Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells.

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.

Mass spectrometric identification of 2-hydroxy-sebacic acid in the urines of patients with neonatal adrenoleukodystrophy and Zellweger syndrome.

The urines of children with neonatal adrenoleukodystrophy and Zellweger syndrome contained an excess of unusual even- and odd-numbered dicarboxylic acids with a chain length of from 5 to 15 carbon atoms, as well as 2-hydroxy-compounds, including 2-hydroxy-isocaproate, 2-hydroxy-glutarate and 2-hydroxy-sebacate. The latter product, not previously found in metabolic diseases, appears as an additional useful marker of these peroxisomal disorders.

Synergistic toxicity of pyrazofurin and cytidine in cytidine deaminase deficient lymphoid cells (Raji).

The intermediary metabolism of pyrimidine nucleosides was studied in a line of human B lymphoblasts (Raji) in which pyrimidine de novo synthesis deficiency was pharmacologically induced by pyrazofurin. It was found that Raji cells are cytidine deaminase deficient that cytidine has a synergistic effect on the toxicity of pyrazofurin towards these cytidine deaminase deficient cells, affecting both the proliferation and the viability of the cells. Indirect evidences suggest that this synergistic toxicity is not mediated by an effect on nucleoside diphosphate reductase nor on the first steps of pyrimidine de novo synthesis.

Interleukin 2 liberation and absorption capacities of rat T lymphocytes in conditions of severe adenylic nucleotide pool depletion due to adenosine deaminase deficiency.

In rat lymph node lymphocytes stimulated for 24 h by concanavalin A in the presence of 10(-5) M 2'-deoxycoformycin (a potent inhibitor of adenosine deaminase) and 10(-5) M 2'-deoxyadenosine the adenylic nucleotide pool was reduced by 55.5% without modification of either the adenylic energy charge or the ability of the cells to liberate interleukin 2. In the same conditions, the ability of rat spleen cells to bind exogenous interleukin 2 activity was not modified. The proliferative response to concanavalin A stimulation was completely inhibited after a 86-h culture period under adenosine deaminase deficiency conditions. It could not be restored by elimination of 2'-deoxyadenosine after a 20-h pretreatment, when adenylic nucleotide pool depletion was 72.4% whereas the interleukin 2 liberation ability was not suppressed. These results suggest that among the early consequences of adenosine deaminase deficiency conditions, which occur before S phase of the cell cycle, the depletion of adenylic nucleotide pool, rather than the impairment of interleukin 2 liberation and absorption capacities, may account for the inability of the lymphocytes to respond to mitogenic stimulation.

Cytidine deaminase activity of human normal and malignant lymphoid cells.

Cytidine deaminase activity was determined by a radioisotopic assay in extracts of peripheral blood mononuclear cells of normal individuals and of patients with acute lymphoblastic leukaemia. The normal enzyme activity had a broad pH optimum between pH 6.5 and 8.0; apparent Km values for cytidine and deoxycytidine were 3.6 +/- 0.6 mumol/l and 26.5 +/- 3.5 mumol/l, respectively; the activity was resistant to heat inactivation; of the various effectors tested, only uridine, deoxyuridine and tetrahydroxyuridine had inhibitory effects. Cytidine deaminase activity was markedly decreased in lymphoblasts of patients with acute lymphoblastic leukaemia; enzyme activity was related to the percentage of circulating blast cells, and not to the clinical, cytological or immunological characters of the leukaemia.

Mass spectrometric identification of abnormal aromatic compounds in the urine of a child with Reye's like syndrome.

The urine of a young child with hypoglycemia and a Reye's like syndrome contained an excess of unusual aromatic products with a three carbon chain, phenylpropionylglycine and 3- and 4-(hydroxyphenyl)propionic and 3-(3-methoxy-4-hydroxyphenyl)propionic acids, as well as of organic acids usually found in fatty acid beta-oxidation defects: the mono- and dicarboxylic acids derived from the respective (omega-1) and omega-oxidation of C6 to C10 fatty acids.

Microdetermination of 2-ketoglutaric acid in plasma and cerebrospinal fluid by capillary gas chromatography mass spectrometry; application to pediatrics.

Quantification of 2-ketoglutaric acid in plasma and cerebrospinal fluid as its O-trimethylsilyl++-quinoxalinol derivative by gas chromatography chemical ionization mass spectrometry is described with benzoylformic acid as internal standard. This technique, with ammonia as reactant gas, only detects the protonated molecular ions. The recovery of 2-ketoglutarate from perchloric-deproteinized plasma is 99.7 +/- 1.2%. The normal value of 2-ketoglutarate in children is 8.6 +/- 2.6 mumol l-1 (mean +/- standard deviation) in plasma (n = 25) and 4.8 +/- 1.4 mumol l-1 in cerebrospinal fluid (n = 20). The plasma level of 2-ketoglutarate is correlated with urea concentration (r = 0.96; p less than 0.001) in healthy subjects and in patients with chronic renal insufficiency. Increased values are found in one case of pyruvate carboxylase deficiency, and inconstantly in diabetes; physiological variations are described during fasting and after an oral glucose load.

Uridine as the only alternative to pyrimidine de novo synthesis in rat T lymphocytes.

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10(-5) M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [3H]thymidine incorporation. Proliferation is completely restored by 5 X 10(-5) M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 X 10(-5) M each, fail to re-establish proliferation but produce an isotropic dilution of [3H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (less than 0.001 nmol X min-1 X 10(-6) cells).

Biochemical study of a case of hemolytic anemia with increased (85 fold) red cell adenosine deaminase.

An increased red cell adenosine deaminase (ADA) activity (85-fold) was found in a 10-year-old male child suffering from severe hemolytic disease. Evidence is given that the excessive ADA activity in erythrocytes is due to an abnormal amount of a catalytically and immunologically normal enzyme. Metabolic studies with the patient's erythrocytes show that the low ATP concentration in these cells (64% of comparably reticulocyte-rich blood) is due both to a diminished synthesis of adenylic nucleotides from adenosine, and to an excessive catabolism of AMP.

Isopycnic centrifugation as method for the separation of rat thymocyte subpopulations.

Rat thymocytes were separated into five density layers by isopycnic centrifugation on a discontinuous-density Ficoll gradient and each fraction was tested for metabolic functions at different times during a 96-h culture period in various conditions. Immediately after separation the rate of oxygen consumption per unit of cellular volume is virtually the same in all fractions. However, the capacity to synthesize nucleic acids, proteins, purine and pyrimidine nucleotides by the de novo pathway and the activity of the salvage pathway of purine nucleotides is much greater in the low density fractions (fractions 1 and 2). In in vitro cell culture conditions (1) in the absence of mitogens this metabolic activity (which demonstrates a high spontaneous mitotic capacity of cells in the low-density fractions) decays rapidly to a level which is practically negligible after 24 h of culture; (2) in the presence of mitogens alone: PHA, Con A, PWM or PNA, with or without prior neuraminidase treatment, after 24 h an induction of the synthesis of nucleic acids and protein is observed concomitant to the apparition of blast cells. This blastogenesis peaks at 72 h and occurs in fractions 1 and 2 only; (3) in the presence of Con A and 2-mercaptoethanol the blast transformation corresponding to the response of the thymocytes to TCGF is observed in all of the thymocyte fractions and extends beyond 72 h of culture. It remains much more intense in the low-density fractions 1 and 2.

Consequences of adenosine deaminase deficiency on thymocyte metabolism.

Using 2'-deoxycoformycin inhibition of adenosine deaminase as a model of adenosine deaminase deficiency, the effects of 10 microM 2'-deoxyadenosine (dAdo) on the metabolism of concanavalin A (Con A)-stimulated rat thymocytes were studied. When dAdo and Con A were added simultaneously, a strong inhibition of the incorporation of [3H]thymidine (84%); [3H]uridine (98%) and L-[3H] leucine (46%) in the acid-insoluble fraction, and of [14C]formate (78%) and H14CO-3 (43%) uptake is observed after 48 h of incubation. When dAdo is added after 12 h of Con A stimulation, no such inhibition is observed, but when added after 24 h of stimulation, there is an enhancement of blastogenesis as measured by nucleic acid, protein, and purine and pyrimidine base synthesis. More detailed studies of thymocytes stimulated by Con A for 0-72 h, followed by short-term incubation periods with dAdo (1-5 h), revealed that thymocyte metabolism becomes progressively less sensitive to dAdo-mediated inhibition during the course of blastogenesis. These results suggest that (a) the inhibition of ribonucleotide reductase is not the only mechanism involved in the inhibition of blastogenesis by dAdo and that (b) such inhibition of thymocyte metabolism is essentially dependent upon the activation state of the cell.