Determination of five nucleosides by LC‐MS/MS and the application of the method to quantify N6‐methyladenosine level in liver messenger ribonucleic acid of an acetaminophen‐induced hepatotoxicity mouse model

Ribonucleic acid N⁶‐methyladenosine methylation plays an important role in a variety of biological processes and diseases. Acetaminophen‐induced hepatotoxicity is one of the major challenges faced by clinicians. To date, the link between N⁶‐methyladenosine and acetaminophen‐induced hepatotoxicity has not been studied. In this study, a simple ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of five nucleosides (adenosine, uridine, cytidine, guanosine, and N⁶‐methyladenosine) in messenger ribonucleic acid. After enzymatic digestion of messenger ribonucleic acid, the nucleosides sample was separated on an Acquity UPLC column with gradient elution using methanol and 0.02% formic acid water, and detected by a Qtrap 4500 mass spectrometer with an electrospray ionization mode. The method was validated over the concentration ranges of 4–800 ng/mL for adenosine, uridine, cytidine, and guanosine and 0.1–20 ng/mL for N⁶‐methyladenosine. It was successfully applied to the determination of N⁶‐methyladenosine levels in liver messenger ribonucleic acid in an acetaminophen‐induced hepatotoxicity mouse model and a control group. This study offers a method for the determination of nucleoside contents in epigenetic studies and constitutes the first step toward the investigation of ribonucleic acid methylation in acetaminophen‐induced hepatotoxicity, which will facilitate the elucidation of its mechanism.     

Antioxidant Extract from Cleistocalyx nervosum var. paniala Pulp Ameliorates Acetaminophen-Induced Acute Hepatotoxicity in Rats

The indigenous purplish red fruit, Cleistocalyx nervosum var. paniala (CN), is grown in northern Thailand. The aqueous extract of CN pulp is known to exhibit antioxidant and anticarcinogenic properties. To search for an antioxidant fraction separated from CN, various hydroalcoholic extractions were performed. The acidified ethanolic extract of CN obtained from 0.5% (v/v) citric acid in 80% (v/v) ethanol yielded greater polyphenol content and DPPH radical scavenging activity when compared with other hydroethanolic extracts. Cyanidin-3-glucoside is a major anthocyanin present in the acidified ethanolic extract of CN (AECN). At a dose of 5000 mg/kg bw, an anthocyanin-rich extract was found to be safe when given to rats without any acute toxicity. To examine the hepatoprotective properties of AECN, an overdose of acetaminophen (APAP) was induced in a rat model, while silymarin was used as a standard reference. The administration of AECN at a dose of 300 mg/kg bw for 28 days improved hepatocyte architecture and modulated serum alanine aminotransferase levels in APAP-induced rats. Furthermore, it significantly decreased serum and hepatic malondialdehyde levels but increased hepatic glutathione content, as well as glutathione peroxidase and UDP-glucuronosyltransferase activities. In conclusion, AECN may effectively reduce oxidative stress induced acute hepatotoxicity in overdose APAP-treated rats through the suppression of oxidative stress and the enhancement of the antioxidant system in rat livers.     

A new compound from the fruit of Amorpha fruticosa and activity against acetaminophen-induced hepatotoxicity

A new isoflavone was obstained from the medicinal herb Amorpha fruticosa.It was elucidated asbenzopyran-12-one,1,4,10,11- tetrahydro-6′-[8′-(hydroxymethyl)ethenyl]-2,3-dimethoxy-8′-O-β-D-glucopyranosyl-O-α-D-arabinoside by spectroscopic methods including UV,IR,1D NMR and 2D NMR techniques.And the activity against acetaminophen-induced hepatotoxicity of this compound was also studied,and found this compound can protect liver obviously from hepatotoxicity induced by acetaminophen (AAP).     

Modulation of antioxidant systems by subchronic exposure to the aqueous extract of leaves from Achillea millefolium L. in rats

We determined the effects of subchronic exposure to aqueous extract of leaves from Achillea millefolium (AE) on enzyme- and non-enzyme-dependent antioxidant systems in rats. Seven days treatment with AE (1 g/kg/twice a day, p.o.) altered the reduced glutathione (GSH) levels and antioxidant enzyme activities in several organs of the animals. Amount of GSH in uterus was increased (73%) while in kidneys it was decreased (23%). Besides, NAD(P)H quinone oxidoreductase 1 (NQO1) activity was increased in forestomach (26%) and in liver (64%), while glutathione S-transferase activity was decreased in the forestomach (32%) and increased in the liver (41%), kidney (35%) and uterus (37%). In preliminary experiments targeting the interaction of AE with acetaminophen (600 mg/kg, p.o.), we observed augmentation of acetaminophen-induced increase of the plasmatic alanine aminotransaminase, aspartate aminotransaminase and lactate dehydrogenase. Overall, the results indicate a potential toxic interaction of AE compounds with xenobiotics that use the glutathione pathway.     

A reliable method of liquid chromatography for the quantification of acetaminophen and identification of its toxic metabolite N-acetyl-p-benzoquinoneimine for application in pediatric studies

The aim of the present study was to develop a simple, selective and reliable method to quantify acetaminophen and its toxic metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI) for pediatric studies using 100 µL plasma samples, by reverse‐phase HPLC and UV detection. The assay was performed using a C₁₈ column and an isocratic elution with water–methanol–formic acid (70:30:0.15; v/v/v) as mobile phase. Linearity of the method was assayed in the range of 1–30 µg/mL for acetaminophen and 10–200 µg/mL for NAPQI, with a correlation coefficient r = 0.999 for both compounds, and inter‐ and intra‐day coefficients of variation of less than 13%. Several commonly co‐administered drugs were analyzed for selectivity and no interference with the determinations was observed. The detection and quantification limits for acetaminophen and NAPQI were 0.1 and 1 µg/mL, and 0.1 and 10 µg/mL respectively. The present method can be used to monitor acetaminophen levels using 100 µL plasma samples, which may be helpful when very small samples need to be analyzed, as in pharmacokinetics determination or drug monitoring in plasma in children. This assay is also able to detect the NAPQI for drug monitoring in patients diagnosed with acetaminophen intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of glutathione, as the dextran conjugate, on acetaminophen-induced hepatotoxicity

Glutathione was covalently attached to dextran (T-40) by the CNBr activation method. In mice given a lethal dose of acetaminophen, the 30-d survival rate increased progressively with coadministration of the conjugate, whereas little improvement was found when free glutathione was given. The dextran conjugate of glutathione maintained the serum transaminase activities at lower levels after acetaminophen administration, giving effective protection against acetaminophen hepatotoxicity.     

Comparative study on Artemisia halodendron Turcz. and its two related plants by GC-MS analysis and protective effect against carbon tetrachloride-induced hepatotoxicity in mice

A rapid method had been used for comparative study on Artemisia halodendron Turcz. and its two related plants by gas phase-mass spectrometry (GC-MS). The comparison of the volatile oils obtained in three plants by GC-MS were similar in 20 compositions. However, n-Hexadecanoic acid (10.40%), Biphenyl (7.867%) and 9,12-Octadecadienoic acid (7.25%) were the predominant in the volatile oils of A. halodendron Turcz., whereas these constituents did not exist in the other two plants. And the study investigated the effect of three plants against CCl₄-induced hepatotoxicity in mice. 70% ethanol extracts of A. halodendron Turcz. showed weaker protective effect than the other two plants. It suggested that they provide a basis for the identification of the A. halodendron Turcz. from the other two plants and the ethanol extract from three plants exerted a protecting effect against hepatotoxicity.     

Hepatoprotective effect of chiisanoside against acetaminophen-induced acute liver injury in mice

This study was designed to investigate the hepatoprotective effect of chiisanoside (CSS) and its possible mechanisms on acetaminophen (APAP)-induced acute liver damage in mice. The serum activities of alanine transaminase (ALT), aspartate transaminase (AST), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and the hepatic levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) were determined using the commercially available assay kits. The hepatic mRNA levels were measured by RT-PCR. The hepatic protein expressions of nuclear factor-kappa B (NF-κB), MAPK and their phosphorylated isoforms were evaluated by western blot assays. The results indicated that CSS (240 mg/kg) exhibited the hepatoprotective effects by inhibiting oxidative stress and inflammation on APAP-induced acute liver injury. Furthermore, the anti-inflammatory activity of CSS is largely related to the regulation of the NF-κB and MAPKs signaling pathways. These findings suggested that CSS possessed hepatoprotective effect against APAP-induced hepatotoxicity in mice.     

Polysulfonylamine. CLXII [1] Kristallstrukturen von Metall‐di(methansulfonyl)amiden. 11 [2] Das dreidimensionale Koordinationspolymer K[(CH3SO2)2N] II: Eine chirale Struktur mit vier unabhängigen Formeleinheiten,Pseudozentrosymmetrie der Kristallpackung und Konformationsisomorphie des Anions

Stick‐Polysulfonylamines. CLXII. Crystal Structures of Metal Di(methanesulfonyl)amides. 11. The Three‐Dimensional Coordination Polymer K[(CH₃SO₂)₂N] II: A Chiral Structure with Four Independent Formula Units, Pseudo‐Centrosymmetry of the Crystal Packing, and Conformational Isomorphism of the Anion For potassium dimesylamide, K[(CH₃SO₂)₂N] or KA, two monoclinic polymorphs have been isolated from different media at ambient temperature and characterized by single crystal X‐ray diffraction at —100 °C. In the previously reported centrosymmetric structure of KA I (P2₁/c, Z′ = 1), the anion adopts an extremely rare conformation in point group C₁, whereas the chiral structure of KA II (P2₁, Z′ = 4) displays three independent anions of normal C₂ symmetry and one anion substantially distorted to C₁. In addition, the independent ions of KA II are pair‐wise related by a pseudo‐inversion centre conferring pseudo‐P2₁/c symmetry and Z′ = 2 upon the packing. Both dimorphs form three‐dimensional coordination polymers, in which the oxygen and nitrogen atoms of the anions are exhaustively involved in κ¹‐ or μ₂‐type bonding to either five or six cations, whereby each of the five independent anions develops a different connectivity pattern. The coordination polyhedra of the cations comprise in KA I an O₆N environment approximating to a distorted octahedron with twofold occupancy of one coordination site, in KA II two O₇ spheres forming distorted monocapped octahedra and two O₄N₂ spheres of trigonal‐prismatic geometry. In conclusion, the Z′ = 4 structure of KA II exhibits both conformational and coordination isomorphism, whereas the KA I/KA II pair represents a case of simultaneous conformational and coordination dimorphism. Weak C—H···O hydrogen bonds are observed in both structures.     

Polysulfonylamine. CLX [1] Kristallstrukturen von Metall‐di(methansulfonyl)amiden. 10 [2] Die dreidimensionalen Koordinationspolymere M[(CH3SO2)2N] mit M = Kalium,Rubidium, Caesium (isotype Strukturen für M = K,Rb)

Polysulfonylamines. CLX. Crystal Structures of Metal Di(methanesulfonyl)amides. 10. The Three‐Dimensional Coordination Polymers M[(CH₃SO₂)₂N], where M is Potassium, Rubidium, Cesium (Isotypic Structures for M = K, Rb) Low‐temperature X‐ray crystal structures are reported for KA (monoclinic, space group P2₁/c, Z′ = 1), RbA (isotypic and isostructural with KA), and CsA (monoclinic, P2₁/n, Z′ = 1), where A^— denotes the anion obtained by deprotonation of the strong nitrogen acid (MeSO₂)₂NH. In KA and RbA, the anion is distorted into a rare C₁ conformation, whereas the standard C₂ conformation is retained in the cesium complex. The structures consist of three‐dimensional coordination networks, in which each cation adopts an irregular (O₆N)‐heptacoordination by forming close contacts to one (O, N)‐chelating, one (O, O)‐chelating and three κ¹O‐bonding ligands; however, the coordination number for Cs⁺ is effectively increased to 8 by a very short Cs···Cs contact distance of 422.5 pm. The crystal packings of the isotypic compounds KA and RbA display lamellar layer substructures that involve six independent ligand‐metal bonds and comprise an internal cation lamella and peripheral regions built up from anion monolayers; the 3D framework is completed by one independent M—O bond cross‐linking the layer substructures. In contrast, CsA features anion monolayers that intercalate planar zigzag chains of cations (Cs···Cs alternatingly 422.5 and 487.5 pm, Cs···Cs···Cs 135.7°), whereby each chain is surrounded and coordinated by four anion stacks and each anion stack connects two cation chains. All structures exhibit close C—H···A interanion contacts consistent with weak hydrogen bonding.     

Gas Liquid Chromatographic Determination of Acetaminophen in Blood Plasma

Abstract

An improved procedure for the GLC determination of acetaminophen (N-acetyl-p-aminophenol) in the presence of N-butyryl-p-aminophenol (internal standard) is described. The method is based on the extraction of acetaminophen from plasma with ethyl acetate containing a known amount of N-butyryl-p-aminophenol. Following a clean-up step with a basic buffer solution and neutralization with acid, both compounds are reextracted into ethyl acetate. The ethyl acetate is evaporated to dryness and the residue dissolved in 5 μl of pyridine and 15 μl of acetic anhydride at 42°C. One to 2 μl samples are injected directly into the gas chromatograph. This extraction process does not give rise to troublesome interfering peaks in the chromatogram. In addition, it prevents late-eluting peaks which inhibit efficient processing of samples. The recovery of acetaminophen is approximately 54%, and the limit of quantitation 0.5 μg/ml of plasma. Data are presented to illustrate the practicality of the method for bioavailability evaluation from acetaminophen plasma levels after oral administration of 325 mg of an acetaminophen dosage form.     

Simultaneous Determination of Tramadol and Acetaminophen in Human Plasma by LC–ESI–MS

This study presents a high-performance liquid chromatography–electrospray ionization–mass spectrometric (LC–ESI–MS) method for the simultaneous determination of tramadol and acetaminophen in human plasma using phenacetinum as the internal standard. After alkalization with saturated sodium bicarbonate, both compounds were extracted from human plasma with ethyl acetate and were separated by HPLC on a Hanbon LiChrospher CN column with a mobile phase of 10 mM ammonium acetate buffer containing 0.5% formic acid–methanol (40:60, v/v) at a flow rate of 1 mL min⁻¹. Analytes were determined using electrospray ionization in a single quadrupole mass spectrometer. LC–ESI–MS was performed in the positive selected-ion monitoring (SIM) mode using target ions at [M+H]⁺ m/z 264.3 for tramadol, [M+H]⁺ m/z 152.2 for acetaminophen and [M+H]⁺ m/z 180.2 for phenacetinum. Calibration curves were linear over the range of 5–600 ng mL⁻¹ for tramadol and 0.03–16 μg mL⁻¹ for acetaminophen. The inter-run relative standard deviations were less than 14.4% for tramadol and 12.3% for acetaminophen. The intra-run relative standard deviations were less than 9.3% for tramadol and 7.9% for acetaminophen. The mean plasma extraction recovery for tramadol and acetaminophen were in the ranges of 82.7–85.9 and 83.6–85.3%. The method was applied to study the pharmacokinetics of a new formulation of tramadol/acetaminophen tablet in healthy Chinese volunteers.     

Effects on feeding rate and biomarker responses of marine mussels experimentally exposed to propranolol and acetaminophen

Environmental risk assessments of human pharmaceuticals and other ‘emerging contaminants’ should integrate both population-relevant endpoints and biomarkers of potential modes of action in a range of species. Adult Mytilus galloprovincialis were exposed to the beta-adrenergic receptor blocker propranolol or to the anti-inflammatory drug acetaminophen (paracetamol), both commonly used therapeutic drugs present in aquatic ecosystems. Mussels were exposed under semi-static conditions for 10 days to either acetaminophen (CAS number 103-90-2; mean measured concentrations 23 and 403 μg/L) or propranolol hydrochloride (CAS number 318-98-9; mean measured propranolol concentrations 11 and 147 μg/L) at 15 ± 1 °C sea water. Feeding rate was assessed as an indicator of general toxicity. For propranolol, the 10-day no-observed effect concentration (^{feeding rate}NOEC) and lowest observed effect concentration (^{feeding rate}LOEC) were 11 and 147 μg/L, respectively. For acetaminophen, feeding rate was increased at both 23 and 403 μg/L, suggesting a 10-day ^{feeding rate}NOEC of 403 μg/L. Primarily, phase I carboxylesterase (CbE), phase II glutathione S-transferase (GST) and the anti-oxidant catalase activities were evaluated in digestive gland. Gill GST and acetylcholinesterase (AChE) activities were also measured. Lipid peroxidation (LPO) levels were measured in both tissues to assess oxidative stress. Some enzymatic activities in liver were also reduced after propranolol exposure whilst acetaminophen enhanced them (CbE p < 0.05). Acetaminophen exposure significantly increased hepatic LPO levels and inhibited AChE activity in gill (10-day NOEC and LOEC of 23 and 403 μg/L, respectively), whereas propranolol (11 μg/L) enhanced gill GST.     

Simultaneously screening multiple UGT1A1 inhibitors from Polygonum multiflorum root using ultrafiltration LC–MS

Liver injury induced by Polygonum multiflorum root (PMR) is an immediate issue requiring global attention. UDP-glucuronosyltransferase 1A1 (UGT1A1) inhibitors are suspected to additively contribute to the hepatotoxicity of PMR. This study was deliberately designed to simultaneously screen UGT1A1 inhibitors from PMR, and their co-contribution to hepatotoxicity was determined. Using ultrafiltration coupled to LC–MS method, four compounds, namely cis-2,3,5,4′-tetrahydroxystilbene-2-O-β-glucoside, trans-2,3,5,4′-tetrahydroxystilbene-2-O-β-d -glucoside, emodin-8-O-β-d -glucoside, and emodin, were screened, exhibiting the in vitro inhibitory activities against UGT1A1 with IC₅₀ values of 76.23, 18.70, 62.18, and 34.02 μM, respectively. The varying activities of the screened UGT1A1 inhibitors were demonstrated by performing a molecular docking simulation. Finally, zebrafish larvae and mice assays demonstrated that the UGT1A1 inhibitors co-contributed to the hepatotoxicity of PMR. These findings are conducive to understand the role of UGT1A1 inhibitors in PMR-induced hepatotoxicity.     

A statistical model for the optimization of dsc performance in the evaluation of drugs for preformulation studies

Differential scanning calorimetry (DSC) is a thermal analytical tool for preformulation studies. Extrapolated melting temperature (T_P) and heat of fusion (ΔH_f) can be used as parameters for optimizing the DSC performance. Two model pharmaceuticals acetaminophen and nicotinamide are used in this study. Using a factorial design for the experimental model and matrix analysis the results, the effect of sample mass, heating rate and the nitrogen flow rate were evaluated on the ΔH_f values and T_P values. Two levels for each of the procedural variables were used as a balanced experimental design with two sample sizes, two heating rates and two nitrogen flow rates. It was found that the change in the heating rate caused significant changes in the ΔH_f values but not the T_p values for acetaminophen. However, no significant effect was found for the T_p value but ΔH_f value was affected to a certain extent for nicotinamide.     

Phosphonium ionic liquids as new, reactive extractants of lactic acid

Solvent properties of ionic liquids with trihexyltetradecylphosphonium cation and bis(2,4,4-trimethylpentyl)phosphinate anion (Cyphos IL-104) or chloride anion (Cyphos IL-101) were studied. IL-104 effectively extracted lactic acid (LA) with distribution coefficients above 40 at low acid concentrations. IL-104 extracted only undissociated acid (LAH) what supported the coordination mechanism of lactic acid extraction via H-bonding. In the extraction of lactic acid by phosphonium chloride (IL-101) an ion-exchange mechanism contributed remarkably to the extraction especially at basic pH where anionic form of this acid predominated. A high solubility of water in hydrophobic IL-104 up to 14.4 mass % was connected with the formation of reverse micelles. A dual mechanism of water extraction to phosphonium ionic liquids was identified, which consisted of water incorporation into reverse micelles and the inclusion of water into the hydrated complex of lactic acid with ionic liquid (IL). The extraction of lactic acid caused splitting of reverse micelles with liberation of water from the solvent. In the saturated solvent only hydration water remained in the complex of lactic acid with phosphonium ionic liquid, with the suggested structure (LAH)_p(IL)(H₂O)₂, where the value of p ranged from 1 to 3.     

Monitoring the pH fluctuation of lysosome under cell stress using a near-infrared ratiometric fluorescent probe

Cell stress responses are associated with numerous diseases including diabetes, neurodegenerative diseases, and cancer. Several events occur under cell stress, in which, are protein expression and organelle-specific pH fluctuation. To understand the lysosomal pH variation under cell stress, a novel NIR ratiometric pH-responsive fluorescent probe (BLT) with lysosomes localization capability was developed. The quinoline ring of BLT combined with hydrogen ion which triggered the rearrangement of π electrons conjugated at low pH medium, meanwhile, the absorption and fluorescent spectra of BLT showed a red-shifts, which gived a ratiometric signal. Moreover, the probe BLT with a suitable pK_a value has the potential to discern changes in lysosomal pH, either induced by heat stress or oxidative stress or acetaminophen-induced (APAP) injury stress. Importantly, this ratiometric fluorescent probe innovatively tracks pH changes in lysosome in APAP-induced liver injury in live cells, mice, and zebrafish. The probe BLT as a novel fluorescent probe possesses important value for exploring lysosomal-associated physiological varieties of drug-induced hepatotoxicity.     

超高效液相色谱-质谱联用技术在药物肝损伤代谢组学研究中的应用

药物引起的肝损伤是全世界关注的健康问题,药源性肝损伤的早期诊断仍然是临床治疗肝损伤的一大难题。本研究中,首先利用对乙酰氨基酚、四氯化碳、大黄素、雷公藤甲素和马兜铃酸构建不同类型的肝细胞损伤模型,利用超高效液相色谱-质谱联用技术(UPLC-MS)分别得到正常组和损伤组的细胞代谢轮廓谱。然后利用模式识别构建分类模型,筛选肝损伤相关的差异代谢物。结果显示,不同类型肝损伤在肝细胞代谢谱上可以被区分开,最终鉴定出14种差异代谢物。损伤组肝细胞经保肝阳性药联苯双酯干预后,差异代谢物水平均趋向于正常组,在一定程度上验证了差异标志物与肝损伤的相关性。实验结果表明细胞代谢组学是研究药物肝损伤的有效工具之一。     

Electrochemical sensor for acetaminophen based on an imprinted TiO2 thin film prepared by liquid phase deposition

An electrochemical sensor based on a molecularly imprinted TiO₂ thin film is proposed for the determination of acetaminophen. The imprinted TiO₂ films were obtained by liquid phase deposition (LPD) in the presence of acetaminophen, the functional monomer and the aqueous solution of (NH₄)₂TiF₆ and H₃BO₃. The results show that acetaminophen is embedded into the imprinted film in the presence of p-tert-butylcalix[6]arene as a functional monomer, and can be removed completely by washing with ethanol. The surface morphology, spectral properties and electrochemical characterizations of the imprinted sensor were investigated in detail. The combination of molecularly imprinted and LPD technique was shown to be a general strategy for constructing a molecular recognition system.