Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples.

We report a reverse phase chromatography mass spectrometry (LC-MS) method for simultaneous quantification of nucleosides and nucleotides from biological samples, where compound identification was achieved by a tier-wise approach and compound quantification was achieved via external calibration. A total of 65 authentic standards of nucleosides and nucleotides were used for the platform development. The limit of detection (LOD) of those compounds ranged from 0.05 nmol/L to 1.25 µmol/L, and their limit of quantification (LOQ) ranged from 0.10 nmol/L to 2.50 µmol/L. Using the developed method, nucleosides and nucleotides from human plasma, human urine, and rat liver were quantified. Seventy-nine nucleosides and nucleotides were identified from human urine and 28 of them were quantified with concentrations of 13.0 nmol/L-151 µmol/L. Fifty-five nucleosides and nucleotides were identified from human plasma and 22 of them were quantified with concentrations of 1.21 nmol/L-8.54 µmol/L. Fifty-one nucleosides and nucleotides were identified from rat liver and 23 were quantified with concentrations of 1.03 nmol/L-31.7 µmol/L. These results demonstrate that the developed method can be used to investigate the concentration change of nucleosides and nucleotides in biological samples for the purposes of biomarker discovery or elucidation of disease mechanisms.

On-line electrochemistry/electrospray ionization mass spectrometry (EC-ESI-MS) system for the study of nucleosides and nucleotides oxidation products.

The main aim of present investigation was to study the oxidation products of nucleosides and nucleotides with the use of on-line electrochemistry/electrospray ionization mass spectrometry (EC-ESI-MS) system. The conditions applied in the system were optimized in complex manner, involving study of the impact of working electrodes or sample solvent on the oxidation of tested compounds and their ionization in mass spectrometry. Finally 5 mM of ammonium acetate was used selected and pH 3 was used for positive ionization mode, while pH 7 was applied for negative ionization in mass spectrometry. It was shown that utilization of both ionization modes is indispensable in order to detect and identify all of oxidation products. Furthermore the identification of compounds obtained using the EC-ESI-MS system was done and results were compared with known metabolites of studied compounds. These products are associated with specific disease states, or may be a potential metabolites. Moreover the analysis of urine samples by liquid chromatography coupled with mass spectrometry confirmed the possibility of using EC-ESI-MS technique to simulate the metabolism of nucleosides and nucleotides, since the oxidation products have also been identified in urine samples.

Preparation of a silica stationary phase co-functionalized with Wulff-type phenylboronate and C12 for mixed-mode liquid chromatography.

A silica stationary phase was designed and synthesized through the co-functionalization of silica with Wulff-type phenylboronate and C12 for mixed-mode liquid chromatography applications. The as-synthesized stationary phase was characterized by elemental analysis and Fourier Transform-InfraRed Spectroscopy (FT-IR). Retention mechanisms, including boronate affinity (BA), reversed-phase (RP) and anion-exchange (AE), were involved. Retention mechanism switching was easily realized by adjustment of the mobile phase constitution. Cis-diol compounds could be selectively captured under neutral conditions in BA mode and off-line separated in RP mode. Neutral, basic, acidic and amphiprotic compounds were chromatographed on the column in RP chromatography, while inorganic anions were chromatographed in AE chromatography to characterize the mixed-mode nature of the prepared stationary phase. In addition, the RP performance was compared with an octadecyl silica column in terms of column efficiency (N/m), asymmetry factor (Af), retention factor (k) and resolution (Rs). The prepared stationary phase offered multiple interactions with analytes in addition to hydrophobic interactions under RP elution conditions. Based on the mixed-mode properties, off-line 2D-LC, for selective capture and separation of urinary nucleosides, was successfully realized on a single column, demonstrating its powerful application potential for complex samples.

Evaluating effects of penicillin treatment on the metabolome of rats.

Penicillin (PEN) V, a well-known antibiotic widely used in the treatment of Gram-positive bacterial infections, was evaluated in this study. LC/MS- and NMR-based metabolic profiling were employed to examine the effects of PEN on the host's metabolic phenotype. Male Sprague Dawley rats were randomly divided into groups that were orally administered either 0.5% methylcellulose vehicle, 100 or 2400mg PEN/kg body weight once daily for up to 14 consecutive days. Urine, plasma and tissue were collected from groups sacrificed at 6h, 24h or 14d. The body fluids were subjected to clinical chemistry and metabolomics analysis; the tissue samples were processed for histopathology. The only notable clinical chemistry observation was that gamma glutamyltransferase (GGT) significantly decreased at 24h for both dose groups, and significantly decreased at 14d for the high-dose groups. Partial least squares discriminant analysis scores plots of the metabolomics data from urine and plasma samples showed dose- and time-dependent grouping patterns. Time- and dose-dependent decreases in urinary metabolites including indole-containing metabolites (such as 3-methyldioxyindole sulfate generated from bacterial metabolism of tryptophan), organic acids containing phenyl groups (such as hippuric acid, phenyllactic acid and 3-hydroxyanthranilic acid), and metabolites conjugated with sulfate or glucuronide (such as cresol sulfate and aminophenol sulfate) indicated that the gut microflora population was suppressed. Decreases in many host-gut microbiota urinary co-metabolites (indole- and phenyl-containing metabolites, amino acids, vitamins, nucleotides and bile acids) suggested gut microbiota play important roles in the regulation of host metabolism, including dietary nutrient absorption and reprocessing the absorbed nutrients. Decreases in urinary conjugated metabolites (sulfate, glucuronide and glycine conjugates) implied that gut microbiota might have an impact on chemical detoxification mechanisms. In all, these results clearly show that metabolic profiling is a useful tool to better understand the effects of the antibiotic penicillin has on the gut microbiota and the host.

Tamm-Horsfall protein-associated nucleotides in patients with interstitial cystitis.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The nucleotides associated with Tamm-Horsfall protein (THP) identified in this study are 2'(3') isomers, which are uncommon and very little is known about their biochemical pathway and role in interstitial cystitis (IC). The current study provides additional evidence of our earlier finding that THP is abnormal in IC patients. This can adversely affect THP's protective function, and suggests that THP plays a role in the pathogenesis of the disease. OBJECTIVE: To identify and characterise Tamm-Horsfall protein (THP)-associated metabolites present in patients with interstitial cystitis (IC). Identification of these metabolites would give us insight into the complex interaction of THP with urinary metabolites and its effect on the protective function of THP. PATIENTS, SUBJECTS AND METHODS: THP was isolated from the urine specimens of 146 patients with IC and 87 control subjects, and was analysed for total sialic acid (SA) content by 1,2-diamino-4,5-methylenedioxybenzene. 2HCl (DMB)-high performance liquid chromatography (HPLC). THP-associated metabolites were isolated by Superdex-200 size-exclusion chromatography (SEC) as a second peak (SP2) and the SP2 was further fractionated into six major components by C18 reverse phase-HPLC. Ion-pair HPLC analysis was performed to identify and quantify THP-associated metabolites. The metabolite structures were confirmed by reversed-phase HPLC combined with electrospray ionization (ESI), liquid chromatography-tandem mass spectrometry (LC-MS and LC-MS/MS) and by high-resolution ESI-time of flight mass spectrometry (HR-ESI-TOFMS). RESULTS: The THP-associated metabolites (SP2) were more prevalent in patients with IC than in the controls (40.4% vs 12.6%, P < 0.001) as determined by Superdex-200 SEC. Superdex-200 SEC showed higher SP2 content in patients with IC than in controls, as determined by area under the peak/100 µg of THP. The mean (sem), for patients was 84.3 (8.1) vs 18.0 (2.4) milli-absorption unit*min, for controls (P < 0.001). Total SA content of THP was much lower in SP2-positive patients with IC than those who were SP2-negative. The mean (sem) was 41.6 (3.2) vs 92.6 (2.2) nmol/mg THP, respectively (P < 0.001). Ion-pair HPLC identified SP2 metabolites as nucleotides, namely 5'-CMP, 3'-UMP, 3'-AMP, 3'-GMP, 2'-AMP and 2'-GMP. The total nucleotide content of SP2 was significantly higher in patients with IC than in controls. The mean (sem) was 25.3 (2.9) vs 2.2 (0.2) µg/mg THP, respectively (P < 0.001). LC-MS and LC-MS/MS confirmed the nucleotides based on retention time on HPLC, and mass to charge ratio (m/z) of the parent ion and the daughter ions. HR-ESI-TOFMS gave further confimation of the nucleotide sturctures with high mass accuracy. CONCLUSIONS: In the THP of patients with IC, there is a direct correlation between reduced SA levels and high prevalence of nucleotides associated with it. The THP of patients with IC has a much higher content of these nucleotides than control, and these unique nucleotide isomers identified are very consistent in all SP2-positive patients with IC, suggesting biological significance. This study provides additional evidence that THP is abnormal in patients with IC.

Metabolic influence of acute cyadox exposure on Kunming mice.

Cyadox is an antibiotic drug and has the potential to be used as a feedstuff additive in promoting the growth of animals. However, the toxicity of cyadox should be fully assessed before application, and this has prompted the current investigation on the metabolic responses of mice to cyadox exposure, using a metabonomic technique. Three groups of Kunming mice were respectively given a single dose of cyadox at three different concentrations (100, 650, and 4000 mg/kg body weight) via gavage. We present here the metabolic alterations of urine, plasma, liver, and renal medulla extracts induced by cyadox exposure. The metabolic alterations induced by cyadox exposure are dose-dependent, and metabolic recovery is achieved only for low and moderate levels of cyadox exposure during the experimental period. Cyadox exposure resulted in a disturbance of gut microbiota, which is manifested in depleted levels of urinary hippurate, trimethylamine-N-oxide (TMAO), dimethylamine (DMA), and trimethylamine (TMA). In addition, mice exposed to cyadox at high levels caused accumulations of amino acids and depletions of nucleotides in the liver. Furthermore, marked elevations of nucleotides and a range of organic osmolytes, such as myo-inositol, choline, and glycerophosphocholine (GPC), and decreased levels of amino acids are observed in the renal medulla of cyadox-exposed mice. These results suggest that cyadox exposure causes inhibition of amino acid metabolism in the liver and disturbance of gut microbiota community, influencing osmolytic homeostasis and nucleic acids synthesis in both the liver and the kidney. Our work provides a comprehensive view of the toxicological effects of cyadox, which is important in animal and human food safety.

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC.

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ≥ 98 %). All samples were analyzed following injection (100 µl) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 µm, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients.

A metabolomic comparison of urinary changes in type 2 diabetes in mouse, rat, and human.

Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.

Determination of glycated nucleobases in human urine by a new monoclonal antibody specific for N2-carboxyethyl-2'-deoxyguanosine.

Sugars and sugar degradation products react in vivo readily with proteins (glycation) resulting in the formation of a heterogeneous group of reaction products, which are called advanced glycation end products (AGEs). AGEs notably change the structure and function of proteins so that extended protein-AGE formation is linked to complications such as nephropathy, atherosclerosis, and cataract. DNA can be glycated in vitro in a similar way as proteins, and the two diastereomers of N(2)-carboxyethyl-2'-deoxyguanosine (CEdG(A,B)) were identified as major DNA AGEs. It was postulated that DNA AGEs play an important role in aging, diabetes, and uremia. However, at the moment, sensitive methods to measure the extent and impact of DNA AGEs in vivo do not exist. In this study, we developed a monoclonal antibody, which recognized CEdG(A,B) with high affinity and specificity (MAb M-5.1.6). The I(50) value for CEdG(A,B) was 2.1 ng/mL, whereas other modified nuclueobases and AGE proteins showed negligible cross-reactivity. Unmodified 2'-deoxyguanosine was only weakly recognized with an I(50) value > 600,000 ng/mL, which is the limit of solubility. MAb M-5.1.6 was then used to measure the urinary excretion of AGE-modified nucleobases in a competitive enzyme-linked immunosorbent assay. The recovery of CEdG(A,B) from human urine was between 87.4 and 99.7% with coefficients of variations between 8.0 and 22.2%. The detection limit was 0.06 ng/mL, and the determination limit was 0.15 ng/mL with a linear range between 0.3 and 100 ng/mL. CEdG equivalents were analyzed in urine samples from 121 healthy volunteers, and concentrations between 1.2 and 117 ng CEdG equiv/mg creatinine were detected.

Estimation of oxidative DNA damage in man from urinary excretion of repair products.

DNA is constantly damaged and repaired in living cells. The repair products of the oxidative DNA lesions, i.e. oxidised nucleosides and bases, are poor substrates for the enzymes involved in nucleotide synthesis, are fairly water soluble, and generally excreted into the urine without further metabolism. Among the possible products, 8-oxo-2'-deoxyguanosine, 8-oxoguanine, thymine glycol, thymidine glycol and, 5-hydroxymethyluracil have so far been identified in urine. It should be emphasised that the excretion of the repair products in urine represents the average rate of damage in the total body whereas the level of oxidised bases in nuclear DNA is a concentration measurement in that specific tissue/cells in the moment of sampling. The rate of oxidative DNA modifications has been studied in humans by means of the repair products as urinary biomarkers, particularly with respect to 8-oxo-2'-deoxyguanosine. The data obtained so far indicate that the important determinants of the oxidative damage rate include tobacco smoking, oxygen consumption and some inflammatory diseases whereas diet composition, energy restriction and antioxidant supplements have but a minimal influence, possibly with the exception of yet unidentified phytochemicals, e.g. from cruciferous vegetables. The data are consistent with the experimentally based notion that oxidative DNA damage is an important mutagenic and apparently carcinogenic factor. However, the proof of a causal relationship in humans is still warranted. In the future the use of biomarkers may provide this evidence and allow further investigations on the qualitative and quantitative importance of oxidative DNA modification and carcinogenesis in man, as well as elucidate possible preventive measures.

Human cyclic hematopoiesis is associated with aberrant purine metabolism.

It appeared possible that abnormal purine or pyrimidine metabolism could cause cyclic hematopoiesis by analogy with the defective lymphopoiesis associated with inherited deficiencies of adenosine deaminase and purine nucleoside phosphorylase. Therefore, we examined erythrocyte purine and pyrimidine nucleotide levels, as well as plasma purine and pyrimidine nucleosides and bases in three patients and in normal controls. These studies showed that during neutropenia there was a significant elevation in the levels of guanosine triphosphate (P = 0.005) and adenosine triphosphate (P less than 0.001) in the patients' red cells not attributable to reticulocyte variation. Serial analysis of a patient's plasma showed a fivefold elevation of hypoxanthine (10.6 mumol/L) during neutropenia, with a return to normal values (1.4 mumol/L) as neutrophil numbers increased. Plasma inosine was also significantly elevated in comparison with normal control values (2.0 mumol/L vs. 0.8 mumol/L), whereas plasma and urinary uric acid were within the normal range. Serial analysis of red cells and plasma from two patients with chronic neutropenia showed no elevations of purine or pyrimidine metabolites. These results provide evidence of a link between abnormal concentrations of purine metabolites and cyclic hematopoiesis, and permit the speculation that aberrant purine metabolism is primarily related to the defective hematopoietic cell proliferation that is characteristic of this disease.

[Treatment of amyotrophic lateral sclerosis with ribonucleotides]. / Lechenie bokovogo amiotroficheskogo skleroza ribonukleotidami

In the treatment of 31 patients with lateral amyotrophical sclerosis the authors used the preparation ENKAD containing a complex of mono- and oligoribonucleotides. In 6 cases there was aggravation of the condition while in 18 of the observed cases--improvement. In 7 patients the condition was not altered. The treatment was convened under biochemical control (the assessment of the uric acid in the blood serum and urine and the excretion of oxypurine in the urine). It was possible to depict definite clinicobiochemical correlations. The conclusion is made that ENKAD can be used in the early stages of the disease and mainly in focal segmentary-nuclear lesions. The preparation is contraindicated in generalization of the process and in impetuous development of the disorder.