ers, made with the highest-grade chemicals, can contain trace amounts of transition metals, and great care should be made to minimize this when performing studies of isolated tissues, membranes, and enzyme systems. In our experience, if this is not done, fully one half of the O2 formed in a study of isolated tissue homogenates can be attributed to transition metal contaminants. This artifact can be eliminated by treating buffers for several hours with chelex and by using diethylenetriamine-penta-acetic acid in near millimolar concentrations. Experiments with the buffer as a background control are also important to assure that the signals are arising from the tissue sample and not the buffer. One should also be cautioned about the literature regarding assays for ROS, which is replete with in vitro experiments using very contrived experimental situations to disprove the validity of an assay for ROS. 2–5 In any biological assay, it is possible to generate artifacts using extreme conditions that are not encountered in vivo. Future experiments should be designed to study these assays in the context of intact tissues and under proper physiological conditions, rather than in artificial “test tube” situations. Results from studies such as these should not be used to refute the use of these assays under properly controlled physiological circumstances. Finally, there have been attempts to examine activities of various enzymes by adding their substrates to intact tissues. For example, NADPH added to intact vessels has been used as a measure of NADPH oxidase activity. These studies are hard to interpret, because it is unclear how extracellular

NADPH enters the cell to be used as a substrate for the NADPH oxidase. There is no established mechanism for cellular uptake of NADPH, and the binding domain to the Nox proteins (the catalytic subunits of the NADPH oxidase) is intracellular. In fact, a recent article showed that NADPH exerts its effects by stimulating purinergic receptors. 6 Thus, one is not studying NADPH oxidase activity using this approach but rather purinergic signaling. When subcellular enzyme fractions, such as membranes, are used, receptor signaling pathways are disrupted, and the added substrates are more likely to reach their respective enzyme binding sites to promote ROS production.