THERAPEUTIC AND STABLE ISOTOPIC INVESTIGATION OF THE UREA CYCLE DISORDERS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The urea cycle is required for excretion of excess nitrogen compounds generated by dietary intake and protein catabolism (1). Human genetic deficiencies of urea cycle enzymes are well known and usually present in the neonatal period or early infancy with metabolic crises and subsequent neurological impairment. Each disease has significant variability in severity. The most common urea cycle disorder is ornithine transcarbamylase (OTC) deficiency. The other urea cycle disorders are carbamyl phosphate synthetase (CPS), argininosuccinic acid synthetase (ASS), argininosuccinate lyase (ASL), and arginase deficiencies. Argininosuccinic acide lyase defiency is also known as Argininosuccinic aciduria (ASA). ASA has an incidence of one in seventy thousand live births and commonly presents in the neonatal period with hyperammonemic metabolic crisis. The clinic picture is that of a healthy appearing neonate who, after a short period of health, develops vomiting, lethargy and anorexia. These symptoms rapidly progress to coma and death if not treated. If the hyperammonemia is prolonged, there is severe and permanent neurolgical impairment. "Neonatal rescue" by heodialysis and alternative pathway drugs is typically followed by life long episodic hyperammonemia usually precipitated by minor infections or dietary imbalance. Recrudescence of hyperammonemia leads to further neurological injury2. Less severe forms of ASA and other urea cyc cycle disorders may present during infancy, childhood, or adulthood and are a consequence of mutation heterogeneity. Treatment of urea cycle disorders relies on two strategies(2,3). The first is reproduction of nitrogen load through the use of a protein-restricted diet. The second approach uses "alternate" or laten enzymatic pathways of the liver to conjugate amino acids to carrier molecules (exogenously administered drugs) and arginine supplementation to increase urinary excretion of nitrogenous products. Currently, ASA patients are treated only with diet and arginine therapy. The principle of arginine therapy is that by replacing the product of downstream of the impaired reaction in the urea cycle (argininosuccinic acid is converted to arginine and fumarate by argininosuccinic acid lyase); the cycle is "reprimed" to continue to produce additional argininosuccinic acid. Because of its extremely high renal clearance, it acts effectively as an efficient nitrogen sink in place of urea. A FDA approved therapy for other disorders earlier in the urea cycle, i.e., ornithine transcarbamylase deficiency and citrullinemia, is sodium phenylbutyrate (Buphenyl). At present there is little quantitative information as to the specific effect of sodium phenylbutyrate on the ability to reduce frequency of hyperammonemic crisis, hepatic transaminase levels, and citrulline/argininosuccinate levels in ASA. Sodium phenylbutyrate is rapidly converted to phenylacetate after administration. Phenylacetate is a metabolically active compound that conjugates with glutamine via acetylation to form phenylacetylglutamine. This compound is water-soluble and is then excreted in the urine. In this way, phenylbutyrate serves as an alternative vehicle for nitrogen excretion. Marked hepatomegaly is a hallmark of ASA and is not found to such a degree in the other urea cycle disorders. Hepatic fibrosis has been documented by liver biopsy of these patients and generally begins early in the disease (4,5). The majority of these patients also have elevations of hepatic transaminases (ALT and AST) to > 2x normal levels. The etiology of these elevations is not known but it has been shown that they occur independently of ammonia control. It is probable that the degree of liver fibrosis correlates to transaminase levels. As children with this disorder survive for longer periods of time with better methods of medical management, it will become more important to better control transaminase levels to avoid fibrosis, which may lead to life threatening cirrhosis. Liver transplantation has been performed in cases of severe cirrhosis. Since the unique metabolite in this condition is arininosuccinic acid and/or its breakdown products, we hypothesize that argininosuccinic acid and/or its metabolites may be the offending agent causing hepatic inflammation. Ironically, the current therapy of high dose arginine treatment is aimed at effectively decreases the frequency of hyperammonemia, hence protecting the brain, it may increase the occurrence of hepatic inflammation. Based on these observations, we hypothesize that by stimulating alternative disposal of nitrogen by diverting nitrogen flux away from the production of argininosuccinic acid, we may observe decreased hepatic inflammation as evidence by decreased LFT s, stabilization or improvement of hepatic fibrosis as measured by MRI and histology where clinically available. Moreover, we may also observe a greater tolerance for dietary peripheral nitrogen and hence decrease the frequency and magnitude of hyperammonemia and decreased steady state level of ASA.