Association Of VKORC1 And CYP4F2 With Plasma Vitamin K1 Concentration And Afffect On Anticoagulation Of Warfarin Enantiomer Concentration And Pharmacodynamics Index

Objective: Warfarin(WF)is the most widely prescribed anticoagulant.Due to the large individual variability in WF response,drug therapy with WF requires frequent and regular monitoring by measurement of the prothrombin time expressed as the international normalized ratio(PT-INR).Multiple clinical and environmental factors contribute to the variability in WF maintenance dose,including race,age,gender,body weight,dietary intake,smoking,comorbidities,comedications and polymorphisms of the gene encoding the enzyme associated with the WF pharmacokinetics and pharmacody namics.The aim of this study was to analysis association of genotype variations VKORC1 and CYP4F2 with plasma vitamin K1(VK1)concentrations in subjects;relationship Between plasma VK1 concentration and WF sensitivity index in patients;relationship between genotype variations and warfarin enantiomer concentration and pharmacodynamics index in patients and.In order to provide reference of warfarin individualized medication in clinical and to improve anticoagulation control,reduce risk of bleeding,So as to achieve the purpose of improving clinical doctors confidence in WF treatment use.Method:The determination of VK1 concentration in human plasma using HPLC-MS/MS:Analytes were ionized in positive mode.The LC-APCI-MS/MS method used Vitamink1-d7 as internal standard,Mass spectra were recorded at m/z 451.0 ?187.0 and 458.6?194.4 for VK1 and Vitamin K1-d7(IS),respectively.After protein precipitation with ethyl alcohol,a single-step extraction with cyclohexane,the samples were separated by HPLC on a reversed-phase Synergl 4u Fusion C18(100mm�2.1 mm;5?m)column with a mobile phase of 0.5% formic acid-methanol,at a flow rate of 0.3m L/min.The determination of WF enantiomer concentration in human plasma using HPLC-MS/MS:Analytes were ionized in electron spray ionization(ESI)negative mode,the method used naproxen as internal standard,mass spectra were recorded at m/z 307.2?160.9 and 228.9?185.1 for WF and naproxen(IS),respectively.With 1mol/L hydrochloric acid as the buffer environment,liquid-liquid extraction with ethyl acetate,the samples were separated by HPLC on a Lux Cellulose-3 Chiral(100mm�2.1mm,2?m)column with a mobile phase of 0.5% formic acid-methanol,at a flow rate of 0.2m L?min-1.Collection and detection of Clinical sample:(1)recruited a total of 21 patients treated with a daily dose of WF for trial fibrillation in the Affiliated Hospital of Southeast University,Nan Jing,China,and 55 unrelated healthy subjects.Predefined inclusion criteria was followed:patients on anticoagulation therapy with WF for at least 3 months;having a stable anticoagulation status,which was determined from the results(within the therapeutic range)of a minimum of 3 consecutive INR measurements;and having a stable daily dosage during 1 month before blood sampling.We excluded patients who were coadministered amiodarone and/or anticancer agents.All subjects were given the experimental standard meal Uniformly.In the early morning of the experiment,9.0m L peripheral blood was taken from patients with atrial fibrillation before administration of WF,and 5.0 m L of per ipheral blood was taken from healthy subjects.This study was approved by the Ethics Committee of the University of Affiliated Hospital of Southeast University.Written informed consent was obtained from all subjects.The concentrations of VK1 and of WF enantiomer in the plasma were measured by HPLC-MS/MS.The levels of coagulation factors,PT/INR,VKORC1,CYP4F2,CYP2C9 Genotypes were measured by external.Based on the measured concentrations of VK1 and warfarin enantiomer,binding to genotype?levels of coagulation factor and PT-INR,using Origin to analysis the correlation of gene polymorphism and VK1 with WF enantiomer concentration and pharmacodynamics index.Results:The HPLC-MS/MS method for the determination of VK1 and WF enantiomers in plasma was proved to be accurate and reliable through methodological validation.Because of the limited sample size and WF individual differences,the VK1 concentration and WF sensitivity index were significantly correlated in the study,the VK1 and polymorphisms of the VKORC1 and CYP4F2 genes were significantly affected in the Chinese Han population.In the VK1 concentration.The results showed that the VK1 concentration in patients with VKORC1G/A genotype was significantly higher than that in patients with the A/A genotype(p<0.05);the VK1 concentration in patients with CYP4F2C/T and T/T genotype was significantly higher in patients with C/C genotype(p<0.05).However,the correlation between VKORC1 gene polymorphism and VK1 concentration was not significant in healthy subjects,In contrast,the VK1 concentration of CYP4F2(T/T+C/T)genotype was significantly higher than that of C/C genotype(p<0.05).Combined with the literatures suggested that clinicians in the WF treatment process,carrying VKO RC1G/G+CYP4F2T/T genotype,patients with safe WF treatment window;carrying VKORC1G/A genotype+CYP4F2C/T genotype,patients;It is noteworthy that patients who carry the VKORC1A/A genotype CYP4F2C/C genotype are particularly sensitive to WF dose,not only real-time detection of INR;also need to adjust the diet,strict control of VK1 intake in Clinical;if necessary,with a new type of oral anticoagulant to ensure the safety of patients with medication.Conclusion HPLC-MS/MS method for the determination of VK1 and WF enantiomers in plasma has been successfully applied to clinical trials of VK1 and WF enantiomer concentrations in patients with atrial fibrillation,and the method is Simple and easy,worthy of clinical application to further promote.It is the first time proved that VKORC1 and CYP4F2 gene polymorphism significantly affected plasma VK1 concentrations in the Chinese Han population.The results of this study suggest that the use of WF should pay attention to VKORC1 and CYP4F2 gene polymorphism,vitamin K1 levels and the level of INR,coagulation factors and other factors to better and faster play the role of WF anticoagulant.