Golgi α-mannosidases regulate cell surface N-glycan type and ectodomain shedding of the transmembrane protease corin.

Corin is a transmembrane protease that activates natriuretic peptides on the cell membrane. Reduced cell surface targeting or increased ectodomain shedding disrupts cell membrane homeostasis of corin, thereby impairing its cell surface expression and enzyme activity. N-glycans are essential in corin ectodomain shedding. Lack of N-glycans promotes corin ectodomain shedding in the juxtamembrane and frizzled-1 domains. The nascent N-glycans, transferred onto the polypeptide of corin, undergo multistep N-glycan processing in the endoplasmic reticulum and Golgi. It remains unclear how trimming by Golgi α-mannosidases, the critical N-glycan processing steps in N-glycan maturation, may regulate corin biosynthesis. In this study, we examined the effects of kifunensine and swainsonine, the inhibitors for α-mannosidases I and II, on corin expression and function. Western analysis of corin proteins in cell lysates and conditioned media from the inhibitor-treated corin-stable HEK293 cells and AC16 cells showed that both α-mannosidases I and II were required to maintain complex N-glycans on cell surface corin and protect corin from ectodomain shedding in the juxtamembrane and frizzled-1 domains. Cell viability analysis revealed that inhibition of α-mannosidase I or II sensitized cardiomyocytes to hydrogen peroxide-induced injury via regulating corin. Moreover, either one of the two coding genes was sufficient to perform Golgi α-mannosidase I trimming of N-glycans on corin. Similarly, this sufficiency was observed in Golgi α-mannosidase II-coding genes. Inhibition of ectodomain shedding restored corin zymogen activation from kifunensine- or swainsonine-induced reduction. Together, our results show the important roles of Golgi α-mannosidases in maintaining cell membrane homeostasis and biological activities of corin.

VOCs emission from a final landfill cover system induced by ground surface air temperature and barometric pressure fluctuation.

Volatile organic compounds (VOCs) emission flux and their concentration profiles were measured at a final municipal solid waste (MSW) landfill cover in Hangzhou, China. The influencing parameters, especially ground surface air temperature and pressure were monitored concomitantly. Furthermore, a numerical model incorporating coupled thermo-hydro-chemical interaction to assess VOCs emission from this final landfill cover (LFC) system was developed and validated with the field test results. The tested total VOC emission flux from the final cover is 0.0124 µg/m2/s, which indicates that the total amount of VOCs emitted into the atmosphere is 391 mg/m2 annually. Among these, dichloromethane (DCM) dominated VOCs emission flux during May, comprising 51.8% of the total emission flux. The numerical simulation results indicated that the diffusive emission flux of VOCs varied consistently with the fluctuation of atmospheric temperature. Whereas, the advective flux varied inversely with the fluctuation of barometric pressure. The highest difference in diffusive emission flux induced by temperature variation is 183 µg/m2/day and occurred in spring. Moreover, the results demonstrated that the impact of atmospheric temperature and pressure fluctuation on the emission of VOC from final covers is non-negligible when reasonably assessing the risks of landfill and landfill gas emission budget.

Field investigation of temporal variation and diffusion of hydrogen sulfide on waste working face and intermediate landfill cover.

This paper presents the study on the variation, influencing factors and diffusion regularity of hydrogen sulfide (H2S) concentration and surface flux on the working face and intermediate geomembrane cover of a landfill. Field investigations were conducted using static chambers at a large-scale municipal solid waste landfill in Hangzhou, China, from January 2019 to June 2021. The analytical models of H2S transport in the working face and intermediate cover were developed to investigate the surface flux under various conditions. The CALPUFF model was used to demonstrate the diffusion path. The H2S surface flux on the working face ranged from 7.1 × 10-3 to 1.7 mg/m2/h, whereas the range was found to be 1.5 × 10-4 to 0.9 mg/m2/h on the intermediate geomembrane cover. This observation indicated that the geomembrane can reduce H2S emissions. In addition, the H2S surface fluxes at the HDPE GMB seams and near the gas collecting wells were generally 1-2 orders of magnitude larger than that in the intact GMB. The analytical model estimates that the intact GMB exhibits a diffusion coefficient of H2S ranging from 2.7 × 10-11 to 2.2 × 10-10 m2/s. However, the diffusion coefficient increases significantly to a range of 3.3 × 10-11-9.8 × 10-7 m2/s on the GMB seams. According to CALPUFF results, only the H2S diffusion from the working face had areas exceeding the standard concentration.

Development and clinical application of a liquid chromatography-tandem mass spectrometry-based assay to quantify eight tyrosine kinase inhibitors in human plasma.

Introduction: Tyrosine kinase inhibitors (TKIs) are widely used in tumor treatment. The detection of these medicines by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can avoid the interference of structurally similar compounds. Objectives: This study aimed to develop and validate a new LC-MS/MS assay for the quantification of eight tyrosine kinase inhibitors in human plasma and to preliminarily evaluate the clinical utility of the therapeutic drug monitoring method. Methods: Plasma samples were prepared by simple protein precipitation and separated using an ultra-high-performance reversed phase column. Detection was achieved using a triple quadrupole mass spectrometer in the positive ionization mode. The assay was validated against standard guidelines. We reviewed and analyzed the results of 268 plasma samples obtained from patients administered imatinib and other TKIs collected from January 2020 to November 2021 at Zhongshan Hospital. The analytes were separated and quantified within 3.5 min. Results: The newly developed method demonstrated linearity for the detected drug concentration in the range of 20 to 2000 ng/ml for gefitinib (r2 = 0.991) and crizotinib (r2 = 0.992), 50 to 5000 ng/ml for nilotinib (r2 = 0.991) and imatinib (r2 = 0.995), 1500-150,000 ng/ml for vemurafenib (r2 = 0.998), 1000-100,000 ng/ml for pazopanib (r2 = 0.993), 0.5-100 ng/ml for axitinib (r2 = 0.992) and 5-500 ng/ml for sunitinib (r2 = 0.991) and N-desethyl sunitinib (r2 = 0.998). The lower limit of quantification (LLOQ) was 20 ng/ml for gefitinib and crizotinib, 50 ng/ml for nilotinib and imatinib, 1500 ng/ml for vemurafenib, 1000 ng/ml for pazopanib, 0.5, and 5 ng/ml for sunitinib and N-desethyl sunitinib, respectively. Specificity, precision, accuracy, and stability were tested, and met the requirements of the guidelines. At the same dose, there was no significant difference in plasma drug concentration between the original imatinib medicine and the generic medicine after patent expiration. Conclusion: We developed a sensitive and reliable method for the quantification of eight TKIs.

A LC-MS/MS method to simultaneously profile 14 free monosaccharides in biofluids.

Monosaccharides are important players in cell metabolism and potential biomarkers. An effective tool to quantify monosaccharides is required in basic research and healthcare. In this study, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that could simultaneously quantify 14 free monosaccharides and evaluated its performance according to clinical guidance. The LC-MS/MS step separated and quantified 14 monosaccharides with 6 min. The coefficient of variation at the lower limit of quantification was less than 20% for each analyte. The R square values from linear regression analyses were all greater than 0.995. The validated assay was employed to profile free monosaccharides in conditioned media from cell culture and patient sera from glucose tolerance test. Both LO2 cells and HEK293 cells consumed D-glucosamine, D-glucose and produced D-glucuronic acid, N-acetyl-D-glucosamine. Additionally, LO2 cells produced D-mannose and L-fucose, whereas HEK293 cells consumed D-mannose. In patient sera from glucose tolerance test, the level of D-glucose increased significantly after glucose intake, but the levels of other monosaccharides didn't. In conclusion, the LC-MS/MS assay we developed for 14-monosaccharide profiling met clinical requirements. The monosaccharide profiling results showed the distinct monosaccharide metabolism between liver and kidney cells, and the ignorable diet effect on 6 serum monosaccharides.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based assay for the simultaneous quantification of 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol in human plasma.

18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18-OXOF) are important biomarkers for the diagnosis of subtypes of primary aldosteronism. The detection of these three analytes by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is free from structurally similar compounds. The aim of this study was to develop and validate a new LC-MS/MS assay for the simultaneous quantification of 18-OHB, 18-OHF and 18-OXOF in plasma and to establish a reference intervals for apparently healthy population. Plasma samples were prepared by solid phase extraction and separated in an ultra-high performance reversed phase column. MS detection was achieved using a triple quadrupole mass spectrometer in both positive and negative ionization modes. The developed assay was then validated against standard guidelines. We collected 691 plasma samples from apparently healthy individuals (M:398, F:293) to establish the reference intervals. The analytes were separated and quantified within 5 min. The newly developed method demonstrated linearity for the detected steroid concentration in range of 5 to 3000 pg/ml for 18-OXOF (r2 = 0.999) and 20 to 3000 pg/ml for 18-OHB (r2 = 0.997) and 18-OHF (r2 = 0.997). The lower limit of quantification (LLOQ) was 2.5 pg/ml, 20 pg/ml and 20 pg/m for 18-OXOF, 18-OHB and 18-OHF respectively. Specificity, precision, accuracy and stability were tested, and met the requirements of the guidelines. 18-OHB was higher in females than in males, but 18-OHF were higher in males than females. The reference intervals of 18-OHB, 18-OHF and 18-OXOF for both genders together were 90.5-1040.6 pg/ml, 224.4-1685.2 pg/ml, 4.0-70.5 pg/ml, respectively. Age was also an important factor influencing the levels of these three hormones. We have developed a sensitive and reliable method for the simultaneous quantification of 18-OHB, 18-OHF, and 18-OXOF. Our work provides a reference interval for the clinical application of these three steroid hormones.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for simultaneous quantification of aldosterone, renin activity, and angiotensin II in human plasma.

Accurate quantification of plasma aldosterone (ALD) and renin activity (PRA)is critical for the diagnosis of primary aldosteronism (PA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the "gold standard" method for the determination of ALDand PRA. The aim of this study is to develop a new LC-MRM/MS assay for quantifying plasma ALD, PRA, and angiotensin II (Ang II) simultaneously and validate its effectiveness. To be more specific, plasmasamples were prepared by solid-phase extraction and separated in an ultra-performance reversed-phase column. MS detection was performed via a triple quadrupole mass spectrometer containing both positive and negative ion monitoring modes. The developed assay was then validated according to the standard guidelines and the influence of sample incubation on ALD and Ang II concentration was evaluated. In addition, the variation of endogenous Ang I was explored. The proposed LC-MRM/MS method was compared another LC-MS/MS method, which detects ALD, Ang I, and Ang II separately. Analyteswere separated and quantified within 5 min. The assay wasvalidated to be linear up to 5000 pg/ml for ALD and Ang II and 33.3 ng/ml/h for PRA.The lower limit of quantification (LLOQ) was 15 pg/ml, 15 pg/ml, and 0.1 ng/ml/hfor ALD, Ang II, and PRArespectively. Specificity, precision, accuracy, and stability were tested to meet the requirements of the guidelines. Significant changes were not found in ALD and Ang II concentrations over the 3 h-incubation. In addition, it was demonstratedthat the resultof PRA was not stronglyinfluenced by the endogenous Ang I. Comparison with another LC-MS/MS method was performed using the same apparatusand the proposed method was proved to be in good coincidence with the correlation coefficients rangingfrom 0.955to0.996. A sensitive and reliable method for simultaneousquantification of ALD, PRA, and Ang II has been developed and this study will significantly promote laboratory workflow efficiency and throughput.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders.

Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays are employed in more and more clinical laboratories to quantify steroids. The steroid quantification by LC-MS/MS shows great value in screening or diagnosing endocrine disorders; however, the number of functional steroids included in the LC-MS/MS methods is still limited. Methods: Here, we describe the performance and validation of a 20-steroid plasma panel by LC-MS/MS. The panel included progestogens (including mineralocorticoids and glucocorticoids), androgens and estrogens biosynthesized in steroid metabolic pathways. The LC-MS/MS method was validated according to guidance documents, and subsequently employed to profile steroid changes in endocrine disorders. Results: Using LC-MS/MS, 20 steroids were separated and quantified in 8 min. Coefficients of variation (CVs) of the 20 analytes at the lower limit of quantification (LLoQ) were all less than 15% (ranging from 1.84% to 14.96%). The linearity of the assay was demonstrated by all the R2 values greater than 0.995. Individual plasma steroids changed significantly in patients with subclinical Cushing's syndrome (SCS) and polycystic ovary syndrome (PCOS) - 17-hydroxypregnenolone (17-OH-PR), testosterone (T) and dihydrotestosterone (DHT) were significantly decreased in SCS patients, while in PCOS patients, pregnenolone, corticosterone (CORT), androstenedione (A4) and T were significantly increased and DHT was decreased. Conclusions: The LC-MS/MS method we developed for the quantification of 20 plasma steroids is clinical practicable. The steroid profiling data using this assay indicate its screening value for endocrine disorders. To further explore the value of the assay, more investigations are however needed.

[Establishment and clinical application of liquid chromatography-tandem mass spectrometric method for simultaneous determination of plasma 5-fluorouracil].

OBJECTIVE: To develop a robust liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of 5-fluorouracil (5-FU) in blood plasma and evaluate the use of 5-FU for treatment response surveillance as well as toxicity prediction in malignant gastrointestinal tumors. METHODS: A LC-MS/MS method was used, with signal linearity, lower limits of quantitation, precision, accuracy and stability being evaluated according to guideline US Food and Drug Administration (FDA)'s guidance for industry bioanalytical method validation.Analysis of 5-FU was performed in 35 gastrointestinal cancer patients admitted to Zhongshan Hospital of Fudan University from April 2013 to December 2013. The relationship between 5-FU with toxicity and treatment effect was compared. RESULTS: The linear ranges of 5-FU was 49-9 800 ng/ml, the lower limit of quantitation was 49.0 ng/ml. The within-run and between-run coefficients of variation (CV) of 5-FU was <3% and <6%.The recovery rates of low, medium and high level quality controls were 103.36%, 88.12% and 91.26% respectively; with standard added recovery of 109.69%, 91.06% and 88.81% respectively. The control added recoveries were 112.16%, 99.12% and 92.28% respectively. The bias was -11.69%, 2.42% and -8.09% when samples were repeated freezing and thawing twice (-80 ℃). The results had a bias -11.97%, 1.42%, -10.91% and 0.56%, 0.14%, 3.82% when samples were kept at 2-8 ℃ for 2 days and 14 days. In 35 gastrointestinal cancer patients, there was no correlation between initial dose of 5-FU and 44 h AUC (concentration 3.44-53.43 mg/L·h). The risk of chemotherapy-related adverse effect in 5-Fu 44 h AUC> 30 mg/L·h group was significantly higher than 44 h AUC < 30 mg/L·h group (χ(2)=12.600, P<0.01). While the chemotherapy response of AUC > 20 mg/L·h group was significantly satisfactory than AUC < 20 mg/L·h group (χ(2)=5.358, P<0.05). CONCLUSIONS: A robust and reliable LC-MS/MS method for the determination of 5-FU in blood plasma has been developed and it is suitable for clinical application. Detecting 5-FU may guide individualized treatment and predict adverse events.