Analysis of purine and pyrimidine bases,ribonucleosides, and ribonucleotides by high-pressure liquid chromatography

A high-pressure liquid chromatographic method has been developed for the separation and quantitation of purine and pyrimidine bases, ribonucleosides, and ribonucleotides. The procedure is carried out on a 1.8 × 700-mm column packed with Aminex-A-25 anion-exchange resin. The column is eluted with a linear gradient of ammonium chloride. The elution buffer contained borate also to complex the sugar phosphates and ethanol to improve the separation of bases and nucleosides. The analysis is completed in about 160 min. The potential application of this method for the quantitation of acid-soluble metabolites in fibroblasts is described.     

Reversed-phase high-performance liquid chromatography of purine compounds for investigation of biomedical problems: application to different tissues and body fluids

An overview of high-performance liquid chromatographic separation techniques (reversed-phase and ion-pair reversed-phase) used in the analysis of purine ribonucleotides, ribonucleosides and nucleobases, including procedures for sample preparation, is given. Coverage of the separation techniques is extended to the measurement of specific radioactivities of these compounds in tracer kinetic experiments for metabolic flux rate analyses. This article is focused on the development and adaptation of reversed-phase separation techniques for nucleotides, nucleosides and bases that are used to examine a variety of biomedical problems. The investigation of purine nucleotide metabolic disorders or physiological transition in the pathomechanisms of different diseases and syndromes or in cell maturation processes, respectively, requires the application of chromatographic separation to a multitude of tissues and body fluids. These samples vary greatly in concentrations of purine compounds with low molecular mass, from ca. 5 mM to ca. 0.5 μM. The advantages and disadvantages of different techniques are critically discussed.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. III. The determination of 14C-labeled 6-thiopurines in L5178Y cell extracts using high-pressure liquid cation-exchange chromatography.

A method is presented for the separation of 6-thiopurine bases and ribonucleosides, of sulphate anions and of common purine bases and oxidized purines by means of high-pressure liquid cation-exchange chromatography using a 0.18 X 100 cm column, filled with Beckman M71 resin, and eluted with 0.4M ammonium formate, pH 4.6, at a linear flow velocity of 5.2 cm/min at 50 degrees C. The method has been applied to the separation and quantitative determination of 14C-labeled 6-mercaptopurine metabolites in HClO4 extracts of L5178Y murine lymphoma cells. Distribution patterns of 14C radioactivity within the cells after a 24 h incubation period with (8-14C)-labeled 6-mercaptopurine have been established. The indentification of 6-mercaptopurine metabolites, such as 6-thioxanthosine ribonucleotide, 6-thioinosinic acid, 6-thioguanylic acid, 6-methylthioinosinic acid, and 6-thiouric acid, after the digestion of the extracts with alkaline phosphatase has been confirmed using the behaviour of each compound in enzymatic peak-shifting analyses with purine nucleoside phosphorylase and the corresponding elution volumes of 6-thiopurine bases and ribonucleosides as proofs. According to the specific radioactivity of the (8-14C)-labeled 6-mercaptopurine batch, the amounts of the various 6-mercaptopurine metabolites in about 6% of the total HClO4 extract of 1.6 . 10(8) labeled cells have quantitatively been determined as 1--130 pmol. The intracellular concentration of 6-thiopurines was determined at 1.4 . 10(-5)mol/1.     

Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography–electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC™) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 μmol/L (UMP and AMP) to 1.3 μmol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.     

Low-level expression of purine bases in BALB/3T3 cells monitored by ultrasensitive graphene-based glass carbon electrode

Using an ultrasensitive chemically reduced graphene oxide and ionic liquid modified glass carbon electrode (RGI–GCE), separated electrochemical signals of adenine and hypoxanthine in both human breast cancer (MCF-7) and mouse embryonic fibroblast (BALB/3T3) cells were observed. For the first time, low-level expression of purine bases in noncancerous BALB/3T3 cells can be electrochemically monitored. The metabolism of purine bases in carcinogen agent-contaminated BALB/3T3 cells was also investigated through the change of electrochemical signals ascribed to different purine bases, which opens a new electrochemical approach to the exploration of a low-level purine mechanism in noncancerous cells.     

Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines

ABSTRACT

Using Caco-2 cells and our previously developed high-performance liquid chromatography method for quantification of purine bases, nucleosides, and nucleotides, we evaluated cellular purine transport and uptake. The analytes were separated using YMC-Triart C18 column with gradient elution. We used Caco-2 cells as intestinal model cells and monitored purine transport across a monolayer for 2 h. The degree of change of purine concentrations in the permeate was very slight; however, it was possible to simultaneously determine these parameters for all purines because of our method's high sensitivity. In the present study, the purine bases (adenine, guanine, hypoxanthine, and xanthine) showed a relatively high permeability as compared with the nucleosides (adenosine, guanosine, inosine, and xanthosine). Increased concentration of metabolites in the permeate was also observed following the addition of purines. In a cell uptake assay, both the cell culture medium (extracellular) and the cells extracted from Caco-2 with acetonitrile:water (7:3) (intracellular) were measured. The additional nucleoside did not increase significantly within the cells. On the other hand, we observed that nucleotide, such as ATP, increased in the cell in a time-dependent manner following the addition of nucleoside. The additional nucleosides were considered to be rather recycled via the salvage pathway than metabolized to purine bases and/or uric acid in the cell. Such differences might have affected the increase in the serum uric acid levels depending on purine form.     

A set of procedures for resolving purine compounds by reversed-phase high performance liquid chromatography: application to the study of purine nucleotide and nucleic acid metabolism

A set of simple procedures for the separation of major purine 5'-ribonucleotides including diguanosine polyphosphates, purine and pyrimidine bases, and 2'- and 3'-nucleotide monophosphates using reversed-phase high-performance liquid chromatography and isocratic elution study of purine nucleotide and nucleic acid biosynthesis in Artemia is presented.     

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).     

Extraction and measurement of myocardial nucleotides, nucleosides, and purine bases by high-performance liquid chromatography

Nucleotides, nucleosides, and purine bases were extracted from human endomyocardial biopsies, freeze-clamped rat hearts, and porcine coronary sinus plasma. Perchloric acid extracts were neutralized with Freon-trioctylamine and analyzed at 250 nm by reverse-phase ion-pairing high-performance liquid chromatography. To achieve the sensitivity necessary for analyzing small (1-3 mg wet wt) tissue samples, a small-bore, 2.1-mm-internal-diameter, C18, 5-micron reverse-phase column and a flow rate of 0.2 ml/min were used. All of the myocardial nucleotides and AMP degradation products were resolved in a total separation time of 27 min with 30 mM KH2PO4, 7.5 mM tetrabutylammonium phosphate buffers, and binary pH and acetonitrile gradients.     

Investigation of the formation and intracellular stability of purine.(purine/pyrimidine) triplexes.

In a previous work we showed that a short triple helix-forming oligonucleotide (TFO) targeted to the murine c-pim-1 proto-oncogene promoter gives a very stable triple helix under physiological conditions in vitro . Moreover, this triplex was stable inside cells when preformed in vitro . However, we failed to detect triplex formation for this sequence inside cells in DMS footprinting studies. In the present work, in order to determine whether our previous in vivo results are limited to this particular short triplex or can be generalized to other purine.(purine/pyrimidine) triplexes, we have tested three other DNA targets already described in the literature. All these purine.(purine/pyrimidine) triplexes are specific and stable at high temperature in vitro . In vivo studies have shown that the preformed triplexes are stable inside cells for at least 3 days. This clearly demonstrates that intracellular conditions are favourable for the existence of purine. (purine/pyrimidine) triplexes. The triplexes can also be formed in nuclei. However, for all the sequences tested, we were unable to detect any triple helix formation in vivo in intact cells by DMS footprinting. Our results show that neither (i) chromatinization of the DNA target, (ii) intracellular K+concentration nor (iii) cytoplasmic versus nuclear separation of the TFO and DNA target are responsible for the intracellular arrest of triplex formation. We suggest the existence of a cellular mechanism, based on a compartmentalization of TFOs and/or TFO trapping, which separates oligonucleotides from the DNA target. Further work is needed to find oligonucleotide derivatives and means for their delivery to overcome the problem of triplex formation inside cells.     

Metabolomic Method: UPLC-q-ToF Polar and Non-Polar Metabolites in the Healthy Rat Cerebellum Using an In-Vial Dual Extraction

Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.     

Differential control of cell cycle,proliferation, and survival of primary T lymphocytes by purine and pyrimidine nucleotides

Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G(1) to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.     

Purine metabolite concentrations in portal and peripheral blood of steers, sheep and rats.

1. The concentration of purine derivatives in portal and peripheral blood of steers, sheep and rats was measured by reverse-phase high performance liquid chromatography. 2. Nucleotides, nucleosides (apart from inosine), adenine and guanine were not found in the plasma samples. Allantoin, uric acid, hypoxanthine and xanthine accounted for virtually all purine metabolites in plasma samples. 3. Non-oxidized derivatives (hypoxanthine and xanthine) were consistently detected in sheep but not in steer or rat plasma samples showing a differential availability of reutilizable purine derivatives between species.     

Quantitation of individual molecular species of phosphatidylcholines by reversed-phase high-performance liquid chromatography with fluorometric detection

The multiplicity of phosphatidylcholines is caused by the presence of different pairs of fatty acids in their individual molecular species and at least 27 miscellaneous fatty acids were identified in phosphatidylcholines in the serum of healthy individuals by combined gas–liquid chromatography and mass spectrometry in our present experiments. A method is described for the separation and quantitation of molecular species of phosphatidylcholine in human serum. Total phosphatidylcholine is isolated from lipids extracted from the serum with chloroform–methanol (2:1) by reversed-phase liquid–liquid extraction and subjected to reversed-phase high-performance liquid chromatography with a discontinuous descending gradient of water. Separation is monitored by fluorometry (340/460 nm) and absorption at 205 nm, if required. Up to 25 different molecular species of phosphatidylcholine may be quantified with a satisfactory reproducibility (±5–8%). Data on the distribution of individual molecular species in phosphatidylcholine of 53 normal serums are presented. The method may be used for quantitation of these phospholipids also in other biological materials (cell lines, leukemic cells from patients), and on a micropreparative scale to isolate individual compounds. The speed of separation as well as a satisfactory reproducibility are its principal advantages.     

An isotopic assay of cyclic 3',5'-nucleotide phosphodiesterase with aluminum oxide columns

A method is described for separating purine bases and nucleosides from cyclic 3′,5′-nucleotides on aluminum oxide columns. Purine bases and nucleosides were found to pass through columns equilibrated with ammonium acetate buffer at pH 4.0 while the cyclic nucleotides were retarded enough to permit separation. Optimal conditions and factors affecting separation are described. The method was shown to be applicable in the isotopic assay of cyclic 3′,5′-nucleotide phosphodiesterase activity over a broad range of substrate concentrations. The advantages of this method and its possible use in a simultaneous cyclase-phosphodiesterase assay are described.     

Analysis of radioactive and nonradioactive purine bases,nucleosides, and nucleotides by high-speed chromatography on a single column

The concentrations of radioactive and nonradioactive purine bases, purine nucleosides, purine mono-, di-, and trinucleotides in acid extracts of fibroblasts were determined by anion-exchange column chromatography. The concentrations of nonradioactive components were determined by computerized integration of the signal from a double-beam uv-detector. The radioactive metabolites were quantitated by high-efficiency, continuous liquid scintillation counting, employing a discrete sample transport system.     

Enucleation of phagocytic cells with adenine, guanine, and their nucleosides in combination with centrifugation

Several purine compounds, such as adenine, guanine, adenosine, guanosine, and their related compounds, exhibited enucleation activity on adherent mouse peritoneal exudate cells (macrophages) during centrifugation at 25,000 and 35,000 g for 60 min at 34 degrees-36 degrees C in medium containing one of these compounds. Enucleation activity, however, did not occur in cells treated with adenine nucleotides, inosine, xanthine, or any of the tested pyrimidines. The purine compounds also had enucleation activity on mouse macrophage-like cell lines (P388D1 and RAW 264) and mouse polymorphonuclear leukocytes, but not on other typical cell lines such as a human epithelial cell line (HeLa S-3) or a mouse fibroblast cell line (L929). Cytochalasin B (CB) treatment, however, resulted in the enucleation of all cell types tested, even at a centrifugal force as low as 5,000 g. The process of macrophage enucleation was observed by both light microscopy and scanning electron microscopy. In enucleated macrophages that had been treated with purine compounds, but not with CB, a newly formed cytoplasmic crater-like structure (about 3-9 microns in diameter) was observed at the original site of the nucleus. Surface structures, such as microvilli and membrane ruffles, remained relatively intact in macrophages that had been enucleated by treatment with purine compounds. By contrast, these surface structures were markedly changed in CB-treated macrophages. Purine compounds may affect cytoskeletal elements in ways similar to the well characterized effects of CB, and thus result in the enucleation of phagocytes. However, the characteristic differences in the enucleation activity exhibited by purine compounds and CB may indicate that purines have a mechanism of action different from that of CB.     

A high-performance liquid chromatography method for separation of purine bases, nucleosides and ureides: application to studies on purine catabolism in higher plants

Mixtures of purine nucleotides, nucleosides, nucleobases, uric acid, allantoin and allantoic acid were fractionated by high-performance liquid chromatography on a polyvinyl alcohol gel column, Asahipak GS-320H, with isocratic elution by sodium phosphate. Application of this system to the determination of the sizes of cellular pools of purine derivatives in plant cells and of the activity of related enzymes, as well as to the purification of enzymatically synthesized radioactive compounds, is described.     

Three-dimensional protein map according to pI,hydrophobicity and molecular mass

A three-dimensional method has been developed to map the protein content of cells according to pI, M(w) and hydrophobicity. The separation of complex protein mixtures from cells is performed using isoelectric focusing (IEF) in the liquid phase in the first dimension, non-porous silica (NPS) RP-HPLC in the second dimension and on-line electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) detection in the third dimension. The experimentally determined pI, M(w) and hydrophobicity can then be used to produce a three-dimensional map of the protein expression of a cell, where now each protein can be tagged by three independent parameters. The ESI-TOF-MS provides an accurate M(w) for the intact protein while the hydrophobicity dimension results from the RP-HPLC component of the separation. The elution time, or percent acetonitrile at time of elution, of the protein is related to the hyrophobicity, which is an inherent property of the protein. 3D protein maps can thus be generated showing pI, M(w) and % acetonitrile at time of elution as well as pI, M(w) and hydrophobicity. The potential of the 3D plot for effective mapping of proteins from cells compared to current 2D methods is discussed.     

Effects of purine riboside on nucleic acid synthesis in ascites cells.

Purine riboside (nebularine, 9-beta-ribofuranosylpurine) is a naturally occurring base analog which closely resembles adenosine. It inhibits carcinogenic growth. Purine riboside strongly inhibits RNA and DNA synthesis in different cancer ascites cells. Gel electrophoretic analysis of RNA synthesis in vivo in the presence of purine riboside shows the ribosomal components to be inhibited the most. A method for assaying purine riboside or its phosphates intracellularly has been devised, and by using this it has been shown that purine riboside is extensively phosphorylated in the cells. The triphosphate derivative of purine riboside has been isolated and tested in the Escherichia coli RNA polymerase assay. It appears not to be incorporated into this type of RNA and to competitively inhibit this reaction with regard to ATP.