Defining Oxidative Stress

The first step in addressing the role of oxidative stress (OxS) in diabetic complications is to define OxS. It is often defined as a shift in the pro-oxidant-antioxidant balance in the pro-oxidant direction. This definition of OxS is more descriptive than quantitative and chemical in nature. Philosophically, it implies a null point, a balance point at which there is no OxS—OxS occurs only when the balance is shifted toward the pro-oxidant direction. There is a conceptual flaw in this definition because it fails to recognize that OxS is a constant feature of biological systems. Peroxides, superoxide, hydroxyl radicals, and other reactive oxygen species (ROS), the mediators of OxS, are being formed continuously in the body and always exist at some steady-state concentration. The resulting oxidative damage to protein, DNA, and other biomolecules is a ubiquitous and universal consequence of life under aerobic conditions.

OxS might be better defined as "a measure of the prevailing level of ROS in a biological system." This definition acknowledges the continuous presence of ROS in biological systems, at some level determined by the relative rates of their formation and consumption. It accepts OxS as a normal feature of cellular metabolism rather than a disturbance in an equilibrium— OxS waxes and wanes but never disappears from the biological scene. Like metabolites, levels of ROS may differ at different stages in the feeding-fasting and diurnal cycles, among different subcellular compartments, among different cell types, in a cell at different stages in its growth and development, and even among cells of the same type but in different regions of a tissue. ROS are mediators of hormone action and growth factor and cytokine activity, and variations in ROS concentrations and OxS in intracellular and extracellular environments appear to be a central feature of regulatory biology (1).

From a quantitative viewpoint, OxS may be considered the sum of the products of the concentration and reactivity of numerous ROS in the cell. Should cells in which redox coenzyme systems are off-balance or in which reduced glutathione (GSH) is depleted also be considered oxidatively stressed? This is not a trivial question because alterations in ascorbate or GSH homeostasis are often cited as evidence of OxS. However, even a poor defense may be adequate in the absence of an oxidative challenge. Persons with glucose 6-phosphate dehydrogenase deficiency, for example, are asymptomatic until they are challenged by drugs or infection, leading to hemolytic anemia. Thus, a shift in the set point or concentration of redox coenzymes in a cell may predispose to oxidative stress, but the perturbation per se does not necessarily indicate that the cell is oxidatively stressed.

At this time it is not possible to quantify OxS, but this may eventually be achievable. The total radical antioxidant potential (TRAP) of plasma can now be estimated, for example, as the sum of a variety of antioxidant concentrations in plasma, including ascorbate, tocopherols, uric acid, and protein (2). Plasma TRAP is commonly expressed relative to that of a concentration of an antioxidant standard, such as Trolox (3). It may eventually be possible to develop a standard, such as "H202 equivalents" for assessing OxS in cells and tissues or actually to measure the concentration of specific oxyradicals by electron paramagnetic resonance spectroscopy (4).

Because of the many components and factors affecting OxS (Fig. 1), it is difficult, if not impossible, to assess the overall status of OxS in a biological system by measurement of the status of an individual or even several enzymes or antioxidant systems. Indeed, the interpretation of these data is often gratuitous. A low level of antioxidant enzyme is often interpreted as evidence of OxS, but high levels of superoxide dismutase are associated with OxS in the lungs in response to hyperbaric oxygen. Similarly, high plasma levels of uric acid are associated with inflammation in gout, whereas high levels of ascorbic may be pro-oxidant in the presence of free or heme iron.

In the absence of unambiguous assays or standards for measuring OxS, measurement of the consequences of OxS has been used as a surrogate. One approach for assessing the status of OxS is to measure the rate of excretion of products of oxidation of DNA, such as thymi(di)ne glycol or 8-oxodeoxy-guan(os)ine (5,6). Another is to measure the level of activation or expression of protein kinases, activator protein-1 or nuclear factor kappa B, growth factors (transforming growth factor-P, insulin-like growth factor-1, and vascular en-

Oxidative Stress a measure of the steady state level of reactive oxygen species a measure of the steady state level of reactive oxygen species



Hyperbaric oxygen Metals overload decompartmentalization Metabolic hyperglycemia glycation & AGEs polyol pathway activity Immunological inflammation

Complement activation autoimmune disease phagocytosis NADPH oxidase myeloperoxidase Drugs & Xenobiotics smoking alcohol

Antioxidant enzymes

SOD, CAT, GPx Antioxidant vitamins

A, C, E Other antioxidants bilirubin glutathione taurine ubiquinol urate Metal sequestration albumin transferrin ferritin hemopexin Dietary factors flavonolds micronutrients selenium

Figure 1 Some factors determining the status of OxS in biological systems.

dothelial growth factor) or heme oxygenase, all of which are involved in the response to oxidative stress (1,7) but also respond to other stresses such as reductive, thermal (heat/cold), and osmotic shock. A third approach, the focus of our research and of this article, is the measurement of the extent of oxidative damage to long-lived proteins, such as collagen, which integrates the time-averaged ambient level of OxS.

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