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High-throughput asparaginase activity assay in serum of children with leukemia
Christian A Fernandez1, Xiangjun Cai, Allie Elozory
1Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital 262 Danny Thomas Place, Memphis, TN 38105, USA.
Insights
A new, fast, and sensitive assay accurately measures asparaginase activity in patient serum, crucial for monitoring leukemia treatment effectiveness and identifying patients with low drug exposure.
Area of Science:
- Biochemistry
- Pharmacology
- Clinical Chemistry
Background:
- Asparaginase is a key antineoplastic agent in acute lymphoblastic leukemia (ALL) therapy.
- Monitoring serum asparaginase activity is vital due to significant inter-individual pharmacokinetic variability.
- Accurate measurement is needed to identify patients with suboptimal drug exposure during leukemia treatment.
Purpose of the Study:
- To develop a rapid, sensitive, and high-throughput assay for quantifying asparaginase activity in patient serum.
- To establish a reliable method for monitoring therapeutic drug levels in leukemia patients.
Main Methods:
- Asparaginase activity was measured using an enzymatically-coupled assay monitoring NADH oxidation to NAD+.
- The assay was adapted to a 96-well format for high-throughput analysis.
- Assay linearity, precision, and reportable range were rigorously evaluated.
Main Results:
- The assay demonstrated a linear range from 0.025 to 2.2 IU/mL (R² = 0.998), extendable to 4.0 IU/mL.
- Excellent inter-assay (CV% 8.8-9.0) and intra-assay (CV% 2.7-3.3) precision were achieved.
- The method proved to be fast, sensitive, and suitable for various asparaginase formulations.
Conclusions:
- The developed 96-well assay is a high-throughput, accurate, and sensitive tool for serum asparaginase activity assessment.
- This assay facilitates close monitoring of asparaginase levels in children undergoing leukemia treatment.
- It enables timely identification of patients requiring dose adjustments for optimal therapeutic outcomes.
Abstract:
Asparaginase is an antineoplastic agent used in combination therapy for acute lymphoblastic leukemia (ALL). The asparaginase activity measured in serum reflects the effectiveness of the drug. However, the wide inter-individual variability in the pharmacokinetics of asparaginase suggests that the serum activity should be closely monitored in patients during therapy. In order to identify patients with low asparaginase exposure during treatment, a fast, sensitive, and high-throughput assay is required for measuring asparaginase activity in patient sera. In this study, asparaginase activity was determined by monitoring the enzymatically-coupled oxidation of reduced nicotinamide adenine dinucleotide (NADH) to NAD(+) in a 96-well format. The rate of disappearance of NADH (ΔmOD/minute) was directly proportional to the activity of asparaginase, and the linear range of the assay was established from 0.025 to 2.2 IU/mL (R(2) = 0.998) with a reportable range that was extended to 4.0 IU/mL by dilution with serum albumin. Inter-assay precision was established (low control CV% = 8.8, high control CV% = 9.0), as was intra-assay precision (low control CV% = 3.3, high control CV% = 2.7). The method is high-throughput and provides a broader linear range of detection compared to previously described assays. The speed, ease, and accuracy of the assay make it suitable for assessing serum asparaginase activity after standard doses of native E. coli, Erwinia, and PEGylated E. coli asparaginase given to children during the treatment of leukemia.
