Novel method for detection of reactive oxygen species in vivo in human skeletal muscle

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.