Zhelev Zhivko, Matsumoto Ken-Ichiro, Gadjeva Veselina, Bakalova Rumiana, Aoki Ichio, Zheleva Antoaneta, Anzai Kazunori
Department of Biophysics, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
Gen Physiol Biophys. 2009 Dec;28(4):356-62. doi: 10.4149/gpb_2009_04_356.
The present study is focused on the mechanism(s) of electron-paramagnetic resonance (EPR) signal reduction kinetic of several nitroxyl radicals and nitroxyl-labeled anticancer drugs in physiological solutions in the context of their application for evaluation of oxidation/reduction status of blood and tissues--an important step in biomedical diagnostics and planning of therapy of many diseases. The nitroxyl derivatives were characterized with different size and water-solubility. Some of them are originally synthesized. In buffer, in the absence of reducing and oxidizing equivalents, the EPR signal intensity of all nitroxyls was constant with the time. In serum and cell cultured medium, in an absence of cells and in a negligible amount of reducing and oxidizing equivalents, there was no significant EPR signal reduction, too. In vitro (in freshly isolated blood samples), the EPR signal intensity was characterized with slow decrease within 30 min, presumably as a result of interaction between the nitroxyl derivative and blood cells. The EPR spectrum of hydrophobic nitroxyls showed a slight anisotropy in cell-containing solutions and it did not changed in non-cell physiological solutions. This suggests for a limited motion of more hydrophobic nitroxyls through their preferable location in cell membranes. In vivo (in the bloodstream of mice under anesthesia), the EPR signal reduction kinetic was characterized by two phases: i) a rapid enhancement within 30 s as a result of increasing of nitroxyl concentration in the bloodstream after its intravenous injection, followed by ii) a rapid decrease (approximately 80-100%) within 2-5 min, presumably as a result of transportation of nitroxyl in the tissues. The hydrophobic nitroxyls were characterized with stronger and faster decrease in EPR signal intensity in the blood in vivo, as a result of their higher cell permeability, rapid clearance from the bloodstream and/or transportation in the surrounding tissues. The hydrophilic nitroxyls persist in the bloodstream (in their radical form) for a comparatively long time. The data suggest that the hydrophobic cell-permeable nitroxyl derivatives are most appropriate for evaluation of cell and tissue oxidation/reduction status, while the hydrophilic nitroxyls (impermeable for cell membranes or with very slow cell permeability) are most appropriate for evaluation of oxidation/reduction status of blood using EPR imaging.
本研究聚焦于几种硝酰自由基和硝酰基标记抗癌药物在生理溶液中电子顺磁共振（EPR）信号衰减动力学机制，这与它们用于评估血液和组织的氧化/还原状态相关——这是生物医学诊断和多种疾病治疗规划中的重要一步。这些硝酰衍生物具有不同的大小和水溶性。其中一些是最初合成的。在缓冲液中，在没有还原和氧化当量的情况下，所有硝酰的EPR信号强度随时间保持恒定。在血清和细胞培养基中，在没有细胞且还原和氧化当量可忽略不计的情况下，EPR信号也没有显著降低。在体外（在新鲜分离的血液样本中），EPR信号强度的特征是在30分钟内缓慢下降，这可能是硝酰衍生物与血细胞相互作用的结果。疏水性硝酰在含细胞溶液中的EPR谱显示出轻微的各向异性，而在无细胞生理溶液中则没有变化。这表明疏水性更强的硝酰通过其在细胞膜中的优先定位而具有有限的运动。在体内（在麻醉小鼠的血流中），EPR信号衰减动力学具有两个阶段：i）静脉注射后，由于血流中硝酰浓度增加，在30秒内快速增强，随后ii）在2 - 5分钟内快速下降（约80 - 100%），这可能是硝酰在组织中运输的结果。由于其更高的细胞通透性、从血流中快速清除和/或在周围组织中运输，疏水性硝酰在体内血液中的EPR信号强度下降更强且更快。亲水性硝酰（以其自由基形式）在血流中持续相对较长时间。数据表明，疏水性细胞可渗透的硝酰衍生物最适合评估细胞和组织的氧化/还原状态，而亲水性硝酰（细胞膜不可渗透或细胞通透性非常缓慢）最适合使用EPR成像评估血液的氧化/还原状态。
