Abstract
A procedure is described for the rapid determination of the intra-erythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10(8) cells) in 350 microl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 microl 70% perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100 degrees C for 45 min. After cooling, 100 microl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (r(2) > 0.998), and the analytical recoveries were 73.2% for 6-TG, 119.1% for 6-MP and 97.4% for 6-MMP. The intra- and inter-assay variations were highest for 6-MP (9.6 and 14.3%, respectively). The lowest detectable concentrations were 3, 3 and 25 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The quantification limits (coefficients of variation <15%) were 8, 10 and 70 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The method was applied to the analysis of 183 samples from 36 children under chemotherapy for acute lymphoblastic leukemia. The concentrations of the metabolites in the red cells of the patients ranged from 0 to 1934 pmol/8 x 10(8) erythrocytes for 6-TGN, and from 0 to 105.8 and 0 to 45.9 nmol/8 x 10(8) erythrocytes for 6-MP and 6-MMP, respectively. The procedure gave results that were in agreement with those obtained with other methods designed to detect cases of non-compliance with treatment, including patient interviews and medical evaluation, among others, demonstrating its applicability to monitoring the treatment of leukemic children.
Highlights
Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children
We describe a quantitation procedure based on the publications of Dervieux and Boulieu [27] and Lennard and Singleton [26], which permits the rapid quantitation of the intraerythrocyte levels of 6-MP, 6-thioguanine nucleotides (6-TGN) and 6MMP
TG), 6-MP, and 6-MMP were purchased from Sigma; perchloric acid was from Carlo Erba and Hank’s balanced salt solution was from Gibco BRL; triethylamine, phosphoric acid and all other chemicals were purchased in analytical grade from Merck (Rio de Janeiro, RJ, Brazil)
Summary
Prior to the discovery and application of chemotherapeutic agents, the majority of children suffering from ALL survived for only one or two months after diagnosis [1]. Procedures to assess compliance with treatment, especially with respect to the effective delivery of chemotherapy, are of great importance to ALL patients and can even determine the outcome of the disease. Such assessments may be carried out by various indirect methods including patient interviews, medical evaluation, drug audits, etc. Such assessments may be carried out by various indirect methods including patient interviews, medical evaluation, drug audits, etc. [4], more direct methods involving quantitation of the drug and its metabolites in urine and erythrocytes have been proposed [5,6,7,8,9]
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Schedule a callMore From: Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas
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