Evaluation of L-asparagine metabolism in animals and man.

acid (13) has been modified to permit its application to the analysis of L-asparagine and L-aspartic acid in tissues and body fluids. The method involves a sequence of enzymic reactions which result ultimately in the oxidation of NADH. With this method it has been possible to determine the concentration of L-asparagine in various tissues, to follow the disposition of L-asparagine in animals and human subjects under various conditions, and to assess the deficiency of L-asparagine in plasma and tissues after the administration of L-asparaginase from Escherichia coli. most commercial preparations by chromatography through a column (2 x 10 cm) of Dowex l-X4 (formate) (200 to 400 mesh). Even at 4° such purified solutions of L-asparagine were slowly hydrolyzed to L-aspartic acid and ammonia and were freshly prepared. The L-asparaginase (EC 3.5 .1.1) E.C.-2 used in these studies had a specific activity of approximately 150 i.u./mg protein and was designated TAD6A by the manufacturer, E. R. Squibb and Sons, New Brunswick , N. J. The 6C3HED lymphoma was maintained by subcutaneous transfer in C3H mice. The L5 178Y and P8 1SY leukemias were propagated in the ascitic form in AKD2F1 mice. Unless otherwise indicated, all animals were maintained on a diet of Purina laboratory chow and tap water ad libitum. Hypophy sectomized rats were purchased from Charles River Breeding Laboratories, Inc., Boston, Mass.; albino rats were partially hepatectomized under ether anesthesia in the standard man ner. Sham operations were performed on the controls. Preparation of Tissues. Blood was drawn into heparinized tubes from humans by venipuncture, from mice by cardiac aspiration after Nembutal anesthesia, and from rats by decapitation. Tubes containing blood were iced and the plasma was separated at 4° in an International clinical centrifuge at 2500 rpm for 15 mm. Erythrocytes were either processed directly or washed with 0.9% NaCl solution at 4° prior to being processed. The separated plasma was pipetted into 10-ml polycarbonate centrifuge tubes and heated in a boiling water bath for 20 mm. Apart from an initial 1:1 SUMMARY A rapid, sensitive, and specific method for the determina tion of L-asparagine and L-aspartic acid has been developed based on an enzymatically coupled oxidation of reduced pyridine nucleotide. With this spectrophotometric or fluori metric method, the concentration of these amino acids has been determined in normal plasma, erythrocytes, tumors, urine, and cerebrospinal fluid. A modification of this method permitted measurement of 0.001 i.u. L-asparaginase. A variety …