Measurement of the total purine contents and free nucleosides, nucleotides, and purine bases composition in Japanese anchovies (Engraulis japonicus) using high-performance liquid chromatography with UV detection.

Dietary purine restrictions are recommended for patients with hyperuricemia and gout. While measuring the purine contents of various foods in our laboratory using high-performance liquid chromatography (HPLC), we observed and reported changes in purine composition. In this study, we measured the total purine content and free purine of raw anchovies as well as after fermentation, using two methods by HPLC. Method 1 involved acid hydrolysis of all purines, such as nucleic acids and nucleotides, to form four corresponding purine bases. Method 2, which is a non-hydrolysis method, is used to measure the amount of free purines (nucleotide, nucleoside, purine base). As a result of method 1, after fermentation, adenine-related and hypoxanthine-related purines and the total purine levels decreased significantly. Regardless of being raw or fermented, each anchovy contained mainly hypoxanthine- and guanine-related purines. Among the hypoxanthine-related purines, the results of method 2 revealed that the raw anchovies contained a lot of inosine monophosphate (IMP), while after fermentation contained more inosine. In guanine-related and adenine-related purines, those nucleotides decreased by fermentation and nucleosides and bases increased. Measurements of free purines revealed that those reductions after fermentation observed in method 1 were derived from decreased nucleotides. These results indicate that purines are affected by the fermentation bacteria and period.

Monitoring of the influence of long-term oxidative stress and ischemia on the condition of kidneys using solid-phase microextraction chemical biopsy coupled with liquid chromatography-high-resolution mass spectrometry.

The limiting factor in conventional quality assessments of transplanted organs, namely the invasiveness of tissue sample collection, has prompted much research on the field of transplantology to focus on the development of alternative evaluation methods of organ quality. In the present project, we undertake the challenge to address the need for a new analytical solution for graft quality assessments by using a novel metabolomic diagnostic protocol based on low-invasive solid-phase microextraction. Solid-phase microextraction probes of ca. 0.2 mm coated with 4 mm long mixed-mode extraction phase were inserted into rabbit kidneys immediately following euthanasia and after 2, 4, 6, and 21 h of preservation. Liquid chromatography-mass spectrometry analysis of the extracts was performed with the use of a reversed phase column and a Q-Exactive Focus mass spectrometer operated in positive ionization mode. Statistical analysis of significantly changing compounds revealed metabolic profile changes in kidneys induced by ischemia and oxidative stress as a function of the duration of cold storage. The most pronounced alterations were reflected in levels of essential amino acids and purine nucleosides. Our findings demonstrate that the proposed approach may be successfully used to monitor changes in the metabolic profile of organs over time of preservation.

Investigation of 8-Aza-7-Deaza Purine Nucleoside Derivatives.

Glycosylation of 6-amino-4-methoxy-1H-pyrazolo[3,4-d]pyrimidine and its iodo- and bromo- analogues with the protected ribofuranose and 2'-deoxyribofuranose under different conditions resulted in the synthesis of N8- and N8-glycosylated purine nucleosides. Five key intermediate nucleosides, having 6-methoxy, 7-iodo, and 2-bromo groups, were further derivatized to 23 final 8-aza-7-deazapurine nucleoside derivatives. The structures of N8- and N8-glycosylated products were assigned based on UV and NMR spectra. HMBC analysis of 2D NMR spectra and X-ray crystallographic studies of the representative compounds unambiguously verified the connection of ribose ring to N8- or N8-position of the purine ring. The anticancer activity of these new compounds was evaluated.

Chromatographic behavior of new deazapurine ribonucleosides in hydrophilic interaction liquid chromatography.

The chromatographic behavior of new biogenic purine nucleosides in hydrophilic interaction liquid chromatography was examined on three different stationary phases, namely bare silica, and amide- and cyclofructan-based stationary phases. The effects of buffer concentration, pH and acetonitrile-to-aqueous-part ratio in the mobile phase on retention and peak shape were assessed. The retention coefficients and peak symmetry values substantially differed with respect to analytes´ structures, stationary phase properties and mobile phase composition. The bare silica column was unsuitable for these compounds under the chromatographic conditions tested due to very broad and asymmetrical peaks. Furthermore, the cyclofructan-based stationary phase provided almost Gaussian peak shapes of all deazapurine nucleosides under most conditions tested. Therefore, the cyclofructan-based stationary phase is the most suitable choice for the chromatographic analysis of nucleosides.

Hepatic xanthine oxidase activity and purine nucleosides levels as physiological mediators to analyze a subcutaneous treatment with (PhSe)2 in mice infected by Toxoplasma gondii.

The aim of this study was to evaluate the levels of purine nucleosides and xanthine oxidase (XO) activity in the liver of mice chronically infected by Toxoplasma gondii and treated with diphenyl diselenide (PhSe)2. For this experiment, forty Swiss mice were used. Twenty animals were orally infected by approximately 50 bradizoites of a cystogenic ME-49 strain of T. gondii, and the same number of uninfected mice was used as a control group. Ten infected and ten uninfected mice were subcutaneously treated twice (days 1 and 20 post-infection (PI)) with 5 µmol kg-1 of (PhSe)2. On day 30 PI, liver samples were collected to measure the levels of hypoxanthine (HYPO), xanthine (XAN), uric acid (UA), and XO activity. Infected animals showed increased (P < 0.05) levels of hepatic XAN and UA, as well as XO activity compared to uninfected animals. The use of (PhSe)2 in healthy mice increased the levels of all nucleosides, but decreased XO activity compared to healthy untreated animals. The group of infected and treated animals showed increased XAN and UA levels, and XO activity compared to the healthy control group, however infected and treated mice showed a decrease in the XO activity compared to the infected untreated group. We conclude that chronic infection caused by T. gondii can induce hepatic changes, such as increased UA levels and XO activity, that can increase the pro-oxidative profile. The (PhSe)2 treatment of healthy animals altered the levels of nucleosides, possibly due to low XO activity that decreased nucleoside degradation. Finally, (PhSe)2 treatment decreased XO activity in the infected group and increased nucleoside levels; however it was unable to reduce the UA levels found during the infection.

Effects of purine nucleotide administration on purine nucleotide metabolism in brains of heroin-dependent rats

ABSTRACT Heroin is known to enhance catabolism and inhibit anabolism of purine nucleotides, leading to purine nucleotide deficiencies in rat brains. Here, we determined the effect of exogenous purine nucleotide administration on purine nucleotide metabolism in the brains of heroin-dependent rats. Heroin was administrated in increasing doses for 9 consecutive days to induce addiction, and the biochemical changes associated with heroin and purine nucleotide administration were compared among the treated groups. HPLC was performed to detect the absolute concentrations of purine nucleotides in the rat brain cortices. The enzymatic activities of adenosine deaminase (ADA) and xanthine oxidase (XO) in the treated rat cortices were analyzed, and qRT-PCR was performed to determine the relative expression of ADA, XO, adenine phosphoribosyl transferase (APRT), hypoxanthine-guaninephosphoribosyl transferase (HGPRT), and adenosine kinase (AK). Heroin increased the enzymatic activity of ADA and XO, and up-regulated the transcription of ADA and XO. Alternatively, heroin decreased the transcription of AK, APRT, and HGPRT in the rat cortices. Furthermore, purine nucleotide administration alleviated the effect of heroin on purine nucleotide content, activity of essential purine nucleotide metabolic enzymes, and transcript levels of these genes. Our findings therefore represent a novel, putative approach to the treatment of heroin addiction.

Determination of pyrimidine and purine bases by reversed-phase capillary liquid chromatography with at-line surface-enhanced Raman spectroscopic detection employing a novel SERS substrate based on ZnS/CdSe silver-quantum dots.

We have developed a new SERS substrate based on the reduction of silver nitrate in the presence of ZnS-capped CdSe quantum dots. This substrate showed higher sensitivities as compared to a hydroxylamine-reduced silver sol. On the basis of this new substrate, at-line SERS detection was coupled with capillary liquid chromatography (cap-LC) for the separation and selective determination of pyrimidine and purine bases. For this purpose, wells of a dedicated microtiter plate were loaded with 20 µL of the SERS substrate and placed on an automated x,y translation stage. A flow-through microdispenser capable of ejecting 50 pL droplets, at a frequency 100 Hz, was used as the interface to connect the cap-LC system to the wells loaded with SERS substrate. A detailed study of the dependence of both the separation and the surface-enhanced Raman spectra of each base on the pH was performed to optimize the system for maximum sensitivity and selectivity. Highly satisfactory analytical figures of merit were obtained for the six investigated bases (cytosine, xanthine, hypoxanthine, guanine, thymine, and adenine) with detection limits ranging between 0.2 and 0.3 ng injected on the capillary LC column, and the precisions were in the range of 3.0-6.3%.

8-(p-CF3-cinnamyl)-modified purine nucleosides as promising fluorescent probes.

Natural nucleotides are not useful as fluorescent probes because of their low quantum yields. Therefore, a common methodology for the detection of RNA and DNA is the application of extrinsic fluorescent dyes coupled to bases in oligonucleotides. To overcome the many limitations from which fluorescent nucleotide-dye conjugates suffer, we have developed novel purine nucleosides with intrinsic fluorescence to be incorporated into oligonucleotide probes. For this purpose we synthesized adenosine and guanosine fluorescent analogues 7-25, conjugated at the C8 position with aryl/heteroaryl moieties either directly, or via alkenyl/alkynyl linkers. Directly conjugated analogues 7-14, exhibited high quantum yields, φ >0.1, and short λ(em) (<385 nm). Alkynyl conjugated analogues 22-25, exhibited low quantum yields, φ <0.075, and λ(em)<385 nm. The alkenyl conjugated analogues 15-21, exhibited λ(em) 408-459 nm. While analogues 15,16, and 20 bearing an EDG on the aryl moiety, exhibited φ <0.02, analogues 17, and 21 with EWG on the aryl moiety, exhibited extremely high quantum yields, φ ≈ 0.8, suggesting better intramolecular charge transfer. We determined the conformation of selected adenosine analogues. Directly conjugated analogue 8 and alkynyl conjugated analogue 22, adapted the syn conformation, whereas alkenyl conjugated analogue 15 adapted the anti conformation. Based on the long emission wavelengths, high quantum yields, anti conformation and base-paring compatibility, we suggest analogues 17 and 21 for further development as fluorescent probes for the sensitive detection of genetic material.

Breast fine-needle aspiration malondialdehyde deoxyguanosine adduct in breast cancer.

This study has analysed the generation of 3-(2-deoxy-ß-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine adduct [M1dG], a biomarker of oxidative stress and lipid peroxidation, in breast fine-needle aspirate samples of 22 patients with breast cancer, at different clinical stages, in respect to 13 controls. The multivariate analysis show that M(1)dG adduct was higher in cases than in controls (Mean Ratio (MR) = 5.26, 95% CI = 3.16-8.77). Increased M1dG was observed in women with a tumour grade 3 and a pathological diameter 2 (MR = 7.61, 95% CI = 3.91-14.80 and MR = 5.75, 95% CI = 3.13-10.59, respectively). A trend with increasing tumour grade and pathological diameter was present (MR = 1.98, 95% CI = 1.57-2.50 and MR = 2.44, 95% CI = 1.71-3.48, respectively). Not significant effects of age and smoking habit were found (MR = 1.58, 95% CI = 0.92-2.72 and MR = 1.68, 95% CI 0.88-3.20, respectively). An increment over the background frequency of M1dG can contribute to breast cancer development. Increasing severity of breast tumour can influence DNA damage level.

Nucleoside composition of Heloderma venoms.

Venoms of Heloderma horridum and Heloderma suspectum were analyzed for the possible presence of purine and pyrimidine nucleosides. Adenosine, cytidine, guanosine, hypoxanthine, inosine, and uridine were found in mug quantities. These amounts are much smaller than those seen in many elapid or viperine venoms, but greater and more varied than those found in crotaline venoms. While their contribution to the hypotension induced by Heloderma venoms may be minor, venom nucleosides nonetheless act in concert with kallikreins/hemorrhagins, alkaline phosphomonoesterase, 5'-nucleotidase, helodermin, helospectins, helothermine, and serotonin. The use of nucleosides as toxins is therefore a generalized squamate strategy, rather than the exclusive province of snakes. Both Heloderma venoms were found to be devoid of NADase and phosphodiesterase activities. Enzymes to release endogenous purines in the prey, are not significant components of Heloderma venoms.

Biophysical stability and enzymatic recognition of oxanine in DNA.

Oxanine (Oxa), which is one of the major products generated from guanine by nitrosative oxidation and is as long-lived as Gua in DNA, has been thought to be one of the major causes for NO-induced DNA damage. In the present study, using several synthetic Oxa-containing oligodeoxynucleotides, biophysical stability and enzymatic recognition of Oxa was investigated in DNA strands. It was found that Oxa did not mediate marked distortion in the whole DNA structure although Oxa pairing with 4 normal bases decreased thermal stability of the DNA duplexes compared to Gua:Cyt base pair. Regarding the responses of the DNA-relevant enzymes to Oxa, it was determined that Oxa was recognized as Gua except that DNA polymerases incorporated Thy as well as Cyt opposite Oxa. These results imply that Oxa tends to behave as a kind of naturally occurring base, Gua and therefore, would be involved in the genotoxic and cytotoxic threats of NO in cellular system.

Determination of purine and pyrimidine bases in natural and cultured Cordyceps using optimum acid hydrolysis followed by high performance liquid chromatography.

A method based on optimum acid hydrolysis followed by high-performance liquid chromatography (HPLC) with diode array detection was developed for quantitative determination of bio-available nucleosides, present as purine and pyrimidine bases including adenine, cytosine, guanine, hypoxanthine, thymine and uracil, in natural and cultured Cordyceps. It was found that the optimum conditions was hydrolyzing Cordyceps sample in eight folds of pure commercial perchloric acid for 1h at 95-100 degrees C. The determination was achieved by using a Zorbax SB-AQ analytical column (250 mm x 4.6 mm i.d., 5 microm) at gradient elution with diode-array detection. All calibration curves showed good linearity (r2>0.999) within test ranges. The developed method showed good repeatability for the quantification of six investigated nucleobases in Cordyceps with intra- and inter-day variations of less than 9.0 and 9.1%, respectively. The validated method was successfully applied to quantify bio-available nucleosides in natural and cultured Cordyceps, which is helpful to control their quality.

Ribavirin: analytical determinations since the origin until today.

Ribavirin (RV) (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), is a synthetic purine nucleoside analog with a broad spectrum of antiviral activity. To better understand the mechanism of action of RV, as well as its pharmacokinetic characteristics, an assay that can allow specific, sensitive, and accurate measurement of RV in biologic samples is critical. In this way, diverse analytical methods have been established. In this work, we have recompiled these methods with the aim to present the different options for the RV determination.

Taxonomic distribution and quantitative analysis of free purine and pyrimidine nucleosides in snake venoms.

The nucleoside content of 32 elapid and viperid venoms was examined. Free purines, principally adenosine (ADO), inosine (INO), and guanosine (GUA), comprised as much as 8.7% of the solid components of some venoms. Thus, purines are far more abundant in some venoms than many proteinaceous toxins. Hypoxanthine (HYP) was found in about half of elapid and viperine venoms, in which it is a relatively minor constituent (<60 microg/g). Adenosine monophosphate (AMP) was tentatively identified in only three elapid and two viperid venoms. The pyrimidines, uridine (URI) and cytidine (CYT), were also found in most elapid and viperine venoms. In most of these, the amount of uridine was substantially greater than that of cytidine. Thymidine (THY) was not found in any venom, indicating that DNA from disintegration of glandular cells is not the source of venom nucleosides. In contrast to elapid and viperine venoms, most crotaline venoms are devoid of free nucleosides. Elapid and viperine venoms also contained other minor, low molecular weight constituents that could not be positively identified. Some had spectra identical to those of adenosine, nicotinamide adenine dinucleotide (NAD), inosine, xanthosine (XAN), and guanosine, while others had unique spectra. There is no apparent correlation between quantities of venom nucleosides and literature values for the three dominant venom enzymes that release endogenous nucleosides, 5'-nucleotidase (5NUC), phosphodiesterase (PDE), and alkaline phosphomonoesterase (PME).

Azoles as Suzuki cross-coupling leaving groups: syntheses of 6-arylpurine 2'-deoxynucleosides and nucleosides from 6-(imidazol-1-yl)- and 6-(1,2,4-triazol-4-yl)purine derivatives.

[reaction: see text] 6-(Imidazol-1-yl)-, 6-(benzimidazol-1-yl)-, and 6-(1,2,4-triazol-4-yl)purine nucleosides undergo a nickel-mediated C-C cross-coupling of azole-substituted purine derivatives with arylboronic acids to give good yields of 6-arylpurine nucleosides.

Mass spectrometric investigation of N-sulfonylated purine nucleic bases and nucleosides.

The gas/phase behaviour of N-sulfonylated purine nucleic bases and nucleosides towards electron impact (EI) and matrix-assisted laser desorption/ionization (MALDI) occurring in a ion trap of a Fourier transform ion cyclotron resonance mass spectrometer is investigated. The influence of the storage time on the protonated molecule ([M+H](+)) abundance under EI conditions confirms that the formation of these ions proceeds through ion/molecule reactions. Using stored-waveform inverse Fourier transform (SWIFT) selective isolation of M(+.) or H(3)O(+), self-chemical ionization, M(+.)/M, and chemical ionization, H(3)O(+)/M, are detected. Investigation of specific EI expulsion of SO(2), SO(2)H and/or SO(2)H(2) from M(+.) and/or [M+H](+) shows that oxygen protonation in bond;SO(2)bond; proceeds faster than nitrogen protonation. Expulsion of SO(2) from molecular ions is not observed in MALDI mass spectra of nucleosides.

Nucleic acid related compounds. 118. Nonaqueous diazotization of aminopurine derivatives. Convenient access to 6-halo- and 2,6-dihalopurine nucleosides and 2'-deoxynucleosides with acyl or silyl halides.

Treatment of 9-(2,3,5-tri-O-acetyl-beta-d-ribofuranosyl)-2-amino-6-chloropurine (1) with TMS-Cl and benzyltriethylammonium nitrite (BTEA-NO2) in dichloromethane gave the crystalline 2,6-dichloropurine nucleoside 2, and acetyl chloride/BTEA-NO2 was equally effective ( approximately 85%, without chromatography). TMS-Br/tert-butyl nitrite/dibromomethane gave crystalline 2-bromo-6-chloro analogue 3 (85%). (Chloro or bromo)-dediazoniation of 3',5'-di-O-acetyl-2'-deoxyadenosine (4) gave the 6-[chloro (5, 63%) or bromo (6, 80%)]purine deoxynucleosides, and 2',3',5'-tri-O-acetyladenosine (8) was converted into the 6-chloropurine nucleoside 9 (71%).

On-line fourier transform infrared detection in capillary electrophoresis.

The coupling of Fourier transform infrared (FT-IR) spectroscopy as a new on-line detection principle in capillary electrophoresis (CE) is presented. To overcome the problem of total IR absorption by the fused-silica capillaries that are normally employed in CE separations, a micromachined IR-transparent flow cell was constructed. The cell consists of two IR-transparent CaF2 plates separated by a polymer coating and a titanium layer producing an IR detection window, 150 microm wide and 2 mm long, with a path length of 15 microm. The IR beam was focused on the detection window using an off-axis parabolic mirror in an optical device (made in-house) attached to an external optical port of the spectrometer. The connections between the fused-silica capillaries and the flow cell were made by a small O-ring of UV-curing epoxy adhesive on the sharply cut ends of the capillaries, allowing the capillaries to be easily replaced. Aqueous solutions comprising mixtures of adenosine, guanosine, and adenosine monophosphate were used to test the system's performance. Conventional on-line UV detection was employed to obtain reference measurements of analytes after the IR detection flow cell. The limit of FT-IR detection for all analytes (in absolute amounts) was in the nano- to picogram range corresponding to concentrations in the low-millimolar range.

A new stable isotope method enables the simultaneous measurement of nucleic acid and protein synthesis in vivo in mice.

We developed a method based on the incorporation of 13C2-units derived from [U-13C]glycine that allows the simultaneous quantification of tissue protein and RNA synthesis in vivo. Two groups of 26 mice were fed diets containing a high (HF diet) or a low quantity of fiber (LF diet). After 6 d, [U13C]glycine was added to the diet and groups of four mice were killed after 2, 4, 6, 8, 12 and 24 h. Hepatic and intestinal mucosal free and RNA-bound purine nucleosides were extracted and enzymically degraded to allantoin. Allantoin was degraded to glyoxylate, which was then reductively aminated to glycine, which contains the two 13C-atoms incorporated via de novo synthesis. Ingestion of the HF diet was associated with significantly (P < 0.05) higher rates of total RNA synthesis in both the liver ( HF diet, 29%/d; LF diet, 21%/d) and mucosa (HF diet, 27%/d; LF diet, 17 %/d). The mean rates of RNA synthesis in each tissue were significantly (P < 0.01) lower than the respective rates of protein synthesis (liver, 67%/d; mucosa, 74%/d). The isotopic enrichment of the free purine nucleotide pool increased rapidly and exponentially, but the steady-state value was substantially (P < 0. 001) lower than that of the RNA-bound purines. The results suggest that the free nucleotide pool consists of two kinetically distinct compartments, one of which is small and has a rapid rate of turnover. This, we propose, acts as the RNA precursor pool. The other is large, has a low rate of turnover and, we believe, is the pool of adenosine triphosphate involved in cellular energetics.