La Favor J D, Anderson E J, Hickner R C
Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Physiol Res. 2014;63(3):387-92. doi: 10.33549/physiolres.932587. Epub 2014 Feb 24.
Excessive production of reactive oxygen species (ROS) are implicated in the pathogenesis of numerous disease states. However, direct measurement of in vivo ROS in humans has remained elusive due to limited access to appropriate tissue beds and the inherently short half-lives and high reactivity of ROS. Herein, we describe a novel technique by which to measure in vivo ROS in human skeletal muscle. Microdialysis probes were inserted into the vastus lateralis of eight healthy volunteers. Amplex Ultrared, a highly specific fluorogenic substrate for hydrogen peroxide (H(2)O(2)), and horseradish peroxidase (HRP), were perfused through microdialysis probes, and outflowing dialysate was collected and fluorescence was measured. Extracellular H(2)O(2) that crossed the microdialysis membrane was measured via fluorescence of the dialysate. Superoxide dismutase (SOD) was then added to the inflowing perfusion media to convert any superoxide crossing the microdialysis membrane to H(2)O(2) within the microdialysis probe. Fluorescence significantly increased (P=0.005) upon SOD addition. These data demonstrate the feasibility of measuring both in vivo H(2)O(2) and superoxide in the extracellular environment of human skeletal muscle, providing a technique with a potential application to a wide range of circulatory and metabolic studies of oxidative stress.
活性氧（ROS）的过度产生与多种疾病状态的发病机制有关。然而，由于难以获取合适的组织床，以及ROS固有的短半衰期和高反应性，在人体中直接测量体内ROS仍然难以实现。在此，我们描述了一种测量人体骨骼肌中体内ROS的新技术。将微透析探针插入八名健康志愿者的股外侧肌。通过微透析探针灌注对过氧化氢（H₂O₂）具有高度特异性的荧光底物Amplex Ultrared和辣根过氧化物酶（HRP），收集流出的透析液并测量荧光。通过透析液的荧光测量穿过微透析膜的细胞外H₂O₂。然后将超氧化物歧化酶（SOD）添加到流入的灌注介质中，以将任何穿过微透析膜的超氧化物转化为微透析探针内的H₂O₂。添加SOD后荧光显著增加（P = 0.005）。这些数据证明了在人体骨骼肌细胞外环境中测量体内H₂O₂和超氧化物的可行性，为氧化应激的广泛循环和代谢研究提供了一种具有潜在应用价值的技术。
