Invited critical reviewUrinary biomarkers of oxidative status
Highlights
► Urine presents a valuable resource to study redox balance in human populations. ► F2-isoprostanes and 8-oxodG are recommended for monitoring individuals over time. ► F2-isoprostanes are recommended for comparisons between individuals. ► Allantoin, acrolein-lysine, and dityrosine are recommended as promising biomarkers.
Introduction
The focus of this review is to evaluate the applicability of existing biomarkers of oxidative status to human studies or epidemiological research. This involves consideration of many factors (which are discussed later, Table 1); and therefore, such evaluation can only be conducted for already studied, as opposed to novel, biomarkers. For this reason, we focus on oxygen-derived damage to biological molecules, because biomarkers of damage produced by reactive nitrogen species are less studied.
Reactive oxygen species (ROS) are constantly produced in aerobic organisms by normal metabolic processes, such as cellular respiration, antibacterial defense, and others [1]. In addition, external exposures (such as ionizing radiation, smoking, and toxins) also induce production of ROS [1]. As a result, exposure to ROS is ubiquitous, and a certain level of oxidative damage is always present in any individual. To counteract their damaging effects, aerobic organisms have developed multiple defense systems [1]. These antioxidant agents include enzymes (such as superoxide dismutase, catalase, glutathione peroxidases), sequesters of metal ions, and endogenous antioxidants (e.g. glutathione, ubiquinol, bilirubin, uric acid, α-tocopherol, and ascorbic acid). The two opposing processes – ROS production and antioxidant defense – set constitutive levels of ROS within the tissues and at the systemic level. Differences in both the intensity of ROS generation and the effectiveness of the antioxidant defense produce variability in oxidative status between individuals [2]. Variability in oxidative status within an individual between tissues as well as between individuals results from a complex interaction of multiple factors, including genetic [3], [4] and epigenetic differences, endogenous promoters of ROS (such as iron and copper) [1], chronic inflammation [5], [6] or other chronic conditions. It should be noted that most chronic conditions occur at the tissue level, while most biomarkers consider oxidative stress at the systemic level, with the implicit assumption that greater tissue-specific ROS production will be reflected by an increased systemic oxidative status. Although there is no evidence to support or refute this assumption, a distinction between tissue-specific and systemic oxidative status should be acknowledged.
The term “oxidative stress” is widely used, but, as noted by Halliwell, this term “is vaguely defined”, referring to “a serious imbalance between production of reactive species and antioxidant defense” [7]. Because some levels of oxidative damage are present in every individual, the question arises as to which levels represent a “normal” (non-stress) range versus pathological elevation, which could be defined as oxidative stress. Because of this uncertainty, we believe that the term “oxidative status” is a term that can be more logically and consistently applied to both stress and non-stress states of oxidative load. For example, relatively large scale human studies (n ≥ 100) reveal a wide variation of any oxidative status biomarker in human populations. For example, in 2828 subjects of the Framingham Heart Study, urinary levels of iPF 2α-III (a marker of lipid peroxidation) ranged from 10 to 1845 ng/mmol creatinine [8]. In 100 healthy children and adolescents, the range 8-OHdG (a marker of DNA oxidative damage) levels in urine was 4.6–27.2 ng/mg creatinine [9]. It is not clear which levels should be considered “normal” (non-stress) and which represent a serious imbalance between ROS generation and antioxidant defense (stress). The term “oxidative status” therefore seems more applicable.
Because ROS have short lifetimes and cannot be directly detected in humans [10], a reasonable alternative approach is the measurement of biomarkers that are the products of non-enzymatic reactions between biological molecules and ROS [1], [7]. The involvement of enzymes in the formation of biomarkers would introduce an inaccessible level of variability, and so these products do not make good biomarker candidates. Assessment of non-enzymatically formed biomarkers circumvents this problem and provides a direct index of the extent of oxidative modifications produced by ROS. Although the levels of such oxidative modifications do not measure the ROS levels per se, they are assumed to be proportional to the ROS levels. Therefore, the core requirement for a biomarker of oxidative status is its validation in vivo against a known oxidative stressor, i.e. a compound that produces ROS in biological systems as measured by electron spin resonance spectroscopy directly. In response to this well-recognized need, the National Institute of Environmental Health Sciences (NIEHS) has established an initiative to conduct a comparative study of biomarkers of oxidative stress (BOSS). The BOSS project tests responsiveness and specificity of the commonly used oxidative indices in an established model of oxidative stress — carbon tetrachloride (CCl4) poisoning in rodents [11], [12], [13], [14]. Similar to this approach, we developed a clinical model of oxidative stress, based on doxorubicin (DOX)-based chemotherapy [15], [16]. DOX has been demonstrated to generate superoxide and hydrogen peroxide in vitro; this ROS production has been observed in animals, at pharmacological levels, using electron spin resonance spectroscopy [17], [18]. This and other important characteristics for evaluation of biomarkers are presented in Table 1. Currently, only a handful of oxidative status biomarkers have been validated in either animal or clinical models.
This review focuses on urinary biomarkers because they represent the least invasive way to assess individual oxidative status and can be used in large-scale human studies. Also, urine is a better matrix than blood/plasma for measurement of oxidative modifications of biological molecules, because it has a much lower organic as well as inorganic metal content, i.e. lower levels of the material that can be oxidized as well as lower levels of the ROS promoters. Therefore, urine is less liable for artificial increase of oxidative markers during sample collection and storage.
Section snippets
F2-isoprostanes
F2-isoprostanes are formed during non-enzymatic oxidation of arachidonic acid by different types of free radicals, including reactive oxygen species [19], [20]. Depending on the position where the oxygen molecule is added to arachidonic acid, four regioisomers are formed, giving each of the four F2-isoprostane series. Furthermore, each series comprises 16 stereoisomers. Mainly two nomenclatures are used for isoprostanes (Taber et al. [21] and Rokach et al. [22]). However, other nomenclatures of
Protein adducts produced by lipid peroxidation products
These biomarkers have been partially discussed in Section 2.2. In this section, we summarize the mechanism of their formation and their qualities as biomarkers. As indicated in the previous section, the more commonly studied products of lipid peroxidation include MDA, 4-HNE, 4-ONE, and acrolein. Each of these products can form adducts to the amino acid residues of the protein, predominately targeting cysteines, histidines, and lysines [72], [73], [74]. Many of these reactions occur via Michael
Oxidative modifications of DNA in urine
ROS attack on DNA produces a large number of purine and pyrimidine-derived lesions [117]. The most studied biomarker is 8-hydroxy-2′-deoxyguanosine (8-oxodG), a stable end product of non-enzymatic DNA oxidation. Urinary 8-oxodG levels have been validated as a sensitive biomarker of oxidative stress in an animal model using the administration of CCl4 [12]. Similar validation against a known oxidative stressor in humans has not been conducted, although an increase in urinary 8-oxodG was
Allantoin
Allantoin is the predominant product of non-enzymatic oxidation of uric acid by many types of free radicals [7], [137], [138]. Whereas in most mammals allantoin is formed by enzymatic oxidation of uric acid (that is catalyzed by urate oxidase), in humans allantoin is formed only by non-enzymatic oxidation of urate (because urate oxidase is silenced in humans by several mutations) [139]. Thus, uric acid presents as the terminal product of purine metabolism in humans, with basal urate levels much
Conclusions
Urinary biomarkers present a great opportunity to conduct large-scale studies because specimen collection is non-invasive and the organic and metal content is low, minimizing sample oxidation during collection and storage. Also, urinary levels of the biomarkers present intergraded indices of redox balance over a longer period of time compared to blood levels, which may make them more sensitive to predicting chronic conditions while also decreasing intra-individual variability of the
Acknowledgments
We thank Katherine Zeph (DCI, Duke University Medical Center) for help in editing the text. This research was supported by the National Institutes of Health grants 5P50CA108786, 1R01DK081028 and 5R25-CA126938-02.
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