Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 µg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-ß-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

[Separation and identification of impurities from intermediates of istradefylline].

Istradefylline is a novel selective adenosine A2A receptor antagonist that is used to treat Parkinson's disease and improve motor dysfunction in the early stage of this disease. During the synthesis of intermediate A1 (6-amino-1,3-diethyl-2,4-(1H,3H)-pyrimidinedione), at least two by-products were formed under alkaline or high-temperature conditions. In a previous study, one of the by-products in the synthesis of the intermediate was studied, and its structure was identified as (E)-N-ethyl-2-cyano-3-ethylamino-2-butene amide. In this study, we used high performance liquid chromatography (HPLC) to analyze another impurity formed during the synthesis of A1, and the following steps were executed: 0.4 g of intermediate was weighed and added to a 50 mL beaker, followed by the sequential addition of 8 mL water and 8 mL acetonitrile, and then, ultrasonic dissolution was performed. Finally, the solution was filtered through a 0.45-µm organic membrane and the test sample solution was obtained. We used the Agilent zorbax C18 chromatography column, with acetonitrile (A)/water(B) as the mobile phase under gradient elution ((tmin/A∶B)=t0/20∶80, t15/60∶40, t20-t50/90∶10). The detector wavelength is 268 nm. In order to separate the impurity from A1, we used a Ceres B preparative column, with acetonitrile-water (30/70, v/v) as the mobile phase. The flow rate was set at 30 mL/min, and the detection wavelength was 268 nm. The structure of the impurity was confirmed by high-resolution mass spectrometry (HRMS), one-dimensional nuclear magnetic resonance (NMR), and two-dimensional nuclear magnetic resonance (2D NMR), and characterized by single-crystal X-ray diffraction (XRD). In MS experiments, an electrospray ionization (ESI) source was used with positive ion scanning. In the NMR experiments, we used tetramethylsilane (TMS) as the internal standard and deuterated dimethyl sulfoxide (DMSO-d6) as the solvent to obtain the spectra. The results of preparative high performance liquid chromatography (Prep-HPLC) showed that good separation effect could be achieved by isocratic elution, and the impurity was perfectly separated. The1H-NMR spectral data are as follows:1H-NMR (600 MHz, DMSO): δ 1.01 (q, J=6.9 Hz, 3H), 1.02 (q, J=6.9 Hz, 3H), 1.07 (t, J=6.9 Hz, 3H), 3.04 (p, J=6.8 Hz, 2H), 3.74 (q, J=7.0 Hz, 2H), 3.94 (q, J=7.1 Hz, 2H), 5.85 (s, 1H). The 13C-NMR spectral data are summarized as follows: 13C-NMR (150 MHz, DMSO): δ13.9, 14.1, 15.9, 34.6, 34.9, 36.9, 81.9, 152.2, 153.3, 159.3, 162.0. The impurity was characterized by single-crystal XRD, and its spatial structure was further verified and determined as 1-(1,3-diethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-3-ethylurea. Based on the chemical structure of the impurity, we propose the following mechanism for the impurity: when A1 is synthesized under alkaline conditions or at high temperature, excessive diethylurea continues to undergo amidation with A1 to obtain this by-product. Although the formation mechanism of the impurity studied in this paper is different from that of the intermediate A1 impurity (E)-N-ethyl-2-cyano-3-ethylamino-2-butene amide, both the impurities are formed at high temperatures. Both will be accompanied by A1 in the subsequent reaction of istradefylline synthesis. The relationship between drug impurities and drug safety is a complex relationship that is affected by many factors. Generally, most impurities in drugs have potential biological activities, and some even interact with the drugs, thus affecting their efficacy and safety and inducing toxic effects. Therefore, to ensure the quality of istradefylline, it is necessary to control the impurity content during the production. The findings of this paper may provide guidelines for controlling the impurity content in istradefylline.

A Highly Sensitive Nonextraction-Assisted HPLC Method with Fluorescence Detection for Quantification of Duvelisib in Plasma Samples and its Application to Pharmacokinetic Study in Rats.

BACKGROUND: Duvelisib (DUV) is a new oral phosphoinositide-3-kinase (PI3K)-δ and PI3K-γ inhibitor. It has been recently granted an accelerated approval for treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). It is also effective in therapy of T-cell lymphoma, solid tumors, and non-Hodgkin's lymphoma. In literature, there is no method valid for quantitation of DUV in human plasma for its therapeutic monitoring and pharmacokinetic studies. PURPOSE: The purpose of this study is the establishment of a highly sensitive HPLC method with fluorescence detection for quantitation of DUV in plasma for its therapeutic monitoring and pharmacokinetic studies of DUV. METHODS: The resolution of DUV and the internal standard (IS) olaparib (OLA) was achieved on Nucleosil CN column, with a mobile phase composed of acetonitrile:water (25:75, v/v) at a flow rate of 1.7 mL min-1. The fluorescence of both DUV and OLA was detected at 410 nm after excitation at 280 nm. The method was validated according to the guidelines of bioanalytical method validation. RESULTS: The method was linear in the range of 5-100 ng mL-1, and its limit of detection (LOD) and limit of quantitation (LOQ) were 2.12 ng mL-1 and 7 ng mL-1, respectively. The precisions of the method were ≤ 8.26%, and its accuracies were ≥ 95.32%. All the other validation parameters were satisfactory. The proposed method was successfully employed to the investigation of the pharmacokinetic profile of DUV in rats following a 25 mg/kg single dose of oral administration. CONCLUSION: The method is characterized with high sensitivity, accuracy, simple sample pretreatment, rapidity, eco-friendly as it consumes low volumes of organic solvent in the mobile phase and has high analysis throughput as its run time was short (~ 10 min).

DBS Assay with LC-MS/MS for the Determination of Idelalisib, A Selective PI3K-δ Inhibitor in Mice Blood and Its Application to a Pharmacokinetic Study.

Idelalisib is a selective and second-generation PI3K-δ inhibitor, approved for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this paper, we present a fully validated dried blood spot (DBS) method for the quantitation of idelalisib from mice blood using an LC-MS/MS, which was operated under multiple reaction monitoring mode. To the punched DBS discs, acidified methanol enriched with internal standard (IS; larotrectinib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of idelalisib and the IS was achieved on an Atlantis dC18 column using a mixture of 10 mM ammonium formate:acetonitrile (25:75, v/v). The flow-rate and injection volume were 0.80 mL/min and 2.0 µL, respectively. Idelalisib and the IS were eluted at ~0.98 and 0.93 min, respectively and the total run time was 2.00 min. Idelalisib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 416.1→176.1 and 429.1→342.1, respectively was used for the quantitation. The calibration range was 1.01-4 797 ng/mL. No matrix effect and carry over were observed. Haematocrit did not influence DBS idelalisib concentrations. All the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mice pharmacokinetic study.

The Association between Purine-Rich Food Intake and Hyperuricemia: A Cross-Sectional Study in Chinese Adult Residents.

OBJECTIVE: To explore the correlation between purine-rich food intake and hyperuricemia in Chinese adult residents. METHOD: A cross-sectional study was conducted on the purine-rich food intake of Chinese adult residents based on the China Health and Nutrition Survey (CHNS) in 2009. The subjects were divided into hyperuricemia group and nonhyperuricemia group according to serum uric acid level, and the differences of the sociodemographic information (age, gender, and region), health status (weight status, blood pressure, blood sugar status), living habits (alcohol consumption, smoking status) and food intake (purine-rich food, other food) were compared between the two groups. Logistic regressions investigated the associations between the daily intake of purine-rich food (animal-derived food and legumes) and hyperuricemia. RESULTS: Eventually, 6813 subjects were included in our study, 1111 of them had hyperuricemia. The intake of seafood, legumes, red meat, and poultry all increased the risk of hyperuricemia (p < 0.05), while the intake of purine-rich fungi and purine-rich vegetables did not affect the occurrence of hyperuricemia. Animal-derived food was the main source of purine-rich food consumed by Chinese adult residents (140.67g/day), which had a great impact on hyperuricemia. Finally, after adjusting for gender, age, region, body mass index (BMI), alcohol consumption, hypertension, and refined grains intake, the risk of hyperuricemia increased by 2.40% and 1.10% for each increase of 10 g in animal-derived food intake (OR = 1.024, 95% CI: 1.018-1.030) and legumes intake (OR = 1.011, 95% CI: 1.003-1.019), respectively. CONCLUSION: The intake of animal-derived food and legumes were positively correlated with the occurrence of hyperuricemia. Controlling the intake of animal-derived food and legumes would be more beneficial to controlling the risk of hyperuricemia.

Time of flight mass spectrometry based in vitro and in vivo metabolite profiling of ribociclib and their toxicity prediction.

The metabolic investigation in the drug discovery process is an imperative aspect for selection of drug candidates with excellent therapeutic efficacy and safety profile. Ribociclib (RIBO), an orally active Cyclin dependent kinases inhibitor recently approved by USFDA for its clinical efficacy against human epithelial growth factor receptor negative and hormonal receptor positive advanced breast cancer. Although an in vitro metabolite identification study of RIBO is available in literature, no systematic metabolic investigation including detailed structural characterization and toxicity prediction of the metabolites generated in in vivo system is reported till date. Therefore, in this study, we focused on the characterization of its entire metabolites generated in in vitro as well as in vivo matrices. In vitro study includes incubation of RIBO in rat and human liver microsomes and human S9 fraction, while in vivo study was carried out using plasma, urine and faeces samples of male Sprague Dawley rats. A total of 22 metabolites were successfully separated on Agilent SB C18 (100 × 4.6 mm, 2.7µ) column using ammonium formate (pH 3.5) and acetonitrile as mobile phase. Metabolites were identified with the help of UHPLC-ESI-Q-TOF-MS/MS by accurate mass measurement. RIBO was found to be metabolised by N- dealkylation, sulphation, acetylation, oxidation, hydroxylation, carbonylation, dehydrogenation and by a combination of these reactions. The in silico toxicity profiling of all the metabolites was carried out with the help of ProTox-II software. Ten out of twenty two newly identified metabolites showed to have potential for possessing immunotoxicity. Novelty of this investigation can be justified by the unavailability of any previously published literature on complete in vitro and in vivo metabolite profiling of RIBO. Moreover, in silico toxicity of the metabolites were also not known till date.

Development of a novel and quick UPLC-MS/MS method for the pharmacokinetic analysis of duvelisib in beagle dogs.

Duvelisib, a new oral phosphoinositide-3-kinase (PI3K)-δ and PI3K-γ inhibitor, was recently approved in the USA as the therapeutic drug for patients with the diseases of relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In the present study of our research, a quick and simple bioanalytical method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique was fully explored and established for the quantification of plasma duvelisib concentrations from beagle dog in which gilteritinib was used as the internal standard (IS). After a simple and quick protein precipitation treated with acetonitrile, the chromatographic separation of the analyte was carried out on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm) conducted in a gradient elution procedure where acetonitrile (solvent A) and 0.1 % formic acid in water (solvent B) consisted as the mobile phase. The measurements of the analyte and IS were explored using a XEVO TQS triple quadrupole tandem mass spectrometer, which was comprised with electrospray ionization (ESI) source in positive ion mode. Selected reaction monitoring (SRM) mode was employed to detect the parent-to-daughter ion transitions as follows: m/z 416.88 → 281.88 for duvelisib, and m/z 553.09 → 436.01 for IS, respectively. The assay was successfully established in the calibration range from 0.5 to 3000 ng/mL for duvelisib, where the lower limit of quantification (LLOQ) was set at 0.5 ng/mL. The precisions of intra-day and inter-day for duvelisib were all below 12.6 %, and the accuracies were from -2.5% to 14.1%. Both matrix effect and mean recovery of the analyte and IS were all acceptable, and the analyte was stable during the assay and storage in dog plasma samples. The novel established bioanalytical method based on UPLC-MS/MS technique was effectively employed to the investigation of the pharmacokinetic profile of duvelisib in beagle dogs following a 1.34 mg/kg single dose of oral administration.

Metabolomics reveals the toxicological effects of polar compounds from frying palm oil.

Polar compounds from frying oils have been found to be harmful to health. However, the mechanisms underlying this phenomenon have largely remained elusive. In this study, mass spectrometry-based metabolomics was used to investigate the toxicological effects of polar compounds. The serum and hepatic metabolites from polar compound-treated mice were measured using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Multi-variate statistical analysis showed that a total of 36 serum metabolites and 18 hepatic metabolites were altered in the polar compound-treated mice as compared with that for normal diet-fed animals. These metabolic changes suggested novel alterations in lipid metabolism with the increase in phospholipids, fatty acids, and cholesterol and the decrease in choline, betaine and l-acetylcarnitine. The TCA cycle and carbohydrate, amino acid and purine metabolism were also impaired, with a significant elevation of d-glucose, d-maltose, ß-mannobiose, branched chain amino acids, aromatic amino acids, and uric acid and a decline in succinate, serine, aspartate, arginine and ornithine. Pearson correlation analysis demonstrated the strong correlations between specific metabolic alterations and the redox index. Our overall findings reveal that polar compounds may progressively cause lipid deposition, impaired energy metabolism and oxidative stress, resulting in toxicological effects on the mammalian health.

Plasma-treated carbon-fiber microelectrodes for improved purine detection with fast-scan cyclic voltammetry.

Here, we developed N2 and O2 plasma-treated carbon-fiber microelectrodes (CFME) for improved purine detection with fast-scan cyclic voltammetry (FSCV). Plasma treatment affects the topology and functionality of carbon which impacts the electrode-analyte interaction. CFME's are less sensitive to purines compared to catecholamines. Knowledge of how the electrode surface drives purine-electrode interaction would provide insight into methods to improve purine detection. Here, plasma-treated CFME's with N2 and O2 plasma was used to investigate the extent to which the surface functionality and topology affects purine detection and to improve purine sensing with FSCV. On average, O2 plasma increased the oxidative current for adenosine and ATP by 6.0 ± 1.2-fold and 6.4 ± 1.6-fold, and guanosine and GTP by 2.8 ± 0.47-fold and 5.8 ± 1.4-fold, respectively (n = 9). The O2 plasma increased the surface roughness and oxygen functionality. N2 plasma increased the oxidative current for adenosine and ATP by 1.5 ± 0.15-fold and 1.9 ± 0.23-fold, and guanosine and GTP by 1.4 ± 0.20-fold and 1.5 ± 0.20-fold, respectively (n = 11). N2 plasma increased the nitrogen functionality with minimal increases in roughness. Both plasma treatments impacted purines more than dopamine. Langmuir isotherms revealed that both plasma gases impact the theoretical surface coverage and adsorption strength of purines at the electrode. Overall, we show that purine detection is improved at surfaces with increased surface roughness, and oxygen and amine functionality. Plasma-treated CFMEs could be used in the future to study the analyte-electrode interaction of other neurochemicals.

Mycoplasma infection of cultured cells induces oxidative stress and attenuates cellular base excision repair activity.

Mycoplasma contamination is a major concern for in vitro cell culture models as its resistance to most antibiotics, which makes the prevention and treatment of infection challenging. Furthermore, numerous studies show that Mycoplasma infection alters a variety of cellular processes, in a wide range of cell lines. However, there is a lack of information pertaining to the effects of Mycoplasma infection on genomic stability. In this study, a dopaminergic neuronal cell line (BE-M17), a popular in vitro model for Parkinson's disease, was used to evaluate the effect of Mycoplasma infection on genomic instability, and base excision repair (BER) activity, using single cell gel electrophoresis (the comet assay). The results showed that Mycoplasma infection induced oxidative stress in the absence of an inflammatory response, with markedly increased levels of DNA damage [strand breaks/alkali-labile sites (SB/ALS), and oxidised purines], compared to uninfected cells. The source of the oxidative stress may have been increased ROS generation, or attenuation of cellular antioxidant capacity (or a combination of both). Uninfected cells initially repaired SB/ALS more rapidly than infected cells, although SB/ALS were fully repaired in both uninfected and infected cells 2 h after H2O2 challenge. However, while uninfected cells showed complete repair of oxidised purines within 24 h, for the infected cells, these were not fully repaired even after 30 h. In conclusion, this study showed that not only does Mycoplasma infection induce oxidative stress and DNA damage, but it also decreases the efficiency of the main pathway responsible for the repair of oxidatively damaged DNA i.e. BER. In this in vitro model, there is no mechanism for infection-induced inflammation, which could be a source of increased ROS production. Therefore, further studies are needed to evaluate how Mycoplasma infection causes oxidatively damaged DNA, and how it modulates cellular DNA repair.

Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells.

Purine metabolites have been implicated as clinically relevant biomarkers of worsening or improving Parkinson's disease (PD) progression. However, the identification of purine molecules as biomarkers in PD has largely been determined using non-targeted metabolomics analysis. The primary goal of this study was to develop an economical targeted metabolomics approach for the routine detection of purine molecules in biological samples. Specifically, this project utilized LC/MS/MS and LC/QTOF/MS to accurately quantify levels of six purine molecules in samples from cultured N2a murine neuroblastoma cells. The targeted metabolomics workflow was integrated with automated label-free digital microscopy, which enabled normalization of purine concentration per unit cell in the absence of fluorescent dyes. The established method offered significantly enhanced selectivity compared to previously published procedures. In addition, this study demonstrates that a simple, quantitative targeted metabolomics approach can be developed to identify and quantify purine metabolites in biological samples. We envision that this method could be broadly applicable to quantification of purine metabolites from other complex biological samples, such as cerebrospinal fluid or blood.

Organic and Peptidyl Constituents of Snake Venoms: The Picture Is Vastly More Complex Than We Imagined.

Small metabolites and peptides in 17 snake venoms (Elapidae, Viperinae, and Crotalinae), were quantified using liquid chromatography-mass spectrometry. Each venom contains >900 metabolites and peptides. Many small organic compounds are present at levels that are probably significant in prey envenomation, given that their known pharmacologies are consistent with snake envenomation strategies. Metabolites included purine nucleosides and their bases, neurotransmitters, neuromodulators, guanidino compounds, carboxylic acids, amines, mono- and disaccharides, and amino acids. Peptides of 2⁻15 amino acids are also present in significant quantities, particularly in crotaline and viperine venoms. Some constituents are specific to individual taxa, while others are broadly distributed. Some of the latter appear to support high anabolic activity in the gland, rather than having toxic functions. Overall, the most abundant organic metabolite was citric acid, owing to its predominance in viperine and crotaline venoms, where it chelates divalent cations to prevent venom degradation by venom metalloproteases and damage to glandular tissue by phospholipases. However, in terms of their concentrations in individual venoms, adenosine, adenine, were most abundant, owing to their high titers in Dendroaspis polylepis venom, although hypoxanthine, guanosine, inosine, and guanine all numbered among the 50 most abundant organic constituents. A purine not previously reported in venoms, ethyl adenosine carboxylate, was discovered in D. polylepis venom, where it probably contributes to the profound hypotension caused by this venom. Acetylcholine was present in significant quantities only in this highly excitotoxic venom, while 4-guanidinobutyric acid and 5-guanidino-2-oxopentanoic acid were present in all venoms.

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism.

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.

SERS of cells: What can we learn from cell lysates?

Surface-enhanced Raman spectroscopy (SERS) is a promising and emerging technique to analyze the cellular environment. We developed an alternative, rapid and label-free SERS-based method to get information about the cellular environment by analyzing cells lysates, thus avoiding the need to incorporate nanoparticles into cells. Upon sonicating and filtrating cells, we obtained lysates which, mixed with Au or Ag nanoparticles, yield stable and repeatable SERS spectra, whose overall profile depends on the metal used as substrate, but not on the buffer used for the lysis process. Bands appearing in these spectra were shown to arise mostly from the cytosol and were assigned to adenine, guanine, adenosine and reduced glutathione (GSH). Spectral differences among various cell types also demonstrated that this approach is suitable for cell type identification.

Metabolite identification and pharmacokinetic profiling of PP242, an ATP-competitive inhibitor of mTOR using ultra high-performance liquid chromatography and mass spectrometry.

PP242 is a second generation novel selective ATP-competitive inhibitor of mTOR that displayed promising anti-cancer activity over several cancer types by inhibiting both the complexes of mTOR (mTORC1 and mTORC2). The purpose of this study is to identify the possible metabolites and to evaluate the pharmacokinetic profile of PP242 after a single oral administration to Sprague-Dawley (SD) rats. Two metabolites, including one phase I and one phase II, were identified by in vitro and in vivo studies using rat liver microsomes (RLMs) as well as rat plasma, urine and feces, respectively, through ultra high-performance liquid chromatography-linear ion trap quadrupole-orbitrap-mass spectrometry (UHPLC-LTQ-Orbitrap-MS). The major biotransformation pathways of PP242 were hydroxylation and glucuronide conjugation. Additionally, a simple and rapid quantification method was developed and validated. The method recovery was within 79.7-84.6%, whereas the matrix effect was 78.1-96.0% in all three quality control (QC) concentrations (low, medium and high) including the LLOQ. Other parameters showed acceptable results according to the US food and drug administration (FDA) guidelines for bioanalytical method validation. Afterwards, pharmacokinetic parameters were evaluated in rat plasma by successfully applying the validated method using liquid chromatography-tandem mass spectrometry (LC-MS/MS). After a single oral administration at a dose of 5mg/kg, the maximum plasma concentration (Cmax) of PP242 was 0.17±0.08µg/mL, while the elimination was moderately fast (T1/2: 172.18±45.54min). All of the obtained information on the metabolite identification and pharmacokinetic parameter elucidation could facilitate the further development of PP242.

Poly-l-cysteine/electrospun copper oxide nanofibers-zinc oxide nanoparticles nanocomposite as sensing element of an electrochemical sensor for simultaneous determination of adenine and guanine in biological samples and evaluation of damage to dsDNA and DNA purine bases by UV radiation.

A new nanocomposite film constructed of poly-l-cysteine/zinc oxide nanoparticles-electrospun copper oxide nanofibers (PLC/ZnO-NPs-CuO-NFs) was prepared on the surface of the graphite electrode (GE). The novel electrode was successfully applied for the simultaneous determination of guanine (G) and adenine (A), two of the most important components of DNA and RNA. The PLC/ZnO-NPs-CuO-NFs/GE enhanced the anodic peak currents of the purine bases conspicuously and could determine them sensitively and separately in 0.1 M phosphate buffer solution at the physiological pH (7.0). The synthesized nanofibers, nanoparticles and nanocomposite were characterized by different methods such as Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and energy dispersive X-ray analysis (EDS). Under the optimum operating conditions, linear calibration curves were obtained in the range of 0.05-6.78 and 0.01-3.87 µM with a detection limit of 12.48 and 1.25 nM for G and A, respectively. The proposed method was applied to quantify A and G in three different DNA samples with satisfactory results. In addition, damage to human blood double-stranded DNA (dsDNA) and DNA purine bases (liberated in previously hydrolyzed human blood dsDNA) caused by UV-C and UV-B were evaluated. The results demonstrated that the proposed biosensing platform not only provides a novel and sensitive approach to detecting DNA damage, but also can be used for simultaneous determination of purine bases and major products of DNA oxidative damage.

Construction and application of a new cell electrochemical detecting system based on the hyposmotic principle.

A new cell electrochemical detecting system has been constructed based on the hyposmotic principle, in which the electrochemical signals have been strengthened by about 109.75% for the signal at about +0.70 V and 532.94% for the signal at about +1.03 V. The electrochemical detection limits of the cells have been improved by one order of magnitude. The individual concentrations of intracellular purines have been obtained.

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food.

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages.

Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells.

Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies. Graphical abstract Metabolomics reveals mycoplasma contamination changes the metabolome of PANC-1 cells.

Identification of sensory attributes, instrumental and chemical measurements important for consumer acceptability of grilled lamb Longissimus lumborum.

In this study, important eating quality attributes that influence consumer liking for grilled lamb loin have been identified using preference mapping techniques. The eating quality attributes identified as driving the consumer liking of lamb loin steaks were "tenderness", "sweet flavour", "meaty aftertaste", "roast lamb flavour" and "roast lamb aftertaste". In contrast, the texture attribute "rubbery" and the flavour attributes "bitter flavour" and "bitter aftertaste" had a negative influence on consumer perceptions. Associations were observed between eating quality and a number of instrumental and chemical measurements. Warner Bratzler Shear Force showed an association with "rubbery" texture and a negative association with "tenderness" and consumer liking scores. The compounds, glucose, glucose-6-phosphate, inosine, inosine monophosphate and adenosine monophosphate were associated with the attributes, "sweet flavour","meaty aftertaste", "roast lamb flavour", "roast lamb aftertaste" and with consumer scores for liking of lamb which is probably caused by the role some of these compounds play as precursors of flavour and as taste compounds.