By Stephen Byrnes, ND, RNCP

Oxidative stress (OS) is fast becoming the nutritional and medical buzzword for the 21st century. Implicated in a growing list of diseases, from cataracts to cancer, health-conscious people should take steps to protect themselves against the ravages of free radicals, the active criminals in OS. Despite the growing dangers of OS, there are some simple, but powerful, weapons against it. An avoidance of factors that contribute to OS; a diet of whole, organic, unprocessed foods; and supplemental anti-oxidants, afford the best protection against this serious and insidious condition.

Oxidative Stress (OS) is not, in and of itself, a disease but a condition that can lead to or accelerate it. OS occurs when the available supply of the body’s antioxidants is insufficient to handle and neutralize free radicals of different types. The result is massive cell damage that can result in cellular mutations, tissue breakdown and immune compromise.

What are free radicals? They are highly unstable molecules that interact quickly and aggressively with other molecules in our bodies to create abnormal cells. They are capable of penetrating into the DNA of a cell and damaging its “blueprint” so that the cell will produce mutated cells that can then replicate without normal controls. Free radicals are unstable because they have unpaired electrons in their molecular structure. This causes them to react almost instantly with any substance in their vicinity. Oxygen, or oxyl, free radicals are especially dangerous.

Surprisingly, however, free radicals are involved in many cellular functions and are a normal part of living. When, for example, a mitochondria within a cell burns glucose for fuel, the mitochondria oxidizes the glucose and in so doing generates free radicals. White blood cells also use free radicals to attack and destroy bacteria, viruses and virus-infected cells. The detoxifying actions of the liver also require free radicals.

Although free radicals have useful functions in the body under controlled conditions, they are extremely unstable molecules that can damage cells if left uncontrolled. Free radicals destroy cellular membranes; enzymes and DNA. They accelerate aging and contribute to the development of many diseases, including cancer and heart disease.

Its important to note here that free radicals are also released in the body from the breaking down or detoxification of various chemical compounds. Additionally, certain foods contain free radicals which, when eaten, enter the body and damage it. The major sources of dietary free radicals are chemically-altered fats from commercial vegetable oils, vegetable shortening and all oils heated to very high temperatures.

Antioxidants to the Rescue

Fortunately, the body maintains a sophisticated system of chemical and biochemical defenses to control and neutralize free radicals. Chemical antioxidants scavenge free radicals, that is, they stabilize the unstable free radicals by giving them the electron they need to “calm down.” The antioxidants are usually consumed or used up in this process–they sacrifice themselves.

The main antioxidants are vitamins A, E and C, betacarotene, glutathione, bioflavonoids, selenium, zinc, CoQ10 (ubiquinone), and various phyto-chemicals from herbs and foods. Green tea, for example, is rich in polyphenols–powerful antioxidants that help fight cancer.

Biochemical antioxidants not only scavenge free radicals, but also inhibit their formation inside the body. These include lipoic acid, and repair enzymes such as catalase, superoxide dismutase (SOD), glutathione peroxidase. Melatonin, a hormone produced by the pineal gland, is also a potent antioxidant. Cholesterol, produced by the liver, is another major antioxidant, which the body uses to repair damaged blood vessels. It is probably for this reason that serum cholesterol levels rise as people age. With age comes more free radical activity and in response the body produces more cholesterol to help contain and control the damage.

Of all the antioxidants, glutathione appears to be pivotal. Made up of three amino acids (cysteine, glycine, and glutamic acid), glutathione is part of the antioxidant enzyme glutathione peroxidase and is THE major liver antioxidant. It is a basic tenet of natural medicine that health cannot exist if the liver is toxic. Not surprisingly, extremely low levels of glutathione are found in people suffering from severe OS. People with AIDS, cancer and Parkinson’s disease, for example, typically have very low glutathione levels.

As noted earlier, oxidative stress occurs when the amount of free radicals in the body exceeds its pool of available antioxidants. Obviously, knowing the varied sources of free radicals and avoiding them is an important part of minimizing their harmful effects.

Where Do They Come From?

As noted above, diet can be a major source of free radical stressors with processed or highly heated oils being the main offenders. If you are still using “foods” like refined vegetable oils, margarine or shortening (or “foods” made with them such as all commercial baked goods and “snack” chips), you need to remove them from your diet. Replace these harmful fats with natural, cold pressed oils such as olive oil (which can be used for cooking) and small amounts of flax oil or walnut oil (which should never be heated). Food grade, unrefined coconut oil and organic butter are also excellent choices, especially for cooking. Both of these naturally saturated fats are rich in certain fatty acids that have proven activity against bacteria, harmful yeasts, fungi and tumor cells.

Additionally, since saturated fats (from animal foods and the tropical oils) and monounsaturated oils (from olive oil and cold-pressed nut oils) are more chemically stable, they are much less susceptible to oxidation and rancidity than their polyunsaturated cousins, which are mostly found in vegetable oils. As a general rule, then, although the body does require a small amount of naturally occurring polyunsaturated oils in the diet each day, it’s best not to consume too much of them as they are more prone to free radical attack in the body. As Linus Pauling, PhD noted: “A diet high in unsaturated fatty acids, especially the polyunsaturated ones, can destroy the body’s supply of vitamin E and cause muscular lesions, brain lesions, and degeneration of blood vessels. Care must be taken not to include a large amount of polyunsaurated oil in the diet

The best food sources for polyunsaturates are fish, flax oil, sesame oil, walnut oil and dark green, leafy vegetables. One caveat: canola oil is not recommended due to its chemical instability and its content of trans-fatty acids (TFAs), formed during processing. TFAs are increasingly being linked wtih cancer, immune system dysfunction and heart disease.

Excessive sugar intake can also contribute to free radical damage. White and brown sugars, and even sugar from so-called natural sources, such as fruit and fruit juices, maple syrup and honey, get converted into triglycerides by the liver and are subject to free radical damage. These damaged fats then promptly attack your arteries and directly contribute to cardiovascular disease. Additionally, cancer and tumor cells feed off of sugar. It is for this reason that excessive sugar intake correlates very strongly with heart disease, cancer and a host of other ailments.

Poor nutrition in general contributes to OS. When the body is fed poorly, it slowly starves and all of its systems suffer. Weak organ systems are prime targets for free radical attack.

Free radicals are also released in the body from the detoxification of drugs (whether legal or illegal), artificial food colorings and flavorings, smog, preservatives in processed foods, alcohol, cigarette smoke, chlorinated drinking water, pesticides, radiation, cleaning fluids, heavy metals such as cadmium and lead, and assorted chemicals such as solvent traces found in processed foods and aromatic hydrocarbons such as benzene and naphthalene (found in moth balls).

Even psychological and emotional stress can contribute to OS. When the body is under stress, it produces certain hormones that generate free radicals. Moreover, the liver must eventually detoxify them and that process also generates free radicals.

Heightened OS has also been observed in athletes after intensive workouts due to the physical stress placed on the body. Both physical and emotional stress also prompt the release of endogenous cortisol, an adrenal hormone that reduces inflammation, but also suppresses the immune system.

It should be obvious that all of us are exposed to free radicals from a variety of sources. Those of us living in cities are exposed to very high levels due to increased smog and pollution. Certainly, all of us need to take preventive action. If not, we could face the following conditions in our futures.

Determining OS

When OS occurs, certain by-products are left behind that are excreted by the body, mostly in the urine. These by-products are oxidized DNA bases, lipid peroxides, and malonidialdehyde from damaged lipids and proteins. The higher the levels of these various markers, the greater the chance there is of an OS-induced disease, or the aggravation and acceleration of an existing one. People with Down’s Syndrome, for example, a genetic disorder, are subject to enormous OS due to increased cellular production of hydrogen peroxide, a potent oxidising agent, and frequently develop Alzheimer’s-like conditions in their 30s.

These tests can be ordered by a doctor, naturopath or nutritionist. If you are concerned, ask your health care provider.

Even if you do not have access to formal testing, anyone can do the following simple test to see how much the body has been affected by free radicals: hold out your hand, palm down, in a relaxed position. Pinch the skin on the back of the hand, lift up the fold and then release it. If you have minimal free radical damage, the skin will snap back into place quickly. If the skin takes a few seconds to go back into place, this is not a good sign and action must be taken.

Illnesses Associated With Oxidative Stress

GI Tract: Diabetes, pancreatitis, liver damage, and leaky gut syndrome
Brain and Nervous System: Parkinson’s disease, Alzheimer’s disease, hypertension and multiple sclerosis
Heart & Blood Vessels: Atherosclerosis, coronary thrombosis.
Lungs: Asthma, emphysema, chronic pulmonary disease.
Eyes: Cataracts, retinopathy, macular degeneration.
Joints: Rheumatoid arthritis
Kidneys: Glomerulonephritis
Skin: “Age spots,” vitiligo, wrinkles.
Body in General: Accelerated aging, cancer, autoimmune diseases, inflammatory states, AIDS and lupus.

Food sources of Antioxidants

CoQ10 (ubiquinone): Beef heart, beef liver, sardines, spinach, peanuts
Betacarotene: All orange and yellow fruits and vegetables; dark green vegetables
Zinc: Oysters, herring, lamb, whole grains
Selenium: Butter, meats, seafood, whole grains
Vitamin A: Cod liver oil, butter, liver, all oily fish
Vitamin E: Cold-pressed, unrefined nut and seed oils; wheat germ oil
Vitamin C: Berries, greens, broccoli, kale, kiwi, parsley, guava
Glutathione (GSH): Fresh fruits and vegetables, fresh meats, low-heat dried whey
Bioflavonoids: Most fruits and vegetables, buckwheat
Polyphenols: Green tea, berries.
Herbal Sources: Milk thistle, ginkgo biloba, tumeric, curry (Padma 28, a packaged Ayurvedic herbal formula, is a special blend of herbal antioxidants.)

NOTE: Try to purchase organic foods to minimize pesticide residues.

Oxygen metabolism, although essential for life, imposes a potential threat to cells because of the formation of partially reduced oxygen species.1,2 One electron reduction of oxygen produces superoxide whereas two electron reduction produces hydrogen peroxide. Therefore, electron flow through oxygen, utilizing processes such as the mitochondrial electron transport chain, flavoproteins, cytochrome P450 and oxidases, is tightly coupled to avoid partial reduction of oxygen.3

Normal cellular homeostasis is a delicate balance between the rate and magnitude of oxidant formation and the rate of oxidant elimination. Oxidative stress can, therefore, be defined as the pathogenic outcome of the overproduction of oxidants that overwhelms the cellular antioxidant capacity. Experimental support for oxidative stress as a mediator of cell death was provided recently by the finding that PC12 cells die following downregulation of Cu/Zn superoxide dismutase.4

Antioxidant defenses fall into two categories; enzymatic and nonenzymatic.1-3 Superoxide dismutases are metalloproteins that dismutate the superoxide radical (O2•) to hydrogen peroxide (H2O2) and molecular oxygen. Three types of superoxide dismutases are found in eukaryotic cells; Cu/Zn superoxide dismutase, predominantly located in the cytosolic fractions; Mn superoxide dismutase, located in the mitochondria, and EC superoxide dismutase, which is found in the extracellular space.1 Catalase, a heme protein located predominantly in peroxisomes and the inner mitochondrial membrane, catalyzes the conversion of H2O2 to H2O. In mammalian cells, the conversion of H2O2 to H2O is also accomplished by the reaction with glutathione catalyzed by glutathione peroxidases, a family of cytosolic selenoenzymes. Non-enzymatic defenses include small molecules such as membrane associated a-tocopherol, ascorbate and glutathione.
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Figure 1. Antioxidant Network

Biochemistry of Reactive Species: Free Radicals vs. Oxidants

The term free radicals has been equated with reactive species or oxidants. By definition, a radical is a molecule possessing an unpaired electron. Superoxide, nitric oxide, hydroxyl, alkoxyl and alkyl-peroxyl (lipid) are radicals. However, with the exception of hydroxyl radical none of these radicals are strong oxidants. Thus, not all radicals are strong oxidants and not all oxidants are radicals.

A critical function of reactive species is immunological host response. Generation of reactive species and strong oxidants by inflammatory cells is essential for killing invading microorganisms. However, experimental evidence has implicated reactive species in the pathogenic mechanism of several diseases. It is, therefore, important to understand the biochemical pathways for the induction of oxidative stress by reactive species. The most reasonable biochemical hypothesis is the reactive species-mediated modification of critical cellular targets.

Iron-sulfur enzymes are direct targets for superoxide and toxicity can be derived from the inactivation of these enzymes.1 Hydrogen peroxide at low mM levels does not react with many biological targets at an appreciable rate. However, the reaction of hydrogen peroxide with reduced divalent redox active metals such as iron can lead to the formation of strong oxidants. This reactivity of hydrogen peroxide may be important in biological oxidations of proteins and lipids that take place at the sites of metal binding. Divalent redox active metals can also catalyze the formation of the highly reactive hydroxyl by the metal-catalyzed Haber-Weiss reaction.5,6

O2 + Fe3+
→ O2 + Fe2+

However, hydroxyl radical reacts with almost all biological targets at rates exceeding 109 M-1sec-1 and therefore its diffusion distance inside a cell is minimal. Thus, in order for hydroxyl radical to cause toxicity it must be formed within a few Angstroms from a biological target.

An alternative pathway of superoxide toxicity is the formation of peroxynitrite by the reaction with nitric oxide.7 Nitric oxide is synthesized by nitric oxide synthases and mediates important physiological functions such as vasorelaxation, platelet aggregation, long term potentiation, and immune responses.8-11 The principal biological target of nitric oxide is guanylate cyclase and/or other iron-containing heme proteins. Nitric oxide is a radical but a weak one electron oxidant. Since both •NO and O2• are radicals they react rapidly to form peroxynitrite:

H2O2 +Fe2+
→ • OH + -OH + Fe3+

The second order rate constant of the reaction between nitric oxide and superoxide is 6.7 x 109 M-1sec-1 which is nearly three times faster than the reaction of superoxide with superoxide dismutase (2.9 x 109 M-1sec-1) and nearly thirty times faster than the reaction of •NO with heme proteins. This implies that the formation of peroxynitrite can out-compete the major scavenging pathways for •NO and O2•. Peroxynitrite is not a free radical but a strong one or two electron oxidant and nitrating agent.12-15 Although peroxynitrite can oxidize most biological molecules similar to the hydroxyl radical, the rate constants of the biological oxidations of peroxynitrite are 10,000 fold slower than the rate of hydroxyl radical. This implies that peroxynitrite will diffuse much further than the hydroxyl radical and will react with selective targets. The targets are determined for the most part by the rate by which they react with peroxynitrite. The fastest reactions for peroxynitrite presently are the reactions with Zn-S and Fe-S centers with metalloproteins and carbon dioxide.12,15 Whereas the Zn-S and Fe-S centers will be oxidized, the last two reactivities will promote nitration of tyrosine residues on proteins. Protein nitrotyrosine has been detected in human diseases and experimental models of disease that do not involve an inflammatory process.7

However, hydroxyl radical reacts with almost all biological targets at rates exceeding 109 M-1sec-1 and therefore its diffusion distance inside a cell is minimal. Thus, in order for hydroxyl radical to cause toxicity it must be formed within a few Angstroms from a biological target.

An alternative pathway of superoxide toxicity is the formation of peroxynitrite by the reaction with nitric oxide.7 Nitric oxide is synthesized by nitric oxide synthases and mediates important physiological functions such as vasorelaxation, platelet aggregation, long term potentiation, and immune responses.8-11 The principal biological target of nitric oxide is guanylate cyclase and/or other iron-containing heme proteins. Nitric oxide is a radical but a weak one electron oxidant. Since both •NO and O2 are radicals they react rapidly to form peroxynitrite:

• NO + O2
ONOO

The second order rate constant of the reaction between nitric oxide and superoxide is 6.7 x 109 M-1sec-1 which is nearly three times faster than the reaction of superoxide with superoxide dismutase (2.9 x 109 M-1sec-1) and nearly thirty times faster than the reaction of •NO with heme proteins. This implies that the formation of peroxynitrite can out-compete the major scavenging pathways for •NO and O2•. Peroxynitrite is not a free radical but a strong one or two electron oxidant and nitrating agent.12-15 Although peroxynitrite can oxidize most biological molecules similar to the hydroxyl radical, the rate constants of the biological oxidations of peroxynitrite are 10,000 fold slower than the rate of hydroxyl radical. This implies that peroxynitrite will diffuse much further than the hydroxyl radical and will react with selective targets. The targets are determined for the most part by the rate by which they react with peroxynitrite. The fastest reactions for peroxynitrite presently are the reactions with Zn-S and Fe-S centers with metalloproteins and carbon dioxide.12,15 Whereas the Zn-S and Fe-S centers will be oxidized, the last two reactivities will promote nitration of tyrosine residues on proteins. Protein nitrotyrosine has been detected in human diseases and experimental models of disease that do not involve an inflammatory process.7
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Figure 2. Targets of Reactive Species

Cellular Responses to Reactive Species: Time and Magnitude of Exposure

The flux and the time of exposure are critical factors in determining the outcome of oxidative stress. Aging can be considered the result of a continuous exposure to a low flux of reactive species over the life span. Although the antioxidant networks maintain the critical balance towards physiology, a few reactive species escape the surveillance of the antioxidant network and react with biological targets. Oxidation of biological targets will not necessarily translate to expression of a phenotype because repair processes may sustain normal physiologic function. However, as the frequency of oxidation of biological targets increases (and possibly as repair processes slow), detection of oxidized proteins, lipids and even DNA becomes apparent with aging and other reactive-species mediated pathologies.16-18

Severe oxidative stress results in necrotic cell death. Generation of reactive species during hyperoxia (breathing of >95% oxygen) or reperfusion of an ischemic tissue leads to tissue necrosis.19-20 A moderate exposure to reactive species can also result in cell death that usually occurs 20-24 hours after the initial insult. In most cases delayed cell death resembles apoptosis since DNA fragmentation and other features of apoptosis are evident. It is not clear how reactive species can induce delayed cell death or apoptosis. Potential pathways that once altered by reactive species will lead to delayed cell death include energy sources (mitochondria, activation of Poly- ADP ribosyl synthase), ionic homeostasis, signal transduction and membrane structural integrity.4,21,22

Overall, the inherent ability of cells to withstand oxidative stress is dependent upon several factors: their antioxidant capacity, the ability to sustain metabolic requirements by deriving energy from alternate pathways, efficiency to repair oxidatively modified biomolecules, and availability and utilization of trophic support.

Reactive Species and Signal Transduction

Recently, evidence has suggested that reactive species can be utilized in signal transduction events.23-27 Signal transduction for the most part is viewed either as a specific interaction between proteins or events mediated by second messenger molecules such as Ca2+ and cyclic nucleotides. Nitric oxide can be clearly considered a signal transducing molecule because it specifically activates guanylate cyclase. However, except for nitric oxide, specific targets that can be utilized in signal transduction are not known for other reactive species. Moreover, the steady state levels of reactive species such as superoxide and hydrogen peroxide are under the control of enzymatic pathways. For example, the steady state levels of superoxide in superoxide dismutase deficient E. coli is 5 x 10-7M (taking into account scavenging by glutathione) whereas in superoxide dismutase proficient E. coli the levels are 2 x 10-10 M.28 Therefore, the lack of specificity and the low intracellular levels creates difficulty in explaining how reactive species can be utilized in signal transduction.

The answer to this question in part can be found in the biochemical reactivities and the cellular targets for reactive species. Superoxide, nitric oxide and peroxynitrite react with Fe-S and Zn-S centers. Fe-S and Zn-S centers are not only found in enzymes regulating bioenergetics but also in transcription factors and in iron regulatory proteins. Peroxynitrite is also a nitrating agent that nitrates tyrosine residues in proteins. Nitration alters the pKa of tyrosine residues and interferes with the ability of tyrosine kinases to phosphorylate.29,30 The activity of different kinases, transcription factors and ion channels is redox sensitive and is dependent on a critical cysteine residue which can be modified by reactive species. Finally, reactive species can indirectly induce signal transduction events by inducing mitochondrial Ca2+ release and lipid peroxidation. These signaling pathways may be critical in mediating apoptosis or delayed cell death.

How to Detect and Quantify Reactive Species

The short half life of most reactive species in biological systems does not permit for their direct detection and quantification.3,5,6,23 Therefore, detection of reactive species relies on indirect measurement of modified targets. If you will, consider reactive species as sharks. Their presence in biological systems is therefore determined by the “bite marks” formed on critical cellular targets. In simple in vitro assays, the task of detection and quantification of reactive species is relatively well established. However, as one moves from the simple test tube assay, to cells in culture, to isolated organs, to whole animals or humans, the difficulty in detecting these “bite marks” increases exponentially. The ability to detect and quantify reactive species is a function of the amount of modified molecules present at a given time and the sensitivity of the assay. Biological targets that have been utilized for detection of oxidative modification include lipids, proteins, thiols and DNA. Reactive species react with more than one biological target and since the concentration of biological targets varies among cells, it is difficult to predict which target will be preferentially modified. Therefore, in more complex systems, it may be necessary to measure more than one end-point modification of biological targets. For example, measurement of the reduced to oxidized glutathione ratio will reflect a degree of oxidative stress but will not be useful in elucidating potential pathways responsible for the oxidation. In some models interference with the formation of the potential reactive species maybe useful in elucidating the reactive pathways.

Another method for detecting reactive species is the use of “reporter” compounds that are oxidized by reactive species to either chromogenic, fluorescent, luminescent or Electron Paramagnetic Resonance products. These probes have been utilized in cells, isolated organs and whole animal models and fall in two categories: cell permeable and non-permeable compounds. Intracellular detection requires a substrate that has a reasonably fast rate of reaction with reactive species and can be delivered at high enough concentrations to out-compete antioxidant and scavenging pathways. Extracellular detection represents the fraction of reactive species that are either generated very close to cell membrane or escape the antioxidant and scavenging networks and have not been reacted with cellular targets. This implies that the magnitude of the stress inside the cell could be significantly higher compared to what is measured extracellularly.