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During the pandemic, Favipiravir (FVP) and Molnupiravir (MPV) have been widely used for COVID-19 treatment, leading to their presence in the environment. A green synchronous spectrofluorimetric method was developed to simultaneously detect them in environmental water, human plasma, and binary mixtures. Maximum fluorescence intensity was achieved at pH 8, with MPV exhibiting two peaks at 300 and 430 nm, and FVP showing one peak at 430 nm. A fluorescence subtraction method effectively removed interference, enabling direct determination of MPV at 300 nm and FVP at 430 nm. The method showed linearity within 2-13 ng/mL for FVP and 50-600 ng/mL for MPV, with recoveries of 100.35% and 100.12%, respectively. Limits of detection and quantification were 0.19 and 0.57 ng/mL for FVP and 10.52 and 31.88 ng/mL for MPV. Validation according to ICH and FDA guidelines yielded acceptable results. The method demonstrated good recoveries of FVP and MPV in pharmaceuticals, tap water and Nile water (99.62% ± 0.96% and 99.69% ± 0.64%) as per ICH guidelines and spiked human plasma (94.87% ± 2.111% and 94.79% ± 1.605%) following FDA guidelines, respectively. Its environmental friendliness was assessed using Green Analytical Procedure Index (GAPI) and the Analytical Greenness Metric (AGREE) tools., (© 2024 John Wiley & Sons Ltd.)

Decitabine is a DNA methyltransferase inhibitor used in the treatment of acute myeloid leukemia and myelodysplastic syndrome. The notion that ongoing trials are presently exploring the combined use of decitabine, with or without the cytidine deaminase inhibitor cedazuridine, and other antileukemic drugs necessitates a comprehensive understanding of pharmacokinetic properties and an evaluation of drug-drug interaction liabilities. We report here the development and validation of a sensitive UHPLC-MS/MS method for quantifying decitabine in mouse plasma, which should be useful for such studies. The method involved a one-step protein precipitation extraction, and chromatographic separation on an XBridge HILIC column using gradient elution. The method was found to be robust, accurate, precise, and sufficiently sensitive (lower limit of quantitation, 0.4 ng/mL) to determine decitabine concentrations in microvolumes of plasma from mice receiving the agent orally or intravenously in the presence or absence of cedazuridine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Metabolic reprogramming is an integral part of the growth-promoting program driven by the MYC family of oncogenes. However, this reprogramming also imposes metabolic dependencies that could be exploited therapeutically. Here we report that the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is an attractive therapeutic target for MYCN-amplified neuroblastoma, a childhood cancer with poor prognosis. Gene expression profiling and metabolomic analysis reveal that MYCN promotes pyrimidine nucleotide production by transcriptional upregulation of DHODH and other enzymes of the pyrimidine-synthesis pathway. Genetic and pharmacological inhibition of DHODH suppresses the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines. Furthermore, we obtain evidence suggesting that serum uridine is a key factor in determining the efficacy of therapeutic agents that target DHODH. In the presence of physiological concentrations of uridine, neuroblastoma cell lines are highly resistant to DHODH inhibition. This uridine-dependent resistance to DHODH inhibitors can be abrogated by dipyridamole, an FDA-approved drug that blocks nucleoside transport. Importantly, dipyridamole synergizes with DHODH inhibition to suppress neuroblastoma growth in animal models. These findings suggest that a combination of targeting DHODH and nucleoside transport is a promising strategy to overcome intrinsic resistance to DHODH-based cancer therapeutics., (© 2021. The Author(s).)

The effect of uridine on the myocardial ischemic and reperfusion injury was investigated. A possible mechanism of its cardioprotective action was established. Two rat models were used: (1) acute myocardial ischemia induced by occlusion of the left coronary artery for 60 min; and (2) myocardial ischemia/reperfusion with 30-min ischemia and 120-min reperfusion. In both models, treatment with uridine (30 mg/kg) prevented a decrease in cell energy supply and in the activity of the antioxidant system, as well as an increase in the level of lipid hydroperoxides and diene conjugates. This led to a reduction of the necrosis zone in the myocardium and disturbances in the heart rhythm. The blocker of the mitochondrial ATP-dependent potassium (mitoK ATP ) channel 5-hydroxydecanoate limited the positive effects of uridine. The data indicate that the cardioprotective action of uridine may be related to the activation of the mitoK ATP channel. Intravenously injected uridine was more rapidly eliminated from the blood in hypoxia than in normoxia, and the level of the mitoK ATP channel activator UDP in the myocardium after uridine administration increased. The results suggest that the use of uridine can be a potentially effective approach to the management of cardiovascular diseases., (© 2021. The Author(s).)

Background and Objectives: Metabolomics facilitates the discovery of biomarkers and potential therapeutic targets for CKD progression., Design, Setting, Participants, & Measurements: We evaluated an untargeted metabolomics quantification of stored plasma samples from 645 Chronic Kidney Disease in Children (CKiD) participants. Metabolites were standardized and logarithmically transformed. Cox proportional hazards regression examined the association between 825 nondrug metabolites and progression to the composite outcome of KRT or 50% reduction of eGFR, adjusting for age, sex, race, body mass index, hypertension, glomerular versus nonglomerular diagnosis, proteinuria, and baseline eGFR. Stratified analyses were performed within subgroups of glomerular/nonglomerular diagnosis and baseline eGFR., Results: Baseline characteristics were 391 (61%) male; median age 12 years; median eGFR 54 ml/min per 1.73 m 2 ; 448 (69%) nonglomerular diagnosis. Over a median follow-up of 4.8 years, 209 (32%) participants developed the composite outcome. Unique association signals were identified in subgroups of baseline eGFR. Among participants with baseline eGFR ≥60 ml/min per 1.73 m 2 , two-fold higher levels of seven metabolites were significantly associated with higher hazards of KRT/halving of eGFR events: three involved in purine and pyrimidine metabolism (N6-carbamoylthreonyladenosine, hazard ratio, 16; 95% confidence interval, 4 to 60; 5,6-dihydrouridine, hazard ratio, 17; 95% confidence interval, 5 to 55; pseudouridine, hazard ratio, 39; 95% confidence interval, 8 to 200); two amino acids, C-glycosyltryptophan, hazard ratio, 24; 95% confidence interval 6 to 95 and lanthionine, hazard ratio, 3; 95% confidence interval, 2 to 5; the tricarboxylic acid cycle intermediate 2-methylcitrate/homocitrate, hazard ratio, 4; 95% confidence interval, 2 to 7; and gulonate, hazard ratio, 10; 95% confidence interval, 3 to 29. Among those with baseline eGFR <60 ml/min per 1.73 m 2 , a higher level of tetrahydrocortisol sulfate was associated with lower risk of progression (hazard ratio, 0.8; 95% confidence interval, 0.7 to 0.9)., Conclusions: Untargeted plasma metabolomic profiling facilitated discovery of novel metabolite associations with CKD progression in children that were independent of established clinical predictors and highlight the role of select biologic pathways., (Copyright © 2021 by the American Society of Nephrology.)

Sofosbuvir is an NS5B nucleotide inhibitor for the treatment of hepatitis C viral infection. In this study the pharmacokinetics (PK) and safety of single and multiple doses of generic sofosbuvir were investigated in healthy Chinese subjects. Twelve subjects (6 male and 6 female) were enrolled in this study. The PK parameters of sofosbuvir and its metabolite (GS-331007) in both blood and urine samples were analyzed after dosing by the established liquid chromatography tandem mass spectrometry analytical method. The safety/tolerability assessment consisted of documenting adverse events, vital signs, electrocardiogram, and laboratory test results. Sofosbuvir was well tolerated. Major PK parameters of the generic formulation of sofosbuvir were similar to those found in previous reports. These data support further clinical evaluation of this generic formulation of sofosbuvir., (© 2019, The American College of Clinical Pharmacology.)

We have previously identified associations of 2 circulating secondary bile acids (glycocholenate and glycolithocolate sulfate) with atrial fibrillation (AF) risk in 1,919 blacks in the Atherosclerosis Risk in Communities cohort. We aimed to replicate these findings in an independent sample of 2,003 white and black Atherosclerosis Risk in Communities participants, and performed a new metabolomic analysis in the combined sample of 3,922 participants, followed between 1987 and 2013. Metabolomic profiling was done in baseline serum samples using gas and liquid chromatography mass spectrometry. AF was ascertained from electrocardiograms, hospitalizations, and death certificates. We used multivariable Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) of AF by 1 standard deviation difference of metabolite levels. Over a mean follow-up of 20 years, 608 participants developed AF. Glycocholenate sulfate was associated with AF in the replication and combined samples (HR 1.10, 95% CI 1.00, 1.21 and HR 1.13, 95% CI 1.04, 1.22, respectively). Glycolithocolate sulfate was not related to AF risk in the replication sample (HR 1.02, 95% CI 0.92, 1.13). An analysis of 245 metabolites in the combined cohort identified 3 additional metabolites associated with AF after multiple-comparison correction: pseudouridine (HR 1.18, 95% CI 1.10, 1.28), uridine (HR 0.86, 95% CI 0.79, 0.93) and acisoga (HR 1.17, 95% CI 1.09, 1.26). In conclusion, we replicated a prospective association among a previously identified secondary bile acid, glycocholenate sulfate, and AF incidence, and identified new metabolites involved in nucleoside and polyamine metabolism as markers of AF risk., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: This study aimed to determine the effect of food intake on uracil and dihydrouracil plasma levels. These levels are a promising marker for dihydropyrimidine dehydrogenase activity and for individualizing fluoropyrimidine anticancer therapy., Methods: A randomized, cross-over study in 16 healthy volunteers was performed, in which subjects were examined in fasted and fed state on two separate days. In fed condition, a high-fat, high-caloric breakfast was consumed between 8:00 h and 8:30 h. Whole blood for determination of uracil, dihydrouracil and uridine plasma levels was drawn on both test days at predefined time points between 8:00 h and 13:00 h., Results: Uracil levels were statistically significantly different between fasting and fed state. At 13:00 h, the mean uracil level in fasting state was 12.6 ± 3.7 ng ml -1 and after a test meal 9.4 ± 2.6 ng ml -1 (P < 0.001). Dihydrouracil levels were influenced by food intake as well (mean dihydrouracil level at 13:00 h in fasting state 147.0 ± 36.4 ng ml -1 and in fed state 85.7 ± 22.1 ng ml -1 , P < 0.001). Uridine plasma levels showed curves with similar patterns as for uracil., Conclusions: It was shown that both uracil and dihydrouracil levels were higher in fasting state than in fed state. This is hypothesized to be an direct effect of uridine plasma levels, which were previously shown to be elevated in fasting state and reduced after intake of food. These findings show that, when assessing plasma uracil and dihydrouracil levels for adaptive fluoropyrimidine dosing in clinical practice, sampling should be done between 8:00 h and 9:00 h after overnight fasting to avoid bias caused by circadian rhythm and food effects., (© 2018 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: The pharmacokinetics and safety of velpatasvir, a potent pangenotypic hepatitis C virus NS5A inhibitor, were evaluated in two hepatic impairment studies: a phase I study in hepatitis C virus-uninfected subjects and a phase III study (ASTRAL-4) in hepatitis C virus-infected patients., Methods: In the phase I study, subjects with moderate or severe hepatic impairment (Child-Pugh-Turcotte Class B or C), and demographically matched subjects with normal hepatic function received a single dose of velpatasvir 100 mg. Pharmacokinetics and safety assessments were performed, and pharmacokinetic parameters were calculated using non-compartmental methods and summarized using descriptive statistics and compared statistically by geometric least-squares mean ratios and 90% confidence intervals. In ASTRAL-4, subjects with decompensated cirrhosis (Child-Pugh-Turcotte Class B) were randomized to receive treatment with either sofosbuvir/velpatasvir ± ribavirin for 12 weeks or sofosbuvir/velpatasvir for 24 weeks. Pharmacokinetic and safety assessments were performed and pharmacokinetic parameters were calculated using a non-compartmental analysis and summarized using descriptive statistics and were compared to pharmacokinetics from ASTRAL-1 [subjects without cirrhosis or with compensated (Child-Pugh-Turcotte Class A) cirrhosis]., Results: In the phase I study, plasma exposures (area under the concentration-time curve) were similar in subjects with Child-Pugh-Turcotte Class B (n = 10) or Child-Pugh-Turcotte Class C hepatic impairment (n = 10) compared with normal hepatic function (n = 13). Percent free velpatasvir was similar in subjects without or with any degree of hepatic impairment. In the phase III study, velpatasvir overall exposure (area under the concentration-time curve over the 24-h dosing interval; AUC tau ) was similar and sofosbuvir exposures were higher (~ 100%) for patients with Child-Pugh-Turcotte Class B hepatic impairment compared with the ASTRAL-1 population, which was not considered clinically relevant., Conclusions: No sofosbuvir/velpatasvir dose modification is warranted for patients with any degree of hepatic impairment.

Purpose: The purpose of this study was to investigate the safety, tolerability, and pharmacokinetic profile of sofosbuvir and its metabolites after a single dose of sofosbuvir 400mg and once daily dosing of sofosbuvir 400mg for 7days in healthy Chinese subjects., Methods: This Phase I, open-label, single- and multiple-dose study enrolled 14 Chinese subjects aged 18 to 45years with an approximately even distribution of healthy male (n = 9) and nonpregnant, nonlactating female subjects (n = 5). Subjects received a single oral dose of sofosbuvir 400mg (one tablet) (morning, fasted conditions; single-dose treatment). After a 3-day washout, subjects received oral sofosbuvir 400mg (one tablet) (morning, fasted) for 7days (multiple dose treatment)., Findings: No significant accumulation of sofosbuvir, GS-566500, or GS-331007 was observed. Steady state of the major metabolite GS-331007 was achieved after 4days of consecutive dosing with sofosbuvir 400mg once daily. Sofosbuvir was generally well tolerated., Implications: Overall, this study supports the further evaluation of sofosbuvir 400mg in the Chinese population. The pharmacokinetic properties of sofosbuvir, GS-556500, and GS-311007 were found to be broadly similar in healthy Chinese subjects compared with non-Chinese subjects in previous sofosbuvir studies. ChinaDrugTrials.org.cn identifier: CTR20150249., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent investigations have focused on the potential role of gastrointestinal (GI) abnormalities in the pathogenesis of Parkinson's disease (PD). The 'dual-hit' hypothesis of PD speculates that a putative pathogen enters the brain via two routes: the olfactory system and the GI system. Here, we investigated (1) whether local exposures of the neurotoxin rotenone in the gut or the brain of mice could induce PD-like neurological and GI phenotypes as well as a characteristic neuropathology in accordance with this 'dual-hit hypothesis' and (2) the effects of a diet containing uridine and fish oil providing docosahexaenoic acid (DHA), in both models. Mice were given rotenone either orally or by an injection in the striatum. Dietary interventions were started 1 week before rotenone exposures. We found that (1) both oral and intrastriatal administration of rotenone induced similar PD-like motor deficits, dopaminergic cell loss, delayed intestinal transit, inflammation, and alpha-synuclein accumulation in the colon; (2) the uridine and DHA containing diet prevented rotenone-induced motor and GI dysfunctions in both models. The models suggest possible bidirectional communication between the gut and the brain for the genesis of PD-like phenotype and pathology. The dietary intervention may provide benefits in the prevention of motor and non-motor symptoms in PD.

DUP-785 (Brequinar sodium) is a potent inhibitor of the mitochondrial dihydroorotate dehydrogenase (DHO-DH), a rate-limiting enzyme in the pyrimidine de novo nucleotide synthesis. In phase I clinical studies at the maximum tolerated dose (MTD) Brequinar induced a long-term inhibition of DHO-DH in white blood cells (WBC) and a long-term depletion of plasma uridine. These two parameters were related to severe myelosuppression, so that in Phase II studies the dose of Brequinar was decreased considerably. We further characterized the mechanism of DHO-DH enzyme inhibition while in blood samples of patients entered into Phase II studies we evaluated DHO-DH inhibition in WBC and plasma uridine depletion. With Electron Spin Resonance it was demonstrated that DHO-DH produced oxygen radical formation, which was inhibited by Brequinar. In the Phase II study depending on the dose (600 to 2000 mg/m 2 ), uridine decreased to 20% (at the highest dose) or to 80-85% (at the middle dose) or did not change, which was associated with inhibition of DHO-DH (1% activity left vs 11 and 24% left). Inhibition of DHO-DH in the tumor of the latter patient was moderate as well (12% activity left). Brequinar was inactive in all tumor types evaluated possibly because of high uridine levels in the tumor. In conclusion, Brequinar was inactive against solid tumors, but DHO-DH inhibition was associated with myeloid toxicity, which may explain its potential for treatment of leukemia or inflammatory diseases.

Purpose: Milk provides a significant source of calcium, protein, vitamins and other minerals to Western populations throughout life. Due to its widespread use, the metabolic and health impact of milk consumption warrants further investigation and biomarkers would aid epidemiological studies., Methods: Milk intake assessed by a validated food frequency questionnaire was analyzed against fasting blood metabolomic profiles from two metabolomic platforms in females from the TwinsUK cohort (n = 3559). The top metabolites were then replicated in two independent populations (EGCUT, n = 1109 and KORA, n = 1593), and the results from all cohorts were meta-analyzed., Results: Four metabolites were significantly associated with milk intake in the TwinsUK cohort after adjustment for multiple testing (P < 8.08 × 10 -5 ) and covariates (BMI, age, batch effects, family relatedness and dietary covariates) and replicated in the independent cohorts. Among the metabolites identified, the carnitine metabolite trimethyl-N-aminovalerate (β = 0.012, SE = 0.002, P = 2.98 × 10 -12 ) and the nucleotide uridine (β = 0.004, SE = 0.001, P = 9.86 × 10 -6 ) were the strongest novel predictive biomarkers from the non-targeted platform. Notably, the association between trimethyl-N-aminovalerate and milk intake was significant in a group of MZ twins discordant for milk intake (β = 0.050, SE = 0.015, P = 7.53 × 10 -4 ) and validated in the urine of 236 UK twins (β = 0.091, SE = 0.032, P = 0.004). Two metabolites from the targeted platform, hydroxysphingomyelin C14:1 (β = 0.034, SE = 0.005, P = 9.75 × 10 -14 ) and diacylphosphatidylcholine C28:1 (β = 0.034, SE = 0.004, P = 4.53 × 10 -16 ), were also replicated., Conclusions: We identified and replicated in independent populations four novel biomarkers of milk intake: trimethyl-N-aminovalerate, uridine, hydroxysphingomyelin C14:1 and diacylphosphatidylcholine C28:1. Together, these metabolites have potential to objectively examine and refine milk-disease associations.

Uridine, a pyrimidine nucleoside present at high levels in the plasma of rodents and humans, is critical for RNA synthesis, glycogen deposition, and many other essential cellular processes. It also contributes to systemic metabolism, but the underlying mechanisms remain unclear. We found that plasma uridine levels are regulated by fasting and refeeding in mice, rats, and humans. Fasting increases plasma uridine levels, and this increase relies largely on adipocytes. In contrast, refeeding reduces plasma uridine levels through biliary clearance. Elevation of plasma uridine is required for the drop in body temperature that occurs during fasting. Further, feeding-induced clearance of plasma uridine improves glucose metabolism. We also present findings that implicate leptin signaling in uridine homeostasis and consequent metabolic control and thermoregulation. Our results indicate that plasma uridine governs energy homeostasis and thermoregulation in a mechanism involving adipocyte-dependent uridine biosynthesis and leptin signaling., (Copyright © 2017, American Association for the Advancement of Science.)

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.

Background: Miller syndrome (post-axial acrofacial dysostosis) arises from gene mutations for the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). Nonetheless, despite demonstrated loss of enzyme activity dihydroorotate (DHO) has not been shown to accumulate, but paradoxically urine orotate has been reported to be raised, confusing the metabolic diagnosis., Methods: We analysed plasma and urine from a 4-year-old male Miller syndrome patient. DHODH mutations were determined by PCR and Sanger sequencing. Analysis of DHO and orotic acid (OA) in urine, plasma and blood-spot cards was performed using liquid chromatography-tandem mass spectrometry. In vitro stability of DHO in distilled water and control urine was assessed for up to 60h. The patient received a 3-month trial of oral uridine for behavioural problems., Results: The patient had early liver complications that are atypical of Miller syndrome. DHODH genotyping demonstrated compound-heterozygosity for frameshift and missense mutations. DHO was grossly raised in urine and plasma, and was detectable in dried spots of blood and plasma. OA was raised in urine but undetectable in plasma. DHO did not spontaneously degrade to OA. Uridine therapy did not appear to resolve behavioural problems during treatment, but it lowered plasma DHO., Conclusion: This case with grossly raised plasma DHO represents the first biochemical confirmation of functional DHODH deficiency. DHO was also easily detectable in dried plasma and blood spots. We concluded that DHO oxidation to OA must occur enzymatically during renal secretion. This case resolved the biochemical conundrum in previous reports of Miller syndrome patients, and opened the possibility of rapid biochemical screening., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A rapid and simple LC-MS/MS method was developed and validated for the simultaneous estimation of sofosbuvir (SF) and its metabolite GS-331007 (GS) using famotidine as an internal standard (IS). The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Extraction with ethyl acetate was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC HSS C₁₈ (50 mm × 2.1 mm, 1.8 μm) column by pumping 0.1% formic acid and acetonitrile (50:50, v/v) in an isocratic mode at a flow rate of 0.3 ml/min. Method validation was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 10-2500 ng/ml for both SF and its metabolite. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1.2 min made it possible to analyze more than 300 human plasma samples per day. The developed assay method was successfully applied to a bioequivalence study in human volunteers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an essential scaffold protein in folate-dependent de novo thymidylate synthesis in the nucleus. SHMT1-deficient mice provide a model to investigate folic acid-responsive NTDs wherein disruption of de novo thymidylate synthesis impairs neural tube closure., Objective: We examined the effects of maternal supplementation with the pyrimidine nucleosides uridine, thymidine, or deoxyuridine with and without folate deficiency on NTD incidence in the Shmt1 mouse model., Design: Shmt1(+/+) and Shmt1(-/-) female mice fed folate-replete or folate-deficient diets and supplemented with uridine, thymidine, or deoxyuridine were bred, and litters (n = 10-23 per group) were examined for the presence of NTDs. Biomarkers of impaired folate status and metabolism were measured, including plasma nucleosides, hepatic uracil content, maternal plasma folate concentrations, and incorporation of nucleoside precursors into DNA., Results: Shmt1(+/-) and Shmt1(-/-) embryos from dams fed the folate-deficient diet were susceptible to NTDs. No NTDs were observed in litters from dams fed the folate-deficient diet supplemented with deoxyuridine. Surprisingly, uridine supplementation increased NTD incidence, independent of embryo genotype and dietary folic acid. These dietary nucleosides did not affect maternal hepatic uracil accumulation in DNA but did affect plasma folate concentrations., Conclusions: Maternal deoxyuridine supplementation prevented NTDs in dams fed the folate-deficient diet, whereas maternal uridine supplementation increased NTD incidence, independent of folate and embryo genotype. These findings provide new insights into the metabolic impairments and mechanisms of folate-responsive NTDs resulting from decreased Shmt1 expression., (© 2015 American Society for Nutrition.)

Studies have shown that uridine concentration in plasma may be an indicator of uric acid production in patients with gout. It has been also postulated that uridine takes part in blood pressure regulation. Since physical exercise is an effective tool in treatment and prevention of cardio-vascular diseases that are often accompanied by hyperuricemia and hypertension, it seemed advisable to attempt to evaluate the relationship between oxypurine concentrations (Hyp, Xan and UA) and that of Urd and BP after physical exercise in healthy subjects. Sixty healthy men (17.2+/-1.71 years, BMI 23.2+/-2.31 kg m(-2), VO(2max) 54.7+/-6.48 ml kg(-1) min(-1)) took part in the study. The subjects performed a single maximal physical exercise on a bicycle ergometer. Blood for analyses was sampled three times: immediately before exercise, immediately after exercise, and in the 30th min of rest. Concentrations of uridine and hypoxanthine, xanthine and uric acid were determined in whole blood using high-performance liquid chromatography. We have shown in this study that the maximal exercise-induced increase of uridine concentration correlates with the post-exercise increase of uric acid concentration and systolic blood pressure. The results of our study show a relationship between uridine concentration in blood and uric acid concentration and blood pressure. We have been the first to demonstrate that a maximal exercise-induced increase in uridine concentration is correlated with the post-exercise and recovery-continued increase of uric acid concentration in healthy subjects. Thus, it appears that uridine may be an indicator of post-exercise hyperuricemia and blood pressure.

Purpose: 5-fluorouracil (5-FU) has been broadly used to treat solid tumors for more than 50 years. One of the major side effects of fluoropyrimidines therapy is oral and intestinal mucositis. Human uridine phosphorylase (hUP) inhibitors have been suggested as modulators of 5-FU toxicity. Therefore, the present study aimed to test the ability of hUP blockers in preventing mucositis induced by 5-FU., Methods: We induced intestinal mucositis in Wistar rats with 5-FU, and the intestinal damage was evaluated in presence or absence of two hUP1 inhibitors previously characterized. We examined the loss of weight and diarrhea following the treatment, the villus integrity, uridine levels in plasma, and the neutrophil migration by MPO activity., Results: We found that one of the compounds, 6-hydroxy-4-methyl-1H-pyridin-2-one-3-carbonitrile was efficient to promote intestinal mucosa protection and to inhibit the hUP1 enzyme, increasing the uridine levels in the plasma of animals. However, the loss of body weight, diarrhea intensity or neutrophil migration remained unaffected., Conclusion: Our results bring support to the hUP1 inhibitor strategy as a novel possibility of prevention and treatment of mucositis during the 5-FU chemotherapy, based on the approach of uridine accumulation in plasma and tissues.

Brain neurons form synapses throughout the life span. This process is initiated by neuronal depolarization, however the numbers of synapses thus formed depend on brain levels of three key nutrients-uridine, the omega-3 fatty acid DHA, and choline. Given together, these nutrients accelerate formation of synaptic membrane, the major component of synapses. In infants, when synaptogenesis is maximal, relatively large amounts of all three nutrients are provided in bioavailable forms (e.g., uridine in the UMP of mothers' milk and infant formulas). However, in adults the uridine in foods, mostly present at RNA, is not bioavailable, and no food has ever been compelling demonstrated to elevate plasma uridine levels. Moreover, the quantities of DHA and choline in regular foods can be insufficient for raising their blood levels enough to promote optimal synaptogenesis. In Alzheimer's disease (AD) the need for extra quantities of the three nutrients is enhanced, both because their basal plasma levels may be subnormal (reflecting impaired hepatic synthesis), and because especially high brain levels are needed for correcting the disease-related deficiencies in synaptic membrane and synapses.

Background: Studies on the systemic availability of nutrients and nutritional status in Alzheimer's disease (AD) are widely available, but the majority included patients in a moderate stage of AD., Objective: This study compares the nutritional status between mild AD outpatients and healthy controls., Methods: A subgroup of Dutch drug-naïve patients with mild AD (Mini-Mental State Examination (MMSE) ≥20) from the Souvenir II randomized controlled study (NTR1975) and a group of Dutch healthy controls were included. Nutritional status was assessed by measuring levels of several nutrients, conducting the Mini Nutritional Assessment (MNA®) questionnaire and through anthropometric measures., Results: In total, data of 93 healthy cognitively intact controls (MMSE 29.0 [23.0-30.0]) and 79 very mild AD patients (MMSE = 25.0 [20.0-30.0]) were included. Plasma selenium (p < 0.001) and uridine (p = 0.046) levels were significantly lower in AD patients, with a similar trend for plasma vitamin D (p = 0.094) levels. In addition, the fatty acid profile in erythrocyte membranes was different between groups for several fatty acids. Mean MNA screening score was significantly lower in AD patients (p = 0.008), but not indicative of malnutrition risk. No significant differences were observed for other micronutrient or anthropometric parameters., Conclusion: In non-malnourished patients with very mild AD, lower levels of some micronutrients, a different fatty acid profile in erythrocyte membranes and a slightly but significantly lower MNA screening score were observed. This suggests that subtle differences in nutrient status are present already in a very early stage of AD and in the absence of protein/energy malnutrition.

Uridine is postulated to participate in the development of insulin resistance. Since exercise is an effective tool in the treatment of insulin resistance it appeared justified to assess the impact of maximal exercise on plasma uridine and insulin sensitivity indices (e.g. insulin and HOMA-IR) in healthy subjects. The study included forty-four healthy males (18.5+/-2.92 years, VO₂max 50.2+/-6.26 ml kg⁻¹ min⁻¹). Subjects performed a single maximal exercise on a bicycle ergometer. Blood samples were taken three times: immediately before exercise, immediately after exercise and at the 30(th) min of rest. Uridine concentrations were determined in the whole blood using high-performance liquid chromatography. Serum insulin levels were measured by a specific ELISA method. Insulin sensitivity was assessed by homeostasis model assessment method (HOMA-IR). A maximal exercise-induced increase in the concentration of uridine correlated with post-exercise increases in insulin levels and HOMA-IR. Our results indicate a relationship between the concentration of uridine in the blood and indicators of insulin sensitivity in healthy subjects. We are the first to demonstrate that a maximal exercise-induced increase in the concentration of uridine is correlated with post-exercise increases in insulin levels and HOMA-IR in healthy subjects. It appears that uridine may be an indicator of insulin resistance.

Experiments on rats have shown that preventive treatment with uridine stabilizes energy metabolism in the heart under conditions of 60-min left coronary artery occlusion. The preparation also prevented antioxidant system dysfunction and LPO hyperactivation. 5-Hydroxydecanoate, a selective blocker of mitochondrial ATP-dependent K(+)-channels, abolished the protective effect of uridine, which attested to the involvement of these channels into mechanisms of the cardioprotective effect of the preparation. The elimination of intravenously administered uridine from the blood of animals with acute ischemia was accelerated in comparison with that in intact animals, which could suggest the participation of this nucleoside in the processes of activation of intracellular anti-ischemic defense mechanisms.

Recently a pyrimidine nucleoside, uridine, has been show to have a protective effect on cultured human corneal epithelial cells, and on dry eye animal model and patients. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry method for the determination of uridine in rabbit plasma and urine. After protein precipitation with methanol including methaqualone (internal standard), the analyte was chromatographed on a reversed-phase column with a mobile phase of 0.1% formic acid aqueous solution and methanol (1:4, v/v). The accuracy and precision of the assay were in accordance with Food and Drug Administration regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of uridine in plasma and urine after a single oral administration of 450 mg/kg uridine in rabbits., (Copyright © 2011 John Wiley & Sons, Ltd.)

Uridine is a pyrimidine nucleoside that plays a crucial role in synthesis of RNA, glycogen, and biomembrane. In humans, uridine is present in plasma in considerably higher quantities than other purine and pyrimidine nucleosides, thus it may be utilized for endogenous pyrimidine synthesis. Uridine has a number of biological effects on a variety of organs with or without disease, such as the reproductive organs, central and peripheral nervous systems, and liver. In addition, it is used in clinical situations as a rescue agent to protect against the adverse effects of 5-fluorouracil. Since the biological actions of uridine may be related to its plasma concentration, it is important to examine factors that have effects on that concentration. Factors associated with an increase in plasma concentration of uridine include enhanced ATP consumption, enhanced uridine diphosphate (UDP)-glucose consumption via glycogenesis, inhibited uridine uptake by cells via the nucleoside transport pathway, increased intestinal absorption, and increased 5-phosphribosyl-1-pyrophosphate and urea synthesis. In contrast, factors that decrease the plasma concentration of uridine are associated with accelerated uridine uptake by cells via the nucleoside transport pathway and decreased pyrimidine synthesis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aim: Treating uridine as a product of UTP degradation and hypoxanthine as a degradation product of ATP, we assessed the concentration of uridine and hypoxanthine in the blood of children with newly diagnosed type 1 diabetes. We also sought to define the relationship between indicators of the degree of metabolic control of diabetes (fructosamine, HbA1c) and the concentration of the tested catabolites., Methods: This study was carried out on 33 children aged 12.26 ± 4.49 with newly diagnosed type 1 diabetes during their first hospitalization. The concentration of uridine and hypoxanthine was determined by high-performance liquid chromatography (HPLC)., Results: The results showed significantly elevated levels of hypoxanthine and uridine in the blood. We further show that blood uridine level is associated with purine metabolism and hyperglycaemia, and we demonstrate a significant positive correlation between the concentration of uridine and (i) the percentage of HbA1c and (ii) fructosamine levels, which indicate the role of hyperglycaemia in the pathogenesis of pyrimidine nucleotide metabolism in type 1 diabetes prior to diagnosis., Conclusion: The results confirm the existence of a relationship between the degree of metabolic control of diabetes and pyrimidine metabolism. Presumably, the analysis of uridine could be used as an adjunct marker of the severity of diabetic complications in newly diagnosed patients., (© 2011 The Author/Acta Paediatrica © 2011 Foundation Acta Paediatrica.)

Background: Uridine is a therapy for hereditary orotic aciduria and is being investigated in other disorders caused by mitochondrial dysfunction, including toxicities resulting from treatment with nucleoside reverse transcriptase inhibitors in HIV. Historically, the use of uridine as a therapeutic agent has been limited by poor bioavailability. A food supplement containing nucleosides, NucleomaxX®, has been reported to raise plasma uridine to supraphysiologic levels., Methodology/principal Findings: Single- and multi-dose PK studies following NucleomaxX® were compared to single-dose PK studies of equimolar doses of pure uridine in healthy human volunteers. Product analysis documented that more than 90% of the nucleoside component of NucleomaxX® is in the form of triacetyluridine (TAU). Single and repeated dosing with NucleomaxX® resulted in peak plasma uridine concentrations 1-2 hours later of 150.9 ± 39.3 µM and 161.4 ± 31.5 µM, respectively, levels known to ameliorate mitochondrial toxicity in vitro. C(max) and AUC were four-fold higher after a single dose of NucleomaxX® than after uridine. No adverse effects of either treatment were observed., Conclusions/significance: NucleomaxX®, containing predominantly TAU, has significantly greater bioavailability than pure uridine in human subjects and may be useful in the management of mitochondrial toxicity.

Background: Uridine has been advocated for the treatment of HIV-1/HAART-associated lipodystrophy (HALS), although its metabolism in HIV-1-infected patients is poorly understood., Methods: Plasma uridine concentrations were measured in 35 controls and 221 HIV-1-infected patients and fat uridine in 15 controls and 19 patients. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Uridine was measured by a binary gradient-elution HPLC method. Analysis of genes encoding uridine metabolizing enzymes in fat was performed with TaqMan RT-PCR., Results: Median plasma uridine concentrations for HIV-1-infected patients were 3.80 µmol/l (interquartile range: 1.60), and for controls 4.60 µmol/l (IQR: 1.8) (P = 0.0009). In fat, they were of 6.0 (3.67), and 2.8 (4.65) nmol/mg of protein, respectively (P = 0.0118). Patients with a mixed HALS form had a median plasma uridine level of 4.0 (IC95%: 3.40-4.80) whereas in those with isolated lipoatrophy it was 3.25 (2.55-4.15) µmol/l/l (P = 0.0066). The expression of uridine cytidine kinase and uridine phosphorylase genes was significantly decreased in all groups of patients with respect to controls. A higher expression of the mRNAs for concentrative nucleoside transporters was found in HIV-1-infected patients with respect to healthy controls., Conclusions: HIV-1 infection is associated with a decrease in plasma uridine and a shift of uridine to the adipose tissue compartment. Antiretroviral therapy was not associated with plasma uridine concentrations, but pure lipoatrophic HALS was associated with significantly lower plasma uridine concentrations.

During prolonged maximal exercise, oxygen deficits occur in working muscles. Progressive hypoxia results in the impairment of the oxidative resynthesis of ATP and increased degradation of purine nucleotides. Moreover, ATP consumption decreases the conversion of UDP to UTP, to use ATP as a phosphate donor, resulting in an increased concentration of UDP, which enhances pyrimidine degradation. Because the metabolism of pyrimidine nucleotides is related to the metabolism of purines, in particular with the cellular concentration of ATP, we decided to investigate the impact of a standardized exercise with increasing intensity on the concentration of uridine, inosine, hypoxanthine, and uric acid. Twenty-two healthy male subjects volunteered to participate in this study. Blood concentrations of metabolites were determined at rest, immediately after exercise, and after 30 min of recovery using high-performance liquid chromatography. We also studied the relationship between the levels of uridine and indicators of myogenic purine degradation. The results showed that exercise with increasing intensity leads to increased concentrations of inosine, hypoxanthine, uric acid, and uridine. We found positive correlations between blood uridine levels and indicators of myogenic purine degradation (hypoxanthine), suggesting that the blood uridine level is related to purine metabolism in skeletal muscles.

Objectives: The assessment of the clinical significance of creatine, cytosine, cytidine, uridine, thymine, thymidine, and 2'-deoxyuridine concentrations in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) for the detection of the relationship between pyrimidine metabolites and disease., Design and Methods: The study group consisted of 119 subjects, which were divided to three groups: control (n=31), type 2 diabetes without nephropathy (DM, n=23), and with nephropathy (DN, n=65). Levels of related metabolites were measured in plasma of all participants., Results: There is a significant increase in levels of cytosine (P<0.001), cytidine (P<0.001), and thymidine (P=0.016) with DN compared to DM. The levels of uridine, thymine, 2'-deoxyuridine, and creatine did not change., Conclusions: The levels of cytosine, cytidine, and thymidine may be useful for monitoring the progression of DM and evaluating the treatment., (2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Objective: It has been demonstrated that uridine infusion induces insulin resistance in rats. Furthermore, it was recently reported that plasma uridine is correlated with homeostasis model assessment of insulin resistance (HOMA-R) in hypertensive patients. Therefore, we investigated whether plasma uridine was correlated with HOMA-R in patients with non-insulin-dependent diabetes mellitus (NIDDM)., Subjects and Methods: The subjects were 23 male patients with NIDDM (average age 63 years) and 18 healthy males (average age 60 years). Blood samples were drawn after an overnight fast, plasma uridine was then measured using high-performance liquid chromatography., Results: The average plasma uridine concentration in patients with NIDDM was higher than that in healthy subjects (P < 0.05). Furthermore, plasma uridine values were positively correlated with HOMA-R (r = 0.48, P < 0.05), serum insulin (r = 0.46, P < 0.05), and serum C-peptide radioimmunoreactivity (CPR) (r = 0.44, P < 0.05) values, whereas they were not significantly correlated with fasting blood glucose or hemoglobin A1c values., Conclusion: We found a positive relationship between plasma uridine value and HOMA-R, serum insulin, and CPR, suggesting that plasma uridine is a marker of insulin resistance in patients with NIDDM.

To investigate whether the concentration of uridine in plasma is related to the urinary excretion of urea, 45 healthy male subjects with normouricemia and normal blood pressure were studied after providing informed consent. Immediately after collection of 24-hour urine, blood samples were drawn after an overnight fast except for water. The contents of ingested foods during the 24-hour urine collection period were described by the subjects and analyzed by a dietician. Simple regression analysis showed that plasma uridine was correlated with the urinary excretions of urea (R = 0.41, P < .01), uric acid (R = 0.36, P < .05), and uridine (R = 0.30, P < .05), as well as uric acid clearance (R = 0.35, P < .05) and purine intake (R = 0.30, P < .05). In contrast, multiple regression analysis showed a positive relationship only between plasma uridine and urinary excretion of urea. These results suggest that an increase in de novo pyrimidine synthesis leads to an increased concentration of uridine in plasma via nitrogen catabolism in healthy subjects with normouricemia and normal blood pressure., ((c) 2010 Elsevier Inc. All rights reserved.)

Unlabelled: Uridine-cytidine kinase (UCK) 2, an enzyme normally expressed in human placenta and testis and highly overexpressed in many neoplasias of blood and solid tissues, catalyzes monophosphorylation of pyrimidine ribonucleosides with efficiency 15- to 20-fold higher than that of ubiquitously expressed isozyme UCK1. In this paper, we report the synthesis of 3'-(E)-(2-iodovinyl)uridine (IV-14) and its preclinical evaluation as a new radiotracer derived from a UCK2-selective antitumor agent, 3'-(ethynyl)uridine., Methods: Radioiodinated IV-14 was prepared from the respective stannyl precursor. (131)I-IV-14 was studied in cellular uptake assays and tested for stability in serum as well as for stability to thymidine phosphorylase, liver-, and mucosa-specific murine uridine phosphorylases. UCK1 and UCK2 expression levels in different tumor cell lines were determined by Western blot. Cellular distribution of (131)I-IV-14 was determined in HL60 cells. Biodistribution studies and gamma-camera scintigraphy were performed on an HL60-xenografted severe combined immunodeficiency (SCID) mouse model., Results: (131)I-IV-14 demonstrated excellent stability in serum. It was stable to human thymidine phosphorylase and to liver- and mucosa-specific murine uridine phosphorylases. Cellular uptake after 24 h of incubation with (131)I-IV-14 was 4.27 +/- 0.21, 3.66 +/- 0.13, 2.69 +/- 0.07, 2.24 +/- 0.18, and 3.26 +/- 0.18 percentage injected dose per 5 x 10(5) Mia-PaCa-2, CX-1, HL60, Capan-1, and Panc-1 cells, respectively. Uptake and retention of IV-14 were regulated by 2 factors: UCK2 expression level and intracellular transport mediated partially via human equilibrating nucleoside transporter 1. A biodistribution study of (131)I-IV-14 in an HL60-xenografted SCID mouse model showed that at 4 h after injection the greatest amount of retained radioactivity was in tumor. The tissue-to-tumor ratio 4 h after injection was 1.0 +/- 0.24 for tumor, 0.40 +/- 0.18 for spleen, 0.25 +/- 0.12 for colon, 0.14 +/- 0.07 for small intestine, and less than 0.1 for other sites. Scintigraphy with (123)I-IV-14 4 h after injection showed the tumor well. In addition, high accumulation of radioiodide in the stomach content was observed and was presumably due to metabolic degradation of IV-14., Conclusion: IV-14 is a UCK2-specific marker, allowing for in vivo addressing of tumors with high RNA synthesis independent of proliferation rate.

Purpose: Although the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine (FdCyd), is being evaluated clinically, it must be combined with the cytidine deaminase inhibitor tetrahydrouridine (THU) to prevent rapid metabolism of FdCyd to the pharmacologically active, yet unwanted, metabolites 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorouracil (FU), and 5-fluorouridine (FUrd). We assessed plasma concentrations of FdCyd and metabolites in patients receiving FdCyd and THU., Methods: We validated an LC-MS/MS assay, developed for a preclinical study, to quantitate FdCyd and metabolites in human plasma. Patients were treated with five daily, 3-h infusions of FdCyd at doses of 5-80 mg/m(2) with 350 mg/m(2) THU. Plasma was obtained during, and before the end of infusions on days 1 and 5., Results: The lower limits of quantitation for FU, FdUrd, FUrd, FC and FdCyd were 1, 1.5, 10, 3, and 10 ng/ml, respectively. Plasma FdCyd increased with dose, from 19-96 ng/ml at 5 mg/m(2) to 1,600-1,728 ng/ml at 80 mg/m(2). FdUrd was undetectable in patients treated with FdCyd doses <20 mg/m(2), and increased from 2.3 ng/ml at 20 mg/m(2) to 3.5-5.7 ng/ml at 80 mg/m(2). FU increased from 1.2-5.5 ng/ml at 5 mg/m(2) to 6.0-12 ng/ml at 80 mg/m(2)., Conclusions: By co-administering FdCyd with THU, FdCyd plasma concentrations were achieved that are known to inhibit DNA methylation in vitro. The accompanying plasma FU and FdUrd concentrations are <10% those observed after therapeutic infusions of FU or FdUrd, while FdCyd levels are well above those required to inhibit methylation in vitro. Therefore, inhibition of DNA methylation with FdCyd and THU appears feasible.

Kinetics of the hydrolytic reactions of four bisphosphonate derivatives of nucleoside antimetabolites, viz., 5-fluorouridine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 4), 5-fluorouridine 5'-beta,gamma-methylene triphosphate ( 5), ara-cytidine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 6), and ara-cytidine 5'-beta,gamma-methylene triphosphate ( 7), have been studied over a wide pH range (pH 1.0-8.5) at 90 degrees C. With each compound, the disappearance of the starting material was accompanied by formation of the corresponding nucleoside 5'-monophosphate, the reaction being up to 2 orders of magnitude faster with the beta,gamma-(1-hydroxyethylidene) derivatives ( 4, 6) than with their beta,gamma-methylene counterparts ( 5, 7). With compound 7, deamination of the cytosine base competed with the phosphate hydrolysis at pH 3-6. The measurements at 37 degrees C (pH 7.4) in the absence and presence of divalent alkaline earth metal ions (Mg (2+) and Ca (2+)) showed no sign of metal ion catalysis. Under these conditions, the initial product, nucleoside 5'-monophosphate, underwent rapid dephosphorylation to the corresponding nucleoside. Hydrolysis of the beta,gamma-methylene derivatives ( 5, 7) to the corresponding nucleoside 5'-monophosphates was markedly faster in mouse serum than in aqueous buffer (pH 7.4), the rate-acceleration being 5600- and 3150-fold with 5 and 7, respectively. In human serum, the accelerations were 800- and 450-fold compared to buffer. In striking contrast, the beta,gamma-(1-hydroxyethylidene) derivatives did not experience a similar decrease in hydrolytic stability. The stability in human serum was comparable to that in aqueous buffer (tau 1/2 = 17 and 33 h with 4 and 6, respectively), and on going to mouse serum, a 2- to 4-fold acceleration was observed. To elucidate the mineral-binding properties of 4- 7, their retention on a hydroxyapatite column was studied and compared to that of zoledronate ( 1a) and nucleoside mono-, di-, and triphosphates.

We investigated the effects of allopurinol on beer-induced changes in the plasma concentration and urinary excretion of purine bases. Five healthy subjects underwent three studies: ingestion of beer after taking 300 mg allopurinol (combination study); ingestion of beer alone; ingestion of allopurinol alone. Increased plasma concentrations and urinary excretion of hypoxanthine were greater in the combination study than the beer alone study. However, increases in total plasma purine base concentrations were greater in the beer alone study, even though increases in plasma uridine concentrations did not differ. Beer-induced increases in plasma concentrations of purine bases appear partially offset by increased urinary excretion of hypoxanthine after allopurinol, which also controls increases in plasma uric acid levels caused by alcoholic beverage ingestion.

In the present study, we investigated the cardiovascular effects of intravenously injected uridine or cytidine, and the role of adenosine receptors in mediating these effects, in conscious normotensive rats. Intravenous (i.v.) administration of uridine (124, 250, 500 mg/kg) dose-dependently decreased arterial pressure and heart rate. Cytidine (124, 250, 500 mg/kg; i.v.) produced slight dose-related hypotension without changing heart rate. Plasma uridine and cytidine concentrations increased time- and dose-dependently while plasma adenosine levels did not change after injection of the respective nucleosides. Pretreatment with intravenous caffeine (20 mg/kg), 8-phenyltheophylline (8-PT) (1 mg/kg), nonselective adenosine receptor antagonists, or 8-p-sulfophenyltheophylline (8-SPT) (20 mg/kg), a nonselective adenosine receptor antagonist which does not cross the blood-brain barrier, abolished the cardiovascular effects of uridine (250 mg/kg; i.v.) or cytidine (250 mg/kg; i.v.). Intracerebroventricular (i.c.v.) caffeine (200 microg) or 8-SPT (50 microg) pretreatment did not change the magnitude of the cardiovascular responses induced by nucleosides. Intravenous 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX) (5 mg/kg), a selective adenosine A(1) receptor antagonist, greatly attenuated the cardiovascular responses to uridine and cytidine. Pretreatment with 3,7,-dimethyl-1-propargylxanthine (DMPX) (2 mg/kg), an adenosine A(1)/A(2) receptor antagonist, attenuated hypotension induced by uridine and blocked the arterial pressure decrease in response to cytidine. Uridine-induced bradycardia was blocked by DMPX. 4-(2-[7-amino-2-(2-furyl[1,2,4]-triazolo[2,3-a[1,3,5]triazin-5-yl-aminoethyl)phenol (ZM241385) (1 mg/kg; i.v.), a selective adenosine A(2A) receptor antagonist, pretreatment produced an only very small blockade in the first minute of the hypotensive effects of uridine without affecting the bradycardia. ZM241385 pretreatment completely blocked cytidine's hypotensive effect. In Langendorff-perfused rat heart preparation, uridine (10(-3) M), but not cytidine, decreased the heart rate. Our results show that intravenously injected uridine or cytidine is able to decrease arterial pressure by activating peripheral adenosine receptors. The data also implicates that the mainly adenosine A(1) receptor activation is involved in the uridine-induced cardiovascular effects, while both adenosine A(1) and A(2A) receptor activations mediate the cytidine's effects.

Background: Since grape juice contains considerable amounts of fructose, which may increase the plasma concentration of urate, the combination of exercise and grape juice may increase the plasma concentration of urate to a greater degree than grape juice or exercise alone., Methods: We performed 3 experiments with 6 healthy male Japanese. The first was exercise alone (exercise alone experiment), the second was grape juice ingestion alone (grape juice alone experiment), and the third was a combination of exercise and grape juice ingestion (combination experiment)., Results: In the exercise alone experiment, the concentrations of purine bases and uridine in plasma, and lactate in blood, as well as the urinary excretion of oxypurines were increased, whereas the urinary excretion of uric acid and fractional excretion of purine bases were decreased. In the grape juice alone experiment, the concentrations of purine bases and uridine, as well as lactate in blood were increased, whereas the fractional excretion of uric acid was decreased. In the combination experiment, the concentrations of purine bases and uridine in plasma, and lactate in blood, as well as the urinary excretion of oxypurines were increased, whereas the urinary excretion of uric acid and fractional excretion of hypoxanthine, xanthine, and uric acid were decreased. The increase in plasma concentration of urate by the combination of exercise and grape juice was greater than that by each alone, though it was not significantly different from the sum of increases in those 2 experiments., Conclusion: Increases in adenine nucleotide degradation and lactic acid production caused by both exercise and grape juice ingestion play an important role in the increase in plasma concentration of urate, while those in combination have an additive effect on that concentration.

Background: Uridine adenosine tetraphosphate (Up4A) was been recently characterized as a potent vasoconstrictor. Up4A occurs in plasma from healthy subjects at concentrations sufficient to cause strong vasoconstrictive effects. In this study, Up4A concentrations in plasma from juvenile hypertensives and normotensives were determined., Methods and Results: Up4A was purified to homogeneity by preparative reverse phase high performance liquid-chromatography (HPLC), affinity chromatography HPLC, and analytic reverse phase HPLC from deproteinized plasma of juvenile hypertensives and normotensives. Mean total plasma Up4A concentration was significantly increased in juvenile hypertensives compared with juvenile normotensives (33.0+/-25.4 versus 3.7+/-0.9 nmol/L; mean+/-SEM, n=40 and 38, respectively; P<0.005). Accordingly, Up4A showed a significant association with juvenile hypertension (OR for ln(Up4A): 1.82; 95% CI 1.12, 2.95). Plasma Up4A concentrations correlated with left ventricular mass (Kendall-tau correlation coefficient 0.220, n=40; P<0.05) and intima media wall thickness (Kendall-tau correlation coefficient 0.296, n=40; P<0.05) in the hypertensives. Because the increased intima media thickness may be related to proliferative effects of Up4A, we studied the effects of Up4A on human vascular smooth muscle cell proliferation. The maximum proliferative effect of Up4A was 80.0+/-24.0% % above control (P<0.01). The proliferative effect of Up4A on smooth muscle cells is cell cycle-dependent, involving stimulation of S phase entry., Conclusion: Circulating levels of Up4A are strongly associated with juvenile hypertension. The endothelium-derived vasoconstrictor Up4A may contribute to the early development of primary hypertension and is moreover an important risk factor of juvenile hypertension.

Background: The relationship between plasma uridine levels and blood pressure (BP), and indicators of muscular purine degradation and insulin resistance (IR) has been evaluated in hypertensive (HT) patients., Methods and Results: In 36 HT patients and 10 normotensive subjects, seated BP was measured, and blood samples were drawn after overnight fast. In 18 of the HT patients, the semi-ischemic forearm test was performed to examine the release of hypoxanthine, ammonium and lactate. Plasma uridine levels were significantly higher than in the normotensive subjects. Fasting plasma insulin levels and homeostasis model assessment of IR correlated with plasma uridine levels in the HT patients. Plasma uridine levels showed a significant correlation with hypoxanthine, ammonia and lactate released from the semi-ischemic exercising muscles of the HT patients., Conclusions: Taken together with the positive correlation with indicators of IR, it is suggested that plasma uridine levels in HT are responsible for purine degradation and IR in skeletal muscles.

Objective: 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., Material and Methods: 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)., Results: 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., Conclusion: 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.

Purpose: To elucidate the in vivo metabolic fate of zebularine (NSC 309132), a DNA methyltransferase inhibitor proposed for clinical evaluation in the treatment of cancer., Experimental Design: Male, CD(2)F(1) mice were dosed i.v. with 100 mg/kg 2-[(14)C]zebularine. At specified times between 5 and 1,440 minutes, mice were euthanized. Plasma, organs, carcass, urine, and feces were collected and assayed for total radioactivity. Plasma and urine were also analyzed for zebularine and its metabolites with a previously validated high-pressure liquid chromatography assay. A similar experiment was done with 2-[(14)C]uridine, the proposed primary metabolite of zebularine., Results: Maximum plasma concentrations were 462, 306, 33.6, 21.7, and 11.5 mumol/L for total radioactivity, zebularine, uridine, uracil (each at 5 minutes), and dihydrouracil (at 15 minutes), respectively. Total radioactivity, zebularine, uridine, uracil, and dihydrouracil were rapidly eliminated from plasma, and after 45 minutes, none of the individual compounds could be quantitated by high-pressure liquid chromatography. Plasma data were consistent with sequential conversion of zebularine to uridine, uracil, and dihydrouracil. 2-Pyrimidinone was not observed. Prolonged retention of radioactivity, at concentrations higher than in plasma, was observed in tissues. Recovery of given radioactivity in urine (30.3% of dose), feces (0.4% of dose), cage wash (7.9% of dose), and tissues and carcass (6.1% of dose) after 24 hours implied that up to 55% of radioactivity was expired as (14)CO(2). Comparison of zebularine and uridine pharmacokinetic data indicated that approximately 40% of the zebularine dose was converted to uridine., Conclusions: Zebularine is extensively and rapidly metabolized into endogenous compounds that are unlikely to have effects at the concentrations observed.

To determine the effects of allopurinol on beer-induced increases in plasma and urinary excretion of purine bases (hypoxanthine, xanthine, and uric acid), we performed three experiments on five healthy study participants. In the first experiment (combination study), the participants ingested beer (10 ml/kg body weight) eleven hours after taking allopurinol (300 mg). In the second experiment (beer-only study), the same participants ingested beer (10 ml/kg body weight) alone, while in the third experiment (allopurinol-only study), they took allopurinol (300 mg) alone. There was a two-week interval between each of the studies. Beer-induced increases in plasma concentration and urinary excretion of hypoxanthine in the combination study were markedly higher than those in the beer-only study. On the other hand, the sum of increases in plasma concentrations of purine bases in the beer-only study was greater than in the combination study, whereas the increase in plasma uridine concentration in the combination study did not differ from the beer-only study. In addition, allopurinol administration inhibited the beer-induced increase in plasma concentration of uric acid. These results suggest that abrupt adenine nucleotide degradation may increase plasma concentration and urinary excretion of hypoxanthine under conditions of low xanthine dehydrogenase activity, which is mostly ascribable to allopurinol. Further, the difference in the sum of increases in plasma concentrations of purine bases between the combination study and beer-only study was largely ascribable to a greater increase in urinary excretion of hypoxanthine in the combination study. In addition, allopurinol intake seems to be effective in controlling the rapid increase in plasma uric acid caused by ingestion of alcoholic beverages.

The metabolism of zebularine (NSC 309132), a novel agent that inhibits DNA methyltransferases, is still uncharacterized. To examine the in vivo metabolism of zebularine, an analytical method was developed and validated (based on FDA guidelines) to quantitate 2-[(14)C]-zebularine and its major metabolites in murine plasma. Zebularine and its metabolites uridine, uracil and dihydrouracil were baseline-separated based on hydrophilic interaction chromatography by using an amino column. The assay was accurate and precise in the concentration ranges of 5.0-100 microg/mL for zebularine, 2.5-50 microg/mL for uridine, 1.0-10 microg/mL for uracil and 0.5-5.0 microg/mL for dihydrouracil. This assay is being used to quantitate zebularine and its metabolites in ongoing pharmacokinetic studies of zebularine.

We examined the biochemical pathways whereby oral uridine-5'-monophosphate (UMP) increases membrane phosphatide synthesis in brains of gerbils. We previously showed that supplementing PC12 cells with uridine caused concentration-related increases in CDP-choline levels, and that this effect was mediated by elevations in intracellular uridine triphosphate (UTP) and cytidine triphosphate (CTP). In the present study, adult gerbils received UMP (1 mmol/kg), a constituent of human breast milk and infant formulas, by gavage, and plasma samples and brains were collected for assay between 5 min and 8 h thereafter. Thirty minutes after gavage, plasma uridine levels were increased from 6.6 +/- 0.58 to 32.7 +/- 1.85 microM (P < 0.001), and brain uridine from 22.6 +/- 2.9 to 89.1 +/- 8.82 pmol/mg tissue (P < 0.001). UMP also significantly increased plasma and brain cytidine levels; however, both basally and following UMP, these levels were much lower than those of uridine. Brain UTP, CTP, and CDP-choline were all elevated 15 min after UMP (from 254 +/- 31.9 to 417 +/- 50.2, [P < 0.05]; 56.8 +/- 1.8 to 71.7 +/- 1.8, [P < 0.001]; and 11.3 +/- 0.5 to 16.4 +/- 1, [P < 0.001] pmol/mg tissue, respectively), returning to basal levels after 20 and 30 min. The smallest UMP dose that significantly increased brain CDP-choline was 0.05 mmol/kg. These results show that oral UMP, a uridine source, enhances the synthesis of CDP-choline, the immediate precursor of PC, in gerbil brain.

Purpose: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in improving the pharmacokinetics and bioavailability of oral uridine. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. PTAU is fully absorbed after oral administration with 100% oral bioavailability., Methods: Uridine (330, 660 or 1320 mg/kg) and/or PTAU (30, 45, 60, 120, 240 or 480 mg/kg) were orally administered to mice. The plasma levels of uridine, its catabolite uracil, and PTAU were measured using HPLC, and pharmacokinetic analysis was performed., Results: Oral PTAU up to 480 mg/kg per day is not toxic to mice. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg has a prolonged plasma half-life of 2-3 h, and peak plasma PTAU concentrations (C(max)) of 41, 51, 74, 126 and 161 microM with AUCs of 70, 99, 122, 173 and 225 micromol h/l, respectively. Coadministration of uridine with PTAU did not have a significant effect on the pharmacokinetic parameters of plasma PTAU at any of the doses tested. Coadministration of PTAU (30, 45, 60 and 120 or 240 mg/kg) with uridine (330, 660 or 1320 mg/kg) elevated the concentration of plasma uridine over that following the same dose of uridine alone, a result of reduced metabolic clearance of uridine as evidenced by decreased plasma exposure (C(max) and AUC) to uracil. Plasma uridine was elevated with the increase of uridine dose at each PTAU dose tested and no plateau was reached. Coadministration of PTAU at 30, 45, 60, 120 and 240 mg/kg improved the low oral bioavailability (7.7%) of uridine administered at 1320 mg/kg by 4.3-, 5.9-, 9.9-, 11.7- and 12.5-fold, respectively, and reduced the AUC of plasma uracil (1227.8 micromol h/l) by 5.7-, 6.8-, 8.2-, 6.3-, and 6.9-fold, respectively. Similar results were observed when PTAU was coadministered with lower doses of uridine. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg improved the oral bioavailability of 330 mg/kg uridine by 1.7-, 2.4-, 2.6-, 5.2- and 4.3- fold, and that of 660 mg/kg uridine by 2.3-, 2.7-, 3.3-, 4.6- and 6.7-fold, respectively., Conclusion: The excellent pharmacokinetic properties of PTAU, and its extraordinary effectiveness in improving the oral bioavailability of uridine, could be useful to rescue or protect from host toxicities of 5-fluorouracil and various chemotherapeutic pyrimidine analogues used in the treatment of cancer and AIDS, as well as in the management of medical disorders that are remedied by the administration of uridine including CNS disorders (e.g. Huntington's disease, bipolar disorder), liver diseases, diabetic neuropathy, cardiac damage, various autoimmune diseases, and transplant rejection.

5-Fluorouracil (5-FU) is a common anticancer agent used in the treatment of solid tumours, with a reported variability in the pharmacokinetic profile and inter-patient differences in efficacy and toxicity. Since 5-FU is intracellularly metabolised to active cytotoxic fluoronucleotides, some authors suggested it would be useful to determine the plasma levels of its main metabolites 5-fluoro-5,6-dihydrouracil (5-FUH2), 5-fluorouridine (5-FUrd) and 5-fluoro-2'-deoxyuridine (5-FdUrd), in order to better characterise population pharmacokinetics-pharmacodynamics (PK-PD) of this drug. We developed and validated an HPLC method to simultaneously determine plasma concentrations of 5-FU and the three main metabolites, and we analysed the plasma concentration-time curves of the first dose of 18 colon cancer patients treated with folinic acid and 5-FU 400 mg m(-2) by intra-venous bolus injection as adjuvant chemotherapy. Non-compartmental PK analysis has been applied to 5-FU and 5-FUH2 concentrations, estimating the following parameters (median values): Cmax 55.44 and 6.23 microg ml(-1), respectively, AUC(0-2 h) 11.59 and 5.94 hx microg ml(-1), CLTB 30.64 and 51.81 lh(-1) m(-2), 5-FUH2/5-FU AUC ratio 0.47 (range 0.29-1.12). We verified the patient covariables which could influence the inter-patient variability in the area under the time-concentration curves, and we observed that age, sex, weight, body surface area, cycle of therapy, toxicity development and 5-FUrd or 5-FdUrd detectability did not have statistical influence on 5-FUH2/5-FU AUC ratio. In eight subjects, we compared the PK data of the first and the fifth day of dose administration, and we found stable 5-FU values, but the 5-FUH2 disposition decreased with lower AUC(0-2 h) (7.90 hx microg ml(-1) versus 5.99 hx microg ml(-1)) and, particularly, Cmax (8.38 microg ml(-1) versus 5.50 microg ml(-1)) at day 5. This fact, evident in almost every patient, could suggest a possible reduction in the catabolic pathway of 5-FU leading to 5-FUH2, with a possible increase of the therapeutic pathway. For this reason, we tried to detect 5-FUrd and 5-FdUrd and, in fact, in our patients these metabolites were detected only in few samples, but most of them at day 5. In conclusion, our study confirms the relevance of pharmacokinetic analysis of 5-FU main metabolites and especially 5-FUH2, to better understand the metabolism and to improve the therapeutic efficacy., (Copyright 2004 Elsevier Ltd.)

To evaluate uridine levels in humans we developed a very sensitive and specific high-performance liquid chromatographic method for the determination of uridine in serum. We use techniques which are available in a standard analytical laboratory. Chromatographic analysis was carried out on a Phenomenex Aqua C18 5 micro 125A column protected by a guard cartridge system. Potassium dihydrogen phosphate buffer-acetonitrile was used as an eluent and oxypurinol as the internal standard. All sample preparation steps were done at 4 degrees C and the autosampler was cooled down to 4 degrees C. The calibration curve was linear throughout the calibration range from 0.25 to 100 micromol/l. This method was primarily established to evaluate uridine serum levels in patients with HIV infection since patients on highly active antiretroviral therapy (HAART) might develop metabolic disturbances that could lead to severe and fatal lactic acidosis due to mitochondrial toxicity. It is suggested that a limited or inadequate uridine supply is at least in part responsible for the onset of such deterioration.

To determine whether purine-free and regular low-malt liquor beverages (happo-shu) increase the plasma concentration and urinary excretion of purine bases (hypoxanthine, xanthine, uric acid) and uridine, 6 healthy males were given regular (10 ml/kg of body weight) and purine-free happo-shu (10 ml/kg of body weight). Plasma concentration-time curves were plotted, and the areas under the curves for uric acid and total purine bases (the sum of hypoxanthine, xanthine, and uric acid) were greater in the regular than in the purine-free happo-shu ingestion experiment (both p < 0.05). In addition, the total urinary excretion of xanthine, total purine bases, and uridine was greater in the regular than in the purine-free happo-shu ingestion experiment (p < 0.05 in all cases), although the total urinary excretion of hypoxanthine and uric acid was no different between the regular and the purine-free happo-shu ingestion experiments. These results suggest that uridine contained in regular happo-shu might contribute to an increase in the urinary excretion of uridine along with ethanol, and that the purines contained in regular happo-shu may contribute to the increase in plasma concentration of uric acid due to purine degradation.