Nucleoside Concentrations Research Articles

Metabolic significance of bisphenol A‐induced oxidative stress in rat urine measured by liquid chromatography–mass spectrometry

Modified nucleosides are formed by DNA repair as a result of oxidative DNA damage and post-transcriptionally modified tRNA in cells. In the present study, profiling of 14 nucleoside was investigated in rat urine to evaluate bisphenol A (BPA)-induced oxidative stress after intraperitoneally injecting rats with 0, 10 or 50 mg kg(-1) per day of BPA for four consecutive days. The urinary concentrations of individual nucleosides were measured by liquid chromatography-tandem mass spectrometry combined with column switching on-line extraction. Increased levels of 5-hydroxymethyl-2'-deoxyuridine (P < 0.01 on first, P < 0.005 on second, P < 0.001 on third and P < 0.01 on fourth day) and 8-hydroxy-2'-deoxyguanosine (P < 0.005 on second, P < 0.001 on third and P < 0.001 on fourth day) were found. Also, the patterns of urinary nucleosides in three dosage groups (control, BPA1, BPA2) were significantly different. Statistical significance was observed between BPA1 (5-hydroxymethyl-2'-deoxyuridine, P < 0.05; 8-hydroxy-2'-deoxyguanosine, P < 0.005) and BPA2 (5-hydroxymethyl-2'-deoxyurindine, P < 0.005; 8-hydroxy-2'-deoxyguanosine, P < 0.001) during the treatment period. Supervised analysis with partial least-squares-discrimination analysis led to discrimination between the three dosage groups. Quantitative alterations showed the metabolic trajectories responsible for physiological responses. The described methods could be used to evaluate and monitor BPA-induced oxidative stress early after exposure.

Characterization of intestinal absorption of mizoribine mediated by concentrative nucleoside transporters in rats

Mizoribine, an imidazole nucleoside, is an inhibitor of purine synthesis and has been used as an orally available immunosuppressive agent in human renal transplantation. In the present study, the intestinal absorption of mizoribine was characterized by examining the contribution of concentrative nucleoside transporters (CNT1, CNT2) in rats. When mizoribine was administered orally in conscious rats, the bioavailability of mizoribine estimated by urinary excretion percentage of unchanged mizoribine was a dose dependent: 53.1 ± 6.0% at 5 mg/kg and 24.0 ± 5.1% at 20 mg/kg. In in-situ loop studies, the disappearance rate, or absorption rate, of mizoribine from the intestinal lumen was comparable between 1 and 5 mg/kg, but significantly lower at 25 mg/kg. Coadministration of adenosine (a substrate of both CNT1 and CNT2), thymidine (a CNT1 substrate) and inosine (a CNT2 substrate) significantly suppressed the intestinal mizoribine absorption, depending on the nucleoside concentrations coadministered. Gemcitabine (a pyrimidine nucleoside analogue, a CNT1 substrate) and ribavirin (a purine nucleoside analog, a CNT2 substrate) also significantly suppressed the mizoribine intestinal absorption. Bile salts such as sodium cholate and sodium glycocholate (10 mM) also significantly suppressed the intestinal mizoribine absorption, but not ribavirin absorption. Mizoribine is an amphoteric compound, however, the suppression of intestinal absorption by bile salts was not ascribed to the electrostatic interaction or micellar formation between mizoribine and bile salts. In conclusion, the intestinal absorption of mizoribine is mediated by CNT1 and CNT2, and nucleoside-derived drugs such as gemcitabine and ribavirin can suppress the intestinal absorption of mizoribine. Bile salts such as sodium glycocholate were also found to cause interaction with mizoribine.

Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH(3) or l-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH(3)-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP approximately 6-thio-GTP > ITP approximately guanosine 5'-tetraphosphate > O(6)-methyl-GTP > 2'-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH(2) group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH(2) group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH(3)- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC(50) approximately 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC(50) approximately 4-12 mM). 8-Oxoguanosine was the best inhibitor identified (IC(50) = 80 microM). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5'-triphosphate synthase inhibitors.

Intense exercise increases adenosine concentrations in rat brain: Implications for a homeostatic sleep drive

Intense exercise and sleep deprivation affect the amount of homeostatically regulated slow wave sleep in the subsequent sleep period. Since brain energy metabolism plays a decisive role in the regulation of behavioral states, we determined the concentrations of nucleotides and nucleosides: phosphocreatine, creatine, ATP, ADP, AMP, adenosine, and inosine after moderate and exhaustive treadmill exercise as well as 3 and 5 h of sleep deprivation and sleep in the rat brain using the freeze-clamp technique. High intensity exercise resulted in a significant increase of the sleep-promoting substance adenosine. In contrast, following sleep, inosine and adenosine levels declined considerably, with an accompanied increase of ADP after 3 h and ATP after 5 h. Following 3 h and 5 h sleep deprivation, ADP and ATP did not differ significantly, whereas inosine increased during the 3 and 5-h period. The concentrations of AMP, creatine and phosphocreatine remained unchanged between experimental conditions. The present results are in agreement with findings from other authors and suggest that depletion of cerebral energy stores and accumulation of the sleep promoting substance adenosine after high intensity exercise may play a key role in homeostatic sleep regulation, and that sleep may play an essential role in replenishment of high-energy compounds.

Thymidine and deoxyuridine accumulate in tissues of patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease due to ECGF1 gene mutations causing thymidine phosphorylase (TP) deficiency. Analysis of post-mortem samples of five MNGIE patients and two controls, revealed TP activity in all control tissues, but not in MNGIE samples. Converse to TP activity, thymidine and deoxyuridine were absent in control samples, but present in all tissues of MNGIE patients. Concentrations of both nucleosides in the tissues were generally higher than those observed in plasma of MNGIE patients. Our observations indicate that in the absence of TP activity, tissues accumulate nucleosides, which are excreted into plasma.

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches

Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE.

Novel approach for developing urinary nucleosides profile by capillary electrophoresis–mass spectrometry

A simple, rapid and efficient capillary electrophoresis–mass spectrometry (CE–MS) method was developed to analyze urinary nucleosides for the first time. The composition of CE buffer and MS parameters were systematically optimized. The optimum buffer was 150 mM acetic acid containing 15% methanol and 15% ethanol. The optimum MS parameters were: methanol containing 0.5% acetic acid was selected as the sheath liquid and the flow rate was 5 μL/min; the flow rate and temperature of drying gas were 6 L/min and 150 °C, respectively; the pressure of nebulizing gas was 2 psig; and the fragmentor and ESI voltage were 100 V and 4000 V, respectively. Under the optimum CE–MS conditions, the urinary nucleosides were separated within 18 min. The linearity between the relative peak areas and the corresponding concentration of nine nucleosides markers were excellent. The limits of detection (S/N = 3) of markers were 0.00862–3.82 nmol/mL. The optimum CE–MS method was applied to analyze urine from 20 bladder cancer patients and 20 healthy volunteers. Considering the standards of many nucleosides cannot be obtained, it is not the ratios of the concentrations of nucleosides to that of creatinine in the literatures, but the ratios of the relative peak area of nucleosides to the concentration of creatinine that used for pattern recognition. And, the statistical analysis result indicated this method was feasible.

Oxypurine and Purine Nucleoside Concentrations in Renal Vein of Allograft Are Potential Markers of Energy Status of Renal Tissue

Impairment of organ function derived from ischemia-reperfusion injury is an important problem in solid organ transplantation. Cell alterations induced by ischemia prime the tissue for subsequent damage that occurs during the reperfusion phase. Purine nucleosides and oxypurines are products of adenine nucleotides degradation. Reperfusion and reoxygenation are accompanied by production of reactive oxygen species and free radicals, which lead to damage of graft tissue. The aim of this study was to measure concentrations of adenine nucleotides and their metabolites in renal allograft vein as well as in recipient's peripheral veins during the reperfusion period and to evaluate their usefulness as markers of tissue metabolism in kidney allografts. The study enrolled 20 renal transplant recipients. The first blood sample was taken from the recipient's ulnar vein before anastomosing of the kidney graft's vessels with recipient's iliac vessels. Samples were then taken from the renal allograft and ulnar veins 5 min after total graft reperfusion measured with an infrared camera. High-performance liquid chromatography (HPLC) was performed to measure whole blood and plasma concentrations of adenosine triphosphate (ATP), adenosine monophosphate (AMP), guanosine (Guo), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), uric acid (UA), and uridine (Urd). Hyp and Xan concentrations were significantly increased in renal allograft vein after reperfusion as compared with peripheral vein during the pre- and post-reperfusion periods. The results of the present study suggest that differences in Hyp and Xan concentrations between renal and peripheral veins reflect metabolic alterations in renal tissue during reperfusion and may be useful for graft function monitoring during reperfusion.

Oxypurine and nucleoside concentrations in renal veins during reperfusion are predictors of early graft function

To examine whether purine and pyrimidine nucleoside concentrations as well as oxypurine concentrations in renal and peripheral veins during reperfusion correlate with graft function parameters. The study population comprised 25 recipients of cadaver kidney transplant. A first blood sample was taken from the recipient's peripheral vein before anastomosing the kidney allograft vessels with the recipient's iliac vessels. Subsequent samples were taken from the allograft renal vein and the recipient's peripheral vein 5 min after beginning reperfusion. High-performance liquid chromatography was done to measure plasma concentrations of the oxypurines hypoxanthine (Hyp), xanthine (Xan) and uric acid and the nucleosides guanosine, inosine (Ino) and uridine (Urd). Concentrations of Hyp, Xan and Ino were significantly higher in the renal than the peripheral vein. The differences between the Xan, Hyp, Ino and Urd plasma concentrations in the renal and peripheral veins before and 5 min after reperfusion correlated positively and significantly with serum creatinine concentrations 24 and 72 h after graft transplantation. Moreover, the concentrations of Hyp were significantly increased in renal transplant recipients with delayed graft function. The results of this study suggest that the concentration of Hyp in the kidney allograft vein can be a useful predictor of early graft function.

Concentration of nucleosides and related compounds in cerebral and cerebellar cortical areas and white matter of the human brain.

1. Nucleosides potentially participate in the neuronal functions of the brain. However, their distribution and changes in their concentrations in the human brain is not known. For better understanding of nucleoside functions, changes of nucleoside concentrations by age and a complete map of nucleoside levels in the human brain are actual requirements. 2. We used post mortem human brain samples in the experiments and applied a recently modified HPLC method for the measurement of nucleosides. To estimate concentrations and patterns of nucleosides in alive human brain we used a recently developed reverse extrapolation method and multivariate statistical analyses. 3. We analyzed four nucleosides and three nucleobases in human cerebellar, cerebral cortices and in white matter in young and old adults. Average concentrations of the 308 samples investigated (mean+/-SEM) were the following (pmol/mg wet tissue weight): adenosine 10.3+/-0.6, inosine 69.5+/-1.7, guanosine 13.5+/-0.4, uridine 52.4+/-1.2, uracil 8.4+/-0.3, hypoxanthine 108.6+/-2.0 and xanthine 54.8+/-1.3. We also demonstrated that concentrations of inosine and adenosine in the cerebral cortex and guanosine in the cerebral white matter are age-dependent. 4. Using multivariate statistical analyses and degradation coefficients, we present an uneven regional distribution of nucleosides in the human brain. The methods presented here allow to creation of a nucleoside map of the human brain by measuring the concentration of nucleosides in microdissected tissue samples. Our data support a functional role for nucleosides in the brain.

Direct determination of nucleosides in the urine of patients with breast cancer using column-switching liquid chromatography–tandem mass spectrometry

We developed an analytical method for a simple, sensitive and simultaneous determination of oxidized nucleosides in urine using column-switching liquid chromatography-electrospray/tandem mass spectrometry (LC-ESI/MS/MS). We connected two columns through a six-way switching valve and effectively separated nucleosides in the urine from the interference by column-switching liquid chromatography. We monitored separated nucleosides using positive ionization tandem mass spectrometry in selective reaction monitoring (SRM) mode. The calibration ranges of nucleosides were 0.2-100 nmol/mL. The linearity of the method was 0.994-0.999, and the limits-of-detection (LOD) at a signal-to-noise (S/N) ratio of 3 were 0.1-0.2 nmol/mL. The coefficients of variation were in the range 2.28-11.74% for within-day variation and 4.36-11.15% for day-to-day variation, respectively. To explore the relationship between breast cancer and the nucleosides level in human urine, we measured the concentrations of nucleosides in female patients with breast cancer (n = 30) and in normal female subjects (n = 30). The concentration of nucleosides was significantly increased in patients with breast cancer when compared with the normal controls (1-methyladenosine; p < 0.005, N(2),N(2)-dimethylguanosine; p < 0.01, 5-hydroxymethyl-2'-deoxyuridine; p < 0.001, 8-hydroxy-2-deoxyguanosine; p < 0.001). Therefore, the elevated levels of nucleosides could be used as an important biomarker for breast-cancer research.

Identification and determination of nucleosides in Cordyceps sinensis and its substitutes by high performance liquid chromatography with mass spectrometric detection

Cordyceps sinensis (Cs) is a well-known traditional Chinese medicine (TCM) and Cordyceps mycelia (Cm), a cultured Cordyceps, is a substitute for Cordyceps sinensis. The most important active components in them are nucleosides. A high selective, sensitive and accurate high performance liquid chromatography method with photodiode array detection (DAD) and mass spectrometric detection has been developed for simultaneous separation, identification and quantification of nucleosides in Cs and Cm using a mobile phase including (A) ammonium acetate (40 mM, pH 5.2) and (B) methanol with a gradient system on a 2.0 mm × 150 mm Shimadzu VP-ODS column. The presence of each nucleoside in Cs and Cm was ascertained by comparison of MS data, UV spectra and retention time with standards. LC/ESI-MS in selective ion monitoring (SIM) mode were used for the quantification of nucleosides in Cs and Cm. 2-Chloroadenosine was used as internal standard for this assay. The precisions and accuracies were in the range of 1.5–5.3% and −3.5 to 5.0%, respectively. The limits of detection and quantification for nucleosides were in the order of 0.1–0.6 μg ml −1 and 0.5–2.0 μg ml −1, respectively. The recoveries were in the range of 92.0–107.0%. With the developed method, the concentrations of nucleosides in Cs and Cm from different sources were determined. Cs, characterized with far lower concentration of adenosine and cordycepin than Cm, can be very easy to distinguish from Cm. This reliable method would be useful for the study and quality control of Cordyceps sinensis and its substitutes.

Myocardial and coronary sinus purines as indicators of pig heart energy metabolism during reperfusion after extracorporeal circulation

The precise understanding of myocardial metabolism is crucial for the optimization of cardiosurgical procedures. We attempted to gain a comprehensive insight into the purine metabolism of the porcine heart during reperfusion by measuring concentrations of nucleotides, nucleosides and oxypurines simultaneously in the myocardium and coronary sinus. Twenty-five pigs were subjected to sham cardiosurgery with extracorporeal circulation and cold cardioplegic arrest of 60 min. Myocardial biopsies, as well as coronary sinus and arterial blood samples were taken before aortic clamping and at 5, 20, 60 and 120 min of reperfusion. HPLC was used to measure concentrations of 17 purines in the bioptates and of 5 in plasma. Reperfusion rapidly normalized the ischaemic decrease in the adenylate energy charge of the myocardium, but during 120 min failed to restore the reduced adenylate pool, because of irreversible loss of nucleosides by cardiomyocytes. Low adenylate energy charge and depletion of the adenylate pool were accompanied by analogous changes in the guanylates and growing deficit of NAD and NADP. Reperfusion was marked by significant release of inosine and guanosine from the heart, without any noticeable effect on hypoxanthine and xanthine. Coronary sinus concentrations of purines provide only a limited insight into the metabolism of the porcine heart. Repeated biopsies of the heart muscle and HPLC determinations of purine profiles represent a comprehensive and unique method for the study of purine metabolism during ischaemia and reperfusion. Future research on myocardial metabolism in disease and during cardiosurgical procedures should additionally be oriented to deficits in guanine and pyridine nucleotides.

Post mortem degradation of nucleosides in the brain: Comparison of human and rat brains for estimation of in vivo concentration of nucleosides

There is an increasing attention paid for nucleoside metabolism and changes of nucleoside concentrations in human brain because of its pathological and physiological relevance. In order to determine the post mortem degradation of nucleosides and nucleoside metabolites, the concentrations of four nucleosides and three nucleobases were measured in rat and neurosurgical human cerebral cortical samples with 30 s to 24 h post mortem delay. Adenosine degradation coefficient (a multiplying factor for calculating concentrations of investigated substances for the living state) was 0.886 for human brain at 2 h post mortem time, while it was 1.976 for rats. Hypoxanthine, an adenosine degradation product had coefficients 0.564 for human brain and 0.812 for the rat brain. We provide data and degradation coefficients for the concentrations of adenosine, guanosine, inosine, uridine, uracil, hypoxanthine and xanthine with 2, 4, 6 and 24 h post mortem delay. We also report a method how to validate human neurosurgical brain samples in terms of sample preparation and statistical analysis.

Urinary nucleosides as biological markers for patients with colorectal cancer

Fourteen urinary nucleosides, primary degradation products of tRNA, were evaluated to know the potential as biological markers for patients with colorectal cancer. The concentrations of 14 kinds of urinary nucleosides from 52 patients with colorectal cancer, 10 patients with intestinal villous adenoma and 60 healthy adults were determined by column switching high performance liquid chromatography method. The mean levels of 12 kinds of urinary nucleosides (except uridine and guanosine) in the patients with colorectal cancer were significantly higher than those in patients with intestinal villous adenoma or the healthy adults. Using the levels of 14 kinds of urinary nucleosides as the data vectors for principal component analysis, 71% (37/52) patients with colorectal cancer were correctly classified from healthy adults, in which the identification rate was much higher than that of CEA method (29%). Only 10% (1/10) of patients with intestinal villous adenoma were indistinguishable from patients with colorectal cancer. The levels of m1G, Pseu and m1A were positively related with tumor size and Duke's stages of colorectal cancer. When monitoring the changes in urinary nucleoside concentrations of patients with colorectal cancer associated with surgery, it was found that the overall correlations with clinical assessment were 84% (27/32) and 91% (10/11) in response group and progressive group, respectively. These findings indicate that urinary nucleosides determined by column switching high performance liquid chromatography method may be useful as biological markers for colorectal cancer.

Cell entry and export of nucleoside analogues

Some nucleoside analogues currently used as antiretroviral agents might promote mutagenesis besides their putative ability to interfere with endogenous nucleotide metabolism and/or inhibit viral transcription. The intracellular concentration of nucleosides and nucleobases is to some extent the result of the metabolic background of the specific cell line used for infection studies, its particular suit of enzymes and transporters. This review focuses on the transporter-mediated pathways implicated in either the uptake or the efflux of nucleoside- and nucleobase-derivatives. From a biochemical point of view, four different types of transport processes for nucleoside-related antiviral drugs have been described: (1) equilibrative uniport, (2) substrate exchange, (3) concentrative Na +- or H +-dependent uptake and finally, (4) substrate export through primary ATP-dependent active efflux pumps. These mechanisms are mainly related to the following set of transporter families: Concentrative Nucleoside Transporter (CNT), Equilibrative Nucleoside Transporter (ENT), Organic Anion Transporter (OAT) and Organic Cation Transporter (OCT), Peptide Transporter (PEPT) and Multidrug Resistance Protein (MRP). The basic properties of these carrier proteins and their respective role in the transport across the plasma membrane of nucleoside-derived antiviral drugs are reviewed.

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography

Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b 0 + b 1 (1/ x) with the regression coefficients b 0 and b 1, the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1–2 months, when they reach their absolute maximum for all age periods, and then decrease.

Intrapericardial Adenine Nucleosides Induce Elevation of Endothelin-1 Concentration in the Pericardial Space of the Dog Heart

The adenine nucleosides, adenosine (ADO) and inosine (INO), and the endothelin-1 (ET-1) play an important role in the regulation of coronary and myocardial functions. The pericardial fluid accumulates these regulatory agents and reflects well their levels in the myocardial interstitium. This offers a possibility to examine the local adenine nucleoside-endothelin interactions in heart tissue. We have previously demonstrated that increased levels of intrapericardial (i.p.) ET-1 enhanced significantly the adenine nucleoside concentrations of the pericardial fluid samples. In this study the effects of i.p.-administered ADO and INO were investigated on the pericardial concentrations of ET-1 in the in situ dog heart. The ET-1 concentrations were determined (enzymelinked immunosorbent assay) in the samples obtained from the pericardial fluid and from the arterial plasma before and after i.p. administration of increasing doses of ADO (10, 100 and 1000 muM, n = 8) and INO (1, 10 and 100 mM, n = 8). Standard hemodynamic variables were recorded continuously. We found that i.p. ADO and INO induced dose-dependent increases of pericardial ET-1 levels after a 15-minute incubation period in pericardial (ET-1max,ADO, +121 +/- 26%, P < 0.02; and ET-1max,INO, +84 +/- 27%, P < 0.05) but not in the plasma samples. The i.p. nucleosides elicited slight but characteristic alterations in the heart rate and ventricular contractility (dP/dt). The results suggest that the pericardial adenine nucleosides interact with the myocardial ET-1 and this effect may be provoked from, and can also be detected in, the pericardium.

Clinical significance and prognostic value of urinary nucleosides in breast cancer patients

Objective: Thirteen urinary nucleosides, primarily degradation products of tRNA, were evaluated as potential tumor markers for breast cancer patients. Design and methods: The micellar electrokinetic chromatography (MEKC) method has been used to analyze the urinary nucleosides in 41 healthy controls, 20 patients with benign breast tumors, and 26 breast cancer patients. Results: Urinary nucleoside concentrations of breast cancer patients were found to increase significantly compared to those of patients with benign breast tumors and healthy controls. By using 13 nucleoside concentrations as data vectors for principal component analysis (PCA), 73% (19/26) of breast cancer patients were correctly identified from healthy controls, while only 20% (4/20) of patients with benign breast tumors were indistinguishable from breast cancer patients. The mean level of all forms of urinary nucleosides in patients with metastatic breast cancer was higher than that in patients with primary breast cancer. The levels of modified nucleosides tended to decrease and return to normal after surgery. Conclusion: The results indicate that urinary nucleosides may be useful as tumor markers for breast cancer.

Biochemical brain markers and purinergic parameters in rat CSF after seizure induced by pentylenetetrazol

Cellular and molecular mechanisms involved in the generation of seizures and the magnitude of neural cells injury are not fully understood. We evaluated astrocyte and/or neuronal injury in rats in the pentylenetetrazol model of acute seizures by measuring S100B and NSE levels in cerebrospinal fluid. Additionally, we determined ADP and GDP hydrolysis by soluble nucleoside triphosphate diphosphohydrolase in the cerebrospinal fluid, and the concentration of nucleosides adenosine, inosine and guanosine as putative markers of brain injury. After pentylenetetrazol-induced seizures: (i) S100B values increased from 10 to 30 min, returning to control levels at 24 h; NSE levels presented a biphasic increase: an increase at 10 to 30 min returning to control levels , and again at 240 min followed by a decline at 24 h; (ii) nucleotidase activities increased from 10 min, returning to control levels at 240 min; (iii) guanosine and inosine levels increased exclusively after 30 min. In summary, this study showed biochemical changes in the cerebrospinal fluid occurring after seizures induced by pentylenetetrazol. Such events may have a modulating effect upon seizure expression, particularly nucleoside triphosphate diphosphohydrolase activities and nucleoside concentrations, but are nevertheless followed by neural death as evidenced by the increase in NSE and S100B levels.