GSH and Horses

Understanding Nature's Strategy for Oxidative Balance in the Horse: The Glutathione Story

1. GSH and the Secret of Mother's Milk

 
Horse and baby horse

 

Why are nursing babies generally healthier and better-developed than those fed formula? Scientists working with an isolate of raw milk discovered an important answer to this question. Now available as GlutaSyn™, this unique milk serum isolate has been extensively studied for its positive impact on immune response, muscle cell metabolism, longevity, and antioxidant protection. Researchers believe that mother's milk delivers these benefits by improving the cells' levels of glutathione, or GSH, the body's "master antioxidant."

Intense Medical Interest in GSH Nearly a thousand medical research papers a year explore the antioxidant impact of GSH on health and disease. GSH is the most important factor protecting the interior of the cell from the constant damage of oxidative (free) radicals. A flexible antioxidant able to quench many types of radicals, GSH also keeps other antioxidants (like vitamins C and E) in their active states. GSH is a major detoxifier in the liver, and helps conserve protein substrate in the liver for energy and growth. GSH is found in foods, but dietary GSH does not improve GSH activity in most cells. GlutaSyn™ is the first veterinary supplement to provide reliable, patented nutritional support for cellular GSH.

What is GlutaSyn? GlutaSyn™ is a unique milk serum isolate that was developed with over fifteen years of rigorous, peer-reviewed research. Made under stringent quality controls with advanced low-temperature ultrafiltration technology, it's an all natural product that concentrates the essential benefits of fresh, raw mother's milk. This milk serum isolate has been tested many times in successful studies exploring its impact on immune system activity. These benefits are due to an even more important property of raw milk serum; its special ability to support the synthesis of cellular glutathione (GSH), the body's master antioxidant and detoxifier. This compound has been tested in Phase I, II, and III clinical trials, and has been awarded three U.S. Patents and one Canadian Patent. This breakthrough finally became a human supplement (Immunocal™) in 1997.

 

2. What does GlutaSyn® do?

The next few pages explore what science has learned about the special benefits of GlutaSyn during two decades of peer-reviewed research.

Here's what people who feed GlutaSyn to their horses have to say about it:

"I can honestly say that this year, having placed all my horses on your product, from aged race horses to yearlings, I have not lost a day of training due to sickness! It is really incredible. No sniffles, no coughing, no nothing! Now with the stakes season upon us I see and hear about many horses unable to race because of sickness..but none of mine." — John Stark, Jr. Gansevoort, NY

"I've tried dozens of supplements on my 17-year old barrel racing Quarter horse, but nothing compares with GlutaSyn. His health, movement and flexibility are all phenomenal. For the first time, I've received numerous compliments from complete strangers that want to know what I am doing to make him look so good and move so well. Thank you, GlutaSyn!" —Michelle Thompson, Sharon, Ontario

"The effects of GlutaSyn were very apparent in my 15-year old barrel racing mare: increased energy, stamina, flexibility and length of stride. Now, after 8 weeks on the product, I can see improvement in muscle tone, less irritability while riding, requires less feed to meet her needs, and has recovered full use of her shoulder (deltoid) muscles which were found to be torn 4 years ago. I have to learn how to ride her again: the years have been wiped away." — Jan Middleditch, Erin, Ontario

 

 

3. About Dr. Gustavo

 

Dr Gustavo 

Gustavo Bounous, MD 

Photo courtesy of Immunotec Research. Ltd.


Dr. Gustavo Bounous, who discovered the remarkable qualities of the milk serum isolate in GlutaSyn, is a respected authority on the problems of nutrient absorption in disease and trauma. Dr. Bounous emigrated from Italy to the U.S. in the 1950's to do research at Indiana University Medical Center, where he soon made important contributions in the emerging science of hemodynamics.

Continuing his research at McGill University in Montreal, Dr. Bounous made a breakthrough in the treatment of hemorrhagic shock.1 This work earned him the 1965 Medal of the Royal College of Physicians and Surgeons of Canada. It also led him to develop the science of enteral nutrition and the invention of the "elemental diet", now used in hospitals throughout the world.

In 1978, Dr. Bounous began his first experiments feeding a special whey protein concentrate to mice. Whey is dried milk serum, the protein-rich liquid that is left over from the manufacture of cheese. Dr. Bounous was surprised to find that when this concentrate was added to the diet, the animals showed a much stronger immune response when challenged with an injection of foreign blood cells.2 This led him to conduct a number of trials comparing his milk serum isolate to other proteins across the spectrum of plant and animal sources. None of the other proteins, including a variety of whey concentrates, had a positive effect on immunity. Only the serum isolate demonstrated significantly improved immune response.3,4,5

Dr. Bounous and his colleagues pressed on. In one study, mice that were fed the isolate and then challenged with sheep red blood cells produced almost 5 times more splenic Plaque Forming Cells than controls.6 It was found that when the B-cells of milk serum-fed animals responded to antigens with typical bursts of oxidative cloning, they had much better levels of the antioxidant GSH (glutathione), and many more viable cells were produced.7 There was also better non-specific response of splenic cells to mitogens,8 and dramatically improved resistance to strains of Salmonella,9Streptococcus pneumoniae, and E. coli bacteria.10 This led to several studies of the isolate’s anti-tumor benefits in animals and humans.111213141516

Scientists often noticed that laboratory animals fed the milk isolate seemed to survive longer than their counterparts fed other whey proteins or standard diets. Trials showed that feeding milk serum isolate, from weaning, produced lifespans up to 50% longer than lifespans of animals fed standard diets.1718

Further research shed light on how the milk serum isolate works. The common thread linking the variety of benefits turned out to be one rather small molecule: glutathione, or GSH. The cells of animals fed the isolate were making more GSH than those of animals fed other diets.19 The milk serum contains a rare GSH precursor (gamma-glutamyl-cysteine (GGC)) and high levels of GSH amino acids (cysteine and glutamic acid). GSH is now believed to be the most important antioxidant in the body, as well as a vital detoxifier in many branches of cellular and hepatic (liver) metabolism. Science continues to explore the role of GSH in basic body processes.20


U.S. and International Patents Awarded
to GlutaSyn®:


U.S. Patent 5,230,902:

Undenatured Whey Protein Concentrate to Improve Active Systemic Humoral Immune Response.

U.S. Patent 5,290,571:

Biologically Active Whey Protein Concentrate.

U.S. Patent 5,451,412:

Biologically Active Undenatured Whey Protein Concentrate as Food Supplement.

Canadian Patent 1,338,682:

Biologically Active Undenatured Whey Protein Concentrate as a Food Supplement.


4. Taking it to the People - Production and Clinical Trials

From the laboratory to the marketplace As Dr. Bounous became involved in human clinical studies, he realized that to make his isolate available to others, he had to move it from the laboratory to sustained, commercial production. But normal pasteurization temperatures completely destroy the active proteins in milk serum, and unpasteurized serum spoils quickly due to its high bacteria count. How could enough be made available for all the people and animals that might benefit?

Unique low-impact process developed Dr. Bounous and a team of researchers, engineers, and investors formed Immunotec Research, Ltd. The company's mission was to perfect the milk serum isolate for large-scale production and bring the product to the world market. Using advanced low-impact pasteurization techniques, prolonged gentle rinsing, and microfilters and ultrafilters, Immunotec's research team eventually created a patented process to produce an extremely clean and pure milk serum isolate that delivers the key milk proteins in their natural, undenatured state. The product is sold for human nutrition and health as Immunocal™, and for animals as GlutaSyn™.

Human research discoveries Dr. Bounous' discovery has been the subject of a variety of successful human clinical trials, with additional studies in progress as of this writing. In just one example, Dr. Larry Lands, a cystic fibrosis researcher at Montreal Children's Hospital, explored the isolate's effects on muscle performance. Dr. Lands' team recently studied twenty healthy young adults performing cycle exercise tests. They found that isolate-fed subjects showed a 35.5% increase in the GSH levels of circulating lymphocytes, accompanied by improvements of 13% in both greater peak power and sprint work capacity.

One of a kind Dr. Bounous' discovery of milk serum proteins is the only method, known to date, to naturally and directly boost cellular glutathione levels. For his work, Dr. Bounous has been awarded five U.S. Patents, a Canadian Patent, and two Patents in Australia. Fellow scientists around the world have justly acclaimed his discovery. Dr. Luc Montagnier, discoverer of the role of HIV in AIDS, advised fellow AIDS researchers that Dr. Bounous' work is "an important line of research which should be expanded."

One man's dedication to the truth Dr. Bounous has dedicated his life to the painstaking pursuit of rigorous scientific research. Because Dr. Bounous took the time to scientifically validate and develop the use of his milk serum isolate step by step, we have today a product made with advanced methods that maximize yield and biological activity, backed by eight patents and two decades of clinical research and development. Now in his seventies, and retired from McGill, Dr. Bounous continues his studies at the laboratories of Immunotec Research, Ltd. in Quebec, Canada.

Exercise Research:

A research team from Montreal Children's Hospital recently reported that healthy young adults fed the isolate showed a 35.5% increase in GSH levels of circulating lymphocytes, with 13% greater peak power and sprint work capacity in cycle exercise tests.

New Research:
(click to view abstracts)

Treatment of Obstructive Airway Disease With a Cysteine Donor Protein Supplement

Lymphocyte Glutathione Levels in Children With Cystic Fibrosis

 

5. Oxygen Never Sleeps

You are probably familiar with the saying, "Rust never sleeps." Rust is the oxidation of iron through free radical exchanges with oxygen. The biologist would describe it more accurately: "Oxygen never sleeps." Oxygen always invites interaction with electrons of other molecules, either taking an electron (becoming a "superoxide" [O2O-] radical) or donating one of its loosely held electrons to another molecule (becoming singlet [O2O+] oxygen). We depend on this "reactive" quality of oxygen to produce the biological energy of life itself. But the energy we gain from oxygen comes with a price.

Oxygen's tendency to stimulate electron transfer creates a lot of compounds that are either missing an electron (giving them positive charge) or that have an extra electron (negative charge). The most energized of these are the free radicals (or oxyradicals, or oxidants). Each radical can generate a chain reaction of electron transfers that can alter hundreds of normal body molecules per second.

The external and internal membranes of the cells are especially vulnerable to radicals. Unchecked, radicals can rapidly alter the structures of membrane lipids and proteins, ruining membrane integrity as well as vital transport operations. These reactions can in turn stimulate chain reactions that spread damage deep within the cell, leading to cell mutation or death.23

Other molecules (such as hydrogen peroxide and singlet oxygen) encourage radical-forming reactions. They are called proradicals because they spontaneously create superoxide, the radical that initiates oxidative chain reactions. Oxygen itself, which makes up 1/5 of the air your horse breathes and lives in, is by far the most abundant of the proradicals.

Oxygen is the "fire" that burns most of our energy, the universal activator required for thousands of essential biomolecular reactions. Oxygen has tremendous chemical power–to feed life, but also to damage and destroy it, when the "fire" rages out of control, and oxidizing radical exchanges overwhelm the body's antioxidant supplies.

 

Superoxidative Radical

 

Acquired Electron     Missing Election


Superoxidative Radical

Superoxidative Radical

Missing Electron     Missing Election

 

Hydroxyl Radical

Missing Electron     Missing Election

Hydroxyl Radical 

                                Missing Electron



6. Radicals in Action

s everyone knows, genetics research grabs most of the headlines in medical science today. Yet even the most astounding advances in genetics will require a thorough understanding of biochemistry in order to be used effectively. A "quieter" revolution is occurring in biochemistry, where scientists are finding that most abnormalities in the body result in increased free radical activity, and most of the symptoms, metabolic disturbances and damage caused by disease, are created by oxidative stress. Here are some "little picture" examples of this new understanding:

• If a respiratory enzyme that is crucial for energy release is missing, whether due to poor nutrition or a genetic fault, the electrochemical potential of the process will be diverted to free radical exchanges. This chaotic oxidation will further block metabolism by destroying other enzymes.24

• If levels of the electrolyte potassium are too low or too high, large numbers of free radicals will emerge and cause most of the symptoms of these potentially fatal imbalances.25

• If an infection occurs, the virus or bacteria will cause free radical damage in its attempt to feed off the body. The immune system dramatically increases radical activity to fight back. This oxidative stress stimulates the production of the prostaglandins, leukotrienes, and interleukins. They make the body feel "sick," and generate more radicals to add to the symptoms and damage of illness.26

• During exercise, oxidative stress increases sharply. As the cells begin to lose their power to generate energy, they also lose their ability to make and recycle antioxidants to combat oxidative stress. This, in turn, accelerates the breakdown of energy metabolism. Radicals can damage metabolism in both direct and indirect ways. For instance, radicals inside the cells disable the sulfur-bearing proteins that transport calcium ions within the cell.27,28 The failure of calcium ion transfer is a cardinal sign of muscle exhaustion and may be directly involved in the process of cramping and tying up.29

Unchecked, radicals can rapidly alter the structures of membrane lipids and proteins, ruining membrane integrity as well as vital transport operations.

Other molecules (such as hydrogen peroxide and singlet oxygen) encourage radical-forming reactions. They are called proradicals because they spontaneously create superoxide, the radical that initiates oxidative chain reactions. Oxygen itself, which makes up 1/5 of the air your horse breathes and lives in, is by far the most abundant of the proradicals.

Oxygen is the "fire" that burns most of our energy, the universal activator required for thousands of essential biomolecular reactions. Oxygen has tremendous chemical power–to feed life, but also to damage and destroy it, when the "fire" rages out of control, and oxidizing radical exchanges overwhelm the body's antioxidant supplies.

 

 

7. Antioxidants: Department of Defense

What are the antioxidants in the horse's body that can control this continual onslaught of oxidation? Some of the horse's antioxidant molecules are made within the body, while others are supplied by the diet. Both types are needed to manage free radical interactions and keep them from inhibiting metabolism or causing lasting damage. The antioxidants include vitamin C, vitamin E, coenzyme Q10, lipoic acid, carotenoids, enzymes derived from B complex vitamins, superoxide dismutase (SOD), catalase, glutathione (GSH), and glutathione peroxidase (GPX), to name a few. Some other molecules play transient roles as antioxidants in specific situations.

Early antioxidant research focused mainly on the dietary antioxidants. The effects of molecules like vitamin C, vitamin E, and beta carotene were measured in the nutrition of humans and animals. Antioxidants that could not be provided in the diet did not gain as much attention as these vitamin antioxidants.

Nowadays, researchers explore the inner workings of the cells in far greater detail, and monitor the level and nature of antioxidant protection at each step. Today's scientists are finding that the most important single factor for radical control inside the cells is GSH.30 

Preventing Chain Reactions

GSH, as glutathione peroxidase (GPX), quenches peroxide radicals and proradicals, including hydrogen peroxide and free fatty acid and phospholipid hydroperoxides:

Antooxidants

 

 

8. GSH: Barometer of Oxidative Stress

In oxidative protection, it seems that all roads lead to GSH, the fundamental "reducing" agent in the body. Many scientists now use GSH levels as a general measure of oxidative stress. For instance, when GSH quenches a radical, it typically oxidizes to GSSG (glutathione disulfide). The measurement of the oxidative balance of a cell or a cellular process is usually based directly on the ratio of GSH to GSSG.31

GSH is found in every cell in the body.

  • GSH can be incorporated into antioxidant enzymes, such as glutathione peroxidase (GPX).
  • GSH is the prime protector of all sulfur-bearing proteins and enzymes, thanks to its sulfhydryl unit (-SH).
  • GSH is present in essentially all (mammalian) cells.
  • GSH can support SOD activity by filling in for SOD's usual partner, catalase.32
  • GSH recycles vitamins C and E and other antioxidants to their active forms.33
  • GSH is the "antioxidant-of-choice" for immune cells and other highly oxidative tissues.34
  • GSH is a detoxifier within many cells.
  • GSH plays a major role in the detoxifying activity of the liver.35
  • GSH is the "partner" of vitamin E in protecting membranes from radical damage.
  •  

     

    9. GSH Nutrition

     

    What does this mean for horses? How do horses get their GSH, and how do they cope with oxidative stress?

    At the beginning of life, nature provides mammals and birds with a special GSH precursor called gamma-glutamylcysteine (GGC). With GGC, the cells can produce GSH simply by adding one more amino acid. This allows them to bypass a major "rate-imiting" step in GSH production. Infant mammals get the GGC dipeptide from their mothers' milk. A much lower level of GGC is present in the raw white of birds' eggs. Since these are the only two food sources of GGC, after weaning or hatching animals must produce GSH internally from other nutrients in a regulated, self-limiting process.37 Like all animals, weaned horses produce their GSH from amino acids they get from protein in their food.

    GSH is made of three amino acids, so it is called a tripeptide. The aminos are glutamic acid, cysteine, and glycine. Glutamic acid, or glutamate, occurs in many feeds and is heavily used by the horse’s body to make other amino acids for protein building (transamination). Glycine is the simplest of the amino acids and, like glutamate, is usually available for GSH synthesis.

    Cysteine, on the other hand, is more likely to be in short supply, especially in the required GGC form (gamma-glutamyl-cysteine). It is this rate-limited step in GSH synthesis that most researchers have tried to overcome in their attempts to increase GSH production.38

    Cysteine by itself is very unstable. It rapidly auto-oxidizes into cystine, a dipeptide that combines two molecules of cysteine in an unusually fragile disulfide bond. Because cystine and cysteine are unstable, bioactive food forms of these amino acids are rare. Most of the cysteine in the body is produced from a much more common amino acid, methionine. Unfortunately, increasing the dietary level of methionine does not substantially increase GSH production in the cells.39

    Glutathione's unique antioxidant and conjugating power comes from cysteine and its sulfhydryl unit (-SH), which enables GSH to protect a broad variety of tissues from free radical damage. The three-dimensional (tertiary) structure of GSH is also important to its flexibility and power as an antioxidant and detoxicant.40

    Synthesis of Glutathione:

    Step 1:

    L-Glutamic acid

    +
    L-Cysteine

    (via GGC Synthetase,
    a rate-limited step)

    GGC
    (gamma-Glutamylcysteine)

    Step 2:

    GGC
    +
    L-Glycine

    (via GSH Synthetase,
    a non-limited step)


    GSH
    (glutathione)



    10. GSH Facts and Fallacies

    There is substantial confusion in the commercial literature about the absorption of GSH from the diet, and about the way the body makes and distributes GSH. A broad review of the science on the subject can give us a more complete picture.

    • Many foods contain some GSH, especially fruits, vegetables, and meat.

    • GSH is readily absorbed from food or supplements. The absorption is essentially passive. Cells lining the intestines absorb dietary GSH temporarily, then pass it on into circulation.41

    • 80% of GSH in circulation is broken down by the kidneys into separate amino acids.42

    • Most cells cannot efficiently absorb GSH from circulation. The few that can use circulating GSH include the cells lining the digestive, respiratory, and urinary tracts (epithelial tissues), and some blood cells. These are the only tissues likely to get pre-formed GSH from the diet.43

    • Plasma (blood fluid) levels of GSH are normally quite low. In trauma or acute infection, plasma levels increase as the liver releases more GSH into circulation, but the kidney continues to rapidly metabolize serum GSH.44

    • Highly oxidative tissues such as the liver, kidney, and heart contain the highest concentrations of GSH.45Heavy demand for GSH is also found in the cells of the muscles, nerves, lungs, and immune system.

    Although GSH enters circulation from the diet, only epithelial and blood cells can absorb it directly. They must grab it quickly; the kidneys rapidly scavenge circulating GSH and break it down for the amino acid pool.

    Nutritional support of GSH production inside the cells seems to offer the best way to enhance GSH activity throughout the body.

    • For the vast majority of tissues, GSH is not supplied by the diet, made in the liver, or delivered by the blood; it is made inside the cells. In mammals, after weaning, the cells produce GSH through two main pathways:

    1. The enzymes GGC (gamma-glutamylcysteine) synthetase and GSH synthetase assemble GSH from amino acids.
    2. Glutathione reductase reduces oxidized GSH (called GSSG, or glutathione disulfide) to recreate active GSH.46

    • A few so-called "prodrugs" can temporarily increase GSH levels in the cells. For instance, NAC (N-acetylycysteine) is used in emergency medicine to treat acetaminophen poisoning. It works by raising liver GSH, which substantially increases liver detoxification capacity. All of these drugs cause moderate to severe side-effects due to their tendency to increase oxidation even while they raise GSH levels.47

    • Supplements of l-cysteine have little effect on GSH levels. Mild to moderate side-effects at physiologically active dosages are generated by the spontaneous auto-oxidation of this unstable free amino acid.48

    • The production of GSH in the cells, from amino acids, is rate-limited by "negative inhibition feedback." As cellular GSH levels increase, the production of the enzyme GGC synthetase slows down, which reduces creation of GGC. Without GGC, GSH cannot be produced. This typically places an upper limit on cellular GSH values.

    • Providing pre-formed GGC, as found in mother's milk, offers the cells a way to produce GSH without the rate-limitng step. This gives the cells the potential to achieve higher cellular GSH levels than would otherwise be possible.49

    • A variety of whey protein products are available for human and animal nutrition. They include whey protein concentrates, whey protein isolates, and ion-exchange whey proteins. Though such products may contain some undenatured whey proteins, they are made with pasteurization and handling procedures that destroy the natural forms of the more fragile milk proteins and dipeptides, including the vital GGC. Extensive testing of a broad range of whey products has shown that none of them provide the unique benefits of patented milk serum isolate.50,51,52,53,54



    11. Oxidative Stress in Horses

     

    Like all multicelled animals, horses face the constant challenge of controlling oxidation to maintain proper metabolism and cellular defenses. A number of factors can impact the horse's capacity to defend against free radicals and excessive oxidation. Understanding these conditions can help the horseman to design horse care programs that enhance antioxidant support and avoid unnecessary oxidative stress.

    Infections 
    The progress of the typical viral or bacterial infection can be seen as one inter-related free radical cascade, one that ends (we hope) in a return to oxidative balance, or health. Horses like people, are more vulnerable to infection when oxidative stress is high. Travel, heavy exercise, poor nutrition, toxicity and many other factors can contribute to a horse's oxidative stress. The relationship between antioxidant activity and immune function is close, and highly interdependent.

    Trailering
    Veterinary researchers are discovering more about the stresses of trailering, but we'll probably never understand what the horse experiences it until we stand, upright and unassisted, in the bed of a moving truck for several hours. The oxidative threat for horses is generated by a combination of psychological stress and prolonged aerobic output in restricted conditions.

    Inflammation 
    Inflammation is a natural process that creates high levels of free radical activity through many different pathways. Successful resolution of inflammation depends on the body's antioxidant defenses to restore balance and avoid degenerative changes.

    Rapid Growth 
    Growth is an energy-intensive process. During rapid development, oxidative stresses can contribute to the disruption of enzyme-dependent growth processes. This can result in inflammation and/or tissue deformation, creating new, potentially permanent sources of oxidative stress.

    Environmental Toxins 
    Toxicologists are familiar with the central role free radicals in the toxicity of hazardous chemicals, drugs, and pollution. Exposure to insecticides, organic solvents, ozone, smoke, and other toxins in the air, feed and water puts horses at risk for damaging levels of oxidative stress.

    Psychological Stress 
    Biologists have long recognized the importance of radicals in generating the physical effects of emotional stress. The demand for antioxidant support is likely to be higher during periods of psychological stress.

    Protozoa and Mycotoxins 
    Microscopic protozoa generate damaging levels of free radicals. In horses afflicted with EPM, the protozoal radicals "eat" through the conductive myelin sheath of the nerves. The fungal mycotoxins and aflatoxins found in contaminated pastures, hay, and grains also due much of their damage through free radical attack.

    Exercise 
    Exercise requires high levels of oxidation as fuel is consumed to meet the demand for work. Both aerobic and anaerobic exercise significantly increase oxidative stress, through somewhat different pathways. Exercise-induced oxidative stress is especially high inside the muscle cells, the heart, blood, and the linings of the lungs.

    Trauma and Burns 
    When horses suffer cuts, injuries, or burns, free radicals are extremely active at the damage site. Antioxidants help by countering the radicals and restoring balance so healing can proceed.

    About Selenium and GSH 
    Horses need selenium to make GPX (GSH peroxidase), a vital antioxidant enzyme. Selenium and GSH have a mutual affinity, and can combine spontaneously to form GPX. Selenium should never be over-fed.

     

    Oxidative Stress in Horses

     

     

     

    12. References

     
    1. Bounous G., Hampson L.G., Gurd, F.N. Cellular nucleotides in hemorrhagic shock. Relationship of intestinal metabolic changes to hemorrhagic enteritis and the barrier function of intestinal mucosa. Ann. Surg. 160:650; 1964. 
    2. Bounous G., Kongshavn P.A., The effect of dietary amino acids on immune reactivity. Immunology 35(2):257-66; 1978.
    3. Bounous G., Letourneau L., Kongshavn P.A.L. Influence of dietary protein type on the immune system of mice. J. Nutr. 113(7):1415-21; 1983. 
    4. ibid 
    5. ibid 
    6. ibid 
    7. Baruchel S., Bounous G., Gold P. Place for an antioxidant therapy in human immunodeficiency virus (HIV) infection. Oxidative Stress, Cell Activation and Viral Infection. Pasquier C, et al. (eds). Basel: Birkhauser Verlag. 312-313; 1994. 
    8. Bounous, G., Kongshavn, P.A.L., Gold, P. The immunoenhancing property of dietary whey protein concentrate. Clinical and Investigative Medicine 11(4): 271-278; 1988. 
    9. Bounous G., Stevenson M.M., Kongshavn P.A.L. Influence of dietary lactalbumin hydrolysate on the immune system of mice and resistance to Salmonellosis. Journal of Infectious Diseases 144:281; 1981. 
    10. Bounous G., Kongshavn P.A.L. The influence of protein type in nutritionally adequate diets on the development of immunity. In Absorption of Amino Acids, Friedman, M. (ed.) C.R.C. Press, Boca Raton, Florida, USA, Vol. 2, pp. 219-233; 1989. 
    11. Bounous G., Kongshavn P.A.L. The effect of dietary amino acid on the growth of tumors. Experientia 37:271-272; 1981. 
    12. Bounous G., Sadarangani C., Pang K.C., Kongshavn P.A.L. Effect of dietary amino acid on tumor growth and cell mediated immunity. Clinical and Investigative Medicine 4:109-115; 1981. 
    13. Visek W.J. Dietary Protein and experimental carcinogenesis. Adv. Exp. Biol. 206:163-186; 1986. 
    14. Bounous G., Papenburg R., Kongshavn P.A.L., Gold P., Fleiszer D. Dietary whey protein inhibits the development of dimethylhydrazine induced malignancy. Clinical and Investigative Medicine 11:213-217; 1988. 
    15. McIntosh G.H., Regester G.Q., Le Leu R.K., Royle P.J. Dairy proteins protect against dimethylhydrazine-induced intestinal cancer in rats. J. Nutr. 125:809-816; 1995. 
    16. Bounous G., Batist G., Gold P. Whey proteins in cancer prevention. Cancer Letters 57:91-94; 1991. 
    17. Birt D.F., Baker P.Y., Hruza D.S. Nutritional evaluations of three dietary levels of lactalbumin throughout the lifespan of two generations of Syrian hamsters. J. Nutr. 112:2151-60; 1982. 
    18. Birt D.F., Schuldt G.H., Salmasi S. Survival of hamsters fed graded levels of two protein sources. Lab. Anim. Sci. 32:363-6; 1982. 
    19. Bounous G., Gervais F., Amer V., Batist G., Gold P. The influence of dietary whey protein on tissue glutathione and the diseases of aging. Clinical and Investigative Medicine 12:343-349; 1989. 
    20. Lomaestro B.M., Malone M., Glutathione in Health and Disease: Pharmacotherapeutic Issues. The Annals of Pharmacotherapy 29: 1263-1273; 1995. 
    21. Lands L.C., Gray V.L., Smountas A.A., The Effect of Supplementation With a Cysteine Donor on Muscular Performance. Presented at the Association Palmonaire due Quebec, Quebec City, October 1998. In revision, J. Appl. Physiol. 1999. 
    22. Levine S.A., Kidd P.M., Antioxidant Adaptation - Its Role in Free Radical Pathology. San Leandro, CA. Biocurrents Division, Allergy Research Group. 1985. pp. 13-20. 
    23. ibid pp. 38-41. 
    24. ibid pp. 121-123. 
    25. ibid pp. 136-140. 
    26. ibid pp. 160-168. 
    27. ibid pp. 140-141. 
    28. Bellomo G., Orrenius S. Altered Thiol and Calcium Homeostasis in Oxidative Hepatocellular Injury. Hepatology 5(5): 876-882; 1985. 
    29. Valberg S.J. Muscular Causes of Exercise Intolerance in Horses. Vet. Clin. North Am. Equine Pract. 12(3):495-515; 1996. 
    30. Lomaestro, B.M., Malone, M. Glutathione in Health and Disease: Pharmacotherapeutic Issues. Ann. Pharma. 29:1263-73; 1995. 
    31. ibid 
    32. Levine, S.A, Kidd P.M., pp. 48-51. 1985. 
    33. Meister, A. The Antioxidant Effects of Glutathione and Ascorbic Acid. In Pasquier et al., (eds.) Oxidative Stress, Cell Activation and Viral Infection. Basel: Birkauser Verlag, 1994. 101-110. 
    34. Baruchel S, Bounous G, Gold P. Place for an antioxidant therapy in human immunodeficiency virus (HIV) infection. Oxidative Stress, Cell Activation and Viral Infection. Pasquier C, et al. (eds). Basel: Birkhauser Verlag, 1994. 312-313. 
    35. Levine and Kidd, pp. 114-115. 1985 
    36. Dr. John T. Pinto, in: Gutman J., Scehettini S., The Ultimate GSH Handbook. Montreal, Canada. Gutman & Scehettini, Enr.,1998. p. 11. 
    37. Bounous G., Gold P. The biological activity of undenatured whey protein: Role of Glutathione.. Clin. Inves. Med. 14:296-309; 1991. 
    38. Anderson M.E., Meister A. Transport and direct utilization of gamma-glutamylcyst(e)ine for glutathione synthesis. Proc. Natl. Acad. Sci. USA,. 80:707-711; 1983. 
    39. Dringen R, Hamprecht B. N-acetylcysteine, but not methionine or 2-oxothiazolidine-4-carboxylate, serves as cysteine donor for the synthesis of glutathione in cultured neurons derived from embryonal rat brain. Neurosci. Lett. 259(2):79-82; 1999. (abstract) 
    40. Levine and Kidd, p. 227. 1985 
    41. Lomaestro B.M., Malone M. Glutathione in Health and Disease: Pharmacotherapeutic Issues. Ann. Pharma. 29:1263-73; 1995. 
    42. ibid 
    43. ibid 
    44. Bray TM, Taylor CG. Enhancement of tissue glutathione for antioxidant and immune functions in malnutrition. Biochem Pharm 47(12):2115-2117; 1994. 
    45. Hazelton, G.A., Lang, C.A. Glutathione Content of Tissues in the Aging Mouse. Biochem. J. 188:25-31; 1980. 
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