Immediate cooling does not prevent the ex vivo hydrolysis of l-asparagine by asparaginase

Background: Monitoring of asparagine (ASN) during asparaginase (ASE) treatment directly links to the antileukemic effect of ASE but is challenging because of ASE-induced ex vivo hydrolysis of ASN. Assuming that ASE is not active at 4°C, immediate cooling of blood samples became the accepted method for ASN determination during ASE therapy. Methods: To evaluate the effect of immediate sample cooling on the ex vivo hydrolysis of ASN by ASE the degradation of ¹³C₄-ASN in whole blood, spiked with different ASE concentrations were analyzed HPLC-MS. ¹³C₄-ASN and ASE were added either to blood at 4°C or to blood at 37°C, which was instantly cooled down to 4°C. Results: Immediate cooling did not prevent the ex vivo hydrolysis of ASN by ASE. The rate of ASN degradation to aspartic acid depended on the amount of ASE, ASE preparation, and time. Spiked into blood at 4°C 100 U/L native E. coli ASE already immediately degraded 100% of ¹³C₄-ASN, whereas 10 U/L reduced the amount of ¹³C₄-ASN by 30%. Spiked into blood at 37°C, which was immediately cooled thereafter, 10 U/L native E. coli ASE hydrolyzed 60% of C4-ASN and 1 U/L between 5% and 10% of C4-ASN. Concentrations of aspartic acid increased in parallel with ASN degradation. In addition, the ex vivo hydrolysis also affected concentrations of glutamine and glutamic acid. Conclusions: Cooling of blood samples did not inactivate ASE. Thus, to evaluate the precise pharmacodynamics of ASE, alternative methods for effective ASE inactivation at the time of blood withdrawal are needed.