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

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 leukemia. 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. Measurement 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.     

Use of automated chromatography on the amino acid analyzer with lithium citrate buffers to separate nucleic acid bases, nucleosides, nucleotides and their precursors

A simple and sensitive method has been developed to separate nucleic acid bases, nucleosides, nucleotides and their precursors by automated chromatography using the amino acid analyzer with lithium citrate buffers. The method is sensitive to a concentration of 5 nmol, linear in the range of 5--100 nmol, and resolves almost all the bases, nucleosides, nucleotides and their precursors of physiologic importance.     

Purification and fractionation of free nucleotides from Euglena gracilis Z by a combined procedure of ligand-exchange and anion-exchange gel chromatography

A simple and rather sensitive method for the purification, fractionation, and assay of acid-soluble nucleotides in Euglena cells was developed. The perchloric acid-soluble extract is purified by ligand-exchange chromatography on copperloaded chelating resin, Wofatit MC 50. The narrow nucleotide peak thus obtained is fractionated by anion-exchange gel chromatography on DEAE-Sephadex A-25 with a linear gradient of 0.0–0.3 m (NH₄HCO₃ + NH₂COONH₄). This paper presents (a) the elution positions of 60 known nucleotides, nucleosides, and bases and (b) the quantitative composition of the acidsoluble nucleotide pool of 48-hr-old photoorganotrophically grown Englena cells in order to demonstrate the advantages of the method described.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

Single-run high-performance liquid chromatography of nucleotides, nucleosides, and major purine bases and its application to different tissue extracts

A high-performance liquid chromatography (HPLC) method is described for the separation and quantitation of nucleotides, nucleosides, purine bases, and related compounds in one single run. The separation of a standard mixture of at least 24 components is achieved within 35 min on glass columns (30 cm, 3-mm i.d.) with C-18 reversed-phase particles of 5 micron, and ammonium dihydrogen phosphate (0.15 M, pH 6.00) and a slow linear gradient of methanol/acetonitrile (to 15%) as eluting solvent. The method has been applied to microsamples of different cells and tissues. Samples (2.5 mg dry wt) were cooled in liquid nitrogen, lyophilized, and extracted with 0.6 N perchloric acid. After neutralization with potassium bicarbonate, the extract (20 microliter) was directly injected into the column. To illustrate the wide applicability of the method, representative chromatograms are shown of extracts of biopsies from heart tissue, skeletal muscle, and brain and liver and from hepatocytes, erythrocytes, and yeast cells, under different conditions, known to induce changes in purine metabolism.     

A thin-layer chromatographic method for separation of thymidine and deoxyuridine nucleotides

A thin-layer chromatographic method for the separation of thymidine and deoxyuridine nucleotides and nucleosides is described. This procedure involves the following sequence of steps: (i) Ion-exchange thin-layer chromatography to afford separation into fractions of increasing degree of phosphorylation, (ii) conversion of each fraction into an equivalent mixture of thymine and uracil through the combined actions of alkaline phosphatase and thymidine phosphorylase, and (iii) partition thin-layer chromatographic separation of thymine and uracil. A key feature of the method is the specificity afforded by the second step which converts only thymidine and deoxyuridine nucleotides and nucleosides to the corresponding pyrimidine bases. An application of the method to the study of [³H]deoxyuridine metabolism in L1210 cells, as well as the effect of methotrexate on this metabolism is also described.     

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.     

A sensitive method for determining quinolinic acid in animal tissues

A sensitive chromatographic method for isolation and measurement of quinolinic acid from rat liver and kidney is described. The method is based on the isolation of quinolinic acid by ion-exchange chromatography. The extraction of quinolinic acid consisted of the freeze clamping of the organ in liquid nitrogen, followed by deproteinization in perchloric acid. The neutralized extract was concentrated by freeze-drying and submitted to the action of concentrated perchloric acid to hydrolyze the nucleotides which interfered in the chromatographic separation of quinolinic acid. The sample was applied to a column of Dowex (HCOO^?) and eluted with a linear gradient of formic acid. The eluted fraction containing quinolinic acid was quantitatively measured by its absorbance at pH 2 and 268 nm in a spectrophotometer.     

Isotachophoresis of nucleic acid constituents

Determination methods for purine and pyrimidine bases, nucleosides, nucleotides and related compounds using analytical capillary isotachophoresis are reviewed. First, the isotachophoretic characterization of these compounds, as well as methods for sample preparation prior to analysis, and the different ways of detecting unknown substances in complex biological systems are described. Then applications of isotachophoretic analysis in medical diagnosis and biomedical research are reviewed. In particular, the analysis of purines, pyrimidines, nucleosides and related compounds in blood and serum for the diagnosis and treatment of inherited diseases and cancer is described. Selected applications of nucleotide analysis in biomedical research using different tissue extracts are also reviewed, and some examples of nucleotide-dependent enzymic reactions, which were performed by means of analytical isotachophoresis, are presented.     

Contribution of hydrolyzed nucleic acids and their constituents to the apparent amino acid composition of biological compounds.

Acid hydrolysis of protein-free mixtures of nucleotides, nucleosides, and nucleic acids yields amino acids, free bases, and possibly other unidentified fragments when analyzed by thin-layer chromatography and by standard amino acid analysis. Glycine is the predominant amino acid detected, which may constitute 47–97% of the apparent amino acid composition, depending on the type of material subjected to hydrolysis. Obviously, hydrolyzed nucleic acids or their constituents can therefore contribute to the apparent amino acid composition of a supposedly pure peptide or of other more complex mixtures of compounds mistakenly believed to contain only protein. To circumvent this problem, we suggest that nucleotides or nucleic acid moieties should be removed from any product for which the amino acid composition is desired, and that whenever a large glycine peak is noted in a hydrolyzed sample, the presence of nucleic acids or their constituents should be suspected.     

HPLC determination of the base composition of yeast tRNA

Summary A relatively simple procedure was developed to quantitate normal and methylated tRNA bases by isocratic HPLC. tRNA was extracted with phenol from lyophilyzed cells, purified and precipitated with isopropanol. After perchloric acid hydrolysis, the samples were subjected to HPLC analysis. The mole % composition of normal and methylated bases was determined in yeasts grown on unusual carbon sources including hydrocarbons and light alcohols.Normal and methylated base composition of total tRNA depends on the microorganism and on the carbon source used.     

Quantitative changes in the content of nucleic acid bases and their derivatives in a culture ofPenicillium sizowi

Acid-soluble (“free”) nucleotides, nucleosides and bases were analyzed in the mycelium and in the culture filtrate of the fungusPenicillium sizowi, using micro-thin-layer chromatography on alumina and densitometry of the zones of the individual components. It was found that the levels of the various components underwent complicated changes, the corresponding curves exhibiting from one to three maxima which occur at different periods of cultivation. It was observed that a substantial amount of nucleotides, nucleosides and bases occurs in the medium as early as at the beginning of the exponential phase of growth. An attempt was made to elucidate some peculiarities of the nucleotide pool ofPenicillium species, using enzymes responsible for the individual transformations of nucleotides, nucleosides and bases.     

Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography

A high-performance liquid-chromatographic (HPLC) method for the rapid separation of purine and pyrimidine nucleotides, NAD+, NADP+, FAD, FMN, UDP-Glc, UDP-glucuronate, and ADP-ribose found in neutralized perchloric acid extracts of rat liver is described. Separation was achieved within 26 min on a radially compressed column of Partisil 10-SAX. The column was eluted with a gradient of sodium phosphate and sodium chloride. The sodium phosphate was purified by passage through tandem columns of anion- and cation-exchange resins to remove uv-absorbing impurities. The sensitivity of this procedure is such that an amount of ATP contained in 10 micrograms of liver can be measured. The recoveries of all nucleotides were between 87 and 107%. In extracts of rat liver interfering substances were found to elute with GDP, and UDP eluted with NADP. Consequently, the tissue contents of UDP and GDP were determined in a second run by measuring the increase in UTP and GTP, respectively, following sample pretreatment with pyruvate kinase (PK). The tissue level of NADP+ was calculated as the difference between the total UDP and NADP+ peak and the increase in UTP following PK treatment. In those nucleotides amenable to enzymatic analysis, namely NAD+, AMP, UDP-Glc, UTP, and ATP, the tissue contents measured enzymatically were not significantly different from those determined by HPLC. However, ADP as measured with PK was found to be 15% higher compared to the HPLC determination.     

A new highly selective physicochemical assay to measure NAD+ in intact cells

A simple, fast, and highly specific chromatographic method for measuring the content of NAD+ in intact cells has been developed. This procedure involves the separation of NAD+ from the bulk of acid-soluble nucleosides, nucleotides, and other pyridine containing molecules by affinity chromatography on dihydroxyboronyl-Bio-Rex. The boronate purified preparations were utilized for the quantification of NAD+ by strong anion exchange high-pressure liquid chromatography under isocratic conditions using a low salt buffer system. The overall recovery of the method exceeded 80%. This new method was applied to determine the extent of NAD+ consumption in intact hepatocytes following treatment with two different DNA damaging agents. A major advantage of this method is that it allows for the simultaneous determination of poly(ADP-ribose) in the acid-insoluble fraction of the same sample.     

Optimal extraction conditions for high-performance liquid chromatographic determination of nucleotides in yeast

Different extraction methods of nucleotides from the yeast Saccharomyces cerevisiae were compared. A new extraction solution--formic acid saturated with 1-butanol--was found to be more effective than the commonly used solutions of trichloroacetic acid, perchloric acid, or formic acid alone. Using this solution the optimal extraction conditions were established. Nucleotide recovery was evaluated by adding standard nucleotides to the extraction medium and carrying them together with the cells through the whole extraction procedure. Nucleotides were separated and quantitated by high-performance liquid chromatography on an anion-exchange column.     

Identifying decomposition products in extracts of cellular metabolites

Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, then, it would seem that the best method for extracting a particular metabolite is the one that gives the largest yield. In extracting Escherichia coli with different methanol:water mixtures, we observed that >or=50% water gave an increased yield of nucleosides and bases compared with 相似文献   

Rapid radioassay for metabolites of adenosine and deoxyadenosine in erythrocytes

A radioassay has been developed to quantify the uptake and initial metabolism of adenosine (Ado) or deoxyadenosine (dAdo) by human erythrocytes. Cell suspension and [³H]Ado are mixed at 3-s intervals with a novel dual-syringe apparatus, and uptake and metabolism of Ado is stopped by centrifuging the cells through a dibutylphthalate layer into perchloric acid. The neutralized cell extract is analyzed by two-dimensional chromatography on poly(ethyleneimine)-cellulose plates by two procedures using combinations of solvents optimised for the separation of nucleosides and nucleobases, and for nucleotides derived from the exogenous [³H]Ado.     

Nucleotide,nucleoside and base nutritional requirements of the mosquito Culex pipiens

Nucleic acid subcomponents needed to satisfy the dietary nucleic acid requirement of Culex pipiens were studied in growth experiments using synthetic media in which nucleosides, bases and alternative nucleotides were variously substituted in mixtures of 3 nucleotides (adenylic acid, thymidylic acid, and either cytidylic or uridylic acid) previously shown to be adequate replacements for whole nucleic acid. Any or all 3 nucleotides could be replaced by corresponding nucleosides without adverse effect, except that adenosine substitution moderately delayed pupation. All base substitutions were unsatisfactory: substitution of thymine for thymidylic acid allowed development to the adult stage but at a greatly reduced rate; single substitution of adenine, cytosine or uracil for the corresponding nucleotides allowed scarcely more development than in the total absence of nucleic acid derivatives. Inosinic acid or inosine were adequate substitutes for adenylic acid, but orotic acid or orotidine were ineffective in place of the pyrimidine ribonucleotides, cytidylic or uridylic acids. Deoxyadenylic acid could take the place of adenylic acid, though inefficiently, but deoxycytidylic and deoxyuridylic acids were very poor replacements for the corresponding ribonucleotides. The minimal required nucleic acid derivatives thus appear to be a purine ribonucleotide (adenylic or inosinic acids), a pyrimidine ribonucleoside (either uridine or cytidine), and the pyrimidine deoxyribonucleoside, thymidine.     

A coupled optical assay for determination of adenine in mixtures.

A rapid and specific spectrophotometric assay for the determination of adenine is described. The method is based on the absorbance change at 265 nm which accompanies the ribose 1-phosphate-dependent conversion of adenine into inosine, catalyzed by the successive action of adenosine phosphorylase and adenosine deaminase. Common purine and pyrimidine bases, nucleosides, and nucleotides do not interfere. The assay was tested in various biochemical situations, in which there was both adenine formation and utilization.