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.

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 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.