HPLC-MS/MS间接测定类风湿关节炎患者红细胞中多聚谷氨酸化甲氨蝶呤的浓度

目的建立检测类风湿关节炎患者红细胞中多聚谷氨酸化甲氨蝶呤（methotrexate polyglutamates,MTXPGs）浓度的HPLC-MS/MS法。方法采用间接测定法,以同位素甲氨蝶呤-d3为内标,分别测定游离甲氨蝶呤（methotrexate,MTX）和总MTX（MTX+MTXPGs）,从而计算出红细胞中MTXPGs浓度。为测定游离MTX,全血样品经反复冻融、甲醇沉淀蛋白后,经LC-MS/MS进样分析,采用电喷雾离子源（ESI）正离子模式,多反应监测（MRM）。为测定总MTX,样品（反复冻融后）与200mmol·L-1抗坏血酸37℃孵育2. 5 h,酶促MTXPGs转化为MTX,蛋白沉淀后进样分析。结果全血MTX质量浓度在1～300ng·m L-1内线性关系良好（r=0. 999 4）,检测限（LOD）为0. 5 ng·m L-1,准确度（RE）在±5%内,日内和日间精密度（RSD）分别低于7. 96%和10. 88%。结论本方法快速、灵敏,具有高专属性和重现性,成功应用于临床上类风湿关节炎患者红细胞中MTXPGs浓度的监测

Effects of CYP3A4 and MDR1 genetic polymorphism on dosage and concentra- tion of tacrolimus in allogeneic hematopoietic stem cell transplantation patients

目的探讨异基因造血干细胞移植（HSCT）患者中CYP3A4S18B和MDRl C3435T基因多态性与他克莫司血药浓度、稳态剂量和不良反应之间的关系。方法采用直接测序法检测CYP3A4。18B和MDRl C3435T基因型，比较不同基因型患者之间他克莫司初始血药浓度／剂量×体表面积（p0／D0）、稳态剂量／体表面积（D’）及不良反应发生率的差异。结果16例HSCT患者CYP3A4S18B和MDRlC3435T等位基因突变频率分别为34．38％和43．75％。CYP3A4S1／S1基因型患者他克莫司初始风／D0’明显高于％18B等位基因携带者（P〈0．05）；稳态时D’显著低于＊18B等位基因携带者（P〈0．05）。MDRlC3435TTT型患者p0／D0’、D’与C等位基因携带者相比差异无统计学意义（P〉0．05）。CYP3A4S18B和MDRIC3435T基因多态性与急性移植物抗宿主病及他克莫司所致不良反应无关（P〉0．05）。结论HSCT患者CYP3A4＊18B基因多态性与他克莫司的p0／D0、D’相关。AIM To evaluate the effects of CYP3A4 and MDR1 genetic polymorphism on the concentration, dosage and adverse drug reaction of tacrolimus in allogeneic hematopoietic stem cell transplantation patients. METHODS The CYP3A4 ＊ 18B and MDR1 C3435T genotype were detected by direct sequencing method. Thedifferences ofpo/Do＇ ratio, D＇, acute graft versus host disease and adverse drug reaction were compared among dif- ferent genotype groups treated with tacrolimus. RESULTS Totally 16 patients were involved in this study. The frequencies of CYP3A4 ＊ 18B and MDR1 C3435T alleles were 34.38% and 43.75% , respectively. The po/Do＇ of tacrolimus in CYP3A4 ＊ 1/ ＊ 1 genotype groups was significantly higher than that of ＊ 18B allele carriers （P 〈 0.05） , and the D＇ was lower than ＊ 18B allele carriers obviously （P 〈0.05）. No significant association was found between the po/Do＇,D＇ of tacrolimus and MDR1 C3435T genotypes （P 〉 0.05）. There was also no significant correla- tion between CYP3A4 ＊ 18B and MDR1 C3435T gene polymorphisms and acute graft versus host disease and adverse drug reactions induced by tacrolimus （P 〉 0.05）. CONCLUSION CYP3A4 ＊ 18B genetic polymorphism in hemato- poietic stem cell transplantation patients is closely correlated to the po/Do＇ratio and D＇ of tacrolimus.厦门市科技惠民计划（编号3502820144023