ammonia) into urea, which can readily be excreted.8 Urea is made up of ammonia, nitrogen from aspartate, and carbon dioxide in the form of bicarbonate, allowing approximately 90% of waste nitrogen-containing compounds to be expelled in urine. In a healthy individual, the uric acid cycle goes through six enzyme reactions and two mitochondrial transporters to convert ammonia into a less toxic substance, such as urea or uric acid.2
The urea cycle, its steps, and essential enzymes. From Mitchell et al., 2009.
The cycle begins in the mitochondria where ammonia and bicarbonate react to produce carbamoyl phosphate.10 Once catalyzed by carbamoyl phosphate synthetase I (CPS1), the anion is able to enter the urea cycle.11 Throughout the cycle, carbamoyl phosphate is converted to citrulline, and then argininosuccinase with the help of a few specialized enzymes.1 In order to cleave the argininosuccinate in the fourth stage of the urea cycle, an enzyme called argininosuccinase must be present. This stage is situated in the cytosol of hepatic cells. If the enzyme is present, argininosuccinate is cleaved to generate arginine and fumarate. The arginine produced by this reaction is cleaved by arginase to form urea, which can be readily excreted.
The Urea Cycle in Diseased Individuals
If an individual has a deficiency in any of the six essential enzymes, they are diagnosed with a urea cycle disorder as a consequence.2 In the case of an argininosuccinate lyase deficiency, the enzyme argininosuccinase is scarce. As a key enzyme for the completion of stage four of the urea cycle, if it is lacking, the cycle is halted at this phase and unable to continue processing toxins properly. Without its specialized enzyme, argininosuccinate is unable to be processed into fumarate and arginine.1 Arginine is a precursor for the production of urea, nitric oxide (NO), glutamate, polyamines, creatine, proline and agmatine.12 If it is absent, not only will toxins accrue in the body but the body will also suffer from a deficiency of nitric oxide and the other metabolites mentioned. Argininosuccinate lyase deficiency, as well as the other urea cycle disorders, are not contagious and are a result of genetic inheritance.13
Genetic Inheritance Patterns
When a deficiency in the argininosuccinate enzyme occurs, the urea cycle is unable to proceed properly and leads to an accumulation of toxins in the blood.2 This deficiency is caused by an error in an individual’s genome. This error is inherited in an autosomal recessive manner, meaning if an individual is affected by the condition, then their sibling has a 25% chance of inheriting it as well.14 In addition, their sibling has a 50% chance of being a carrier for the condition and a 25% chance of being unaffected.14 Even though it is a recessive condition, ASL deficiency has a pretty significant prevalence of approximately 1 in every 70,000 births.1 The human ASL gene is located on chromosome 7q11.21 and contains 17 exons.15 Typically, the ASL gene encodes a homotetramer enzyme with four identical subunits and four active sites. Argininosuccinate lyase deficiency is caused by multiple heterogeneous mutations in the ASL gene, including nonsense, missense, deletions, and insertion mutations.16 The most common mutations causing an ASL deficiency tend to occur in the fourth, fifth, and seventh exons. These mutations lead to argininosuccinase with reduced or absent activity.16 An argininosuccinate lyase deficiency has been associated with several life-threatening conditions, including the following: elevated plasma ammonia, liver dysfunction, hypertension, neurocognitive deficiencies, hepatic disease, and electrolyte imbalances.1 With argininosuccinate lyase being the only enzyme in the body with the ability to generate arginine, if its activity is limited, the body is unable to produce it.12 In this case, arginine would need to be supplemented in the diet.
There are two manifestations of argininosuccinate deficiency: neonatal onset and late-onset.17 There are a number of screening methods that can detect these two onset forms. Firstly, individuals can be screened for the genetic disorder before they are even born through neonatal screening. This procedure identifies the neonatal form of the disorder. Mutational analysis, performed on amniocytes, measures the levels of argininosuccinic acid in the amniotic fluid.11 If the levels are elevated, the fetus is affected. 11 These procedures are often performed on women who are affected by the condition argininosuccinic aciduria (ASA), which is caused by an ASL deficiency.18 Due to its heritable properties, there is a greater chance a child with inherit an ASL deficiency if either parent carries a homozygous recessive gene for it.18
After birth, the neonatal form of an ASL deficiency can be recognized by newborn screening in conjunction with various symptoms including tiredness, poor feeding, fluctuating body temperature, and vomiting.12 Newborn screenings use tandem mass spectrometry (TMS) to measure the amino acid levels of argininosuccinic acid and citrulline in the blood.11 Blood samples may be obtained through a simple prick to the skin or through a sterile needle depending on the preference of the professional performing the examination. If the levels are elevated, they generally indicate an argininosuccinate lyase deficiency.The late-onset form may manifest itself with similar symptoms to the neonatal form but extends to hyperammonemia, cognitive impairment, abnormal behaviour, brittle hair, and cirrhosis.1 Sometimes the condition may not present any symptoms at all, which is when regular TMS in newborns is advantageous. Due to the unpredictable nature of apparent symptoms, a variety of treatments have been created to manage current symptoms and prevent the development of others.
There have been two principal treatments generated specifically for argininosuccinate lyase deficiencies.13 An ASL deficiency is currently incurable and treatments tend to focus on regulation. The first is aimed towards controlling the elevated levels of ammonia in the body, while the second focuses on preventing hyperammonemia and long-term complications associated it. When ammonia has the chance to accumulate and minor hyperammonemic periods occur, oral protein intake is limited or halted.15 Reducing oral protein intake minimizes intestinal ammonia production and prevent further accretion of toxins. In order to treat the symptoms of hyperammonemia, the individual is then supplemented with glucose, lipids, and arginine supplementation to stimulate and complete the urea cycle.15
For chronic treatment, limited protein consumption and supplementation of arginine are managed through the diet.15 Alternative treatments include the use of sodium benzoate and sodium phenyl butyrate, chemicals which cause nitrogen to be excreted as phenylacetylglutamine and hippuric acid in the urine.13 Currently, the only long-term modifications of argininosuccinate lyase deficiencies can be achieved by liver transplantation.13 This type of treatment is only used in patients who have experienced resistance to the other treatments and recurrent hyperammonemia.13 However, due to the genetic heritability of urea cycle disorders, future treatments may include gene therapy techniques.19 By transplanting normal genes into cells and replacing the defective ones, the genetic urea cycle disorders could be corrected and curable.
Argininosuccinate lyase deficiency is caused by a mutation in the gene that encodes the enzyme and is inherited as an autosomal recessive disorder.1 Argininosuccinate lyase is an enzyme that is an essential enzyme for the completion of the urea cycle. Without it, the urea cycle is unable to be performed properly and an accumulation of toxins develops in patient’s blood and tissues.2 Individuals with this disorder can be diagnosed both before and after birth, each stage of life demonstrating its own set of symptoms.17 Multiple treatments have been introduced for the disorder to decrease the severity of symptoms and to regulate the urea cycle.13 Currently, the only long-term modifications for ASL deficiencies are liver transplants.20 Unfortunately, these modifications are difficult to obtain and not available for every individual suffering from an ASL deficiency. With the increasing interest in gene therapy, there is still a chance that a potential cure for urea cycle disorders will be discovered that is more accessible, cost-effective and produces long-term effects.19