High-throughput spectrophotometric assay of reactive oxygen species in serum
High-throughput spectrophotometric assay of reactive oxygen species in serum
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DOI：
10.1016/j.mrgentox.2007.04.006
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发表时间：
2007-07-10
影响因子：
1.9
通讯作者：
Hayashi, Tomonori
中科院分区：
文献类型：
--
作者：
Hayashi, Ikue;Morishita, Yukari;Hayashi, Tomonori
活性氧代谢物衍生物（D-ROM）试验已被开发用于测定血液样品中氧中心自由基的量，作为氧化应激的标志物。本研究旨在改进D-ROM测试，并利用临床化学分析仪开发自动化检测系统。将5微升血清加入到96孔微量滴定板的1孔中，得到总共240微升含有烷基胺和金属的反应溶液。然后自动混合、孵育并使用具有过渡金属催化能力的分光光度计在505 nm处显色测量活性氧物质（ROS）水平。举例来说，该测定系统用于测量吸烟者和从不吸烟者的ROS血清水平。通过该系统测定的血清ROS水平与自由基和过氧化物的量相关，自由基和过氧化物与体内的各种分子反应并形成稳定的代谢物。该测试可以使用冷冻血清以及新鲜血清。该系统的内部和内部偏差在5%以内，并在4和500 mg/l过氧化氢的范围内显示出一致的线性。吸烟者的血清ROS水平随着每天吸烟的数量而增加（每天每包增加36.5%; P < 0.0001）。该检测系统将是一种简单，廉价，可靠的工具，用于评估人群中的氧化应激。我们对吸烟的初步研究结果表明，该检测系统在各种流行病学和临床环境中具有应用潜力。(c)2007年Elsevier B. V.。All rights reserved.
The derivatives of reactive oxygen metabolites (D-ROM) test has been developed to determine the amount of oxygen-centered free radicals in a blood sample as a marker of oxidative stress. This study aims to improve the D-ROM test and develop an automated assay system by use of a clinical chemistry analyzer. Five microliters of serum was added to 1 well of a 96-well microliter plate for a total 240 mu l of reaction solution containing alkylamine and metals. This was followed by automatic mixing, incubation and measurement of reactive oxygen species (ROS) levels as a color development at 505 nm using a spectrophotometer with catalytic capability for transition metals. This assay system was used to measure serum levels of ROS in cigarette smokers and never-smokers, by way of example. The levels of serum ROS determined by this system correlate with the amounts of free radicals and peroxides, which reacted with various molecules in the body and formed stable metabolites. This test can use frozen sera as well as fresh ones. The inter- and intra-deviation of this system was within 5% and showed consistent linearity in the range between 4 and 500 mg/l of hydrogen peroxides. Serum ROS levels among smokers increased with the number of cigarettes smoked per day (36.5% increment per pack per day; P < 0.0001). This assay system will be a simple, inexpensive, and reliable tool for assessing oxidative stress in human populations. Our preliminary results on cigarette smoking imply that this assay system has potential for application in various epidemiological and clinical settings. (c) 2007 Elsevier B.V.. All rights reserved.