Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in conjunction with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10³ concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.     

Quantitative high-performance liquid chromatography of nucleosides and bases in human plasma

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the estimation of purines, pyrimidines and their congeners in biological fluids. An initial HPLC separation allowed the collection of a number of effluent fractions, each of which contained a single component of interest. The re-application of these fractions to a second HPLC separation permitted the resolution and quantification of nanogram amounts of these components. Isocratic elution with volatile buffers renders the samples amenable to automatic sampling procedures or lyophilisation. Data are presented on the application of the method to the analysis of nucleosides and bases in human plasma.     

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.     

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.     

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.     

Gold complexes of purine and pyrimidine nucleosides

The reactions of chloroauric acid (HAuCl₄) with inosine=ino, guanosine=guo, triacetylinosine=trino, triacetylguanosine=trguo, and cytidine=cyd were studied. Complexes of Au(III) and Au(I) with these nucleosides have been isolated from reactions at different pH values in aqueous and in methanolic solutions. The Au(I) complexes were obtained by reducing Au(III) with 1-ascorbic acid in aqueous solutions. All the isolated complexes were characterized by elemental analyses, conductivity measurements, IR, ¹H nmr, and esr spectra. The Au(III) complexes correspond to the general formulae [Au(nucl)₂Cl₂] Cl, Au(nucl)Cl₃, and Au(nucl-H⁺)Cl₂, while the Au(I) complexes are of the Au(nucl)₂Cl type, where nucl represents the above nucleosides. In the complex with the composition [AucydCl₂]₂ that was isolated from aqueous solutions, the Au atom is believed to be in the (II) oxidation state.Possible structures for all the isolated complexes based on the experimental data are proposed and discussed.     

4'-C-methyl-beta-D-ribofuranosyl purine and pyrimidine nucleosides revisited

In order to evaluate their antiviral properties, a series of 4'-C-methyl-beta-D-ribofuranosyl purine and pyrimidine nucleosides has been prepared. Unfortunately, none of these 4'-branched nucleosides showed any antiviral activity or cytotoxcity when tested against HIV, HBV, and Yellow Fever virus.     

Synthesis of purine and pyrimidine bases and nucleosides under the cold plasma conditions (IX)

Living-matter precursors are obtained by the action of the high-frequency electrical discharges on a methane-ammonia-water mixture. Some purine and pyrimidine bases as well as nucleosides are detected and estimated quantitatively by column chromatography, one- and two-dimensional paper chromatography, paper chromatography with standard addition, colour tests as well as by optical density measurement within the UV region.     

Synthesis of purine and pyrimidine bases and nucleosides under the cold plasma conditions (IX)

Living-matter precursors are obtained by the action of the high-frequency electrical discharges on a methane-ammonia-water mixture. Some purine and pyrimidine bases as well as nucleosides are detected and estimated quantitatively by column chromatography, one- and two-dimensional paper chromatography, paper chromatography with standard addition, colour tests as well as by optical density measurement within the UV region.     

Metabolism of pyrimidine bases and nucleosides in Neisseria meningitidis.

In Neisseria meningitidis, uridine, deoxyuridine, cytosine, cytidine, or deoxycytidine could not be used by uracil-requiring mutants as pyrimidine sources. Consistent with these findings, only 5-fluorouracil of the different fluoropyrimidine bases and nucleosides showed any inhibitory effect on the growth of four prototrophic strains of N. meningitidis. Likewise, only radioactive uracil was readily incorporated into nucleic acids, whereas uptake of radioactive uridine, cytosine, or cytidine could not be demonstrated. Uracil was converted to uridine 5'-monophosphate by uracil phosphoribosyltransferase, whereas enzyme activities for conversion of cytosine or any of the nucleosides were not detectable in meningococcal extracts.     

Metabolism of pyrimidine bases and nucleosides in Bacillus subtilis.

In Bacillus subtilis, uracil (Ura), uridine (Urd), and deoxyuridine (dUrd) are metabolized through pathways similar to those of enteric bacteria. Ura is probably converted to uridine 5'-monophosphate by uridine 5'-monophosphate pyrophosphorylase. More than 95% of dUrd added to cultures is converted to Ura and deoxyribose-1-phosphate. Although dUrd kinase activity is detectable in vitro, this enzyme does not seem to play an important role in the metabolism of dUrd. The metabolism of cytosine (Cyt), cytidine (Cyd), and deoxycytidine (dCyd) in B. subtilis appears to be different from that in enteric bacteria. Cytosine cannot be used by Ura-requiring mutants as pyrimidine source. dCyd is deaminated by dCyd-Cyd deaminase or phosphorylated to dCyd nucleotides by dCyd kinase. Cyd is deaminated by dCyd-Cyd deaminase of phosphorylated by Cyd kinase. This Cyd kinase activity has never been reported for B. subtilis.     

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.     

Transport of purine and pyrimidine bases and nucleosides from endosperm to cotyledons in germinating castor bean seedlings

During germination and early growth of castor bean (Ricinus communis), all cellular constituents of the endosperm are eventually transferred to the growing embryo. The present results bear on the transport of breakdown products of nucleic acids. The total content of nucleic acids and nucleotides declines rapidly between day 4 and day 8 of seedling development. Concomitant with this decline, a secretion of adenosine, guanosine, and adenine from excised endosperms into the incubation medium takes place, accompanying a much more extensive release of sucrose and amino acids. Release of nucleotides could not be detected. The rates of release were linear for at least 5 hours for all compounds measured, indicating that they were liberated due to a coordinated metabolism. Uptake studies with cotyledons removed from the seedling showed that these have the ability to absorb all the substances released from the endosperm. Besides sucrose and amino acids, both nucleosides and free purine and pyrimidine bases were taken up by the cotyledons with high efficiency. AMP was also transported whereas ATP was not. Kinetic analyses were carried out to estimate the maximal uptake capacities of the cotyledons. Rates of uptake were linear for at least 1 to 2 hours and saturation kinetics were observed for all substances investigated. It is concluded that nucleosides can serve best as transport metabolites of nucleic acids, inasmuch as they are taken up by the cotyledons with the highest efficiency, the V_max/K_m ratios being considerably higher than those found for free purine and pyrimidine bases. For both adenosine and adenine transport, the V_max was about 2 micromoles per hour per gram fresh weight, and the K_m values were 0.12 and 0.37 millimolar, respectively. The rates of metabolite release from the endosperm and the capacity of the absorption system in the cotyledons are shown to account for the observed rates of disappearance of nucleic acids from the endosperm and efficient transport to the growing embryo.     

Entamoeba histolytica: uptake of purine bases and nucleosides during axenic growth.

A comparison was made of the uptake mechanisms of selected purine bases and nucleosides by axenically grown Entamoeba histolytica. Adenine, adenosine, and guanosine were taken up, in part, by a “carrier”-mediated system. Guanine, hypoxanthine, and inosine entered amoebas via diffusion. Inhibitor studies support the presence of individual transport sites for adenine-adenosine and adenosine-guanosine. Additional sites for transport of adenine, adenosine, and guanosine are implied by “non-productive binding” involving guanine, hypoxanthine, and inosine. Uptake of adenine, adenosine, and guanosine was reduced by iodoacetate and N-ethylmaleimide. Ribose failed to inhibit uptake of purine nucleosides.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.