High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

Abreu, Ronney A. De; Baal, John M. van; Bakkeren, J. A. J. M.; Bruyn, C. H. M. M. de; Schretlen, E. D. A. M.

doi:10.1016/s0378-4347(00)80354-0

Cited by 65 publications

(3 citation statements)

References 10 publications

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“…Previously, v/e published HPLC methods for the detection of purine and pyrimidine nu cleotides [8], nucleosides and bases [9] and thiopurine nucleosides and bases [10,11], In the present article we describe two HPLC methods, one for measurement of methylthiopurine and thiopurine nucleosides and bases, and another for measurement of (methyl)thiopurine nu cleotides. Both methods are used in the clinical randomized ALL-8 trial of the Dutch Childhood Leukaemia Study Group.…”

Section: W Ken' En K Amp Jansen Ct At I J Ch Rom a Togr B 672mentioning

confidence: 99%

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Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

Keuzenkamp-Jansen¹,

Abreu²,

Bökkerink³

et al. 1995

Journal of Chromatography B: Biomedical Sciences and Applicatio

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The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukaemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di-and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measure ment of the low concentrations of mono-, di-and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/109 cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.

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Section: W Ken' En K Amp Jansen Ct At I J Ch Rom a Togr B 672mentioning

confidence: 99%

“…Chroma tography was carried out with a minor modi fication of the method described earlier [8]. The gradient, indicated in Table 2, allows a reduced runtime as compared to the method described before [8] without loss of resolution.…”

Section: 11 Chromatography O F (Methyl)thiopurine Nucleotidesmentioning

confidence: 99%

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

Keuzenkamp-Jansen¹,

Abreu²,

Bökkerink³

et al. 1995

Journal of Chromatography B: Biomedical Sciences and Applicatio

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“…In addition, we incubated a 0.9 mM stock solution of (deoxy)adenosine and a 9.0,mM stock solution of deoxyguanosine in culture medium containing 20% AB serum for 4 days at 37°C. The concentrations of adenosine and deoxyguanosine remained unchanged and that of deoxyadenosine was 90% of the initial value (assessed by high-performance liquid chromatography [1]). On the basis of these results we used 20% AB serum in the culture.…”

Section: Purine Interconversion Enzyme Aciivities In Unseparated Thymmentioning

confidence: 99%

Lymphocyte Maturation in the Human Thymus

et al. 1983

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The combination of centrifugal elutriation as an efficient and reproducible method to separate thymocytes by size, micromethods to assess purine interconversion enzymes, and assessment of purine (deoxy)nucleoside inhibition of mitogen responses enabled us to study purine metabolism at the intrathymic level. Out of six fractions, four (nos. 3-6), containing medium- and large-sized lymphocytes, showed a proliferative response after stimulation with phytohaemagglutinin (PHA). In fractions 1-6 the number of cells with an immature immunological phenotype gradually decreased, and cells with the phenotype of mature cells gradually increased. The enzyme activity ratio of adenosine deaminase to purine nucleoside phosphorylase gradually decreased from 21 in fraction 1 to 7 in the last fraction (blood T-cell value, 0.7). We conclude that this enzyme activity ratio is a useful marker for intrathymic T-cell maturation stages. In PHA-responsive cell fractions (3-6), the sensitivity to inhibition of the PHA response by (deoxy)adenosine and deoxyguanosine was inversely related to the enzyme activity ratio of ecto-5'-nucleotidase to deoxycytidine kinase. These findings are compatible with the hypothesis that intracellular concentrations of phosphorylated (deoxy)nucleosides are related to this inhibition. We conclude that the differences in purine metabolism among the various (mitogen-responsive) human thymocyte fractions are related to lymphoid cell function. Since the number of cells contributing to the enzyme activities and the number of cells contributing to the proliferative response (about 15% of unseparated cells) differ considerably, it is not possible to evaluate enzyme activities in unseparated thymocytes in terms of relationships between purine metabolism and lymphocyte function.

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Biochemical consequences of 2′-deoxycoformycin treatment in a patient with T-cell lymphoma. Some unusual findings

Korte

Haverkort

Leeuwen

et al. 1987

Cancer

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The nucleotide content of the various blood cells and the urinary excretion of purine and pyrimidine metabolites were studied in a patient with a T-cell lymphoma (early T-cell phenotype) before and during treatment with deoxycoformycin (dCF; given intravenously [iv] during 3 days, biweekly). During and after the administration of dCF, high amounts of dATP were found in the lymphoid cells and the erythrocytes (maximally, 480 pmol/106 lymphocytes and 5.5 nmol/106 erythrocytes), but not in the polymorphonuclear leukocytes. The amount of dATP in the erythrocytes, however, was significantly lower than described in the literature. During each administration of dCF, the number of blast cells in the peripheral blood rose initially, followed by a rapid decrease. After three courses, a hematologic remission was achieved and maintained for 6 weeks; then an autologous bone-marrow transplantation was performed. During the first dCF course a large amount of deoxyadenosine was found in the urine. During the second course, this excretion was much lower, but still higher than in healthy individuals. In the patient described, dCF showed a highly specific toxicity for the immature T-lymphoblast; hardly any changes were seen in the numbers of the other hematopoietic cells, both in the blood and in the bone marrow.

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High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

Cited by 65 publications

References 10 publications

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

Lymphocyte Maturation in the Human Thymus

Biochemical consequences of 2′-deoxycoformycin treatment in a patient with T-cell lymphoma. Some unusual findings

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