1.1 What is Oxidative Stress? . . . 2
1.2 Oxidative Stress . . . 2
1.3 Measurement of Oxidative Stress . . . 3
1.4 Biomarkers of Oxidative Stress . . . 3
References . . . 4
Contents Chapter
Oxidative Stress
Barry B. Halliwell and Henrik E. Poulsen
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2 Barry Halliwell and Henrik E. Poulsen
1 1.1 What is Oxidative Stress?
Oxygen was discovered by the Swedish scientist Carl Wilhelm Scheele and reported in his thesis Luft und dem Feuer (Air and Fire) from Uppsala and Leipzig in 1777. Later it was realized that in higher animals, breathing supplies the cells with oxygen and serves to eliminate the carbon dioxide formed from cellular metabolism. The well-known reac- tion between oxygen and fuel (e.g., carbon in wood) requires high temperatures. How- ever, it was discovered that special proteins in the cells—enzymes—are able to catalyze this combustion at body temperature. The trick is that the enzymes can bind both oxygen and the substrate and bring them into close proximity so that chemical reaction can oc- cur and the liberated energy can be stored as ATP for use elsewhere and later in the cell.
Oxygen was consequently considered a good thing. However, experience from ex- posure to high-oxygen concentrations in deep-sea divers and premature babies showed that organ damage could be a result of exposure to too much oxygen. As we learned to measure oxidative damage better, we realized that it happens in vivo even at normal atmospheric O2 levels.
It is now well established that free radical chemistry occurs in biology, and it is also becoming increasingly clear that free radicals not only function in cellular respiration, as damaging species, but also in the signaling systems within cells. As an example of the ac- ceptance of these phenomena, the journal Nature Medicine has “oxidative stress” among its limited number of keywords for paper submission.
1.2 Oxidative Stress
Oxidative stress was initially defined by Sies (1985, 1986) as a serious imbalance between oxidation and antioxidants, “a disturbance in the prooxidant–antioxidant balance in fa- vor of the former, leading to potential damage.” The definition seems simple; however, it builds on definitions about oxidation, antioxidants, and balance.
The definition of oxidation seems also simple: loss of electrons by a species, gain of oxygen, or loss of hydrogen. However, if something is oxidized, something else must be reduced. The effect depends on the context. As put forward by Buettner (1993), there is a pecking order of oxidants. In biology, substances very high in the pecking order (e.g., the hydroxyl radical) will almost always be an oxidant; other substances (e.g., NO· or H2O2, can act as oxidants or reductants, depending on whether they react with sub- stances lower or higher in the pecking order.
An antioxidant is more difficult to define. A popular (but not comprehensive) defini- tion was put forward by Halliwell: An antioxidant is any substance that, when present at low concentrations as compared with those of an oxidizable substrate, significantly delays or prevents the oxidation of that substrate. (Its shortcomings are discussed in an upcoming book, Free Radicals in Biology and Medicine, 4th ed., Halliwell and Gut- teridge, 2006.) As argued above, the chemical terms are oxidation and reduction, and an antioxidant is clearly different from a reducing agent. A reducing agent may even be a prooxidant if it reduces oxygen to free radicals or converts transition metal ions to lower oxidation states that react more readily with peroxides. Many biological reducing agents are Janus-faced: They can be anti- or prooxidants, depending on the levels of O2 and transition metal ions around.
Balance or imbalance is poorly defined. Generally, we think of our environment as
3 Chapter 1 Oxidative Stress an oxidative environment, and this presumably is true for outer surfaces of the body.
However, our knowledge is limited regarding intracellular conditions. They are gener- ally reducing, but with some subcellular variations (e.g., the endoplasmic reticulum is more oxidized than the mitochondria). Even within the cytosol there might be consider- able differences between locations close to the cell membrane and close to the nuclear membrane. It could be that there is a balance between oxidants and antioxidants, but it seems rather unlikely. The cell must rapidly and transiently modulate its redox state to send signals.
1.3 Measurement of Oxidative Stress
The term oxidative stress rests on definitions that are not always sufficiently clear; con- sequently, oxidative stress is a somewhat vague term, as is oxidative damage. Halliwell and Whiteman (2004) have defined the latter as “the biomolecular damage that can be caused by direct attack of reactive species during oxidative stress.”
In very simple noncompartmentalized systems, e.g., an in vitro system with a limited number of oxidants and targets for oxidation, it is often self-evident how oxidative stress is defined and measured: by simply measuring antioxidants, free radicals, and other re- active species (RS) and doing a balance sheet. Care must be taken: What is seen depends on what is measured. RS can be measured directly (e.g., by electron spin resonance or various trapping methods), or indirectly by examining end products of their reaction with biomolecules (oxidative damage).
More-complicated systems need much more careful approaches. A cell is compart- mentalized with many different molecular targets for oxidation. Lipids in the outer cell membrane most probably have quite a different oxidative environment as compared with the inner mitochondrial membranes. Nucleic acids also exist in different compartments, and the oxidative environment is quite different between transcribing and nontranscrib- ing DNA in the nucleus, DNA in the mitochondria, and the different types of RNA.
Likewise for protein oxidation, plasma proteins and cellular proteins exist in different compartments and thereby in different oxidative environments.
At the next level of complexity, different organs and different parts of the organs may present quite different conditions. For example, the liver receives a mixture of arterial and venous blood and thereby much lower oxygen concentrations than most other or- gans, and even within the liver, cells in the first part of the sinusoid live in a quite differ- ent oxidative environment than those in the end of the sinusoid.
That the structure and organization is complicated should not make researchers re- frain from trying to define the system under investigation. Rather, it should in many cases make us more humble in the interpretation of data obtained in complicated sys- tems, and more careful in defining and understanding the limitations of simple methods used to investigate complicated systems.
1.4 Biomarkers of Oxidative Stress
When considering the effect of increased oxidative stress (or decreased for that matter), the issue of target is mandatory. From a general point of view, lipids, proteins, carbohy- drates, and DNA are considered important macromolecules. For measurement of the
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oxidation of these molecules, the biomarker approach is most often used. A biomarker of disease is defined as a molecular indicator of a specific biological property, a biochem- ical feature or facet that can be used to measure the progress of disease or the effects of treatment. The reader should be aware that there are other forms of biomarkers, e.g., biomarkers of exposure.Such a biomarker should fulfill certain criteria, as given in Table 1.1.
Table 1.1 Criteria for the ideal biomarker of oxidative stress No. Criterion
1 It should be predictive of development of the disease or condition under investigation (ex- ample: lipid peroxidation in plasma should predict arteriosclerotic events or cardiovascular death).
2 It should reflect biological event(s) that can be related to the pathogenesis of the disease or condition.
3 It should be stable over short periods (weeks, months) in stable individuals.
4 It should produce identical results when the same sample is measured in different laborato- ries.
5 The sample from which it is measured should be stable on storage.
6 The biomarker should relate to immediate events within short periods or should reflect integration of events over a well-defined period.
7 Preferentially, the biomarker measurement should be noninvasive or measurable in an easily available biological specimen (example: urine, sputum) or in minimally invasively obtainable biological specimen (example blood or plasma).
8 The cost of sample analysis should be low, and it should be possible to perform a large num- ber of analyses within a reasonable time.
Whereas the methods to measure events that are related to oxidative stress—be it oxi- dation, free radicals, or antioxidants—are numerous, it should be realized that very few, if any, of them fulfill the criteria in Table 1.1, and hence cannot yet be considered bio- markers of oxidative stress. To our knowledge, there are no publications that in a proper scientific way fulfill criterion 1, namely predictive of development of disease. Nonethe- less, recent studies with F2-isoprostanes and 3-nitrotyrosine look promising in this di- rection.
References
Buettner GR (1993) The pecking order of free radicals and antioxidants: lipid peroxidation, α-to- copherol, and ascorbate. Arch Biochem Biophys 300:535–543
Halliwell B, Whiteman M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142:231–255 Sies H (1985) Introductory remarks. In: Sies H (ed) Oxidative stress. Academic, London, pp 1–8 Sies H (1986) Biochemistry of oxidative stress. Angew Chem Int Ed Eng 25:1058–1071
