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.

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.

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:

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.

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

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.

• 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}

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.

12. References