Exploring Oxidative Stress And Nitrosative Stress With OxisResearch®
I. Oxidative and Nitrosative Stress Overview
Every cell has chemical reactions involving the oxidation and reduction of molecules. These reactions or redox pathways can lead to the production of free radicals.
A free radical is any chemical species capable of independent existence possessing one or more unpaired electrons. Biological free radicals are thus highly unstable molecules that have electrons available to react with various organic substrates.
Many free radicals are the result of naturally occurring processes such as oxygen metabolism and inflammatory processes. For example, when cells use oxygen to generate energy, free radicals are created as a consequence of ATP production by the mitochondria. Exercise can increase the levels of free radicals as can environmental stimuli such as ionizing radiation (from industry, sun exposure, cosmic rays, and medical X-rays), environmental toxins, altered atmospheric conditions (e.g. hypoxia and hyperoxia), ozone and nitrous oxide (primarily from automobile exhaust). Lifestyle stressors such as cigarette smoking and excessive alcohol consumption are also known to affect levels of free radicals. Radical species may combine to form other more damaging or toxic species such as peroxynitrite (O=NOO¯), a product of superoxide and nitric oxide radical reaction.
Free radicals react with key organic substrates such as lipids, proteins, and DNA. Oxidation of these biomolecules can damage them, disturbing normal functions and may contribute to a variety of disease states.
It has been noted that certain organ systems are predisposed to greater levels of oxidative or nitrosative stress. Those organ systems most susceptible to damage are the pulmonary system (exposed to high levels of oxygen), the brain (exhibits intense metabolic activity yet has lower levels of endogenous antioxidants), the eye (constantly exposed to damaging UV light), circulatory system (victim to fluctuating oxygen and nitric oxide levels) and reproductive systems (at risk from the intense metabolic activity of sperm cells). Nearly every organ system can be found to have an Oxidative or Nitrosative “Achilles heel”. With the current understanding that free radicals can act as cell signaling or “messenger” agents it is likely that they also play a role in normal cellular function as well as various disease etiologies.
Reactive Oxygen Species (ROS) is a term collectively describing radicals and other non-radical reactive oxygen derivatives. These intermediates may participate in reactions giving rise to free radicals or that are damaging to organic substrates. ROS in living organisms include the following:
Reactive Nitrogen Species (RNS) are radical nitrogen-based molecules that can act to facilitate nitrosylation reactions. Reactive Nitrogen Species (RNS) include:
| Nitrous oxide
Many other radical species can be formed by biological reactions, for example: phenolic and other aromatic species are often formed during xenobiotic metabolism as part of natural detoxification mechanisms.
Oxidative stress occurs when the generation of ROS in a system exceeds the system’s ability to neutralize and eliminate them. The imbalance can result from a lack of antioxidant capacity caused by disturbance in production, distribution, or by an over-abundance of ROS from an environmental or behavioral stressor. If not regulated properly, the excess ROS can damage a cell’s lipids, protein or DNA, inhibiting normal function. Because of this, oxidative stress has been implicated in a growing list of human diseases as well as in the aging process.
Nitrosative stress occurs when the generation of RNS in a system exceeds the system’s ability to neutralize and eliminate them. Nitrosative stress may lead to nitrosylation reactions that can alter protein structure thus inhibiting normal function.
An extensive, highly effective group of protective agents and defense mechanisms referred to collectively as the Antioxidant Defense System (ADS), acts to regulate oxidative reactions.
The ADS includes enzymes and antioxidants to prevent the start of oxidative damage and/or control its spread. There are also enzymes to repair oxidative damage, and mechanisms to target damaged molecules for destruction and replacement. Essential antioxidants are either endogenous (internally synthesized) or exogenous (consumed). They are typically categorized as scavenger antioxidants and prevention antioxidants.
Scavenger antioxidants remove ROS and include the following categories:
- Small molecule antioxidants include both water-soluble compounds such as Vitamin C or glutathione and lipid soluble compounds such as Vitamin E, carotenes, lipoic acid, and Coenzyme Q10.
- Large molecule “enzyme” antioxidants include superoxide dismutase (SOD) that detoxifies the superoxide ion, catalase, which deals with hydrogen peroxide (H2O2), and glutathione peroxidase (GPx) whose function is to detoxify cellular peroxides. These enzymes must be synthesized by cells and are subject to genetic and/or macromolecular regulatory mechanisms.
Preventative antioxidants hinder the formation of new ROS. These antioxidants are proteins that bind ROS to protect essential proteins. The group includes albumin, metallothionine, transferrin, ceruloplasmin, myoglobin, and ferritin.
ROS and RNS today:
Researchers are now making rapid progress in understanding the role of oxidative stress and nitrosative stress in cardiovascular diseases such as atherosclerosis, ischemia/reperfusion injury, restenosis and hypertension; cancer; inflammatory diseases such as acute respiratory distress syndrome (ARDS), asthma, inflammatory bowel disease (IBD), dermal and ocular inflammation and arthritis; metabolic disease such as diabetes; and diseases of the central nervous system (CNS) such as amyotrophic lateral sclerosis (ALS), Alzheimer’s, Parkinson’s, and stroke. The increased awareness of oxidative stress related to disease and the need to measure the delicate balance that exists between free radicals and the systems in place to regulate them has given rise to a demand for new research tools.
II. Quantifying Oxidative and Nitrosative Stress
Researchers wishing to assess the level and/or effect of oxidative or nitrosative stress on a given system typically have two basic methods at their disposal:
1. Measure levels of specific ROS and RNS present in the system
2. Measure the effect (including damage and ADS remodeling) of ROS and RNS on a given system.
Since ROS and RNS are generally highly reactive, exacting Spin Trap methodologies are typically required to adequately detect and quantify their existence in biological samples. For this reason the second method is the most often employed strategy in the study of oxidative and nitrosative stress. To accomplish this, researchers can look at modifications to a system’s regulatory mechanisms (e.g. endogenously expressed antioxidants), or look at by-products of the applied stressor (e.g. damage/modification of lipids, proteins or DNA). Researchers may also wish to look at causative factors such as the events facilitating an inflammatory cascade such as neutrophil activation and subsequent release of myeloperoxidase and/or lactoferrin.
The OxisResearch® product line provides a “toolbox” for researchers to accomplish the basic goals as described earlier. Generally speaking, we offer researchers four basic types of tools...
...directed against four major areas of biomarkers:
3) Antioxidant (regulatory)
4) Inflammatory (pro-oxidant)
Tools for assaying the presence and quantity of:
1. Oxidative Biomarkers:
a) Lipids (MDA, HAE, LOOH, 8-Isoprostane, 8-iso- metabolite)
b) Proteins (Aconitase, a1-Antiproteinase, Nitrotyrosine)
c) DNA (8-OHdG)
2. Nitrosative Biomarkers:
a) Nitric Oxide (Enzymatic and non-enzymatic Griess methods)
b) Nitric Oxide Synthase (Colorimetric and Radioactive methods)
c) Peroxynitrite (Nitrotyrosine)
3. Antioxidant Biomarkers:
a) Glutathione (GSH/GSSG, cGPx, plGPx, GR)
b) Superoxide Dismutase (SOD-525)
c) Catalase (Catalase-520)
4. Inflammatory Biomarkers:
a) Neutrophils (Myeloperoxidase, Lactoferrin)
Tools for semi-quantitative (e.g. blotting and/or IHC) detection of:
1. Oxidative Biomarkers:
a) Lipids (Anti-4-Hydroxynonenal)
b) Proteins (Anti-Nitrotyrosine)
c) DNA (Anti-8-Hydroxydeoxyguanasine)
2. Nitrosative Biomarkers:
a) Nitric Oxide Synthase (Anti-iNOS, Anti-cNOS, Anti-bNOS)
b) Peroxynitrite (Anti-Nitrotyrosine)
3. Antioxidant Biomarkers:
a) Glutathione (Anti-GST M1-1, Anti GST A1-1, Anti GST P1-1)
b) Superoxide Dismutase (Anti-Cu/ZnSOD, Anti-MnSOD)
c) Catalase (Anti-Catalase)
d) Cytochromes (Anti P450-2E1, Anti-P450 3A4)
4. Inflammatory Biomarkers:
a) Neutrophils (Anti-Myeloperoxidase, Anti-Lactoferrin)
1. Oxidative Biomarkers:
a) Proteins (Anti-Nitrotyrosine)
b) DNA (Anti-8-hydroxydeoxyguanasine)
2. Nitrosative Biomarkers: (NO associated enzymes)
a) Nitric Oxide Synthase (iNOS, cNOS, bNOS)
3. Antioxidant Biomarkers:
a) Glutathione (GST M1-1, GST A1-1, GST P1-1)
b) Superoxide Dismutase (bovine Cu/ZnSOD, MnSOD)
c) Catalase (Catalase)
4. Inflammatory Biomarkers:
a) Neutrophils (Myeloperoxidase)
1. Oxidative Biomarkers:
a) Lipids (4-HNE, 4-HNE-diethylacetal, 4-HHE-diethylacetal)
b) Proteins (3-Nitro-L-Tyrosine)
c) DNA (8-OHG 8-OHdG)
2. Nitrosative Biomarkers:
a) Peroxynitrite (3-nitro-L-Tyrosine)
3. Antioxidant Biomarkers:
a) Glutathione (GR-control, cGPx-control)
b) Superoxide Dismutase (Cu/ZnSOD-control)
c) Catalase (Catalase-control)
III. Selecting OxisResearch® Products
Catalog Product Information
OXIS International, Inc. through its OxisResearch® line of products has been addressing the need for measuring the balance that exists between free radicals, and the systems in place to regulate them. OXIS offers an array of innovative products for testing of Antioxidant, Oxidative, Nitrosative and Inflammatory Biomarkers. To help you more effectively evaluate our products for your particular system, this catalog thoroughly describes each OxisResearch® kit including useful applications and reference materials for all. Each assay listing shows the following information in detail:
If unable to locate information specific to your model or sample type, the OXIS application specialists are available for consultation and can recommend a product or technique best suited for your particular application.
- Product Description
- Summary of Specifications
- Method of Use
- Selected Research Applications
- Product References
OXIS products are playing a key role in what is now known about the role of reactive oxygen (ROS) and nitrogen (RNS) species in physiology and disease. Our products have been cited in over 150 peer reviewed international scientific journals. We hope the selected citations listed in this catalog will convey the multiplicity of current uses for our products and perhaps inspire you in finding new ones.
For more OxisResearch® product information and assistance, visit our web site at www.oxisresearch.com, email us at firstname.lastname@example.org, or call OXIS International at 800.547.3686 (USA) to speak with one of our expert application specialists.
An immunoglobulin molecule that has a specific amino acid sequence by virtue of which it interacts only with the antigen that induced its synthesis in cells of the lymphoid series (especially plasma cells) or with antigen closely related to it. Antibodies are classified according to their ode of action as agglutinins, bacteriolysins, haemolysins, opsonins, precipitins, etc.
Antibodies produced by clones of cells such as those isolated after hybridization of activated b lymphocytes with neoplastic cells. These hybrids are often referred to as hybridomas.
Immunoglobulins induced by antigens specific for tumours other than the normally occurring histocompatibility antigens.
2 - any of a large number of proteins of high molecular weight that are produced normally by specialized B cells after stimulation by an antigen and act specifically against the antigen in an immune response, that are produced abnormally by some cancer cells, and that typically consist of four subunits including two heavy chains and two light chains -- called also immunoglobulin
Antibodies produced by human or animal cells following clinical or experimental exposure to parasitic protozoan antigens.
Immunoglobulins produced as a response to viral antigens; includes all classes of immunoglobulins elicited by all viral components.
antioxidant (or anti-oxidant)
One of many synthetic or natural substances added to products to prevent or delay their deterioriation by action of oxygen in air. In biochemistry and medicine, antioxidants are enzymes or other organic substances, such as vitamin e or beta-carotene, that are capable of counteracting the damaging effects of oxidation in animal tissue. (anti-oxidant)
2 - any of various substances (as beta-carotene, vitamin C, and alpha-tocopherol) that inhibit oxidation or reactions promoted by oxygen and peroxides and that include many held to protect the living body from the deleterious effects of free radicals. (anti-oxidant)
<biochemistry> A specific biochemical in the body which has a particular molecular feature that makes it useful for measuring the progress of disease or the effects of treatment.
2 - a distinctive biological or biologically derived indicator (as a biochemical metabolite in the body) of a process, event, or condition (as aging or exposure to a toxic substance) <age-related biomarkers of disease and degenerative change.
<enzyme> Tetrameric haem enzyme (245 kD) that breaks down hydrogen peroxide.
2 - a red crystalline enzyme that consists of a protein complex with hematin groups and catalyzes the decomposition of hydrogen peroxide into water and oxygen.
Decomposition of H2O2 to O2 and H2O, as in the action of catalase; analogous to peroxidase reaction.
enzyme-linked immunoabsorbent assay - <investigation> The enzyme-linked immunoabsorbent assay is serologic test used as a general screening tool for the detection of antibodies to the HIV virus. Reported as positive or negative. Since false positive tests due occur (for example recent flu shot), positives will require further evaluation using the western blot. ELISA technology links an a measurable enzyme to either an antigen or antibody. In this way, it can then measure the presence of an antibody or an antigen in the bloodstream.
2 - a quantitative in vitro test for an antibody or antigen in which the test material is adsorbed on a surface and exposed either to a complex of an enzyme linked to an antibody specific for the antigen or an enzyme linked to an anti-immunoglobulin specific for the antibody followed by reaction of the enzyme with a substrate to yield a colored product corresponding to the concentration of the test material.
<biochemistry> A protein molecule produced by living organisms that catalyses chemical reactions of other substances without itself being destroyed or altered upon completion of the reactions.
Enzymes are classified according to the recommendations of the Nomenclature Committee of the International Union of Biochemistry. Each enzyme is assigned a recommended name and an Enzyme Commission (EC) number.
2 - any of numerous complex proteins that are produced by living cells and catalyze specific biochemical reactions at body temperatures.
They are divided into six main groups:
oxidoreductases - <enzyme> The class of all enzymes catalyzing oxidoreduction reactions. The substrate that is oxidised is regarded as a hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The recommended name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be used. Oxidase is only used in cases where o2 is the acceptor.
transferases - <enzyme> Transferases are enzymes transferring a group, for example, the methyl group or a glycosyl group, from one compound (generally regarded as donor) to another compound (generally regarded as acceptor). The classification is based on the scheme "donor:acceptor group transferase".
hydrolases - <enzyme>
lyases - <enzyme> A class of enzymes that catalyze the cleavage of c-c, c-o, and c-n, and other bonds by other means than by hydrolysis or oxidation.
isomerases - <enzyme> A class of enzymes that catalyze geometric or structural changes within a molecule to form a single product. The reactions do not involve a net change in the concentrations of compounds other than the substrate and the product.
ligases - <enzyme> A class of enzymes that catalyze the formation of a bond between two substrate molecules, coupled with the hydrolysis of a pyrophosphate bond in ATP or a similar energy donor.
A crystalline betaine C9H15N3O2S that is found especially in ergot and blood -- called also thioneine
A chemically active atom or molecular fragment containing a chemical charge due to an excess or deficient number of electrons. Radicals seek to receive or release electrons in order to achieve a more stable configuration, a process that can damage the large molecules within cells.
2 - an especially reactive atom or group of atoms that has one or more unpaired electrons ; especially : one that is produced in the body by natural biological processes or introduced from outside (as in tobacco smoke, toxins, or pollutants) and that can damage cells, proteins, and DNA by altering their chemical structure.
Highly reactive molecules with an unsatisfied electron valence pair. Free radicals are produced in both normal and pathological processes. They are proven or suspected agents of tissue damage in a wide variety of circumstances including radiation, damage from environment chemicals, and aging. Natural and pharmacological prevention of free radical damage is being actively investigated.
free radical reductase
<enzyme> Catalyses conversion of carbon-centreed lipid radicals into an inactive species by utilizing vitamin e at one end and glutathione at the other
<biochemistry> The tripeptide _ glutamylcysteinylglycine. It contains an unusual peptide linkage between the _ carboxyl group of the glutamate side chain and the amine group of cysteine.
The concentration of glutathione in animal cells is _5mM and its sulphydryl group is kept largely in the reduced state. This allows it to act as a sulphydryl buffer, reducing any disulphide bonds formed within cytoplasmic proteins to cysteines. Hence, few, if any, cytoplasmic proteins contain disulphide bonds.
Glutathione is also important as a cofactor for the enzyme glutathione peroxidase, in the uptake of amino acids and participates in leucotriene synthesis.
2 - a peptide C10H17N3O6S that contains one amino acid residue each of glutamic acid, cysteine, and glycine, that occurs widely in plant and animal tissues, and that plays an important role in biological oxidation-reduction processes and as a coenzyme.
Biological molecules soluble in apolar solvents, but only very slightly soluble in water. They are an heterogenous group (being defined only on the basis of solubility) and include fats, waxes and terpenes.
2 - any of various substances that are soluble in nonpolar organic solvents (as chloroform and ether), that with proteins and carbohydrates constitute the principal structural components of living cells, and that include fats, waxes, phospholipids, cerebrosides, and related and derived compounds.
Peroxidase catalyzed oxidation of lipids using hydrogen peroxide as an electron acceptor.
Peroxides produced in the presence of a free radical by the oxidation of unsaturated fatty acids in the cell in the presence of molecular oxygen. The formation of lipid peroxides results in the destruction of the original lipid leading to the loss of integrity of the membranes. They therefore cause a variety of toxic effects in vivo and their formation is considered a pathological process in biological systems. Their formation can be inhibited by antioxidants, such as vitamin e, structural separation or low oxygen tension.
<enzyme> Peroxidase found in the lysosomal granules of myeloid cells, particularly macrophages and neutrophils, responsible for generating potent bacteriocidal activity by the hydrolysis of hydrogen peroxide produced in the metabolic burst) in the presence of halide ions. A metallo enzyme containing iron. Deficiency of myeloperoxidase is not fatal and it is reportedly absent entirely in chickens.
2 - a green peroxidase of phagocytic cells (as neutrophils and monocytes) that is held to assist in bactericidal activity by catalyzing the oxidation of ionic halogen to free halogen.
nitric oxide (NO)
<biochemistry> This compound is produced from L arginine by the enzyme nitric oxide synthase. Acts as a potent vasorelaxant via elevation of intracellular cGMP in vascular smooth muscle.
Synthesis of nitric oxide is not confined to endothelium, isoforms of nitric oxide synthase are also found in brain, neutrophils and platelets.
Synonym: endothelium derived relaxation factor.
Oxidizing agents or electron-accepting molecules in chemical reactions in which electrons are transferred from one molecule to another (oxidation-reduction). In vivo, it appears that phagocyte-generated oxidants function as tumour promoters or cocarcinogens rather than as complete carcinogens perhaps because of the high levels of endogenous antioxidant defenses. It is also thought that oxidative damage in joints may trigger the autoimmune response that characterises the persistence of the rheumatoid disease process.
Classically, one of a group of enzymes, now termed oxidoreductases (EC class 1), that bring about oxidation by the addition of oxygen to a metabolite or by the removal of hydrogen or of one or more electrons. Oxidase is now used for those cases in which O2 acts as an acceptor (of H or of electrons); those removing hydrogen are now termed dehydrogenases. For individual oxidases, see the specific names.
Direct oxidase, originally, an oxidase catalyzing the transfer of O2 directly to other bodies; now termed oxygenase.
Indirect oxidase, originally, an oxidase that acts by reducing a peroxide; now termed peroxidase.
Terminal oxidase, the last protein in the electron transport, respiratory chain. In mammals this is cytochrome c oxidase.
<biochemistry> The process whereby fatty acids are degraded in steps, losing 2 carbons as (acetyl) CoA. Involves CoA ester formation, desaturation, hydroxylation and oxidation before each cleavage.
<chemistry> A reactant that accepts electrons from another reactant. The oxidizing agent is the species getting reduced.
A highly oxidized environment within cells that is thought to promote HIV replication because cells are forced into a highly activated state due to loss of control of their regulatory systems.
2 - physiological stress on the body that is caused by the cumulative damage done by free radicals inadequately neutralized by antioxidants and that is held to be associated with aging.
<enzyme> A haem enzyme that catalyses reduction of hydrogen peroxide by a substrate that loses two hydrogen atoms. Within cells, may be localised in peroxisomes. Coloured reaction products allow detection of the enzyme with high sensitivity, so peroxidase coupled antibodies are widely used in microscopy and ELISA. Lactoperoxidase is used in the catalytic surface labelling of cells by radioactive iodine.
<chemistry> Term used interchangeably for the superoxide anion or the weak acid HO2(.).
Superoxide is generated both by prokaryotes and eukaryotes and is an important product of the metabolic burst of neutrophil leucocytes. A very active oxygen species, it can cause substantial damage and may be responsible for the inactivation of plasma antiproteases that contributes to the pathogenesis of emphysema.
2 - any of various toxic oxygen-containing free radicals ; especially : the monovalent anion O2- or a compound containing it <potassium superoxide KO2>
<chemistry> A harmful derivative of oxygen capable of oxidative destruction of cell components.
superoxide dismutase (SOD)
<enzyme> Any of a range of metalloenzymes that catalyses the formation of hydrogen peroxide and oxygen from superoxide and thus protects against superoxide induced damage.
2 - a metal-containing antioxidant enzyme that reduces potentially harmful free radicals of oxygen formed during normal metabolic cell processes to oxygen and hydrogen peroxide.
Usually has either iron or manganese as the metal cation in prokaryotes, copper or zinc in eukaryotes.
V. A Letter To Our Patrons
Our catalog introduces fourteen new assay kits to compliment our already broad range of oxidative stress related testing products. In the past two years we have identified new biomarkers of relevance to oxidative and nitrosative stress while creating simpler, more robust tests for existing biomarkers where possible.
With the knowledge that commercially available tests related to oxidative protein modifications were lacking in the marketplace we tasked our researchers with identifying biomarkers that may be useful in this area. The result of this program was the development of two new assays for detection of enzyme activities subject to oxidative modification (i.e. Aconitase-340 for mitochondrial and tissue related studies and α1-Antiproteinase- 410 for plasma relevant studies). The activity of both of these enzymes has been shown to be mediated by oxidative stressors and in the case of the α1-antiproteinase, the deactivation is thought to be reversible via the methionine sulfoxide/methionine sulfoxide reductase pathway. We also brought to market a new Nitrotyrosine ELISA kit based on its relevance to peroxynitrite mediated damage to proteins.
In the area of lipid peroxidation we sought to improve upon existing methods such as our popular LPO-586 assay. With the release of our MDA-586 and HAE-586 assays we made it possible for the first time to measure highly reactive hydroxyalkenal by-products without the contribution of the more readily detectable malondialdehyde (MDA). At the same time we improved upon the MDA side of the assay by providing validated methods for detection of “free” and “total” MDA in biological samples. We also provide third derivative spectroscopy procedures for those interested in a more definitive test for plasma MDA using our MDA-586 product. We recommend our MDA-586 for plasma and tissue samples and our HAE-586 for detection of HAE formation in low protein samples such as oils and other lipid extracts.
We now offer improved methods for determining 8-epi-prostaglandin F2α levels in various biological samples. Two new assays we introduced, one for determination of urinary 8-isoprostane that does not require solid phase extraction and an entirely new assay for the metabolite of 8-isoprostane in urine. We believe the metabolite assay may offer significant advantages over traditional measurements of the parent compound.
Other additions to our assay portfolio include a glutathione-S-transferase assay, glucose-6-phosphate de- dehydrogenase hydrogenase assay, and a nitric oxide assay.
We expect that the next two years will yield more and better technologies for the study of oxidative and nitrosative stress. We are committed to quickly identifying and facilitating their commercialization in order that we might remain your preferred provider in this area.
Thank you for you patronage, we look forward to being of continued service to you and hope we can be an integral part of your successful investigation.
Our very best regards,
The OXIS Team
"Dedicated to helping researchers understand the relevance
of Oxidative and Nitrosative Stress on biological systems”