Final Diagnosis -- arginosuccinase deficiency (hyperammonemia)


Quantitative plasma amino acid levels reveal marked elevation of arginosuccinnic acid and its anhydrides. Citruline is also elevated. These results support the diagnosis of ARGINOSUCCINASE DEFICIENCY.

Contributor's notes:

The urea cycle is a six step biochemical pathway that the body uses to eliminate nitrogenous wastes (excess ammonia). The cycle is diagrammed below. The essential points are that nitrogen enters the cycle both in the form of ammonia and aspartate, and leaves the cycle as urea, which is rapidly and effectively cleared by the kidney. The nitrogens of interest are highlighted in red, and the enzymes that run the cycle are abreviated as follows:

NAGS = N-acetyl-glutamate synthetase
CPS = carbamyl phosphate synthetase
OTC = ornithine transcabamylase
AS = argininosuccinic acid synthetase
AL = argininosuccnase (argininosuccinate lyase)
AR = arginase

If there is an inborn error of urea metabolism (ie., a deficiency of one of these enzymes), then the cycle does not run and urea is not generated. Rather, there is a build up of the urea cycle intermediates that precede the block and a toxic build up of ammonia within the blood. In the current case, the deficiency of arginosuccinase (sometimes referred to as arginosuccinic acid lyase) resulted in a build up of arginosuccininc acid, citrulline, glutamine and ammonia.

While there is a wide range of expression in urea cycle abnormalities, including neonatal, childhood and adult presentations, many of the unfortunate individuals with these disorders present early and fare poorly. In such cases, liver transplantation may be considered.

Inborn errors of metabolism are associated with all six enzymes. The incidence of arginosuccinase deficiency is reported as 1 in 75,000. All of the urea cycle disorders have follow an autosomal recessive inheritence pattern, except for the ornithine transcarbamylase deficiency, which is X linked.


The ammonia level obtained in this case was done using a colorimetric assay on the Kodak Ektachem Analyzer (Eastman Kodak Company, Rochester, NY) using an Ektachem NH3/Amon Clinical Chemistry Slide (Johnson and Johnson Clinical Diagnostics, Rochester, NY). The procedure employs a multilayered analytical system fixed onto a polyester support (below).

Essentially, the 10 microliter patient sample is added to a reagent layer consisting of a pH 9.2 buffer which effectively converts whatever ammonium ion (NH4+) is present to ammonia(NH3). Uncharged, ammonia then passes through a semipermiable membrane where it reacts with bromophenol blue to produce a blue dye which is read at a wavelenght of 600 nanometers. The amount of ammonia present in the sample is proportional to the amount of blue dye produced.

The serum amino acid levels were determined by High Performance Liquid Chromatography using Perkin-Elmer HPLC instrumentation (Perkin Elmer, Norwalk, Connecticut 06859) and the Pickering Amino Acid Module (Pickering Laboratories, Inc., Mountain View, CA 94043). Data were analyzed using Turbochrom 3 software (PE Nelson, Cuppertino, CA 95014) on a DEC station 325 personal computer (Digital Equipment Corporation, Acton, MA)

The basic premise behind liquid chromatography is the separation of multiple different solutes within a solution by passage through a column. The type of column employed in this case uses an ion-exchange mechanism. One can think of the column as having two components, one stationary and the other mobile. The stationary component is polar and the mobile component starts out non-polar. Each solute (ie., amino acid) will have a certain degree of affinity for the stationary versus the mobile component based upon such considerations as the pH and ionic strength of the mobile phase, and the polarity of the individual amino acid and its tendency to donate or accept protons (ie., its pk', or its acid-base titration curve). Then as a function of time, the ionic strength of the mobile phase is increased and the bound solute is competitively released from the mobile phase.

Since the time it takes for a given amino acid to pass through the column is a function of its affinity for the mobile versus the stationary phase, there results a separation of the different amino acids. That is, they will exit the column at different times. The time each amino acid takes to travel through the collum is characteristic for each amino acid under any given conditions, and is termed its Retention Time.

Of course, separating out the amino acids is only half the problem. Additionally, for the diagnosis of inborn errors of metabolism, one must quantify them as well. This is done colorimetrically. As the amino acid eluate leaves the column it passes through a reaction chamber where reacts with ninhydrin to produce a purple pigment (Ruhemann's Purple), which is read at a wavelength of 500 nanometers. One can therefore plot absorbance versus time, and generate a graph which has peaks that correspond to the amino acid retention times. The identity of each amino acid is determined by the retention time of given peak, and the quantity is determined by the area underneath that peak. A specific amount of norleucine (a non-naturally occurring amino acid) is added to the sample to add as an internal standard.

The overall setup follows:

Again, the results for this patient follows:


  1. Blitzer and Cowan, Inborn Metabolic Errors chapter in Clinical Laboratory Medicine, edited by Kenneth McClatchey, Williams and Wilkins, p. 494-495, 1994.
  2. Bowers, Ullman and Burtis, Chromatography chapter in Tietz Textbook of Clinical Chemistry, second edition, edited by Burtis and Ashwood, Saunders, p. 670, 1986.
  3. Brusilow and Howich, Urea Cycle Enzymes chapter in The Metabolic and Molecular Bases of Inherited Disease, seventh edition, edited by Scriver, Beaudet, Sly and Valle, p. 1187-1232, 1995.
  4. Ektachem Clinical Chemistry Slides (NH3/AMON), Test Methodology, publication No. MP2-90, CAT No. 185 1401, Johnson and Johnson Clinical Diagnostics, May 1995.
  5. Silverman and Christenson, Amino Acids and Proteins chapter in Tietz Textbook of Clinical Chemistry, second edition, edited by Burtis and Ashwood, Saunders, p. 670, 1986.

Contributed by Eric Schubert, MD, Jeff Martens, MD, and Warren Diven, PhD


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