Fernandez Christian A, Cai Xiangjun, Elozory Allie, Liu Chengcheng, Panetta J Carl, Jeha Sima, Molinelli Alejandro R, Relling Mary V
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital 262 Danny Thomas Place, Memphis, TN 38105, USA.
Int J Clin Exp Med. 2013 Aug 1;6(7):478-87. Print 2013.
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.
天冬酰胺酶是一种用于急性淋巴细胞白血病（ALL）联合治疗的抗肿瘤药物。血清中测得的天冬酰胺酶活性反映了该药物的疗效。然而，天冬酰胺酶药代动力学存在较大的个体间差异，这表明在治疗期间应对患者的血清活性进行密切监测。为了识别治疗期间天冬酰胺酶暴露量低的患者，需要一种快速、灵敏且高通量的检测方法来测定患者血清中的天冬酰胺酶活性。在本研究中，通过监测96孔板中还原型烟酰胺腺嘌呤二核苷酸（NADH）酶促偶联氧化为NAD(+)来测定天冬酰胺酶活性。NADH的消失速率（ΔmOD/分钟）与天冬酰胺酶的活性直接成正比，该检测方法的线性范围为0.025至2.2 IU/mL（R(2)=0.998），通过用血清白蛋白稀释可将报告范围扩展至4.0 IU/mL。建立了批间精密度（低对照CV%=8.8，高对照CV%=9.0）以及批内精密度（低对照CV%=3.3，高对照CV%=2.7）。与先前描述的检测方法相比，该方法具有高通量且检测线性范围更广。该检测方法的速度、简便性和准确性使其适用于评估白血病治疗期间给予儿童标准剂量的天然大肠杆菌、欧文氏菌和聚乙二醇化大肠杆菌天冬酰胺酶后的血清天冬酰胺酶活性。
