Static and Dynamic Redox State-Related Parameters

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This entry is adapted from the peer-reviewed paper 10.3390/antiox11050864

Oxidative stress refers to an imbalance between oxidant and antioxidant molecules, which is usually associated with oxidative damage to biomolecules and mitochondrial malfunction. Redox state-related parameters include (1) the direct measurement of ROS, (2) the assessment of the antioxidant defense status, and (3) the analysis of the resulting oxidative damage to molecules. Directly measuring ROS appears to be the preferred method among scientists, but most ROS are extremely unstable and difficult to measure. The processes of determining both the oxidative damage to biomolecules and the antioxidant system status, although both are indirect approaches, provide a reliable method to measure oxidative stress on a given sample. Recently, the Seahorse XF and the Oroboros O2k systems have provided new insights into the redox state from a more dynamic point of view. These techniques assess mitochondrial oxidative phosphorylation function and bioenergetics on isolated mitochondria, cultured cells, or specific tissues such as permeabilized fibers.

oxidative stress redox state ROS seahorse oroboros in vivo imaging

1. Direct Measurement of ROS

Major ROS that can be measured include the superoxide radical (·O₂⁻), hydrogen peroxide (H₂O₂), singlet oxygen, hydroxyl radical (·OH), and peroxyl radicals (·ROO⁻). Nitric oxide (·NO) and peroxynitrite (ONOO⁻) interact with ROS, and altogether, they form a group of species called RONS (reactive oxygen and nitrogen species). In physiological conditions, a major source of ROS production is the mitochondria; however, other cellular organelles or compartments can also produce ROS, such as peroxisomes, the endoplasmic reticulum (ER), and the plasma membrane. These compartments contain oxidative enzymes such as NADPH oxidases (in peroxisomes and ER) and xanthine oxidases (in peroxisomes). Also, other enzymes, including nitric oxide synthases, lipoxygenases, cyclooxygenases, monooxygenases, and myeloperoxidases, found in different intracellular spaces can produce ROS ^[1].

The most common way to estimate ROS intracellular levels is using a fluorescence methodology associated with suitable probes ^[2]. Some probes detect total cellular ROS levels, and some probes can be mitochondria specific. Thus, the measurement of non-mitochondrial ROS can be performed by determining the difference between total and mitochondrial ROS levels. Another possibility is to block the mitochondria before measuring the total ROS production. Also, imaging probes are helpful to confirm locations.

ROS determination is, however, hampered due to their extremely short lifespan; peroxyl radicals and hydrogen peroxide are relatively stable molecules with half-lives of seconds to minutes, but hydroxyl radicals are very reactive and their half-life is limited to nanoseconds ^[3].

There are also limitations to fluorescence probes which can be attributed to their lack of specificity and sensitivity and the difficulty associated with quantification and data interpretation ^[4]^[5]. Indeed, according to Forman et al., measuring ROS with fluorescent dyes is an inappropriate methodology. Reporter dyes are very unspecific, such as in the case of the 1,3-diphenylisobenzofuran (DPBF) probe, which has been used to detect singlet oxygen, hydroxyl radicals, and H₂O₂ ^[6]. Therefore, the proper identification, separation, and quantification of the specific oxidation product are required, in addition to the performing of appropriate controls using inhibitors ^[7].

Despite being the most widely used probes, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydrorhodamine-123 (DHR123) cannot provide a reliable measurement when it comes to intracellular H₂O₂ because of their lack of specificity; i.e., DCFH-DA reacts with hydroxyl radical (·OH) and alkoxyl radicals (·RO⁻) and DHR123 reacts with horseradish peroxidase (HRP) and with hypochlorous acid (HOCl) ^[8]^[9]. However, DCFH-DA is a suitable redox indicator of intracellular changes in iron signaling or peroxynitrite formation ^[8], and DHR123 can be used as a nonspecific indicator of intracellular ONOO⁻ and HOCl formation ^[10]. The detection of extracellular H₂O₂ can be performed using the AmplexRed probe, which is highly oxidized by horseradish peroxidase (HRP) and H₂O₂ to the fluorescent resorufin ^[11]. The advantage of this probe relies on its specificity to H₂O₂, but it is tissue unspecific and resorufin is light sensitive.

Mitochondria-targeted hydroethidine (MitoHE) and MitoSox react with superoxide radicals, giving rise to several red-fluorescent products with overlapping spectra. Therefore, these probes are not a reliable indicator of mitochondrial superoxide radical formation ^[12]. To overcome this spectral overlap limitation, ethidium and ethidine can be separated by HPLC or LC-MS/MS, which in turn increases the specificity for free radicals ^[13].

Another interesting approach is to monitor the heterogeneous cellular redox state in a spatiotemporal manner. The cellular redox state is governed by pyridine nucleotides (NADPH/NADP⁺ and NADH/NAD⁺), thiols (glutathione and thioredoxin systems), and ROS. Genetically encoded fluorescent sensors can be applied to target specific organelles using specific amino acid extensions. Some of these probes are yellow fluorescent protein (rxYFP)-based redox biosensors, such as iNap (to monitor NADPH/NAD⁺), SoNar and Frex (to assess NADH/NAD⁺), and Hyper (to detect H₂O₂). Other probes are based on the green fluorescent proteins (roGFP) to target thiol groups (such as glutathione and thioredoxin systems) ^[14]. Indeed, Hyper probes have been used to track H₂O₂ changes in mitochondria and peroxisomes ^[15]. Similarly, roGFP sensors have been targeted to various positions of the cytosol, thereby revealing the heterogeneity of glutathione ^[16]. Taken together, these probes offer a dynamic approach to assess the different biological processes in which pyridine nucleotides, thiol redox systems, and H₂O₂ are involved. However, the main drawback of these probes is the overlap of their excitation/emission spectra and their pH-dependency ^[17].

One step beyond is the in vivo imaging system (IVIS) spectrum, where optical imaging enables the study of molecular mechanisms in living animals using bioluminescent or fluorescent reporters in a non-invasive manner. Bioluminescence imaging (BLI) can be used to detect and monitor biological targets, whereas fluorescence imaging (FLI) is indicated for monitoring and quantifying the cell behavior of biological targets. Therefore, ROS can be detected using BLI. A bioluminescence probe, namely L-012 (an analog of luminol), was initially described for measuring the production of superoxide anion ^[18]. Subsequent studies revealed that L-012 is nonspecific and can be used to detect other free radicals, such as reactive nitrogen species, and monitor NADPH oxidase activity ^[19]^[20]^[21]. Another more chemoselective bioluminescent probe is Peroxy Caged Luciferin-1 (PCL-1), which is a boronic luciferin molecule that selectively reacts with H₂O₂ to release bioluminescent luciferase. This probe has been used for the real-time detection of basal endogenous levels of H₂O₂ in mice ^[22]. Taken together, bioluminescent probes can be sensitive enough to detect H₂O₂ in major organ systems such as the brain, the liver, the bladder, or the heart, thus giving rise to the possibility of monitoring several diseases, including neurodegenerative diseases, cirrhosis, and heart disease.

2. Products of Oxidative Damage

Products of oxidative damage mainly include protein oxidation, lipid peroxidation, and DNA damage, which can be measured in vitro (monolayer cell cultures) and ex vivo (tissues and organs). The following paragraphs describe the most used and accepted methods and technologies for the determination of each type of oxidative damage to products.

2.1. Markers of Protein Oxidation

When proteins are attacked by ROS, three main modifications may occur hydrogen atom abstraction from C–H, S–H, N–H, or O–H bonds; electron abstraction from electron-rich sites; and the addition of electrons to electron-rich centers ^[23]. These modifications entail conformational and structural alterations; therefore, a complete analysis of protein oxidation should include the gross modifications of parent proteins, the detection of protein oxidation intermediates, and the detection of end products ^[24].

One study analyzed parent protein conformational changes subjected to increased oxidative stress levels using size exclusion chromatography (SEC). The results show that SEC elutions were a mixture of protein aggregates and fragments, which were later identified using LC/MS ^[25]. Other studies have used immunoblotting (such as the western blot (WB) and enzyme-linked immunosorbent assay (ELISA)) techniques with specific antibodies to detect protein structural modifications in Huntington’s disease ^[26]^[27] and Multiple Sclerosis ^[28]. However, detecting minor changes in parent proteins against a large background of unaltered proteins can be very challenging.

Protein oxidation intermediates include radicals, hydroperoxides, chloramines, bromamines, and sulfenic acids. These molecules are transient and often present at low concentrations. Nonetheless, they can be determined by electron spin resonance spectroscopy. Indeed, using this methodology, some peptide-derived radicals have been detected following hydroxyl radical attack on amyloid-β (1–40) and α-synuclein to unravel the protein conformational disorders observed in Alzheimer’s disease and Parkinson’s disease, respectively ^[29].

The detection of stable protein products such as sulfur-containing amino acids, the oxidation of aromatic amino acids, and protein carbonyls can yield higher quality data and enable quantification. Protein carbonyls can be quantified via their reaction with 2,4-dinitrophenylhydrazine (DNPH) to provide the hydrazone, which is then measured by optical absorbance or by antibodies against standards ^[30]^[31]^[32]. Indeed, oxyblot analysis has been used to detect protein carbonylation in senescent fibroblasts treated with H₂O₂ ^[33]. Moreover, protein carbonyls are very stable and can therefore be considered biomarkers in oxidative stress-related diseases. However, the biological and clinical relevance of protein oxidation as a biomarker is only useful if the employed methodology can identify and quantify the specific modification. MS is probably the most accurate technique since it provides both conformational and quantitative information. Indeed, it is critical to correlate the observed modification of the protein with the observed biological and functional defects and to assess the relation of causality.

2.2. Markers of Lipid Peroxidation

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals or nonradical species attack lipids containing carbon-carbon double bond(s). During ROS attack, hydrogen is abstracted from carbon with oxygen insertion, resulting in the formation of lipid peroxyl radicals and hydroperoxides, which are short-lived molecules. Once lipid peroxidation is initiated, a propagation of chain reactions will take place until termination products are produced. The final products can be formed depending on the original fatty acid chain attacked and the number of oxidation events, leading to a complex variety of products ^[34].

Lipid peroxyl radicals are the first products of lipid peroxidation and are essentially unstable. A recent study developed a fluorogenic antioxidant probe bearing a BODIPY reporter chromophore associated with a-tocopherol as a trap segment that detects lipid peroxyl radicals ^[35]. However, this study did not specify the particular radical that was being measured, which can only be measured through electron spin resonance (ESR) ^[36]. Similarly, lipid hydroperoxides are also unstable molecules and can be detected and identified by HPLC ^[37]^[38].

Many aldehydes are produced during lipid peroxidation, although the most widely studied is malondialdehyde (MDA), owing to the superficial simplicity of the assay with thiobarbituric acid (TBARS assay). However, in complex biological samples, TBARS reacts with many oxidation products and not only with MDA ^[7]. Therefore, many derivatization methods have been developed for the specific analysis of MDA by HPLC with visible (HPLC-UV/Vis) or fluorescence (HPLC-FL) detection ^[39] and mass spectrometry coupled with gas/liquid chromatography (GC-MS, GC-MS/MS, or LC-MS/MS) ^[39]^[40]^[41].

Another common aldehyde used as a marker of lipid peroxidation is HNE. Classical methods for HNE determination have mainly been based on various spectrophotometric and/or chromatographic approaches ^[40]. Other methodologies include immunochemical techniques. On the one hand, immunohistochemistry and immunocytochemistry are qualitative, morphological techniques that allow for the visualization of HNE within cells (immunocytochemistry) or in tissues (immunohistochemistry) ^[41]. On the other hand, immunoblotting (such as WB and ELISA) assays offer a more quantitative approach to detect HNE-protein adducts ^[42]. However, the antibodies used in ELISA are not specific for HNE, and, therefore, a large-scale MS methodology must be performed for HNE detection ^[43].

Lastly, F2-Isoprostanes are chemically and metabolically stable and nonreactive and are considered the best indicators of nonenzymatic lipid peroxidation. Once again, measurement with ELISA is nonspecific, but several methods have been developed to specifically measure F2-Isoprostanes including LC-MS and GC–MS ^[44]. Indeed, a study demonstrated that both HNE and F2-Isoprostanes measured by HPLC/MS perfectly correlate with inflammation and oxidative stress in renal disease ^[45].

When analyzing lipid peroxidation, the original lipidic profile of the sample must be taken into consideration. The oxidation of plasma happens preferentially to that of polyunsaturated fatty acids (PUFAs), and once these are depleted, the oxidation of cholesterol proceeds. Moreover, the composition of PUFAs in the blood is dependent on diet, which hampers comparisons between subjects. Another challenge associated with lipid peroxidation measurements is that hundreds of products are formed. Each product is produced in a different yield and is metabolized and excreted at different rates, which hampers the identification and quantification of all of them ^[34]. Despite all these issues, the main problem associated with the use of lipid peroxidation as a biomarker of oxidative stress is that it does not provide information on the in vivo origin or trigger of the lipid peroxidation.

2.3. Markers of DNA Oxidation

Under oxidative stress conditions, DNA lesions can be produced directly or indirectly ^[46]. In the direct lesion, guanine gives an electron to the original radical cation, which is then “chemically” repaired ^[47]. In turn, the guanine cation undergoes hydration to form 8-oxo-2′-deoxyguanosine (8-oxo-dG). The attack of hydroxyl radical on the DNA (indirect lesion) affects either DNA bases (70%) or deoxyribose moieties (30%). The latter gives rise to single-strand brakes (SSB).

Direct and indirect methods have been developed to measure DNA lesions. Direct methods detect specific DNA lesions, whereas indirect methods usually measure strand breaks, which can be assessed by analyzing DNA repair enzymes that convert lesions into strand breaks.

8-oxo-dG, as well as 5-hydroxy-2′-deoxycytidine (5-HO-dCyd) and 8-oxo-dAdo, can be detected using HPLC coupled to electrochemical detection (HPLC-ECD) ^[48]^[49]. Another method consists of the derivatization of the DNA bases to make them volatile enough to be separated and analyzed by GC-MS. This technique enables the detection of all DNA lesions. Nowadays, HPLC-MS/MS is the most used method for the detection of specific DNA lesions ^[50]^[51]^[52]^[53]. Indeed, some studies have reported a positive correlation between 8-oxo-dG levels measured by HPLC-MS/MS and chronic liver inflammation ^[54] and chronic kidney disease ^[55]. Alternative and simpler ways to measure DNA damage include ELISA and immunohistochemical analysis ^[56]^[57]^[58], although these methods can be nonspecific, and results should not be reported only based on this type of technical measurement.

The alkaline elution (AE) and the “Comet” assay are indirect methods commonly used to detect DNA strand brakes ^[59]. When the DNA is damaged, the cell activates the DNA damage response (DDR), which can also be monitored. This approach targets DNA repair proteins such as tumor suppressor p53 (TP53), γ-Histone 2AX (γ-H2AX), ataxia-telangiectasia related kinase (ATR), ataxia-telangiectasia mutated kinase ATM, X-ray repair cross-complementing protein 1 (XRCC1), human 8-oxoguanine-DNA-glycosylase (hOGG-1), and the xeroderma pigmentosum group D (XPD) helicase, which can be measured by flow cytometry, immunoblot, IHC, or HPLC ^[60]^[61]^[62]. The study performed by Martinet et al. showed a complete analysis of oxidative DNA damage where they analyzed 8-oxo-dG levels by ELISA, DNA strand breaks using the Comet assay, and DNA repair enzymes by immunoblot. They found increased levels of all three markers in human atherosclerotic plaques ^[63].

3. Antioxidants

Endogenous oxidative stress can be modulated by the prevention of ROS formation or by the quenching of ROS with antioxidants, which can be divided into enzymatic and nonenzymatic components.

Mitochondrial ROS production is regulated, in part, by the MMP. It has been suggested that small increases in the MMP induce ROS formation, whereas slight decreases can reduce ROS formation. Hence, a mild uncoupling of the mitochondrial ETC may represent the first line of defense against oxidative stress ^[64]. Mitochondrial uncoupling proteins (UCP), specially UCPs 2 and 3, have been found to reduce ROS production from mitochondria. Indeed, the accumulation of ROS leads to the activation of UCPs, which in turn produces a proton leak within the ETC, thereby creating a negative feedback loop that modulates ROS formation ^[65].

UCPs can be measured by qPCR, WB, and ELISA, although their activity can be determined by assessing the mitochondrial ETC function, and in particular, the proton leak, using respirometers, such as the Seahorse extracellular flux analyzer and the Oroboros Oxygraph-2k system, which are explained in the following sections.

Enzymatic components refer to antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST). SOD enzymes catalyze the conversion of superoxide anion into the potential second messenger hydrogen peroxide, which in turn is detoxified by CAT or GPx ^[66]. The expression of these enzymes can be determined by qPCR and the protein amount can be qualitatively determined by WB; however, their activities are measured using colorimetric assays. SOD activity can be assayed by monitoring the rate of inhibition concerning the reduction of nitroblue tetrazolium (NBT) ^[67]. CAT activity can be determined by monitoring the disappearance of H₂O₂ at 240 nm ^[68]. GPx activity can be assayed by measuring the decomposition of H₂O₂ and GSH ^[69], and GR activity can be measured by monitoring NADPH depletion ^[70]. GST activity can be determined using chlorodinitrobenzene as substrate ^[71]. A proper study of the antioxidant status must report data on the enzymes’ gene expression and protein levels as well as their activities to evaluate their antioxidant potential or capacity. Accordingly, a study reported all these data to suggest that lifelong soya consumption increases the antioxidant defense system in diabetic rats ^[72].

Non-enzymatic components include glutathione (GSH/GSSG) and vitamins A, C, and E. Within cells, glutathione exists in both reduced (GSH) and oxidized (GSSG) states. In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form (GSH) while less than 10% exists in the disulfide form (GSSG). An increased GSSG/GSH ratio is considered indicative of oxidative stress. GSH levels are measured spectrophotometrically using the DTNB reagent, which reacts with sulfhydryl groups ^[73]^[74]. Indeed, it has been demonstrated that GSH levels, GSSG levels, and the GSH/GSSG ratio are significantly altered immediately after intense physical exercise in sedentary adults and return to basal levels after recovery, thus demonstrating that this type of physical exercise induces oxidative stress ^[75]. However, measurement is hampered due to an underestimation of GSH and an overestimation of GSSG because of the auto-oxidation of GSH. Therefore, fluorometric assays have also been developed to measure glutathione levels using fluorescent probes such as ortho-phthalaldehyde (OPA) and monochlorobimane (MCB) ^[76]. Glutathione detection using only fluorescent probes is not specific enough, but a combination of fluorescence and HPLC could overcome such spectrophotometric limitations. Indeed, a recent study showed that glutathione-OPA adduct is a valid method for the simultaneous measurement of GSH and GSSG to assess the redox status in biological samples ^[77].

The levels of vitamin A, retinoids and carotenoids, vitamin C (ascorbic acid), and vitamin E (tocopherols), all-powerful antioxidants, are mostly determined by HPLC ^[78]. It is important to mention that for a molecule to be considered a ROS scavenger, it would need to overcome all the other potentially reactive molecules in the sample ^[7]. Thus, increased vitamin levels do not necessarily represent an increased antioxidant status. An exception is vitamin E, which reacts rapidly with lipid hydroperoxyl radicals overcoming the propagation reaction, and can therefore be considered as a marker of lipid peroxidation.

In addition, the total antioxidant capacity (TAC) can also be measured to evaluate the oxidative state of a given sample ^[79]. The different methods used for the evaluation of the antioxidant capacity are grouped into three distinct categories: spectrometry, electrochemical assays, and chromatography. The spectrometry technique includes several tests, among them the ORAC, HORAC, TRAP, CUPRAC, and FRAP tests, which have been described elsewhere ^[80]. A unique test that measures the total antioxidant capacity is an attractive idea; this fast and easy method would be very useful in clinical analysis to gain a general idea of the antioxidant status. Nonetheless, assessing TAC is associated with some challenges and limitations. Data reported by the different spectrometric tests do not correlate with each other because each antioxidant molecule reacts differently in each test. These TAC tests are very sensitive to the oxidant insult, but in vivo, several oxidant insults are produced at the same time (such as cancer and tobacco smoke), and the TAC of a sample will be different depending on the nature of the oxidant stimulus ^[81]. Therefore, TAC tests are a complementary measurement to each specific antioxidant enzyme measurement.

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Cristina Mas-Bargues

Esther García-Domínguez

Consuelo Borras

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Update Date: 31 May 2022

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