两种检测方法测定甲氨蝶呤血药浓度的比较分析

目的:比较荧光偏振免疫分析法(FPIA)和酶增强免疫法(EMIT)监测甲氨蝶呤(MTX)血浆药物浓度的相关性。方法:收集接受大剂量甲氨蝶呤化疗后的患儿血液样品,分别用FPIA和EMIT法进行测定,考察2种测定方法的相关程度。结果:在MTX血浆浓度≥0.30μmol·L-1时,以FPIA法测定结果(X)和EMIT法测定结果(Y)所作的线性回归方程如下:Y=1.0451 X+0.1153,两组结果间存在较好的相关性(Spearmen相关系数为0.968),EMIT方法的测定结果高于FPIA法所测结果,差异有统计学意义(P<0.05)。结论:MTX血浆药物浓度≥0.30μmol·L-1时,EMIT法测定结果高于FPIA法,且和后者相关性良好,经相互换算后可用于指导四氢叶酸钙(CF)的解救。     

EMIT和FPIA测定丙戊酸、甲氨喋呤和环孢霉素A血药浓度的比较研究

目的比较均相酶放大免疫分析法(EMIT)和荧光偏振免疫分析法(FPIA)测定丙戊酸(VPA)、甲氨喋呤(MTX)和环孢霉素A(CsA)血药浓度结果,并评价两种测定方法所得结果的相关性。方法收集患者服药后的血样,采用EMIT和FPIA同时测定VPA、MTX和CsA血药浓度;以FPIA测定值为X,以EMIT测定值为Y,进行线性回归,评价其相关性。结果所得线性回归方程如下:YVPA=1.894 1+1.190 3X,r=0.983;YMTX=0.099 24+1.136X,r=0.992;YCsA=1.146 5+0.846 1X,r=0.971。EMIT测定血浆中VPA血药浓度较FPIA高11.2μg.mL 1,EMIT测定MTX血药浓度较FPIA高0.22μmol.L 1,EMIT测定CsA血药浓度较FPIA低20.6 ng.mL 1,差异有统计学意义(P<0.05)。结论 EMIT与FPIA测定VPA、MTX和CsA血药浓度差异具有统计学意义,在治疗药物监测中应予以注意。     

高效液相色谱法、荧光偏振免疫分析法和质谱法监测丙戊酸血药浓度的比较

目的 比较高效液相色谱法(HPLC)、荧光偏振免疫分析法(FPIA)和质谱法(HPLC-MS)监测丙戊酸血药浓度的相关性。方法 收集规律服用丙戊酸制剂达稳态后的血清样本207例,分别用3种方法进行测定,考察3种测定方法的相关性和差异性。结果 3种测定方法的相关性良好,但总体水平上HPLC法与FPIA法测定结果相比较差异有统计学意义。FPIA法测定丙戊酸血药浓度较HPLC法高0.7μg·m L-1(95%CI:-8.6~9.9μg·m L-1),当血药浓度数据处于低、中浓度(<90μg·m L-1)时,2种方法所测定结果均值相近,且差异无统计学意义;当血药浓度数据处于高浓度(>90μg·m L-1)时,2种测定方法所获得的数据存在显著性差异。结论 临床用不同方法监测丙戊酸血药浓度时,对不同测定方法的差异应予以关注并作相应调整,尤其是当血药浓度范围>90μg·m L-1时,HPLC法与FPIA法所测定结果之间不可直接进行比较分析。     

色谱法测定人血浆中万古霉素的浓度

目的：建立HPLC法和液相色谱-串联质谱（LC-MS/MS）法检测人血浆中万古霉素的浓度,并分别与FPIA法进行比较。方法：HPLC法：100μL血浆样品加入等体积10%的高氯酸沉淀,振荡离心后取上清液进样。采用Agilent Eclipse XDB-C18（250 mm×4.6 mm,5μm）,柱温为35℃,流动相为0.05 mol·L-1 KH2PO4（pH=3.2）-甲醇（74∶26,v/v）,流速为1 mL·min-1,检测波长为230 nm。 LC-MS/MS法：100μL血浆样品用300μL乙腈沉淀,振荡离心后取上清液进样。色谱柱为Agilent Eclipse XDB-C18（100 mm ×2.1 mm,3.5μm）,柱温为40℃,流动相为水（含0.1%甲酸）-乙腈（90∶10,v/v）,流速为0.20 mL·min-1。采用电喷雾化离子源（ESI）,多离子反应模式（MRM）检测,万古霉素和内标去甲万古霉素的监测离子对分别为m/z 725.0→144.0和718.5→144.0。采用建立的HPLC法和LC-MS/MS法测定95份临床样本,并与FPIA法进行相关性和测定方法的偏倚分析。结果：HPLC法和LC-MS/MS法测定万古霉素的线性范围分别为1.2~96μg·mL-1和0.4~96μg·mL-1,低、中、高三种浓度质控品日内和日间相对标准差（RSD）均〈15%。两种方法与FPIA法有很强的相关性（r=0.9621,P〈0.0001和r=0.9466,P〈0.0001）,无明显偏倚。结论：建立的HPLC和LC-MS/MS法快速、灵敏、准确,样本测定结果与FPIA法无显著差异,适用于万古霉素的常规血药浓度监测及人体药物代谢动力学研究。     

HPLC法与EMIT法检测血样中甲氨蝶呤浓度的比较研究

目的:比较HPLC法和酶放大免疫法（EMIT）检测血样中甲氨蝶呤（MTX）浓度的相关性。方法:分别用HPLC法和EMIT法测定患者使用大剂量MTX 44 h后的血样,考察2种测定方法的区别和相关程度。结果:HPLC法和EMIT法检测的MTX血浓度差异有统计学意义（P<0.05）,EMIT法测定结果较HPLC法高0.12μmol·L^-1,通过passing-badlok回归分析2种方法具有良好的相关性（r=0.996 2）。结论:HPLC法和EMIT法测定MTX血浆药物浓度结果具有明显差异,在临床进行MTX药物浓度监测中应予以关注并作相应调整。     

HPLC-MS/MS法测定白血病患者血浆甲氨蝶呤浓度

目的:建立测定白血病患者使用大剂量甲氨蝶呤化疗后血浆中甲氨蝶呤浓度的HPLC-MS/MS法,并应用于临床治疗药物监测,为临床合理用药提供可靠依据。方法:采用多索茶碱作内标,血浆样品经含内标甲醇沉淀蛋白处理。色谱柱为Ultimate XB-C18,柱温40℃,流速0.75 m L·min-1,流动相为含0.1%甲酸和2 mmol·L-1乙酸铵的水溶液和含0.1%甲酸的甲醇溶液,梯度洗脱。质谱检测方式为电喷雾离子阱正离子模式,MRM扫描,监测甲氨蝶呤m/z 455.200→308.200,多索茶碱m/z 267.100→181.100。结果:甲氨蝶呤浓度在0.022~2.200μmol·L-1内线性关系良好,Y=4.56 x+0.081(r=0.996 0)。结论:该方法简便、准确、快速,检测的特异性及灵敏度高,适用于甲氨蝶呤的血药浓度测定。     

HPLC-MS/MS法和EMIT法分析人血浆中霉酚酸浓度的相关性

目的:建立高效液相色谱串联质谱法(HPLC-MS/MS)测定人血浆中霉酚酸的浓度,并与酶放大免疫分析法(EMIT)测定的结果进行对比。方法:经霉酚酸治疗的患者血浆样本共111份,以EMIT法进行常规监测后再用HPLC-MS/MS法复测,采用Kolmogorov-Smirnova检验、配对t检验、Pearson相关分析、Passing-Bablok回归法和Bland-Altman一致性限度图研究2种方法测定结果的相关性和一致性,并进行经济学分析。结果:EMIT法和HPLC-MS/MS法测定霉酚酸血浆药物浓度结果的差异有统计学意义,EMIT法检测值(C_EMIT)比HPLC-MS/MS法检测值(C_HPLC-MS/MS)高(27.28±28.62)%,Passing-Bablok回归方程为C_HPLC-MS/MS＝0.931×C_EMIT -0.367,r=0.988。此外,HPLC-MS/MS法检测单个样本的成本比EMIT法低35.4元。结论:该研究建立的HPLC-MS/MS法与EMIT法具有较好的相关性,与EMIT法相比,HPLC-MS/MS法成本低,减轻了患者的经济负担,可替代EMIT法用于霉酚酸的常规监测。     

LC-MS/MS法测定WY-42在大鼠血浆中的质量浓度

目的建立HPLC-MS/MS法测定灌胃给药后大鼠血浆中WY-42的质量浓度。方法色谱柱为Waters TerraMS C18柱(150 mm×2.1 mm,5μm),流动相为甲醇-体积分数2%甲酸水溶液(体积比90∶10),柱温40℃,流速为0.2 mL·min-1,WY-42和内标格列吡嗪检测离子对分别为m/z377.2→215.1和m/z 446.2→321.2。结果 WY-42质量浓度在5.0⁵00.0μg·L-1内线性关系良好,回归方程为A=1.35×10-2ρ-1.06×10-2(r=0.993 3),日内精密度和日间精密度的RSD不大于15.0%。结论该方法简便、灵敏,适用于测定大鼠血浆中WY-42的质量浓度。     

三种检测方法测定甲氨蝶呤血药浓度的比较分析

目的比较高效液相色谱法（HPLC）、荧光偏振免疫分析法（FPIA）和酶增强免疫法（EMIT）监测甲氨蝶呤（MTX）血浆药物浓度的相关性。方法收集接受大剂量甲氨蝶呤化疗后的患者的血液样品,分别用HPLC、FPIA和EMIT法进行测定,考察3种测定方法的相关程度。结果多配对样本Friedman检验提示3种方法间差异有显著性,Wilcoxon检验结果提示HPLC法和FPIA法检测结果之间差异无显著性,EMIT法结果与另外两种方法之间差异有显著性。3种检测方法的的回归分析结果为：Y HPLC=1.00X FPIA＋0.015（r=0.978,P〈0.000 1）;Y HPLC=0.94X EMIT-0.079（r=0.956,P〈0.000 1）;Y FPIA=0.95X EMIT-0.089（r=0.927,P〈0.000 1）。结论 3种甲氨蝶呤检测方法之间还是存在差异,但这种差异是可以预见性的,且可以通过换算消除,临床治疗和研究工作要予以注意。     

液相色谱-质谱联用法监测急性淋巴细胞白血病患儿中甲氨蝶呤血药浓度及临床应用

目的:建立以液相色谱-质谱联用(LC/MS)法测定血清中甲氨蝶呤(MTX)的方法并用于临床适时监测,进一步探讨MTX剂量-血药浓度与药物不良反应(ADR)发生的相关性。方法:建立以ESI离子源,SCI(+)扫描方式,离解电压1.1 KV,雾化氮气1.5 L·min-1,干燥氮气15 L·min-1,干燥温度200℃,模块加热温度400℃,加氢目标离子m/z455,碎片离子455,456的LC/MS法测定血清中MTX的方法并进行方法学验证,对59例临床ALL患儿接受大剂量甲氨蝶呤(HD-MTX)化疗后48,72,96 h时间点271例次MTX血药浓度监测和ADR发生情况观察。结果:方法的回归方程Y=(6.139 28E7)X-528120(n=6,r=0.999 7),MTX血清浓度在0.01～160μmol·L-1范围线性良好,最低定量限0.01μmol·L-1(S/N≥3),通过方法学验证符合临床生物样本分析要求;对HD-MTX患儿化疗后48 h与72 h点血药浓度监测。结果组内两监测点血药浓度变异较大(CV%>97%),经独立样本t检验存在显著性差异(P=0.000),然而不同病理诊断分组间两时间点血药浓度无显著性差异(P>0.05);MTX剂量与ADR发生经多元变量非参数的偏相关分析结果相关系数rs=0.133。结论:该检测方法具有快捷、准确、痕量测定血中MTX浓度的特点,可用于临床及科研MTX血药浓度的监测;四氢叶酸钙(CF)的救援应通过监测MTX血药浓度并根据结果调整剂量,监测C48h和C72h点用于预判MTX蓄积中毒并指导CF救援有一定的临床实际意义;通过MTX监测血药浓度制定个体治疗方案,ADR发生与MTX剂量之间并非强相关。     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg.kg) or i.p. (50 mg.kg) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) l.h. kg in the male rat and 10.6 (95% CI: 7.5, 15.0) l.h. kg in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p 0.001) in plasma obtained from the male (8.8 2.0%) compared with the female rat (11.7 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg x kg(-1)) or i.p. (50 mg x kg(-1)) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) 1 x h(-1) x kg(-1) in the male rat and 10.6 (95% CI: 7.5, 15.0) 1 x h(-1) x kg(-1) in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was approximately 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p < 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p < 0.001) in plasma obtained from the male (8.8 +/- 2.0%) compared with the female rat (11.7 +/- 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Interindividual variability in metabolic activation in humans in vivo

In assessing interindividual variability in metabolic activation, the toxic metabolite is often too unstable for conventional analysis. Possible alternatives include a stable product of the reactive metabolite e.g. cysteinyl derivatives of N-acetyl-4-benzoquinoneimine, the toxic metabolite of paracetamol, adducts with DNA or protein, and indirect measurement of the activity of the enzyme(s) producing the active metabolite. An example of the last approach is the use of furafylline, a highly specific inhibitor of human CYP1A2, to determine the extent of the metabolic activation of the cooked food mutagens PhIP and MeIQx. The extent of inhibition, determined from levels of unchanged amine in urine, is an indirect measure of the activity of the activation pathway. Further refinement of this approach, allied to improved measures of the biological process of interest should prove of value in evaluating interindividual variability and its role in the risk assessment process.     

Theophylline kinetics in peripheral tissues in vivo in humans

Several biochemical and cellular effects have been described for methylxanthines under in vitro conditions. However, it is unknown, whether threshold concentrations required to exert these effects are attained in target tissues in vivo. We therefore employed the microdialysis technique for measuring theophylline concentrations in peripheral tissues under in vivo conditions.Following in vitro and in vivo calibration, microdialysis probes were inserted into the medial vastus muscle and into the periumbilical subcutaneous adipose layer of healthy volunteers. Following single oral dose administration of 300 mg or i.v. infusion of 240 mg theophylline, in vivo time courses of theophylline concentrations were monitored in tissues and plasma. Major pharmacokinetic parameters (c_max, t_max, AUC) were calculated for plasma and tissue time courses. The mean AUC_tissue /AUC_plasma-ratio was 0.56 (p.o.) and 0.55 (i.v.) for muscle and 0.55 (p.o.) and 0.72 (i.v.) for subcutaneous adipose tissue.We conclude that microdialysis provides important information on the distribution and the tissue pharmacokinetics of theophylline.Abbreviations FPIA Fluorescence polarisation immuno assay - AUC Area under the curve - t_max Time to peak concentration - c_max Peak concentration     

休克时血中氧自由基的变化

本实验测定10名休克患者血浆和红细胞的丙二醛(MDA)、血浆总抗的氧化活性(AOA)的含量。结果表明:休克病人红细胞膜和血浆 MDA 含量(4.298±0.722;5.348±0.834)与对照组(3.235±0.682;4.356±1.081)比较明显增高(P<0.05);血浆 AOA(39.65±7.858)与对照组(48.21±10.81)比较明显降低(P<0.01)。提示:休克时,患者机体内自由基反应增强是引起组织细胞损伤的原因之一。     

Changes in angiopoietin expression in glomeruli involved in glomerulosclerosis in rats with daunorubicin-induced nephrosis

AIM: To study the potential pathological role of endogenous angiopoietins in daunorubicin-induced progressive glomerulosclerosis in rats. METHODS: Seventy male Wistar rats were allocated randomly into a daunorubicin group (DRB; n=40) or a control group (n=30). The rats in the DRB group were injected with DRB (15 mg/kg), in their tails. Subsequently, at intervals of 1, 2, 4, 6, 8, and 12 weeks, 5 male Wistar rats in each group were chosen randomly for 24 h urinary protein quantitative measurements (24 h UPQM), and determination of plasma tumor necrosis factor alpha (TNF-alpha), angiopoietin-1 (Ang1), and angiopoietin-2 (Ang2) levels. Kidney sections were examined by electron microscopy, Periodic Acid Schiff (PAS) staining, immunohistochemical staining and in situ hybridization histochemistry. RESULTS: As glomerulosclerosis progressed in the DRB group, expression of Ang1 mRNA and protein in glomeruli decreased and expression of TNF-alpha protein, Ang2 mRNA and protein in glomeruli increased. Expression of Ang1 mRNA and protein in glomeruli were negatively correlated with 24 h UPQM, Fn protein expression, and mean area of extracellular matrix (MAECM). In comparison, expression of Ang2 mRNA and protein in glomeruli were positively correlated with 24 h UPQM, Fn protein expression and MAECM; furthermore, there was a positive correlation between plasma Ang2 and 24 h UPQM. Plasma TNF-alpha and expression of TNF-alpha in glomeruli were positively correlated with expression of Ang2 mRNA and protein in glomeruli. There was a negative correlation between Ang1 protein expression and Ang2 protein expression in glomeruli. CONCLUSION: During DRB-induced glomerulosclerosis, podocyte injury led to a shift in the balance of Ang1 and Ang2 in glomeruli. Increased TNF-alpha in plasma and glomeruli may upregulate Ang2 expression in glomeruli. Elevated Ang2 in both plasma and glomeruli may mediate protein permeability through the glomerular filtration barrier. Moreover, local expression of Ang2 may facilitate the progress of glomerulosclerosis by upregulating a component expression of extracellular matrix.     

Trichinellosis in immigrants in Switzerland

We describe a case of trichinellosis diagnosed at the Division of Infectious Diseases, Hospital of Lugano, in January 2009. This case was associated with a cluster of cases and was traced to the consumption of contaminated meat after a wild boar hunt in Bosnia.