Evidence and possible consequences of the phosphorylation of nucleoside reverse transcriptase inhibitors in human red blood cells
Evidence and possible consequences of the phosphorylation of nucleoside reverse transcriptase inhibitors in human red blood cells
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DOI：
10.1128/aac.00831-06
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发表时间：
2007-06-01
影响因子：
4.9
通讯作者：
Grassi, Jacques
中科院分区：
文献类型：
--
作者：
Durand-Gasselin, Lucie;Da Silva, David;Grassi, Jacques
核苷类逆转录酶抑制剂（NRTI）在单核细胞中的细胞内代谢已被深入研究，但在红细胞（RBC）中的代谢却被忽视。然而，其他类似核苷（特别是利巴韦林）的磷酸化先前已经描述过。在这项研究中，我们首次研究了NRTI在人红细胞中的磷酸化。检测了人类免疫缺陷病毒感染患者红细胞中细胞内齐多夫定（AZT）单磷酸、AZT三磷酸、拉米夫定（3TC）三磷酸和替诺福韦（TFV）二磷酸以及内源性dATP、dGTP和dTTP的存在。我们观察到3TC、TFV和内源性嘌呤脱氧核苷选择性磷酸化以产生它们的三磷酸部分的证据。相反，没有发现AZT磷酸盐代谢物的痕迹，只有微弱的dTTP信号可见。红细胞和外周血单核细胞（PBMC）细胞内TFV二磷酸和3TC三磷酸水平的比较进一步强调了NRTI在每种细胞类型中的代谢特异性。这些发现提出了红细胞参与药物-药物相互作用、药物药代动力学和药物诱导毒性的问题。此外，PBMC样品的典型制备方法是梯度密度离心，并不能防止它们被RBC污染。我们证明了PBMC中RBC的存在阻碍了临床PBMC样本中细胞内TFV二磷酸和dATP水平的准确测定。因此，我们建议在PBMC制备过程中使用氯化铵溶液去除红细胞，以提高细胞内药物监测的准确性和精密度。
The intracellular metabolism of nucleoside reverse transcriptase inhibitors (NRTI) in mononuclear cells has been thoroughly studied, but that in red blood cells (RBC) has been disregarded. However, the phosphorylation of other analogous nucleosides (in particular, ribavirin) has been described previously. In this study, we investigated for the first time the phosphorylation of NRTI in human RBC. The presence of intracellular zidovudine (AZT) monophosphate, AZT triphosphate, lamivudine (3TC) triphosphate, and tenofovir (TFV) diphosphate, as well as endogenous dATP, dGTP, and dTTP, in RBC collected from human immunodeficiency virus-infected patients was examined. We observed evidence of a selective phosphorylation of 3TC, TFV, and endogenous purine deoxynucleosides to generate their triphosphate moieties. Conversely, no trace of AZT phosphate metabolites was found, and only faint dTTP signals were visible. A comparison of intracellular TFV diphosphate and 3TC triphosphate levels in RBC and peripheral blood mononuclear cells (PBMC) further highlighted the specificity of NRTI metabolism in each cell type. These findings raise the issue of RBC involvement in drug-drug interaction, drug pharmacokinetics, and drug-induced toxicity. Moreover, the typical preparation of PBMC samples by gradient density centrifugation does not prevent their contamination with RBC. We demonstrated that the presence of RBC within PBMC hampers an accurate determination of intracellular TFV diphosphate and dATP levels in clinical PBMC samples. Thus, we recommend removing RBC during PBMC preparation by using an ammonium chloride solution to enhance both the accuracy and the precision of intracellular drug monitoring.