Non-invasive biomarkers of oxidative stress: reproducibility and methodological issues

Oxidative stress is the hallmark of various chronic inflammatory lung diseases. Increased concentrations of reactive oxygen species (ROS) in the lungs of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples or to examine the affected compartments, to the patient's discomfort. As a consequence, there is a need for less or non-invasive approaches to measure oxidative stress. The collection of exhaled breath condensate (EBC) has recently emerged as a non-invasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lower respiratory tract airways. The biomarkers of oxidative stress such as H2O2, F2-isoprostanes, malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress such as nitrate/nitrite and nitrosated species have been successfully measured in EBC. The reproducibility, sensitivity and specificity of the methodologies used in the measurements of EBC oxidative stress biomarkers are discussed. Oxidative stress biomarkers also have been measured for various antioxidants in disease prognosis. EBC is currently used as a research and diagnostic tool in free radical research, yielding information on redox disturbance and the degree and type of inflammation in the lung. It is expected that EBC can be exploited to detect specific levels of biomarkers and monitor disease severity in response to appropriate prescribed therapy/treatment.     

Nickel exposure and plasma levels of biomarkers for assessing oxidative stress in nickel electroplating workers

Context: The mechanism of nickel-induced pathogenesis remains elusive.

Objective: To examine effects of nickel exposure on plasma oxidative and anti-oxidative biomarkers.

Materials and methods: Biomarker data were collected from 154 workers with various levels of nickel exposure and from 73 controls. Correlations between nickel exposure and oxidative and anti-oxidative biomarkers were determined using linear regression models.

Results: Workers with a exposure to high nickel levels had significantly lower levels of anti-oxidants (glutathione and catalase) than those with a lower exposure to nickel; however, only glutathione showed an independent association after multivariable adjustment.

Discussion and conclusion: Exposure to high levels of nickel may reduce serum anti-oxidative capacity.     

Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure

This study investigated how Cd exposure affected oxidative biomarkers in Japanese flounder, Paralichthys olivaceus, at early life stages (ELS). Fish were exposed to waterborne Cd (0–48 µg L^− 1) from embryonic to juvenile stages for 80 days. Growth, Cd accumulation, activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18), and levels of glutathione (GSH) and lipid peroxidation (LPO) were investigated at three developmental stages. Flounder growth decreased and Cd accumulation increased with increasing Cd concentration. In metamorphosing larvae, CAT and SOD activities were inhibited and GSH level was elevated, while LPO was enhanced by increasing Cd concentrations. CAT and GST activities of settling larvae were inhibited but GSH level was elevated at high Cd concentrations. In juveniles, SOD activity and LPO level were increased but GST activity was inhibited as Cd concentration increased. Antioxidants in flounder at ELS were able to develop ductile responses to defend against oxidative stress, but LPO fatally occurred due to Cd exposure. These biochemical parameters could be used as effective oxidative biomarkers for evaluating Cd contamination and toxicity in marine environments: CAT, SOD, GSH, and LPO for metamorphosing stage; CAT, GSH, and GST for settling stage; and SOD, GST, and LPO for juvenile stage.     

Evaluation of urinary biomarkers of oxidative/nitrosative stress in adolescents and adults with Down syndrome

Urinary biomarkers of oxidative stress have been little studied in adults with Down syndrome (DS), usually no more than two biomarkers have been measured in the population studied and controversial results are reported in literature. Thus, we aimed to assess a set of oxidative and nitrosative stress biomarkers in urine samples of adolescents and adults with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2′-deoxyguanosine (8-OHdG), isoprostane 15-F_2t-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H₂O₂) and nitrite/nitrate (NOx). Fluorimetric and spectrophotometric assays were performed in DS (n = 78), some of them taking levothyroxine for hypothyroidism (n = 24), and in their healthy age-matched controls (n = 65). We found that levels of AGEs, diTyr, H₂O₂ and NOx are increased in DS patients in any or in all age groups, whereas Cr levels were lower in DS than in controls in all age groups. Besides, correlations with age in DS were positive for diTyr and negative for Cr, TBARS, 15-F_2t-IsoP and NOx. We also found lower levels of Cr from 15 to 19 years, higher levels of TBARS and AGEs from 20 to 40 years and higher levels of diTyr from 15 to 40 years in DS patients receiving levothyroxine than in DS without hypothyroidism diagnosed. We conclude that AGEs, diTyr, H₂O₂ and NOx could be used as oxidative stress biomarkers in DS in contrast to 8-OHdG, 15-F_2t-IsoP and TBARS, at least with the methods used. However, renal impairment could occur in DS and Cr adjustment may bias the results, particularly in hypothyroid patients.     

Development and application of oxidative stress biomarkers

Abstract

Oxidative stress may cause a wide variety of free radical reactions to produce deleterious modifications in membranes, proteins, enzymes, and DNA. Reactive Oxygen Species (ROS) generated by myeloperoxidase (MPO) can induce lipid peroxidation and also play an important role in the generation of reactive chlorinating and brominating species. As the universal biomarkers, chemical, and immunochemical approach on oxidatively modified and halogenated tyrosines has been carried out. As amido-type adduct biomarkers, chemical, and immunochemical evaluation of hexanoyl- and propanoyl-lysines, hexanoyl- and propanoyl-dopamines and phospholipids were prepared and developed for application of evaluation of novel antioxidative functional food factors. We have also involved in application of oxidatively modified DNAs such as 8-hydroxy- and 8-halogenated deoxyguanosines as the useful biomarkers for age-related diseases using both in vitro and in vivo systems. Application of these oxidative stress biomarkers for novel type of functional food development and recent approach for development of novel evaluation systems are also discussed.     

Peroxiredoxins as biomarkers of oxidative stress

Background

Peroxiredoxins (Prxs) are a class of abundant thiol peroxidases that degrade hydroperoxides to water. Prxs are sensitive to oxidation, and it is hypothesized that they also act as redox sensors. The accumulation of oxidized Prxs may indicate disruption of cellular redox homeostasis.

Scope of review

This review discusses the biochemical properties of the Prxs that make them suitable as endogenous biomarkers of oxidative stress, and describes the methodology available for measuring Prx oxidation in biological systems.

Major conclusions

Two Prx oxidation products accumulate in cells under increased oxidative stress: an intermolecular disulfide and a hyperoxidized form. Methodologies are available for measuring both of these redox states, and oxidation has been reported in cells and tissues under oxidative stress from external or internal sources.

General significance

Monitoring the oxidation state of Prxs provides insight into disturbances of cellular redox homeostasis, and complements the use of exogenous probes of oxidative stress. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Salivary biomarkers of oxidative stress: A critical review

Human saliva is an increasingly attractive medium for biomarker discovery due to its amenability to noninvasive and repeated sampling, ease of collection and processing, and suitability for single analyte or metabolomic measurements. Salivary biomarkers of oxidative stress reflect local and systemic pathologies and may inform on the diagnosis, prognosis, and therapeutic responsiveness of numerous human diseases. However, for many of the disorders investigated, data reporting on alterations in salivary redox homeostasis are often highly conflicted across studies. We surveyed the available biomedical literature on this topic and noted significant discrepancies in the study designs, target populations, and operating procedures which likely contribute to the discordant data sets reported. Based on these observations, guidelines are provided to minimize interlaboratory variability in redox biomarker discovery based on human saliva.     

Tumor invasion and oxidative stress: biomarkers and therapeutic strategies

Tumor invasion is paradigmatic of the complex interactions connecting a carcinoma with its environment, and a reflex of the cellular and molecular heterogeneity that defines the initiation of dissemination and metastasis. The hostile situation generated by a growing carcinoma and a reactive stroma is at the basis of the promotion of carcinoma invasion and metastasis, with oxidative stress emerging as a main player in the acquisition of an aggressive tumor phenotype. In this review, we present this complex scenario with a focus on the contribution of the reactive environment and the oxidative stress to the cellular and molecular events associated with carcinoma invasion and metastasis. We also discuss the potential of oxidative stress as a source of biomarkers of advance disease, and as supplier of a therapeutic armamentarium against the initial steps of metastatic dissemination.     

Variability of oxidative stress biomarkers in hemodialysis patients

Abstract

Oxidative stress biomarkers may have a role in the future to assist clinical decisions regarding the use of antioxidant therapies and their efficacy. The aims of this study were to evaluate the within and between-individual variability of plasma oxidative stress biomarkers and investigate factors affecting their variability. Plasma F2-isoprostanes and protein carbonyls were measured in 14 hemodialysis patients every 2 weeks for 10 weeks. Within-individual coefficients of variation (CVs) were isoprostanes?=?30.4% (range?=?6.1–66.7%) and protein carbonyls?=?16.3% (8.4–29.5%). Between-individual CVs were isoprostanes?=?34.4% (28.9–40.2%) and protein carbonyls?=?19.5% (15.6–24.5%). There were no significant (p?>?0.05) relationships between the oxidative stress biomarkers and dietary antioxidant intake, medications, clinical and demographic parameters.     

Associations between functional polymorphisms in antioxidant defense genes and urinary oxidative stress biomarkers in healthy, premenopausal women

Functional polymorphisms in endogenous antioxidant defense genes including manganese superoxide dismutase (MnSOD), catalase (CAT), and glutathione peroxidase (GPX-1) have been linked with risk of cancer at multiple sites. Although it is presumed that these germline variants impact disease risk by altering the host’s ability to detoxify mutagenic reactive oxygen species, very few studies have directly examined this hypothesis. Concentrations of 8-isoprostane F2α (8-iso-PGF2α) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxoxdG)—sensitive indicators of lipid peroxidation and DNA oxidation, respectively—were measured in 24-h urine samples obtained from 93 healthy, premenopausal women participating in a dietary intervention trial. In addition, DNA was extracted from blood for genotyping of MnSOD Val16Ala, CAT-262 C > T, and GPX1 Pro198Leu genotypes by Taqman assay. Although geometric mean concentrations of 8-iso-PGF2_α and 8-oxoxdG varied across several study characteristics including race, education level, body mass index, and serum antioxidant levels, there was little evidence that these biomarkers differed across any of the examined genotypes. In summary, functional polymorphisms in endogenous antioxidant defense genes do not appear to be strongly associated with systemic oxidative stress levels in young, healthy women.     

Long-term stability of oxidative stress biomarkers in human serum

The storage time and storage temperature might affect stability of oxidative stress biomarkers, therefore, they have to be analyzed after long-term storage of serum samples. The stability of three biomarkers reflecting oxidative stress: reactive oxygen metabolites (ROM) for hydroperoxides, total thiol levels (TTL) for the redox status and biological antioxidant potency (BAP) for the antioxidant status, was investigated at several time points during 60 months of storage at ?20 and ?80?°C. Biomarkers ROM and BAP showed a very good stability during storage for 60 months at both temperatures. In addition, the correlation of the data after 60 months of storage compared with the starting data was very good with correlation coefficients >0.9. The TTL assay showed good results in serum samples stored at ?80?°C, but not in samples stored at ?20?°C. Serum samples for analysis of the set of oxidative stress biomarkers ROM, BAP and TTL can be stored up to 60 months at ?80?°C. ROM and BAP can also be stored at ?20?°C during this period. The present results are very important for the biomarker-related epidemiological studies that make use of biobanks with samples stored for many years and for new project planning, including sample storage conditions.     

Candidate salivary biomarkers associated with alveolar bone loss: cross-sectional and in vitro studies

This cross-sectional study evaluated the association between radiographic evidence of alveolar bone loss and the concentration of host-derived bone resorptive factors (interleukin-1 beta, tumor necrosis factor-alpha, interleukin-6, prostaglandin-E2), and markers of bone turnover [pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), osteocalcin, osteonectin] in stimulated human whole saliva collected from 110 untreated dental patients. Alveolar bone loss scores for each patient were derived from radiographic examination. Variables positively associated with increased bone loss score were: age, current smoking, use of bisphosphonate drugs, and salivary interleukin-1beta levels above the median. Salivary osteonectin levels above the median were associated with a decreased bone loss score. Additional in vitro studies were carried out to determine the fate of interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha added to whole and parotid saliva. All cytokines added to saliva were detected in significantly lower concentrations than when added to buffer alone. Protease inhibitors added to saliva did not prevent the reduction in detection of biomarkers. Variation in time of incubation, repeated cycles of freezing and thawing, or exposure to dimethylsulfoxide did not appreciably affect the measurement of cytokines in saliva. These results suggest that detection of biomarkers by conventional immunoassays may underestimate the actual quantity of molecules in saliva.     

Clinical application of breath biomarkers of oxidative stress status

Isolation and quantification of volatile breath biomarkers indicative of relevant alterations in clinical status has required development of new techniques and applications of existing analytical chemical methods. The most significant obstacles to successful application of this type of sample have been reduction in required sample volume permitting replicate analysis (an absolute requirement for all clinical studies), separation of the analyte(s) of interest from background molecules, water vapor and other molecules with similar physical properties, introduction of automation in analysis and the use of selective detection systems (electron impact mass spectrometry, flame photometric, thermionic detectors), and automated sample collection from the human subject. Advances in adsorption technology and trace gas analysis have permitted rapid progress in this area of clinical chemistry.     

Stress proteins as biomarkers of oxidative stress: effects of antioxidant supplements

The potential benefits to health of the supply of antioxidants, either through dietary intake or as supplements, is equivocal. There is a need to develop biomarkers that may act as monitors of cellular defense as influenced by antioxidant status. Thirty-two individuals participated in the project and 19 received supplements for 5 weeks in the form of a capsule containing a defined mixture of antioxidants. No change was noted in levels of superoxide dismutase and glutathione peroxidase following antioxidant supplementation. On the other hand, increase in total antioxidant status and decrease in malondialdehyde, protein carbonyl formation, and erythrocyte hemolysis were noted. In lymphocytes isolated from individuals receiving antioxidant supplements and subjected to a heat shock in the presence of the free radical generator 2, 2'-azobis-(2-amidinopropane)-dihydrochloride, enhanced synthesis of heat shock proteins hsp 105, hsp 90, hsp 70, and hsp 40 by contrast with decreased synthesis of heme oxygenase HO-1 (hsp 32) were noted. We conclude that antioxidant status modulates the synthesis of stress proteins.     

Chronically and acutely exercised rats: biomarkers of oxidative stress and endogenous antioxidants.

The responses to oxidative stress induced by chronic exercise (8-wk treadmill running) or acute exercise (treadmill running to exhaustion) were investigated in the brain, liver, heart, kidney, and muscles of rats. Various biomarkers of oxidative stress were measured, namely, lipid peroxidation [malondialdehyde (MDA)], protein oxidation (protein carbonyl levels and glutamine synthetase activity), oxidative DNA damage (8-hydroxy-2'-deoxyguanosine), and endogenous antioxidants (ascorbic acid, alpha-tocopherol, glutathione, ubiquinone, ubiquinol, and cysteine). The predominant changes are in MDA, ascorbic acid, glutathione, cysteine, and cystine. The mitochondrial fraction of brain and liver showed oxidative changes as assayed by MDA similar to those of the tissue homogenate. Our results show that the responses of the brain to oxidative stress by acute or chronic exercise are quite different from those in the liver, heart, fast muscle, and slow muscle; oxidative stress by acute or chronic exercise elicits different responses depending on the organ tissue type and its endogenous antioxidant levels.     

The effects of oxidative stress in urinary tract infection

We aimed to determine the effects of oxidative stress in urinary tract infection (UTI). One hundred sixty-four urine samples obtained from patients with the prediagnosis of acute UTI admitted to the Faculty of Medicine, Kahramanmaras Sutcu Imam University, were included in this study. Urine cultures were performed according to standard techniques. Urinary isolates were identified by using API ID 32E. The catalase and superoxide dismutase activity and the lipid peroxidation levels known as oxidative stress markers were measured in all urine samples. Thirty-six pathogen microorganisms were identified in positive urine cultures. These microorganisms were as follows: 23 (63.8%) E coli, 5 (13.8%) P mirabilis, 4 (11.1%) K pneumoniae, 2 (5.5%) Candida spp, 1 (2.7%) S saprophyticus, and 1 (2.7%) P aeruginosa. It was observed that lipid peroxidation levels were increased while catalase and superoxide dismutase activities were decreased in positive urine cultures, compared to negative cultures. We conclude that urinary tract infection causes oxidative stress, increases lipid peroxidation level, and leads to insufficiency of antioxidant enzymes.     

Uptake and clearance of PCB congeners in Chamaelea gallina: response of oxidative stress biomarkers

PCB uptake and clearance by clams, Chamaelea gallina, were studied in specially designed flow-through channels. After 8 weeks exposure to 10 ppb Aroclor 1254 in water, clams were depurated for 10 weeks, in the same exposure channel or after transfer to clean systems. Accumulation of the 20 congeners studied depended on its initial abundance and physicochemical properties. A linear relationship was found between log bioconcentration factor and log octanol/water partition coefficient of each form. Clearance of each PCB depended also on its initial load and solubility, being faster in clams transferred to clean systems. Exposure significantly enhanced catalase and 6-P-gluconate dehydrogenase activities, but not other antioxidative enzymes. Superoxide dismutase, low during the exposure phase, increased seven-fold during depuration. Aroclor-treated clams had higher GSH levels than controls, but decreased to 15-35% after 2 days clearance, rose to 150% after 12 days, and declined to low levels by the end of the experience. Biotransformation of PCBs to quinones and redox cycling-promoted oxidative stress might explain the increased antioxidative defenses. The biochemical changes observed at the beginning of clearance could be attributed to clam handling, by adaptation to and recovery from hypoxic/anoxic stress.