Nucleoside Profiles Research Articles

Synthesis of Platinum(II) N-Heterocyclic Carbenes Based on Adenosine.

Organometallic derivatization of nucleosides is a highly promising strategy for the improvement of the therapeutic profile of nucleosides. Herein, a methodology for the synthesis of metalated adenosine with a deprotected ribose moiety is described. Platinum(II) N-heterocyclic carbene complexes based on adenosine were synthesized, namely N-heterocyclic carbenes bearing a protected and unprotected ribose ring. Reaction of the 8-bromo-2′,3′,5′-tri-O-acetyladenosine with Pt(PPh3)4 by C8−Br oxidative addition yielded complex 1, with a PtII centre bonded to C-8 and an unprotonated N7. Complex 1 reacted at N7 with HBF4 or methyl iodide, yielding protic carbene 2 or methyl carbene 3, respectively. Deprotection of 1 to yield 4 was achieved with NH4OH. Deprotected compound 4 reacted at N7 with HCl solutions to yield protic NHC 5 or with methyl iodide yielding methyl carbene 6. Protic N-heterocyclic carbene 5 is not stable in DMSO solutions leading to the formation of compound 7, in which a bromide was replaced by chloride. The cis-influence of complexes 1–7 was examined by 31P{1H} and 195Pt NMR. Complexes 2, 3, 5, 6 and 7 induce a decrease of 1JPt,P of more than 300 Hz, as result of the higher cis-influence of the N-heterocyclic carbene when compared to the azolato ligand in 1 and 4.

Processed velvet antler: nutritional profile, in vitro antioxidant capacities, and alleviating symptoms of kidney-yang deficiency in mice

We present the nutritional composition, antioxidant activity, and pharmacological effects of three processed velvet antler products [antler powder (AP), antler slice (AS), and honey antler slice (HAS)] prepared in our laboratory. The nutrient profile of crude protein, fatty acids, amino acids, mineral elements, and nucleosides were established. The antioxidant activity was measured. The effects of three extracts on kidney-yang deficiency, induced by hydrocortisone, in mice were investigated. The nutrition composition of HAS was relatively low owing to its high moisture. AP, AS, and HAS extracts all exhibited concentration-dependent activity in DPPH*, ABTS, and FARP antioxidant assays. HAS had similar effect with AP and AS in improving oxidative stress in mice with kidney-yang deficiency, moreover, it could improve the serum cAMP level much significantly than the others. The processing without repeated heating might contribute in preserving more bioactive components in HAS, which could be the explanation of its excellent efficiency in relieving kidney-yang deficiency.

Temporal variations of nucleosides and nucleotides in rabbit milk

Nucleotides and nucleosides have a preeminent role in physiological and biochemical processes for newborns, the major source of these during early development is the breast milk. Different biomolecules exhibit daily fluctuations in maternal milk that could transfer temporal information that synchronize newborn circadian system. As a first approach, we characterized the diurnal profile of nucleotides and nucleosides contained in maternal milk of rabbits during the first week of lactation. It is possible that some nucleosides, such as adenosine, play a relevant role in setting up the emerging circadian rhythmicity, whereas uridine and guanosine could participate in the maintenance of rhythmicity.

Profile of Nucleosides and Nucleotides in Donkey’s Milk

Nucleotides play a crucial role to cellular functions; they can be obtained from the diet or through the nucleotide salvage pathway, however, in particular situations (occurring mainly in newborns) the metabolic demand of nucleotides exceeds the capacity of their synthesis. These molecules, are receiving attention from a nutraceutical point of view because of their potential direct role in regulating metabolism and infant body condition. Donkey's milk may be considered a good replacer for cow's milk in feeding children with severe Ig-E mediated cow's milk protein allergy, due to its high similarity with human milk. In this study, the presence of cytidine, uridine, CMP, UMP, guanosine, and adenosine, involved in numerous biochemical and physiological activities, were detected for the first time through a RP-HPLC method.

Rapid determination of nucleosides, nucleobases and free amino acids in brown seaweeds using ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

In this study the contents of nucleosides, nucleobases and free amino acids (FAAs) in common brown seaweeds Saccharina japonica, Sargassum pallidum, S. fusiforme, S. thunbergii and S. muticum from major production areas in China were determined by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQ-MS). The total contents of nucleosides and nucleobases varied from 48.46 to 4,105.00 μg g−1. Among the five seaweeds, levels of nucleosides and nucleobases in S. japonica were higher than the other species. The contents of ribonucleosides in these brown seaweeds were higher than those of deoxynucleosides. Uridine was the predominant constituent in all samples while 2′-deoxycytidine was the least in most samples. FAAs contents ranged from 0.14 to 22.76 mg g−1. Among the five seaweeds, levels of FAAs in S. muticum were higher than the other species. Essential amino acids accounted for 3.26–51.49 % of total FAAs contents in different species. Glutamic acid was the major constituent in most samples. Four non-protein amino acids, γ-aminobutyric acid, taurine, hydroxyproline and citrulline, were found in the seaweed samples. Furthermore, principal component analysis (PCA) showed that chemical profiles of nucleosides, nucleobases and FAAs in S. pallidum and S. fusiforme were different from S. muticum, S. thunbergii and S. japonica. Common brown seaweeds could be promising natural sources for future industrial research into nucleosides, nucleobases and FAAs with potential benefits for human health.

Liquid chromatography tandem mass spectrometry study of urinary nucleosides as potential cancer markers

The aim of this work was a comprehensive analysis of metabolite profiles of nucleosides from biological samples obtained from patients with urogenital cancer disease as well as an interpretation of cancer-related patterns of the analyzed profiles. In our study we proposed a targeted approach that was focused on the simultaneous determination of twelve nucleosides from over a hundred urine samples. For analytes’ quantification high performance liquid chromatography technique hyphenated with tripple quadrupole mass spectrometer was applied. The developed method was validated in terms of linearity, precision, accuracy as well as in terms of limit of quantification and limit of detection. The obtained, normalized data set was analyzed using univariate statistical analysis. The achieved results revealed statistically significant (p<0.05) differences between levels of five nucleosides determined in urine samples from cancer patients and healthy volunteers, namely: 6-methyladenosine, inosine, N-2-methylguanosine, 3-methyluridine and N,N-dimethylguanosine. Basing on the putative markers we built the discrimination models using partial least squares discriminant analysis as well as K-nearest neighbor method. The sensitivity and specificity of the markers calculated from the obtained models were in the range of 61.9–88.89% and 27.78–50%, respectively. The proposed procedure can be considered as a holistic approach for metabolites’ analysis and includes clinical, analytical and bioinformatics sections.

Mass spectrometry based analysis of nucleotides, nucleosides, and nucleobases—application to feed supplements

In this work, accurate MS-based methods for quantitative profiling of nucleotides, nucleosides, and nucleobases in yeast extracts used as additives in animal feedstuff are presented. Reversed-phase chromatography utilizing a stationary phase compatible with 100% aqueous mobile phases resulted in superior analytical figures of merit than HILIC or ion-pair reversed-phase separation. The novel separation method was combined with both molecular and elemental mass spectrometry. By use of RP-LC-MS-MS, excellent limits of detection <1 μmol L(-1) could be obtained for all the compounds investigated. The elemental speciation analysis approach enabled determination of nucleotides by phosphorus detection. Sensitivity of LC-ICP-MS was 1-2 orders of magnitude lower than that of LC-MS-MS. Quantitative analysis of yeast products using complementary MS detection furnished values in good agreement.

Profile of Nucleotides and Nucleosides in Taiwanese Human Milk

Human milk-borne nucleotides and their related metabolic products have been reported to have important physiological roles in breast-fed infants. The purpose of this study was to measure the concentrations of free nucleotides and nucleosides in human milk from Taiwanese women. A total of 24 individual milk specimens were collected from women in Taipei and Kaohsiung, at four stages of lactation. Vegetarian or non-vegetarian dietary patterns were recorded. The samples were analyzed for nucleotides and nucleosides by high-performance liquid chromatography. The mean (±standard deviation) free nucleotide and nucleoside concentrations in Taiwanese human milk were 213.13 ± 76.26 μmol/L and 16.38 ± 7.11 μmol/L. The predominant nucleotide was cytidine diphosphate for almost all samples, regardless of the location, stage of lactation, or dietary status of the subjects. Overall, the mean concentrations of cytidine diphosphate, cytidine monophosphate, uridine monophosphate, guanosine monophosphate, adenosine monophosphate, and inosine monophosphate, in milk samples were 129.86 μmol/L, 49.10 μmol/L, 5.60 μmol/L, 0.82 μmol/L, 2.96 μmol/L, and 25.25 μmol/L, respectively (equivalent to 61.0%, 23.1%, 2.6%, 0.4%, 1.0%, and 11.9% of free nucleotide composition). In free nucleosides, cytidine and uridine were predominant during all stages of lactation. The average concentrations of cytidine, uridine, adenosine, guanosine, and inosine, in milk samples were 9.25 μmol/L, 6.33 μmol/L, 0.18 μmol/L, 0.36 μmol/L, and 0.23 μmol/L, respectively (equivalent to 56.5%, 38.7%, 1.1%, 2.2%, and 1.4% of free nucleoside composition). Comparing vegetarian and non-vegetarian statuses, it was found that the total free nucleotide concentration was high in the vegetarian group (p=0.037). Our data showed a wide range of concentrations of individual nucleotides and nucleosides in Taiwanese human milk. Unique dietary status could affect the nucleotide and nucleoside levels in human milk, especially the nucleotides in our study. However, the mechanism of modulation of nucleotide and nucleoside levels in human milk is not clear.

Targeted serum metabolite profiling of nucleosides in esophageal adenocarcinoma

Nucleosides are indicators of the whole-body turnover of transfer RNA. Based on the activity of cancer cells these molecules could potentially be used as cancer biomarkers, and several studies have determined that the metabolic levels of nucleosides are significantly altered in cancer patients compared to control groups. Here we report a targeted metabolite investigation of serum nucleosides in esophageal adenocarcinoma specimens. We quantified eight nucleosides using high-performance liquid chromatography/triple quadrupole mass spectrometry (HPLC/TQMS) and determined that the metabolic levels of 1-methyladenosine (p <2.14 × 10(-7)), N(2),N(2)-dimethylguanosine (p <2.78 × 10(-7)), N(2)-methylguanosine (p <2.48 × 10(-6)) and cytidine (p <6.98 × 10(-4)) were significantly elevated while the concentration of uridine (p <3.74 × 10(-3)) was significantly lowered in serum samples from cancer patients compared to those of control group. Our results suggest that nucleosides could potentially serve as useful biomarkers to identify esophageal adenocarcinoma.

Altered levels of nucleoside metabolite profiles in urogenital tract cancer measured by capillary electrophoresis

Metabolic profiles of nucleosides studied on the level of urine are closely related to the pathophysiological status of the organism. Posttranscriptional modifications of RNA (mostly tRNA) in cell nucleus are responsible for change of nucleoside levels during malignant disease. In this paper, 256 metabolite profiles from 160 urogenital tract cancer patients and 96 healthy controls, composed of 19 nucleosides were collected and studied with the application of such an approach. This approach comprised of the analysis of urine extracts and the investigation of collected nucleoside and modified nucleoside profiles by advanced statistical data processing tools such as principal component analysis (PCA), hierarchical cluster analysis (HCA), K-Nearest Neighbor method (kNN) and partial least squares-discriminant analysis with probabilistic function (p-PLS-DA). It has been shown that alterations of metabolite profiles in cancer diseases are mainly expressed by the fold change of the urine levels of most nucleosides. In addition, observed metabolite-to-metabolite ratios differ in urogenital cancer patients compared to healthy controls. The obtained relationships between urinary nucleoside profiles and the presence of cancer diseases have been evaluated. Discrimination of the cancer patients and the non-cancer healthy subjects is with 76.5% sensitivity and 80.2% specificity. The presented results prove the usefulness of the metabolomic approach in studying urinary nucleoside profiles with high diagnostic potency in urogenital cancer diseases. Profiles of urinary nucleosides might be employed as a reliable and convenient tool in the diagnostics of urogenital tract cancer diseases.

Prediction of breast cancer by profiling of urinary RNA metabolites using Support Vector Machine-based feature selection

BackgroundBreast cancer belongs to the most frequent and severe cancer types in human. Since excretion of modified nucleosides from increased RNA metabolism has been proposed as a potential target in pathogenesis of breast cancer, the aim of the present study was to elucidate the predictability of breast cancer by means of urinary excreted nucleosides.MethodsWe analyzed urine samples from 85 breast cancer women and respective healthy controls to assess the metabolic profiles of nucleosides by a comprehensive bioinformatic approach. All included nucleosides/ribosylated metabolites were isolated by cis-diol specific affinity chromatography and measured with liquid chromatography ion trap mass spectrometry (LC-ITMS). A valid set of urinary metabolites was selected by exclusion of all candidates with poor linearity and/or reproducibility in the analytical setting. The bioinformatic tool of Oscillating Search Algorithm for Feature Selection (OSAF) was applied to iteratively improve features for training of Support Vector Machines (SVM) to better predict breast cancer.ResultsAfter identification of 51 nucleosides/ribosylated metabolites in the urine of breast cancer women and/or controls by LC- ITMS coupling, a valid set of 35 candidates was selected for subsequent computational analyses. OSAF resulted in 44 pairwise ratios of metabolite features by iterative optimization. Based on this approach ultimately estimates for sensitivity and specificity of 83.5% and 90.6% were obtained for best prediction of breast cancer. The classification performance was dominated by metabolite pairs with SAH which highlights its importance for RNA methylation in cancer pathogenesis.ConclusionExtensive RNA-pathway analysis based on mass spectrometric analysis of metabolites and subsequent bioinformatic feature selection allowed for the identification of significant metabolic features related to breast cancer pathogenesis. The combination of mass spectrometric analysis and subsequent SVM-based feature selection represents a promising tool for the development of a non-invasive prediction system.

The detection of genetic markers of bladder cancer in urine and serum

To review the most recent publications focusing on the use of genetic markers (DNA, RNA and nucleosides) in urine and serum and provide an opinion on their potential utility for screening, diagnosis and prognosis of urothelial carcinoma. Several studies have shown the diagnostic utility of urine tests based on improved microsatellite analysis of loss-of-heterozygosity, detection of fibroblast growth factor receptor 3 mutations, detection of single RNA and multiple gene signatures as well as nucleoside profiles. Although of interest, all these studies lack appropriate controls and validation before being considered as serious candidate clinical biomarkers. The presence of fibroblast growth factor receptor 3 mutations in tumors was identified as a hallmark of tumors of low malignant potential. Serum DNA analysis of hypermethylation of a set of genes shows promise as an indicator of cancer progression and mortality. Finally, a case-control study of 775 patients and 397 controls showed that DNA hypomethylation of blood cells combined with smoking habits can provide stratification of cancer risk that may be very helpful in conceiving bladder cancer screening studies. The challenge is not so much to identify better markers or methods but rather to commit to implementing them into clinical practice by stimulating and funding the clinical trials required.

Metabonomics in cancer diagnosis: mass spectrometry-based profiling of urinary nucleosides from breast cancer patients

Modified nucleosides are formed post-transcriptionally in RNA. In cancer disease, the cell turnover and thus RNA metabolism is increased, yielding higher concentrations of excreted modified nucleosides. In the presented study, urinary ribonucleosides were used to differentiate between breast cancer patients and healthy volunteers. The nucleosides were extracted from urine samples using affinity chromatography and subsequently analyzed via liquid chromatography ion trap mass spectrometry (LC-IT-MS). The peak areas were related to the internal standard isoguanosine and to the urinary creatinine level. For bioinformatic pattern recognition we used the support vector machine. We examined 113 urine samples from breast cancer patients (stage Tis-T4) and 99 control samples from healthy volunteers. We achieved a sensitivity of 87.67% and a specificity of 89.90% when including 31 nucleosides. The medical metabonomics concept based on the urinary nucleoside profile reveals a significantly improved classification compared with currently applied breast cancer biomarkers such as CA15-3.

Evaluation of different warping methods for the analysis of CE profiles of urinary nucleosides

Nowadays, numerous metabolite concentrations can readily be determined in a given biological sample by high-throughput analytical methods. However, such raw analytical data comprise noninformative components due to many disturbances normally occurring in the analyses of biological material. To eliminate those unwanted original analytical data components, advanced chemometric data preprocessing methods might be of help. Here, such methods are applied to electrophoretic nucleoside profiles in urine samples of cancer patients and healthy volunteers. In this study, three warping methods: dynamic time warping (DTW), correlation optimized warping (COW), and parametric time warping (PTW) were examined on two sets of electrophoretic data by means of quality of peaks alignment, time of preprocessing, and way of customization. The application of warping methods helped to limit shifting of peaks and enabled differentiation between whole electropherograms of healthy and cancer patients objectively by a principal component analysis (PCA). The evaluation of preprocessed data and raw data by PC analysis confirms differences between the applied warping tools and proves their suitability in metabonomic data interpretation.

Development and validation of urinary nucleosides and creatinine assay by capillary electrophoresis with solid phase extraction

For the analysis of metabolite nucleoside profiles, capillary electrophoretic (CE) methods preceded by appropriate solid phase extraction procedures have been developed. The approach has been proposed for the determination of 13 nucleosides and creatinine in human urine. A background solution composed of 100 mM borate–72 mM phosphate–160 mM SDS and a fused silica capillary of 70 cm length to detector and 50 μm i.d. were used. The methods developed were statistically validated for their linearity, trueness, precision and selectivity. Stability of the analyzed nucleoside profiles in urine during storage was checked. Validation parameters of solid phase extraction procedures for urinary nucleosides were evaluated. The developed analytical methods were employed for the analysis of 22 urine samples from healthy patients and cancer patients from the urological ward. Nucleoside profiles were compared among the subjects. It was proved that the methods proposed were suitable for a fast and reliable determination of urinary creatinine and modified nucleoside profiles, which can be further submitted for the metabonomic analysis of cancer patients.

Artificial neural networks based on principal component analysis input selection for clinical pattern recognition analysis

Two clinical data sets were applied for pattern recognition in order to discover the correlation between urinary nucleoside profiles and tumours. One data set contains 168 clinical urinary samples, of which 84 specimens are from female thyroid cancer patients (malignant tumour group), and the other samples were collected from healthy women (normal group). However, 168 clinical urinary samples comprised the second data set, too. In all the specimens, each number of the samples for both uterine cervical cancer patients (malignant tumour group) and healthy females (normal group) is 60, and the other 48 samples were collected from uterine myoma patients (benign tumour group). For the two data sets, the separation and quantitative determination of the clinical urinary nucleosides were performed by capillary electrophoresis (CE). The pattern recognition was achieved applying multiple layer perceptron artificial neural networks (MLP ANN) based on conjugate gradient descent training algorithm. Moreover, applying the proposed principal component analysis (PCA) input selection scheme to MLP ANN, the accuracy rate of the pattern recognition was improved to some extent (or without any deterioration) even by much simpler structure of MLP ANN. The study showed that MLP ANN based on PCA input selection was a promising tool for pattern recognition.

Increasing conclusiveness of metabonomic studies by cheminformatic preprocessing of capillary electrophoretic data on urinary nucleoside profiles

Nowadays, bioinformatics offers advanced tools and procedures of data mining aimed at finding consistent patterns or systematic relationships between variables. Numerous metabolites concentrations can readily be determined in a given biological system by high-throughput analytical methods. However, such row analytical data comprise noninformative components due to many disturbances normally occurring in analysis of biological samples. To eliminate those unwanted original analytical data components advanced chemometric data preprocessing methods might be of help. Here, such methods are applied to electrophoretic nucleoside profiles in urine samples of cancer patients and healthy volunteers. The electrophoretic nucleoside profiles were obtained under following conditions: 100 mM borate, 72.5 mM phosphate, 160 mM SDS, pH 6.7; 25 kV voltage, 30 °C temperature; untreated fused silica capillary 70 cm effective length, 50 μm I.D. Different most advanced preprocessing tools were applied for baseline correction, denoising and alignment of electrophoretic data. That approach was compared to standard procedure of electrophoretic peak integration. The best results of preprocessing were obtained after application of the so-called correlation optimized warping (COW) to align the data. The principal component analysis (PCA) of preprocessed data provides a clearly better consistency of the nucleoside electrophoretic profiles with health status of subjects than PCA of peak areas of original data (without preprocessing).

Conformational analysis of a nucleoside of 1,4-dihydro-4-oxoquinoline-3-carboxylic acid analogue

The synthesis of new ribonucleosides is an essential research area in the investigation of new therapeutically useful agents, particularly those used in the treatment of HIV infection. The conformation of these nucleosides may have direct implications for their ability to bind to receptor targets. We have prepared the 7-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic acid derivatives and used the ensemble of low-energy minima to develop conformational profiles of quinolonic nucleosides and verify their accuracy in different calculations of structural parameters. Results are compared with experimental data obtained by X-ray and NMR analysis. Finally, we intend to test the applicability of these methods to conformational analysis of other nucleosides and verify if the preferential conformation is the one which gives the best anti-HIV or antiviral activity.

Capillary electrophoretic profiling and pattern recognition analysis of urinary nucleosides from uterine myoma and cervical cancer patients

Capillary electrophoretic (CE) profiling analysis combined with pattern recognition methods is described for the correlation between urinary nucleoside profiles and uterine cervical cancer. Nucleosides were extracted from urine specimens by solid-phase extraction in affinity mode using phenylboronic acid gel. CE separation was carried out with an uncoated fused-silica capillary (570 mm×50 μm I.D.) maintained at 20°C, using 25 m M borate–42.5 m M phosphate buffer (pH 6.7) containing 200 m M sodium dodecyl sulfate as the run buffer under the applied voltage of 20 kV. A total of 15 nucleosides were positively identified in urine samples (2 ml) from eight uterine myoma (benign tumor group), 10 uterine cervical cancer (malignant tumor group) patients and 10 healthy females (normal group) studied. The star symbol plots drawn based on each mean concentration of nucleosides normalized to that in normal group enabled one to discriminate malignant and benign groups from normal group. In addition, canonical discriminant analysis performed on the nucleoside data of 28 individual urine specimens correctly classified into three separate clusters according to groups in the canonical plot.

Structure-inhibitory profiles of nucleosides for the human intestinal N1 and N2 Na+-nucleoside transporters.

To determine the structure-inhibitory profiles of nucleosides for the N1 and N2 Na+-nucleoside transporters of the human intestine. The uptake of 3H-labeled prototypic substrates of the N1 (inosine) and N2 (thymidine) transporters into human intestinal brush border membrane vesicles was measured by a rapid filtration technique in the presence and absence of various uridine and adenosine analogs and antiviral and anticancer nucleoside drugs (100 and 1000 microM). In the ribose ring, the 3'-oxygen is required for inhibition of uptake of nucleosides by both the N1 and N2 transporters. The structural requirements for such inhibition differ with respect to modifications on the 5' position of the sugar ring or on the base. The N2 transporter is more tolerant to these substitutions than is the N1 transporter. The 6 position on uracil and the 8 position on adenine are critical for inhibition of uptake of nucleosides by both the N1 and N2 nucleoside transporters. These data are the first evidence that the binding site(s) of the human N1 and N2 transporters differ in their interaction with analogs of their common substrates, uridine and adenosine. Such studies can provide insight into the critical structural determinants of the substrate necessary for recognition by the Na+-nucleoside transporters of the human intestine.