Iannone A, Bini A, Swartz H M, Tomasi A, Vannini V
Department of Biophysics, University of Illinois College of Medicine at U.C.
Biochem Pharmacol. 1989 Aug 15;38(16):2581-6. doi: 10.1016/0006-2952(89)90541-8.
Paramagnetic nitroxide spin labels have been extensively used to probe various biophysical and biochemical properties of the cellular environment. Recently nitroxides have been proposed as contrast enhancing agents in proton magnetic resonance imaging and contrast enhancement has been demonstrated in animal studies. Nitroxides, possessing a stable unpaired electron, increases the relaxation rates of protons, providing an enhancement of contrast. Nitroxides are metabolized intracellularly principally via reversible reduction to hydroxylamines. Rates of reduction depend on the physical characteristics of the nitroxides, in general 5-membered pyrrolidine ring are reduced more slowly than those with a 6-membered piperidine ring. Oxidation back to the nitroxide is relevant for lipid soluble hydroxylamines, while is low for water soluble ones. It is known that nitroxides are metabolized by subcellular fractions (cytosol, mitochondria, microsomes), though the enzymatic and non-enzymatic systems involved are poorly characterized. In the present study, the first of the necessary steps toward a systematic study of the metabolism of nitroxides by subcellular organelles, we have chosen to study the metabolism of 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl in isolated rat liver microsomes. Microsomes were able to reduce Tempol slowly without any substrate addition; when NADPH was added, the reduction rate substantially increased. In phenobarbitone induced rats the reduction rate was significantly higher than in not-induced microsomes. NADPH-dependent reduction rate was inhibited by thallium chloride (an inhibitor of the flavin-centered cytochrome P-450 reductase), superoxide dismutase, and by N-ethylmaleimide; menadione increased it. The Tempol reduction rate was not significantly affected by various cytochrome P-450 inhibitors with the sole exception of metyrapone. A solution containing purified cytochrome P-450 reductase and NADPH readily reduced Tempol. Microsomes fortified with NADPH were able to reduce Tempol at an appreciable rate. In order to distinguish between reduction of nitroxides to hydroxylamine or destruction of nitroxides following nitroxide reduction, microsomal suspensions were treated with a mild oxidant (ferricyanide 0.5-10 mM). The recovery varied from 40 to 60%, indicating a process of probe destruction leading to as yet unknown metabolites. The present study clearly indicates that, in this model system, cytochrome c (P-450) reductase and not cytochrome P-450 is responsible for the observed Tempol metabolism; along with hydroxylamine formation, other Tempol derived metabolites are formed during the process.
顺磁性氮氧化物自旋标记已被广泛用于探究细胞环境的各种生物物理和生化特性。最近，氮氧化物已被提议作为质子磁共振成像中的造影剂，并且在动物研究中已证实了造影增强效果。氮氧化物具有稳定的未配对电子，可提高质子的弛豫率，从而增强对比度。氮氧化物主要通过可逆还原为羟胺在细胞内代谢。还原速率取决于氮氧化物的物理特性，一般来说，五元吡咯烷环比六元哌啶环还原得更慢。对于脂溶性羟胺，氧化回氮氧化物是相关的，而对于水溶性羟胺则较低。已知氮氧化物可被亚细胞组分（胞质溶胶、线粒体、微粒体）代谢，尽管所涉及的酶促和非酶促系统的特征尚不明确。在本研究中，作为对亚细胞器中氮氧化物代谢进行系统研究的必要第一步，我们选择研究4-羟基-2,2,6,6-四甲基哌啶-N-氧化物在分离的大鼠肝微粒体中的代谢。微粒体在不添加任何底物的情况下能够缓慢还原Tempol；当添加NADPH时，还原速率大幅增加。在苯巴比妥诱导的大鼠中，还原速率明显高于未诱导的微粒体。NADPH依赖的还原速率受到氯化铊（黄素中心细胞色素P-450还原酶的抑制剂）、超氧化物歧化酶和N-乙基马来酰亚胺的抑制；甲萘醌则使其增加。除美替拉酮外，各种细胞色素P-450抑制剂对Tempol还原速率没有显著影响。含有纯化的细胞色素P-450还原酶和NADPH的溶液能够快速还原Tempol。用NADPH强化的微粒体能够以可观的速率还原Tempol。为了区分氮氧化物还原为羟胺或氮氧化物还原后氮氧化物的破坏，微粒体悬浮液用温和的氧化剂（0.5 - 10 mM铁氰化物）处理。回收率在40%至60%之间变化，表明存在导致未知代谢物的探针破坏过程。本研究清楚地表明，在该模型系统中，细胞色素c（P-450）还原酶而非细胞色素P-450负责观察到的Tempol代谢；在此过程中，除了形成羟胺外，还形成了其他源自Tempol的代谢物。
