Clinical application of enzyme immunoassay in the analysis of citrullinemia.

We have developed a sensitive enzyme immunoassay (EIA) for the quantification of argininosuccinate synthetase (ASS) in the range of 0.05-1 ng/tube using a Fab'-peroxidase conjugate prepared with maleimide compound devised by Ishikawa et al. Amounts of hepatic ASS in control subjects were determined by this method with the purified enzyme as a standard and their specific activities were calculated to be 1.3-1.4 mumol product/min (U)/mg of ASS, which was quite similar to that of the purified enzyme. Amounts of ASS in the liver of patients with three types of citrullinemia, type I, II and III according to our arbitrary classification, were determined by the EIA method. The following results were obtained: hepatic ASS from a patient diagnosed as type I citrullinemia because of its abnormally large Km values for citrulline and aspartate was calculated to have a specific activity of 0.18 U/mg of ASS, indicating that the enzyme is a variant; specific activities of ASS in the hepatic extract of type II citrullinemic patients were 1.2-1.3 U/mg of ASS, confirming the finding obtained by single radial immunodiffusion method that the decrease of the activity in type II is caused by a decrease in the total amount of ASS protein; a very small amount of immune cross-reactive material was detected in the liver of a patient diagnosed as type III citrullinemia based on findings of undetectable ASS activity in the liver and cultured skin fibroblast.