OBJECTIVETo study the CPS-II mechanism underlying the pathological process of elevated blood ammonia leading to liver injury.

METHODSAn in vitro hyperammonemia hepatocyte cell model was constructed by exposure to various concentrations of NH4Cl. The subsequent changes to cellular morphology were observed by microscopy. to cell apoptosis were determined by flow cytometry, and to mRNA and protein expression of CPS-II were examined by real-time PCR and western blotting, respectively.

RESULTSExposure to NH₄Cl led to dose-dependent morphological damage, apoptosis and necrosis of the hepatocytes. The apoptosis rate was significantly higher for the high-dose group than for the control (no exposure) group (24.7% ± 2.39% vs. 4.1% ± 0.78%, q =8.06, P less than 0.05). Expression of the CPS-II mRNA was significantly elevated in response to NH₄Cl exposure (vs. the control group; F=191.881, P < 0.05).The CPS-II mRNA expression level increased with increasing NH₄Cl concentration (grey values: 1.040 ± 0.045, 1.641 ± 0.123, 2.285 ± 0.167 and 3.347 ± 0.124, respectively). The CPS-II protein expression level was also significantly enhanced in response to the NH₄Cl exposures (CPS-II protein and internal GAPDH grey value ratios: 0.099 ± 0.0130, 0.143 ± 0.025, 0.161 ± 0.036 and 0.223 ± 0.042, respectively; t=3.825, 3.968 and 6.908, P less than 0.05).

CONCLUSIONCPS-II mRNA and protein expression levels become elevated with increase in the NH₄Cl concentrations, suggesting that in addition to the urea cycle, CPS-II may play an important role in the ammonia metabolism under the condition of hyperammonemia.

Ammonia ; Apoptosis ; Hepatocytes ; Humans ; Hyperammonemia ; Liver ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Somatostatin

Objective A simple,specific and rapid LC-MS/MS method was established to determine flumatinib and its two major metabolites in human plasma for clinical therapeutic drug monitoring.Methods The determination was performed on an ACQUITY UPLC HSS T3 column(2.1 mm×50 mm,1.8 μm)with mobile phases consisting of acetonitrile and 10 mmol·L-1 ammonium formate(containing 0.1%formic acid)with gradient elution at the flow rate of 0.5 mL·min-1.The elution time was 6 min.The temperature of the column was 38℃.The ion source was electrospray ion source and the scanning mode was multiple reaction monitoring scanning in positive ion mode.Results The mass concentrations of flumatinib and its metabolites(flumatinib M1 and flumatinib M3)have a good linear relationship within the concentration range investigated.The precision and stability of the method are good.The precision is less than 15%,and the relative deviation is within±15%.The extraction recoveries of flumatinib and its metabolites approach nearly 100%.Conclusion The method is simple and sensitive,and can accurately determine the plasma concentration of flumatinib and its metabolites,providing a basis for clinical rational drug use.

Tyrosine kinase inhibitors （TKIs） represent a class of small-molecule targeted drugs that improve the survival time of patients with gastrointestinal stromal tumor （GIST）. Imatinib， sunitinib， regorafenib， ripretinib， and avapritinib are commonly used TKIs in the clinical treatment of various types of GIST. This article provides a comprehensive review of the pharmacokinetics and therapeutic drug monitoring （TDM） of these five drugs， finding that there is significant individual variability in the pharmacokinetics of these drugs. Among them， the absorption of imatinib， regorafenib， and avapritinib are influenced by food intake. Imatinib should be taken with meals and 200 mL of water， regorafenib is taken with a low-fat meal， while avapritinib is taken on an empty stomach. TKIs are mainly metabolized by cytochrome P450 3A4 （CYP3A4）， and when used in combination with CYP3A4 inducers or inhibitors， drug exposure levels will significantly change； apart from metabolic enzymes， the exposure levels of TKIs are also influenced by interactions with the transporter proteins P-glycoprotein and breast cancer resistance protein. Currently， research on TDM for TKIs is still in the exploratory stage， with a substantial amount of literature reporting the effective concentrations of imatinib， sunitinib and regorafenib. However， the precise relationship between exposure levels and efficacy/ toxicity needs further exploration. Currently， there is a lack of research on the correlation between exposure levels and efficacy/ toxicity of ripretinib and avapritinib. It is recommended to implement TDM in patients taking these drugs and explore their therapeutic window in combination with pharmacokinetic models. The commonly used methods for clinical TDM of TKIs include immunoassay， chromatography， and surface-enhanced Raman spectroscopy， providing a technical basis for clarifying the therapeutic window of TKIs.

OBJECTIVE To establish a method for concentration determination of caffeine and its three metabolites， theophylline， paraxanthine and theobromine in urine， and apply it in clinical practice. METHODS Using caffeine-13C3-d3 as internal standard （IS）， and the urine samples were protein precipitated with acetonitrile； HPLC-MS/MS method was adopted to determine the concentrations of caffeine and its three metabolites. The determination was performed on Waters ACQUITY UPLC® BEH HILIC column with mobile phase consisting of 60 mmol/L ammonium acetate （A）-acetonitrile （B） （gradient elution） at the flow rate of 0.5 mL/min. The column temperature was set at 38 ℃ ， and the sample size was 2 μL. The electrospray ionization detection was operated in a positive mode by multiple reaction monitoring. The detection ions for quantitative analysis were m/z 195.1→110.0 for caffeine， m/z 181.1→124.0 for theophylline， m/z 181.1→124.0 for paraxanthine， m/z 181.1→138.0 for theobromine， and m/z 198.1→ 140.1 for IS. The above method was used to determine the concentrations of caffeine and its three metabolites in the urine of 19 infants with apnea of prematurity （AOP）. RESULTS The linear ranges of mass concentration of caffeine， theophylline， paraxanthin and theobromine were 0.200-200， 0.050-50.0，0.050 0-50.0， and 0.100-100 μg/mL， respectively. The lower limits of quantification were 0.200， 0.050， 0.050 and 0.100 μg/mL （r＞0.990）， respectively. RSDs of intra-day and intra- day precision were not above 10.37%， and matrix factors were 85.68%-109.90%； extraction recoveries were 93.53%-109.40% （RSD≤15%）， and RSDs of stability tests were all lower than 15%. The concentrations of caffeine and its three metabolites in the urine of 19 cases were （27.346±7.951）， （0.351±0.223）， （0.428±0.395） and （0.472±0.374） μg/mL， respectively. CONCLUSIONS The established HPLC-MS/MS method is simple， sensitive and can be used for the determination of caffeine and its three metabolites in urine samples of AOP.