IJCEM Copyright © 2008-All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711
Int J Clin Exp Med 2013;6(7):478-487

Original Article
High-throughput asparaginase activity assay in serum of children with leukemia

Christian A Fernandez, Xiangjun Cai, Allie Elozory, Chengcheng Liu, J Carl Panetta, Sima Jeha, Alejandro R Molinelli, Mary V Relling

Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA;
Department of Oncology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA

Received June 11, 2013; Accepted July 9, 2013; Epub August 1, 2013; Published August 15, 2013

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 (R2 = 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. (IJCEM1306007).

Keywords: Asparaginase activity, asparaginase, acute lymphoblastic leukemia, high throughput, clinical assay

Address correspondence to: Dr. Mary V Relling, Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, 262
Danny Thomas Place, MS 313, Memphis, TN 38105, USA. Phone: 901-595-2348; Fax: 901-595-8869; E-mail: mary.relling@stjude.org