Development and Validation of a Single HPLC Method for Analysis of Purines in Fish Oil Supplements

Gout is one of the most common forms of inflammatory arthritis, affecting over 8 million adults in the US. Individuals with gout are advised to avoid the habitual intake of purine-rich foods such as meats, seafood, purine-rich vegetables, and animal protein. An increased risk of developing or having subsequent attacks of gout is particularly associated with the consumption of seafood. However, clinical studies have shown that certain seafood and fish oil supplements that contain large amounts of omega-3 fatty acids provide important cardiovascular benefits. Individuals who might benefit from omega-3 fatty acid supplementation may therefore avoid fish oil products because they contain purines. Currently, there are no distinct high-performance liquid chromatography (HPLC) methods available in the literature that are validated as per the International Conference on Harmonisation (ICH) guidelines for the analysis of omega-3 fatty acid oils or fish oil containing products for purine content. A robust, fast, and efficient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the specific analysis of the naturally occurring purines guanine, purine, theobromine, and adenine. These purines are often found in fish oil and seafood. The analytical method reported herein quantifies all four purines in fish oil in about 20 minutes.

Tetra-n-butylammonium Hydroxide:an Efficient Catalyst for N-Alkylation of Pyrimidines and Purines

An efficient procedure for N-alkylation of pyrimidines and purines in the presence of tetra-n-butylammonium hydroxide（TBAH） is described. The method is very practical and the alkylation can occur at room temperature and the yields of the N-alkyl pyrimidines and purines were found to be excellent.

Visible light induced oxidative coupling of purines with arenes

The photocatalyzed synthesis of 9-arylpurines has been developed using 9H-purines and non-activated arenes.This method is highly atom economical using an acridinium photocatalyst induced by visible light under air atmosphere at room temperature.It employs no metal or external oxidant for the synthesis of9-arylpurine derivatives.

Purines:From Diagnostic Biomarkers to Therapeutic Agents in Brain Injury

The purines constitute a family of inter-related compounds that serve a broad range of important intracellular and extracellular biological functions.In particular,adenosine triphosphate(ATP)and its metabolite and precursor,adenosine,regulate a wide variety of cellular and systems-level physiological processes extending from ATP acting as the cellular energy currency,to the adenosine arising from the depletion of cellular ATP and responding to reduce energy demand and hence to preserve ATP during times of metabolic stress.This inter-relationship provides opportunities for both the diagnosis of energy depletion during conditions such as stroke,and the replenishment of ATP after such events.In this review we address these opportunities and the broad potential of purines as diagnostics and restorative agents.

Probiotics administration alleviates cognitive impairment and circadian rhythm disturbance induced by sleep deprivation

Gut microbiome is indispensable for maintaining normal brain function.Specifically,gut microbiota plays a causal role in sleep deprivation(SD)-induced cognitive impairment.In this study,neurobehavioral effects of the Bifidobacterium breve strain(CCFM1025)were assessed in sleep-deprived mice.CCFM1025 improved the body weight and food and water intake of the mice.It also alleviated SD-induced cognitive behavioural abnormalities(in the novel object recognition test),but did not show beneficial effects on mood-and spatial memory-related behaviours.CCFM1025 significantly altered the gut microbial composition and genome function.Key microbial metabolites that may regulate sleep function were also identified,such as isovaleric acid andγ-aminobutyric acid in the gut and purine metabolites in the serum.Those metabolites may participate in gutbrain communication by acting on the striatal melatonin system,for example to increase melatonin levels,and by regulating the expression of circadian clock genes such as those encoding the adenosine A2A receptor and period circadian regulator 1.Collectively,administration of probiotics alleviated cognitive impairment and circadian rhythm disturbance induced by SD via modulation of gut microbiome and its metabolites.These findings may help guide the treatment of insomnia or other sleep disorders via dietary strategies.

A novel strain of Levilactobacillus brevis PDD-5 isolated from salty vegetables has beneficial effects on hyperuricemia through anti-inflammation and improvement of kidney damage

Hyperuricemia is a metabolic disorder caused by abnormal purine metabolism,resulting in abnormally high serum uric acid.In this study,a novel Levilactobacillus brevis PDD-5 isolated from salty vegetables was verified with the function of alleviating hyperuricemia.The relevant effects of L.brevis PDD-5 in lowering uric acid were analyzed by in vitro and in vivo experiments.The results showed that the L.brevis PDD-5 has(68.86±15.46)%of inosine uptake capacity and(95.75±3.30)%of guanosine uptake capacity in vitro.Oral administration of L.brevis PDD-5 to hyperuricemia rats reduced uric acid,creatinine,and urea nitrogen in serum,as well as decreased inosine and guanosine levels in the intestinal contents of rats.Analysis of relevant markers in the kidney by ELISA kits revealed that L.brevis PDD-5 alleviated oxidative stress and inflammation.Moreover,the gene expression of uric acid transporter 1(URAT1)and glucose transporter 9(GLUT9)was down-regulated,and the gene expression of organic anion transporter 1(OAT1)was up-regulated after treatment with L.brevis PDD-5.Western blot analysis showed that L.brevis PDD-5 alleviated hyperuricemia-induced kidney injury through the NLRP3 pathway.The se findings suggest that L.brevis PDD-5 can lower uric acid,repair kidney damage,and also has the potential to prevent uric acid nephropathy.

Purines Change at Acupoints along the Pericardium Meridian in Healthy and Myocardial Ischemic Rats

Objective: To quantify the purine concentrations of the acupoints along the pericardium and nonpericardium meridians under healthy and myocardial ischemia conditions to investigate the relationship between acupoint purine change and body functional status in rats.Methods: A total of 70 rats underwent an operation for myocardial ischemia,while 40 of them survived.They were randomly assigned to the following 5 subgroups:Neiguan(PC 6),Quze(PC 3),Tianquan(PC 2),Quchi(LI 11),and Jianyu(LI 15).Simultaneously,another40 healthy rats were also randomized into the same 5 subgroups as the control group.The tissue ?uids at the acupoints were collected by microdialysis for 30 min.Subsequently,the concentration of adenosine triphosphate(ATP),adenosine diphosphate(ADP),adenosine monophosphate(AMP),and adenosine(ADO) were quanti?ed using the high-performance liquid chromatography method.Results: Compared with the healthy group,the ADO at PC 6(P=0.012),PC 3(P=0.038),PC 2(P=0.024),and LI 15(P=0.042) obviously increased in the model group,while no signi?cant difference was observed at LI 11(P=0.201).However,ATP,ADP,and AMP manifested no significant changes in these areas,except for ATP at LI 15(P=0.036).Conclusions: Myocardial ischemia could induce an increase in ADO at acupoints of the upper arm and shoulder area,suggesting that the body functional status could affect the responsiveness of acupoints.The status of these acupoints could be pathogenically activated by disease,and distribution following some speci?c courses.

Synthesis of C8-alkyl-substituted purine analogues by direct alkylation of 8-H purines with tetrahydrofuran catalyzed by CoCl2·6H2O

C8-Alkyl-substituted purine analogues were synthesized through direct alkylation of 8-H purine with tetrahydrofuran in the presence of Co catalyst in one step. The reactions gave a series of novel C8-oxygen heterocyclic alkyl purine compounds in good yields under mild reaction conditions by the readily available alkylating reagents, providing a complementary route to the classical coupling reactions for the synthesis of C8-alkyl-substituted purine analogues.

Molecular approaches for spinal cord injury treatment

Injuries to the spinal cord result in permanent disabilities that limit daily life activities.The main reasons for these poor outcomes are the limited regenerative capacity of central neurons and the inhibitory milieu that is established upon traumatic injuries.Despite decades of research,there is still no efficient treatment for spinal cord injury.Many strategies are tested in preclinical studies that focus on ameliorating the functional outcomes after spinal cord injury.Among these,molecular compounds are currently being used for neurological recovery,with promising results.These molecules target the axon collapsed growth cone,the inhibitory microenvironment,the survival of neurons and glial cells,and the re-establishment of lost connections.In this review we focused on molecules that are being used,either in preclinical or clinical studies,to treat spinal cord injuries,such as drugs,growth and neurotrophic factors,enzymes,and purines.The mechanisms of action of these molecules are discussed,considering traumatic spinal cord injury in rodents and humans.

Combinatorial co-expression of xanthine dehydrogenase and chaperone XdhC from Acinetobacter baumannii and Rhodobacter capsulatus and their applications in decreasing purine content in food

This study investigated the combinatorial expression of xanthine dehydrogenase(XDH)and chaperone XdhC from Acinetobacter baumannii and Rhodobacter capsulatus and their applications in decreasing purine content in the beer,beef and yeast.Naturally occurring xdhABC gene clusters of A.baumannii CICC 10254 and R.capsulatus CGMCC 1.3366 as well as two refactored clusters constructed by exchanging their xdhC genes were overexpressed in Escherichia coli and purified to near homogeneity.RcXDH chaperoned by AbXdhC showed nearly the same catalytic performance as that by RcXdhC,except for the decreased substrate affinity.While the AbXDH co-expressed with RcXdhC displayed enhanced acidic adaptation but weakened catalytic activity.All the XDHs degraded purines in beer,beef and yeast extract effectively,indicating potential applications in low-purine foods to prevent hyperuricemia and gout.The study also presents a method for exploiting the better chaperone XdhC and novel XDHs by functional complement activity using existing XdhCs such as RcXdhC.

Integrating UHPLC-MS/MS quantitative analysis and exogenous purine supplementation to elucidate the antidepressant mechanism of Chaigui granules by regulating purine metabolism

Chaigui granules(CG)are a compound composed of six herbal medicines with significant antidepressant effects.However,the antidepressant mechanism of CG remains unclear.In the present study,we attempted to elucidate the antidepressant mechanism of CG by regulating purine metabolism and purinergic signaling.First,the regulatory effect of CG on purine metabolites in the prefrontal cortex(PFC)of chronic unpredictable mild stress(CUMS)rats was analyzed by ultra high-performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS)targeted quantitative analysis.Meanwhile,purinergic receptors(P2X7 receptor(P2X7R),A1 receptor(A1R)and A2A receptor(A2AR))and signaling pathways(nod-like receptor protein 3(NLRP3)inflammasome pathway and cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)pathway)associated with purine metabolism were analyzed by western blotting and enzyme-linked immunosorbent assay(ELISA).Besides,antidepressant mechanism of CG by modulating purine metabolites to activate purinergic receptors and related signaling pathways was dissected by exogenous supplementation of purine metabolites and antagonism of purinergic receptors in vitro.An in vivo study showed that the decrease in xanthine and the increase in four purine nucleosides were closely related to the antidepressant effects of CG.Additionally,purinergic receptors(P2X7R,A1R and A2AR)and related signaling pathways(NLRP3 inflammasome pathway and cAMP-PKA pathway)were also significantly regulated by CG.The results of exogenous supplementation of purine metabolites and antagonism of purinergic receptors showed that excessive accumulation of xanthine led to activation of the P2X7R-NLRP3 inflammasome pathway,and the reduction of adenosine and inosine inhibited the A1R-cAMP-PKA pathway,which was significantly ameliorated by CG.Overall,CG could promote neuroprotection and ultimately play an antidepressant role by inhibiting the xanthine-P2X7R-NLRP3 inflammasome pathway and activating the adenosine/inosine-A1R-cAMP-PKA pathway.

Separation of Purine and Its Derivatives by Capillary Zone Electrophoresis

The separation of a group of 17 purine and its derivatives by capillary zone electrophoresis is presented. A systematic approach was used to study the effect of pH, buffer type, organic modifiers, applied potential, sodium dodecyl sulfate (SDS) and cyclodextrins on the separation of these purine derivatives. An ideal condition was found for their separation, which was 30 mmol/L sodium borate buffer (pH 9–9.5), 10%(V/V) methanol buffer modifier and 20 kV. Under this condition, the 17 purine derivatives were baseline separated and the linear correlation coefficient for adenine, uric acid and 2-thioxanthine was 0. 99 over two orders of magnitude. The variation of peak areas was less than 4. 6%(n=5) and that of migration times was in the range of 0%–3%, while the samples were injected hydrodynamically at a height of 15 cm and an injection time of 8–10 s. In addition, alcohol, 1-propanol, 1-butanol and acetonitrile were also effective additives in the separation. However, SDS and various β-cyclodextrin (β-CDs) were found to do no good to their separation.

Evolutionary impacts of purine metabolism genes on mammalian oxidative stress adaptation

Many mammals risk damage from oxidative stress stemming from frequent dives(i.e., cycles of ischemia/reperfusion and hypoxia/reoxygenation),high altitude and subterranean environments, or powered flight. Purine metabolism is an essential response to oxidative stress, and an imbalance between purine salvage and de novo biosynthesis pathways can generate damaging reactive oxygen species(ROS). Here, we examined the evolution of 117 purine metabolism-related genes to explore the accompanying molecular mechanisms of enhanced purine metabolism in mammals under high oxidative stress. We found that positively selected genes,convergent changes, and nonparallel amino acid substitutions are possibly associated with adaptation to oxidative stress in mammals. In particular, the evolution of convergent genes with c AMP and c GMP regulation roles may protect mammals from oxidative damage. Additionally, 32 genes were identified as under positive selection in cetaceans, including key purine salvage enzymes(i.e., HPRT1), suggesting improved re-utilization of non-recyclable purines avoid hypoxanthine accumulation and reduce oxidative stress. Most intriguingly, we found that six unique substitutions in cetacean xanthine dehydrogenase(XDH), an enzyme that regulates the generation of the ROS precursor xanthine oxidase(XO) during ischemic/hypoxic conditions, show enhanced enzyme activity and thermal stability and diminished XO conversion activity. These functional adaptations are likely beneficial for cetaceans by reducing radical oxygen species production during diving. In summary, our findings offer insights into the molecular and functional evolution of purine metabolism genes in mammalian oxidative stress adaptations.

Purine nucleotides and their metabolites in patients with type 1 and 2 diabetes mellitus

We measured the erythrocyte levels of principal nucleotides (ATP, ADP, AMP, GTP, GDP, GMP, IMP), nucleosides (Ado, Guo, Ino) and Hyp with HPLC. Purine concentrations were determined in the erythrocytes of 36 type 1 and 40 type 2 diabetic patients. The increased dephosphorylation of adenine and guanine nucleotides, indicated by increased Ado, Ino, Guo and Hyp concentrations as the products of purine nucleotide degradation, suggests serious energy metabolism disruptions in diabetes. An increase in AMP, GMP, IMP concentrations, as well as a decrease in AEC and GEC values, points to significant alterations in erythrocyte purine nucleotide concentration.

A rice XANTHINE DEHYDROGENASE gene regulates leaf senescence and response to abiotic stresses

Xanthine dehydrogenase, a member of the molybdenum enzyme family, participates in purine metabolism and catalyzes the generation of ureides from xanthine and hypoxanthine. However, the mechanisms by which xanthine dehydrogenase affects rice growth and development are poorly understood. In the present study, we identified a mutant with early leaf senescence and reduced tillering that we named early senescence and less-tillering 1(esl1). Map-based cloning revealed that ESL1 encodes a xanthine dehydrogenase, and it was expressed in all tissues. Chlorophyll content was reduced and chloroplast maldevelopment was severe in the esl1 mutant. Mutation of ESL1 led to decreases in allantoin, allantoate, and ABA contents. Further analysis revealed that the accumulation of reactive oxygen species in esl1 resulted in decreased photosynthesis and impaired chloroplast development, along with increased sensitivity to abscisic acid and abiotic stresses. Ttranscriptome analysis showed that the ESL1 mutation altered the expression of genes involved in the photosynthesis process and reactive oxygen species metabolism.Our results suggest that ESL1 is involved in purine metabolism and the induction of leaf senescence.These findings reveal novel molecular mechanisms of ESL1 gene-mediated plant growth and leaf senescence.

ANALYSIS OF N-METHYL-N-NITROSOUREA-INDUCED MUTATIONS IN A SHUTTLE VECTOR PLASMID PROPAGATED IN MOUSE O^6-METHYLGUANINE-DNA METHYLTRANSFERASE-DEFICIENT CELLS IN COMPARISON WITH PROFICIENT CELLS.

To investigate a contribution of O-methylguanineto mutagenesis in mouse cells,we constructed ashuttle vector plasmid,pYZ289,from a part ofpZ189 plasmid and polyoma virus DNA.The plasmidcontains a supF gene as a marker of mutation andcan replicate in both E.coli and mouse cells.ThepYZ289 treated with N-methyl-N-nitrosourea (MNU)

The Stimulatory Effects of the Antimicrobial Agents Bavistin, Cefotaxime and Kanamycin on <i>In Vitro</i>Plant Regeneration of <i>Centella asiatica</i>(L.)—An Important Antijaundice Medicinal Plant

Antimicrobial agents such as bavistin, cefotaxime and kanamycin were evaluated for their effects on the rapid shoot regeneration from nodal explants of Centella asiatica (L.). Filter sterilized bavistin (250 mg/L) was augmented alone and in combination with cytokinins such as BAP and TDZ into the media to trace the effect on regeneration. On this basis, the potential use of bavistin (150 mg/L) along with BAP (2.0 mg/L) was evaluated which showed the maximum shoot number (6.6) and shoot length (4.4 cm) respectively. Cefotaxime at the concentration of 100 μM/L was found to be effective to obtain the maximum shoot number formation (5.8) with the regeneration frequency (90%). Kanamycin at the concentration of 80 μM/L induced maximum shoot regeneration (5.12). Kanamycin at 100 μM/L or at higher concentrations reduced the shoot regeneration. The best rooting response was noticed when in vitro regenerated microshoots were transferred to the rooting medim which was supplemented with IBA (2.0 mg/L). This combination generates 90% of regeneration frequency and maximum number of roots per shoot (14.2) and high root length (4.2 cm). The rooted plants were acclimatized and transferred to field for survivalance. The addition of antibiotics was found to be more effective and safer for using since their effects on regeneration were found to be negligible.

Four New 6-Oxy Purine Alkaloids from the South China Sea Sponge,Haliclona cymaeformis

In this study, the chemical analysis of the marine sponge spieces, Haliclona cymaeformis, collected from the South China Sea was carried out, Two pairs of regioisomers of alkyl substitutional 6-oxy purine alkaloids(1a/1b and 2a/2b) were isolated. All of them possess two structural moieties, a 6-oxy purine nucleus and a pentan-2-one or hexan-2-one alkyl chain. Among them, 1a and 2a are the major N-9-substitutional regioisomers, and 1b and 2b are the minor N-7-substitutional regioisomers.

NMR STUDIES ON THE WEAK INTERACTION OF FOUR-MONONUCLEOTIDES WITH TRYPTAMIDE AND LEUCINAMIDE

NMR spectroscopy analysis was introduced to investigate the weak interaction of four 5'-mononucleotides with tryptamide and leucinamide. Assuming a 1∶1 complex formation, the association constants were determined by nonlinear regression analysis. The association affinity of 4 mononucleotides with tryptamide decreased in the order: G>A>C>U, indicating that purine bases stack more strongly with tryptamide than pyrimidine bases. But with leucinamide the affinity decreases in the order: G>C>A=U, suggesting an amino acid-anticodonic preferential interaction. The implication of the present data to the origin of genetic code was briefly discussed.

Silencing of Os XDH reveals the role of purine metabolism in dark tolerance in rice seedlings

Xanthine dehydrogenase(XDH) is a crucial enzyme involved in purine metabolism. To evaluate the effect of XDH deficiency on rice growth during dark treatment, wild type(WT) Nipponbare(Oryza sativa L.) and two independent transgenic lines with severe RNAi suppression(xdh3 and xdh4) were used in the present experiment. Under normal growth conditions, chlorophyll levels and biomass were indistinguishable between WT and the two RNAi transgenic lines, but XDH enzyme activity and ureide levels were suppressed in XDH RNAi transgenic lines. When XDH RNAi transgenic lines were subjected to dark treatment, chlorophyll content and biomass were significantly decreased, while O~–· production rate and malonaldehyde(MDA) were significantly increased compared to WT. The spraying test of exogenous allantoin raised chlorophyll content and biomass and reduced O~–· production rate and MDA in WT and both transgenic lines, and it also simultaneously reduced differences between RNAi and WT plants caused by XDH deficiency in growth potential and anti-oxidative capacity under dark treatment. These results suggested that fully functional purine metabolism plays an important role in reducing the sensitivity of rice seedlings to dark stress.