Thursday, June 16, 2022

How Do You Measure Oxidative Stress

Kidney Diseases And Oxidative Stress

Oxidative Stress Part 1

Oxidative stress is involved in a plethora of diseases affecting renal apparatus such as glomerulo- and tubule-interstitial nephritis, renal failure, proteinuria, and uremia . The kidneys are negatively affected by oxidative stress mainly because of the fact that ROS production induces the recruitment of inflammatory cells and proinflammatory cytokine production, leading to an initial inflammatory stage. In this early phase, a predominant role is played by TNF-alpha and IL-1b, as proinflammatory mediators, as well as by NF-B as transcriptional factor required to sustain the inflammatory process. The latter stage is characterized by an increase in TGF-beta production, which orchestrates the extracellular matrix synthesis. So, when the oxidative stress stimuli act chronically on kidney tissues, the results will be an initial stage of inflammation and later the formation of abundant fibrotic tissue that impairs organ function potentially leading to renal failure. Certain drugs, such as cyclosporine, tacrolimus, gentamycin, and bleomycin, are known to be nephrotoxics mainly because of the fact that they increase free radical levels and oxidative stress via lipid peroxidation . Heavy and transition metals , acting as powerful oxidative stress inducers, are responsible for various forms of nephropathy, as well as for some types of cancers .

How Should We Measure The Output Of These Probes

The simplest technique is the fluorescence microplate reader, where data are presented as increases or decreases in relative fluorescence. However, the quality and sensitivity of the machines commercially available vary tremendously, and the requirement for additional excitation and emission filters often makes them expensive. Newer models are available that do not require expensive filters but act on a dual monochromator principle and offer greater flexibility. Simultaneous dual wavelength excitation and emission measurements are particularly useful in determining the fluorescence ratio changes required for C-11-BODIPY581/591 or the product of reaction of O2 with DHE. It is important to check if the machine being used is a top-reading fluorescence machine, since this requires the cells to be in suspension. Bottom-reading machines have the advantage that the cells can be measured in situ without the need for trypsinization or cell scraping, processes that themselves generate cellular oxidative stress and result in artifactual changes in fluorescence. Plate readers measure total fluorescence, that is, they do not distinguish between intracellular and extracellular fluorescence from chemical reactions in the culture medium. We have already alluded to this problem in the case of DCFDA.

Effects Of Rs On Other Probes

A wide range of probes is available to study many cellular events, such as pH changes and ion movements and to identify various cellular organelles . For example, rises in Ca2+ frequently accompany oxidative stress . Several papers have indicated that ion probes can be affected by the generation of RS or can themselves contribute to RS formation, such as calcein . MitoTracker Red, a fluorescent probe used to identify mitochondria, can stimulate mitochondrial ROS generation , for example.

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Detect & Quantify Biomarkers Of Oxidative Stress

As a trusted manufacturer of thousands of widely cited and thoroughly validated ELISA kits and antibodies, we understand quality means delivering sensitivity, specificity, and consistency. Over 300 immunoassays and 3000 antibodies enable sensitive & specific detection of protein and small molecule biomarkers of oxidative stress. Our StressgenĀ® assay kits and reagents are some of the most widely cited products for quantification of critical stress-regulating metabolic enzymes and stress-responsive pathways.

Approaches And Methods To Measure Oxidative Stress In Clinical Samples: Research Applications In The Cancer Field

Optimization of the oxidative stress conditions. (A) Cell growth in the ...

Penelope Duerksen-Hughes

1Department of Basic Sciences, Loma Linda University School of Medicine, 11021 Campus Street, 101 Alumni Hall, Loma Linda, CA 92354, USA


1. Introduction

Reactive oxygen species are chemically reactive molecules containing oxygen that play several beneficial roles for the organism. At low/moderate concentrations, they are needed for physiological activities such as intracellular cell signaling and homeostasis, cell death, immune defense against pathogens, and induction of mitogenic response . These free radicals are produced endogenously as a natural by-product of the normal cellular metabolism of oxygen. Additionally, they can be induced by exogenous sources such as UV light, ionizing radiation, lifestyle, diet, stress, and smoking. Maintaining equilibrium between the reducing and oxidizing states is crucial for proper physiological functions therefore, living organisms are equipped with antioxidant defense systems, consisting of both enzymatic and nonenzymatic antioxidants, to regulate the levels of these free radicals .

Biomarkers of oxidative stress are therefore important tools in the assessment both of disease status and of the health-enhancing effects of antioxidants in humans. In this review, we aim to discuss different methods and approaches used for the evaluation of oxidative stress in clinical samples, as well as to review recent findings of oxidative stress in patients with different types of cancer.

2.2.1. Protein Damage

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Data Extraction And Analysis

For each study, the study characteristics , participant characteristics , and the interventions were extracted and identified by an author and verified by another author based on running-induced changes on the biochemical parameters of oxidative stress and inflammationwhich is related to inducing oxidative stress. All the parameters were evaluated in blood samples collected during or after the running program. Disagreements were resolved through discussions with other authors.

Versatile Methods To Measure The Anti

Fourteen convenient kits to measure the total antioxidant capacity of biological fluids, cells, and tissue . They can also be used to assay the antioxidant activity of naturally occurring or synthetic compounds for use as dietary supplements, topical protection, and therapeutics. Lets take a look at them.

5 fluorescence based assays

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Measuring Changes In Blood Pressure And Vascular Tone

The free radical NO plays a key role in vasodilatation. Its action can be antagonized by O2, which removes NO by reacting fast with it to give ONOO . In the healthy arterial wall, there is a balance between NO and O2, but increased oxidative stress can diminish the bioactivity of NO . Sources of O2 in and around the vessel wall include the vascular endothelium , fibroblasts, lymphocytes, phagocytes and the enzyme xanthine oxidase . Upregulation of O2-generating NADH oxidases may play a key role in causing impaired vascular tone and hypertension .

Many papers have claimed that high doses of infused or orally administrated antioxidants can scavenge ROS, restore NO bioavailability and ameliorate human endothelial dysfunction , although negative reports are now accumulating . The timing of antioxidant administration and the state of the vascular bed may be critical variables . Hence it is possible that examination of short-term vascular effects, which is easily achievable in human studies by measurements of forearm blood flow, can gather evidence about localized scavenging of RS in blood vessel walls by antioxidants.

However, it must not be assumed that any beneficial effects of antioxidants on vascular function are necessarily due to free radical scavenging. For example, ascorbate may have a direct stimulating effect on eNOS activity by increasing endothelial tetrahydrobiopterin levels .

Exogenous Antioxidants And Human Health

Manage Your Oxidative Stress & Antioxidant Capacity | Prof Jin-Xiong She Interview Series Ep 5

Human body put in place several strategies to counteract the effects of free radicals and oxidative stress, based on enzymatic and nonenzymatic antioxidant molecules, all of them being endogenous antioxidants. Beside these, there are several exogenous antioxidant molecules of animal or vegetal origin, mainly introduced by diet or by nutritional supplementation.

Here, we will discuss the most relevant nutritional antioxidants and their protective effects for human health.

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Oxidative Stress In Clinical Samples Of Cancer Patients

ROS is a contributing factor in the natural process of aging as well as in various pathological diseases including cancer. Oxidative stress has been reported in almost all types of cancers, promoting many aspects of tumor development and progression . During the process of carcinogenesis, an increase in ROS levels can occur due to elevated metabolic activity, oncogene activation, increased cellular receptor signaling, or mitochondrial dysfunction. Overproduction of ROS can also be induced exogenously by carcinogenic insults such as cigarette smoke, heavy metals, ionizing radiation, and asbestos . Alternatively, oxidative stress observed in cancer cells can arise from low levels or inactivation of antioxidant defense mechanisms as a result of mutations in tumor suppressor genes. For instance, mutant BRCA1 and p53 have been shown to attenuate the activation and function of nuclear factor -like 2 , a transcription factor that stimulates a stress response pathway by inducing expression of ROS-detoxifying enzymes .

Detrimental Effects Of Free Radicals On Human Health

As stated before, if in excess, free radicals and oxidants give rise to a phenomenon known as oxidative stress this is a harmful process that can negatively affect several cellular structures, such as membranes, lipids, proteins, lipoproteins, and deoxyribonucleic acid . Oxidative stress emerges when an imbalance exists between free radical formation and the capability of cells to clear them. For instance, an excess of hydroxyl radical and peroxynitrite can cause lipid peroxidation, thus damaging cell membranes and lipoproteins. This in turn will lead to malondialdehyde and conjugated diene compound formation, which are known to be cytotoxic as well as mutagenic. Being a radical chain reaction, lipid peroxidation spreads very quickly affecting a large amount of lipidic molecules . Proteins may as well being damaged by oxidative stress, undergoing to conformational modifications that could determine a loss, or an impairment, of their enzymatic activity .

If not strictly controlled, oxidative stress can be responsible for the induction of several diseases, both chronic and degenerative, as well as speeding up body aging process and cause acute pathologies .

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Is There A Single Biomarker Of Oxidative Stress Or Oxidative Damage

No, there is not , although biomarkers of damage to several different molecules frequently rise in parallel in cells subjected to severe oxidative stress. Why is there no such correlation in healthy tissues? Levels of 8OHdG in DNA are a steady-state balance between rate of oxidative DNA damage and rate of removal of lesions by DNA repair mechanisms. Isoprostanes, once formed, are metabolized quickly. Oxidized proteins are degraded, mostly by the proteasome system, and appear to turn over more slowly. Thus even if all biomolecules are damaged, the extents and time courses of the biomarkers of such damage can be very different. Hence, no correlation was observed between levels of plasma isoprostanes and oxidative DNA damage products in healthy human subjects .

Measuring Rs In Vivo: Basic Principles

Analysis of oxidative stress. (A, B) ROS production measurement by flow ...

Some fascinating techniques such as L-band electron spin resonance with nitroxyl probes and magnetic resonance imaging spin trapping are under development to measure RS directly in whole animals , but no probes are currently suitable for human use. Most RS persist for only a short time in vivo and cannot be measured directly. There are a few exceptions: examples include H2O2 , and perhaps, NO, in the sense that serum levels of NO2 have been claimed to measure vascular endothelial NO synthesis , despite the fact that NO2 is quickly oxidized to NO3in vivo . Essentially, there are two approaches to detecting transient RS:

  • attempting to trap these species and measure the levels of the trapped molecules and
  • measuring the levels of the damage done by RS, that is, the amount of oxidative damage.

Depending on the method that is used to measure it, the plasma or serum total antioxidant capacity’ usually involves major contributions from urate, ascorbate and sometimes albumin SH groups , although different methods measure different things . Thus, for example, if plasma albumin levels fall, TAC will fall. If urate levels rise, TAC will rise. The multiple changes in blood chemistry that occur in sick people mean that TAC changes should be interpreted with caution. TAC is also influenced by diet, often because consumption of certain foods may produce changes in plasma ascorbate and/or urate levels .

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Assessment Of Antioxidant Status

The human body is equipped with an antioxidant system that serves to counterbalance the deleterious effects of oxidative free radicals. When the balance between antioxidants and ROS species, referred to as redox homeostasis, is disturbed, oxidative stress can occur. The disturbance of this prooxidant and antioxidant balance can be a result of increased free radical production, antioxidant enzyme inactivation, or excessive antioxidant consumption. Assessment of the antioxidant status can thereby be correlated to the extent of oxidative stress in clinical samples.

Redox homeostasis is regulated by two arms of antioxidant machineries: enzymatic components and nonenzymatic, low molecular compounds. Several approaches have been developed to measure the different activities or levels of these antioxidants. Alternatively, the total antioxidant status can be evaluated to assess the oxidative state of clinical samples.

2.3.1. Enzymatic Antioxidants

Superoxide Dismutase. Superoxide dismutase is a family of antioxidant enzymes that regulate ROS levels by catalyzing the conversion of superoxide to hydrogen peroxide and molecular oxygen .

2.3.2. Nonenzymatic Antioxidants

Vitamin E. Vitamin E is a lipid-soluble vitamin which acts as a lipid peroxyl radical scavenger, preventing lipid peroxidation chain reactions in the cell membranes .

2.3.3. Total Antioxidant Capacity

Total Phenolic Content Assay

Phenolic compounds are present in fruits, vegetables and medicinal plants and can impart antioxidant capacity to reduce the effects of reactive oxygen species. These compounds also can provide UV protection, are anti-inflammatory and anti-microbial. The ZenBio Total Phenolic Content Assay uses the Folin-Ciocalteu Reagent to react with phenolic compounds in a test sample producing a highly colored molybdenum species . The phenolic content of a sample can be normalized to equivalent Gallic Acid units. The total phenolic content assay is an endpoint assay measuring absorbance at = 765nm .

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So How Will You Measure The Anti

Besides the kits mentioned in this post, you can also choose to outsource your oxidative activity measurement, to labs such as tebu-bio who perform this service .

Interested in finding the best way to measure the anti-oxidative potential of your samples? Leave your questions and comments below, Ill be pleased to get in touch with you!

An Introduction To Oxidative Stress Assays

Oxidative Stress Blood Test and Shocking Results After Using Protandim

Oxidative stress occurs when there is an increased production of reactive oxygen species or a decrease in the effectiveness of cellular antioxidant defenses. Cells generate ROS as a result of increased metabolism or a stress response. Glutathione, an antioxidant, is used to mitigate the damage of ROS. Assays to determine oxidative stress may measure levels of toxic reactive oxygen species or levels of cellular antioxidants.

Glutathione is an antioxidant found in eukaryotic cells, most often in the reduced form . A small portion of cellular glutathione is in the oxidized state . GSSG is an excellent indicator of oxidative stress in cells.

The GSH/GSSG-Glo Assay is a luminescence-based system used to detect and quantify glutathione in both the oxidized and reduced states. The stable luminescence is produced by the conversion of Luciferin-NT, a GSH probe, to luciferin by a glutathione S-transferase enzyme. This conversion to luciferin is coupled to a firefly luciferase reaction that produces a luminescent signal proportional to the amount of GSH present.

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How Do You Know If You Have Oxidative Stress

Here are a few signs to look out for:

  • Fatigue
  • Headaches and sensitivity to noise
  • Susceptibility to infections
  • Oxidative stress has also been associated with numerous health conditions including chronic fatigue syndrome, fibromyalgia, diabetes, Alzheimers disease, anxiety, insomnia, cancer, and more. I feel so strongly about helping you address and prevent these conditions that I decided to write a whole blog series about oxidative stress covering a different condition in each article. Find that blog series here.

    Cardiovascular Disease And Oxidative Stress

    Cardiovascular diseases are clinical entities with a multifactorial etiology, generally associated with a very large amount of risk factors, the most broadly recognized of which are hypercholesterolaemia, hypertension, smoking habit, diabetes, unbalanced diet, stress, and sedentary life . During the last years, research data pointed out that oxidative stress should be considered either a primary or a secondary cause for many CVDs . Oxidative stress acts mainly as a trigger of atherosclerosis. It is well known that atheromatous plaque formation results from an early endothelial inflammation, which in turn leads to ROS generation by macrophages recruited in situ. Circulating LDL are then oxidized by reactive oxygen species, thus leading to foam cell formation and lipid accumulation. The result of these events is the formation of an atherosclerotic plaque. Both in vivo and ex vivo studies provided evidences supporting the role of oxidative stress in atherosclerosis, ischemia, hypertension, cardiomyopathy, cardiac hypertrophy, and congestive heart failure .

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    Antioxidant Peptides Exhibit Acei Activity

    Oxidative stress caused by either increased production of reactive oxygen or compromising the biological antioxidant defense system is an issue in the development and progression of CVD.30 Since increased oxidative stress or free radicals cause oxidative damage to a number of intracellular or extracellular molecules, such nucleic acids, proteins, carbohydrates or lipids, or even other smaller physiologically important molecules, they have been implicated in other diseases such as diabetes, cancer, or obesity, conditions that may aggravate the pathogenesis of CVD. Owing to the fact that hypertension is associated with oxidative stress, antioxidant-rich diets reduce the arterial blood pressure in hypertensive subjects and modulate endothelial function.

    M. Mendez-Armenta, C. Rios, in, 2011

    How Do You Diagnose Oxidative Stress

    Acetate inhibits oxidative stress in LPS

    Tests to measure oxidative stress of cells are specialized tests and may not be available in all laboratories. There are several techniques to measure OS. Standard OS measurement methods include direct and indirect assessment of OS. The direct methods directly estimate ROS levels while the indirect methods measure the levels of the products formed due to oxidation of cellular components by free radicals and an indirect measure of reactive oxygen species .

    Direct Methods
    • Chemiluminescence to measure ROS levels
    • ROS levels in white cells and platelets by flow cytometry
    • Cytochrome c reduction test
    • evels of products of lipid peroxidation, protein oxidation and DNA oxidation
    • Chemokine levels
    • Levels of antioxidants, micronutrients and enzymes
    • Redox potential
    • Thiobarbituric acid assay

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