Sensitive on-line radioactivity measurements with a heterogeneous flow-cell: application to lymphoid cell ribonucleotides separated by high-performance liquid chromatography

Korte, Dirk de; Marijnen, Yolanda M. T.; Haverkort, Willem A.; Gennip, Albert H.; Roos, Dirk

doi:10.1016/s0378-4347(00)83231-4

Cited by 13 publications

(3 citation statements)

References 4 publications

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“…The separations were performed at a flow rate of 2 ml/min. The radioactivity incorporated in the various compounds was monitored by an on-line heterogeneous system [22], the Ramona D (Isomess, Straubenhardt, FRG) with a siliconized yttrium flow cell (550 pi.…”

Section: Nucleotide Analysismentioning

confidence: 99%

Preparation of Leukocyte-Poor Platelet Concentrates from Buffy Coats

Pietersz¹,

Korte²,

Reesink³

et al. 1988

Vox Sanguinis

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To study the influence of contaminating leukocytes on the storage conditions of platelet concentrates (PC), various amounts of leukocytes were added to identical PC. From 12 blood donations, 12 leukocyte-poor PC were prepared and pooled. Subsequently, the pool was divided into 12 identical PC. The plasma volume of the PC was 58.6±0.6 ml, the platelet concentration was 1.01±0.04x10^9/ml (mean ± SD) and the red cell contamination did not exceed 10^7 per PC. To 4 groups of 3 PC, pooled leukocytes were added from the same 12 blood donations. The leukocyte contamination for each group of 3 PC was 0.14±0.05, 1.96±0.09, 5.53±0.98 and 13.0±0.93x10^6/ml (mean ± SD) for groups I-IV, respectively. The PC were stored for 7 days at 22 °C in normal polyvinylchloride bags. A significant correlation was found between increasing concentrations of leukocytes in the PC and the drop in pH (r=-0.93), glucose consumption (r=-0.91), lactic acid production (r=0.93) and release of lactate dehydrogenase (r=0.92) during storage of the PC. The excretion of β-thromboglobulin, depletion of platelet adenine nucleotides, decreased ability to incorporate 3H-adenosine into metabolic nucleotides and poor morphology of the platelets were also significantly correlated with an increased number of leukocytes in the PC. These data show that high concentrations of leukocytes in PC have a significant detrimental effect on the viability of platelets during storage at 22 °C. We conclude that for good storage conditions of PC, the upper limit of leukocytes per PC should not exceed 10^7.

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Section: Nucleotide Analysismentioning

confidence: 99%

Preparation of Leukocyte-Poor Platelet Concentrates from Buffy Coats

Pietersz¹,

Korte²,

Reesink³

et al. 1988

Vox Sanguinis

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“…In the first step the amount of radiolabel in CTP or UTP was divided by the specific activity of the radiolabeled uridine. Next, the calculated amount of radiolabeled CTP or UTP was divided by the total amounts of CTP or UTP, detected by absorption at 254 nm [14].…”

Section: Distribution Of Radiolabeled Uridine Over Nucleotidesmentioning

confidence: 99%

Evidence for transformation‐related increase in CTP synthetase activity in situ in human lymphoblastic leukemia

Berg

Lenthe

Busch

et al. 1993

European Journal of Biochemistry

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To determine the role of the enzyme CTP synthetase (EC 6.3.4.2) in the synthesis in situ of CTP in normal and in malignant lymphoblastic cells, the metabolism of radiolabeled pyrimidine ribonucleosides was studied in proliferating normal T lymphocytes and was compared with that of proliferating MOLT-3 cell-line cells and differentiated (non-proliferating) MOLT-3 cells. Both the incorporation of ['4C]uridine into UTP and CTP and the incorporation of [14C]cytidine in CTP, as well as the fluxes of these labeled nucleosides through the nucleotide pools into nucleic acids, were elevated in proliferating MOLT-3 cells compared to proliferating T lymphocytes. Furthermore, the conversion of UTP into CTP was enhanced in proliferating MOLT-3 cells compared to proliferating T lymphocytes, indicating a higher activity of CTP synthetase in the leukemic cells. In non-proliferating MOLT-3 cells, the pyrimidine ribonucleotide fluxes were decreased compared to proliferating MOLT-3 cells and proliferating T lymphocytes. However, the decreased ratio of uracilkytosine ribonucleotides that was found in proliferating T lymphocytes and proliferating MOLT-3 cells compared to non-proliferating blood lymphocytes, was preserved in the differentiated MOLT-3 cells. Moreover, although the fluxes had decreased, most CTP was still synthesized by CTP synthetase in the differentiated MOLT-3 cells. Thus, the elevated activity of CTP synthetase in MOLT-3 cells was independent of the cell growth and maturation stage. We conclude that the increased activity of CTP synthetase is associated with the process of malignant transformation in MOLT-3 cells. Therefore, CTP synthetase offers an attractive target for selective therapy in human acute T-lymphoid leukemia.Previously, we have shown in our laboratories that peripheral blood cells from patients with acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma contain increased amounts of uracil and cytosine nucleotides and contain more uridinediphosphate-sugar compounds with a changed composition than do peripheral blood leukocytes from healthy donors [l -31. Typically, the ratio between uracil and cytosine ribonucleotides is decreased in the leukemic cells [l -31. In proliferating normal T lymphocytes too, an increase in the uracil and cytosine ribonucleotide pools has been observed, with a decreased ratio compared to non-proliferating peripheral blood lymphocytes 141.CTP synthetase catalyses the conversion of UTP into CTP and is the rate-limiting step in the synthesis of cytosine nucleotides from both the de novo and uridine salvage synthesis routes (see Fig. 1). In mouse S 49 T-lymphoma cellline cells the ratio between uracil and cytosine ribonucleotide phate-buffered saline; BrdU, 5-bromo-2'-deoxyuridine.(EC 6.3.4.2); uridine-cytidine kinase (EC 2.7.1.48).pools is balanced by allosteric feedback inhibition of CTP synthetase by the CTP pool [5]. Furthermore, it has been shown by in vitro enzyme analyses that an increased activity of CTP synthetase in tissue homogenates of...

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“…[14C]CMP (substrate), the extract was analysed on an anion-exchange HPLC column (Partisil-5 SAX, 12.5 cm × 0.46 cm; Partisphere system, Whatman, Clifton, M J, USA). The column was eluted with a buffer system as described ( The radioactivity in the eluate from the column was monitored with an online heterogeneous system (De Korte et al, 1987), the Ramona D (Isomess, Straubenhardt, FRG) with a siliconized yttrium flow cell (550 Ftl).…”

Section: Chemicalsmentioning

confidence: 99%

Deficiency of pyrimidine 5′‐nucleotidase in human leukocytes

Korte

Doorn

Sijstermans

et al. 1988

J of Inher Metab Disea

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Both erythrocytes and leukocytes from a patient with erythrocyte pyrimidine 5'-nucleotidase (P5N) deficiency were shown to contain increased amounts of pyrimidine nucleotides. These findings suggested that the leukocytes were also deficient for P5N. Measurement of the P5N activity in lysates from lymphocytes or granulocytes, in the presence of inhibitors for non-specific 5'-nucleotidase or alkaline phosphatase, indeed showed a deficiency for P5N in lymphocytes and granulocytes of the patient with erythrocyte P5N deficiency. However, the P5N deficiency in the leukocytes did not cause clinical disturbances in addition to the weak haemolytic anaemia.

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Sensitive on-line radioactivity measurements with a heterogeneous flow-cell: application to lymphoid cell ribonucleotides separated by high-performance liquid chromatography

Cited by 13 publications

References 4 publications

Preparation of Leukocyte-Poor Platelet Concentrates from Buffy Coats

Preparation of Leukocyte-Poor Platelet Concentrates from Buffy Coats

Evidence for transformation‐related increase in CTP synthetase activity in situ in human lymphoblastic leukemia

Deficiency of pyrimidine 5′‐nucleotidase in human leukocytes

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