Simultaneous determination of adenosine and inosine using single-wall carbon nanotubes modified pyrolytic graphite electrode

Voltammetric determination of adenosine and inosine has been carried out at single-wall carbon nanotubes (SWNTs) modified pyrolytic graphite electrode (PGE) at pH 7.2 using Osteryoung square wave voltammetry (OSWV). The modified electrode exhibits remarkable electrocatalytic properties towards adenosine and inosine oxidation with a peak potential of approximately 1229 mV and 1348 mV, respectively. Linear calibration curves are obtained over the concentration range 0.5 microM to 1.0 mM in adenosine and 10 microM to 1.0 mM in inosine with sensitivity of 1.0 microA microM(-1) and 1.9 microA microM(-1) for adenosine and inosine respectively. The limit of detection for adenosine and inosine was found to be 0.51x10(-7) M and 2.04x10(-7) M, respectively. The proposed method was also used to estimate these compounds in human blood plasma and urine samples and the method was validated using HPLC.     

Determination of the kinetic parameters of adenosine deaminase by electrophoretically mediated microanalysis

The possibility of determining the Michaelis constant of the irreversible deamination of adenosine to inosine by adenosine deaminase, using capillary electrophoresis, was investigated. This paper describes the use of electrophoretically mediated microanalysis (EMMA) as the technique for carrying out the assay. Initial reaction velocities of the enzymatic reaction were estimated from the peak area of inosine, and the Michaelis constant was calculated according to the Lineweaver-Burk equation. The result (K_m = 5.3 × 10⁻⁵ M ± 8 × 10⁻⁶ M) was consistent with previously reported values. Using the present method, a total amount of as few as 1.2 fmole of enzyme and 9.2 ng of substrate were injected in the capillary for the construction of a Michaelis Menten curve (seven concentrations of substrate, each concentration analyzed in triplicate), which is far smaller than the quantities required in conventional methods.     

Determination of Adenosine and Inosine in Sheep Plasma Using Solid Phase Extraction Followed by Liquid Chromatography with UV Detection

A simple and sensitive liquid chromatography assay following solid phase extraction was developed for simultaneous determination of adenosine and inosine in sheep plasma. The system consisted of a Symmetry C₁₈ column, a mobile phase composed of acetonitrile, 100 mM sodium dihydrogen phosphate and water, and ultraviolet detection at 254 nm. The method showed good sensitivity (limits of detection for adenosine and inosine were 30 and 50 ng/ml, respectively, in the plasma samples), repeatability, and linearity. The developed method was applied to sheep plasma samples from a study examining the cardio active potential of the combination of adenosine and inosine.     

Synthesis and evaluation of N6-substituted azide- and alkyne-bearing N-mustard analogs of S-adenosyl-l-methionine

The synthesis of a family of N-mustard analogs of S-adenosyl-l-methionine (SAM) containing azides and alkynes at the N6-position of the adenosine base has been accomplished from commercially available inosine. Further biochemical analysis of these analogs indicates successful modification of pUC19 plasmid DNA in an enzyme-dependent fashion with DNA methyltransferases M.TaqI and M.HhaI.     

Quantitation of adenosine, inosine and hypoxanthine in biological samples by microbore-column isocratic high-performance liquid chromatography

This paper describes a simple and sensitive high-performance liquid chromatographic method for measuring adenosine, inosine and hypoxanthine in cell suspensions. The method involves direct injection of the filtered sample on a microbore C18 reversed-phase column with UV detection at 259 nm. The mobile phase consisted of 125 mM potassium dihydrogenphosphate, 1.0 mM tetrabutylammonium hydrogen-sulfate, 1.5% acetonitrile and 20 mM triethylamine, pH 6.5. The minimum detectable amounts (signal-to-noise ratio of 3:1) were 2.0 pmol of adenosine, 2.5 pmol inosine and 3.5 pmol of hypoxanthine. The limits of quantitation were 2.9 +/- 0.2 pmol for adenosine, 4.2 +/- 0.3 pmol for inosine and 4.9 +/- 0.4 pmol for hypoxanthine. This method was used to quantitate adenosine release by dispersed rat renal outer medullary cells (tubules) under conditions of normoxia and hypoxia.     

Nucleosides, 45.-Assignment of glycosylation sites in o-hexopyranosyl-ribonucleosides by 13C-NMR

Evidence is presented that glycosylation of a ribose-OH group in nucleosides results in a significant downfield shift for the appended ¹³C nucleus and smaller upfield displacements for the adjacent carbons, providing an efficient tool for differentiating between 2′-$\text{o}$-, 3′-$\text{o}$- and 5′-$\text{o}$-glycosyl-ribonucleosides. Therewith, the products formed on enzymatic galactosylation of uridine, inosine, and adenosine are unequivocally assigned β(1→3)-glycosidic linkages ($\text{3a}$ – $\text{3c}$).     

Determination of concentrations of adenosine and other purines in human term placenta by reversed-phase high-performance liquid chromatography with photodiode-array detection: evidence for pathways of purine metabolism in the placenta.

A robust analytical method, using reversed-phase high-performance liquid chromatography with gradient elution and photodiode-array detection, was used to measure six purines and beta-NAD+ in acid-soluble extracts of samples taken from six different regions of human term placenta. Resolution of the analyte peaks in chromatographic profiles of the extracts, and the use of optimized integration, allowed simultaneous quantitation of all seven analytes from a single chromatogram. Peak purity was confirmed via on-line analysis of peak spectra, utilizing the purity parameter treatment of spectral data. Major placental purines were adenosine, inosine, hypoxanthine and adenine. Except for adenine, concentrations of the purines varied by two-fold or more between different regions of each placenta, but concentration ratios, i.e., adenosine/inosine and inosine/hypoxanthine, were similar. The findings indicate that the pathway of ATP breakdown to hypoxanthine in ischemic human term placenta is via adenosine, and that regional differences in placental concentrations of adenosine and its metabolites may result from regional differences in degree of ischemia.     

Radical-based transformation of vicinal diols to olefins via thioxocarbamate derivatives: a simple approach to 2′,3′-didehydro-2′,3′-dideoxynucleosides

The bis-O-thioxocarbamate derivatives obtained from the reaction of vicinal diols with phenyl isothiocyanate are shown to be reduced with tris(trimethylsilyl)silane in the presence of azobisisobutyronitrile to afford the corresponding olefins in good yields. In this way, 2′,3′-didehydro-2′,3′-dideoxy analogs of adenosine, guanosine, inosine, cytidine and uridine were prepared by the radical-based deoxygenation of the corresponding ribonucleosides via the bis-O-thioxocarbamate derivatives.     

Separation and identification of positively charged and neutral nucleoside adducts by capillary electrochromatography-microelectrospray mass spectrometry

Capillary electrochromatography (CEC) is shown to be capable of separating mixtures containing both positively charged and neutral styrene oxide–adenosine adducts. In a study of the mechanism of deamination of positively charged 1-(2-hydroxy-1-phenylethyl) adenosine using ¹⁸O-labeled water, possible contamination of the chromatographically purified deamination product, 1-(2-hydroxy-1-phenylethyl) inosine, with the positively charged 1-(2-hydroxy-1-phenylethyl) adenosine was observed. Because the deamination product and the presumed contamination have the same molecular weights and similar structures, CEC-microelectrospray mass spectrometry (CEC-μESI/MS) was used to confirm the presence and identity of the suspected impurity. A trace amount of the positively charged 1-(2-hydroxy-1-phenylethyl) adenosine, which could not be observed by either HPLC-UV or CEC-UV, was detected by CEC-μESI/MS. This discriminatory ability of CEC-μESI/MS is attributed to the fact that positive ion mode ESI-MS is a more sensitive detector for a positively charged compound than a UV detector, and that the combination of electroosmotic and electrophoretic flows and hydrophobic interactions with the stationary phase contributes to the separation of the positively charged compound. As a result, the positively charged compound was observed to elute much earlier and with much sharper peaks than the neutral compounds for which electroosmotic flow is the only “pumping” force for the solvent.     

Measurement of adenosine, inosine and hypoxanthine in human term placenta by reversed-phase high-performance liquid chromatography

An analytical method was developed for measuring adenosine, inosine and hypoxanthine in freshly delivered human term placentas. Representative freeze-clamped samples were taken from the sub-maternal surface of each placenta. Acid-soluble extracts of the samples were analyzed by reversed-phase high-performance liquid chromatography on columns packed with 10-micron porous octadecylsilica, using gradient elution with a linear increase in methanol concentration in ammonium phosphate buffer. Resolution of hypoxanthine from xanthine and adenosine from adenine, and quantitation of hypoxanthine and adenosine were achieved using 0.05 M ammonium dihydrogen phosphate, pH 6.5, as the low-strength eluent. Resolution of inosine from a prominent peak of beta-NAD was optimized using 0.02 M ammonium dihydrogen phosphate, pH 5.6, as low-strength eluent. Recovery of standards was greater than 90%. Mean contents (+/- S.D.) of the analytes in placentas from seven normal deliveries were, adenosine 30.6 +/- 11.5 nmol/g, inosine 68.0 +/- 25.8 nmol/g and hypoxanthine 217 +/- 127.5 nmol/g.     

Reverse Phase HPLC For Rapid,Comprehensive Measurement of Nucleotides,Nucleosides and Bases of The Myocardial Adenine Pool

Abstract

A novel reverse phase HPLC method is described for the simultaneous measurement of adenosine tri-, di- and monophosphates (ATP, ADP, AMP), inosine monophosphate (IMP), adenosine, inosine, hypoxanthine, nicotinamide adenine dinucleotide (NAD) and uric acid in cardiac tissues and coronary effluent. The use of a simplified perchloric acid extraction procedure and ODS columns easily modified with Mq⁺⁺, Tris and phosphate buffer, allows considerable saving in analysis time together with extremely good resolution, particularly for ATP and ADP, and provides a very practical tool for the routine assessment of changes in adenine pool metabolites.     

Quality evaluation of Cordyceps through simultaneous determination of eleven nucleosides and bases by RP-HPLC

A new RP-HPLC method has been developed for the simultaneous determination of 11 nucleosides and bases, including adenosine, cordycepin, cytidine, guanosine, inosine, thymidine, uridine, cytosine, guanine, thymine, and uracil in Cordyceps. Determination was achieved on a Zorbax 300SB C18 analytical column (4.6 x 250 mm id, 5 mm) using gradient elution with diode-array detection. All calibration curves showed good linearity (r2 > 0.9995) within the test ranges. The developed method was simple, rapid, and accurate, and showed good reproducibility for the quantification of 11 nucleosides and bases in natural and cultured Cordyceps with both intra- and inter-day variations of less than 1.8%. Furthermore, hierarchical clustering analysis based on the typical peaks of adenosine, cordycepin, and inosine in HPLC profiles from the 11 tested samples showed that natural and cultured Cordyceps were in different clusters, which could provide a means of discriminating between Cordyceps of different origins. Thus, adenosine, cordycepin, and inosine could be used as markers for quality control of Cordyceps.     

Simultaneous determination of six main nucleosides and bases in natural and cultured Cordyceps by capillary electrophoresis

A simple method is described for simultaneous determination of six main nucleosides and bases including adenine, uracil, adenosine, guanosine, uridine and inosine in Cordyceps by capillary electrophoresis (CE). Chemometric optimization based on central composite design was employed to find the optimum resolution. The optimum factor space was defined by three parameters: buffer concentration, pH and concentration of acetonitrile as organic modifier. Resolution (Rs) was employed to evaluate the response function. A running buffer composed of 500 mM boric acid, adjusted pH to 8.6 with sodium hydroxide and 12.2% acetonitrile as modifier was found to be the most appropriate for the separation. The contents of the six components were determined by using adenosine monophosphate as an internal standard. Furthermore, hierarchical clustering analysis based on characteristics of 32 peaks in CE profiles from the tested 12 samples showed that natural and cultured Cordyceps were in different clusters. Adenosine and inosine were extracted as markers for discrimination of natural Cordyceps. The result of clustering based on the two peaks characteristics was in excellent agreement with that based on 32 peaks'. Thus, adenosine and inosine could be used as markers for quality control of natural and cultured Cordyceps.     

Chemical Studies of Formosan Cobra (Naja Naja Atra) Venom: Part II. Isolation and Characterization of Guanosine,Adenosine, and Inosine from Cobra Venom

A non-protein fraction from crude Formosan cobra venom was found to be a nucleoside mixture. The components were identified as guanosine, adenosine and inosine by paper chromatographic and polyamide thin-layer chromatographic correlations and their spectroscopic data. The mole ratio of guanosine/adenosine/inosine was found to be 7:2.5:1.     

A new method for the determination of adenine phosphoribosyltransferase activity in human erythrocytes by reversed phase high performance liquid chromatography

A new method for the determination of adenine phosphoribosyltransferase (APRT) activity in human erythrocytes is described. APRT activity was assayed by a non-radiochemical method in which adenosine monophosphate (AMP) and AMP metabolites produced from a substrate adenine were converted to inosine by alkaline phosphatase and adenosine deaminase. The inosine thus produced was quantitated by reversed phase HPLC. This method was simple, precise, sensitive and free from interference with other co-existing erythrocyte enzymes. Four patients with 2,8-dihydroxyadenine urolithiasis and others with several disorders in purine metabolism have been studied, showing that the present method is clinically useful for the diagnosis and the evaluation of the severity of some human diseases.     

Simultaneous determination of adenosine and its metabolites by capillary electrophoresis as a rapid monitoring tool for 5'-nucleotidase activity

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of micro-molar adenosine, hypoxanthine and inosine in enzyme assays without using radioactive labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. Under the optimal condition, the good separation with high efficiency was achieved in 6 min. Using deoxyadenylate as an internal standard, the linear range of the method was 5-200 microM, and the concentration limits of detection of adenosine, hypoxanthine and inosine were 2.2, 3.6 and 1.4 microM, respectively. Application of the proposed method was demonstrated by the activity assay of 5'-nucleotidase from Hep G2 cells.     

Sensitive determination of inosine RNA modification in single cell by chemical derivatization coupled with mass spectrometry analysis

Inosine is a vital RNA modification across three kingdoms of life. It has been demonstrated that inosine plays important roles in modulation of the fate of RNAs. In the current study, we developed a highly sensitive method to determine inosine in a single cell by N-cyclohexyl-N’-β-(4-methylmorpholinium)ethylcarbodiimide p-toluenesulfonate(CMCT) derivatization in combination with mass spectrometry analysis. The results showed that the detection sensitivity of inosine was increased by 556-fold aft...     

Single Tryptophan of Disordered Loop from Plasmodium falciparum Purine Nucleoside Phosphorylase: Involvement in Catalysis and Microenvironment

Among various tropical diseases, malaria is a major life-threatening disease caused by Plasmodium parasite. Plasmodium falciparum is responsible for the deadliest form of malaria, so-called cerebral malaria. Purine nucleoside phosphorylase from P. falciparum is a homohexamer containing single tryptophan residue per subunit that accepts inosine and guanosine but not adenosine for its activity. This enzyme has been exploited as drug target against malaria disease. It is important to draw together significant knowledge about inherent properties of this enzyme which will be helpful in better understanding of this drug target. The enzyme shows disorder to order transition during catalysis. The single tryptophan residue residing in conserved region of transition loop is present in purine nucleoside phosphorylases throughout the Plasmodium genus. This active site loop motif is conserved among nucleoside phosphorylases from apicomplexan parasites. Modification of tryptophan residue by N-bromosuccinamide resulted in complete loss of activity showing its importance in catalysis. Inosine was not able to protect enzyme against N-bromosuccinamide modification. Extrinsic fluorescence studies revealed that tryptophan might not be involved in substrate binding. The tryptophan residue localised in electronegative environment showed collisional and static quenching in the presence of quenchers of different polarities.     

A Multiple Enzyme Method for Maasurement of K,Na ATPase

Abstract

A multiple enzyme method for spectrophotometrical measurement of K, Na ATPase activity is described. The method includes three enzymatic steps: Hydrolyses of ATP by ATPase, conversion of inosine to hypoxanthine and ribose -1- phosphate by purine nucleoside phosphorylase and finally xanthine oxidase mediated oxidation of hypoxanthine to xanthine with consequent formation of formazan from a tetrazolium salt. The phospholytic cleavage of inosine in the medium is dependent of an ATPase- phosphate complex.     

The yeast <Emphasis Type="Italic">ISN1</Emphasis> (<Emphasis Type="Italic">YOR155c</Emphasis>) gene encodes a new type of IMP-specific 5'-nucleotidase

Background  

The purine salvage enzyme inosine 5'-monophosphate (IMP)-specific 5'-nucleotidase catalyzes degradation of IMP to inosine. Although this enzymatic activity has been purified and characterized in Saccharomyces cerevisiae, the gene encoding IMP 5'-nucleotidase had not been identified.