Redox Biology 1 (2013) 483–491

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Redox Biology
journal homepage: www.elsevier.com/locate/redox

Review Article

Biological markers of oxidative stress: Applications to cardiovascular
research and practice
Edwin Ho a, Keyvan Karimi Galougahi a,b, Chia-Chi Liu a, Ravi Bhindi a,b,
Gemma A. Figtree a,b,n
a
North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Sydney, Australia
b
Department of Cardiology, Royal North Shore Hospital, Sydney, Australia

art ic l e i nf o a b s t r a c t

Article history: Oxidative stress is a common mediator in pathogenicity of established cardiovascular risk factors.
Received 22 July 2013 Furthermore, it likely mediates effects of emerging, less well-defined variables that contribute to residual
Received in revised form risk not explained by traditional factors. Functional oxidative modifications of cellular proteins, both
31 July 2013
reversible and irreversible, are a causal step in cellular dysfunction. Identifying markers of oxidative
Accepted 31 July 2013
stress has been the focus of many researchers as they have the potential to act as an “integrator” of a
multitude of processes that drive cardiovascular pathobiology. One of the major challenges is the
Keywords: accurate quantification of reactive oxygen species with very short half-life. Redox-sensitive proteins with
Biomarker important cellular functions are confined to signalling microdomains in cardiovascular cells and are not
Cardiovascular disease readily available for quantification. A popular approach is the measurement of stable by-products
Glutathionylation
modified under conditions of oxidative stress that have entered the circulation. However, these may not
Oxidative stress
accurately reflect redox stress at the cell/tissue level. Many of these modifications are “functionally
Prognosis
silent”. Functional significance of the oxidative modifications enhances their validity as a proposed
biological marker of cardiovascular disease, and is the strength of the redox cysteine modifications such
as glutathionylation. We review selected biomarkers of oxidative stress that show promise in
cardiovascular medicine, as well as new methodologies for high-throughput measurement in research
and clinical settings. Although associated with disease severity, further studies are required to examine
the utility of the most promising oxidative biomarkers to predict prognosis or response to treatment.
& 2013 The Authors. Published by Elsevier B.V. Open access under CC BY license.

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Biomarkers of oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Isoprostanes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Malondialdehyde. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
Oxidative protein modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
Nitrotyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
S-glutathionylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
Myeloperoxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
Oxidized low-density lipoprotein and oxidized phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
ROS-induced changes in gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488

Abbreviations: CVD, cardiovascular disease; ROS, reactive oxygen species; IsoP, isoprostane; MDA, malondialdehyde; TBARS, thiobarbituric acid reacting substance; O2d  ,
superoxide; dOH, hydroxyl radical; HO2d, hydroperoxyl radical; ONOO  , peroxynitrite; HOCl, hypochlorous acid; H2O2, hydrogen peroxide; NO2, nitrogen dioxide; MPO,
myeloperoxidase; OxLDL, Oxidized low-density lipoprotein; GSH, glutathione (reduced)
n
Correspondence to: North Shore Heart Research Group, Level 12, Kolling Building, Royal North Shore Hospital, St Leonards, NSW 2065, Australia. Tel.: þ61 2 9926 4915;
fax: þ61 2 9926 6521.
E-mail address: gemma.figtree@usyd.edu.au (G.A. Figtree).

2213-2317 & 2013 The Authors. Published by Elsevier B.V. Open access under CC BY license.
http://dx.doi.org/10.1016/j.redox.2013.07.006

. . . . . . . . . . . . . . . . . . . . high-sensitivity troponin I [4] and high-sensitivity C-reactive protein (hs-CRP) for cardiovascular risk prediction [5]. . . . . . . ROS also induce specific post-translational modifications that alter the function of important cellular proteins and signalling IsoPs are a family of stable. . . . . . . organ and system function is see text for other abbreviations. . . . . . specificity. PUFA ¼polyunsaturated fatty acids. . . . .g. . 489 Disclosures. . . . etc. . . . . . . . . . . . . . . . . . . . . . . . . 489 Introduction recognized as a key determinant of the validity of the marker. . . . . . prognosis measure the modification [14]. . . . . or pharmacological responses to a thera. storage conditions and specimen preparation steps. . . . . . . . . . . In addition to generalized oxidation resulting in cell dysfunction. . . .) and evidence for guiding management and targets of oxidation because of their molecular structure abundant improving patient outcome [6]. . . . . . . . . . . . . . Table 1 summarizes the advantages and individualization of therapy in cardiovascular disease (CVD). oxidation resistance assays many sources including mitochondria. . . . . . 1. . . . . . . . . . . . . necrosis or Isoprostanes apoptosis. . . . 1. This is shown schematically in Fig. . Other factors influencing the clinical applicability of a ROS bio- The term biomarker has been defined by The National Insti. . . . . this has proven to be a complex challenge given the evanescent nature of ROS. . . . . . . . . . . its performance characteristics (e. . . . sensitivity and reproducibility of the assay used to peutic intervention” [1]. . . of the oxidative modification on cell. . . . . . . . . . . 489 Sources of funding . . . . . . . . . . . . . . . . . . some are known to have direct effects on function of the molecule (e. . specificity. . . . . . . . . . . . . . . . . . . . . . Fig. . . . . . Factors that The important role of free radical oxidation of cellular compo- determine the clinical utility of a biomarker include the ease and nents in CVD has been recognized since the proposal of the cost of measurement. . . . . . . . . . . . . . . . . . . . . Reactive oxygen species (ROS) are derived from escent products of lipid peroxidation. . . . . . . . . . The short half-life of these species makes them excellent signalling molecules but confounds their measurement in the circulation of complex biological systems by standard approaches such as spin-trapping [7]. . . . . . . . . . . The important role of oxidative erated from the peroxidation of arachidonic acid. . . . . . . . . . Other lipid oxidation products that have role of oxidative stress in the pathophysiology of CVD is well been explored as biomarkers include lipid hydroperoxides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA. . . . . . . . . . . . . . . . . . . the stability of the biomarker throughout various and evaluated as an indicator of normal biological processes. . . . . . . . . . .g. . . . . . . . . . . . . . . . sensi. . Two of the most well studied The most promising biomarkers are the ones that closely markers of lipid peroxidation are isoprostanes (IsoPs) and mal- correlate with the pathophysiological process of the disease. . . . . . . . . . . . . . . . . . . . . . . . with reactive double bonds [17]. . . proteins and carbohydrates are examples of molecules that can be modified by excessive ROS in vivo. . . . . . traditional cardiovascular risk factor analysis. Ho et al. . .8]. . . . nitric oxide synthases and NADPH oxidase [9]. . . . . 488 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . marker include the ease of obtaining an appropriate biological tutes of Health as “a characteristic that is objectively measured specimen. . Instead the focus has been on measuring stable markers in the circulation that may reflect systemic oxidative stress. . . The functional significance or causal role marked with n. . Biomarkers of oxidative stress Biomarkers of oxidative stress can be classified as molecules that are modified by interactions with ROS in the microenviron- ment. . . . . . . . 489 References . . . . . . . . . . . . . . . . . . xanthine oxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lipids (including phos- pholipids). . . The ondialdehyde (MDA). . . . . . . . fluor- established [7. Formation pathways of selected biomarkers of oxidative stress. . and shown to have value in addition to steps for biomarker development for clinical application. . . prostaglandin-like compounds gen- pathways in the heart [10–13]. . . . but others merely reflect the degree of oxidative stress that have been shown to have prognostic significance in cardiovascular disease are in the local environment. / Redox Biology 1 (2013) 483–491 Measuring the net antioxidant capacity of the serum. . . . . . . . a polyunsaturated stress in cardiovascular pathophysiology has encouraged quantifi- cation of ROS as a promising biomarker reflecting the disease process. . . . 488 Future directions . . . . . . include N-terminal pro-B-type natriuretic peptide (NT-proBNP) for heart failure [2]. Of these modifications. . . . .484 E. . . . inhibit enzyme Fig. . and molecules of the antioxidant system that change in response to increased redox stress. . . 2 demonstrates the timeline and required into clinical practice. . . . . . . . . . . oxidative theory of atherogenesis [15. uncoupled and oxysterols. . . . . . . . . . . . . . . . . . Applications include diagnosis. . . . . . . . . . . . and disadvantages of the selected oxidative stress biomarkers Examples of circulating biomarkers that have been incorporated discussed below. . However. This review will discuss current biomarkers of oxidative stress focusing on their advantages and disadvantages in research and clinical setting and future directions in this field. . . . . . . . . . . . . . . . . . .16]. . . GSH¼ glutathione (reduced). . . . . . . glycated haemoglobin (HbA1c) for glycaemic control in diabetes Lipid peroxidation [3]. . . . . . . Biomarkers function). . . . . and the pathogenic processes. . . . . . Lipids are susceptible tivity. . . . . . . . . .

74–76. enzyme-linked immunosorbance assays (ELISA) and myocardial ischaemia/reperfusion [27]. may not reflect that of cellular antioxidant quantification to Commercial kits available to microdomains that are CVD risk need further measure antioxidant capacity. diabetes trometry (GC/MS). Studies show results MDA can predict progression of CAD and carotid atherosclerosis at 3 years. Biomarker Advantages Disadvantages Comments References IsoPs Can be detected in various Current methods of No evidence linking this [22. scaling up. Nitrotyrosine Human studies have Circulating levels are not Nitrotyrosine formation on [43. and can then be quantified in tissues. but significant ex vivo and dOH). IsoPs are elevated in association with risk IsoPs can be measured using gas chromatography–mass spec. cardiovascular tissues. plays a causal role in renal and easy to use but have variable performance and results vasoconstriction and associated renal failure [28].24.69. obesity. MPO Commercial assays available.39.58. in the presence of a range of MS) or requires further CV risk factors. Commercial ring in rhabdomyolysis as a result of redox cycling between ferric immunoassay kits for IsoPs have been developed that are cheap and ferryl forms of myoglobin.55] demonstrated association equivalent to tissue levels. increasing clinical severity. blood and that the utility of plasma F2-IsoPs is limited by time to analysis. however does not the TBARS assay. increasing Reduction in OxLDL by ELISAs for OxLDL detection [80–83] OxLDL correlates with antioxidant pharmacotherapy readily available. A small study revealed formation include: (1) mitochondrial electron transport chain (super. validation (immunoassay kits). E. fatty acid present in phospholipids of cell membranes [18]. IsoPs are for 36 h [25]. for CVD risk prediction. vasculature) where modification occurs presents a clinical obstacle. Serum antioxidant capacity GPX-1 demonstrated to be Antioxidant activity in serum Clinical relevance of [90] inversely proportional to CAD. The independence of circulating IsoPs levels of renal Levels of IsoPs in plasma and urine samples have been shown or hepatic function allows them to more directly reflect IsoPs to correlate with in vivo oxidative stress in a number of animal and production and oxidative stress [22]. that the concentrations of F2-IsoPs in human plasma measured by oxide (O2d ) and hydroxyl radical (dOH)). demonstrated as biomarkers artefact. samples reflect that in microarray technology. Sources of free radicals for IsoPs a biomarker to epidemiological studies. Access to tissue (myocardium. / Redox Biology 1 (2013) 483–491 485 Table 1 Advantages and disadvantages of various biomarkers of oxidative stress. Good reproducibility from frozen samples. [22]. ROS-induced changes to The expression of several Microarray technology can be It is unclear if expression [87. human studies [20. The correlate poorly with mass spectrometric techniques [24]. OxLDL Elevated in CAD. urine. liquid chromatography–mass spectrometry mellitus. so called because they contain F-type prostane rings. has not been matched by Also is predictive of future reduction in CVD severity.88] gene expression genes may be measured manually and computationally profiles of cells in biological simultaneously using expensive.65] eNOS and Na þ –K þ pump prone to methodological investigated as biomarker. important to the pathogenesis investigation Reproducibly quantified of CVD. particular cardiovascular with CAD independent of Current detection methods are proteins have direct effect on traditional risk factors expensive and impractical for function. potentially increasing the power of this biomarker. Ex vivo stability is an important consideration when applying dins from arachidonic acid [19]. MDA Technically easy to quantify TBARS assay is non-specific Shows promise as a clinical [33. F2-IsoP levels . and hyperhomocysteinemia [22].13.40] spectrophotometrically using (can detect aldehydes other biomarker. as well as role in pathogenesis. factors such as cigarette smoking. CAD in healthy population. No study of stability of urinary F2-IsoPs has been published to our are the most stable of the IsoPs family and show the most potential knowledge. S-glutathionylation S-glutathionylation of SERCA. Detection of S-glutationylation Modified Hb currently being [10. which generation of IsoPs from arachidonic acid is independent of the are is still regarded as the gold standard for IsoP quantification cyclooxygenase enzyme that catalyzes the formation of prostaglan. performance. as well as (LC/MS). This is presumably secondary to on-going oxidation subsequently released from the cell membrane into circulation by of lipids in plasma sample during prolonged storage and suggests phospholipases [21]. F2-IsoPs.97–99] Strong evidence that MPO and time to analysis.26]. Influenced by sample storage MPO is a promising biomarker [1. hypercholesterolaemia. Elevated F2-IsoPs occur- radioimmunoassay in plasma and urine samples [23].34. (2) P450 enzymes (O2d GC/MS at 0 and 24 h ex vivo were similar. Ho et al. despite frozen sample storage. as a biomarker. (3) lipoxygenase (hydroperoxyl radical (HO2d)) and (4) artefactual generation of F2-IsoPs occurred in plasma stored on ice transition-metal catalyzed formation of free radicals [20]. urine) and quantification are impractical biomarker to clinical has been shown to be elevated for large-scale screening (GC/ outcomes yet. correlates with CVD risk.25] samples (serum. ELISA kits to than MDA) and sample have a functional impact on detect MDA also have good preparation can influence the pathophysiology of CVD.

Other ways of mentation with various antioxidants including α-lipoic acid and quantifying protein nitration are immunocytochemical and immu- aminoguanidine [36.486 E. Schematic timeline of required steps in biomarker development. from discovery in the Laboratory to clinical application after validation in large scale clinical trials. 2. suitable for high throughput analysis. although mechanism for nitration in vivo have been a matter of controversy. However. Antibodies against specific nitrated pro- with documented coronary artery disease found that serum levels teins have recently been developed and tested [46]. only few are regularly applied to patients in clinical practice. In rats. with aldehydes other than of oxidative stress. However. a small cross- vitamin C and/or E supplementation. [32]. Myeloperoxidase (MPO). / Redox Biology 1 (2013) 483–491 Fig. with its transition metal centre. downside of this is that the presence of nitrite in the sample and induced diabetic models [35–37].37]. MDA interacts with proteins and is itself potentially atherogenic. elevated TBARS levels predicted carotid muscle cells (VSMCs) [19. Clinical trials attempting to decrease atherosclerotic plaque progression over 3 years as assessed by F2-IsoP production in humans with antioxidant therapy. A study of TBARS in 634 patients anti 3-NO2-Tyr antibodies. can react MDA is typically quantified from plasma samples with the most with ONOO  to yield oxo-metal complexes and NO2 thus facilitating popular method being a colorimetric assay based on the reaction the nitration reaction [43]. protein extracts from biological samples can be completely The TBARS assay has been applied as an indicator of oxidative hydrolyzed before quantification of nitrotyrosines by chromatogra- stress in a number of cardiovascular disease models. Although many ROS biomarkers have reached clinical trials level. such as carotid wall thickness on ultrasound [40]. independently of traditional risk factors and inflammatory mar- tion. TBARS phy. Plasma TBARS concentration in the acid precipitation of proteins or acid hydrolysis can influence these experimental models can be normalized through supple. . Potential concentrations are elevated in the plasma of streptozotocin. CVD and response to therapies.26]. Ho et al. and may participate in the actual for a major vascular procedure in a 3-year follow-up period pathogenesis of atherosclerosis through effects on vasoconstric. and proliferation of vascular smooth kers [39]. have generated mixed results sectional study revealed no significant association between ele- [30]. Results are expressed as moles of 3-NO2-Tyr/Tyr. the nitration of tyrosine residues in the sample [45]. Alter- with improved specificity [34]. and OxLDL and macrophages and thereby to promote atherosclerosis results in a nitro group adduct on susceptible tyrosine residues [42]. It is also not known if reduction of IsoP levels correlates with vated TBARS and the presence of CVD after correcting for blood improvement in cardiovascular risk. Protein tyrosine nitration is mediated by reactive nitrogen species and have been postulated to impair the interaction between such as peroxynitrite (ONOO  ) and nitrogen dioxide (NO2). are increased in human atherosclerotic lesions compared with of TBARS could predict major cardiovascular events and the need normal vascular tissue [29]. platelet aggregation. TBARS were found to be elevated in the nohistochemical assays based on either monoclonal or polyclonal serum of cigarette smokers [38]. Further clinical studies are glucose levels [41]. Malondialdehyde MDA is generated in vivo via peroxidation of polyunsaturated Oxidative protein modifications fatty acids. Animal and human studies therefore support a required to determine if IsoPs may be useful in prognostication in potential role of lipid oxidation in predicting the progression of CVD processes or individualization of treatment strategies. natively. These antibody-based assays are typically standard technique [44]. MDA's reaction with lysine residues generates lysine– Nitrotyrosine lysine cross-links [31] which have been identified in apolipopro- tein B (apoB) fractions of oxidized low density lipoprotein (OxLDL). Several ELISA kits to detect MDA are also spectrometry (MS/MS) coupled with GC or HPLC as the current gold commercially available. Free nitrotyrosine (3-NO2-Tyr) represents the MDA reacting with TBA to produce compounds that absorb in the turnover of nitrated proteins and can be measured by tandem mass same range as MDA [33]. this TBA reacting substances measures reflective of tyrosine nitration have been used as indicators (TBARS) assay lacks specificity for MDA. Further studies are required to establish validated against measurement of MDA by high-performance a normal basal range of circulating free 3-NO2-Tyr in healthy liquid chromatography (HPLC) and demonstrate good performance individuals (proposed by Pelluffo and Radi to be 1 nM) [43]. Although the precise intermediates and between MDA and thiobarbituric acid (TBA). Moreover.

macrophages a reactive cysteine residue and the abundant cellular tripeptide and monocytes. Nitrotyr.61]. impairing its the functional significance of glutathionylation of haemoglobin is not function in cholesterol efflux [73]. current methods of detecting nitrotyrosine are relatively biomarker for CVD processes and merits further exploration of expensive and impractical for scaling up for high-throughput accurate quantification methods and predictive value for prognos- screening and analysis. the direct usefulness of measuring glutathionyla. nitration of proteins and lipoproteins may also play S-glutathionylation of susceptible proteins is commonly measured a direct pathophysiological role. be quantified in biological samples using a range of commercially- dium and/or vascular tissue – with altered function resulting in available ELISA plates. Inflamma- S-glutathionylation of haemoglobin has been proposed as a marker of tory cells recruited into the vascular wall release MPO-derived ROS oxidative stress [62].48]. the formation of a disulphide bridge between inflammatory cells such as activated neutrophils. humans with dilated cardiomyopathy compared with healthy controls [47]. in the case of fibrinogen. and is increased in patients with diabetes.g. 3. hypochlorous acid on protein tertiary structure and function in a manner similar to (HOCl). Ho et al. and other key affects the quantification of MPO. However. which has potent antimi- accessing the tissue in which these functionally relevant modifica. For example. E. Elevated circulating MPO levels well established. The impact of glutathionylation of each measured spectrophotometrically [68]. nitrotyrosine is associated with increased activity and acceleration of clot formation [53]. . the circulating cells in parallel with modification of the same molecule cardiovascular events in patients presenting to emergency with in the vasculature or myocardium. / Redox Biology 1 (2013) 483–491 487 Nitrotyrosine formation on enzymes such as sarcoplasmic reticulum Ca2 þ -ATPase (SERCA2a) [47. potentially. there are the use of MS techniques. proteins with important functional consequences is a promising more. make MPO levels one of the most promising and requires careful specimen handling and preparation [65]. [10. In a case control study of 100 patients with established coronary artery disease (CAD). MPO acts as a master enzyme in the generation of glutathione. For example. that it is enriched within atheromatous plaques [70]. and increased risk of myocardial infarction and An additional challenge facing the use of glutathionyated death in patients with acute coronary syndrome [76]. Further. manganese superoxide dismutase (SOD) [49]. prospective studies. chest pain [75]. circulating nitrated proteins and and recently established by our Group for β1 subunit of the lipoproteins may not accurately reflect the degree of nitration of Na þ –K þ pump [67]). biomarkers of oxidative stress for clinical cardiologists [77]. MPO-derived ROS can then modify lipids. regulation of a number of key cellular proteins. SERCA [13] as the formation of guaiacol oxidation products that can be easily and Na þ  K þ pump [10. In contrast to glutathionyla. SERCA [13]. including endothelial MPO function can be measured by peroxidase activity assays such nitric oxide synthase (eNOS) [58]. Measurement of S-glutathionylation of target key proteins in the vessel wall or tissue of interest [57]. many studies have also tions occur. high MPO levels were able to predict finding a susceptible candidate protein whose function is modified in increased risk of developing CAD in healthy individuals [74]. or. as well as the ready availability of glutathionylated proteins is prone to methodological artefact of commercial assays. and Na þ –K þ pump [10.60]. Fig. ryanodine receptor [59]. anti-glutathione antibody (as has been developed for actin [66] In the case of atherosclerosis. phosphorylation [10. biomarker of oxidative stress would be substantially strengthened by In prospective studies. In contrast. tyrosine hydro- xylase [51] and aldolase A [52] inhibits their normal activity. these issues before nitrotyrosine can be adopted as an oxidative stress biomarker ready for the clinic. MPO plays an essential role in host immune defences because tion of these proteins as biomarkers is hampered by difficulty in of its unique ability to generate HOCl. prostacyclin synthase [50]. erythrocytes). non-reducing conditions [14]. This oxidative modification can exert effects (H2O2) to species including dOH. The argument for the use of S-glutathionylation as a have been found to be associated with the presence of CAD [1]. bioavailability of nitric oxide [71]. or gain-of-function effect as with nitrated fibrinogen (for a comprehensive review of nitration products refer to Peluffo and Radi [43]). nitrated LDL is taken up using low-resolution techniques such as Western Blotting under by macrophages leading to foam cell formation [56]. generate atherogenic OxLDL tion of eNOS.64]. ONOO  . heparin in the patient signalling pathways particularly relevant to cardiovascular function or the collection tube could alter measurements [69]. Researchers have therefore investigated the potential of implicated MPO in the pathogenesis of atherosclerosis.60]. More efficient approaches include Despite the pathophysiological role of nitration. 3). The results proteins as biomarkers of oxidative stress is that measurement of these large. Nitrotyrosine formation has been observed in vascular and myo- cardial tissue in both healthy individuals and those with CVD [54]. crobial activity.13] and has been shown to mediate redox lipoproteins and proteins. is a stable yet reversible reaction that confers a 305 Da a range of ROS by catalyzing the conversion of hydrogen peroxide negatively charged group. and NO2. showing S-glutathionylation of proteins in circulating cells (e. SERCA and the Na þ –K þ pump (as illustrated in Fig. handling and processing alterations in intracellular Na þ and Ca2þ handling. Sample collection. Furthermore. Nitration of tyrosine residues in enzymes may affect their function through steric or allosteric hindrance. Schematic illustration illustrating the functional effect of glutathionylation osine formation on SERCA2a is significantly higher in cardiac tissue of of key cardiovascular proteins eNOS [58]. plasma protein-bound nitrotyrosine levels were found to be significantly higher among patients with CAD even after adjustment for traditional risk factors for CVD and CRP [55]. [72]. that can in turn promote endothelial dysfunction by reducing the hyperlipidaemia and renal failure [63. and modify high density lipoprotein (HDL). MPO mass/concentration can of these membrane proteins has been reported in either the myocar. Further studies are needed to address tication in appropriately designed prospective studies. ELISA with monoclonal several challenges in applying nitrotyrosine as a CVD biomarker. Since its discovery in 1994. Myeloperoxidase S-glutathionylation MPO is a haeme enzyme that is abundant in granules of human S-Glutathionylation.

A study of 353 Optimization of such therapies at the level of individual patients healthy subjects revealed that vitamin E supplementation decreased is however the “holy grail” in clinical cardiology. as ROS also play a .11. culmination of years of ongoing insults. Additional examples include the most effective drugs/dose regimens for an individual patient peroxisome proliferator-activated receptor gamma coactivator (Fig. The JUPITER trial tor to this antioxidant protective effect is via the HDL-associated supported the clinical utility of assessing inflammatory status in paraoxanase (PON) which has peroxidase-like activity [85]. particular in those that do not tolerate a combination 1-alpha (PGC-1α) [88] and the thioredoxin family as reviewed by “cocktail” of these proven medications. However. Effective pharmacotherapies with prognostic significance in as well as antioxidant properties (Khera A. failure of niacin to reduce the incidence of vascular events in the as ROS lie downstream from the inflammatory driver. particularly relevant to assessing central role in the pathogenesis of atherosclerosis. and this presents a challenge for clinical applicability of the ROS- kines including adhesion molecules such as intercellular adhesion responsive genes as biomarkers. erythrocyte GPX- Tsimikas [78]). However. Thus. et al. The burden of circulating oxLDL but did not slow down the progression of carotid atherosclerotic disease. collected biological samples (e. rather than reversing the previously laid down One of the mechanisms of protection by high-density lipopro. as well as Heart Protection Study 2 despite achieving significant increases in other non-inflammatory mediators of cardiovascular disease. at least in part. Since increased redox stress is a major paradigm in pathophysiology of these disease states. be explained by a lack of effective biomarker of ROS may have even greater potential. stress and its consequences [10. but uses different monoclo. OxLDL is now more frequently detected using specific Activity of antioxidant enzymes such as catalase. pathophysiology. and increasing Future directions OxLDL levels correlate with increasing severity of disease (e.488 E. atheroma. The OxLDL-E06 ELISA assay quantifies oxidized 1 and not SOD activity was inversely associated with incidence of phospholipids on apoB-100 molecules. A reduction in pathophysiologically- genes involved in regulating cellular and systemic oxidative stress. 61 atherosclerosis and heart failure (as well as other conditions such (10). The OxLDL-4E6 sandwich ELISA assay samples were haemolysed and stored frozen [90]. angiotensin converting enzyme (Nrf-2).g. / Redox Biology 1 (2013) 483–491 Oxidized low-density lipoprotein and oxidized phospholipids Lee and colleagues [89]. 4). Whether gene expression profiling of cells in commonly macrophages via scavenger receptor pathways to form foam cells. The LDL-DLH3 acts in cardiovascular events after adjusting for cardiovascular risk factors reverse to the OxLDL-E06 ELISA assay by quantifying apoB-100 [90]. been shown to decrease rates of cardiovascular events. However. Ho et al.g. adducts being both proatherogenic and proinflammatory [78]. exert at least some of their clinical efficacy by reducing oxidative idant function of the HDL rather than simply increasing the levels. in stored samples using ELISA are technical advantages that enable its application for screening large populations [80]. the utility of oxidative stress biomarkers in prognostication and gui- ROS-induced changes in gene expression dance of individualized treatment is driven by their potential to act as an “integrator”. 5). β-blockers and statins) may provide valuable insight oxidative stress and drives the increased expression of numerous both into the efficacy of the treatment and guidance to selection of cellular antioxidant enzymes [87]. an HDL levels [86] may. The commercial detects MDA-LDL and copper oxidized-LDL epitopes. in one individual is the artery intima-media thickness over a 3-year period [84]. Low level of expression of these genes may reflect a low level of The oxidation of LDL can occur non-enzymatically or can be oxidative stress in the relevant system or individual variation in catalyzed by enzymes such as 12/15-lipoxygenase. From a technical perspective. reflecting the total impact of the many patho- ROS levels have been shown to influence the expression of key physiological processes (Fig. The guiding intervention to limit cardiovascular events [91]. OxLDL levels are higher in patients with CVD [81]. with the cardiovascular redox status. Novel efficacious treatments therefore need to focus as diabetes and hypertension that predispose humans to CVD) on targeting enhancement of the cholesterol efflux and antiox. relevant oxidative biomarkers with a particular combination of A prime example is Nuclear factor (erythroid-derived 2)-like 2 pharmacological therapies (e. E1390). glutathione monoclonal antibodies that directly recognize unique oxidation. most prominently in [83]. It has been proposed that profiling the expression of these ROS-sensitive genes using microarray technol- The oxidation and glycation of LDL and phospholipids plays a ogy may be a valuable tool. stable angina vs. Quantification of both disease load and lipoprotein oxidation and generation of OxLDL. This approach remains to be explored. lowering OxLDL with antioxidant therapies has not atherosclerotic-related diseases and heart failure syndromes. further studies of the clinical utility of this is that which has the biggest effect in halting the ongoing biomarker are required. The stability of OxLDL in biomarker to be evaluated in a large-scale high-throughput storage at 801 and the fact that it can be reproducibly quantified screening. Original studies of OxLDL depended on detection of circulating Measuring the net antioxidant capacity of the serum plasma autoantibodies against various oxidation-specific epitopes of OxLDL. response and may result in a higher level of oxidative cellular tion occurs primarily within vascular walls where it is taken up by damage. and ideally in stabilizing the plaque and reducing the tein (HDL) against the atherosclerotic process is by decreasing “soft plaque” component. the overlying endothelial cells to produce proinflammatory cyto. molecule-1 (ICAM-1).g. There are currently 3 plasma OxLDL ELISAs measures of antioxidant capabilities. availability of antioxidant enzyme assay kits allows this potential mercially available for experimental use. unstable angina vs.92]. JACC. and is com. 2013. OxLDL levels Significant progress has been made in primary and secondary also appear to be predictive of future CAD in apparently healthy men prevention of cardiovascular adverse events. myocardial infarction) [82]. “functionality” of the HDL in regard to both its cholesterol efflux. vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) [79]. peroxidase 1 (GPX-1) and SOD have been quantified in plasma as specific epitopes. blood cells) accurately reflects Accumulation of OxLDL within the vascular walls also stimulates the gene expression in cardiovascular tissue has yet to be resolved. and successful treatment although promising. the enzyme activities of the on oxidized phospholipid molecules. best treatment response is thus very challenging. for example. GPX-1 and SOD remain stable even when the erythrocytes of the nal antibodies for detection. A major contribu. OxLDL forma. In a prospective study of available for research (and not clinical) purposes (reviewed by patients with suspected coronary artery disease. a transcription factor that is upregulated in response to inhibitors.

Munzel. Aviles. International Expert Committee report on the role of the A1C and pathophysiology highlight the importance of identifying the assay in the diagnosis of diabetes. and thus be a useful therapeutic guide in patients who are unable to tolerate a “cocktail” of agents. Conclusion As oxidative stress is a unifying feature of almost all of the cardiovascular risk factors known to drive the atherosclerotic process. Brennan. Oxidative stress and cardiovascular injury: important manner. E. as well as a factor that is increased in response to neurohormonal abnormalities in heart failure and in cardiac ischaemia. S. Association between myeloperoxidase levels and baseline conditions may partially explain the apparent paradox of risk of coronary artery disease.L.J. Clinical application of C-reactive protein for cardiovascular is shown to be of pathophysiological relevance. vitamin E reducing markers of ROS. Fig. However. A. but not improving the rate of [2] M. Sources of funding KKG is supported by a scholarship from Heart Research Aus- tralia. individualization of treatment strategies. Circulation 108 small clinical studies. Circulation 123 (2011) 551–565.K. Nicaud. Griendling. Circulation 107 (2003) 363–369. The demonstrated effects of potent pharmacotherapies (e. large clinical trials of antioxidants have failed to (2003) 1912–1916.J. References physiological role in signalling. Schematic illustration of ROS as a common mediator of cardiovascular specificity.L. Czyz. JAMA 286 (2001) 2136–2142. Diabetes Care 32 (2009) 1327–1334. the lack of specificity may reflect the unique disease.R. Hamm.M.S.g. Ho et al. Despite promising results in experimental and Part I: basic mechanisms and in vivo monitoring of ROS. ability of biomarkers of oxidative stress to integrate these risk factors angiotensin receptor blockers [100. Warnholtz. and imaging biomarkers properties that can alter the disease process in a prognostically.L. Peetz. P.B. D. M. FitzGerald. Hazen.S.A. Post. E. New England Journal of Medicine 361 (2009) 868–877. both the redox biology community and clinical researchers need to team up to design studies that go beyond just validating the association of a marker with severity of disease. Roth. and β-blockers. critical role of ROS in cellular and vascular homoeostasis under D. Sinning. GF is supported by the University of Sydney Medical Foundation. Gillett. E. C. Blankenberg. Heart 92 (2006) 843–849. S. it may also be useful in disease detection and prevention. Wild. R. Jachmann. None. Ridker. S. Zhang. Eleftheriadis. it may be useful in identification of novel effective treatments. M. Fu. well-established pharmacotherapies. G. G. particularly useful for patients intolerant of combi- nation therapies. E.J. N.J. Genth-Zotz. the relationship between a decrease in ROS biomarker and the response to treatment is not simple. M. This may result from difficulties in targeted delivery of antioxidant therapy to the key cellular microdomains [96].S. Novel biomarkers in circulation that reflect pathophysiologically relevant oxidative signalling cascades within critical cellular micro- domains in cardiovascular system have the potential to supersede the currently available biomarkers. Sprecher. for this to occur. a biomarker that reflects oxidative stress may be criticized for its lack of Fig. Lubos. With technical advances in quantification of biomarkers of stress suggest that biomarkers of ROS may be an early measure of the success of oxidative stress and validation in prospective clinical studies of their pharmacotherapy in a particular patient. Topol. making ROS-based biomarkers excellent “integrators” for total cardiovas- cular risk.L. 5. S. Penn. Tzikas. Schnabel.J. Pearce. Zeller. L. C. CCL is supported by a Fellowship (PF 12S 6924) from the National Heart Foundation of Australia.F. 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