Asselin B L, Lorenson M Y, Whitin J C, Coppola D J, Kende A S, Blakley R L, Cohen H J
Strong Children's Research Center, University of Rochester, New York 14642.
Cancer Res. 1991 Dec 15;51(24):6568-73.
The antileukemic activity of L-asparaginase (ASNase), an important component of therapy for acute lymphoblastic leukemia, is thought to result from depletion of serum L-asparagine (Asn). In studies of the pharmacological effects of ASNase, investigators have reported prolonged reduction in the serum concentration of Asn after the administration of ASNase. Such measurements may not be valid because ASNase present in the blood sample may hydrolyze Asn before its determination. We examined recovery of [U-14C]Asn from blood samples with and without various concentrations of added ASNase. In the presence of greater than or equal to 0.01 IU/ml of ASNase, the amount of [U-14C]Asn recovered was less than 15% of that without ASNase. Utilizing this assay, we studied the effect of 2 known inhibitors of ASNase in an attempt to improve Asn recovery. In the presence of aspartic beta semialdehyde (ASA), or 5-diazo-4-oxo-L-norvaline (DONV), and up to 1.0 IU/ml ASNase, Asn levels remained at greater than 90% of control. ASA prevented the hydrolysis of exogenous Asn in blood samples drawn from patients after ASNase injection. We also developed a method to determine Asn in serum utilizing high pressure liquid chromatography. Using this method, we found that the Asn level was greater than 90% of a normal level in the presence of 40 mM DONV and 1.0 IU/ml ASNase. Examination of serum from 4 patients treated with ASNase showed that Asn is detectable 7-19 days sooner when DONV is present in the blood collection system than in its absence. We conclude that: (a) as little as 0.01 IU/ml ASNase can hydrolyze Asn added to blood; (b) continued hydrolysis of Asn by ASNase ex vivo can result in falsely low serum Asn measurements; (c) ASA or DONV present in the collection tubes obviates the problem of continued ASNase activity; and (d) the degree and duration of Asn depletion after ASNase therapy is much less than previously believed. Thus, for accurate measurements of the duration and degree of Asn depletion by ASNase, an ASNase inhibitor such as ASA or DONV should be present in the blood collection system.
L-天冬酰胺酶（ASNase）是急性淋巴细胞白血病治疗的重要组成部分，其抗白血病活性被认为是由于血清L-天冬酰胺（Asn）的消耗所致。在ASNase药理作用的研究中，研究人员报告称，给予ASNase后，血清Asn浓度会持续降低。但由于血样中存在的ASNase可能在测定前就水解Asn，所以这些测量结果可能无效。我们检测了添加不同浓度ASNase和未添加ASNase的血样中[U-14C]Asn的回收率。当ASNase浓度大于或等于0.01 IU/ml时，回收的[U-14C]Asn量不到未添加ASNase时的15%。利用该检测方法，我们研究了2种已知的ASNase抑制剂的作用，试图提高Asn的回收率。在存在天冬氨酸β-半醛（ASA）或5-重氮-4-氧代-L-正缬氨酸（DONV）且ASNase浓度高达1.0 IU/ml的情况下，Asn水平保持在对照的90%以上。ASA可防止在注射ASNase后从患者采集的血样中外源性Asn的水解。我们还开发了一种利用高压液相色谱法测定血清中Asn的方法。使用该方法，我们发现，在存在40 mM DONV和1.0 IU/ml ASNase的情况下，Asn水平大于正常水平的90%。对4例接受ASNase治疗患者的血清检测表明，当采血系统中存在DONV时，与不存在DONV相比，可提前7 - 19天检测到Asn。我们得出以下结论：（a）低至0.01 IU/ml的ASNase就能水解添加到血液中的Asn；（b）ASNase在体外持续水解Asn会导致血清Asn测量值假性偏低；（c）采血管中存在ASA或DONV可避免ASNase持续活性的问题；（d）ASNase治疗后Asn消耗的程度和持续时间远低于先前的认知。因此，为准确测量ASNase导致的Asn消耗的持续时间和程度，采血系统中应存在ASA或DONV等ASNase抑制剂。
