ENZYMATIC TECHNIQUE FOR MEASURING N-PHOSPHONACETYL-L-ASPARTIC ACID IN TISSUES

  • 1 January 1978
    • journal article
    • research article
    • Vol. 62  (10) , 1503-1507
Abstract
An enzymatic technique was presented for measuring N-phosphonacetyl-L-aspartic acid (PALA; NSC-224131) in biologic specimens. Tightly bound PALA was quantitatively detached from its target enzyme, L-aspartic acid transcarbamylase (ATCase), by heating at 95.degree. C for 5 min. Denatured proteins were removed by centrifugation. PALA in the supernatant fluid was quantitated by exposing intact splenic ATCase to representative aliquots or subdilutions of the resultant supernatant in the presence of L-[4-14C]aspartic acid and carbamyl phosphate. After 30 min incubation at 37.degree. C, unreacted L-[4-14C]aspartic acid was dissipated enzymatically and newly formed [4-14C]carbamyl-L-aspartic acid was quantitated by scintillation spectrometry. The percentage inhibition of ATCase responded in a linear way to the logarithm of the concentration of PALA between 0.10 and 1.00 .mu.M. The PALA concentration of an unknown was determined indirectly by matching the percentage inhibition caused by the unknown to the inhibition caused by a known series of standard concentrations of PALA over the linear range. This assay was sensitive, adequately reproducible despite the use of an unpurified enzyme and notably facile. It can be used to measure PALA in plasma, urine, tissues and tumors of subjects treated with this new oncolytic drug.