scholarly journals Development of A LC-MS/MS Method for Simultaneously Determining Nucleosides and Methy-Nucleosides in Mice Liver mRNA of Epimedin C-Induced Liver Injury Model​

2021 ◽  
Author(s):  
Zhizhen Song ◽  
Zeyun Li ◽  
Xueqian Wen ◽  
Ruijuan Liu ◽  
Xin Tian
Keyword(s):  
Liver Injury ◽  
High Performance ◽  
Liver Toxicity ◽  
Enzymatic Digestion ◽  
Control Group ◽  
Good Precision ◽  
Vacuolar Degeneration ◽  
Serum Aminotransferase ◽  
Mrna Methylation ◽  
Mice Liver

Abstract Background Epimedium, the crude drug used in clinical, has been proved to have the potential to cause liver damage in patients. As one of the main active ingredient of Epimedium, Epimedin C is reported to have the potential hepatotoxicity. However, the mechanism of Epimedin C-induced mice liver injury has not been studied. mRNA methylation is implicated in the regulation of many biological processes and diseases. The study of mRNA methylation in Epimedin C-induced liver injury may contribute to clarify the liver toxicity mechanism of Epimedin C. Therefore, an analysis method needs to be established to determine liver mRNA nucleoside and methy-nucleoside levels in epigenetic studies. Methods The Epimedin C groups were intragastrically administered single doses of 10 and 40 mg/kg body weight of Epimedin C for four weeks, and the normal control group were given the same volume of saline. The results of hematoxylin and eosin staining of the liver and serum aminotransferase levels were used to evaluate liver injury. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer. Results In this study, a selective, rapid and sensitive method was established using high performance liquid chromatography-tandem mass spectrometry for the simultaneous determination of six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in mRNA. In this method, linear concentrations of adenosine, cytidine, N6-methyladenosine and N5-methylcytidine were 40-20000 pg/mL, guanosine was 0.2–100 ng/mL, and uridine was 2-1000 ng/mL. N6-methyladenosine and N5-methylcytidine modification levels of the mice liver mRNA increased after Epimedin C treatment. Epimedin C led to the elevation of serum aminotransferase levels, and increased the inflammatory cell infiltration and vacuolar degeneration in liver, indicated the liver injury in mice. Conclusion This method was successfully applied to the detection of six liver mRNA nucleosides levels in Epimedin C-induced liver injury with good precision and accuracy. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study offers a method for the research on the mechanism of Epimedin C-induced liver injury, and will facilitate the research on the hepatotoxicity of herbal medicine in epigenetic studies.

scholarly journals UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Zhizhen Song ◽  
Zeyun Li ◽  
Xueqian Wen ◽  
Ruijuan Liu ◽  
Xin Tian
Keyword(s):  
Liver Injury ◽  
Mouse Model ◽  
High Performance ◽  
Enzymatic Digestion ◽  
Control Group ◽  
Mice Model ◽  
Vacuolar Degeneration ◽  
Serum Aminotransferase ◽  
Mrna Methylation ◽  
Mice Liver

Abstract Background Epimedin C, one of the main active ingredients of Epimedium, has been reported to have potential hepatotoxicity. However, the mechanism of Epimedin C-induced liver injury has not been studied. mRNA methylation, mainly including N6-methyladenosine and N5-methylcytidine, is implicated in the regulation of many biological processes and diseases. The study of quantifying mRNA methylation alterations in Epimedin C-induced liver injury mice may contribute to clarify the mechanism of its hepatotoxicity. Therefore, an analysis method needs to be established to determine nucleoside and methyl-nucleoside levels in liver mRNA. Methods An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously determine six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in liver mRNA. Besides, the Epimedin C-induced liver injury mouse model was studied by intragastrical administration Epimedin C at a daily dose of 10 or 40 mg/kg for 4 weeks. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer. Results In this method, calibration curves of the six nucleosides showed good linearity over their concentration ranges. The linear ranges were 40–20,000 pg/mL for adenosine, cytidine, N6-methyladenosine and N5-methylcytidine, 0.2–100 ng/mL for guanosine, and 2–1000 ng/mL for uridine. Epimedin C-induced liver injury mouse model was successfully established,which could be proved by the elevation of serum aminotransferase levels, and the increased inflammatory cell infiltration as well as vacuolar degeneration in liver. The N6-methyladenosine and N5-methylcytidine levels, and the ratios of N6-methyladenosine to adenosine and N5-methylcytidine to cytidine of the mice liver mRNA were all significantly increased after Epimedin C treatment. Conclusion The established method was successfully applied to the determination of six nucleosides levels in liver mRNA of the Epimedin C-induced liver injury mice model and the control group. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study will facilitate the mechanism research on the hepatotoxicity of Epimedin C.

scholarly journals Enhancement of Liver Toxicity on Diabetes Mellitus by a Universal Chemical Pollutant (Acrylonitrile) in Rats

2019 ◽  
Vol 26 (2) ◽  
pp. 9-17
Author(s):  
Sameer E. Alharthi
Keyword(s):  
Oxidative Stress ◽  
Diabetes Mellitus ◽  
Aqueous Solution ◽  
Liver Toxicity ◽  
Elevated Serum ◽  
Diabetic Rat ◽  
Control Group ◽  
Distilled Water ◽  
Serum Aminotransferase ◽  
Cyp2e1 Activity

The present study was designed to investigate potential liver damage due to acrylonitrile in Streptozotocin induced diabetes in rats. Twenty-four rats were divided into 4 treatment groups. Nondiabetic control rat receiving distilled water, non-diabetic rat receiving acrylonitrile aqueous solution (10 mg/kg/day), diabetic control rat receiving distilled water and diabetic rat receiving acrylonitrile aqueous solution. All groups received the treatment for 4 weeks. The animals were assessed for hepatoxicity markers in serum, oxidative stress markers, CYP2E1 activity and cyanide formation in tissues. Acrylonitrile significantly elevated serum aminotransferase, alanine aminotransferase, total bilirubin levels, triglycerides and total cholesterol in diabetic groups as compared to normal control group. Antioxidant markers like glutathione showed significant decline while a significant increase in malondialdehyde, superoxide dismutase and catalase in diabetic rats treated with acrylonitrile. CYP2E1 activity was observed in acrylonitrile – exposed nondiabetic and diabetic groups as compared to control. Cyanide formation was raised in both the nondiabetic and diabetic groups as compared to control group. Acrylonitriles can produce acute hepatic injury, induction of diabetes mellitus type II, and accomplish the CYP2E1 enzyme which sequentially leads to generation of oxidative stress and its metabolic product–cyanide that may potentiate the oxidative stress posing more deleterious effect.

scholarly journals Epigallocatechin-3-Gallate (EGCG), An Active Constituent of Green Tea: Implications in the Prevention of Liver Injury Induced by Diethylnitrosamine (DEN) in Rats

2019 ◽  
Vol 9 (22) ◽  
pp. 4821 ◽  
Author(s):  
Saleh A. Almatroodi ◽  
Mohammed A. Alsahli ◽  
Hanan Marzoq Alharbi ◽  
Amjad Ali Khan ◽  
Arshad Husain Rahmani
Keyword(s):  
Cell Cycle ◽  
Liver Injury ◽  
Green Tea ◽  
Liver Toxicity ◽  
Oxidative Capacity ◽  
Oxidative Enzymes ◽  
Control Group ◽  
Active Constituent ◽  
Drug Induced ◽  
Oxidative Potential

Liver diseases are one of the most detrimental conditions that may cause inflammation, leading to tissue damage and perturbations in functions. Several drugs are conventionally available for the treatment of such diseases, but the emergence of resistance and drug-induced liver injury remains pervasive. Hence, alternative therapeutic strategies have to be looked upon. Epigallocatechin-3-gallate (EGCG) is a naturally occurring polyphenol in green tea that has been known for its disease-curing properties. In this study, we aimed to evaluate its anti-oxidative potential and protective role against diethylnitrosamine (DEN)-induced liver injury. Four different groups of rats were used for this study. The first group received normal saline and served as the control group. The second group received DEN (50 mg/kg body wt) alone and third group received DEN plus EGCG (40 mg/kg body wt) only. The fourth group were treated with EGCG only. The liver protective effect of EGCG against DEN toxicity through monitoring the alterations in aspartate transaminase (AST), and alanine transaminase (ALT) and alkaline phosphatase (ALP) activities, serum level of pro-inflammatory mediators and anti-oxidant enzymes, histopathological alterations, measurement of cellular apoptosis, and cell cycle analysis was examined. The rats that were given DEN only had a highly significantly elevated levels of liver enzymes and pro-inflammatory cytokines, highly decreased anti-oxidative enzymes, and histological changes. In addition, a significant elevation in the percentage of apoptotic nuclei and cell cycle arrest in the sub- G1 phase was detected. EGCG acts as a hepatoprotectant on DENs by reducing the serum levels of liver functional enzymes, increasing total anti-oxidative capacity, reducing pathological changes and apoptosis, as well as causing the movement of cells from the sub G1 to S or G2/M phase of the cell cycle. In conclusion, EGCG displayed a powerful hepatoprotective additive as it considerably mitigates the liver toxicity and apoptosis induced by DEN.

scholarly journals Hepatoprotective Effects of Taraxacum officinale Root Extract on Permethrin-induced Liver Toxicity in Adult Mice

2020 ◽  
Author(s):  
Fatma Ghorbel Koubaa ◽  
Mariem Chaâbane ◽  
Bochra Choura ◽  
Mouna Turki ◽  
Fatma Makni-Ayadi ◽  
...  
Keyword(s):  
Liver Injury ◽  
Liver Toxicity ◽  
Taraxacum Officinale ◽  
Negative Control ◽  
Control Group ◽  
Root Extract ◽  
Indoor Environments ◽  
Protective Effects ◽  
Sod Activity ◽  
Adult Mice

Background: Globally, permethrin is used as an insecticide for pest control in indoor environments and in agriculture to enhance food production by eradicating undesirable insects and controlling disease vectors.  Objective: The present study investigated the protective effects of Taraxacum officinale (dandelion) on permethrin-induced liver injury in mice.  Methods: Adult mice were divided into four groups. The first group was the negative control group, whereas the second group was the positive control group that received dandelion through the diet at 2% (corresponding to a dose of 5 g/kg bw). The third group received permethrin (96 mg/kg bw) by gavage, whereas the fourth group received permethrin and a diet enriched with dandelion (cotreatment). All mice were sacrificed after 14 days of treatment. Results: Biomarkers of liver toxicity (AST, ALT, ALP, and LDH activities and bilirubin level) increased following permethrin treatment. Permethrin induced oxidative stress, which was indicated by an increase in MDA and GSH levels as well as GPx activity and a decrease in SOD activity. Permethrin treatment caused histological alterations in the liver, whereas co-treatment with dandelion reduced liver injury. Our results revealed that alterations of biochemical parameters and liver histological profile in mice following permethrin exposure were reversed towards normalization by the treatment with dandelion roots extract.  Conclusion: The protective effect of this plant might be due to its antioxidant capacity.

Determination of Verapamil in Exhaled Breath Condensate by Using Microextraction and Liquid Chromatography

2019 ◽  
Vol 15 (5) ◽  
pp. 535-541 ◽  
Author(s):  
Fariba Pourkarim ◽  
Ali Shayanfar ◽  
Maryam Khoubnasabjafari ◽  
Fariborz Akbarzadeh ◽  
Sanaz Sajedi-Amin ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Exhaled Breath Condensate ◽  
Exhaled Breath ◽  
Good Precision ◽  
Dispersive Liquid Liquid Microextraction ◽  
Breath Condensate ◽  
Liquid Microextraction

Background:Developing a simple analysis method for quantification of drug concentration is one of the essential issues in pharmacokinetic and therapeutic drug monitoring studies.Objective:A fast and reliable dispersive liquid-liquid microextraction procedure was employed for preconcentration of verapamil in exhaled breath condensate (EBC) samples and this was followed by the determination with high-performance liquid chromatography-ultraviolet detection.Methods:A reverse-phase high-performance liquid chromatography (RP-HPLC) combined with a dispersive liquid-liquid microextraction method (DLLME) was applied for quantification of verapamil in the EBC samples. The developed method was validated according to FDA guidelines.Results:Under the optimum conditions, the method provided a linear range between 0.07 and 0.8 µg.mL-1 with a coefficient of determination of 0.998. The intra- and inter-day relative standard deviation and relative error values of the method were below 15%, which indicated good precision and accuracy. The proposed method was successfully applied for the analysis of verapamil in two real samples with concentrations of 0.07 and 0.09 µg.mL-1.Conclusion:The established HPLC-UV-DLLME method could be applied for the analysis of verapamil in human EBC samples.

Deproteinizing methods evaluated for determination of uric acid in serum by reversed-phase liquid chromatography with ultraviolet detection.

1987 ◽  
Vol 33 (8) ◽  
pp. 1427-1430 ◽  
Author(s):  
R Sakuma ◽  
T Nishina ◽  
M Kitamura
Keyword(s):  
Uric Acid ◽  
Liquid Chromatography ◽  
Perchloric Acid ◽  
High Performance ◽  
Reversed Phase ◽  
Precipitation Method ◽  
Zinc Hydroxide ◽  
Good Precision ◽  
Ultraviolet Detection

Abstract We evaluated six deproteinizing methods for determination of uric acid in serum by "high-performance" liquid chromatography with ultraviolet detection: those involving zinc hydroxide, sodium tungstate, trichloroacetic acid, perchloric acid, acetonitrile, and centrifugal ultrafiltration (with Amicon MPS-1 devices). We used a Toyosoda ODS-120A reversed-phase column. The mobile phase was sodium phosphate buffer (40 mmol/L, pH 2.2) containing 20 mL of methanol per liter. Absorbance of the eluate was monitored at 284 nm. The precipitation method with perchloric acid gave high recoveries of uric acid and good precision, and results agreed with those by the uricase-catalase method of Kageyama (Clin Chim Acta 1971;31:421-6).

scholarly journals Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2194
Author(s):  
Kamil Łuczykowski ◽  
Natalia Warmuzińska ◽  
Sylwia Operacz ◽  
Iga Stryjak ◽  
Joanna Bogusiewicz ◽  
...  
Keyword(s):  
Thin Film ◽  
Bladder Cancer ◽  
High Performance ◽  
Biomarker Discovery ◽  
Solid Phase ◽  
Biological Fluids ◽  
Reversed Phase ◽  
Control Group ◽  
Metabolic Evaluation ◽  
Metabolomic Profiling

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study’s results may support a better understanding of bladder cancer development and progression mechanisms.

scholarly journals Maresin 1 protects the liver against ischemia/reperfusion injury via the ALXR/Akt signaling pathway

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Da Tang ◽  
Guang Fu ◽  
Wenbo Li ◽  
Ping Sun ◽  
Patricia A. Loughran ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Liver Injury ◽  
Signaling Pathway ◽  
Inflammatory Responses ◽  
Ischemia Reperfusion ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Serum Aminotransferase ◽  
Primary Mouse ◽  
Maresin 1

Abstract Background Hepatic ischemia/reperfusion (I/R) injury can be a major complication following liver surgery contributing to post-operative liver dysfunction. Maresin 1 (MaR1), a pro-resolving lipid mediator, has been shown to suppress I/R injury. However, the mechanisms that account for the protective effects of MaR1 in I/R injury remain unknown. Methods WT (C57BL/6J) mice were subjected to partial hepatic warm ischemia for 60mins followed by reperfusion. Mice were treated with MaR1 (5-20 ng/mouse), Boc2 (Lipoxin A4 receptor antagonist), LY294002 (Akt inhibitor) or corresponding controls just prior to liver I/R or at the beginning of reperfusion. Blood and liver samples were collected at 6 h post-reperfusion. Serum aminotransferase, histopathologic changes, inflammatory cytokines, and oxidative stress were analyzed to evaluate liver injury. Signaling pathways were also investigated in vitro using primary mouse hepatocyte (HC) cultures to identify underlying mechanisms for MaR1 in liver I/R injury. Results MaR1 treatment significantly reduced ALT and AST levels, diminished necrotic areas, suppressed inflammatory responses, attenuated oxidative stress and decreased hepatocyte apoptosis in liver after I/R. Akt signaling was significantly increased in the MaR1-treated liver I/R group compared with controls. The protective effect of MaR1 was abrogated by pretreatment with Boc2, which together with MaR1-induced Akt activation. MaR1-mediated liver protection was reversed by inhibition of Akt. Conclusions MaR1 protects the liver against hepatic I/R injury via an ALXR/Akt signaling pathway. MaR1 may represent a novel therapeutic agent to mitigate the detrimental effects of I/R-induced liver injury.

scholarly journals Development and application of a spectrophotometric method in quality evaluation of benzimidazole anthelminthics in Nairobi city county

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Johnson K. Murage ◽  
Beatrice K. Amugune ◽  
Peter Njogu ◽  
Stanley Ndwigah
Keyword(s):  
Spectrophotometric Method ◽  
High Performance ◽  
Neglected Tropical Diseases ◽  
Developing Economies ◽  
Raw Materials ◽  
Poor Quality ◽  
Prolonged Treatment ◽  
Good Precision ◽  
Market Surveillance

Abstract Background Neglected tropical diseases (NTDs) are a group of communicable diseases which are prevalent in the tropics affecting more than one billion people. Treatment and prevention of these infections is very costly to developing economies. Helminthiases are classified among NTDs. The communities afflicted are poor and have limited access to essential resources for their livelihood. Poor-quality drugs for NTDs may lead to death or prolonged treatment without achieving the desired results. The limited resources used in purchasing poor-quality drugs will therefore be wasted instead of being put to good use. Most of the methods available for the analysis of benzimidazole anthelminthics utilize high-performance liquid chromatography. They are therefore time consuming, require sophisticated and expensive equipment, utilize rare and expensive reagents and solvents, and call for skilled personnel. A simple, rapid, and inexpensive ultraviolet spectrophotometric method of analysis would therefore come in handy especially in the analysis of many samples as occurs during post-authorization market surveillance for quality. Results The suitable solvent for the spectroscopic analysis was established as 0.1 M methanolic HCl. The wavelength of analysis was set at 294 nm. Upon validation, the method was found to have good linearity. The range over which linearity was established was way beyond the 80 to 120% of the working concentration specified by the ICH. The method exhibited good precision. Out of 32 commercial samples analyzed, five (15.6%) did not comply with compendial specifications. Intra-brand batch variation was also observed. Out of three batches of product A002T analyzed, one did not comply with compendial specifications. Conclusion A major limitation in the analysis of benzimidazole anthelminthics is the lack of reliable, simple, rapid, and low-cost methods of analysis with high throughput. The developed method serves to fill this gap. It can be used in the analysis of raw materials and finished products. It can also be used in the establishment of the quality of products prior to registration. The method will prove very useful in post-market surveillance of quality of benzimidazole anthelminthics.

scholarly journals Hepatoprotective effect of galangin on carbon tetrachloride-induced hepatotoxicity via the LKB1/AMPK pathway

2021 ◽  
Vol 19 ◽  
pp. 205873922110008
Author(s):  
Meng Chen ◽  
Xinyan Song ◽  
Jifang Jiang ◽  
Lei Xing ◽  
Pengfei Wang
Keyword(s):  
Carbon Tetrachloride ◽  
Liver Toxicity ◽  
Corn Oil ◽  
Histopathological Examination ◽  
Hepatoprotective Effect ◽  
Control Group ◽  
Group Model ◽  
Protective Effects ◽  
Model Group ◽  
Expression Levels

To investigate the protective effects of galangin on liver toxicity induced by carbon tetrachloride (CCl4) in mice. Mouse hepatotoxicity model was established by intraperitoneal injection (i.p.) of 10 ml/kg body weight CCl4 that diluted with corn oil to a proportion of 1:500 on Kunming mice. The mice were randomly divided into five groups named control group, model group, and 1, 5, and 10 mg/kg galangin group. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed by ELISA. Liver histopathological examination was observed via optical microscopy. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and glutathion (GSSG) were analyzed to assess oxidative stress. Finally, western blot assay was carried out to analyse the expression levels of total AMP-activated protein kinase (AMPK), phospho-AMPK (p-AMPK), total liver kinase B1 (LKB1), and phospho-LKB1 (p-LKB1). Compared with the control group, in the model group, the levels of AST, ALT, MDA, and GSSG increased significantly ( p < 0.01); the activity of SOD and GSH decreased significantly ( p < 0.01); and the histopathological examination revealed liver necrosis. However, treatment with galangin (5 and 10 mg/kg) significantly reversed these CCl4-induced liver damage indicators. Furthermore, treatment with galangin (10 mg/kg) significantly increased the p-AMPK and p-LKB1 expression levels ( p < 0.01). This study supports the hepatoprotective effect of galangin against hepatotoxicity, perhaps occurring mainly through the LKB1/AMPK-mediated pathway.

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