Search

In this article we present a patient with acute lymphoblastic leukemia (ALL) associated with eosinophilia, in which the eosinophilia preceded a meningeal and bone-marrow relapse of ALL. We analysed the purine and pyrimidine nucleotide content of the eosinophils (92% pure) and compared the nucleotide pattern with that of eosinophils from healthy donors and from patients with eosinophilia not associated with leukemia. The ratios of purine : pyrimidine and of uracil :cytosine nucleotides were decreased compared with those in eosinophils from healthy donors and from patients with eosinophilia with other aetiologies. The total nucleotide concentration was increased, especially the concentration of UDP-sugars and pyrimidine nucleotides. The decrease in these ratios and the increase in concentration of the nucleotides and the UDPsugars were also detected in leukemic cells of patients with ALL (de Korte et al., Leukemia Res.10, 389–396 (1986)) compared to normal lymphocytes. We suggest a malignant character of the eosinophils in our patient with ALL associated with eosinophilia, in contrast with the nonmalignant state suggested previously for these cells.

The intracellular purine and pyrimidine ribonucleotide concentrations were determined in the lymphoid cells from peripheral blood and/or bone-marrow of 29 patients with acute lymphoblastic leukemia (ALL), as well as in the mononuclear cells from peripheral blood of 12 patients with ALL in remission. The lymphoid cells of ALL patients showed an imbalance in the nucleotide pool compared with normal lymphocytes, whereas the nucleotide pool of mononuclear cells from patients with ALL in remission had normal values. The imbalance in the lymphoid cells from ALL patients involed decreased ratios of purine:pyrimidine, adenine:guanine and uracil:cytosine nucleotides, and an increased amount, together with a changed composition, of the UDP sugars. When compared with tonsil-derived B lymphocytes and thymocytes, ALL lymphoid cells have an increased amount (absolute and relative) and a change composition of the UDP sugars. Significant differences were found between the mean values for the immunologically defined subgroups of ALL and between the mean values for patients with a high or a low percentage of blass cells. However, individual patients cannot be classified according to their nucleotide pattern, because of the overlapping ranges. The results of this study may be useful for the design of new therapeutic regimens.

An anion-exchange high performance liquid chromatography (HPLC) method is described for the quantitation of intracellular purine and pyrimidine nucleotides. With an ammonium phosphate salt and pH gradient, complete separation is achieved of all major nucleotides and several interfering substances, such as dehydroascorbic acid and NAD. For optimal resolution of the monophosphates, strict control of the equilibration pH is essential. To prevent interference by a degradation product of NADPH with the determination of GDP, the pH of the high-ionic strength buffer has to be in the range of 4.9-5.0. The use of radially compressed, prepacked cartridges filled with Partisil-10 SAX appeared to be a fast and cheap alternative for expensive stainless-steel columns. The use of ammonium phosphate buffers, in combination with precolumns filled with pellicular silica and SAX resin, and interim EDTA washes prevents baseline shift. This allows analysis at 0.01 Absorbance Units Full Scale during the entire column lifetime (about 180 analyses), which is sufficiently sensitive for the quantitation of low levels of nucleotides, especially when the amount of sample is limited. The usefulness of the presented Chromatographie system is demonstrated by the quantitation of the nucleotides in extracts of lymphocytes and neutrophils from the blood of healthy human donors. With this method nucleotide concentrations were measured, with a within-assay variation of 5–10% and an inter-donor variation of 10%.

Recently, several reports have been published about abnormal purine and pyrimidine metabolism in leukemic cells as compared to normal peripheral blood cells. Enzyme activities1,2 as well as metabolite concentrations3,4 have been studied. These studies demonstrated marked differences in the activities of many enzymes controling purine and pyrimidine metabolism in leukemic cells, such as adenosine deaminase, purine nucleoside Phosphorylase and terminal deoxynucleotidyl transferase. De Abreu et al.3 determined significantly increased concentrations of adenine and uracil nucleotides in the lymphoblasts of patients with non-B, non-T acute lymphoblastic leukemia. Liebes et al.4 found increased concentrations of nicotinamide adenine nucelotides in lymphoid cells of patients with chronic lymphatic leukemia.

Abnormalities in purine and pyrimidine metabolism may be associated with cell-cycle phase, proliferation, immaturity or transformation. Therefore, detailed information concerning purine and pyrimidine pattern in malignant cells compared to normal cells is needed. Abnormal enzyme activities (1) as well as abnormal metabolite concentrations (2) have been described for leukemic cells.

The effect of rapid cooling to 20-24 degrees C of whole blood immediately after collection, using 'cooling units' with butane-1,4-diol and prolonged storage up to 24 h at ambient temperature was investigated in the whole blood and the subsequently prepared plasma, buffy coat and buffy-coat-poor red cell concentrate (BC-poor RCC) in saline-adenine-glucose-mannitol (SAG M) solution. Factor VIII:C content of the plasma (n = 10), after 24 h storage was 80 +/- 3% of the initial value. In routine procedures factor VIII:C content in the plasma (n = 129 pools of 20 donor units plasma) was 0.77 +/- 0.078 IU/ml, after storage of the whole blood for 16-20 h. In whole blood (n = 10), the 2,3-diphosphoglycerate (2,3-DPG) content of the red cells decreased from 4.36 +/- 0.55 to 1.47 +/- 0.6 mumol/ml red cells after 24 h storage at 20-24 degrees C. After storage of the BC-poor RCC (n = 10) at 2-6 degrees C for 1 week, the 2,3-DPG had dropped to 0.76 +/- 0.46 mumol/ml red cells. During the first 24 h of storage of whole blood, the adenine triphosphate (ATP) levels of the red cells remained stable. A mean increase of 20% of the initial value was observed after addition of SAG M solution. In the BC-poor RCC the ATP slowly decreased to 81 +/- 5% after 5 weeks and to 68 +/- 6.6% of the initial value after 6 weeks storage.(ABSTRACT TRUNCATED AT 250 WORDS)

A case of eosinophilic meningitis 2 months before the appearance of lymphoblasts in the cerebrospinal fluid is described in a child with acute lymphoblastic leukemia (ALL). The peripheral blood showed no simultaneous eosinophilia. The child was successfully treated for her CNS relapse, and complete remission was easily obtained. The eosinophils and lymphoblasts disappeared quickly after the administration of intrathecal methotrexate. However, 3 1/2 years later hypereosinophilia developed in the blood and bone marrow, heralding bone marrow relapse. Simultaneously, meningeal relapse was diagnosed and this time the cerebrospinal fluid showed a mixture of lymphoblasts and eosinophils. Treatment was reinstituted and complete remission was again obtained. Analysis of the blood eosinophils showed abnormal nucleotide patterns. Similar patterns were previously found in the lymphoblasts from other ALL patients.

Discontinuous mRNA synthesis in trypanosomes is thought to involve a 140-nucleotide precursor, called the mini-exon-derived RNA or medRNA, which contributes its 5' 35 nucleotides to the 5' end of nascent mRNAs. We used in vivo labelling of RNA to show that medRNA has a half-life of less than 6 min, whereas putative high mol. wt intermediates containing the 3' part of the medRNA have an average half-life of less than 1 min. This eliminates priming of pre-mRNA synthesis by intact medRNA as the main mode of discontinuous mRNA synthesis. Potential intermediates of 35 and 105 nucleotides were labelled in parallel with medRNA, but their significance could not be assessed in RNA preparations containing medRNA, as they are also produced by artefactual cleavage of medRNA. We show, however, that high mol. wt RNA, free of medRNA, can release medRNA segments upon a debranching treatment. These results are consistent with a trans splicing mechanism involving short-lived forked intermediates, analogous to lariats in cis splicing systems.

The flux through the purine and pyrimidine metabolism of normal lymphocytes, resting as well as proliferating, and lymphoblastic cell-line cells (MOLT-3) was measured by incubation of the cells with radioactive precursors of either “de novo” or “salvage” pathways. Tonsillar lymphocytes were taken as immature, slowly proliferating lymphocytes (2% in S phase) and PHA-stimulated lymphocytes as more actively proliferating lymphocytes (about 8% and 20% in S phase on the 2nd and 4th day after stimulation, resp.). The incorporation of the precursors in the purine and pyrimidine ribonucleotides was followed by a combination of anion-exchange HPLC and on-line radioactivity measurement. Lymphoblastic cell-line cells incorporated 10 to 200 times more of the various precursors in the ribonucleotides, compared to both resting and proliferating normal lymphocytes. The low adenine:guanine ratio in leukemic cells was reflected in the incorporation pattern of the various precursors. To some extent, this was also true for the low uracil:cytosine ratio. The activities of the branch-point enzymes IMP dehydrogenase and CTP synthetase most likely determine the imbalance in the nucleotide pool of lymphoblastic cells and might provide targets for selective chemotherapy.