Micheliolide ameliorates lipopolysaccharide-induced acute kidney injury through suppression of NLRP3 activation by promoting mitophagy via Nrf2/PINK1/Parkin axis.

BACKGROUND: Sepsis-associated acute kidney injury (SA-AKI) represents a frequent complication of in critically ill patients. The objective of this study is to illuminate the potential protective activity of Micheliolide (MCL) and its behind mechanism against SA-AKI. METHODS: The protective potential of MCL on SA-AKI was investigated in lipopolysaccharide (LPS) treated HK2 cells and SA-AKI mice model. The mitochondrial damage was determined by detection of reactive oxygen species and membrane potential. The Nrf2 silencing was achieved by transfection of Nrf2-shRNA in HK2 cells, and Nrf2 inhibitor, ML385 was employed in SA-AKI mice. The mechanism of MCL against SA-AKI was evaluated through detecting hallmarks related to inflammation, mitophagy and Nrf2 pathway via western blotting, immunohistochemistry, and enzyme linked immunosorbent assay. RESULTS: MCL enhanced viability, suppressed apoptosis, decreased inflammatory cytokine levels and improved mitochondrial damage in LPS-treated HK2 cells, and ameliorated renal injury in SA-AKI mice. Moreover, MCL could reduce the activation of NLRP3 inflammasome via enhancing mitophagy. Additionally, Nrf2 deficiency reduced the suppression effect of MCL on NLRP3 inflammasome activation and blocked the facilitation effect of MCL on mitophagy in LPS-treated HK2 cells, the consistent is true for ML385 treatment in SA-AKI mice. CONCLUSIONS: MCL might target Nrf2 and further reduce the NLRP3 inflammasome activation via enhancing mitophagy, which alleviated SA-AKI.

Simultaneous analysis of natural and artificial sweeteners in sugar-free drinks and urine samples by column-switching UHPLC-charged aerosol detection method.

Sweeteners are considered an alternative to high-calorie foods or drinks and have been widely used globally. However, the simultaneous separation and detection of high-polarity natural and artificial sweeteners are challenging owing to their broad-spectrum physical and chemical properties. Herein, we developed a column-switching UHPLCCAD method and used it for detecting and quantitating 12 sweeteners, including natural sweeteners (erythritol, mannitol, xylitol, sorbitol and stevioside) and artificial sweeteners (acesulfame potassium, saccharin sodium salt, sodium cyclamate, sucralose, aspartame, alitame and neotame). The LOD and LOQ were 0.932-6.25 µg/mL and 3.10-20.83 µg/mL, respectively, and the method demonstrated excellent linearity (R² ≥ 0.9990), good precision (intraday and interday precision was 0.59-6.88 %), and high recovery (average recoveries were 85.16-108.64 %). This method was applied to determine the sweeteners in 15 sugar-free drinks purchased from the local Chinese supermarkets. What's more, natural sweetener erythritol and artificial sweetener acesulfame potassium were suspected over addition in sugar-free drinks. Meanwhile the method was applied to the sweeteners in various sugar-free drinks and the dynamic monitoring of transit and excretion in vivo after drinking. Those prove that the method can be used to the detection of sugar free drinks and quality control of the sweeteners. The study highlights the potential of UHPLC-charged aerosol detection technology in detection of multiple components in food industry.

A novel UHPLC-MS/MS method for the determination of four α-dicarbonyl compounds in wine and dynamic monitoring in human urine after drinking.

α-dicarbonyl compounds (α-DCs) serve as potential biomarkers for oxidative stress-related diseases but are difficult to detect.To study the metabolism of carbonyl compounds, we developed a new mass spectrometry probe, 3-benzyl-2-oxo-4λ3-thiazolidine-4-carbohydrazide (BOTC), containing hydrazyl groups for the targeted detection of carbonyl functional groups.In a novel approach, we used BOTC pre-column derivatization with ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to simultaneously detect four kinds of α-DCs in red wine as well as in urine after drinking. The α-DCs were completely separated (R2 ≥ 0.9995), detection was sensitive (detection limit was 12.5-50 fmol), consistent (intraday and interday precision was 0.1-5.7 %), and efficient (average recoveries were 103.3-110.2 %). The method was applied to the analysis of α-DCs in different wines and the dynamic monitoring of transit and excretion in vivo after drinking. Our novel method provides a new strategy for the detection of α-dicarbonyl compounds in red wine and dicarbonyl compounds produced in oxidative stress-related diseases.

Development of a diphenyl sulfide structure derivatization reagent for amino acid enantiomers analysis: Application of dynamic monitoring in human urine after drinking wine.

We developed a novel chiral mass spectrometry derivatization reagent (S)-(3-(4-carboxythiazolidin-3-yl)-3-oxopropyl) diphenylsulfonium (CTOD) with a positively charged sulfur-containing structure for high-sensitivity detection of the chiral resolution of amino acid enantiomers. CTOD reacted with DL-amino acids at 60oC for 60 min to generate the corresponding diastereomers, fifteen chiral amino acid-derived products were separated. Resolution (Rs) values were of the range 1.54-4.36, except Asn 1.07, achieving good separation. A highly sensitive and selective UHPLC-MS/MS method for the simultaneous determination and chiral separation of five chiral amino acids (Pro, Ala, Glu, Asp, and Phe) based on CTOD derivatization was established and applied to the detection of chiral amino acids in different wines. The diastereomeric resolution of the five amino acids was 1.71-5.42, and an excellent linear relationship was obtained in the range of 0.25-500 pmol (R2 ≥0.9993). The detection limit was 0.05-0.25 pmol. The intra- and inter-day precisions were 0.51-5.76% and 0.78-5.18%, respectively, and the average recovery was 90.03-99.99%. In addition, the metabolic concentration of chiral amino acids was monitored after drinking red wine and white wine, and the fitting curve of metabolic concentration was drawn.

Development and evaluation of a novel fluorescent chiral derivatization reagent DBD-S-M-Pro: first observation of four chiral amino acids in human hair.

This study reports a novel fluorescent chiral derivatization reagent, 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-(2-succinimidoxy)-trans-2-methyl-L-proline (DBD-S-M-Pro), with a benzoxadiazole structure containing an N-hydroxysuccinimide activation group. DBD-S-M-Pro targets chiral amino-functional compounds under alkaline conditions without a condensation agent. Gradient elution was performed on a BEH C18 (100 × 2.1 mm, 1.7 µm) column with a mobile phase of 0.05% formic acid (FA) in 10 mM ammonium acetate (CH3COONH4) and 0.1% FA in acetonitrile or methanol. The efficiency of the chiral resolution was evaluated under excitation and emission wavelengths of 450 nm and 560 nm, respectively. The 19 chiral amino acids were separated in the range of 1.45-14.84. The resolutions of almost all DL-amino acids exceeded 1.5; the exceptions were serine (Ser) and lysine (Lys), with resolutions of 1.45 and 1.46, respectively. In addition, a new approach was devised for the simultaneous analysis of four chiral amino acids (DL-Glu, DL-Ala, DL-Val, and DL-Phe) in human hair. These amino acids were analyzed in the range of 12.5-400 pmol, with R2 ≥ 0.9990, limits of detection (S/N = 3) of 4-10 pmol, and intraday and interday precisions of 0.57-6.23%. The average spikes in the hair recoveries were 89.76-111.54%, and the matrix effects were 92.47-102.40%. Next, the contents of free chiral amino acids in the hair samples of 10 healthy volunteers (five males and five females) were analyzed with this method, and the differences were compared. The developed DBD-S-M-Pro provides a novel strategy for the sensitive determination of free chiral amino acids in living organisms.

Comprehensive characterization of the chemical composition of Lurong dabu decoction and its absorbed prototypes and metabolites in rat plasma using UHPLC-Q Exactive Orbitrap-HRMS.

Lurong Dabu decoction (LRDBD) is an effective traditional Chinese Korean ethnic medicine prescription composed of eight herbs, which is used for treating asthma. However, its material basis has not been studied yet. Herein, the use of a new and highly sensitive UHPLC-Q Exactive Orbitrap-HRMS technique is proposed for the high-resolution and accurate identification of the material basis of LRDBD. We identified 122 compounds belonging to different groups in LRDBD. Among these, 23 ingredients produced by decoction were identified and compared with 8 single herb compounds. Moreover, 39 other significantly different compounds were identified. Additionally, 29 absorbed prototype components and 35 metabolites were identified in rat plasma. Half of the prototype components were originated from antler velvet, it has corroborated the compatibility theory of Sasang medicine. To the best of our knowledge, the material basis of LRDBD was characterized for the first time. Our findings provide basic data and a method for further discovering potential drug targets and revealing the action mechanism of LRDBD in asthma treatment.

Relative quantitation of glycans in cetuximab using ultra-high-performance liquid chromatography-high-resolution mass spectrometry by Pronase E digestion.

Glycans play important roles in the activity and function of monoclonal antibodies (mAbs). In this study, an isotope labeling method for the relative quantitative analysis of glycans in cetuximab, a chimeric human/mouse IgG1 monoclonal antibody that specifically targets epidermal growth factor receptor, via hydrophilic interaction LC-ultra-high-performance LC-HRMS was established based on Pronase E digestion. To this aim, novel isotope MS probes, i.e., 3-benzoyl-2-oxothiazolidine-4-carboxylic acid (d0-BOTC) and 3-(2,3,4,5,6-pentadeuterio-benzoyl)-2-oxothiazolidine-4-carboxylate acid (d5-BOTC), which include a carboxyl group to target the amino functional group in glycosylamine, were developed. The nonspecific Pronase E enzyme could simultaneously digest the peptide bound to the N- and O-glycans into glycosylamine having only one amino acid. Since the mass difference between the light- and heavy-labeled glycans was 5.0 Da, the relative abundance of their MS peaks was used to achieve the qualitative and relative quantitative analysis of glycans. Sialylglycopeptide was used as a complex glycan model to validate the accuracy of the method. The results demonstrated the good linearity (R2 ≥ 0.9994) between the experimentally detected MS intensity ratios and the theoretical molar ratios of the d0-BOTC to the corresponding d5-BOTC derivatives in the dynamic range of 0.03-10 and 0.03-20 of three orders magnitude for the d5-BOTC/d0-BOTC ratios. The reproducibility was between 0.16% and 10.70%, and the limit of detection was 13 fmol. The feasibility of the relative quantification method was investigated by analyzing the glycan content in cetuximab, finding good consistency between experimental and theoretical molar ratios (5:1, 3:1, 1:1, 1:3, 1:5) of d0/d5-BOTC-labeled glycans. Finally, 13 glycans were successfully identified in cetuximab by applying this method using an in-house Tracefinder database. This study provides a novel strategy for the high throughput analysis, identification, and functional study of glycans in mAbs.

Monitoring of salicylic acid content in human saliva and its relationship with plasma concentrations.

Aspirin is a widely used anti-inflammatory drug. It is reported that a relationship may exist between salicylic acid content in plasma and saliva after taking aspirin. This study established a rapid, convenient, and safe method to assess salicylic acid concentration in human saliva. A novel HPLC-ultraviolet detector was used to measure salicylic acid concentrations in human saliva and plasma. A C18 reversed-phase column with an aqueous solution of 0.1% trifluoroacetic acid (TFA)-acetonitrile mobile phase was used, and drug peaks were recorded at 303 nm. Salicylic acid was completely separated in saliva and plasma. Excellent linearity and correlation (r2 ≥ 0.9999) was observed between 0.1 and 2.0 µg/mL. The detection limit (S/N = 3) was 33 ng/mL, and intra- and inter-day recoveries were 103.5-113.3% and 101.1-109.5%, respectively. Salicylic acid was measured within nine hours after administration of acetylsalicylic acid tablets. A positive correlation between salicylic acid content in saliva and plasma was found (r = 0.867, p < 0.001). The proposed method was used successfully to measure salicylic acid concentration in human saliva. Meanwhile, we explored the relationship between salicylic acid levels in plasma and saliva. Saliva might replace blood for monitoring aspirin treatment. In addition, the research provides a reference for application to saliva samples.