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Measurement of Serum L-Asparagine in the Presence of L-Asparaginase Requires the Presence of an L-Asparaginase Inhibitor¹

Strong Children's Research Center and the Cancer Center [B. L. A., J. C. W., D. J. C., H. J. C.], Department of Medicine and Clinical Research Center [M. Y. L.], and Department of Chemistry, College of the Arts and Sciences [A. S. K.], University of Rochester, Rochester, New York 14642, and Department of Biochemical and Clinical Pharmacology [R. L. B.], St. Jude Children's Research Hospital, Memphis, Tennessee 38101

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 0.01 IU/ml of ASNase, the amount of [U-¹⁴C]Asn recovered was <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 ß semialdehyde (ASA), or 5-diazo-4-oxo-L-norvaline (DONV), and up to 1.0 IU/ml ASNase, Asn levels remained at >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 >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.

¹ Supported by Program Project Grant 5P01CA34183-06 from the NIH, Food and Drug Administration Grant 00199-03, Clinical Research Center Grant RR-0044 from the Division of Research Resources, and a Wilmot Cancer Research Fellowship awarded by the James P. Wilmot Foundation (B. L. A.), a Leukemia Society Research Fellowship awarded by the Leukemia Society of America, Inc. (B. L. A.), and USPHS Training Grant HL07111152 (B. L. A.).

² To whom requests for reprints should be addressed, at Department of Pediatrics, Box 777, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642.

Received 2/12/91. Accepted 10/ 2/91.

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