Davis D L, Murthy J N, Napoli K L, Kahan B D, Gallant-Haidner H, Yatscoff R W, Soldin S J
Department of Laboratory Medicine, Children's National Medical Center, Washington, DC 20010, USA.
Clin Biochem. 2000 Feb;33(1):31-6. doi: 10.1016/s0009-9120(99)00088-0.
We have previously identified a minor immunophilin of 52 kDa molecular weight capable of binding tacrolimus and sirolimus. Because immunophilins are capable of binding both parent drug and metabolites and HPLC assays are typically used to assess parent drug in clinical situations, we used this immunophilin in a radioreceptor assay (RRA) to determine if any metabolites not included in the HPLC measurement would bind to the immunophilin and be associated with thrombocytopenia in patients receiving sirolimus.
We tested 51 steady-state trough whole blood samples from non-thrombocytopenic patients and 51 steady-state trough samples from thrombocytopenic patients and compared them to HPLC measurements of parent drug in the same samples. We also tested whole blood samples spiked with authentic sirolimus metabolites using RRA to ascertain the effect these metabolites have on the technique.
We found minimal cross-reactivity in this assay for sirolimus metabolites (binding ranged from <10% to 26%), and good correlation of the radioreceptor assay with HPLC (linear regression slope 0.92, y-intercept 0.79). There was no statistically significant difference between the RRA and HPLC results in two patient groups-thrombocytopenic and non-thrombocytopenic-using the paired t-test (p<0.005) and Bland-Altman analysis.
These findings indicate that although the RRA could be substituted for HPLC in therapeutic drug monitoring, the 52 kDa immunophilin does not offer an advantage in terms of detecting metabolites associated with thrombocytopenia. However, the RRA offers the advantages of shorter turnaround time, smaller sample volume and potential for automation.
我们之前鉴定出一种分子量为52 kDa的小亲免蛋白，它能够结合他克莫司和西罗莫司。由于亲免蛋白能够结合母体药物及其代谢产物，且在临床情况下通常使用高效液相色谱法（HPLC）来评估母体药物，因此我们在放射受体分析（RRA）中使用这种亲免蛋白，以确定HPLC测量中未包含的任何代谢产物是否会与亲免蛋白结合，并与接受西罗莫司治疗的患者的血小板减少症相关。
我们检测了51份来自非血小板减少症患者的稳态谷值全血样本和51份来自血小板减少症患者的稳态谷值样本，并将它们与同一样本中母体药物的HPLC测量结果进行比较。我们还使用RRA检测了添加了真实西罗莫司代谢产物的全血样本，以确定这些代谢产物对该技术的影响。
我们发现该分析中对西罗莫司代谢产物的交叉反应极小（结合率范围为<10%至26%），且放射受体分析与HPLC具有良好的相关性（线性回归斜率为0.92，截距为0.79）。使用配对t检验（p<0.005）和Bland-Altman分析，在血小板减少症和非血小板减少症这两组患者中，RRA和HPLC结果之间没有统计学上的显著差异。
这些发现表明，尽管在治疗药物监测中RRA可以替代HPLC，但52 kDa亲免蛋白在检测与血小板减少症相关的代谢产物方面并无优势。然而，RRA具有周转时间短、样本量小和自动化潜力等优点。
