Changing the Way We Age MEDIA RELEASE _______________________________________________________________________ For Immediate Release 25 September 2001 FITNESS VISIONARY SETS

OUT TO CHANGE THE WAY WE AGE VANCOUVER ; On 30 September 2001, Colin Milner launches the International Council on Active Aging (ICAA), an organization that seeks to change the way we age by uniting professionals in the retirement, assisted living, fitness, rehabilitation and wellness fields. The ICAA will offer a community of like-minded individuals the information, education, and tools needed for success with the aging baby boomer market and beyond. What the ICAA will do, says Milner, is give people the knowledge that will raise the industry standards and level of service when it comes to aging members and residents.What we really offer, he continues, are the tools for success. For instance, the organization is developing an ongoing wellness support package for its members to deliver to their clientele. Serving this market better will lead to more people making healthier choices, says Milner, and that will change the way we age. Research has shown that, other than diet modification, exercise holds the greatest promise for reducing the risk of chronic disease. The Alliance for Aging Research calls chronic disease a threat to the nation's health and economic well-being. With projections indicating that 160 million Americans will have chronic conditions by 2040, the ICAA and its members could make a significant difference to the country's health and bottom line. Our aging population also represents a largely untapped and rapidly growing business opportunity. Between 1987 and 1999 club memberships in the under 55 crowd grew 76%, while memberships in the over 55 crowd grew 245%. This segment already exercises at an average of more than four percent above the general population and, according to the International Health and Racquet Sports Association, represents the centerpiece of the fitness industry?s growth between now and 2010. Professionals who understand and serve the aging adult well have a significant advantage over their competitors. The ICAA aims to prepare and support its members so they can achieve optimal success. Continued../2 International Council on Active Aging Media release page 2 27 August 2001

One of the ICAA's goals is to spread change through society by sharing world-class information on health and wellness. For months, Milner has worked at assembling an advisory board that shares his vision. He has 20 of the foremost authorities in aging research, fitness, gerontology, and senior housing serving on the ICAA's advisory board. Each adviser will be instrumental in helping the organization develop programs, training, information, and tools to share with its members. Milner's long-term goal is to find a partner that will help fund ongoing research into health management and disease prevention. After 30 September, more information about the ICAA will be posted on the organization?s website at www.icaa.cc - 30 For more information, about the ICAA or other aging related issues, contact Colin Milner, CEO, International Council on Active Aging Toll Free: 866-335-9777(in North America), Tel: 604/734-4466, E-mail: cmilner@attglobal.net <mailto:cmilner@attglobal.net>

BACKGROUNDER ______________________________________________________________________ 25 September 2001 VISION The International Council on Active Aging (ICAA) is dedicated to changing the way we age by uniting professionals in the retirement, assisted living, fitness, rehabilitation, and wellness fields to help dispel society's myths about aging. We will also help these professionals to empower aging baby boomers and older adults to improve their quality of life and maintain their dignity. MISSION The ICAA connects a community of like-minded professionals who share the goals of changing society's perceptions of aging and improving the quality of life for aging baby boomers and older adults within the six dimensions of wellness. The council supports these professionals with education, information, resources, and tools, so they can achieve optimal success. *** APPROACH The ICAA will integrate the following elements into a cutting edge approach for professionals working with aging baby boomers and older adults:

1. Wellness programming The ICAA is working on delivering programs for aging baby boomers and older adults in all six dimensions of wellness: physical, emotional, spiritual, vocational, intellectual, and social. For example, one of three people over the age of 65 fall each year. The ICAA is developing with its advisory board a complete fall prevention program that could help minimize this risk. 2. Health behavior changes The ICAA will provide professionals with education and information to help them support mature adults in making healthy living changes. 3. Marketing support The ICAA will provide professionals with specially designed and branded programs plus marketing tools that will make it easier for them to attract an aging clientele. *** FOUNDER'S BIOGRAPHY Industry leader Colin Milner has worked in the fitness business for 19 years. He managed fitness clubs, consulted, and was president/publisher of a trade magazine and directory, before joining Keiser Corporation in 1992. Milner was vice president of sales and marketing at Keiser, as well as chief operating officer of the Keiser Institute on Aging. Prior to establishing the ICAA, Milner was president of IDEA Health and Fitness Association, the world?s largest association of fitness professionals. During his time at Keiser, the Institute on Aging, was awarded the prestigious LIW Award in 1999 for the most innovative product in the industry. The Institute also won three other awards including the Associations Advance America Award of Excellence in 2000 for its international Fall Prevention Campaign. Milner currently sits on the advisory boards of American Senior Fitness Association, Canadian Association of Fitness Professionals, and Club Success Magazine. Canadian Fitness Business magazine has compared Milner to David Foot, bestselling author of Boom, Bust and Echo, for his vision of the impact our aging population will have on society. The author of more than 80 industry articles, Milner has been interviewed extensively for leading publications, such as the New York Times, the Los Angeles Times, the Wall Street Journal, and Business Week. He has also presented for numerous organizations throughout North America on the topic of attracting and serving the older adult market. *** ADVISORY BOARD The ICAA advisory board is made up of the following experts in medicine, gerontology, senior housing, research, and fitness: Bonni L. Kaplan, B.S.

Director of Marketing and Communications Senior Lifestyle Corporation Ben Hurley, Ph.D. Dept of Kinesiology College of Health and Human Performance University of Maryland Beth Zbieg, MA/CCC-SLP National Director of Rehabilitation Services Marriott Senior Living Services Debra Rose, Ph.D. Co-Director, Center for Successful Aging California State University Fullerton Dennis Keiser B.S President/CEO Keiser Corporation Glen Colarossi, M.A. President AgeFit Gloria M. Gutman, Ph.D. President, International Association of Gerontology and Professor and Director, Gerontology Research Centre & Programs, Simon Fraser University, Gwen Hyatt, M.S President Desert Southwest Fitness Center for Continuing Education Jan Montague, M.G.S. President Montague Eippert and Associates Jan Seaman, P.E.D. Executive Director American Association for Active Lifestyles and Fitness Jessie Jones, Ph.D. Professor Kinesiology and Co-director, Center for Successful Aging, Cal State Fullerton John Rude, M.S. President John Rude and Associates Katherine Meacham Hamlin, MBA, B.S. Director, Health & Fitness Services Johnson & Johnson Health Care Systems Inc Kay Van Norman, M.S. President, Seniors Unlimited Kevin Steele, Ph.D. Vice President Health Services/Corporate Accounts 24 Hour Fitness Clubs Sandy Coffman, President Programming for Profit Terry Fay, B.A. Director of Resident Programs Senior Lifestyle Corporation

Terry Ferebee Eckmann, M.S. Faculty Minot State University Human Performance Department Co-Owner, Fitness First. William J. Evans, Ph.D. Director Professor of Geriatrics, Physiology, and Nutrition Nutrition, Metabolism, and Exercise Laboratory Donald W. Reynolds Center on Aging, University of Arkansas for Medical Sciences FACTS ABOUT AN AGING POPULATION ________________________________________________________________________ 50 Plus Market Size Numbers 100 million North Americans Since January 1, 1996, one person turns 50 every eight seconds--this rate will continue for the next 20 years Holds 80% of the nation's wealth Has 55% of the discretionary income Provides 50% of total consumer demand Participation 30% of the 50-plus crowd exercises on a regular basis, as opposed to 24.5% of the general population. The 50-plus group represents 13.9% of the people in health clubs and 43% of those in a hospital wellness setting. Once they join a club, the 50-plus crowd remains a member 4.7 years longer than the average member. 64% of the 50-plus demographic say they want to participate in fitness more. Cost to Society A study by the Alliance for Aging Research found that older Americans who lose their independence cost $26 billion per year more in medical and long-term care than if they could live on their own. The Center for Disease Control projects that 59.4 million people will suffer from arthritis over the next 20 years. This figure represents almost 20% of the population. The United States could save $10 billion in health costs, most stemming from loss of independence, if osteoarthritis could be delayed by five years. One in two women and one in eight men over 50 will have an osteoporosis-related fracture in their lifetimes. One in three people over 65 fall every year; of those people hospitalized, 50% die within the first year. The total direct cost of fall injuries in 1994 for those over 65 was estimated at $20.2 billion.

HEALTH TREND MAKES PHYSICAL AND FISCAL SENSE Active Aging Advocate Lists 10 Trends Supporting Shift to

Preventive Care VANCOUVER, B.C. (December 30, 2003)-The developing public health trend of the decade is the shift in focus to preventive care, according to the International Council on Active Aging (ICAA), the world's largest trade association for the senior fitness and wellness industry. Change is occurring on all levels, from federal, state and municipal governments to corporations and individuals. That's due to the increase in dollars, programs and coalitions dedicated to preventing disease, says Colin Milner, the ICAA's chief executive officer. Efforts to promote prevention among midlife and older adults are especially important, according to Milner, as 2003 estimates by the Federal Hospital Insurance Trust Fund's trustees suggest the Medicare trust fund will be exhausted by 2026-four years earlier than previously thought. "Our society has reached the tipping point in embracing the preventive health model," says Milner, "because prevention makes both physical and fiscal sense given our aging population." The ICAA has identified the following 10 trends that collectively support this shift to a preventive care model: 1. The rapid rise of obesity and diabetes rates 2. The growth of "anti-aging" products 3. The link between health and wealth 4. The increasing calls for preventive screening 5. The rise in research showing that exercise is a key ally in the battle against cancer 6. The power of lifestyle choices 7. The increasing calls for physicians to prescribe exercise 8. The focus on caregivers' health 9. The volume of health initiatives to address older adults' physical activity level 10. The increase in communities designed for activity (SEE ENCLOSED FACT SHEET FOR A MORE IN DEPTH LOOK AT EACH TREND.) Together, the above trends have helped to make disease prevention a public policy priority. The ICAA agrees with the following comment by Health and Human Services Secretary Tommy G. Thompson: "Prevention is the right cause, the right issue, the right time." About the International Council on Active Aging (ICAA) The ICAA is the world largest association dedicated to changing the way we age by uniting and working with professionals in the retirement, assisted living, fitness, rehabilitation and wellness fields. It connects a community of like-minded professionals who share the goals of changing society's perceptions of aging and improving the quality of life for aging baby boomers and older adults within the six dimensions of wellness (emotional, vocational, physical, spiritual, intellectual, social.) The council supports these professionals with education, information, resources and tools, so they can achieve optimal success with this growing market. The ICAA also takes an active role in helping to change the way society perceives aging. The council has recently joined 49 of the nation's most prominent health and aging organizations

to work on the development and implementation of the National Blueprint on Aging. Contributors to the Blueprint's development include AARP, the American College of Sports Medicine, the American Geriatrics Society, the Centers for Disease Control and Prevention, the National Institute on Aging and the Robert Wood Johnson Foundation. - 30 - For more interviews or information about the ICAA or aging related issues, contact: Colin Milner, CEO International Council on Active Aging Toll-free: 866-335-9777 (in North America) Telephone: 604-734-4466 Cell# 604-763-4595 Email: cmilner@attglobal.net Website: www.icaa.cc FACT SHEET ON HEALTH TRENDS 1. The rapid rise in obesity and diabetes rates According to the Centers for Disease Control and Prevention (CDC), more than 44 million Americans are now considered obese (defined as having a Body Mass Index greater than or equal to 30), an increase of 74% since 1991. And obesity rates are highest among those ages 40-69 years. This epidemic of obesity, combined with the aging of the population, has fueled a dramatic rise in the prevalence of diabetes. Worldwide, more than 300 million people are at risk of developing diabetes, states the International Diabetes Federation. In the United States, estimates suggest that 17 million people have diabetes, with almost six million cases still undiagnosed. A further 16 million individuals have prediabetes, a condition that increases the risk of developing the disease. And the cost of diabetes? The American Diabetes Association estimates that the national cost of diabetes in the United States was $132 billion in 2002. The soaring prevalence and costs of diabetes have led the federal government to make preventing obesity and diabetes two cornerstones of its Steps to a HealthierUS initiative. 2. The growth of "anti-aging" products A September 2003 study by market researchers Freedonia Group, Inc., predicts that demand for products to help fight aging will rise more than 11% per year to $29 billion in 2007. This growth is being driven by new or improved products targeted at midlife and older consumers that claim to maintain health and enhance appearance. 3. The link between health and wealth Preventive health makes fiscal sense for everybody. Sedentary adults can save on average $2,200 per year in healthcare costs by starting to exercise moderately for just 90 minutes per week, concludes a recent study by HealthPartners Research Foundation, the subsidiary of a Minnesota-based nonprofit group of healthcare providers. The study is the first to associate changes in physical activity with changes in an individual's healthcare costs. For health insurance companies, a

report published by the American Association of Health Plans (AAHP), an organization that represents more than 1,000 health plans, states that insurance companies could save money "by intervening beforehandwith seven common conditions in older adults." The report continues, "These interventions are often simple to implement and could greatly improve the quality of life for older Americans." For employers, a preliminary estimate of lost productivity time from common pain conditions alone is approximately $80 billion per year, according to a study in the Journal of the American Medical Association (November 12, 2003). "Prevention Makes Common Sense," a September 2003 report by the U.S. Department of Health and Human Services (HHS), shows that corporate health promotion and disease management programs produce a return on investment of $1.49-$4.91 in benefits for every dollar spent. And, finally, HHS estimates that U.S. government spending on healthcare in 2003 will total $1.66 trillion. According to HHS, about 75% of healthcare spending can be attributed to chronic health conditions such as diabetes, obesity and cardiovascular disease-much of which can be avoided or delayed through preventive measures (e.g. early screening) and healthy lifestyle choices (i.e. eating a nutritious diet, engaging in regular physical activity, not smoking and limiting alcohol use). 4. The increasing calls for preventive screening Just 10% of America's current spending on healthcare goes toward prevention, says Dr. Cristina Beato, assistant secretary, U.S. Department of Health and Human Services (HHS). But the federal government aims to change that. In 2003, President Bush, HHS Secretary Tommy Thompson and U.S. Surgeon General Richard H. Carmona all promoted prevention as the desired model for healthcare and self-responsibility as the desired public attitude towards health. A key part of prevention is screening for disease. Prehypertension and prediabetes are new markers of chronic health issues that can be identified through screening. Individuals who learn of their increased risk for disease can make lifestyle changes to avoid or delay developing these conditions. Calls were made in 2003 for increases in screening for depression, and it is likely that screening for this health issue will become more common in coming years. A two-year study led by researchers from Harvard Medical School reports significant numbers of major depressive episodes (MDE) in all segments of the U.S. population. The researchers also note that few of the 13-14 million adults who suffer an MDE in a one-year period receive proper treatment. 5. The rise in research showing that exercise is a key ally in the battle against cancer Several long-term research studies now support the benefits of physical activity for preventing cancer or cancer

recurrences. For instance, the results of a 25-year study by scientists from the University of North Carolina at Chapel Hill suggest that people who keep fit and trim may significantly reduce their likelihood of dying from cancer. And the American Cancer Society's new "Guide for Informed Choices" advises cancer survivors about physical activity and nutrition. This guide builds on the society's first recommendations for physical activity published in 2001. 6. The power of lifestyle choices Healthy lifestyle habits have proven to be effective in preventing or managing chronic conditions, such as osteoporosis, diabetes, hypertension and high cholesterol, without the sometimes serious side effects of medications. The 2003 PREMIER study supported by the National Heart, Lung and Blood Institute shows that individuals can make all the needed lifestyle changes to prevent or control high blood pressure at the same time. Dr. Lawrence J. Appel, professor of medicine at the Johns Hopkins Medical Institutions in Baltimore and study coauthor, calls lifestyle changes "a first-line weapon in the fight against high blood pressure." HHS Secretary Tommy G. Thompson also emphasizes the importance of healthy lifestyle habits. "We cannot overstate how critical physical activity is for our good health," says Thompson, "and we want every American to understand that small steps toward a more physically active life yield significant health benefits." 7. The increasing calls for physicians to prescribe exercise Many organizations are calling upon the medical community to get more involved in disease prevention by recommending physical activity and proper nutrition. These groups include, among others, the Centers for Disease Control and Prevention; American Heart Association; U.S. Surgeon General; and U.S. Preventive Task Force. The International Council on Active Aging and industry partners the American Society on Aging, IDEA Health and Fitness Association and the Medical Fitness Association also urge action. With nearly one billion doctors' visits every year, the ICAA believes the information highway to better health must start with physicians. 8. The focus on caregivers' health A 1997 National Alliance for Caregiving/AARP survey revealed that 22.4 million American households were involved in caring for adults ages 50 and above, and that number could rise to 39 million by 2007. The economic value of services provided by family caregivers has been estimated at about $257 billion annually-a figure more than double the $115 billion spent on nursing home care and home care combined. The increasing longevity of Americans means that more adults in their 50s, 60s or 70s are caring for parents or other family members, while studies have shown that caregivers often become

ill due to stress and lack of time to care for their health. As our population ages, the health of caregivers will continue to grow in importance. Through National Family Caregivers Month, the U.S. Administration on Aging (AoA) aims to create more public awareness of family caregiving and much-needed services available. AoA also provides the National Family Caregiver Support Program, a $155.2 million program that helps states work in partnership with area agencies on aging and service providers in the local community to create services in five basic areas for family caregivers, including respite care. Other groups such as the National Family Caregivers Association and Illinois-based Mather Institute on Aging also focus on providing tools, education and support for caregivers. 9.The volume of health initiatives to address older adults' physical activity level In the last five years or so, health initiatives have been launched at all governmental levels to help older adults become and stay more physically active. National programs include, but a certainly not limited to the following: * Steps to a HealthierUS, the HHS initiative to prevent obesity, diabetes and asthma; * Active for Life, a program funded by the Robert Wood Johnson Foundation that is testing two physical activity interventions for midlife and older adults; * The President's Challenge, an awards program by the President's Council on Physical Fitness and Sport that motivates Americans to incorporate more physical activity into their lives daily; * National Blueprint on Aging, a coalition of more than 50 national organizations working to implement strategies to raise the physical activity level among adults ages 50 and older; and * Active Aging Week, a public awareness program created by the International Council on Active Aging in partnership with international dance fitness company Jazzercise, Inc., to encourage older adults to become more physically active. 10. The increase in communities designed for activity Julie Gerberding, director of the Centers for Disease Control and Prevention, encourages officials to adopt a community-based approach to the health problem. Studies show that the built environment or community planning and design can either aid or create barriers to active living, particularly among older adults. Numerous organizations and programs encourage or help regions to implement strategies for healthy communities. These initiatives include the Robert Wood Johnson Foundation's national program Active Living by Design, which recently awarded grants to 25 community partnerships throughout the U.S. to implement strategies to promote healthier lifestyles.http://www.icaa.cc/PressInfo/healthtrends.htm *******************************************

Imagine this headline in your morning newspaper or the lead story on the TV or radio news. LIFESPAN EXTENDED BY 35% Natural Medical Breakthrough for the 21st Century In the early 1980's, Dr. Gustavo Bounous, a world renowned research scientist, noticed that one group of his lab animals was living, on average, 35% longer than the others. His work in the field of health and nutrition had been comparing the food value of various proteins but this observation redirected his entire course of investigation. He didn't know how it happened or why it happened but that it did happen over and over again. The animals being given one specific whey protein concentrate (WPC) were outliving all others. But Why? The first question that Dr. Bounous attacked was the cause of death in the animals that died first. If he didn't know why the one group lived longer, perhaps he could determine why the other group died at younger ages. His post-mortems revealed an astonishing fact. The animals receiving the WPC protein did not die of the same things that killed the other groups of animals. The younger animals died of strokes and cancers while the WPC fed animals did not. But Why? Why didn't the WPC group have strokes and cancers? Was there something in the WPC that helped prevent them? The next series of tests answered that question. The animals were exposed to a known cancer causing substance. The animals in the WPC group showed much lower susceptibility to tumors. Their tumors were much smaller and less frequent. The WPC fed group had some sort of resistance to the cancers. The doctor then altered his test. He didn't give any animals the WPC protein and exposed the entire group to the same cancer causing substance. For 20 weeks, there was no protective WPC given. Then one group of animals was switched to the WPC protein diet. The post- mortems were astonishing. The WPC fed group again had smaller and fewer tumors. The WPC protein was proven to both reduce the incidence of new cancers (prevention) and to substantially slow or reverse the growth of existing tumors (treatment)! But Why? Dr. Bounous had indeed found something of value, a substance that would inhibit cancer in lab animals but

he still didn't know how or why. One afternoon, during one his very infrequent coffee breaks, Dr. Bounous happened to meet a fellow scientist from the other end of the same medical building complex. During what started as a short break, they discussed their respective work - Dr. Bounous work in nutrition and his remarkable findings and Dr. Batist, a young researcher working on something called glutathione and it's possible role in cancer. Dr. Batist suggested that what was happening appeared to be immune system enhancement of the WPC group animals. Dr. Batist knew that cancer cells had excessive levels of glutathione (GSH) and normal cells had much lower levels. Using Dr. Batist's equipment, the glutathione (GSH) levels of the lab animals were measured and it was determined that the WPC supplement caused the GSH levels in normal cells to go up (which was good) and the GSH levels in cancer cells to go down (which was also good). Glutathione? There was already a great body of work published about GSH in the medical literature. It was already known that GSH is the body's own principal antioxidant - that each cell in the body makes what it needs when it's needed. It was already known that without GSH, the other antioxidants (A, C, E, etc) can not function efficiently. It was already known that GSH is a key component of the liver's detoxification enzyme system, and it was already known that GSH is the rate determining step in the immune response system (the body's response to infection). All this was known because medical science had tested and observed the results of reducing GSH levels but until now, there was no known way of raising GSH levels throughout the body. HERE WAS THE BREAKTHROUGH! For the first time, researchers were able to raise the levels of GSH within the cells in a safe and convenient way. Then it happened - disaster struck!! The WPC stopped working!! But Why? For nearly two years, Dr. Bounous and his team of researchers went back over the results rechecking, reanalyzing, reevaluating. They checked the equipment, the animals, the testing procedures and the whey protein and could find nothing that had changed. One evening, tired from a long and frustrating day, Dr. Bounous was watching a gormet cooking program on TV where two French chefs were discussing cheese. One of them mentioned the difference in the taste and texture of cheese since the pasteurization process was changed. (In the late 1980's there was a salmonella outbreak in Europe and the pasteurization temperature of milk was raised from 63 C to 72 C worldwide to better control the bacteria.) Since whey protein was a byproduct of the cheese industry which was itself a byproduct of milk processing, Dr. Bounous theorized that the temperature could have had an effect.

He decided to create a supply of whey protein using the old method of pasteurization for testing. The results proved the theory - The bioactivity was back! But Why? During the retesting, everything was rechecked including the composition of the whey protein. They contained the same ingredients so what was the difference? The product made at the lower temperature was undenatured. This critical difference sets this WPC product apart from every other WPC or protein supplement on the market. What does undenatured mean? Undenatured means that the conformation or sequence of components remains the same from raw material to finished product. Think of a jar of coloured jellybeans representing the components of whey protein. There are black, blue, red, yellow, green - a rainbow of colours. Now visualize within that jar two orange jellybeans stuck to a black jellybean - three stuck together. This is the way bioactivity is achieved in nature. Two cysteine amino acids are linked by a sulfur (thiol) bond - just like the three jelly beans. For some reason, when the two linked amino acids are swallowed, they are not separated. They pass through digestion and absorption, circulate in the blood stream, and are absorbed into our cells yet if they are ingested separately (denatured), they are digested and consumed before entering the cells. Think again of the jellybeans. Only the two orange beans linked by a black are active. High temperature pasteurization breaks the bond holding them together. You still have a jar of jellybeans. They look the same, they weigh the same and they taste the same. They still have the same nutritional value, but the key sequence is broken. The essential compound (cysteine) is digested and never reaches the cells in active form. By utilizing a patented low temperature / filtration process, Dr. Bounous has developed a bioactive undenatured whey protein concentrate. And Then? Having reestablished the bioactivity of his discovery and reasserted it's effectiveness in cancer prevention and treatment, Dr. Bounous was approached by another researcher, a Dr. Baruchel, recently of the Pasteur Institute in Paris, where he and Dr. Luc Montagnier were credited with the discovery of the virus responsible for AIDS a few years earlier. Dr Baruchel, fresh from his work at the Pasteur Institute, requested authorization to test the newly discovered undenatured WPC on a small group of his AIDS patients to determine if the product could positively affect their immune systems. Dr. Baruchel reports "Over the 3-month period, glutathione levels increased in all 3 cases. His results are quoted here. ... in patients who maintain an adequate total caloric

intake, the addition of "bioactive" whey protein concentrate as a significant portion of total protein intake increases body weight and shows elevation of glutathione (GSH) content of mononuclear cells toward normal levels." These results lead the Center for Disease Control in Atlanta to write "Laboratory studies have shown that a new whey protein concentrate, called Immunocal, can inhibit HIV replication while also stimulating the production of glutathione, an amino acid that helps control the progression of the virus." in their CDC Aids Daily Summary 02/06/97. And Then? For over twenty years, medical science had known that reduced glutathione leads to impaired health but had no way to positively impact on the cells to alter the intracellular production of glutathione. Now, because of this first ever opportunity, clinical trials are in progress all over the world with this remarkable product. Among the trials there are: Hepatitis B & Hepatitis C - Japan Lyme Disease Study - Springfield Illinois Prostatic Cancer Trial - Royal Victoria Hospital Leukemia Trial - King Khalid Hospital, Saudi Arabia Breast Cancer Phase II Trial - New York Breast Cancer Phase II Trial - Nova Scotia Cancer Center HIV Phase III Clinical Trial - Montreal General Hospital Major Surgery & Multiple Trauma Study - University of Munich Cystic Fibrosis Study - Montreal Children's Hospital Full information available in the U.S. Physician's Desk Reference - Year 2000-2011 Editions All of these studies and many more are ongoing, not to determine if raising glutathione levels will help, but to clinically determine how much it will help. Dr Larry Lands and his group at Montreal Children's Hospital recently concluded the first part of their study on oxidative stress and muscle fatigue. Their first task was to create a baseline for comparison. They found something totally unexpected. Not only did raising glutathione levels slow the onset of muscle fatigue (which was expected) but in fact it increased muscle strength and 30 second work capacity by 13% in normal healthy young adults . What does this all mean for you and me? -----------------------------------------------------------I cannot make health claims for this product - it is a

food supplement, not a drug. This product is not intended to diagnose, cure, prevent or treat any disease. These statements have not been reviewed by the FDA. -----------------------------------------------------------Having fulfilled my legal requirements of disclaimer to the FDA, let me make a few personal observations. 1. As of this date (12/06/02) there are over 90,000 articles in the medical literature (MedLine / PubMed) regarding the importance of glutathione in health and disease. There can be no question about the importance of glutathione in the body. 2. Until this discovery, there was no way to safely and effectively elevate glutathione levels over any prolonged period of time. 3. There is only one proven way to increase glutathione in the cells of your body (HMS-90 in Canada & Immunocal in the USA). 4. Immunocal & HMS-90 have been proven and patented to increase the human immune response. 5. Immunocal & HMS-90 have been proven and patented as a method of treatment for HIV-seropositive individuals. 6. Immunocal & HMS-90 have been proven and patented to have anti-cancer therapeutic properties. 7. I can't make specific assertions about increased lifespan in humans since it will be many years before we will have hard data to evaluate BUT, as in the lab animals, if increasing glutathione in our cells decreases the risk of early death from cancers and strokes it stands to reason that lifespan will be significantly increased. 8. A study by the University of Michigan found that "Higher glutathione levels were associated with fewer number of illnesses, higher levels of self- rated health, lower cholesterol, lower body mass, and lower blood pressures." 9. Reduced intracellular glutathione has been implicated in or associated with aging, allergy, ALS, anemia, arthritis, asthma, arteriosclerosis, alzheimers's, auto-immune disease, chronic fatigue, Crohn's, fibromyalgia, diabetes, hepatitis, lupus, MS, neurodegenerative disease, parkinson's, cancer, cataract, stroke, cystic fibrosis, chronic bronchitis, HIV / AIDS, and hundreds of conditions relating to

oxidative stress. If reduced glutathione is implicated / associated with all of these conditions, doesn't it make good sense to get your GSH levels up? 10. Jean Carper, in her book Stop Aging Now!, sums it up the best. "You must get your levels of glutathione up if you want to keep your youth and live longer. High levels of GSH predict good health and long life. Low levels predict disease and early death." There are only two groups of individuals that should not take these products, organ transplant recipients (boosting the immune system increases the chance of rejection) and people allergic to milk protein specifically. The product is safe for lactose intolerant people since it contains less than 1% lactose. People undergoing chemotherapy should consult their oncologist before taking these products since a few chemotherapy drugs do lower GSH levels by design. The products are safe to use before, between, and after chemotherapy but check with your doctor before using them during chemotherapy. The only thing standing between you and death is your immune system. Take good care of it! Don't wait for your risks to become realities! Take Action! Order now! Support your immune system today! http://www.immunotec.com/01.prod How Long Should We Live This interesting article addresses some of the key issues regarding Life Span & Glutathione. A careful reading of this material could make a big difference in how you think about your life.

There's a lot to understand about Life Span & Glutathione. We were able to provide you with some of the facts above, but there is still plenty more to write about in subsequent articles. ******************************************** GSH, like all proteins, consists of amino acids. It is a tripeptide (mixture of three amino acids) of glycine, glutamate (glutamic acid) and cysteine. This sulfer-containing amino acid is absent from many diets and its absence limits the bodys ability to produce GSH. GSH is being depleted from our bodies on a daily basis. Oxidative stress originating from outside the body is a feature of life in the modern world. First, the tens of thousands of confirmed toxic substances in our external environment are invariably sources of free radicals or related oxidants. Add to this substantial burden of the many negative aspects of the modern Westernized lifestyle and a picture emerges of the human organism burdened by chronic stress, disease and increased aging threatening a shorter lifespan than might otherwise be possible. Some of the most important of the exogenous oxidative stressors include cigarette smoking, pharmaceuticals, hydrocarbons, strenuous exercise, dietary deficiency, ionizing radiation, tissue injury, iron overload, bacterial and viral infections and alcohol intake. The human body faces constant attack of free radicals through the simple breathing process due to the pollutants in the air we breath. With our reliance on oxygen, humans cannot escape this ongoing oxidative challenge. It may be the ultimate challenge of being alive. An ever more impressive body of evidence indicates that the cumulative damaging effects of oxygen radicals and other oxidants are principal contributors to degenerative diseases and to the progressive loss of organ functions that we recognize as aging. GSH has three major functions: Antioxidant: An antioxidant is a substance that neutralizes destructive free radicals; some are manufactured by the metabolic processes of the body, others are derived from foods, the air we breath, exercise, stress and disease. GSH is the most powerful antioxidant occurring naturally in the cells of the body. Through its significant reducing power, GSH also makes major contributions to the recycling of other antioxidants that have become oxidized. Healthy cells homeostatically oppose free radicals through the use of antioxidants, of which GSH plays a significant role. The effectiveness of other antioxidants like vitamins C and E depends on the availability of GSH.

Oxidative related diseases: accelerated aging, cell destruction, causes damage to DNA cellular patterns which leads to cancer, arteriosclerosis, coronary artery disease, Parkinsons disease, diseases of the immune system, diabetes, cataract formation, Alzheimers, macular degeneration, COPD, allergy/asthma, stroke. GSH also plays a main role in detoxification..primarily in Phase II Liver detox. It "binds" to many toxins by its sulfur molecules and aids in forming a complex which the body then rids itself of. The research on glutatione is enormous. If you put glutathione in the search engine of either of the research sites below, you will see what I mean. Try putting in glutathione + any issue you are personally concerned with, such as chronic fatigue syndrome and you will receive research abstracts on that subject.

Glutathione (GSH) & Aging The GSH antioxidant system is the body's powerhouse for diffusing and disposing of free radicals that threaten cell, tissue and organ damage, thus slowing the approach of aging. John T. Pinto of Sloan Kettering Cancer Center in New York proclaims GSH "The master antioxidant. " Jean Carper in her bestseller "Stop Aging Now!" highlights the same point: "You must get your levels of GSH up if you want to keep your youth and live longer. High blood levels of GSH predict good health as you age and a long life. Low levels predict early disease and death." These opinions result from convincing, fascinating research and experimentation. Age-specific decreases in GSH are seen in all tissues, including liver, kidney, lung, heart, spleen and the brain. Laboratory studies on the role of GSH in aging show GSH deficiency in all aging creatures, from mosquitoes and houseflies to rats and mice. Similar findings in humans indicate that elderly subjects bear increased risk of disease and impairment. Blood-GSH concentrations in younger people (20-40 years) are 20 to 40% higher than in those aged 60-80 years. Studies by leading experts on aging (C.A. Lang, M. Julius and others) suggest that elevated GSH levels give elderly individuals a physical, psychological and sociological advantage over those with lower levels. Researchers Mara Julius and Calvin Lang measured glutathione concentrations in community-based individuals over the age of 60 years. They mapped these values to their level of health, number of

illnesses, and risk factors for chronic disease (tobacco, alcohol, cholesterol, blood pressure and obesity). Higher glutathione levels corresponded to lessened effects of aging and better general health. Those with 20% greater blood GSH -levels experience about one-third the rate of arthritis, high blood -pressure, heart disease, circulatory difficulties and other maladies. Dr. Lang also looked at glutathione levels in age groups: 20-40, 40-60, 60-80 and 80-100 years. The youngest group had acceptable levels but 14% of the 40-60 year olds and 53% of the 60-80 year olds had critically low levels. Interestingly, only 24% of the 80-100 year olds had low levels, perhaps explaining how they reached such a ripe old age in the first place. The Italians G. Paolisso and M.R. Tagliamonte went one step further, comparing adults under age 50 with those over 50. Both the GSH and antioxidant function were depressed in the older group. However, those over 100 years old had higher GSH levels than the other over-50 group. Again, this may explain their unusual longevity. Several researchers over the years have also shown that life span can be extended by restricting diet and maintaining low body weight. No satisfactory explanation has emerged for this phenomenon, but some scientists have demonstrated that glutathione levels rise in these longer-living individuals. They suggest that glutathione may be involved in a molecular mechanism that contributes to longevity. S.L. Nuttal and his British team published a revealing study in The Lancet, comparing GSH levels in individuals of different ages and states of health. The healthy young had the highest levels, ahead of the healthy elderly. The lowest levels were found in sick, elderly patients. The results clearly showed that GSH levels fall as we age and as we become ill. The more severe the illness, the more evident the decrease. Back in the laboratory, scientists are trying to find out whether elevated GSH levels can actually extend the life span. Aging-expert John Richie Jr. thinks that glutathione deficiency may be a biochemical cause of the aging process. In some of his experiments MgTC-a GSH promoting drug similar to OTC-was fed to mosquitoes. GSH levels were found to be 50 to 100% higher, and life span was increased by almost 40%. In another experiment, Diane Birt at the University of Nebraska fed hamsters the whey-protein concentrate lactalbumin - a GSH-precursor. These animals also lived longer. Interestingly, control hamsters on a diet including casein and cysteine, or methionine did not benefit. In fact high cysteine loads proved

harmful, showing how the bioactivity of these amino acids changes when part of a larger protein, rather than free amino acids. Dr. Gustavo Bounous and other researchers at McGill University demonstrated this anti-aging effect using a natural product to elevate GSH levels. They fed mice a specially developed whey protein isolate-later trademarked Immunocal - and compared their GSH levels and lifespan to mice on a standard diet. Not only were the tissue GSH levels found to be higher, the Immunocal-fed mice had an average life span of 27 months (corresponding to a human age of 80 years) as compared to the control diet average of 21 months (human equivalent of 55 years). This is an astonishing increase of 30%. Further experiments using both cysteine and caseine (another milk protein) neither increased longevity nor raised GSH levels. Health News for June 8, 1999 Antioxidant enzymes low in aging disease NEW YORK Jun 08 (Reuters Health) -- People with progeria -- a rare, rapid aging disease -- have low levels of the antioxidant enzymes believed to fight aging, researchers from the University of Iowa, Iowa City, have found. "We're very certain that the loss of these enzymes is damaging," said one of the team, Dr. Larry W. Oberley, a radiology professor, in an interview with Reuters Health. The results not only suggest that replacement of these enzymes may help treat people with progeria, but they also provide insight into the normal aging process, he said. "This is an extremely devastating disease," he said. People with progeria live on average to the age of 13. "By the time they're 6 years old, they look like they're 60 or 70." In addition to rapid aging, patients suffer delayed growth, a build up of fats and cholesterol in the arteries, cardiovascular disease and other illnesses. Studying progeria also enables researchers to examine the normal aging process. "We think normal cell aging is caused by free radical damage," said Oberley. In their study, funded by the National Institutes of Health, Oberley and colleagues compared skin cells of progeria patients with those of healthy patients. Normally, healthy cells produce antioxidant enzymes that attack damaging free radicals. Free radicals are produced through normal metabolic processes in the body, or as the result of disease. The researchers found that in cells from progeria patients, levels of three important antioxidant enzymes were lower than those found in healthy cells. Activity levels of the enzyme catalase were 50% lower than normal, while glutathione peroxidase activity was

70% less. In addition, the investigators note that progeria cells respond less well to the stress of poor nutrition compared with healthy cells. Although Oberley said it is not clear exactly how lower enzyme levels affect progeria, the study results indicate a potential avenue of treatment. The results suggest two types of treatment for progeria, he said. For short-term treatment, existing drugs that mimic the missing enzymes can be studied. A long-term solution is to use genetic engineering to deliver genes coding for the enzymes into the cells of progeria patients. This way, the genes will be able to produce the missing enzymes in the body. Research in this area is at an early stage, but may be applicable to many diseases including cancer, heart disease, stroke or diabetes, said Oberley. "There are very few diseases that don't have a free radical component," he said. Dietary antioxidants in food or vitamin supplements, such as vitamins C and E, are important but have a smaller effect than the antioxidant enzymes produced by the body, he noted. SOURCE: Biochemical and Biophysical Research Communications 1999;257:163-167. ****************************************** New Understandings about Acetylcysteine and Glutathione: Richard A. Passwater Ph.D. Our understanding of the many nutritional and biochemical roles of glutathione and its precursors is expanding rapidly at this time. We are learning how the precursors to sulfur-containing peptides and amino acids are important in keeping our bodies nourished, our immune systems healthy, and in protecting us against cancer and heart disease. Glutathione and cysteine have been important in my research for over thirty years. Even before that, I believe that some pioneers in the health food profession indirectly realized the importance of these compounds even though they didn't know about the specific compounds. When the "pioneers" spoke so favorably of getting adequate "sulfur" in the diet, I believe that they were really testifying to the importance of these sulfur-containing compounds. Now, the interest in learning how sulfur-containing amino acids and their precursors nourish the body is increasing.

Researchers are rushing to study the roles these nutrients to also discover their role in halting the dreaded Human Immunodeficiency Virus (HIV), breaking up lipoprotein detoxifying harmful chemicals, scavaging free radicals and possibly protecting against some cancer processes. I have discussed cysteine and glutathione several times before. Later in this article, I will discuss the role of glutathione precursors and why they are more effective than glutathione itself in AIDS patients. These non-fuel nutrients are nutritional accessory factorsm,that are normally produced in plants, man and other animals. Thus, they have always been a part of the human diet. Glutathione plays several critical roles in the body. Its more important roles are protecting cells and cellular components against oxidative stress and in detoxification. My interest in glutathione and cysteine began in the 1960's when they were found to be protective against nuclear radiation. I reasoned that the same mechanism of action would make them excellent free-radical scavengers as well. NAC is produced in living organisms from the amino acid cysteine {two compounds that are involved in intracellular glutathione production are N-acetyl-L-cysteine (NAC) and L-2-oxo-thiazolidine 4-carboxylate (Procysteine)}. Thus,NAC is a natural sulfur-containing amino acid derivative found naturally in foods and is a powerful antioxidant. These dual properties help repair oxidative damage in the body. This has made this nutrient of special interest to athletes for some time as heavy exercise increases oxidative damage in the body. But the latest research interests are in AIDS and heart disease. AIDS and Immunity Now the biochemical and medical research communities have taken special interest in this nutrient because of its importance in increasing intracellular glutathione levels and other biochemical properties. Oral glutathione is largely broken down in the digestive system into dipeptides and free amino acids. Although some glutathione is absorbed intact, it still must cross the cell walls to serve many body needs. As will be seen later, HIV infection decreases the transport of glutathione into cells, and as a result, the immune system fails. Thus, even injections of glutathione fail to restore intracellular glutathione levels in AIDS patients. (Later we will discuss the evidence that shows that glutathione is well absorbed and transported across cellular membranes of healthy persons.) NAC, on the other hand, is well absorbed,

readily passes through cellular membranes in AIDS patients, and stimulates glutathione production within the cells. Approximately ten percent of orally ingested NAC shows up in the blood. Earlier research has suggested that NAC suppresses Human Immunodeficiency virus (HIV) in infected cell cultures. NAC is also of interest to AIDS patients as it protects against some of the damage produced by radiation. AIDS patients undergoing radiotherapy for Kaposi's syndrome may have a double benefit from this nutrient. Heart Disease, Another area of interest is that research has pinpointed a specific lipoprotein called Lp(a) as one of the two most reliable indicators of heart disease risk. The other reliable indicator is the level of vitamin E in the blood. Lp(a) is a much more reliable indicator than blood cholesterol level, low density lipoprotein, high-density lipoprotein or their ratios to each other. Now recent research has found that NAC is the most effective nutrient knownto lower Lp(a) levels. NAC reduces Lp(a) by almost 70%. [16-19] Immunity: NAC affects immunity via its role in intracellular glutathione production. This role becomes critical when normal glutathione production pathways are impaired, as for example, by the Human Immunodeficiency Virus (HIV). Eck has shown that reduced intracellular glutathione is the "direct and early consequence of retroviral infection." Intracellular glutathione has a powerful influence on how well T- and B-lymphocyte cells function. In addition, intracellular glutathione availability affects the production of phagocytes (macrophages, monocytes and neutrophils). NAC has been shown to block the AIDS virus (HIV) production in vitro, apparently by increasing glutathione levels in HIV-infected cells. In 1989, Dr. Leonore Herzenberg told Associated Press writer Steve Wilson, "I am really excited about this. Looking at the scientific evidence for what (NAC)does, and the scientific evidence for how AIDS works, our guess is that treatment with (NAC) may be quite good. But, until we get it tested in patients, we won't know if it will work. Detoxification: This food-factor is also gaining new interest because

it protects against toxins and has been widely used to treat bronchopulmonary diseases. NAC detoxifies several poisons including acetaminophen and other drugs, mercury, lead, cadmium, paraquat, urethane, aflatoxin and Escherichia coli. NAC, cysteine and glutathione contain sulfur in the form of sulfhydryl groups. Sulfhydryl groups directly react readily with many toxins, especially heavy metals such as lead, mercury and cadmium. Sulfhydryl groups also help remove toxins indirectly via an enzyme system called the P-450 System. Although NAC is a food component and a nutrient accessory factor, it is also marketed as a drug with approved medical claims. NAC is approved for use in bronchopulmonary diseases and to prevent liver damage from acetaminophen overdose. Either NAC tablets or solutions may be used to protect against acetaminophen overdose. Normally, the 20 percent solution is drank after dilution with a cola drink. The Lancet reports that NAC is also effective in reducing the toxic effects of carbon tetrachloride, chloroform and carbon monoxide. NAC can also reduce the side effects of drugs such as doxorubicin and ifosphamide. NAC has been used for about thirty years to break up mucus in persons having bronchopulmonary diseases including chronic bronchitis, cystic fibrosis,asthma, sinusitis and pneumonia. NAC helps reduce the viscosity of mucus so that it may be more easily coughed up. NAC accomplishes this by converting the disulfide bonds of the mucoproteins into sulfhydryl bondsand cleaving the mucoproteins into smaller molecules. Several companies provide a 10 or 20 percent NAC solution as a nebulizer spray (such as Bristol Laboratories' Mucomyst TM), while others such as Italy's Zambon group provides NAC in tablet form. Optimal Intake Ranges NAC in normal food supplementation ranges is without known toxicity and has been administered by physicians under supervision in doses of two tofour grams daily. Larger quantities used for treating acetaminophen overdoses have produced adverse reactions such as nausea, vomiting, and other gastrointestinal symptoms. Rash, with or without mild fever, has been reported on rare occasions with very large quantities. When administered via nebulizer, adverse effects can include stomatitis, nausea and nasal irritation. Intravenous administration could also produce edema and a rapid heart beat.

Ingestion of more than 150 milligrams per kilogram of body weight (that is nine grams per day for a 132 pound person, twelve grams per day for a 176 pound person, or fifteen grams per day for a 220 pound person) may produce toxic or other undesirable effects. *********************************************************************** 1.Human Aging ResearchPasswater, Richard A. and Welker, Paul A.Amer. Lab. 3(5):21-6 (May 1971) 2.Pharmokinetics of oral acetylcysteine.Rodenstein, D., DeCoster, A. and Gazzaniga, A.Clin. Pharmacokin. 3:247-54 (1978) 3. Toxicological, pharmacokinetics and metabolic studies onacetylcysteine.Bonanomi,L. and Gazzaniga, A.Eur. J. Respir. Dis. 61(Sup):45-51 (1980) 4.Clinical pharmacokinetics of N-acetylcysteine.Borgstrom, L., Kagedal, B. and Paulsen, O.Eur. J. Clin. Pharmacol. 31:217-22 (1986) 5.Pharmokinetics and bioavailability of reduced and oxidizedN-acetylcysteine.Olsson, B., et al.Eur. J. Clin. Pharmocol. 34:77-82 (1988) 6. Acetylcysteine: A drug that is much more than a mucokinetic.Ziment, I.Biomed. Pharmacother. 42:513-20 (1988) 7. Pharmacokinetics and bioavailability of oral acetylcysteinein healthy volunteers.De Caro, L., et al.Arzneim.-Forsch/Drug Res., 39:382 (1989) 8. Suppression of Cytokine-induced Human Immunodeficiency Virus(HIV) expression by N-acetylcysteine (NAC), glutathione(GSH) and Glutathione monoester (GSE)Kinter, Audrey L., et al.75th Annual Meeting of the Federation of American Societiesfor Experimental Biology, Atlanta, Georgia (April 21-5, 1991)J. FASEB 5(5) A1265 (1991) 9. Suppression of human immunodeficiency virus expression inchronically infected monocytic cells by glutathione,glutathione ester, and N-acetylcysteine. Kalebic, Thea, et al.Proc. Natl. Acad. Sci. 88(3):986-90 (1991) 10. Thiol-based compounds may limit AIDS progression.CDC AIDS Weekly p3 (Feb. 25, 1991) 11. Cytokine-stimulated human immunodeficiency virus replicationis inhibited by N-acetylcysteine.Roederer, M., et al.Proc. Natl. Acad. Sci. 87:4884-8(1990) 12. Reducing agents and AIDS - Why are we waiting?Turner, V. F.Med. J. Aust.153/8, 502 (1990) 13. Mercaptoethanol and N-acetylcysteine enhance T-cell colonyformation in AIDS and ARC. Wu, J., et al.Clin. Exp. Immunol. 77/1, 7-10 (1989)

14. AIDS - Drug therapy; Acetylcysteine research.Cooper, MikeCDC AIDS Weekly p4 (Oct. 2, 1989) 15. Glutathione deficiency and radiosensitivity in AIDSpatients.Vallis, K. A.The Lancet 337:918-9 (April 13, 1991) 16 Lipoprotein(a) reduction by N-acetylcysteine.Gavish, Dov and Breslow, Jan L.The Lancet 337:203-4 (Jan. 26, 1991) 17. N-acetylcysteine and lipoprotein.Stalenhoef, A. F. H., et al.The Lancet 337:491 (1991) 18. AcetylcysteineEditorialThe Lancet 337(8749):1069-70 (May 4, 1991) 19. N-Acetylcysteine and immunoreactivity of lipoprotein(a).Scanu, Angelo M.The Lancet 337:1159 (May 4, 1991) 20. Metabolic disorder as early consequence of SimianImmunodeficiency Virus infection in Rhesus macaques.Eck, Hans-Peter, et al.Lancet 338:346-7 (Aug.10, 1991) 21. Mercaptoethanol and N-acetylcysteine enhance T-cell colonyformation in AIDS and ARC.Wu, J., Levy, E. M. and Black, P. H.Clin. Exp. Immunol. 77:7-10 (1989) 22. Clinical application for heavy metal-complexing potential ofN-acetylcysteine.Lorber,A., et al.J. Clin. Pharmacol. 13:332-6 (1973) 23. Treatment of acute methylmercury ingestion byhemodialysis with N-acetylcysteine.Lund,M. E., Clarkson, T. W. and Berlin, M.J. Toxicol. Clin. Toxicol. 22:31-49(1984) 24. N-acetylcysteine therapy of acute heavy metal poisoning inmice. Henderson,P., et al.Vet. Hum. Toxicol. 27:522-5 (1985) 25. Experimental chelation therapy in chromium, lead and boronintoxification with N-acetylcysteine.Tong, T. G.Toxicol. Appl. Pharmacol. 83:142-7 (1986) 26. Mechanism of action of N-acetylcysteine in the protectionagainst hepatotoxicity of acetaminophen in rats in vivo.Lauterburg, B. H., Corcoran, G. B. and Mitchell, J. R.J. Clin. Invest. 71:980-991 (1983) 27. Role of glutathione in prevention of acetaminophen-inducedhepatotoxicity by N-acetyl-L-cysteine in vivo.Corcoran, G. B. and Wong, B. K.J. Pharmacol. Exp. Ther. 238:54-61 (1986) 28. Effect of N-acetylcysteine on plasma cysteine andglutathione following paracetamol administration.Burgunder, J. M., Varriale, A. and Lauterberg, B. H.Eur. J. Clin. Pharmacol. 36:127-31 (1989) 29. Improvement by acetylcysteine of hemodynamics and oxygentransport in fulminant hepatic failure.Harrison, Phillip M., et al.N. Engl. J. Med. 324(26):1852-7(June 27, 1991) 30. Long-term oral acetylcysteine in chronic bronchitis: Adouble-blind controlled study.Multicenter

Study GroupEur. J. Respir. Dis. 61(Sup.):93 (1980) 31. The reduction in vitro in viscosity of mucoproteinsolutions by a new mucolytic agent, N-acetylcysteine.Sheffner, A. L.Ann. NY Acad. Sci. 106:288(1963)

All rights, including electronic and print media, to this article are copyrighted by Richard A. Passwater, Ph.D. and Whole Foods magazine (WFC Inc.) ******************************************** I found @ http://www.redwings.org/HTMLarts/gshhealtha.htm The Annals of Pharmacotherapy 1995; 29:1263-73. Reprinted with permission from The Annals of Pharmacotherapy www.theannals.com http://www.theannals.com/cgi/content/abstract/29/12/1263

GLUTATHIONE IN HEALTH AND DISEASE: PHARMACOTHERAPEUTIC ISSUES Ben M Lomaestro and Margaret Malone OBJECTIVE: To review the current research and importance of glutathione (GSH) therapy in health and disease and to provide a basic overview of the widespread use and interest in this compound. DATA IDENTIFICATION: Articles were obtained via a MEDLINE search of the term glutathione in conjunction with specific disease states mentioned, and via extensive review of references found in articles identified by computer search. STUDY SELECTION: Emphasis was placed on the most recent research, human research, and in discussing multiple disease states. DATA EXTRACTION: The literature was reviewed for methodology, quality, and practical aspects of interest to clinical pharmacists. DATA SYNTHESIS: GSH is a tripeptide of extreme importance as a catalyst, reductant, and reactant. It continues to be investigated in diverse areas such as acute respiratory distress syndrome, toxicology, AIDS, aging, oncology, and liver disease. Despite the widespread clinical interest in GSH, we were not able to identify an in-depth review of this compound in the pharmacy literature. CONCLUSIONS: The list of potential indications for modulation of GSH is extensive and broad. This review

introduces clinicians to what GSH is, its basic chemistry, and some areas of active research. IN OUR INSTITUTION, we have had recent requests for information on manipulation of glutathione (GSH) concen-trations in patients, and have observed the recent and growing body of literature on this topic. A MEDLINE search on GSH, from 1991 to May 1995, identified more than 8000 published works; however, we were unable to find any review articles in the pharmacy literature. Our purpose was to review the recent literature concerning GSH to introduce clinicians to its complex and diverse functions, its widespread role in maintaining health, and its use in the pharmacotherapy of a variety of diseases. GSH is a tripeptide of L-glutamate, L-cysteine, and glycine. It is considered to be the most prevalent and most important intracellular nonprotein thiol/sulfhydryl compound in mammalian cells, and the most abundant low-molecular-weight peptide.1-4 It is a cellular reductant, catalyst, and reactant involved in many biologic processes of transport, metabolism, storage, and protection.5 Some of these metabolic functions are presented in Table 1. The mechanisms of GSH activity are both direct and indirect, the latter by maintaining other cellular antioxidants in a functional state.3,6,7 GSH may be especially important for organs that are exposed to exogenous toxins, such as the liver, kidney, lung, and intestines.8 Reduced GSH occurs in millimolar concentrations intracellularly in humans, but in only trace amounts in plasma and most other body fluids. One exception is fluids lining the lower part of the respiratory tract, where it may help to scavenge inhaled toxins and free radicals produced by activated lung phagocytes.9 GSH can be depleted intracellularly either by forming a direct complex with an electrophilic agent (accomplished investigationally by agents such as bromobenzene or diethyl maleate) via inhibition of synthesis, or by subjecting cells to oxidant stress.3 Increasing GSH supply into cells to enhance protection and minimize injury has been proposed in: (1) oxidative injury to the lung from oxygen therapy, cigarette smoke, and/or atmospheric pollutants; (2) oxidative injury to the skin from ultraviolet radiation; (3) injury to the heart and lung from antitumor therapy; and (4) injury to the kidney and small intestine from reperfusion following ischemic events.10 Interventions to increase tissue GSH concentrations have used GSH itself, monoesters that are more completely absorbed, or alternative precursors. Table 1. Metabolic Functions of Glutathione DNA synthesis and repair Protein synthesis Prostaglandin synthesis Amino acid transport Metabolism of toxins and carcinogens Enhancement of immune system function

Prevention of oxidative cell damage Enzyme activation Depletion of intracellular GSH appears to be critical for subsequent alterations in protein thiol and calcium homeostasis.11 GSH depletion and the subsequent low stores of protein thiol result in both calcium release from intracellular stores and inhibition of calcium extrusion, producing a marked increase in cytosolic calcium concentration, which triggers cytotoxicity.11 Glutathione Synthesis GSH is synthesized from L-glutamate, L-cysteine, and glycine in 2 adenosine triphosphate (ATP)-dependent reactions (Figure 1). The first reaction, catalyzed by gamma-glutamylcysteine synthetase, is effectively rate-limited by GSH biofeedback.1,3,6 The second step involves GSH synthetase, which is not subject to negative feedback by GSH. When GSH is consumed and feedback inhibition is lost, availability of cysteine as a precursor can become the rate-limiting factor.8 Oxidized glutathione (GSSG) is formed in antioxidant reactions that involve GSH, and can accumulate with increased oxidative processing in cells. The ratio of GSSG/ GSH serves as a sensitive index of oxidative stress.12 Because its oxidative functions require GSH to be in its reduced form, GSSG is reduced to regenerate GSH in a reaction catalyzed by GSH reductase that requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a hydrogen donor (Figure 2).3,12 Absorption and Distribution of Glutathione Little is known about the average daily intake of GSH, the amounts of GSH in various food sources, or the importance of dietary GSH in health or disease. The estimated daily intake in humans is about 150 mg of GSH per day.13 Investigations in humans have used 15 mg/kg as an oral bolus to increase the plasma GSH concentration two- to fivefold. The transit of orally administered GSH to tissues is thought to occur via absorption from the intestinal lumen, export from enterocytes into the blood, and uptake from the plasma into cells.10,14 Gastrointestinal transport of GSH appears to be via nonenergy-requiring, sodium-independent, carrier-mediated diffusion.15 The plasma concentration of GSH is low because of its rapid turnover, and more than 80% of plasma GSH is removed by the kidney.16 The serum half-life of GSH after intravenous administration is less than 2 minutes; however, the half-life in epithelial lining fluid is much longer, suggesting independence of respiratory epithelial lining fluid and plasma with regard to GSH metabolism.17 Various epithelial cells, such as enterocytes, alveolar cells, renal proximal tubular cells, endothelial cells, and retinal pigmented epithelial cells, are capable of exogenous GSH uptake, which supports the function of GSH-dependent detoxification

systems.10,13 This allows GSH concentrations to be maintained better than by synthesis alone. Increasing plasma GSH concentrations by oral administration has been shown to increase the availability of GSH for transport into these tissues.13 This provides the basis for augmenting GSH concentrations against a wide variety of pathophysiologic states, including hepatic dysfunction or cirrhosis, or conditions affecting the epithelial cells, which can use exogenous GSH for protection. Most cells, in contrast to epithelial cells, do not have a direct transport capacity for intact GSH.3 Substrates for GSH synthesis are provided either by transport of amino acids into the cells or by transpeptidase activity at the cell surface, which is responsible for salvaging amino acids from circulating GSH for reuse in the intracellular resynthesis of GSH. The cellular concentration of GSH, therefore, is regulated by a complex process of precursor amino acid transport across cell membranes, intracellular synthesizing enzymes, feedback regulation, and intracellular GSH complexing via conjugation of GSH with a variety of electrophilic compounds through GSH transferase reactions. Although GSH is synthesized from precursors in virtually all cells, the liver is the main source of plasma GSH.18 In animal models hepatic concentrations of GSH change diurnally, with the highest concentration at 1000 hours and the lowest at 1800 hours.18,19 The plasma concentration of GSH is a function of hepatic synthesis, oxidation-reduction reactions, extrahepatic uptake and degradation, and GSH absorption.l3 In the liver, distribution of GSH is heterogeneous, which may play a significant role in susceptibility to acute hepatotoxicity from various toxic compounds such as acetaminophen metabolites, redox cycling compounds, peroxides, and others. Most GSH clearance from plasma occurs in the kidneys and the lungs.1,8,20,21 In the kidneys, exposure to toxins and the requirement for detoxification by GSH are high. Steady-state intracellular GSH concentrations vary in different parts of the kidneys, which also may determine the localization of injury by toxins.8 Role of Cysteine Homeostasis in Glutathione Metabolism Cysteine provides the reactive thiol group, which is key to the function of GSH.8 It is required for hepatic GSH synthesis and may be derived from methionine, which serves as a major source of cysteine, via the trans-sulfuration pathway of the liver.18 Cysteine also inhibits hepatic GSH efflux. It cannot be transported in/from plasma or stored within the cell as cysteine, as it would rapidly autooxidize to cystine, producing potentially dangerous oxygen radicals.8 This toxic autooxidation is avoided by storing almost all nonprotein-bound cysteine as

GSH.1,8 Cysteine occurs in plasma in the following forms: the solitary amino acid with its free thiol group (free cysteine), a disulfide between 2 molecules of cysteine (cystine), and a mixed disulfide with cysteinyl residues of albumin or other plasma proteins (protein-bound cysteine).22 The kidneys use cystine as a source of cysteine, and they clear and break significant quantities of plasma GSH into component amino acids.8 Therefore, the kidneys are a major source of plasma cysteme that can be used by the liver for resynthesis of GSH. Major Functions of Glutathione The major functions of GSH can be explained by its role in detoxification, redox reactions, and the storage and transport of cysteine. Because a major physiologic function of GSH is to provide cells with a reducing environment and to destroy the reactive oxygen compounds and free radicals formed in metabolism, organs that have low concentrations of other antioxidants (such as catalase and superoxide dismutase) are thought to be more dependent on GSH for detoxification of reactive oxygen species than are organs that have alternative antioxidants.20 DETOXIFICATION Either by spontaneous conjugation or by reduction, GSH provides the bulk of available sulfhydryl groups for binding and detoxification of reactive endogenous and exogenous compounds such as peroxides and electrophiles.2,23,24 GSH peroxidase is an enzyme present in tissues,25 which converts peroxides, using GSH as a substrate, into less harmful fatty acids, water, and GSH disulfide.26 These reductive reactions generate the oxidized form of GSH, GSH disulfide (GSSG).24 Under normal circumstances, to preserve high inuacellular concentrations of free GSH, GSSG is reduced rapidly back to GSH by the NADPH-dependent GSSG reductase. In this way a high GSH/GSSG ratio is maintained within the cells.24 However, GSSG is actively excreted from the cell when its intracellular concentration exceeds the reductive capacity of the cell, as in conditions of oxidative stress. The GSH/GSSG ratio is hypothesized to affect the redox balance of protein thiols, and to regulate the activity of certain enzymes by disulfide exchange reactions. The capacity for GSH synthesis is insufficient to maintain GSH concentrations when tissues are exposed to certain drugs or their metabolites (e.g., acetaminophen), redox cycling compounds (e.g., menadione), peroxides (e.g., tertbutyl hydroperoxide), X-rays, or ultraviolet radiation.13 Its depletion has been associated with enhanced toxicity of many compounds that cause increased morbidity or death.2 For example, hepatotoxicity of acetaminophen is caused

by the production of a highly reactive intermediate oxygen metabolite (N-acetyl-p-benzoquinoneimine). This metabolite covalently bonds to tissue macromolecules, resulting in tissue injury and cell death (Figure 3).25,27,28 Ito et al.25 have shown that inhibition of the GSH redox cycle enhances acetaminophen cytotoxicity in cultured rat hepatocytes. Excessive amounts of acetaminophen can overwhelm the capacity of GSH for conjugation and lead to toxicity.27 Also, fasting (by shunting acetaminophen metabolism away from glucuronidation or by depletion of intracellular GSH) and chronic ethanol use (via intracellular GSH depletion) can increase susceptibility to acetaminophen hepatotoxicity.28 GSH also may activate parent compounds by their metabolic conversion to active intermediates. For example, the metabolism of nitroglycerin requires interaction with thiols such as cysteine and GSH for transformation to vasoactive metabolites such as nitrosothiols or nitric oxide.29 In addition to activation of nitroglycerin via interaction with thiol compounds such as cysteine and GSH, dilation of small vessels only responsive to nitroglycerin in the presence of exogenous cysteine has been proposed. Finally, N-acetyl-cysteine (NAC) has been shown to partially prevent tolerance development to nitrate-induced antianginal effects. GLUTATHIONE AS AN ANTIOXIDANT Oxygen radical stress occurs in all aerobic organisms as a result of aerobic metabolism, with intermediates such as superoxide and hydrogen peroxide that lead to further production of oxygen radicals, which can cause lipid peroxidation and disrupt metabolic processes.8 Antioxidant defenses are not completely efficient, and increased free-radical formation (oxidative stress) is likely to increase the damage.9 The GSH redox cycle (the balance between GSH and GSSG) has been shown to be more effective than catalase in hydrogen peroxide detoxification in endothelial cell cultures.12 GSH serves as a substrate for 2 antioxidant enzymes: GSH peroxidase and phospholipid hydroperoxide GSH peroxi-dase.12 Glutathione-S-transferase is also a glutathione- dependent enzyme, mainly involved in xenobiotic and lipid peroxide detoxification.12 Although GSH exerts antioxidant properties through antioxidant enzymes, it also provides protection against oxidative stress by nonenzymatic free radical scavenging.12 Reduced GSH is capable of directly scavenging radicals and peroxides by being oxidized to either GSSG or to a mixed disulfide, thereby preventing cell membrane lipid peroxidation and its subsequent deleterious effects on cellular functions.4 Various oxygen radical stresses have been shown to result in GSSG formation and short-term depletion of GSH.3 It has been postulated that the oxidation-reduction status of GSH may act as a third

messenger in either enhancing or diminishing the activities of a number of biologic processes, such as enzyme catalysis, protein synthesis, and receptor binding.30 Relationship of S-adenosyl-L-Methionine and Glutathione The metabolism of GSH and S-adenosyl-L-methionine (SAM) are closely linked, as the liver forms GSH as a product of SAM metabolism (Figure 4). The liver uses as much as 70% of dietary methionine, and most is converted to SAM.31,32 SAM is an important metabolic substrate and is involved in the initiation of 3 major pathways: (1) transmethylation for the synthesis of various proteins and lipids, among them phospholipids for cell membranes; (2) trans-sulfuration to form GSH and sulfated compounds via homocysteine and cysteine; and (3) aminopropylation for polyamine synthesis.33 Glutathione in Disease LIVER DISEASE Decreased SAM use by the liver can result in reduced GSH availability and potential hepatotoxicity.34 Patients with cirrhosis of the liver have hypermethioninemia and a block of the trans-sulfuration pathway leading to delayed methionine clearance and decreased concentration of methionine endproducts (including GSH) following a methionine load.32,33,35 The lack of accumulation of intermediary metabolites suggests that the defect is located in the initial step of methionine transformation regulated by SAM synthe-tase.33,34 Because GSH is vital in detoxification and cell physiology, its depletion has been speculated to represent an important contributing factor of liver injury, and enhanced morbidity in patients with liver injury.2 Chawla et al.22 measured the concentrations of cysteine, GSH, and taurine in 14 healthy subjects and 10 patients with cirrhosis fed either mixed food, nasogastric hyperalimentation with Vivonex (Sandoz Nutrition, Minneapolis, MN), or FreAmine III (McGraw, Irvine, CA) intravenous hyper-alimentation. Vivonex and FreAmine III do not contain cysteine. Subnormal plasma concentrations of GSH and cysteine were observed in patients with cirrhosis independent of their diet. The data support the hypothesis of an acquired dysfunction in the hepatic trans-sulfuration pathway, rather than a change in bioavailability. Because most plasma GSH originates in hepatocytes, the authors hypothesized that decreased plasma GSH also could signify intracellular depletion. This would potentially impair the ability of the hepatocyte to maintain normal redox potential, destroy peroxides and free radicals, and detoxify drugs.22 Loguercio et al.16 evaluated the plasma and erythrocyte GSH and cysteine concentrations of patients with liver cirrhosis with respect to

alcoholic or nonalcoholic etiology, severity of liver disease, and nutritional status. The data demonstrated a four- to eightfold decrease in plasma GSH content in 48 cirrhotic patients versus a control group of 18 healthy volunteers. This decrease was irrespective of cirrhosis etiology and thought to reflect diminished hepatic GSH synthesis. A significant decrease in cysteine in severe cases of cirrhosis also was observed. It is known that plasma concentrations of cysteine are affected by diet composition and fasting. However, cysteine also is provided by methionine through the trans-sulfuration pathway, which if impaired could contribute to the depression of plasma cysteine concentration. This investigation found no correlation between plasma cysteine and nutritional status, pointing to impairment of trans-sulfuration as the major cause. Altomare et al.2 measured reduced and oxidized hepatic GSH concentrations during alcoholic and nonalcoholic liver injury in 35 chronic alcoholics, 20 nonalcoholic patients with liver disease (chronic active hepatitis, chronic persisting hepatitis, steatosis, cirrhosis), and 15 control patients (admitted for uncomplicated abdominal procedures). Decreased GSH concentrations were noted in patients with alcoholic (2.55 0.1 mol/g of liver) and nonalcoholic liver diseases (2.77 0.1 mol/g of liver) compared with controls (4.14 0.1 mol/g of liver) that were speculated to be a contributing factor in liver injury and could enhance the risk of hepatic toxicity from a variety of toxic agents. The GSSG concentration was also significantly higher in alcoholics (8.2 0.3% of total) and nonalcoholics (8.5 0.8% of total) than in control subjects (4.4 0.2% of total), reflecting an abnormal balance between GSH and GSSG in these patients. The investigators concluded that decreased hepatic GSH concentrations in patients with liver disease may represent a contributing factor of liver injury susceptibility and toxicity risk in these patients. Furthermore, excess GSSG is postulated to result in alteration of a variety of cell functions, including enzyme function, protein synthesis, cell integrity, microtubular function, transport processes, and release mechanisms.2 Data suggest that plasma and hepatic GSH also are decreased in patients with acute viral hepatitis, or chronic liver injury from a variety of causes such as chronic hepatitis, nonalcoholic liver cirrhosis, and alcoholic liver disease.36,37 The etiology again is thought to include altered SAM metabolism in patients with chronic liver disease regardless of the stage or cause of the disorder.23 Seifert et al.38 found a correlation between liver disease in chronic alcoholics and a subsequent GSH depletion state. Chronic alcoholic patients between the ages of 21 and 65 years were investigated for

predisposition to acetaminophen hepatotoxicity caused by increased activity of the cytochrome P450 system and decreased hepatic GSH concentrations. Venous blood was analyzed for liver profile, prothrombin time, GSH, and GSSG. Acetaminophen use and diet appeared to have no effect on plasma GSH concentrations. Total GSH in patients with normal gamma-glutamyl transferase (GGT) did not differ significantly from that in healthy volunteers. Acetaminophen toxicity was not observed. However, patients with an elevated GGT (102 IU/L) had significantly lower plasma total GSH concentrations (2.38 2.42 mol/L vs. 5.81 4.11 mol/L, p < 0.0001). These data suggest that in chronic alcoholics with liver disease, GSH deficiency exists, which may predispose to further liver toxicity caused by resultant inadequate defense mechanisms. ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

ROLE OF GLUTATHIONE IN THE IMMUNE SYSTEM: AIDS DATA Many of the pathologic aspects of disease in patients with AIDS are not caused directly by the HIV infection, but are secondary effects caused by the host response to infection.39 One of the more important aspects of this disease is the chronic inflammatory and oxidative stresses that accompany the infection, which eventually contribute to the loss of CD4 (helper) T-cells, increased opportunistic infections, immuno-deficiency in general, wasting disease, and death. Further-more, HIV infection is characterized by a systemic GSH deficiency, which has been postulated to increase viral replication and to increase production of oxidants from inflammatory cells possibly contributing to immune system dysfunction.17 The consequences of GSH depletion in AIDS and the role of thiol replacement therapy have been described.39-44 The progression of HIV infection from its early asymptomatic stage to active late-stage AIDS is thought to begin with the production of inflammatory cytokines that stimulate the production of the latent virus.42 Adjuvant therapy with GSH replacement in patients infected with HIV could offer several potential benefits. For example, stimulation of viral replication by inflammatory cytokines is optimal at decreased glutathione concentrations.39,40 GSH and NAC effectively block the cytokine-induced production of virus and may extend the latency period of early infection.39,40 Restoration of depleted GSH concentrations in T-cells may be critical for restoration of leukocyte function.39 Intracellular GSH has been shown to play an important

role in aspects of T-cell function, including the binding, internalization, and degradation of interleukin-2, as well as DNA synthesis.4 In particular, GSH and sulfhydryl compounds are known to augment the activation of cytotoxic T-cells in mixed lymphocyte cultures, T-cell proliferation in response to mitogens, and the differentiation of T and B lymphocytes.45 In vivo administration of GSH has been demonstrated to enhance the activation of cytotoxic T-cells, but depletion of GSH intracellularly inhibited the activation of lymphocytes, increased susceptibilities of human lymphoid cells to radiation, and suppressed cell-mediated cytotoxic functions, suggesting that intracellular GSH can modulate the function of immune cells. The current hypothesis is that because adequate concentrations of GSH are required for proper lymphocyte function, a deficiency in GSH may contribute to the immunodeficiency seen in the later stages of HIV infection.41 In addition, the action of inflammatory cytokines may mediate cachexia and the wasting that accompanies late stages of AIDS, which also may be alleviated by GSH replacement.40,42 Holroyd et al.17 investigated the effect of administering reduced GSH 600 mg via aerosolization twice daily to 14 HIV-seropositive individuals. GSH concentrations in the lung epithelial lining fluid were compared before and at 1, 2, and 3 hours after administration. Total GSH concentrations increased and remained in the normal range for the 3 hour post-treatment studied. A striking increase in oxidized GSH (GSSG increased from 5% at baseline to about 40% 3 h after treatment) was noted, possibly reflecting the potential value of GSH as an antioxidant providing protection in the lung tissue. NAC is a cysteine prodrug capable of maintaining intra-cellular thiol concentrations and replenishing GSH in deficiency states. It blocks HIV expression in acute and chronic infection models, and HIV replication in normal peripheral blood mononuclear cells.40 The administration of NAC has been mentioned as a "new approach to anti-HIV therapy," which differs from existing antiviral drugs in that it inhibits host-mediated stimulation of viral replication arising in normal immune responses, and may hence extend latency. In vitro, NAC has been shown to block cytokine-stimulated HIV replication in an acutely infected T-cell line and in acutely infected peripheral blood mononuclear cells from healthy individuals.42 PULMONARY DISEASE GSH deficiency has been proposed to have a role in the pathophysiology of a number of lung diseases, including chronic obstructive pulmonary disease,46 acute respiratory distress syndrome (ARDS),47-49 neonatal lung damage,50 and asthma.51 The lung is particularly at risk from oxidative damage as it is

exposed to oxygen, oxygen radicals produced by alveolar macrophages, inhaled environmental and blood-borne toxins, including cigarette smoke and atmo-spheric pollutants.8,10 Free radical induced toxicity is worsened by concomitant GSH deficiency, and free radical production further depletes GSH through use.52 GSH present in the epithelial lining fluid of the lower respiratory tract may be the first line of defense against oxidant stress.8 In idiopathic pulmonary fibrosis, for example, GSH concentrations are only 25% of normal values in the epithelial lining fluid and may be involved in the underlying pathophysiology of the disease.8 Neonates. Pulmonary GSH concentrations have been found to be lower in premature infants with a lower gestational age (25 vs. 39.8 wk average gestational age).53 Theories to explain why pulmonary GSH could be depleted in some infants include decreased cord cysteine concentrations, seen in earlier gestational age, that may impair GSH synthesis as intracellular GSH depends on the availability of cysteine. This hypothesis was investigated by Grigg et al.50 The concentration of GSH in bronchoalveolar lavage fluid was measured on the first day of life in intubated infants born at less than 35 weeks gestation irrespective of initial respiratory status. Lower concentrations of GSH were observed in 7 infants who subsequently developed chronic lung disease when compared with 27 infants who did not require oxygen supplementation at 36 weeks postconceptional age. This investigation provided preliminary evidence that a low lung epithelial lining fluid concentration of GSH on the first day of life is associated with an increased risk of chronic lung disease. However, a method of increasing GSH concentrations in this population was not discussed. Asthma. Airway inflammation in asthma also is associated with the increased generation of reactive oxygen species and pathophysiologic changes.51 In a comparison of 10 adults with mild asthma versus 17 healthy volunteers, concentrations of GSH in bronchoalveolar lavage fluid were increased in the subjects with mild asthma. The mean bronchial GSH concentrations were 13.0 nM/mg of protein in healthy volunteers versus 23.9 nM/mg of protein in mild asthmatics. The mean alveolar GSH concentration was 23.3 nM/mg of protein in volunteers versus 36.5 nM/mg of protein in mild asthmatics. The authors hypothesized that an increasing GSH concentration represents an adaptive mechanism to increase antioxidant defenses, which results in fewer symptoms, reduced airway reactivity, and milder disease in patients with asthma. Acute Respiratory Distress Syndrome. GSH is detected in high concentrations in the extracellular epithelial lining fluid of the lower respiratory tract of healthy

subjects, and could act as a first-time scavenger of toxic oxygen intermediates and protect against lung cell damage and injury.l7 Patients with ARDS and sepsis have a GSH deficiency in the lower respiratory tract extracellular epithelial lining fluid, which could favor oxidative stress and subsequent damage.47,48 Also, greater percentage of total GSH exists in the oxidized form in patients with ARDS than in healthy subjects, possibly indicating increased oxidant stress in the lower respiratory tract of ARDS patients.49 The mechanism for GSH deficiency is thought not to be dilutional (as occurs in cardiogenic pulmonary edema), but representing depletion of tissue stores. GSH depletion in plasma and granulocytes in ARDS is reversible by NAC therapy. Suter et al.47 improved systemic oxygenation and reduced the need for ventilatory support in 32 patients with acute lung injury caused by various underlying diseases, with NAC 40 mg/kg/d administered as a continuous infusion over the first 3 days after admission to the intensive care unit. Patients in the NAC group also had a shorter median stay in the intensive care unit than did those in the placebo group (median 7 vs. 10 d, respectively). ONCOLOGY By acting as an antioxidant and by virtue of binding to cellular mutagens, GSH has the ability to react with peroxides and several electrophiles, including carcinogenic epoxide metabolites.7,54 GSH has been shown to directly modulate proliferation of highly purified T-cells, suggesting that GSH is essential for steps closely involved with DNA synthesis. Depressed intracellular GSH in the liver and in mammary tissue has been shown to promote carcinogen binding to DNA.54 Oral glutamine, for example, has been proposed to have a useful role in increasing host GSH concentrations in the gut, liver, lung, kidneys, heart, and muscle after exposure to radiation or chemotherapy without enhancing tumor growth.55 Flagg et al.7 investigated the association between dietary GSH intake and the risk of oral and pharyngeal cancer in an epidemiologic study of 1830 participants. In this case-control study, the investigators noted an inverse relationship between dietary intake of GSH and the risk of oral cancer, but only in a cohort consuming GSH mostly from raw fruit and vegetables (rather than meat or cooked vegetables, for example). However, the possibility that GSH intake from fruit and vegetables might be protective against oral cancer risk could not be distinguished from the more general benefit of consuming raw fruits and vegetables, such as increased ingestion of fiber. The investigators hypothesized anticarcinogenic protective mechanisms of GSH to include its direct antioxidant function, indirect maintenance of other antioxidants, possible

mediation of DNA synthesis and repair, and the ability to bind with cellular mutagens.3 Resistance to chemotherapy caused by detoxification by GSH has been postulated,56 suggesting that there may be possible benefits of selectively decreasing intracellular GSH in tumors to enhance the therapeutic effect of chemotherapy. Direct bonding to the sulfhydryl group of GSH in the cytoplasm has been shown to inactivate cisplatin, for example. Further, GSH depletion can inhibit DNA repair in bladder carcinoma cell lines after cisplatin or doxorubicin damage. Limited data suggest that GSH and its related enzymes enhance drug resistance, varying with the characteristics of the tumor and the chemotherapy chosen. The use of buthionine sulfoximine, which inhibits gamma-glutamylcysteine synthetase, has been investigated in animals as a method to decrease GSH.3 With elucidation of its mechanism, incidence, and impact on resistance to chemotherapy, interventions to decrease target concentrations of GSH may become part of future oncologic therapeutic regimens. PREECLAMPSIA Walsh and Wang26 determined that GSH peroxidase activity is significantly lower, and lipid peroxides and thromboxane significantly higher, in preeclamptic placentas than in healthy placentas because of an unknown mechanism. They postulated that peroxides stimulate prostaglandin H (PGH) synthase, leading to the generation of oxygen radicals and increased formation of both thromboxane and lipid peroxides. They proposed that PGH synthase converts arachidonic acid into prostaglandin G2 and prostaglandin H2. Thromboxane synthase then converts prostaglandin H2 into thromboxane A2. When PGH synthase is activated, the peroxidase function of the enzyme generates oxygen radicals that could interact with polyunsaturated fatty acids in the placenta to form lipid peroxides. GSH peroxidase inactivates peroxides, using GSH as its cofactor to convert lipid peroxides to less harmful hydroxylated fatty acids, water, and GSH disulfide. If GSH peroxidase activity is deficient, lipid peroxidation could increase in the tissues, leading to increased stimulation of PGH synthase, increased thromboxane production, and further increase lipid peroxides. Deficiency of this enzyme would likely increase morbidity in preeclampsia. PARKINSON'S DISEASE The brains of patients with Parkinson's disease exhibited a reduction of GSH, selective to the substantia nigra (SN). This did not appear to be related to drug therapy. It was postulated to be of significance in the pathogenesis of this disease via production of oxidative damage.57,58 These patients had an increased concentration of the GSH degradative enzyme gamma-glutamyltranspeptidase in the SN, and a normal concentration of the synthetic enzyme

gamma-glutamyl-cysteine synthetase.57 GSH depletion occurred without a change in GSSG, suggesting efflux of GSH out of the glia, perhaps with additional increased conversion of GSH to GSSG in response to increased hydrogen peroxide formation. At this time it is unclear whether free radical involvement in Parkinson's disease is a primary or secondary event in nigral cell death or whether it occurs early or late in the disease process.58 SEPSIS Henderson and Hayes59 reviewed the existing data on NAC as an antioxidant in patients with severe sepsis. They noted that GSH is depleted in severe sepsis, possibly in the defense against active radicals generated by inflammatory cells during systemic inflammatory response syndrome. These inves-tigators are experimenting with a regimen of NAC 150 mg/kg in dextrose 5% infused intravenously over 30 minutes followed by 15 mg/kg/h for 4 days as an antioxidant active radical scavenger. Their hypothesis is that NAC may improve renal function, reduce fluid requirements, and lower tissue edema. Spies et al.60 reported a 2-year investigation of 58 patients with septic shock randomized to receive NAC 150 mg/kg iv over 15 minutes followed by 12.5 mg/h over 90 minutes. Using a 10% or more increase in whole body oxygen con-sumption as their endpoint, 13 patients who received NAC were considered responders, and 16 were considered nonresponders. None of the patients who received placebo (n = 29) exhibited a 10% increase in whole body oxygen consumption. In summary, NAC transiently improved tissue oxygenation in about half of the patients with septic shock to whom it was administered. MYOCARDIAL ISCHEMIA AND REPERFUSION INJURY During myocardial ischemia and reperfusion, both myocardial GSH and the GSH/GSSG ratio within the ischemic tissues are reduced, and the extent of the myocardial injury is inversely dependent on the myocardial GSH content.61-63 Treatment with gamma-glutamylcysteine ethyl ester can increase intracellular reduced GSH concentrations, and has been shown to result in a dose-dependent reduction in infarct size in a canine model of occlusion-reperfusion.61 However, an investigation of coronary artery occlusion in 23 mongrel dogs administered NAC 30 minutes before and continued until 3 hours after reperfusion failed to observe an increase in total GSH or GSH peroxidase activity at the biopsied ischemic zone, or a decrease in myocyte death.64 RENAL DYSFUNCTION AND NEPHROTOXICITY We have alluded to the potential for decreases in GSH concentration to impair GSH conjugation and modify the metabolic fate of many compounds. GSH deficiency may contribute to the nephrotoxicity of ischemic events

and drug toxicity. This type of toxicity may be exhibited by cyclosporine. Although the exact mechanism of cyclosporine nephrotoxicity remains unknown, its administration has been associated with in vivo reduction of GSH concentrations in the livers and kidneys of rats, which may be related to adverse effects of this immunosuppressive agent.65,66 Cyclosporine has peroxidative properties, induces lipid peroxidation in renal microsomes, and may lead to inactivation of microsomal glucose-6-phosphate activity and toxicity.66,67 Therefore, contribution to cyclosporine nephro- and hepatotoxicity has been postulated to be caused by its generation of free radicals and depletion of GSH. Investigators also have hypothesized that cyclosporine can modify resistance to chemotherapy by augmenting the cytotoxic effect of drugs through inducing a GSH deficiency.65 The ability of GSH administration to prevent nephrotoxicity from renal ischemia, and consequent production of oxygen free radicals, has been investigated. A total of 200 mg/kg of GSH (available in Japan as Tathion, Yamanouchi Pharmaceutical, Tokyo, Japan) was administered intra-venously to 10 patients before cardiopulmonary bypass surgery, and the influence on postoperative renal dysfunction was compared with 9 other patients undergoing the same procedure without GSH administration.16 Administration of GSH resulted in a significantly higher urine volume (approximately 20%) on the first and second postoperative days, with a trend to lower blood urea nitrogen and plasma creatinine concentrations in the GSH group that did not reach statistical significance. Also, the mean arterial pressure and systemic vascular resistance index were lower than those in the control group. The investigators concluded that administration of exogenous GSH had a beneficial effect on renal function by virtue of its antioxidant properties, and possibly by a vasodilator action to increase the glomerular filtration rate as well. Role of Glutathione in Aging Although higher GSH concentrations have been associated with good health, the significance of low GSH status in the elderly is inferred from limited data.68,69 Investigators have noted lower GSH concentrations to be associated with the combination of advanced age and increased risk of chronic diseases such as chronic renal failure, malignant disorders, diabetes, alcoholism, Parkinson's disease, and cataract formation. Julius et al.68 measured GSH concentrations in 33 people between 60 and 79 years of age, and related the data to health, the number of illnesses, and other risk factors for chronic disease. There appeared to be a direct relationship between higher GSH concentrations and increasing age with good health. The association of GSH with good health was positive

and independent of age (i.e., volunteers with higher GSH concentrations were healthier than age-matched volunteers with lower GSH concentrations). People with chronic diseases had lower mean GSH concentrations than those who were free of disease. Lang et al.69 compared GSH blood concentrations in healthy young and healthy elderly subjects and found that the reference group of 20- to 39-year-old subjects (n = 40) had a GSH concentration 17% higher on average than the 60- to 79-year-old cohort of 60 subjects (mean SD, 547 53.5 g/1010 vs. 452 86.8 g/1010 erythrocytes, respectively). Caution has been advised in interpreting these small studies to define the association of GSH with aging.70 Further large-scale studies are needed to prove that low relative GSH concentration is an overall risk factor for morbidity among the elderly. Pharmacotherapeutic Interventions to Increase Glutathione Concentrations GLUTATHIONE AND RELATED AGENTS GSH is present to the greatest extent in fruits, vegetables, and meats.71 Agents such as 1-cyano-2-hydroxy-3-butene, present in cabbage, brussels sprouts, broccoli, and cauliflower, have raised GSH concentrations severalfold in animal models.72 The results of studies to date of GSH absorption have been conflicting. Several investigators have shown that orally administered GSH increases plasma concentrations of reduced and protein-bound GSH through intestinal absorption in animal models10,13,15 and humans.73 GSH is not commercially available as an oral or injectable product in the US because of pharmaceutical problems, including poor oral bioavailability and a short halflife (2 min) with intravenous administration. Investigators have used bulk quantities of GSH purchased from various chemical companies. For these reasons, precursors of GSH have been investigated. In mice pretreated with buthionine sulfoxime to inhibit GSH synthesis and induce a deficient state, oral administration of GSH resulted in statistically significant increases in GSH concentrations in kidney, heart, lung, brain, small intestine, and skin, but not in the liver. Administration of the equivalent amount of the constituent amino acids to GSH-deficient mice resulted in little change in GSH concentration in all tissues studied. Mice not pretreated with buthionine sulfoximine, but administered GSH, experienced an increase in plasma GSH, but not tissue GSH.10 In humans, oral doses of GSH 15 mg/kg increased plasma GSH 1.5- to 10-fold over the basal concentration in 4 of 5 volunteers tested.73 The maximum concentration of plasma GSH generally occurred 1 hour after GSH

administration. Equivalent amounts of amino acid constituents of GSH failed to increase plasma GSH concentrations. These data suggest that oral GSH can replete GSH concentrations in several tissues following GSH depletion, such as after toxicologic or pathologic conditions that alter GSH homeostasis.10 It has been speculated that because oral administration of GSH leads to its inactivation by peptidases, it should not be possible to significantly increase plasma GSH concentrations with oral administration.74 Witschi et al.74 investigated the bioavailability of a single dose of oral GSH in 7 healthy volunteers who had fasted. Their data showed a nonsignificant increase in plasma GSH after doses as high as 3.0 g, suggesting negligible systemic availability of oral GSH in humans. Cook and Sherlock75 also were not able to demonstrate a benefit from oral GSH (100 mg tid for 28 d) in 10 patients with hepatic cirrhosis of various etiology. Analysis included measurement of biochemical parameters and an assessment of sense of well-being compared with 10 other patients with cirrhosis not administered GSH. In a separate arm of investigation, another 12 patients with cirrhosis received GSH 200 mg/d im without producing any difference in biochemical parameters. This latter GSH group did note an increased sense of well-being versus the control group from the oral GSH investigation. Evidence suggests that cells export GSH, but evidence that GSH is transported into cells to any appreciable extent is conflicting.5,10,73 Therefore, methyl, ethyl, and isopropyl esters of GSH in which the glycine carboxyl group is esterified have been used and are orally bioavailable via rapid intracellular deesterification with effective transport and hydrolysis intracellularly.5,61 As an example, orally administered GSH ethyl ester is able to significantly raise GSH concentrations in the liver, kidney, spleen, pancreas, heart, and lung of the mouse, and in human red blood cells, skin fibroblasts, and several lymphoid cell lines.5 Various esters and amides of GSH are transported into cells, but some have toxicity caused by cleavage products such as methanol, ammonia, and alcohols.5 None is commercially available in the US at this time. Gamma-glutamylcysteine ethyl ester was studied recently as a GSH precursor used for myocardial protection in dogs with ischemia and reperfusion damage.61 When administered as 3 or 10 mg/kg intravenously immediately before reperfusion in a canine coronary occlusion-reperfusion model of myocardial infarction, a significant dose-dependent reduction in infarct size was observed. Although administration of this ester appeared safe and had protective properties in this model, it also is not commercially available at this time.

CYSTEINE, CYSTINE, AND ACETYLCYSTEINE Cysteine usually is nonessential in the diet because it can be synthesized endogenously from methionine and phenylalanine.39,76 Impaired synthesis of cysteine from methionine may necessitate the provision of a source of cysteine to some patients with cirrhosis; however, supplementation with L-cysteine could lead to hypercysteinemia and potential toxicity.77 Administration of oral L-Cysteine to patients with cirrhosis has been noted to cause a twofold greater maximal plasma cysteine concentration and plasma elimination half-life, and a delayed excretion of metabolic end products when compared with those of controls. However, an impaired cysteine uptake from the plasma has been proposed secondary to a decrease in plasma GSH. Another example of precursor use involves L-2-oxothiazofidine-4-carboxylate, which is converted to S-carboxyl-L-cysteine and undergoes spontaneous decarboxy-lation to liberate L-Cysteine, thereby supporting GSH synthesis.8 In noncirrhotic, malnourished patients receiving parenteral nutrition, deficiencies in cysteine (and other amino acids) also can occur, possibly caused by the loss of the first-pass delivery of methionine to the liver and portal blood flow.31 As mentioned previously, cysteine is oxidized easily into cystine, and therefore, is not easily incorporated (and minimally present) in standard crystalline amino acid solutions used in intravenous hyperalimentation. This has resulted in hypo-cystinemia, with plasma cystine concentrations decreasing to 30% below baseline, as described in 12 patients with cirrhosis who received a FreeAmmine II source of amino acids in their intravenous hyperalimentation.76 NAC is available, as Mucomyst (Apothecon, Division of Bristol-Myers Squibb, Princeton, NJ) and generic products, and has been used for years as an antidote to acetaminophen toxicity. NAC may be a direct source of cysteine following hydrolysis or may reduce plasma cystine through thiol- disulfide exchange, liberating endogenous cysteine.8 Under normal conditions (no GSH deficiency), NAC does not increase total GSH, since the intracellular concentration is under feedback control. Recently, despite administration of NAC 600 mg po tid, a sustained increase in GSH concen-trations could not be found in the plasma, bronchoalveolar lavage fluid, or lung tissue of patients with chronic obstructive pulmonary disease.46 S-ADENOSYL- L-METHIONINE Hepatic GSH concentrations have been restored to nearly normal in liver biopsies of patients with cirrhosis following long-term oral SAM administration.23 Studies have documented improvement in pruritus, jaundice, and bio-chemical parameters in patients with intrahepatic cholestasis of pregnancy treated with SAM.33 The clinical efficacy of SAM in

the treatment of cholestasis associated with hepatic diseases has been reviewed.78 For the treatment of liver disorders, such as intrahepatic cholestasis, the recommended dose of SAM is 800 mg parenterally or 1600 mg/d orally. 23,78 Loguercio et al.78 administered SAM 2 g/d iv for a total of 15 days to 20 patients with biopsy-proven alcoholic cirrhosis. An increase in red blood cell GSH (to 2.20 1.10 mM/L from a baseline of 1.60 0.97 mM/L) and a decrease in cysteine content (to 65 14 M/L from 122 42 M/L) was demonstrated. The cysteine groups of SAM synthetase might be protected from oxidation by a normal concentration of GSH.80 When there is a reduction in liver GSH or increased concentrations of GSSG by toxin or disease, a vicious cycle might start. Depletion of GSH could lead to inactivation of SAM synthetase, with further decrease in GSH concentrations. worsening the deficiency in SAM synthetase. In this context, SAM administration may act as a precursor for GSH synthesis and also bypass the deficiency in SAM synthetase. SAM is not commercially available in the US. Summary The importance of GSH in health and disease is a subject of active research, presentation, and publication. GSH appears to be metabolically important in a wide variety of disease states, only a few of which have been discussed. At our institution, GSH has been the topic of grand rounds, and we have attempted to modify GSH stores in long-term parenteral nutrition patients with cirrhosis. We believe the significance and widespread use of GSH will only increase with time, and have attempted to introduce the topic to a broad range of clinicians. As pharmaceutical manipulation of GSH concen-trations is an intervention likely to increase within the coming years, health clinicians need to be aware of the rationale of such attempts and methods of administering bioavailable forms of GSH or its substrates. References 1. Meister A. Glutathione metabolism and its selective modification. J Biol Chem 1988;263:17205-8. 2. Altomare E, Vendemiale G, Alano O. Hepatic glutathione content in patients with alcoholic and non alcoholic liver diseases. Life Sci 1988;43: 991-8. 3. Deneke SM, Fanburg BL. Regulation of cellular glutathione. Lung Cell Mol Physiol 1989;1:LI63-LI73. 4. Wu D, Meydam SN, Sastre J, Hayek M, Meydani M. In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects. J Nutr 1994;124:655-63. 5. Anderson ME, Powrie F, Puri RN, Meister A. Glutathione monoethyl ester: preparation, uptake by tissues, and conversion to glutathione. Arch Biochem

Biophys 1985;239:538-48. 6. Meister A, Anderson ME. Glutathione. Ann Rev Biochem 1983;52:711 60. 7. Flagg EW, Coates RJ, Jones DP, Byers TE, Greenberg RS, Gridley G, et al. Dietary glutathione intake and the risk of oral and pharyngeal cancer. Am J Epidentiol 1994; 139:453-65. 8. DeLeve LD, Kaplowitz N. Glutathione metabolism and its role in hepatotoxicity. Pharmacol Ther 1991;52:287-305. 9. Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 1994;344:721-4. 10. Aw TY, Wierzbicka G, Jones DP. Oral glutathione increases tissue glutathione in vivo. Chem Biol Interact 1991;80:89-97. 11. Bellomo G, Offhenius S. Altered thiol and calcium homeostasis in oxidative hepatocellular injury. Hepatology 1985;5:876-82. 12. Toborek M, Hennig B. Fatty acid-mediated effects on the glutathione redox cycle in cultured endothelial cells. Am J Clin Nutr 1994;59:60-5. 13. Hagen TM, Wierzbicka GT, Sillau AH, Bowman BB, Jones DP. Bioavailability of dietary glutathione: effect on plasma concentration. Am J Physiol 1990;259:524-9. 14. Hagen TM, Jones DP. Role of glutathione transport in extrahepatic detoxification. In: Sakamoto Y, Higashi T, Taniguchi N, et al., eds. Glutathione centennial: molecular perspectives and clinical implications. New York: Academic Press, 1989:423-33. 15. Hunjan MK, Evered DF. Absorption of glutathione from the gastrointestinal tract. Biochim Biophys Acta 1985;815:184-8. 16. Amano J, Suzuki A, Sunamori M. Salutary effect of reduced glutathione on renal function in coronary artery bypass operation. J Am Coll Surg 1994; 179:714-20. 17. Holroyd KJ, Buhl R, Borok Z, Roum JH, Bokser AD, Grimes GJ, et al. Correction of glutathione deficiency in the lower respiratory tract of H1V seropositive individuals by glutathione aerosol treatment. Thorax 1993;48:985-9. 18. Kaplowitz N, Aw TY, Ookhtens M. The regulation of hepatic glutathione. Ann Rev Pharmacol Toxicol 1985~25:715-44. 19. Shimizu M, Morita S. Effects of feeding and fasting on hepatolobular distribution of glutathione and cadmium-induced hepatotoxicity. Toxicology 1992;75:97-107. 20. Martensson J, Meister A. Mitochondrial damage in muscle occurs after marked depletion of glutathione and is prevented by giving glutathione monoester. Proc Nad Acad Sci U S A 1989;86:471-5. 21. Griffith OW, Meister A. Glutathione: interorgan translocation, turnover, and metabolisrn. Proc Nad

Acad Sci U S A 1979;76:5606- 10. 22. Chawla RK, Lewis FW, Kutner MH, Bate DM, Roy RGB, Rudman D. Plasma cysteine, cystine, and glutathione. Gastroenterology 1984;87: 770-6. 23. Friedel HA, Goa KL, Benfield P. S-adenosyl-L-methionine: a review of its pharmacological properties and therapeutic potential in liver dysfunction and affective disorders in relation to its physiological role in cell metabolism. Drugs 1989;38:389-416. 24. Jochnnann C, Klee S, Ungemach FR, Younes M. The role of glutathione and protein thiols in CBrC13(-) induced cytotoxicity in isolated rat hepatocytes. Pharmacol Toxicol 1994;75:7-16. 25. Ito Y, Suzuki Y, Ogonuki H, Hiraishi H, Razandi M, Terano A, et al. Role of iron and glutathione redox cycle in apap-induced cytotoxicity to cultured rat hepatocytes. Dig Dis Sci 1994;39:1257-64. 26. Walsh SW, Wang Y. Deficient glutathione peroxidase activity in preeclampsia is associated with increased placental production of thromboxane and lipid peroxides. Am J Obstet Gynecol 1993; 169: 1456-61. 27. Sheiner P, De Majo W, Levy GA. Acetylcysteine and fulminant hepatic failure. Hepatology 1992;15:552-4. 28. Whitcomb DC, Block GD. Association of apap, hepatotoxicity with fasting and ethanol use. JAMA 1994;272:1845-50. 29. Boesgaarg S, Iversen HK, Wroblewski H, Poulsen HE, Frandsen H, Kastrup J, et al. Altered peripheral vasodilator profile of nitroglycerin during long-term infusion of N-acetylcysteine. J Am Coll Cardiol 1994;23:163-9. 30. Gilbert HF. Biological disulfides: the third messenger? J Biol Chem 1982;257:12086-9 1. 31. Corrales F, Cabrero C, Pajares MA, Ortiz P, Martin-Duce A, Mato JM. Inactivation and dissociation of S-adenosylmethionine synthetase by modification of sulfhydryl groups and its possible occurrence in cirrhosis. Hepatology 1990; 11:216-22. 32. Chawla RK, Bonkovsky HL, Galambos JT. Biochemistry and pharmacology of S-adenosyl-L-methionine and rationale for its use in liver disease. Drugs 1990;40(suppl 3):98-110. 33. Kaye GL, Blake JC, Burroughs AK. Metabolism of exogenous S-adenosyl-L-methionine in patients with liver disease. Drugs 1990;40 (suppl 3):124-8. 34. Marchesini G, Bugianesi E, Bianchi G, Fabbri A, Marchi E, Zoli M, et al. Effect of S-adenosyl-L-methionine administration on plasma levels of sulphur-containing amino acids in patients with liver cirrhosis. Clin Nutr 1992; 11:303-8. 35. Cabrero C, Martin-Duce A, Ortiz P, Alemany S, Mato JM. Specific loss of the high-molecular weight form of S-adenosyl-L-methionine synthetase in human liver cirrhosis. Hepatology 19W8:15304. 36. Loguercio C, Delvecchio Blanco C, Coltorti M,

Nardi G. Alteration of erythrocyte glutathione, cysteine and glutathione synthetase in alcoholic and non-alcoholic cirrhosis. Scand J Clin Lab Invest 1992;52: 207-13. 37. Shigesawa, T, Sato C, Marurno F. Significance of plasma glutathione determination in patients with alcoholic and non-alcoholic liver disease. J Gasumviterol Hepatol 1992;7:7-11. 38. Seifert CF, Anderson DC, Bui B, Glore SR, Vndracek TG, Raymond T. Correlation of acetarninophen and ethanol use, plasma glutathione concentrations and diet with hepatotoxicity. Pharmacotherapy 1994; 14: 376-7. 39. Roederer M, Staal FIT, Ela SW, Herzenberg LA, Herzenberg LA. N- acetylcysteine: potential for AIDS therapy. Pharmacology 1993;46: 121-9. 40. Roederer M, Ela SW, Staal FJT, Herzenberg LA, Herzenberg LA. N- acetylcysteine: a new approach to anti-HIV therapy. AIDS Res Hum Retroviruses 1992;8:209-17. 41. Staal FIT, Roederer M, Israelski DM, Bubp J, Mole LA, McShane D, et al. AIDS Res Hum Retroviruses 1992;8:305-11. 42. Roederer M, Raju PA, Staal FJT, Herzenberg LA, Herzenberg LA. N-acetylcysteine inhibits latent HIV expression in chronically infected cells. AIDS Res Hum Retroviruses 1991;7:563-7. 43. Roederer M, Staal FIT, Anderson M, Rabin R, Raju PA, Herzenberg LA, et al. Disregulation of leukocyte glutathione in AIDS. Ann N Y Acad Sci 1993;667:113-25. 44. Staal FIT, Roederer M, Raju PA, Anderson MT, Ela SW, Herzenberg LA, et al. Antioxidants inhibit stimulation of H1V transcription. AIDS Res Hum Retrovirus 1993;9:299-306. 45. Buhl R, Holroyd KJ, Mastrarigeli A, Cantin AM, Jaffe HA, Wells FB, et al. Systemic glutathione deficiency in symptom free HIV seropositive individuals. Lancet 1989;2:1294-8. 46. Bridgeman MME, Marsden M, Selby C, Morrison D, MacNee W. Effect of N-acetyl cysteine on the concentrations of thiols in plasma, bronchoalveolar lavage fluid and lung tissue. Thorax 1994;49:670-5. 47. Suter PM, Domenighetti G, Schaller MD, Uverriere MC, Ritz R, Perret C. N-acetylcysteine enhances recovery from acute lung injury in man. Chest 1994;105:190-4. 48. Pacht ER, Timerman AP, Lykens MG, Merola AJ. Deficiency of alveolar fluid glutathione in patients with sepsis and the adult respiratory distress syndrome. Chest 199 1; 100: 1397-403. 49. Bunnell E, Pacht ER. Oxidized glutathione is increased in alveolar fluid of patients with adult respiratory distress syndrome. Am Rev Respir Dis 1993;148:1174-8. 50. Grigg J, Barber A, Silverman M. Bronchoalveolar lavage fluid glutathione in intubated premature

infants. Arch Dis Child 1993;69:49-51. 51. Smith LJ, Houston M, Anderson J. Increased levels of glutathione in bronchoalveolar lavage from patients with asthma. Am Rev RespiR Dis 1993; 147:1461-4. 52. Johnson ME, Sill JC, Uhl CB, Van Dyke RA. Effect of halothane on hypoxic toxicity and glutathione status in cultured rat hepatocytes. Anesthesiology 1993-,79:1061-71. 53. Picone TA, Daniels TA, Ponto KH, Pitard WB. Cord blood tryptophan concentrations and total cysteine concentrations. JPEN J Parenter Enteral Nutr 1989; 13:106-7. 54. Liu JZ, Zhang BZ, Milner JA. Dietary selenite modifies glutathione metabolism and 7,12-dimethylbenz(a)anthracene conjugation in rats. J Nutr 1994;124:172-80. 55. Fahr MJ, Kombluth J, Blossom S, Schaeffer R, Klinberg VS. Glutamine enhances immunoregulation of tumor growth. JPEN J Parenter Enteral Nutr 1994;18:471-6. 56. Ahn H, Lee E, Kim K, Lee C. Effect of glutathione and its related enzymes on chemosensitivity of renal cell carcinoma and bladder carcinoma cell lines. J Urol 1993; 151:263-7. 57. Sian J, Dexter DT, Less AJ, Daniel S, Jenner P, Marsden CD. Glutathione-related enzymes in brain in Parkinson's disease. Ann Neurol 1994;36:356-61. 58. Jenner P. Oxidative damage in neurodegenerative disease. Lancet 1994;344:796-8. 59. Henderson A, Hayes P. Acetylcysteine as a cytoprotective antioxidant in patients with severe sepsis: potential new use for an old drug. Ann PharmacoLher 1994;28:1086-8. 60. Spies CD, Reinhart K, Witt 1, Meier-Hellmann A, Hanneman L, Bredle DL, et al. Influence of N-acetylcysteine on indirect indicators of tissue oxygenation in septic shock patients: results from a prospective, randomized, double-blind study. Crit Care Med 1994;22:1738-46. 61. Hoshida S, Kuzuya T, Yamashita N, Nishida M, Kitahara S, Hori M,.et al. Gamma-glutamylcysteine ethyl ester for myocardial protein in dogs during ischemic and reperfusion. J Am Coll Cardiol 1994;24: 1391-7. 62. Blaustein A, Deneke SM, Stolz RI, Baxter D, Healey N, Fanburg BL. Myocardial glutathione depletion impairs recovery after short periods of ischemia. Circulation 1989;80:1449-57. 63. Singh A, Lee KJ, Lee CY, Goldfarb RD, Tsan MF. Relation between myocardial glutathione content and extent of ischemia-reperfusion injury. Circulation 1989;80:1795-804. 64. Forman MB, Puett DW, Cates CU, McCroskey DE, Beckman JK, Greene HL, et al. Glutathione redox pathway and reperfusion injury. Circulation 1988;78:202-13.

65. Duruibe V, Okonmah A, Blyden GT. Effect of cyclosporin on rat liver and kidney glutathione content. Pharmacology 1989;39:205-12. 66. Inselmann G, Hannemann 1, Baumann K. Cyclosporine A induced lipid peroxidation and influence on glucose-6-phosphate in rat hepatic and renal microsomes. Res Commun Chem Pathol Pharmacol 1990;68: 189-203. 67. Wang C, Salahudeen AK. Cyclosporine nephrotoxicity: attenuation by an antioxidant-inhibitor of lipid peroxidation in vitro and in vivo. Transplantation 1994;58:940-6. 68. Julius M, Lang CA, Gleiberman L, Harburg E, DiFranceisco W, Schork A. Glutathione and morbidity in a community-based sample of elderly. J Clin Epiderniol 1994;47:1021-6. 69. Lang CA, Naryshkin S, Schneider DL, Mills B1, Lindeman RD. Low blood glutathione in healthy aging adults. J Lab Clin Med 1992; 120: 720- 5. 70. Fletcher RH, Fletcher SW. Glutathione and ageing: ideas and evidence (editorial). Lancet 1994;344:1379-80. 71. Jones DP, Coates RJ, Flagg EW, Eley JW, Block G, Greenberg RS, et al. Glutathione in foods listed in the National Cancer Institute's health habits and history food frequency questionnaire. Nutr Cancer 1992; 17: 57-75. 72. Davis MA, Wallig MA, Eaton D, Borroz 1, Jeffery EH. Differential effect of cyanohydroxybutene on glutathione synthesis in liver and pancreas of male rats. Toxicol Appl Pharmacol 1993; 123:257-64. 73. Jones DP, Hagen TM, Weber R, Wierzbicka GT, Bonkovsky HL. Oral administration of glutathione (GSH) increases plasma GSH concentrations in humans (abstract). FASEB J 1989;3:A 1250. 74. Witschi A, Reddy S, Stofer B, Lauterburg BH. The systemic availability of oral glutathione. Eur J Clin Pharmacol 1992;43:667-9. 75. Cook GC, Sherlock S. Results of a controlled clinical trial of glutathione in cases of hepatic cirrhosis. Gut 1965;6:472-6. 76. Rudman D, Kutner M, Ansley J, Jansen R. Chipponi J, Bain RP. Hypotyrosinemia, hypocystinernia, and failure to retain nitrogen during total parenteral nutrition in cirrhotic patients. Gastroenterology 198 1;8 1: 1025-35. 77. Tribble DL, Jones DP, Ardehali A, Feeley RM, Rudman D. Hypercysteinemia and delayed sulfur excretion in cirrhotics after oral cysteine loads. Am J Clin Nutr 1989;50:1401-6. 78. Almasio P, Bortolini M, Pagliaro L, Coltorti M. Role of S-adenosyl- methionine in the treatment of intrahepatic cholestasis. Drugs 1990;40 (suppI 3):111-23. 79. Loguercio C, Del Vecchio C, Coltorti M. Effects of intravenous S- adenosyl-L-methionine on red blood cell

content of glutathionine and cyteine in alcoholic cirrhosis (abstract). Gastroenterology 1993;104: A944. 80. Ortiz P, Moreno J, Puerta JL, Mato JM. S-adenosyl-L-methionine and the liver. Ital J Gastroenterol 1993;25:135-7.

MY UNIQUE COPY *************** What Glutathione (GSH) Is to You and Me. Have you ever wondered what exactly is up with Glutathione? This informative report can give you an insight into everything you've ever wanted to know about Glutathione. It's really a good idea to probe a little deeper into the subject of Glutathione. What you learn may give you the confidence you need to venture into new areas. GSH is a tripeptide of L-glutamate, L-cysteine, and glycine. It is considered to be the most prevalent and most important intracellular nonprotein thiol/sulfhydryl compound in mammalian cells, and the most abundant low-molecular-weight peptide.1-4 It is a cellular reductant, catalyst, and reactant involved in many biologic processes of transport, metabolism, storage, and protection.5 Some of these metabolic functions are presented in Table 1. The mechanisms of GSH activity are both direct and indirect, the latter by maintaining other cellular antioxidants in a functional state.3,6,7 GSH may be especially important for organs that are exposed to exogenous toxins, such as the liver, kidney, lung, and intestines.8 Reduced GSH occurs in millimolar concentrations intracellularly in humans, but in only trace amounts in plasma and most other body fluids. One exception is fluids lining the lower part of the respiratory tract, where it may help to scavenge inhaled toxins and free radicals produced by activated lung phagocytes.9 GSH can be depleted intracellularly either by forming a direct complex with an electrophilic agent (accomplished investigationally by agents such as bromobenzene or diethyl maleate) via inhibition of synthesis, or by subjecting cells to oxidant stress.3 Increasing GSH supply into cells to enhance protection and minimize injury has been proposed in: (1) oxidative injury to the lung from oxygen therapy, cigarette smoke, and/or atmospheric pollutants; (2) oxidative injury to the skin from ultraviolet radiation; (3) injury to the heart and lung from

antitumor therapy; and (4) injury to the kidney and small intestine from reperfusion following ischemic events.10 Interventions to increase tissue GSH concentrations have used GSH itself, monoesters that are more completely absorbed, or alternative precursors. Table 1. Metabolic Functions of Glutathione DNA synthesis and repair Protein synthesis Prostaglandin synthesis Amino acid transport Metabolism of toxins and carcinogens Enhancement of immune system function Prevention of oxidative cell damage Enzyme activation Depletion of intracellular GSH appears to be critical for subsequent alterations in protein thiol and calcium homeostasis.11 GSH depletion and the subsequent low stores of protein thiol result in both calcium release from intracellular stores and inhibition of calcium extrusion, producing a marked increase in cytosolic calcium concentration, which triggers cytotoxicity.11 Glutathione Synthesis GSH is synthesized from L-glutamate, L-cysteine, and glycine in 2 adenosine triphosphate (ATP)-dependent reactions (Figure 1). The first reaction, catalyzed by gamma-glutamylcysteine synthetase, is effectively rate-limited by GSH biofeedback.1,3,6 The second step involves GSH synthetase, which is not subject to negative feedback by GSH. When GSH is consumed and feedback inhibition is lost, availability of cysteine as a precursor can become the rate-limiting factor.8 Oxidized glutathione (GSSG) is formed in antioxidant reactions that involve GSH, and can accumulate with increased oxidative processing in cells. The ratio of GSSG/ GSH serves as a sensitive index of oxidative stress.12 Because its oxidative functions require GSH to be in its reduced form, GSSG is reduced to regenerate GSH in a reaction catalyzed by GSH reductase that requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a hydrogen donor (Figure 2).3,12 Absorption and Distribution of Glutathione Little is known about the average daily intake of GSH, the amounts of GSH in various food sources, or the importance of dietary GSH in health or disease. The estimated daily intake in humans is about 150 mg of GSH per day.13 Investigations in humans have used 15 mg/kg as an oral bolus to increase the plasma GSH concentration two- to fivefold. The transit of orally administered GSH to tissues is thought to occur via absorption from the intestinal lumen, export from enterocytes into the blood, and uptake from the plasma into cells.10,14 Gastrointestinal transport of GSH appears to be via nonenergy-requiring, sodium-independent, carrier-mediated diffusion.15 The plasma concentration of GSH is low because of its rapid turnover, and more than 80% of plasma GSH is removed by the kidney.16 The serum half-life of GSH

after intravenous administration is less than 2 minutes; however, the half-life in epithelial lining fluid is much longer, suggesting independence of respiratory epithelial lining fluid and plasma with regard to GSH metabolism.17 Various epithelial cells, such as enterocytes, alveolar cells, renal proximal tubular cells, endothelial cells, and retinal pigmented epithelial cells, are capable of exogenous GSH uptake, which supports the function of GSH-dependent detoxification systems.10,13 This allows GSH concentrations to be maintained better than by synthesis alone. Increasing plasma GSH concentrations by oral administration has been shown to increase the availability of GSH for transport into these tissues.13 This provides the basis for augmenting GSH concentrations against a wide variety of pathophysiologic states, including hepatic dysfunction or cirrhosis, or conditions affecting the epithelial cells, which can use exogenous GSH for protection. Most cells, in contrast to epithelial cells, do not have a direct transport capacity for intact GSH.3 Substrates for GSH synthesis are provided either by transport of amino acids into the cells or by transpeptidase activity at the cell surface, which is responsible for salvaging amino acids from circulating GSH for reuse in the intracellular resynthesis of GSH. The cellular concentration of GSH, therefore, is regulated by a complex process of precursor amino acid transport across cell membranes, intracellular synthesizing enzymes, feedback regulation, and intracellular GSH complexing via conjugation of GSH with a variety of electrophilic compounds through GSH transferase reactions. Although GSH is synthesized from precursors in virtually all cells, the liver is the main source of plasma GSH.18 In animal models hepatic concentrations of GSH change diurnally, with the highest concentration at 1000 hours and the lowest at 1800 hours.18,19 The plasma concentration of GSH is a function of hepatic synthesis, oxidation-reduction reactions, extrahepatic uptake and degradation, and GSH absorption.l3 In the liver, distribution of GSH is heterogeneous, which may play a significant role in susceptibility to acute hepatotoxicity from various toxic compounds such as acetaminophen metabolites, redox cycling compounds, peroxides, and others. Most GSH clearance from plasma occurs in the kidneys and the lungs.1,8,20,21 In the kidneys, exposure to toxins and the requirement for detoxification by GSH are high. Steady-state intracellular GSH concentrations vary in different parts of the kidneys, which also may determine the localization of injury by toxins.8 Role of Cysteine Homeostasis in Glutathione Metabolism Cysteine provides the reactive thiol group, which is

key to the function of GSH.8 It is required for hepatic GSH synthesis and may be derived from methionine, which serves as a major source of cysteine, via the trans-sulfuration pathway of the liver.18 Cysteine also inhibits hepatic GSH efflux. It cannot be transported in/from plasma or stored within the cell as cysteine, as it would rapidly autooxidize to cystine, producing potentially dangerous oxygen radicals.8 This toxic autooxidation is avoided by storing almost all nonprotein-bound cysteine as GSH.1,8 Cysteine occurs in plasma in the following forms: the solitary amino acid with its free thiol group (free cysteine), a disulfide between 2 molecules of cysteine (cystine), and a mixed disulfide with cysteinyl residues of albumin or other plasma proteins (protein-bound cysteine).22 The kidneys use cystine as a source of cysteine, and they clear and break significant quantities of plasma GSH into component amino acids.8 Therefore, the kidneys are a major source of plasma cysteme that can be used by the liver for resynthesis of GSH. Major Functions of Glutathione The major functions of GSH can be explained by its role in detoxification, redox reactions, and the storage and transport of cysteine. Because a major physiologic function of GSH is to provide cells with a reducing environment and to destroy the reactive oxygen compounds and free radicals formed in metabolism, organs that have low concentrations of other antioxidants (such as catalase and superoxide dismutase) are thought to be more dependent on GSH for detoxification of reactive oxygen species than are organs that have alternative antioxidants.20 DETOXIFICATION Either by spontaneous conjugation or by reduction, GSH provides the bulk of available sulfhydryl groups for binding and detoxification of reactive endogenous and exogenous compounds such as peroxides and electrophiles.2,23,24 GSH peroxidase is an enzyme present in tissues,25 which converts peroxides, using GSH as a substrate, into less harmful fatty acids, water, and GSH disulfide.26 These reductive reactions generate the oxidized form of GSH, GSH disulfide (GSSG).24 Under normal circumstances, to preserve high inuacellular concentrations of free GSH, GSSG is reduced rapidly back to GSH by the NADPH-dependent GSSG reductase. In this way a high GSH/GSSG ratio is maintained within the cells.24 However, GSSG is actively excreted from the cell when its intracellular concentration exceeds the reductive capacity of the cell, as in conditions of oxidative stress. The GSH/GSSG ratio is hypothesized to affect the redox balance of protein thiols, and to regulate the activity of certain enzymes by disulfide exchange reactions. The capacity for GSH synthesis is insufficient to

maintain GSH concentrations when tissues are exposed to certain drugs or their metabolites (e.g., acetaminophen), redox cycling compounds (e.g., menadione), peroxides (e.g., tertbutyl hydroperoxide), X-rays, or ultraviolet radiation.13 Its depletion has been associated with enhanced toxicity of many compounds that cause increased morbidity or death.2 For example, hepatotoxicity of acetaminophen is caused by the production of a highly reactive intermediate oxygen metabolite (N-acetyl-p-benzoquinoneimine). This metabolite covalently bonds to tissue macromolecules, resulting in tissue injury and cell death (Figure 3).25,27,28 Ito et al.25 have shown that inhibition of the GSH redox cycle enhances acetaminophen cytotoxicity in cultured rat hepatocytes. Excessive amounts of acetaminophen can overwhelm the capacity of GSH for conjugation and lead to toxicity.27 Also, fasting (by shunting acetaminophen metabolism away from glucuronidation or by depletion of intracellular GSH) and chronic ethanol use (via intracellular GSH depletion) can increase susceptibility to acetaminophen hepatotoxicity.28 GSH also may activate parent compounds by their metabolic conversion to active intermediates. For example, the metabolism of nitroglycerin requires interaction with thiols such as cysteine and GSH for transformation to vasoactive metabolites such as nitrosothiols or nitric oxide.29 In addition to activation of nitroglycerin via interaction with thiol compounds such as cysteine and GSH, dilation of small vessels only responsive to nitroglycerin in the presence of exogenous cysteine has been proposed. Finally, N-acetyl-cysteine (NAC) has been shown to partially prevent tolerance development to nitrate-induced antianginal effects.

GLUTATHIONE AS AN ANTIOXIDANT Oxygen radical stress occurs in all aerobic organisms as a result of aerobic metabolism, with intermediates such as superoxide and hydrogen peroxide that lead to further production of oxygen radicals, which can cause lipid peroxidation and disrupt metabolic processes.8 Antioxidant defenses are not completely efficient, and increased free-radical formation (oxidative stress) is likely to increase the damage.9 The GSH redox cycle (the balance between GSH and GSSG) has been shown to be more effective than catalase in hydrogen peroxide detoxification in endothelial cell cultures.12 GSH serves as a substrate for 2 antioxidant enzymes: GSH peroxidase and phospholipid hydroperoxide GSH peroxi-dase.12 Glutathione-S-transferase is also a glutathione- dependent enzyme, mainly involved in xenobiotic and lipid peroxide detoxification.12 Although GSH exerts antioxidant properties through

antioxidant enzymes, it also provides protection against oxidative stress by nonenzymatic free radical scavenging.12 Reduced GSH is capable of directly scavenging radicals and peroxides by being oxidized to either GSSG or to a mixed disulfide, thereby preventing cell membrane lipid peroxidation and its subsequent deleterious effects on cellular functions.4 Various oxygen radical stresses have been shown to result in GSSG formation and short-term depletion of GSH.3 It has been postulated that the oxidation-reduction status of GSH may act as a third messenger in either enhancing or diminishing the activities of a number of biologic processes, such as enzyme catalysis, protein synthesis, and receptor binding.30 Relationship of S-adenosyl-L-Methionine and Glutathione The metabolism of GSH and S-adenosyl-L-methionine (SAM) are closely linked, as the liver forms GSH as a product of SAM metabolism (Figure 4). The liver uses as much as 70% of dietary methionine, and most is converted to SAM.31,32 SAM is an important metabolic substrate and is involved in the initiation of 3 major pathways: (1) transmethylation for the synthesis of various proteins and lipids, among them phospholipids for cell membranes; (2) trans-sulfuration to form GSH and sulfated compounds via homocysteine and cysteine; and (3) aminopropylation for polyamine synthesis.33 Glutathione in Disease LIVER DISEASE Decreased SAM use by the liver can result in reduced GSH availability and potential hepatotoxicity.34 Patients with cirrhosis of the liver have hypermethioninemia and a block of the trans-sulfuration pathway leading to delayed methionine clearance and decreased concentration of methionine endproducts (including GSH) following a methionine load.32,33,35 The lack of accumulation of intermediary metabolites suggests that the defect is located in the initial step of methionine transformation regulated by SAM synthe-tase.33,34 Because GSH is vital in detoxification and cell physiology, its depletion has been speculated to represent an important contributing factor of liver injury, and enhanced morbidity in patients with liver injury.2 Chawla et al.22 measured the concentrations of cysteine, GSH, and taurine in 14 healthy subjects and 10 patients with cirrhosis fed either mixed food, nasogastric hyperalimentation with Vivonex (Sandoz Nutrition, Minneapolis, MN), or FreAmine III (McGraw, Irvine, CA) intravenous hyper-alimentation. Vivonex and FreAmine III do not contain cysteine. Subnormal plasma concentrations of GSH and cysteine were observed in patients with cirrhosis independent of their diet. The data support the hypothesis of an

acquired dysfunction in the hepatic trans-sulfuration pathway, rather than a change in bioavailability. Because most plasma GSH originates in hepatocytes, the authors hypothesized that decreased plasma GSH also could signify intracellular depletion. This would potentially impair the ability of the hepatocyte to maintain normal redox potential, destroy peroxides and free radicals, and detoxify drugs.22 Loguercio et al.16 evaluated the plasma and erythrocyte GSH and cysteine concentrations of patients with liver cirrhosis with respect to alcoholic or nonalcoholic etiology, severity of liver disease, and nutritional status. The data demonstrated a four- to eightfold decrease in plasma GSH content in 48 cirrhotic patients versus a control group of 18 healthy volunteers. This decrease was irrespective of cirrhosis etiology and thought to reflect diminished hepatic GSH synthesis. A significant decrease in cysteine in severe cases of cirrhosis also was observed. It is known that plasma concentrations of cysteine are affected by diet composition and fasting. However, cysteine also is provided by methionine through the trans-sulfuration pathway, which if impaired could contribute to the depression of plasma cysteine concentration. This investigation found no correlation between plasma cysteine and nutritional status, pointing to impairment of trans-sulfuration as the major cause. Altomare et al.2 measured reduced and oxidized hepatic GSH concentrations during alcoholic and nonalcoholic liver injury in 35 chronic alcoholics, 20 nonalcoholic patients with liver disease (chronic active hepatitis, chronic persisting hepatitis, steatosis, cirrhosis), and 15 control patients (admitted for uncomplicated abdominal procedures). Decreased GSH concentrations were noted in patients with alcoholic (2.55 0.1 mol/g of liver) and nonalcoholic liver diseases (2.77 0.1 mol/g of liver) compared with controls (4.14 0.1 mol/g of liver) that were speculated to be a contributing factor in liver injury and could enhance the risk of hepatic toxicity from a variety of toxic agents. The GSSG concentration was also significantly higher in alcoholics (8.2 0.3% of total) and nonalcoholics (8.5 0.8% of total) than in control subjects (4.4 0.2% of total), reflecting an abnormal balance between GSH and GSSG in these patients. The investigators concluded that decreased hepatic GSH concentrations in patients with liver disease may represent a contributing factor of liver injury susceptibility and toxicity risk in these patients. Furthermore, excess GSSG is postulated to result in alteration of a variety of cell functions, including enzyme function, protein synthesis, cell integrity, microtubular function, transport processes, and release mechanisms.2 Data suggest that plasma and hepatic GSH also are

decreased in patients with acute viral hepatitis, or chronic liver injury from a variety of causes such as chronic hepatitis, nonalcoholic liver cirrhosis, and alcoholic liver disease.36,37 The etiology again is thought to include altered SAM metabolism in patients with chronic liver disease regardless of the stage or cause of the disorder.23 Seifert et al.38 found a correlation between liver disease in chronic alcoholics and a subsequent GSH depletion state. Chronic alcoholic patients between the ages of 21 and 65 years were investigated for predisposition to acetaminophen hepatotoxicity caused by increased activity of the cytochrome P450 system and decreased hepatic GSH concentrations. Venous blood was analyzed for liver profile, prothrombin time, GSH, and GSSG. Acetaminophen use and diet appeared to have no effect on plasma GSH concentrations. Total GSH in patients with normal gamma-glutamyl transferase (GGT) did not differ significantly from that in healthy volunteers. Acetaminophen toxicity was not observed. However, patients with an elevated GGT (102 IU/L) had significantly lower plasma total GSH concentrations (2.38 2.42 mol/L vs. 5.81 4.11 mol/L, p < 0.0001). These data suggest that in chronic alcoholics with liver disease, GSH deficiency exists, which may predispose to further liver toxicity caused by resultant inadequate defense mechanisms. ROLE OF GLUTATHIONE IN THE IMMUNE SYSTEM: AIDS DATA Many of the pathologic aspects of disease in patients with AIDS are not caused directly by the HIV infection, but are secondary effects caused by the host response to infection.39 One of the more important aspects of this disease is the chronic inflammatory and oxidative stresses that accompany the infection, which eventually contribute to the loss of CD4 (helper) T-cells, increased opportunistic infections, immuno-deficiency in general, wasting disease, and death. Further-more, HIV infection is characterized by a systemic GSH deficiency, which has been postulated to increase viral replication and to increase production of oxidants from inflammatory cells possibly contributing to immune system dysfunction.17 The consequences of GSH depletion in AIDS and the role of thiol replacement therapy have been described.39-44 The progression of HIV infection from its early asymptomatic stage to active late-stage AIDS is thought to begin with the production of inflammatory cytokines that stimulate the production of the latent virus.42 Adjuvant therapy with GSH replacement in patients infected with HIV could offer several potential benefits. For example, stimulation of viral replication by inflammatory cytokines is optimal at decreased glutathione concentrations.39,40 GSH and NAC effectively block the cytokine-induced production of virus and may extend the latency period of early

infection.39,40 Restoration of depleted GSH concentrations in T-cells may be critical for restoration of leukocyte function.39 Intracellular GSH has been shown to play an important role in aspects of T-cell function, including the binding, internalization, and degradation of interleukin-2, as well as DNA synthesis.4 In particular, GSH and sulfhydryl compounds are known to augment the activation of cytotoxic T-cells in mixed lymphocyte cultures, T-cell proliferation in response to mitogens, and the differentiation of T and B lymphocytes.45 In vivo administration of GSH has been demonstrated to enhance the activation of cytotoxic T-cells, but depletion of GSH intracellularly inhibited the activation of lymphocytes, increased susceptibilities of human lymphoid cells to radiation, and suppressed cell-mediated cytotoxic functions, suggesting that intracellular GSH can modulate the function of immune cells. The current hypothesis is that because adequate concentrations of GSH are required for proper lymphocyte function, a deficiency in GSH may contribute to the immunodeficiency seen in the later stages of HIV infection.41 In addition, the action of inflammatory cytokines may mediate cachexia and the wasting that accompanies late stages of AIDS, which also may be alleviated by GSH replacement.40,42 Holroyd et al.17 investigated the effect of administering reduced GSH 600 mg via aerosolization twice daily to 14 HIV-seropositive individuals. GSH concentrations in the lung epithelial lining fluid were compared before and at 1, 2, and 3 hours after administration. Total GSH concentrations increased and remained in the normal range for the 3 hour post-treatment studied. A striking increase in oxidized GSH (GSSG increased from 5% at baseline to about 40% 3 h after treatment) was noted, possibly reflecting the potential value of GSH as an antioxidant providing protection in the lung tissue. NAC is a cysteine prodrug capable of maintaining intra-cellular thiol concentrations and replenishing GSH in deficiency states. It blocks HIV expression in acute and chronic infection models, and HIV replication in normal peripheral blood mononuclear cells.40 The administration of NAC has been mentioned as a "new approach to anti-HIV therapy," which differs from existing antiviral drugs in that it inhibits host-mediated stimulation of viral replication arising in normal immune responses, and may hence extend latency. In vitro, NAC has been shown to block cytokine-stimulated HIV replication in an acutely infected T-cell line and in acutely infected peripheral blood mononuclear cells from healthy individuals.42 PULMONARY DISEASE GSH deficiency has been proposed to have a role in the pathophysiology of a number of lung diseases,

including chronic obstructive pulmonary disease,46 acute respiratory distress syndrome (ARDS),47-49 neonatal lung damage,50 and asthma.51 The lung is particularly at risk from oxidative damage as it is exposed to oxygen, oxygen radicals produced by alveolar macrophages, inhaled environmental and blood-borne toxins, including cigarette smoke and atmo-spheric pollutants.8,10 Free radical induced toxicity is worsened by concomitant GSH deficiency, and free radical production further depletes GSH through use.52 GSH present in the epithelial lining fluid of the lower respiratory tract may be the first line of defense against oxidant stress.8 In idiopathic pulmonary fibrosis, for example, GSH concentrations are only 25% of normal values in the epithelial lining fluid and may be involved in the underlying pathophysiology of the disease.8 Neonates. Pulmonary GSH concentrations have been found to be lower in premature infants with a lower gestational age (25 vs. 39.8 wk average gestational age).53 Theories to explain why pulmonary GSH could be depleted in some infants include decreased cord cysteine concentrations, seen in earlier gestational age, that may impair GSH synthesis as intracellular GSH depends on the availability of cysteine. This hypothesis was investigated by Grigg et al.50 The concentration of GSH in bronchoalveolar lavage fluid was measured on the first day of life in intubated infants born at less than 35 weeks gestation irrespective of initial respiratory status. Lower concentrations of GSH were observed in 7 infants who subsequently developed chronic lung disease when compared with 27 infants who did not require oxygen supplementation at 36 weeks postconceptional age. This investigation provided preliminary evidence that a low lung epithelial lining fluid concentration of GSH on the first day of life is associated with an increased risk of chronic lung disease. However, a method of increasing GSH concentrations in this population was not discussed. Asthma. Airway inflammation in asthma also is associated with the increased generation of reactive oxygen species and pathophysiologic changes.51 In a comparison of 10 adults with mild asthma versus 17 healthy volunteers, concentrations of GSH in bronchoalveolar lavage fluid were increased in the subjects with mild asthma. The mean bronchial GSH concentrations were 13.0 nM/mg of protein in healthy volunteers versus 23.9 nM/mg of protein in mild asthmatics. The mean alveolar GSH concentration was 23.3 nM/mg of protein in volunteers versus 36.5 nM/mg of protein in mild asthmatics. The authors hypothesized that an increasing GSH concentration represents an adaptive mechanism to increase antioxidant defenses, which results in fewer symptoms, reduced airway reactivity, and milder disease in

patients with asthma. Acute Respiratory Distress Syndrome. GSH is detected in high concentrations in the extracellular epithelial lining fluid of the lower respiratory tract of healthy subjects, and could act as a first-time scavenger of toxic oxygen intermediates and protect against lung cell damage and injury.l7 Patients with ARDS and sepsis have a GSH deficiency in the lower respiratory tract extracellular epithelial lining fluid, which could favor oxidative stress and subsequent damage.47,48 Also, greater percentage of total GSH exists in the oxidized form in patients with ARDS than in healthy subjects, possibly indicating increased oxidant stress in the lower respiratory tract of ARDS patients.49 The mechanism for GSH deficiency is thought not to be dilutional (as occurs in cardiogenic pulmonary edema), but representing depletion of tissue stores. GSH depletion in plasma and granulocytes in ARDS is reversible by NAC therapy. Suter et al.47 improved systemic oxygenation and reduced the need for ventilatory support in 32 patients with acute lung injury caused by various underlying diseases, with NAC 40 mg/kg/d administered as a continuous infusion over the first 3 days after admission to the intensive care unit. Patients in the NAC group also had a shorter median stay in the intensive care unit than did those in the placebo group (median 7 vs. 10 d, respectively). ONCOLOGY By acting as an antioxidant and by virtue of binding to cellular mutagens, GSH has the ability to react with peroxides and several electrophiles, including carcinogenic epoxide metabolites.7,54 GSH has been shown to directly modulate proliferation of highly purified T-cells, suggesting that GSH is essential for steps closely involved with DNA synthesis. Depressed intracellular GSH in the liver and in mammary tissue has been shown to promote carcinogen binding to DNA.54 Oral glutamine, for example, has been proposed to have a useful role in increasing host GSH concentrations in the gut, liver, lung, kidneys, heart, and muscle after exposure to radiation or chemotherapy without enhancing tumor growth.55 Flagg et al.7 investigated the association between dietary GSH intake and the risk of oral and pharyngeal cancer in an epidemiologic study of 1830 participants. In this case-control study, the investigators noted an inverse relationship between dietary intake of GSH and the risk of oral cancer, but only in a cohort consuming GSH mostly from raw fruit and vegetables (rather than meat or cooked vegetables, for example). However, the possibility that GSH intake from fruit and vegetables might be protective against oral cancer risk could not be distinguished from the more general benefit of consuming raw fruits and vegetables, such

as increased ingestion of fiber. The investigators hypothesized anticarcinogenic protective mechanisms of GSH to include its direct antioxidant function, indirect maintenance of other antioxidants, possible mediation of DNA synthesis and repair, and the ability to bind with cellular mutagens.3 Resistance to chemotherapy caused by detoxification by GSH has been postulated,56 suggesting that there may be possible benefits of selectively decreasing intracellular GSH in tumors to enhance the therapeutic effect of chemotherapy. Direct bonding to the sulfhydryl group of GSH in the cytoplasm has been shown to inactivate cisplatin, for example. Further, GSH depletion can inhibit DNA repair in bladder carcinoma cell lines after cisplatin or doxorubicin damage. Limited data suggest that GSH and its related enzymes enhance drug resistance, varying with the characteristics of the tumor and the chemotherapy chosen. The use of buthionine sulfoximine, which inhibits gamma-glutamylcysteine synthetase, has been investigated in animals as a method to decrease GSH.3 With elucidation of its mechanism, incidence, and impact on resistance to chemotherapy, interventions to decrease target concentrations of GSH may become part of future oncologic therapeutic regimens. PREECLAMPSIA Walsh and Wang26 determined that GSH peroxidase activity is significantly lower, and lipid peroxides and thromboxane significantly higher, in preeclamptic placentas than in healthy placentas because of an unknown mechanism. They postulated that peroxides stimulate prostaglandin H (PGH) synthase, leading to the generation of oxygen radicals and increased formation of both thromboxane and lipid peroxides. They proposed that PGH synthase converts arachidonic acid into prostaglandin G2 and prostaglandin H2. Thromboxane synthase then converts prostaglandin H2 into thromboxane A2. When PGH synthase is activated, the peroxidase function of the enzyme generates oxygen radicals that could interact with polyunsaturated fatty acids in the placenta to form lipid peroxides. GSH peroxidase inactivates peroxides, using GSH as its cofactor to convert lipid peroxides to less harmful hydroxylated fatty acids, water, and GSH disulfide. If GSH peroxidase activity is deficient, lipid peroxidation could increase in the tissues, leading to increased stimulation of PGH synthase, increased thromboxane production, and further increase lipid peroxides. Deficiency of this enzyme would likely increase morbidity in preeclampsia. PARKINSON'S DISEASE The brains of patients with Parkinson's disease exhibited a reduction of GSH, selective to the substantia nigra (SN). This did not appear to be related to drug therapy. It was postulated to be of significance in the pathogenesis of this disease via

production of oxidative damage.57,58 These patients had an increased concentration of the GSH degradative enzyme gamma-glutamyltranspeptidase in the SN, and a normal concentration of the synthetic enzyme gamma-glutamyl-cysteine synthetase.57 GSH depletion occurred without a change in GSSG, suggesting efflux of GSH out of the glia, perhaps with additional increased conversion of GSH to GSSG in response to increased hydrogen peroxide formation. At this time it is unclear whether free radical involvement in Parkinson's disease is a primary or secondary event in nigral cell death or whether it occurs early or late in the disease process.58 SEPSIS Henderson and Hayes59 reviewed the existing data on NAC as an antioxidant in patients with severe sepsis. They noted that GSH is depleted in severe sepsis, possibly in the defense against active radicals generated by inflammatory cells during systemic inflammatory response syndrome. These inves-tigators are experimenting with a regimen of NAC 150 mg/kg in dextrose 5% infused intravenously over 30 minutes followed by 15 mg/kg/h for 4 days as an antioxidant active radical scavenger. Their hypothesis is that NAC may improve renal function, reduce fluid requirements, and lower tissue edema. Spies et al.60 reported a 2-year investigation of 58 patients with septic shock randomized to receive NAC 150 mg/kg iv over 15 minutes followed by 12.5 mg/h over 90 minutes. Using a 10% or more increase in whole body oxygen con-sumption as their endpoint, 13 patients who received NAC were considered responders, and 16 were considered nonresponders. None of the patients who received placebo (n = 29) exhibited a 10% increase in whole body oxygen consumption. In summary, NAC transiently improved tissue oxygenation in about half of the patients with septic shock to whom it was administered. MYOCARDIAL ISCHEMIA AND REPERFUSION INJURY During myocardial ischemia and reperfusion, both myocardial GSH and the GSH/GSSG ratio within the ischemic tissues are reduced, and the extent of the myocardial injury is inversely dependent on the myocardial GSH content.61-63 Treatment with gamma-glutamylcysteine ethyl ester can increase intracellular reduced GSH concentrations, and has been shown to result in a dose-dependent reduction in infarct size in a canine model of occlusion-reperfusion.61 However, an investigation of coronary artery occlusion in 23 mongrel dogs administered NAC 30 minutes before and continued until 3 hours after reperfusion failed to observe an increase in total GSH or GSH peroxidase activity at the biopsied ischemic zone, or a decrease in myocyte death.64 RENAL DYSFUNCTION AND NEPHROTOXICITY

We have alluded to the potential for decreases in GSH concentration to impair GSH conjugation and modify the metabolic fate of many compounds. GSH deficiency may contribute to the nephrotoxicity of ischemic events and drug toxicity. This type of toxicity may be exhibited by cyclosporine. Although the exact mechanism of cyclosporine nephrotoxicity remains unknown, its administration has been associated with in vivo reduction of GSH concentrations in the livers and kidneys of rats, which may be related to adverse effects of this immunosuppressive agent.65,66 Cyclosporine has peroxidative properties, induces lipid peroxidation in renal microsomes, and may lead to inactivation of microsomal glucose-6-phosphate activity and toxicity.66,67 Therefore, contribution to cyclosporine nephro- and hepatotoxicity has been postulated to be caused by its generation of free radicals and depletion of GSH. Investigators also have hypothesized that cyclosporine can modify resistance to chemotherapy by augmenting the cytotoxic effect of drugs through inducing a GSH deficiency.65 The ability of GSH administration to prevent nephrotoxicity from renal ischemia, and consequent production of oxygen free radicals, has been investigated. A total of 200 mg/kg of GSH (available in Japan as Tathion, Yamanouchi Pharmaceutical, Tokyo, Japan) was administered intra-venously to 10 patients before cardiopulmonary bypass surgery, and the influence on postoperative renal dysfunction was compared with 9 other patients undergoing the same procedure without GSH administration.16 Administration of GSH resulted in a significantly higher urine volume (approximately 20%) on the first and second postoperative days, with a trend to lower blood urea nitrogen and plasma creatinine concentrations in the GSH group that did not reach statistical significance. Also, the mean arterial pressure and systemic vascular resistance index were lower than those in the control group. The investigators concluded that administration of exogenous GSH had a beneficial effect on renal function by virtue of its antioxidant properties, and possibly by a vasodilator action to increase the glomerular filtration rate as well. Role of Glutathione in Aging Although higher GSH concentrations have been associated with good health, the significance of low GSH status in the elderly is inferred from limited data.68,69 Investigators have noted lower GSH concentrations to be associated with the combination of advanced age and increased risk of chronic diseases such as chronic renal failure, malignant disorders, diabetes, alcoholism, Parkinson's disease, and cataract formation. Julius et al.68 measured GSH concentrations in 33 people between 60 and 79 years of age, and related the data to health, the number of illnesses, and other

risk factors for chronic disease. There appeared to be a direct relationship between higher GSH concentrations and increasing age with good health. The association of GSH with good health was positive and independent of age (i.e., volunteers with higher GSH concentrations were healthier than age-matched volunteers with lower GSH concentrations). People with chronic diseases had lower mean GSH concentrations than those who were free of disease. Lang et al.69 compared GSH blood concentrations in healthy young and healthy elderly subjects and found that the reference group of 20- to 39-year-old subjects (n = 40) had a GSH concentration 17% higher on average than the 60- to 79-year-old cohort of 60 subjects (mean SD, 547 53.5 g/1010 vs. 452 86.8 g/1010 erythrocytes, respectively). Caution has been advised in interpreting these small studies to define the association of GSH with aging.70 Further large-scale studies are needed to prove that low relative GSH concentration is an overall risk factor for morbidity among the elderly. Pharmacotherapeutic Interventions to Increase Glutathione Concentrations GLUTATHIONE AND RELATED AGENTS GSH is present to the greatest extent in fruits, vegetables, and meats.71 Agents such as 1-cyano-2-hydroxy-3-butene, present in cabbage, brussels sprouts, broccoli, and cauliflower, have raised GSH concentrations severalfold in animal models.72 The results of studies to date of GSH absorption have been conflicting. Several investigators have shown that orally administered GSH increases plasma concentrations of reduced and protein-bound GSH through intestinal absorption in animal models10,13,15 and humans.73 GSH is not commercially available as an oral or injectable product in the US because of pharmaceutical problems, including poor oral bioavailability and a short halflife (2 min) with intravenous administration. Investigators have used bulk quantities of GSH purchased from various chemical companies. For these reasons, precursors of GSH have been investigated. In mice pretreated with buthionine sulfoxime to inhibit GSH synthesis and induce a deficient state, oral administration of GSH resulted in statistically significant increases in GSH concentrations in kidney, heart, lung, brain, small intestine, and skin, but not in the liver. Administration of the equivalent amount of the constituent amino acids to GSH-deficient mice resulted in little change in GSH concentration in all tissues studied. Mice not pretreated with buthionine sulfoximine, but administered GSH, experienced an increase in plasma GSH, but not tissue GSH.10 In humans, oral doses of GSH 15 mg/kg increased plasma GSH 1.5- to 10-fold over the basal concentration in 4

of 5 volunteers tested.73 The maximum concentration of plasma GSH generally occurred 1 hour after GSH administration. Equivalent amounts of amino acid constituents of GSH failed to increase plasma GSH concentrations. These data suggest that oral GSH can replete GSH concentrations in several tissues following GSH depletion, such as after toxicologic or pathologic conditions that alter GSH homeostasis.10 It has been speculated that because oral administration of GSH leads to its inactivation by peptidases, it should not be possible to significantly increase plasma GSH concentrations with oral administration.74 Witschi et al.74 investigated the bioavailability of a single dose of oral GSH in 7 healthy volunteers who had fasted. Their data showed a nonsignificant increase in plasma GSH after doses as high as 3.0 g, suggesting negligible systemic availability of oral GSH in humans. Cook and Sherlock75 also were not able to demonstrate a benefit from oral GSH (100 mg tid for 28 d) in 10 patients with hepatic cirrhosis of various etiology. Analysis included measurement of biochemical parameters and an assessment of sense of well-being compared with 10 other patients with cirrhosis not administered GSH. In a separate arm of investigation, another 12 patients with cirrhosis received GSH 200 mg/d im without producing any difference in biochemical parameters. This latter GSH group did note an increased sense of well-being versus the control group from the oral GSH investigation. Evidence suggests that cells export GSH, but evidence that GSH is transported into cells to any appreciable extent is conflicting.5,10,73 Therefore, methyl, ethyl, and isopropyl esters of GSH in which the glycine carboxyl group is esterified have been used and are orally bioavailable via rapid intracellular deesterification with effective transport and hydrolysis intracellularly.5,61 As an example, orally administered GSH ethyl ester is able to significantly raise GSH concentrations in the liver, kidney, spleen, pancreas, heart, and lung of the mouse, and in human red blood cells, skin fibroblasts, and several lymphoid cell lines.5 Various esters and amides of GSH are transported into cells, but some have toxicity caused by cleavage products such as methanol, ammonia, and alcohols.5 None is commercially available in the US at this time. Gamma-glutamylcysteine ethyl ester was studied recently as a GSH precursor used for myocardial protection in dogs with ischemia and reperfusion damage.61 When administered as 3 or 10 mg/kg intravenously immediately before reperfusion in a canine coronary occlusion-reperfusion model of myocardial infarction, a significant dose-dependent reduction in infarct size was observed. Although administration of this ester appeared safe and had

protective properties in this model, it also is not commercially available at this time. CYSTEINE, CYSTINE, AND ACETYLCYSTEINE Cysteine usually is nonessential in the diet because it can be synthesized endogenously from methionine and phenylalanine.39,76 Impaired synthesis of cysteine from methionine may necessitate the provision of a source of cysteine to some patients with cirrhosis; however, supplementation with L-cysteine could lead to hypercysteinemia and potential toxicity.77 Administration of oral L-Cysteine to patients with cirrhosis has been noted to cause a twofold greater maximal plasma cysteine concentration and plasma elimination half-life, and a delayed excretion of metabolic end products when compared with those of controls. However, an impaired cysteine uptake from the plasma has been proposed secondary to a decrease in plasma GSH. Another example of precursor use involves L-2-oxothiazofidine-4-carboxylate, which is converted to S-carboxyl-L-cysteine and undergoes spontaneous decarboxy-lation to liberate L-Cysteine, thereby supporting GSH synthesis.8 In noncirrhotic, malnourished patients receiving parenteral nutrition, deficiencies in cysteine (and other amino acids) also can occur, possibly caused by the loss of the first-pass delivery of methionine to the liver and portal blood flow.31 As mentioned previously, cysteine is oxidized easily into cystine, and therefore, is not easily incorporated (and minimally present) in standard crystalline amino acid solutions used in intravenous hyperalimentation. This has resulted in hypo-cystinemia, with plasma cystine concentrations decreasing to 30% below baseline, as described in 12 patients with cirrhosis who received a FreeAmmine II source of amino acids in their intravenous hyperalimentation.76 NAC is available, as Mucomyst (Apothecon, Division of Bristol-Myers Squibb, Princeton, NJ) and generic products, and has been used for years as an antidote to acetaminophen toxicity. NAC may be a direct source of cysteine following hydrolysis or may reduce plasma cystine through thiol- disulfide exchange, liberating endogenous cysteine.8 Under normal conditions (no GSH deficiency), NAC does not increase total GSH, since the intracellular concentration is under feedback control. Recently, despite administration of NAC 600 mg po tid, a sustained increase in GSH concen-trations could not be found in the plasma, bronchoalveolar lavage fluid, or lung tissue of patients with chronic obstructive pulmonary disease.46 S-ADENOSYL- L-METHIONINE Hepatic GSH concentrations have been restored to nearly normal in liver biopsies of patients with cirrhosis following long-term oral SAM administration.23 Studies have documented improvement in pruritus, jaundice, and bio-chemical parameters in

patients with intrahepatic cholestasis of pregnancy treated with SAM.33 The clinical efficacy of SAM in the treatment of cholestasis associated with hepatic diseases has been reviewed.78 For the treatment of liver disorders, such as intrahepatic cholestasis, the recommended dose of SAM is 800 mg parenterally or 1600 mg/d orally. 23,78 Loguercio et al.78 administered SAM 2 g/d iv for a total of 15 days to 20 patients with biopsy-proven alcoholic cirrhosis. An increase in red blood cell GSH (to 2.20 1.10 mM/L from a baseline of 1.60 0.97 mM/L) and a decrease in cysteine content (to 65 14 M/L from 122 42 M/L) was demonstrated. The cysteine groups of SAM synthetase might be protected from oxidation by a normal concentration of GSH.80 When there is a reduction in liver GSH or increased concentrations of GSSG by toxin or disease, a vicious cycle might start. Depletion of GSH could lead to inactivation of SAM synthetase, with further decrease in GSH concentrations. worsening the deficiency in SAM synthetase. In this context, SAM administration may act as a precursor for GSH synthesis and also bypass the deficiency in SAM synthetase. SAM is not commercially available in the US. Summary The importance of GSH in health and disease is a subject of active research, presentation, and publication. GSH appears to be metabolically important in a wide variety of disease states, only a few of which have been discussed. At our institution, GSH has been the topic of grand rounds, and we have attempted to modify GSH stores in long-term parenteral nutrition patients with cirrhosis. We believe the significance and widespread use of GSH will only increase with time, and have attempted to introduce the topic to a broad range of clinicians. As pharmaceutical manipulation of GSH concen-trations is an intervention likely to increase within the coming years, health clinicians need to be aware of the rationale of such attempts and methods of administering bioavailable forms of GSH or its substrates. References 1. Meister A. Glutathione metabolism and its selective modification. J Biol Chem 1988;263:17205-8. 2. Altomare E, Vendemiale G, Alano O. Hepatic glutathione content in patients with alcoholic and non alcoholic liver diseases. Life Sci 1988;43: 991-8. 3. Deneke SM, Fanburg BL. Regulation of cellular glutathione. Lung Cell Mol Physiol 1989;1:LI63-LI73. 4. Wu D, Meydam SN, Sastre J, Hayek M, Meydani M. In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects. J Nutr 1994;124:655-63. 5. Anderson ME, Powrie F, Puri RN, Meister A.

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50. Grigg J, Barber A, Silverman M. Bronchoalveolar lavage fluid glutathione in intubated premature infants. Arch Dis Child 1993;69:49-51. 51. Smith LJ, Houston M, Anderson J. Increased levels of glutathione in bronchoalveolar lavage from patients with asthma. Am Rev RespiR Dis 1993; 147:1461-4. 52. Johnson ME, Sill JC, Uhl CB, Van Dyke RA. Effect of halothane on hypoxic toxicity and glutathione status in cultured rat hepatocytes. Anesthesiology 1993-,79:1061-71. 53. Picone TA, Daniels TA, Ponto KH, Pitard WB. Cord blood tryptophan concentrations and total cysteine concentrations. JPEN J Parenter Enteral Nutr 1989; 13:106-7. 54. Liu JZ, Zhang BZ, Milner JA. Dietary selenite modifies glutathione metabolism and 7,12-dimethylbenz(a)anthracene conjugation in rats. J Nutr 1994;124:172-80. 55. Fahr MJ, Kombluth J, Blossom S, Schaeffer R, Klinberg VS. Glutamine enhances immunoregulation of tumor growth. JPEN J Parenter Enteral Nutr 1994;18:471-6. 56. Ahn H, Lee E, Kim K, Lee C. Effect of glutathione and its related enzymes on chemosensitivity of renal cell carcinoma and bladder carcinoma cell lines. J Urol 1993; 151:263-7. 57. Sian J, Dexter DT, Less AJ, Daniel S, Jenner P, Marsden CD. Glutathione-related enzymes in brain in Parkinson's disease. Ann Neurol 1994;36:356-61. 58. Jenner P. Oxidative damage in neurodegenerative disease. Lancet 1994;344:796-8. 59. Henderson A, Hayes P. Acetylcysteine as a cytoprotective antioxidant in patients with severe sepsis: potential new use for an old drug. Ann PharmacoLher 1994;28:1086-8. 60. Spies CD, Reinhart K, Witt 1, Meier-Hellmann A, Hanneman L, Bredle DL, et al. Influence of N-acetylcysteine on indirect indicators of tissue oxygenation in septic shock patients: results from a prospective, randomized, double-blind study. Crit Care Med 1994;22:1738-46. 61. Hoshida S, Kuzuya T, Yamashita N, Nishida M, Kitahara S, Hori M,.et al. Gamma-glutamylcysteine ethyl ester for myocardial protein in dogs during ischemic and reperfusion. J Am Coll Cardiol 1994;24: 1391-7. 62. Blaustein A, Deneke SM, Stolz RI, Baxter D, Healey N, Fanburg BL. Myocardial glutathione depletion impairs recovery after short periods of ischemia. Circulation 1989;80:1449-57. 63. Singh A, Lee KJ, Lee CY, Goldfarb RD, Tsan MF. Relation between myocardial glutathione content and extent of ischemia-reperfusion injury. Circulation 1989;80:1795-804. 64. Forman MB, Puett DW, Cates CU, McCroskey DE, Beckman JK, Greene HL, et al. Glutathione redox

pathway and reperfusion injury. Circulation 1988;78:202-13. 65. Duruibe V, Okonmah A, Blyden GT. Effect of cyclosporin on rat liver and kidney glutathione content. Pharmacology 1989;39:205-12. 66. Inselmann G, Hannemann 1, Baumann K. Cyclosporine A induced lipid peroxidation and influence on glucose-6-phosphate in rat hepatic and renal microsomes. Res Commun Chem Pathol Pharmacol 1990;68: 189-203. 67. Wang C, Salahudeen AK. Cyclosporine nephrotoxicity: attenuation by an antioxidant-inhibitor of lipid peroxidation in vitro and in vivo. Transplantation 1994;58:940-6. 68. Julius M, Lang CA, Gleiberman L, Harburg E, DiFranceisco W, Schork A. Glutathione and morbidity in a community-based sample of elderly. J Clin Epiderniol 1994;47:1021-6. 69. Lang CA, Naryshkin S, Schneider DL, Mills B1, Lindeman RD. Low blood glutathione in healthy aging adults. J Lab Clin Med 1992; 120: 720- 5. 70. Fletcher RH, Fletcher SW. Glutathione and ageing: ideas and evidence (editorial). Lancet 1994;344:1379-80. 71. Jones DP, Coates RJ, Flagg EW, Eley JW, Block G, Greenberg RS, et al. Glutathione in foods listed in the National Cancer Institute's health habits and history food frequency questionnaire. Nutr Cancer 1992; 17: 57-75. 72. Davis MA, Wallig MA, Eaton D, Borroz 1, Jeffery EH. Differential effect of cyanohydroxybutene on glutathione synthesis in liver and pancreas of male rats. Toxicol Appl Pharmacol 1993; 123:257-64. 73. Jones DP, Hagen TM, Weber R, Wierzbicka GT, Bonkovsky HL. Oral administration of glutathione (GSH) increases plasma GSH concentrations in humans (abstract). FASEB J 1989;3:A 1250. 74. Witschi A, Reddy S, Stofer B, Lauterburg BH. The systemic availability of oral glutathione. Eur J Clin Pharmacol 1992;43:667-9. 75. Cook GC, Sherlock S. Results of a controlled clinical trial of glutathione in cases of hepatic cirrhosis. Gut 1965;6:472-6. 76. Rudman D, Kutner M, Ansley J, Jansen R. Chipponi J, Bain RP. Hypotyrosinemia, hypocystinernia, and failure to retain nitrogen during total parenteral nutrition in cirrhotic patients. Gastroenterology 198 1;8 1: 1025-35. 77. Tribble DL, Jones DP, Ardehali A, Feeley RM, Rudman D. Hypercysteinemia and delayed sulfur excretion in cirrhotics after oral cysteine loads. Am J Clin Nutr 1989;50:1401-6. 78. Almasio P, Bortolini M, Pagliaro L, Coltorti M. Role of S-adenosyl- methionine in the treatment of intrahepatic cholestasis. Drugs 1990;40 (suppI 3):111-23.

79. Loguercio C, Del Vecchio C, Coltorti M. Effects of intravenous S- adenosyl-L-methionine on red blood cell content of glutathionine and cyteine in alcoholic cirrhosis (abstract). Gastroenterology 1993;104: A944. 80. Ortiz P, Moreno J, Puerta JL, Mato JM. S-adenosyl-L-methionine and the liver. Ital J Gastroenterol 1993;25:135-7. So now you know a little bit about Glutathione. Even if you don't know everything, you've done something worthwhile: you've expanded your knowledge. *******************************************

WHEY PROTEIN REPORT Introduction Current Concepts on Whey Protein Usage Prepared for The Cleveland Eye Clinic by David Marshall, Jr., O.D., Ph.D. Consult INTRODUCTION A. What is WHEY? Whey is a by-product of cheese manufacture resulting from drainage of liquid from the curd1. It contains lactose, protein, ash, and lipids. The protein concentration at this stage of processing is about 67% protein. Whey proteins can be fractioned and concentrated via a process called micro-filtration to yield whey protein concentrates (WPC). The protein concentration can be as high as ~ 95% protein after the removal of the fats and lactose. WPC's are an excellent source of nutrition and are high in lactalbumin, minerals, and vitamins. It possesses a number of functional advantages such as solubility, high water retention, foaming and gelation 1. As a result of these qualities, whey protein concentrates (WPC) have been used in a wide range of food products such as the formulation of dairy foods, egg white replacement, beverages, surimi and comminuted meat products 1. In response to the efficacy of whey consumption, Karen Collins, M.S., R.D., a registered dietician from the American Institute for Cancer Research, states that whey is a safe and healthy ingredient in foods since levels of pesticides and hormones are not concentrated in whey 2. Whey protein concentrates are of particular interest

to the practitioner due to their wide-range and near full-blend of essential and non-essential amino acids, which are commonly referred to as the building blocks of life. Linked amino acids combine to form proteins. These protein comprise nearly every tissue and organ in the body, therefore any supplementation of the diet with proteins may be beneficial to injury repair, metabolism, and general health. B. Whey Manufacturing The importance of whey products has gained prominence within the last 5 years. Asia is the leading exporter of whey produce, exporting an estimated 15,680,683 kg in 1992 and 16,184,519 kg in 1995. This represents~ 60% of all worldwide exports. The leading importer of whey is North America, which in 1995 brought in an estimated 26,444.593 kg . This amount represents a 5 fold increase in imported product over a four year span and nearly 90% of all worldwide imports of whey 3. Commonly whey is prepared from cheese products such as cheddar cheese. In accordance with the Ohio State University method11, whey is prepared from milk using lactic acid culture and rennet as a coagulant. The milk is HTST pasteurized (1630 F, 30, seconds) and held overnight at 400C then warmed to 300 C and inoculated with 1.5% cheese starter. After incubation for 30 minutes, 38.7 ml rennet extract is diluted with cold water and added to 50 gallons of milk with stirring. Twenty-five minutes later curd is cut and cooked by gradual warming to 38-400. Cover a thirty minute time interval while the temperature is maintained for 1.5 hours. The whey is then drained through a stainless steel screen and separated at 550 C. WPC is produced by ultrafiltration at 450 C after addition of 20 g of citric acid with the pH adjusted to3. After ultrafiltration to 1/5 the original volume, the retentate is diafiltered with addition of 20 g citric acid at pH3. The concentrated whey is warmed to 400C and spray dried to, provide whey protein concentrates. This WPC (protein 79.9% and calcium 0.136%) is then used to prepare laboratory scale samples and the WPC solutions are freeze dried 3. Additional micro-filtration techniques may be added to increase the protein yield to ~95%. Many whey products are then put through an ion-exchange process to remove fat and lactose. In this procedure the cold-filtered protein concentrate is put through a static electrical charge which separates undesirable fractions from the WPC. However, there is a price to pay for this process. The immunoglobulin fraction is greatly reduced4. The end product is a whey protein isolate that is relatively fat, lactose, and sugar-free product possessing a high amino acid (protein) concentration. In addition to the before-mentioned procedures, some

manufacturers hydrolyze their WPC to produce di-, tri-, oligo-, and polypeptide (long and short-chain amino acids). It also provides for a variety of amino acids, with special emphasis on the branched-chained amino acids (L-leucine, L-isoleucine, and L-valine), in addition to important amino acids such as L-cystine and glutamine, all of which are essential for wound healing, immunity, and cellular nitrogen retention. note: Hydrolyzing breaks apart peptide bonds. The ion-exchange and hydrolyzing process used on whey protein permanently modifies the native structure. The protein is denatured and the biological activity is ended. Current Concepts of Whey Usage A. Nutritional Value The real nutritional value of WPC's lies in their abundance of amino acids. For example the typical WPC contains up to 18 amino acids, which nearly represents the full blend.57 Alanine, arginine, and aspartic acid are three common amino acids found in numerous whey proteins. Alanine aids in the metabolism of glucose, whereas arginine causes retardation of tumors and assists in the release of growth hormones and the maintenance of a healthy immune system. It also provides an environment for an increase in muscle mass and body fat reduction, in addition to being an essential ingredient for protein synthesis. Aspartic acid increases stamina, therefore it is good for fatigue. It also aids in RNA/DNA synthesis. Cysteine/cystine are perhaps two of the more important amino acids found in WPC. The are helpful in detoxifying toxins and are precursors to the body's most potent antioxidant, glutathione. They promote the burning of fat and are useful in the treatment of rheumatoid arthritis and bronchitis. Due to the possible toxic effects of cysteine at high levels, cystine is the preferred supplemental agent. Glutamic acid and glutamine are essential to nervous tissue function. Glutamic acid is a neurotransmitter for retinal neurons and is commonly referred to as "brain fuel" since the brain converts it to a compound that regulates brain cell activity. It is also a precursor to glutathione. Glutamine is important to cellular nitrogen retention and is important in alcoholism, mental ability, impotence and maintaining a healthy digestive tract. Glutathione, a potent antioxidant, is important for the safe metabolism of the hydrogen peroxide free radical. It helps protect against radiation and oxidative damage and is the body's best defense against the formation of cataracts, age-related macular degeneration (ARMD), cancer, and immunity. This important protein will be discussed in

detail later in text. The essential amino acids are well represented in most WPC's. They can't be manufactured by the body and therefore must be obtained through dietary consumption. They consist of the branched-chained amino acids, lysine, phenylalanine, methionine, and tryptophan. The three branched-chained amino acids are leucine, isoleucine, and valine. They are essential to tissue growth and repair. They promote the healing of bones ,skin, and muscle, They also regulate blood sugar levels, so they must be taken in a balance to insure proper regulation. Phenylalanine is often used to treat depression. It is a precursor to the neurotransmitter, norepinephrine and aids in memory. It should be avoided by phenylketonurics (PKU) Lysine, methionine and tryptophan are also found in many WPC's. Lysine is an essential building block for all protein and helps to maintain proper nitrogen balance. The body uses methionine to derive the brain food, choline. It also aids in digestion, as well as serving as a fat burner. It can interact with other substances to detoxify harmful agents, and is essential for the production of cysteine and taurine. L-Tryptophan acts as an sleep aid, as demonstrated by the drowsy feeling we sometimes experience after Thanksgiving dinner (turkey meat has a relatively high amount of tryptophan.). It is also necessary for the production of niacin and is used by the body to make the neurotransmitter, serotonin. The rational for using WPC's to attain this dietary level of nutrition is that one scoop of powder provides approximately 20 grams of amino acids. To acheive this level of nutrition taking tablets or soft gels would require taking 48 to 60 units per day, which would be difficult and expensive. B. Body-building A large number of weightlifters and bodybuilders use WPC's during their training regimen. They sometimes utilize a three step process which consists of stacking, "cycling", and "cutting". The initial stage, stacking involves taking two or more compounds at one time to maximize results. They may take WPC and or creatine or chromium. This is then followed by cycling, in which large doses of a supplement is used to change the level of body fat. Some will use 10 or more one ounce WPC servings a day. This should be approached with caution since large doses of protein consumption may present challenges to the liver. The final stage, cutting, involves maximizing the muscle bundles for visualization. This is the point in which the bodybuilder aspires to achieve that "cut up" appearance. It is important to consult a physician before attempting any of these extreme methods.

C. Clinical Implications 1. Wound Healing Austrialian researchers claim that whey extract could become a standard treatment for chronic wounds, especially ulcerations from diabetes and hypertension. Initial experiments using a whey extract containing a number of natural growth factors excelled at spurring cells to grow thus prompting this essential step in the body's normal repair of injuries32. The compound also encouraged rapid wound healing in rats and pigs32. Scientists working on a joint government-industry project at the Cooperative Research Center for Tissue Growth and Repair in Adelaide, Australia have patented a process for mitogenic bovine whey extract which is unique as a naturally-derived cocktail of growth factors in which 1000 L of milk yields 30 grams of extract. When the extract is mixed with a collagen gel and applied to wounds it enhanced the healing of surgical incisions. Clinical trials are currently underway in the United States at the Veterans Administration Medical Center in Bay Pines, Florida under the direction of Martin Robson, M.D. These observations have their origin in the fact that whey protein contains high levels of amino acids which may be important to wound healing. These amino acid include arginine, glycine, and particularly the branch-chained amino acids (BCAA) leucine, isoleucine and valine, which are essential to promote healing of bones, skin, and muscle tissues. Another amino acid, proline, aids in the production of collagen, heals cartilage and strengthens joints, tendons and cardiac muscle. 2. Glutathione In order to understand the mechanisms beneficial biochemical interactions and possible whey protein potential health benefits of WPC, a brief biochemical review of the protein glutathione is required, since it is this protein more than any other that has been thought to provide a protective function for a number of organ systems, including the crystalline lens of the eye, the retina, prostate gland, and the immune system. a. Glutathione Synthesis For the body to produce glutathione (GSH) six building blocks are required: L-glutamate, L-cysteine, L-glycine, magnesium, potassium, and 5' ATP. Two enzymes are also required and they are L-gamma-glutamyl-cysteine synthetase (equation 1) and glutathione synthetase (equation 2) and the reaction proceeds in the following manner: Mg 2+ (1) L-glutamate + L-cysteine + ATP---------> L-gamma -glutamylcysteine + ADP + P Mg2+

(2) L-gamma-glutamylcysteine + L-glycine +ATP-----------> K+ L-gamma-glutamylcysteinylglycine +ADP + P L-cysteine is the rate limiting substrate in this reaction12 , while the rate controlling enzyme for the reaction is L-gamma -glutamylcysteine synthetase5 . b. Scavenger Pathways The 2 major functions of glutathione are to detoxify hydrogen peroxide (H2O2) and other organoperoxidases (free radicals) and to defend against oxidation within cells via the Glutathione Redux Cycle, or more commonly referred to as the Scavenger Pathways 6,7. Glutathione plays it's role of "scavenger" through out the body. The role of scavenger is primarily accomplished through glutathione peroxidase (GSH px). The peroxidase interacts with the H2O2 to reduce it to harmless water, thus limiting it electron stealing capacity. This is illustrated in the following equation: (2) 2-glutathione-SH +ROOH ------> glutathione disulfide + ROH + H2O The disulfide is then reduced with the co-enzyme NADPH in the presence of the enzyme Glutathione Reductase to yield the original glutathione compound. (3) glutathione reductase +NADPH + H+ -----------> 2-glutathione-SH + NADP Many theories of aging and disease are based upon the interaction of the formation of free-radicals and the subsequent reduction in glutathione levels which allows for an accumulation of free-radicals to remain within a cell and organ or organ system. Free-radicals that remain within cells may cause cell damage, DNA damage and may even cause cell death, cancer transformation or loss of cell immunity to viral or bacterial infection. 3. Ocular Ramifications To fully appreciate the manner in which supplementation with WPC may be beneficial to maintenance of ocular tissues, let us look at the processes of cataractogenesis and oxidative insult to the retina. a. Cataracts 1. Mechanisms of cataractogenesis The exact mechanism of cataract formation follows a strict ordered sequence. Initially, in the pre-cataractous state, a cascade of early cataract-related changes occurs resulting in an oxidative insult to the cell membrane and to the amino acids methionine and cysteine 17-20. This can be caused by x-rays, photochemical insult, hyperbaric oxygen levels 18 and other causes. The remaining cascade of events then proceeds in the following manner: (a) unfolding of protein structures, which exposes the protein thiols making them available for further oxidation, (b) di-sulphide linked aggregate

formation, and (c) a decrease in enzymatic activity (glutathione peroxidase) which protects and repairs damage 17,18,20 . It is at this stage that the cataract truly begins to develop accompanied by a change in the lens Redux ratio, decreased ATP levels, and a change in cation permeability which induces an influx of H2O (water) within the cell membrane17-20 . The site of initial damage according to Spector18 occurs in the lens epithelium. The causative agent at this point is thought to be primarily the hydrogen peroxide radical, which in the absence of glutathione, may now accumulate unopposed. These free-radicals which are defined as molecules with an one or more unpaired electrons in their outer orbit, which attempt to stabilize their charge by stealing an electron from the outer shell of a stable neighboring molecule setting off a chain-reaction sequence6, causes DNA damage to the lens epithelium cells 21. Examination of lens epithelial cells at the time of cataract surgery usually reveals an ongoing pattern of programmed cell death (apoptosis)18 which may documented by evaluation of lens chromatin fragmentation21 . In the case of experimentally UV-induced cataracts, exposure to the UV radiation generated hydrogen peroxide, superoxide and DNA damage21,31 . The consequences of this action is to degrade the lens crystallin, decrease transmembrane voltage, reduce glutathione stores24 , decrease enzyme activity, and increase in prostaglandin production25 . It is interesting to note that no significant change in the non-protein thiols occurred until 85% of the lens epithelium cells were already dead21 . 2. Cataractogenesis and Glutathione The major role of glutathione in the crystalline lens of the eye is to provide lens clarity via maintenance of the limiting anterior epithelial cell layer and to correct and/or halt oxidative damage to the lens5.Arnold was perhaps the first investigator to discover the presence of glutathione in the lens, and it is now clearly evident that the highest concentration of glutathione is found in the lens epithelium6 , which has a 5X greater concentration than in the second most glutathione rich site, the lens cortex. The nuclear region of the lens is primarily devoid of GSH5,6 . It is well is established that lenticular glutathione levels decrease with age5,6,8 ranging from a concentration of 3.5 umol/g at age 20 and decreasing to 1.8 umol/g at age 65. It is inferred that it is this age related reduction in GSH which is in part responsible for cataract formation in the elderly. In fact, Reis was the first to note the lack of GSH in cataractous lenses5. In addition, a number of substances which inhibit GSH synthesis, such as Buthionine Sulfoximine16 produce cataracts in experimental conditions. It is interesting to note

that glutathione levels are unchanged in some reversible forms of cataracts, such as those induced after the administration of diquat5. Reduced GSH levels is a precipitating factor age-related cataract formation7. To further illustrate this point, researchers at Alcon Laboratories have used aldose reductase inhibitors to halt the progression of certain cataracts13. The importance in this finding is that aldose reductase acts competitively to reduce levels of GSH and its inhibition allowed GSH levels to remain high. As mentioned previously, glutathione and it's enzyme, glutathione peroxidase has great importance in regards to the elimination of free-radicals within the lens. Recent reports indicate that glutathione metabolizes chronic low levels of free-radical production26 typical of normal metabolism and the most potent anti-oxidant in the lens system. This point is illustrated by the inability of other enzymes, such as catalase to limit lens damage caused by hyperbaric oxygen27 and H202,28,29. Glutathione is essential for the maintenance of tissue ascorbate (Vitamin C) and alpha-tocopherol (vitamin E) levels 30 according to Mrtensson. He found that as GSH levels decreased, a corresponding decrease in ascorbic acid and vitamin E followed, which led to systematic mitochondrial death, which in turn leads to a cessation of cellular metabolism31. The regional distribution of glutathione in different cataracts is of interest. Pau et al 9 found that in primary nuclear and supranuclear cataracts, there was only a slight decrease in GSH levels relative to the subcapsular and 20 nuclear, implying a different mechanism for various cataract types and possibly a different mode of non-surgical intervention. Experimental data has shown that supplying the lens with additional glutathione, particularly in the form of the peroxidase increases delays cataract development and may even prevent cataract development23. This may be accomplished my enriching the aqueous with the glutathione precursor amino acids or glutathione directly10 . Glutathione in conjunction with vitamin E added as a supplement to a galactose diet did halt opacification of cortical cataracts in the animal model15. Further evidence for GSH infiltration into the lens comes from the identification of glutathione transporters in the lens epithelium11. Taking glutathione orally has not been shown to raise tissue levels of glutathione. Taking WPC which contains high levels of cysteine/cystine, the rate-limiting substitute for the production of glutathione, may be helpful for the prevention of cataracts. b. Macular degeneration It is widely accepted that lipid peroxidation plays a major role in retinal light damage58. The vertebrae

retina is known to contain relatively high levels of antioxidants and anti-oxidant enzymes59 . Among these are three members of the glutathione system, glutathione peroxidase, reductase, and transferase. The others are catalase and superoxide dismutase 60. When levels of these antioxidants begin to decrease with increasing age59,60 , retinal changes associated with macular degeneration begin to develop. This is particularly true for glutathione peroxidase and catalase59,60 . The method oxidative damage occurs is due to light or heat damage and then coupled with the age-related reduction in GSH(px) and catalase allows for the propagation and accumulation of the free radicals, making the retina susceptible to further damage60. Stone and Dratz 61 found evidence of glutathione-dependent enzymes in the outer segments of rats and suggest that since these membranes are rich in polyunsaturated fatty acids, they are susceptible to free-radical induced peroxidation. Therefore when RPE disruption occurs with early ARMD, which limits the amount of phagocytosis occurring, coupled with the age-related reductions in antioxidant activity, adverse retinal changes are manifested and may proceed unchallenged. It may therefore be possible to replenish or revitalize the antioxidant activity of the retina by dietary supplementation primarily with a glutathione or precursor to halt the progressive lipid perioxidation occurring during ARMD. c. Immunity Primarily the process of enhancing the immune response is accomplished through the replenishment of glutathione (GSH). It has been theorized that the ability of lymphocytes (CD4 cells) to correct oxidative damage is determined by their capacity to regenerate intracellular stores of glutathione which allows them to respond vigorously to a wide variety of antigens33. In 1981, researchers discovered that mice fed a non-denatured whey protein concentrate exhibited a marked increase in antibody production in response to T-cell dependent antigens36. Numerous experiments in subsequent years have confirmed this early observation 37,38,39,40,41,42,43,44. Thus, enhanced immunity against colds and hepatitis and most dramatically pneumococcal infection42 could be accomplished through dietary supplementation with whey protein concentrates (WPC's). An interesting peripheral observation was that the immunosustaining effect of the protein mixture found in whey was unrelated to its nutritional efficiency and as a result of this phenomenon this unique property was defined as the bioactivity of the product33. This bioactivity occurs through the ability of the

protein concentrate to help replenish glutathione levels via continuous dietary provision of glutathione precursors, especially cysteine/cystine, during lymphocyte proliferation, thus supporting an optimal immune response. This process seems to not only increase intercellular levels of GSH or GSH precursors at the time of ingestion, but also builds up stores of these substances within the cells which lasts for a substantial post-ingestion time interval33. The proposed bioactivity is dependent upon three (3) bioactive proteins contained in whey, serum albumin, alpha lactalbumin and lactoferrin. These proteins contain a high number of cysteine/cystine residues, an important GSH precursor. Serum albumin contains 17 cystine residues/66,000 MW molecule and 6 glutamylcysteine dipeptides39. Lactoferrin contains 17 cystine residues/ 77,000 MW molecule and four gluatmylcysteine dipeptides, while alpha-lactalbumin contains 4 residues per 14,000 Mw molecules 39. It is these residues which are primarily responsible for replenishment of GSH stores. d. Cancer Two major theories of oncology both implicate GSH as a putative protective factor due to its dual role as a antioxidant and detoxifying agent. Free radical accumulation is thought to be a major factor in tumor formation 45. In fact at least twelve (12) carcinogens have been identified that are detoxified by GSH conjugation. These are: aflaxotoxin B1, N-acetyl-2-aminofluorene, benzanthracene, benzopyrene, benzidine, dimethyl-hydrazine, 1-nitropyrine dimethylnitrosamine, ethylmethane sulfonate, N-methyl-4-aminobenzene, 7-methylbenzanthracene and 3-methyl-cholanthracene 46,47,48. Further evidence supporting the anti-tumor forming capacity of whey protein is illustrated by a University of Wisconsin study in which hormones known as androgens are responsible for depleting GSH levels in the prostate. This relatively GSH-free environment is thought to promote prostate carcino-genesis in men 49. This condition can be reversed in vitro by increasing colonial levels of GSH via continuous whey protein supplementation. e. Diseases of Aging As in carcinogenesis, free-radical accumulation has been implicated in producing a variety of diseases associated with aging 50.These maladies result from the toxic accumulation of these materials due to the absence or reduced levels of GSH. Diseases such as Alzheimer's 51, Parkinson's 52, and arteriosclerosis53 all appear to be preceded or associated with cellular organ or organ system reductions in GSH. Therefore much speculation has arisen regarding the potential benefits supplementation with whey protein may provide in these cases. If results obtained from other organ system studies are of any indication

results could be promising. f. HIV and AIDS The mechanism by which whey protein concentrate yields an enhanced immune response has already been discussed. Recently this knowledge has been applied to the treatment of HIV infected individuals. Staal et al34 reported that HIV-infected individuals have lower GSH concentrations in their blood lymphocytes, while Herzenberg et al35 found that the more glutathione patients carry in their CD4 helper T-cells, the cells primarily targeted by the HIV virus, the greater the chance of increased longevity exists. More recent claims have stated that supplementation with whey protein concentrates (WPC's) may help AIDS patients maintain body weight and in some cases limit wasting syndrome. In a pilot study WPC's were given to a population consisting of 14 AIDS infected children (ages 8 months-15 years) in an attempt to limit determine the efficacy of daily oral dietary ingestion of whey proteins. They found no toxic side effects and an average weight gain of 3.2 - 18% from their entrance weight54. A. Robert Neurath M.D. and his colleagues at the Laboratory of Biochemical Urology at Lindsley F. Kimball Research Institute of the New York Blood Centers has reported that a modified version of protein extracted from whey blocked the aids virus from infecting cells in vitro55. In their National Institutes of Health sponsored study the scientists modified beta- lactoglobulin to produce a substance referred to as B69. They reported that B69 latched onto a protein structure called CD4 on the surface of cells which kept the aids virus from using this site as an entryway into the cell. Newarth further hypothesized that if additional results with B69 were promising, the compound may be formulated into a cream or foam that could be dispensed within condoms to limit the transmission of the virus. Dr. Jefferey Laurance, an AIDS researcher at Cornell Medical College, however, urges caution regarding B69 developments. He states that HIV can infect some cells , including rectal and vaginal cells, without using the CD4 site as an entryway. In summary, animal studies have yielded promising reports regarding prolonged life spans of infected animals in the laboratory, while theoretical data56 and initial clinical observations in humans have produced evidence that dietary supplementation with WPC's can provide definite benefits to the HIV infected individual. Further research, however, is warranted in this area. REFERENCES 1 Choi, MJ. and Mangino, M.. The Effect of Pre-heating Conditions on the Gelation of Whey Proteins. Preliminary Report. Preprint. pgs 1-11, 1997.

2 Collins, K.. Is Whey Good for You? How do Cooking Oils Differ? Medical Tribune News Service, 1997. 3 Exports and Imports of Whey products. Statistics, Canada, Dairy Section, AAFC, 1997. 4 1 Nutrition, Frequently Asked Questions.AST Research Communique, 1997. 5 Rathburn, WB. Glutathione in Ocular Tissues. 469-510. 6 Rathburn, WB. Gutathione Metabolism int the Mammalian Ocular Lens. 194-206. 7 Reddy, VN. Glutathione and its Function in the Lens - An Overview. Exp Eye Res.50:771-778, 1990. 8 Hockwin, O. and Korte, I.Role of Glutathione in the Aging Process of the Lens. 207-214. 9 Pau, H., Graf, P., and Sies, H.Glutathione Levels in Human Lens: Regional Distribution in Different Forms of Cataract. Exp Eye Res 45: 17-20, 1990. 10 Harding, JJ. Free and Protein-Bound Glutathione in Normal and Cataractous Human Lenses. Biochem J. 117, 957-960, 1970. 11 Kannan, R., Yi, JR., Tang, D., Zlokovic, BV., Kaplowitz, N.. Identifcation of a Novel, Sodium-Dependent, Reduced Glutathione Transporter in the Rat Lens Epithelium. Invest Ophthalmol. Vis Sci 37:11: 2269-2275, 1996. 12 Rathburn, WB., and Murray, DL. Age-related Cysteine Uptake as Rate-limiting in Glutathione Synthesis and Glutathione Half-life in the Cultured Human Lens. Exp Eye Res. 53: 205-212, 1991. 13 Lou, MF., Dickerson Jr., JE., Garadi, R., and York Jr., BM.Glutathione Depletion in the Lens of Galactosemic and Diabetic Rats. Exp Eye Res 38:517-530, 1988. 14 Spector, A., Wang GM., and Wang, RR..Photochemically Induced Cataracts in Rat Lenses can be Prevented by AL-3823A, a Glutatthione Peroxidase Mimic. Proc. Natl. Acad. Sci. USA 90: 7485-7489, 1993. 15 Creighton, MO., and Trevithick, JR..Cortical Cataract Formation Prevented byVitamin E and Glutathione. Exp Eye Res. 29: 689-693, 1979. 16 Maitra, I., Serbinova, E., Trishler, H., and Packer, L.. Alpha-lipoic acid Prevents Buthionine Sulfoximide-induced Cataract Formation in Newborn Rats. Free Radical Biology and Medicine 18:4: 823-829, 1995. 17 Spector, A. Oxidation and Cataract.Symposium Paper. 1984 Human Cataract Formation. Ciba Foundation Symposium 106, 48-64, 1984. 18 Spector, A., Wang, GM., Wang, RR., Li, WC., and Kuszak, JR.. A Brief PhotochemicallyInduced Oxidative Insult Causes Irreversible Lens Damage and Cataract I. Transparency and Epithelial Cell Layer. Exp Eye Res. 60: 471-481, 1995. 19 Spector, A., Wang, GW., Wang, RR., Li, WC., and Kleiman, NJ.. A Brief Photchemically Induced Oxidative

Insult Causes Irreversible Lens Damage and Cataract II. Mechanism of Action. Exp Eye Res. 60: 483- 493, 1995. 20 Spector, A.. The Search for a Solution to Senile Cataracts Proctor Lecture. Invest Oph. and Vis Sci. 25: 130-146, 1984. 21 Li, WC., and Spector, A.. Lens Epithelial Cell Apoptosis is an Early Event in the Development of UVB-induced Cataract. Free Radical Biology and Medicine 20:3: 310-311, 1996. 22 Li, WC., Wang, GM., Wang, RR., and Spector, A.. The Redox Active Components of H2O2 and N-Acetyl-L-Cysteine Regulate Expression of c-jun and c-fos in Lens Systems. Eye Exp Res. 59:179-190, 1994. 23 Spector, A., Wang, GM., Wang, RR., Garner, WH., Moll, H.. The Prevention of Cataract Caused by Oxidative Stress in Cultured Rat Lenses. I H2O2 and Photochemically Induced Cataract. Curr Eye Res. 12:2: 163-179, 1993. 24 Hightower, KR., McCready, JP.. Mechanism Involved in Cataract Development FollowingNear-ultraviolet radiation of Cultured Lenses. Curr. Eye Res. 11:679-689, 1992. 25 Andley, UP.,, Herbet, JS., Morrison, AR., Reddan, JR, Pentland, AP.. Mechanism of Lens Epithelial Cell Proliferation by Enhanced Protaglandin Synthesis after UVB Exposure. Invest. Ophthalmol. Vis. Sci. 35 : 374-381, 1994 26 Tumminia, SJ., Chambers, C., Qin, C., Zigler Jr., S., and Russell, P. Curr Eye Res. 15: 845-851, 1996. 27 Giblin, FJ., Schrimscher, L., Chakrapani, B., and Reddy, VN.. Exposure of Rabbit Lens to hyperbaric Oxygen in Vitro: Regional Effects on GSH Level. Invest Ophthalmol. Vis. sci. 26:8: 1312-1319, 1988. 28 Giblin, FJ., Reddan, Jr., Schrimscher, L., Dziedic, DC., and Reddy, VN.. The Relative Roles of the Glutathione Redox Cycle and Catalase in Detoxification of H2O2 by Cultured Rabbit Lens Epithelial Cells.. Exp Eye Res. 51: 795-804, 1990. 29 Garland, D.. Role of Site-specific, Metal-catalyzedOxidation in Lens Aging and Cataract: A Hypothesis. Exp Eye Res. 50: 677-682, 1990. 30 Mrtenssson, J., and Meister, A.. Gluathione Deficiency Decreases Tissue Aswcorbate Levels in Newborn Rats: Ascorbate Spares Glutathione and Protects. Proc. Natl. Acad. Sci. USA 88:4656-46460, 1991. 31 Varma, SD., Kumar, S., and Richards, RD.. Light-induced Damage to Ocular Lens Cation Pump: Prevention by Vitamin C..Proc. Natl. Acad. Sci. USA 76: 3504-3506, 1979. 32 Daily InScight.. A Better Whey to Heal?. Academic Press, 1997. 33 Fidelus, RK., Tsan, MF.. Glutathione and Lymphocyte Activation: A Function of Aging and Auto-immune Disease. Immunology 61: 503-508, 1987.

34 Staal, FJT., Roederer, M., Israelski, DM., Bubp, J.. Intracellular Glutathione levels in T cell Subsets Decreases in HIV-infected individuals. AIDS Res and Hum Retroviruses 8: 305-311, 1992. 35 Herzenberg, L., De Rosa, S., Dubs, G., Roederer, M.. Glutathione Deficiency is Associated with Impaired survival in HIV Disease. Proc Natl Acad Sci USA 94: 1967-1972, 1997. 36 Bounous, G., Stevenson, MM., Konshavn, PAL.. Influence of Dietary Lactalbumin Hydrolsate on the Immune System of Mice and Resistance to Salmonellosis. J. Infect. Dis. 144: 282, 1981. 37 Bounous, G., Kongshavn, PAL.. Influence of Dietary Proteins on the Immune System of Mice. J. Nutr. 112: 1747-1755, 1982. 38 Bounous, G., Letourneau, L., Kongshavn, PAL.. Influence of Dietary Protein Type on the Immune System of Mice. J. Nutr. 113: 1415-1421, 1983. 38 Bounous, G., Kongshavn, PAL.. Differential effect of Dietary Protein Type on the B-cell and T-cell Immune Response in Mice. J. Nutr. 115: 1403-1408, 1985. 40 Bounous, G.,Shenouda, N., Kongshavn, PAL., Osmond, DG.. Mechanism of Altered B-cell Response induced by Changes in Dietary Protein Type in Mice. J. Nutr. 115: 1409-1417, 1985. 41 Bounous, G., Kongshavn, PAL., Gold, P.. The Immunoenhancing Property of Dietary whey Protein Concentrate. Clin. Invest. Med. 11: 271-278, 1988. 42 Bounous, G., Kongshavn, PAL.. Influence of Protein Type in Nutritionally Adequate Diets on the Development of Immunity. In: Friedman, M. ed. Absorption and Utilization of Amino Acids. Boca Raton, Florida: CRC Press. 2: 219-223, 1989. 43 Parker, N., Goodrum, KJ.. A Comparison of Casein, Lactalbumin, and Soy Protein Effect on the Immune Response to a T-dependent antigen. Nutrit. Res. 10: 781-792, 1990. 44 Hirai, R., Nakai, S., Kikuishi, H, Kawai, K.. Evalaution of the Immunological Enhancement Activities of Immunocal. Otsuka Pharmaceutical Co. Cellular Technolgy Insitiute, 1990. 45 Ritchie, JP.. The Role Glutathione in Aging and Cancer. Exp Gerontol 27: 615-626, 1992. 46 Newberne, PM, Bulter, WH.. Acute and Chronic Effects of Aflatoxin B1 on the Liver of Domestic and Laboratory Animal: A Review. Cancer Res 29: 236-250, 1969. 47 Meerman, JHN., Beland, FA., Ketterer, B., Srai, SKF.. Indentification of Glutathione Conjugates Formed from N-hydroxy-2-acetylaminofluorene in the Rat. Chem Biol Interact 39: 149-168, 1982. 48 Boyland, E., Sims, P.. The Metabolism of Benz(a)anthrracene and Dibenz (a,h)anthracene and Their 5,6 di-hydro Derivatives by Rat Liver Homogenates. Biochem J. 97:7-16, 1965.

49 Waterfall, JF., Sims, P.. Epoxy Derivatives of Aromatic Polycyclic Hydrocarbons. The Properties and Metabolism of EpoxidesRelated to Benzo(a) pyrene and to 7-8 and 9-dihydrobenzo(a)pyrene. Biochem J. 128: 265-277, 1972. 50 Blumberg, JB., Meydani, SN.. Role of Dietary Antioxidants in Aging. In: Nutrition and Aging. Hutchinson, MG., Munro, HN. (Eds.) New York: Academic Press, 85-97, 1986. 51 Jeandel, C., Nicholas, MB., dubois, F., Nabey-Belleville, F.. Lipid Peroxidation and Free Radical Scavengers in Alzheimer's Disease. Gerontology 35: 275-282, 1989. 52 Ebadi, M., Srinvasan, SK., Baxi, MD.. Oxidative Stress and Antioxidant Therapy in Parkinson's Disease. Proc Neurobiol 48: 1-19, 1996. 53 Kuzuya, M., Naito, M., Funaki, C., Hayahi, T.. Protective Role of Intracellular Glutathione Against Oxidized Low Density Lipoprotein in Cultured Endothelial Cells. Biochem Biophys Res Commun 163: 1466-1472, 1989. 54 CTN Trial Results. Whey protein Supplementation. Whey Protein Supplementation in HIV-infected Children: A Pilot Study. 1997 55 The Associated Press News Release. Whey Eyed as AIDS Blocker. The Associated Press, New York, New York, 1995. 56 Noelle, RJ., Lawrence, DA.. Determination of Glutathione in Lymphocytes and Possible Association of Redox state and Proliferative Capacity of Lymphocytes. Biochem J. 198: 571-579, 1981. 57 Balch, JF., Balch, PA.. Presciption for Nutritional Healing. Avery Publishing Group, Garden City Park, New York, 27-31, 1990. 58 Taylor, A., Jaques, PF.. Oxidation and Aging: Impact on Vision. In: Sies, H., Erdman, J., Williams, G. (Eds.) Proceedings International Conference on Antioxidants. Princeton, New Jersey, Univ. Press, 1992. 59 Newsome, DA., Miceli, MV., Liles, MR.. Antioxidants in the Retinal Pigment Epithelium. In: Prog Retinal Eye Res. 13: 101-123, 1994. 60 Dovrat, A., and Gershon, D.. Rat Lens Superoxide Dismutase and Glucose-6-phophate dehyrogenase: Studies on the Catalytic Activity and the Fate of Enzyme Antigen as a Function of Age. Exp Eye Res. 33: 651-655, 1981. 61 Stone, WL., and Dratz, EA.. Selenium and Non-selenium Glutathione Peroxidase Activities in Selected Ocular and Non-Ocular Rat Tissues. Exp Eye Res. 35: 405-412, 1982. 62 Rathbun, W.B., Holleschau, A.M., Cohen, J.F., Nagasawa, H.T. Prevention of Acetominophen and Naphthalene - Induced Cataract and Glutathorne Loss by CySSME. Invest Ophthalmol. 37:5: 923-9, 1996. **********************************************

WHEY PROTEIN ISOLATES and SELENIUM Australian Journal of Dairy Technology 1998, 53:37-47 There is evidence from animal and cell culture studies that milk proteins have anti-carcinogenic properties. This review discusses the anti-carcinogenic potential of milk proteins provided by whey protein isolate. Proteins supplying the sulfur amino acids cysteine and methionine appear to play a central role in tumor prevention. Milk components such as bovine serum albumin, alpha-lactalbumin and lactoferrin are also rich sources of gamma-glutamylcysteine, which can be utilized at the cellular level to synthesize glutathione. Selenium is an essential mineral in human nutrition. Selenium Yeast is a natural dietary selenium supplement with clinically proven physiological value in improving the selenium status in man. Selenium is a known anti oxidant and is an essential component of Glutathione peroxidase enzyme, which inactivates tree radicals. Selenium is required in thyroid metabolism while convertingactive thyroid hormone (14) into the inactive thyroid hormone (13). lnactivates heavy metals, especially mercury, lead and cadmium. Glutathione and its dependent enzymes play an importantrole in defense mechanisms that protect against cancer. The provision of glutathione precursors by whey proteins may explain the ability of dietary whey proteins to enhance both humoral and cell-mediated immune responses in laboratory animals. Whey contains growth factors with anti-cancer action at extremely low concentrations, and these compounds may survive digestion in sufficient quantities to generate a physiological response. Whey protein concentrate also contains the membrane phospholipid sphingomyelin, which has a strong ability to suppress tumor growth. These proteins contain exceptional amounts of cysteine (cys-cys) and glutamyl cysteine (glu-cys) (Somersall, and Bounous, 1999). Meister, 1983, demonstrated that the Glu-Cys precursors of GSH can easily enter the cell and therefore be synthesized into GSH. It thus becomes noteworthy that the most labile milk proteins - Serum Albumin and Lactoferrin - are those which contain these putative GSH-promoting peptide components (Baruchel et al, 1996). Interestingly, the Glu-Cys di-pepetide is an exclusive feature of the only obligatory foods in the early life of mammals and oviparous species, i.e. Milk and egg white respectively (Bounous and Gold, 1991) When undenatured, lactoferrin, serum albumin and Alpha lactalbumin contain almost the same number of cystine residues per total amino acid (Eigel et al, 1984;

Goodman and Schanbacher, 1991). Hence, in serum albumin, there are 17 cystine residues per 66,000 MW molecule, and six Glu-Cys dipeptides (Eigel et al, 1984); in Lactoferrin 17 cystine residues per 77,000 MW, and four Glu-Cys dipeptides (Goodman and Schanbacher, 1991); and in Alpha-Lactalbumin, four cystine residues per 14,000 MW molecule (Eigel et al, 1984). On the other hand beta-lactoglobulin has only two cystine residues per 18,400 MW molecule (Eigel et al, 1984), and IgG1, the predominant immunoglobulin in cows milk serum, only four disulfide bridges (cystine) per 166,000MW molecule (Baruchel et al, 1996). Undenatured whey proteins and more specifically, the cystine rich thermolabile proteins, represent an effective cysteine delivery system for the cellular synthesis of glutathione. Cellular GSH depletion has been implicated in the pathogenesis of a number of degenerative conditions and disease states including Parkinson's, Alzheimer's, arteriosclerosis, cataracts, cystic fibrosis, malnutrition, aging, AIDS, and cancer (Bounous and Gold, 1991). When GSH's metabolic functions are considered as a whole, it is easy to see how cellular GSH deficiency compromises health, performance and quality of life. Some of GSH's metabolic functions are as follows: enhancement of immune function, elimination of toxins, elimination of carcinogens, antioxidant cell protection, protection against ionizing radiation, DNA Synthesis and repair, protein synthesis, prostaglandin synthesis, Leukotriene synthesis, amino acid transport, enzyme activation and regulation (Gutman and Schettini, 1998). Whey protein isolate can provide those essential building blocks which are essential to GSH formation. It has been recently reported by Land et al, 1999 that augmenting the availability of cysteine may positively affect immune function. According to Drege and Holm, 1997 conditions that are associated with low cystine and glutamine concentrations also demonstrate decreased natural killer cell activity. GSH plays a central role in the functioning of immune cells, in particular it's creation and maintenance of T-cell lymphocytes, the body's frontline defense against infection (Gutman and Schettini, 1998). Bounous et al, 1993, reported that a proprietary WPI has potential therapeutic benefits in conditions leading to immunodeficiency. It was observed by these researchers that the humoral immune response of mice fed the proprietary WPI diet was almost five times greater than the corresponding values for mice fed a casein diet and a cysteine enriched casein diet. Augmented GSH production under conditions of homeostatic challenge will place demands upon available cysteine resources. Lymphocyte GSH levels and immune responsiveness can be influenced by feeding the rate limiting precursor of GSH, Bounous et al.,

1989. Their studies show that the administration of cysteine in the form of a proprietary WPI mixture was far more effective than when administering the rate limiting precursor of GSH as free cysteine. Fidelus and Tsan, 1986, showed that modulation of intracellular glutathione may indeed affect immune responsiveness. By maintaining high intracellular GSH levels, oxidative damage is minimized (Fidelus and Tsan, 1986; Gougerot-Pocidalo et al, 1988). In fact the capacity of a cell to recover from an oxidative insult is considered to be represented by its ability to regenerate intracellular stores of Glutathione (Noelle and Lawrence, 1981) This can also prevent disease or aid recovery. Even when an animal does not come down with an illness, it's immune system will be primed to fight it. In this way one can take advantage of both approaches to health, prevention and therapeutic. The mechanisms responsible for depletion of glutathione levels are varied and often times need to be corrected prior to establishing pre-depletion levels of glutathione or ultimately, optimum levels. Nutritional deprivation has a major effect on an animal's ability to maintain, and/or increase glutathione. For example, the biosynthetic supply of reduced glutathione is sufficient to withstand an inflammatory challenge in well-nourished animals but not in protein deficient or amino acid imbalanced animals. Also, the amount of free radical exposure or reactive oxygen derive molecules has a significant effect on how quickly glutathione levels can be replenished. The availability of cystine within the cell is the apparent rate limiting factor in the synthesis of glutathione to replenish the cell's store during the immune response or after oxidative molecule challenge. Given the nature of the active peptides, or "bioactive peptides", in whey protein, the oral administration of these peptides induces a rapid replenishment of glutathione (GSH) especially in the lymphocyte during the GSH-depleting development of the immune response. It is likely that immunoactive di-and tri- peptides can pass freely across the intestinal wall and react directly with peripheral lymphocytes. The amount of cellular (GSH) is a tightly self-regulated system because of the feedback inhibition of gamma-glutamylcystine synthetase activity by the GSH level (Richman and Meister, 1975). In reality, the Cystine delivery system of whey protein produces a substantial increase in cellular GSH up to but not above normal values. Whey protein releases Cystine or glutamylcystine in the small intestine and after transport in the blood plasma, these Cystine di-peptides effectively cross the cell's membrane. Cystine di-peptides remain stable in the circulation, unlike glutathione itself.

It is well accepted that the absorption of di- and tri- peptides from the gastrointestinal tract is an important biological phenomenon and a significant proportion of circulating amino acids are in the form of small peptides (Power and Murphy, 1999). Once liberated in the body, bioactive peptides perform an impressive array of regulatory functions and display a wide range of other activities. The bioactivity of whey protein is dependant upon a critical concentration of various bioactive proteins one of which being Lactoferrin. Several antimicrobial peptides can be derived from the protein Lactoferrin. Lactoferrin is a well-characterized iron-binding glycoprotein that occurs in mammalian body fluids, most notably milk. As an intact protein, its is considered to be an important host defense against microbial infections. Enzymatic cleavage of Lactoferrin has been demonstrated to produce three distinct peptides with antibacterial activity towards entertoxigenic E. coli. Two of these peptides display antimicrobial activity toward a number of pathogenic microorganisms, while a third peptide has been reported to inhibit the growth of Listeria monocytogenes. (Power and Murphy, 1999) The innate structure and composition of whey protein enable its bioactive proteins rapid and efficient absorption. The presence of milk derived peptides enables one to make scientifically viable conclusions. It is a well-known fact that part of the importance of dietary protein is manifested through intermediate biologically active peptides formed as a part of the degradation process in the gastrointestinal tract. Significant evidence may be found in the literature to support the notion of nutritionally important, bioactive peptides over free amino acids and intact protein have been reported in the literature (Siemensma et al., 1993). These include: Transport of short peptides across the intestinal wall is facilitated compared to free amino acids. Peptides are less hypertonic than free amino acids and this has the net effect of increasing their efficiency of absorption and reduces osmotic problems. Short peptides, in many cases, are less antigenic than larger polypeptides or the native protein from which they are derived. Selenomethionine is well absorbed and retained.From selenomethionine, all other needed selenium compounds are produced in the body. On the other hand, selenomethionine, like its sulfur analog, methionine, cannot be synthesized efficiently in the human organism. All quality nutritional selenium supplements should therefore contain selenomethionine Borglund, M and Akesson, B. 1988 Effect of selenium supplementation on the distribution of selenium in plasma proteins of health subjects. lnternat. J. Vit.

Nutr. Res. 58, 97-102. Clark, L C. et. al., 1996 Effects of selenium supplementation cancer prevention in patients with carcinoma of the skin. iouri American Medical Assoc. 276 (24), 1957-1963. Clausen, J. and Nielsen, S. A. 1998 Comparison of whole blood se nium values and erythrocytes of glutathione peroxidase activities normal individuals on supplementation with selenate, seleni L-selenomethionine and high selenium yeast. Biol. Trace EIem. R15, 125-138. Kumpulainen, i., Salmenpera, L, Siimes, M.A., Koivistoinen, P., a Perheentupa, i. 1985 Selenium status of exclusively breast-I infants as influences by maternal organic or inorganic selenium si plementation. i. Amer. Clin. Nutr. 42, 829-835. ******************************************

http://www.immunotec.biz/files/IMMUNOTEC/aArticle_18.pdf http://www.freewebs.com/whey/Athletic-Performance-and-Immunocal.pdf Word count: 5676 Glutathione and Exercise Christian Leeuwenburgh and Li Li Ji The Biodynamics Laboratory University of Wisconsin-Madison Madison, WI 53706 During exercise the increase in whole body oxygen consumption of 10-20 fold causes a severe disturbance of various biochemical pathways. The oxygen flux in individual muscle fibers is believed to increase by as much as 100-200 fold. This tremendous increase in oxygen consumption results in an increased leakage of electrons from the mitochondrial respiratory chain, forming various one-electron oxygen intermediates, such as superoxide anion, hydrogen peroxide and hydroxyl radicals. These reactive oxygen species are capable of triggering a chain of damaging reactions in the cell, such as lipid peroxidation, inactivation of certain enzymes, alteration of cellular oxidoreductive status, and oxidative damage to proteins and DNA. Reactive oxygen species are removed constantly in the cell by scavengers known as antioxidants. The most important antioxidants are vitamin E (a-tocopherol),

vitamin C (ascorbic acid), b-carotene, and glutathione (GSH). In addition, specialized enzymes catalyze the metabolism of reactive oxygen species preventing them from causing further damage. It is the balance between the production of reactive oxygen species and the levels of antioxidants that determines the oxidative stress induced by exercise. Biochemistry and Function of Glutathione Glutathione (L-g-glutamyl-L-cysteinyl-glycine; GSH) is a tripeptide found in virtually all animal cells. It has been the subject of much research since its discovery by Rey-Pailhade in 1888. Rey-Pailhade named the structure philothione (Greek for love and sulfur) because of its spontaneous reaction to sulfur. Eventually it was found that the molecule itself contained sulfur, and Hopkins named the structure glutathione. The elucidation of its structure was not resolved until around 1930. Initially it was thought to be a dipeptide containing glutamate and cysteine. The later elucidated tripeptide structure was hypothesized to be a reducing agent that maintains enzymes in an active state, and was involved in keeping various compounds like dehydroascorbate (vitamin C) and a-tocopherol (vitamin E) in the reduced state. The many enzymes involved in GSH metabolism and function were discovered in the following years. Now the studies of GSH and related enzyme systems have become an important area of biochemistry, nutrition, and pharmacology. Much of the work regarding the function, metabolism and regulation of GSH have been carried out in Cornell University by Professor Alton Meister and his colleagues. Although GSH is essential for normal cell function, most organs and tissues cannot synthesize GSH by themselves. Instead, GSH has to be taken up from extracellular source and imported into the cell. This process encompasses seven enzymes and crosses the cell membrane. The modern concept of GSH metabolism is depicted in Figure 1. The synthesis and degradation of GSH involve two separate pathways which form an interrelated cycle to maintain intracellular GSH homeostasis. This cycle is referred to as the g-Glutamyl Cycle. The majority of the various amino acids needed for the intracellular synthesis of GSH are transported into the cell. This first involves the degradation and transport of GSH at the cell membrane. The first enzyme involved in the cross-membrane transport of GSH is g-glutamyltranspeptidase (GGT). This enzyme cleaves the extracellular GSH into a glutamate amino acid bound to either a cystine or other available amino acid. The resulting peptide is then easily transported into the cell. The remaining cysteinylglycine (CysH-Gly) of the GSH molecule is transported into the cell and further cleaved into

CysH and Gly. GGT is predominately an external membrane-bound enzyme but various isoenzymes could exist inside the cell. The transport of GSH is largely dependent on the availability of GSH outside of the cell. The synthesis of GSH in the cell involves a series of reactions requiring a total of 2 ATP. The two major enzymes responsible for this synthesis are g-glutamylcysteine synthetase (GCS) and glutathione synthetase (GS), the former being rate-limiting. If the receptive amino acid is cystine, the so-formed g-glutamylcystine can bypass the GCS reaction and derive GSH directily via the GS reaction. Otherwise the imported g-Glu-AA is further metabolized to 5-oxoproline and then glutamate, which along with cysteine is a substrate for GCS. Feedback inhibition of GSH regulates the activity of GCS which seldom operates at maximal flux. Genetic deficiencies in GSH synthesizing enzymes may lead to life-threatening acidosis because of the accumulation of 5-oxyproline, a cytotoxic compound, which is formed by 5-oxyprolinase reaction. GSH has a broad spectrum of functions which include: (1) detoxification of exogenous and endogenous compounds, such as reactive electrophiles and peroxides with glutathione S-transferase and glutathione peroxidase; (2) reducing disulfide linkages of enzymes and proteins, thereby maintaining the essential thiols at a reduced status; (3) functioning as a nontoxic reservoir of cysteine and forming a vehicle for the transport of cysteine to all organs; (4) being actively involved in leukotriene and prostaglandin metabolism. (5) playing an important role in the reduction of ribonucleotides to deoxyribonucleotides, and (6) serving as a scavenger of certain free radicals. Figure 2 shows the major mechanism by which GSH removes hydroperoxides (including xenobiotic organic peroxide and lipid hydroperoxide). By accepting an electron from the peroxide (or donating a hydrogen ion), GSH is oxidized to half of a disulfide (GSSG) . This reaction is catalyzed by the Se-containing enzyme GSH peroxidase (GPX). The GSSG formed from these oxidizing reactions can be reduced back to GSH by the flavin-containing enzyme glutathione reductase (GR). This important intracellular step requires NADPH which is supplied by a reduction of NADP+, a relatively abundant cellular reducing power, via the hexose monophosphate pathway or an isocitrate dehydrogenase catalyzed reaction. Together, these reactions make GSH a "recyclable" antioxidant in the cell. In the liver, GSH S-transferase (GST) catalyzes the conjugation of GSH with a wide variety of xenobiotic toxins to form water-soluble compounds, thereby reducing oxidative stress inflicted by the metabolism of these prooxidants. When GPX activity is low, such as under

Se-deficiency, the importance of GST may rise due to its ability to remove certain organic peroxides. Recent evidence show that GSH is an efficient scavenger of hydroxyl radicals and singlet oxygen. In addition, GSH may be involved in the recycling of vitamin E radicals. When there is an excessive production of free radicals or reactive oxygen species under various physiological and pathological conditions, the concentration of GSH will fall whereas GSSG will rise, resulting in a decreased GSH/GSSG ratio. This dynamic alteration is indicative of an oxidative stress. Cellular Concentration of Glutathione GSH is the most abundant short-chain peptide and the most important non-protein thiol source in the body. Its concentration in the cell is in the millimolar range for most tissues. However, there is a great variability in GSH content in different body tissues depending on their function and oxidative capacity. The eye lens has the highest GSH concentration of approximately 10 mM among all tissues, probably due to its essential role to protect against photoradiation in this tissue. The concentration of GSH in the liver is between 5-7 mM. It is mainly synthesized within the hepatocytes. Kidney has about 3 mM and Heart about 2 mM, whereas the muscle has about 1-2 mM depending on fiber type. Red oxidative tissue e g. red vastus lateralis has 2 fold higher contents of glutathione (2 mM) then the white vastus lateralis. Skeletal muscles are highly heterogeneous in a variety of metabolic properties, such as oxidative potential, pattern of fuel utilization, and antioxidant enzyme activity. Therefore, the levels of GSH and GSSG as well as the ratio of GSH/GSSG, reflect these differences in metabolic characteristics. Concentration of GSH in the red blood cell is relatively high (~2 mM) compared to that of blood plasma (< 0.05mM). Since the red blood cell carries oxygen directly and hemoglobin contains large amount of iron, an important catalyst for free radical formation, high levels of GSH in the red blood cell is needed for adequate protection against potential oxidative damage. Most of the glutathione in the cell is kept in the reduced state in vivo, giving a high GSH/GSSG ratio. Fluctuations occurs when the cell is under oxidative stress, making the GSH/GSSG ratio a sensitive marker of cellular redox status. Transport of Glutathione Although a number of cells are capable of synthesizing GSH, liver by far is the most important organ for synthesize and transport of GSH de novo. It is estimated that 90% of the plasma GSH comes from

hepatic origin, where the various amino acids (Glu, Cys, Gly) from the portal blood are synthesized to GSH. The majority of the GSH synthesized is exported via exocytosis across the sinusoidal membrane into plasma whereas the remaining is excreted into the bile. Damage to the liver like cirrhosis will lead to a decreased efflux of GSH. Use of GSH by the liver is usually small but it's importance rises when free radicals are produced in metabolizing xenobiotic drugs. Furthermore, import of GSH is rather limited because of the low activity of GGT. Although human liver contains significantly higher amounts of sinusoidal GGT compared to other species such as rat, the amount is still not sufficient for significant import of GSH. The kidney on the other hand is the major consumer of plasma GSH. Anywhere from 70-90% clearance from the plasma occurs during first-pass extraction of GSH. It is also a high capacity system due to a large volume of rennin blood flow. It has been demonstrated that exogenously administered GSH is rapidly cleared within a short period of time, keeping plasma GSH within a narrow range. The constituent amino acids are taken up by cells of the proximal tubule and used for resynthesis of GSH, or returned to the liver and other organs. The lung and intestine also significantly contribute to plasma GSH turnover, because of the recent finding that a high concentration of GGT is present in the lung. Although muscle contains low activity of GGT as well as synthesizing enzymes of GSH, its large mass makes it also a potential consumer of circulating GSH, particularly when the muscle is under oxidative stress and extra antioxidant protection is required. Thus uptake of GSH by different organs and tissues is primarily dependent on the activity of GGT in the cell membrane. This is clearly demonstrated by the finding that an inhibitor of GGT can lead to a marked increase in GSH in blood and eventually the urine (glutathionuria). Humans with an inborn GGT deficiency also experience glutathionuria. Thus, glutathione is subject to constant turnover in the body, with the liver, kidney, lungs, heart, intestine, and muscle being the main organs responsible for GSH homeostasis (See Figure 3). In addition to supplying GSH for interorgan transport, the export of GSH through the membrane appears to also protect the cell membrane of the exporting tissues against oxidative and other types of damage. This is accomplished by maintaining thiols and other antioxidants (e.g. a-tocopherol) required for membrane integrity in the reduced state. The interorgan transport of glutathione is primarily in its reduced form (i.e. GSH) rather than GSSG. However, tissues do export GSSG when GSSG production is drastically increased and exceeds the reducing capacity of GSSG reductase. For example, liver

excretes large amounts of GSSG into the bile duct when a large dose of prooxidant xenobiotic drug is metabolized, thereby alleviating the oxidative stress the liver is exposed to. Increased levels of GSSG are deleterious and will disturb intracellular redox status and cause cross-linkage of proteins and nucleotides to form mixed disulfide. These alterations of cell structure can result in functional impairment of the cell, such as energy production and replication. In addition to liver, several other tissues also exhibit such active export of GSSG, including erythrocytes, eye, and heart, resulting in an increase in plasma GSSG. Plasma GSSG and the ratio of GSH/GSSG are therefore often used as an indicator of total body oxidative stress. It is hypothesized that during intense exercise ATP pool is markedly decreased thereby hampering the export of GSSG and eventually causing an increase of GSSG in the cell. This has been postulated as a potential factor for muscle fatigue. Dietary Requirement and Absorption of GSH It is known that GSH is an important anti-carcinogens and antioxidant in the mammalian cells. It detoxifies a variety of carcinogens and chemical toxins in the gastrointestinal tract. This makes GSH a little-known but important nutrient in the diet. Glutathione is present in many food sources like fresh fruits, vegetables, and meats. It is estimated that a daily human diet high in these food sources contain approximately 150 mg of GSH. At this time there is no dietary requirement of GSH in the US, but in Japan and some other countries GSH is nutritionally supplemented to provide an additional protection against xenobiotics in the diet. The additional GSH also provide an increased antioxidant level in the intestinal cells and in the blood. GSH absorption in the GI tract with GSH supplemented diets is studied quit extensively. Although the results show an increase in GSH in the blood after dietary supplementation, there is still some debate on the extent of the absorption. Scientists in the US have found significant increases in blood plasma concentration of GSH after oral GSH supplementation, indicating that transepithelial transport of intact GSH occurs in the small intestine, and that there might exist a tripeptide transport channel in the intestinal membrane. It is also well known that the bile excretes GSH into the small intestine to assist in detoxification. This process may allow dietary or billary derived GSH to be utilized directly by various organs via circulating blood. Moreover, it has been shown that oral administration of GSH may be therapeutically useful in instances of oxidative stress.

Although GSH can be absorbed by the GI track relatively efficiently, the uptake of GSH by the cell is limited by the cross-membrane transport systems and ultimately by the activities of the various enzymes involved. Thus, other possible route of GSH supplementation has been explored. It has been demonstrated that GSH monoethyl ester can be taken up by many types of cells as well as organelles, such as mitochondria, far more efficiently than GSH. Once absorbed, GSH is deesterified and GSH levels are increased inside the cell. This provide an efficient way to raise intracellular GSH concentration in cells which are deprived of GSH or exposed to an excessive oxidative stress. GSH exhibits a pronounced circadian rhythm. GSH synthesizing enzymes are highly active after a meal; the highest levels of GSH are reached 6 to 7 hours post absorption. This enhancement of GSH synthesis depends on the nutritional composition of the meal and the activity of the enzymes involved. Glutathione as an Antioxidant during Exercise In order to understand the role of GSH in protection against exercise-induced oxidative injury, we need first to understand why exercise can cause an oxidative stress to the body. There are a variety of mechanisms which may contribute to tissue and cell damage during strenuous aerobic exercise and intense physical training. Each of them likely plays an important role in the etiology of cell injury, but oxygen derived free radicals seem to cross link most of the mechanisms involved. The following are the most relevant processes occurring during and after an acute bout of strenuous exercise. (1) increased oxygen flux through the mitochondrial electron transport chain causing excessive leakage of one-electron reductants of oxygen, such as superoxide radicals; (2) direct mechanical damage to muscle protein and ligament stimulating activation of the lysosomal system and attracting polymorpho neutrophils at the site of damage, producing superoxide and hydrogen peroxide. This may contribute to the delayed injury and muscle soreness after exercise; (3) disruption of calcium homeostasis and activation of Ca2+ dependent proteases causing further tissue damage; (4) increased oxidation of intracellular thiols and glutathione leading to altered redox status and possible inactivation of certain enzymes; (5) certain types of exercise may cause redistribution of blood flow and local ischemic conditions resembling that of a heart ischemia-reperfusion induced injury. At present there is no clear consensus as to whether oxygen free radicals are the causes or results of the above-mentioned episodes, however, it is not difficult to perceive that a highly efficient antioxidant system

is critical in protection against the oxidative processes and stopping the vicious cycles occurring during and after heavy exercise. A major role of GSH in protecting tissues from the exercise-induced oxidative damage is to serve as a substrate for the Se-containing enzyme GSH peroxidase (GPX). It has been shown that under Se deficiency wherein GPX activity in muscle and liver tissues are reduced to minimum, increased lipid peroxidation occurs despite a normal level of GSH. The biochemical reactions involved in the GSH antioxidant function are summarized in Figure 2. During moderate exercise, the formation of GSSG as a result of the removal of hydroperoxide can be reversed by the GSSG reductase reaction, at the expense of NADPH. Since most cells have relatively abundant sources of reducing power (NADPH), accumulation of GSSG does not occur and the GSH/GSSG ratio is maintained at a high level (estimated to be 50-100). These mechanisms make GSH a "rechargeable" antioxidant. However, the balance can be disrupted if (1) hydroperoxide production is excessive, such as during exhaustive exercise; (2) GR-catalyzed reaction is impaired due to a lack of flavin protein (essential for GR synthesis) or when GR is inhibited by GSSG or NADP+. In addition to these enzymatically catalyzed functions, GSH is also an efficient scavenger to "quench" superoxide (O2.-) and hydroxyl radical (.OH), which can be formed during exercise. These processes are illustrated in the following equations. O2.- + 2GSH 2GS. + H2O2 .OH + GSH GS. + H2O Finally, GSH is capable of scavenging singlet oxygen (1O2). Because of the significant role of GSH in cellular protection against reactive oxygen species (a term used to include oxygen radicals and other toxic oxygen intermediates not qualified to be called free radicals), numerous studies have been conducted to elucidate its response, regulation and adaptive changes during acute and chronic exercise. For purposes of discussion, we will summarize these findings in the following categories: (1) an acute bout of exercise; (2) chronic training; and (3) GSH depletion and supplementation. An Acute Bout Of Exercise As early as 1985, GSH status (a term used to address the concentrations of GSH and GSSG, and the ratio of GSH/GSSG) of various body tissues was measured in rats following an acute bout of exhaustive exercise. It was found that GSSG was significantly increased after exercise in skeletal muscle, liver, and plasma. The investigators proposed that GSSG is an useful indicator of oxidative stress in the body. GSH was

slightly decreased in the liver and muscle whereas it was increased in plasma. The total glutathione levels (GSH+GSSG) in the blood was consistently higher after exercise. They hypothesized that the increase of blood GSH was due to an increase in GSH efflux from the liver. It was further suggested that GSH export from the liver could be stimulated by vasopressin, a hormone released during physical exercise. These authors later showed that during an acute bout of exhaustive exercise in rats, running time correlated significantly (r= - 0.79) with the GSH content in the liver and that depletion of hepatic GSH under these conditions was approximately 20%. The findings in the above-mentioned study have been confirmed by a number of subsequent studies in principle, although some discrepancies have also been generated. These controversies were primarily caused by the fact that different exercise intensities and durations were used by various investigators, causing the tissue responses to be different. It is now clear that, at least in skeletal muscle, GSH oxidation to GSSG is proportional to work load and there seems to be a threshold beyond which significant accumulation of GSSG in tissue can occur. This finding seems to be consistent with the scenario of GSH-GSSG recycle mechanism and the higher tissue GSSG reflects the inability of the cycle to cope with the excessive amount of hydroperoxide produced during exercise. Exercise duration seems to be an important factor in determining whether oxidative stress will take place. It was demonstrated in humans that maximal graded bicycle exercise lasting 10-15 min did little to alter blood GSH status, whereas sub maximal exercise at ~65% VO2max caused a significant increase in blood GSSG and a decrease in GSH. These findings were confirmed by experiments in rats. Consistent with the scenario that liver is exporting GSH during prolonged exercise, the authors laboratory has found that skeletal muscle content of GSH was increased along with a significant increase in GSSG, and that this increase in GSH was proportional to exercise intensity. It was hypothesized that exercising muscle takes up GSH from circulation to cope with an increased oxidative stress in muscle cells. To further support this notion, the authors showed that the ingredient amino acid of GSH, e.g. glutamate and cysteine, were also significantly elevated in muscle. GSH and GSSG responses to exercise also seemed to differ in various muscle fiber type, with the slow-twitch oxidative (type 1) muscle being more resistant to oxidation than the fast-twitch oxidative (IIa) fibers. This fiber-specific pattern may be related to the higher intrinsic GSH concentration in the former (~3 mM) compared to the latter (1-2 mM). Recruitment pattern during exercise may also play a role as the fast-twitch glycolytic

(IIb) does not show substantial alteration of GSH during prolonged exercise despite its low GSH level (<1 mM). In order to investigate the possible mechanisms involved in the regulation of hepatic and blood GSH status, an experiment was carried out in the authors' laboratory wherein human subjects rode on a bicycle to exhaustion with or without carbohydrate supplementation. It was found that subjects who had repeated oral glucose ingestion had blunt blood GSH response, whereas the controls demonstrated elevated GSH during the two and a half hour ride. Glucose supplemented subjects also had higher blood antioxidant enzyme activity and showed signs of greater oxidative stress. It was hypothesized that carbohydrate supplementation probably altered the normal hormonal responses during prolonged exercise, such as an increased glucagon and decreased insulin levels, thereby disrupting hepatic export of GSH into the blood. Although these studies provided evidence that interorgan GSH transport may occur during prolonged heavy exercise, two crucial pieces of information remain to be identified: (1) there is an activation of membrane transport of GSH, e.g., an increased GGT activity or a decreased Km for GSH, or both, during acute exercise; and (2) the elevated GSH in muscle cells is indeed of hepatic origin. So far there is little data to substantiate these hypothesis. It also has to be pointed out that some investigators believe that muscles export GSH into the plasma, instead of importing GSH from the plasma, during exercise to assist coping with the oxidative stress in the body . This hypothesis was supported by the notion that muscle and liver both have low GGT activity, and by their findings that plasma GSH levels were sustained during prolonged exercise in hepectomised rats. Given the large volume of muscle mass, this hypothesis seems to be attractive. However, the mechanism to export GSH from muscle cells has not been postulated and the signals stimulating muscle GSH efflux remain to be identified in this scenario. Chronic Training The effect of chronic exercise training on tissue GSH status and regulation has not been studied extensively and data in this topic is rather limited. Although the already published research suggests that training may have a profound impact on GSH status in various individual tissues and total body GSH homeostasis, the conclusions drawn from these studies were not consistent. Our previous studies show that highly trained human subjects, both in males and females, do not demonstrate significant disturbance in blood GSH status during prolonged exercise. This is in contrast

with the results with less trained or untrained individuals who had increased GSSG and decreased GSH/GSSG ratio. Similar results have been found in the rats wherein GSH levels in various major organs were better preserved in response to an acute bout of submaximal treadmill running after 10 weeks of training. It seems plausible that training has induced some mechanisms in the body which can maintain GSH homeostasis under acute oxidative stress. Because trained individuals have a higher rate of hepatic blood flow at a given work intensity compared to untrained individuals, a greater hepatic release of GSH is one of the most likely reason. An increase in GSH synthesis in the liver and a decrease of GSH consumption by other organs have also been postulated, but these explanations are largely speculative. With regard to skeletal muscle GSH status, an early study indicates that GSH concentration is significantly increased as a result of training in the red but not in the white skeletal muscle in rats. Recently, it has been reported that in Beagle dogs GSH content increased substantial in several muscle types after a rigorous training program (55 weeks). Consistent with these findings was an elevation of muscle GGT activity, an indication of enhanced GSH transport, and an increase in GSH synthesizing enzymes in the exercised skeletal muscle. The effect of training on muscle GSH status has recently been investigated in our laboratory. Using young, adult and old rats, we found GSH response to training was muscle fiber specific. The deep portions of the vastus lateralis muscle (type IIa) showed a significant adaptations of GPX and SOD activities and had no change in GSH content or GSH/GSSG ratio. Soleus muscle, on the other hand, showed a significant decrease of GSH content and the GSH/GSSG ratio, associate with a significant reduction of GGT activity with training. The discrepancies between our data and the others are not readily explainable, however, it points out the possibility that muscles involved in high intensity chronic exercise may have a deficit of GSH and that supplementation of GSH or analogs may be advisable in physically active individuals. Glutathione Depletion And Supplementation Because of the important role of GSH in the total body antioxidant function, depletion of GSH would be expected to exacerbate oxidative stress during prolonged exercise. Several methods are currently available to deplete the body GSH pool, each involving a different mechanism. The most effective one which has the least side effect is to use buthionine sulfoximine (BSO), developed initially by Dr. Meister. BSO inhibits the rate-limiting enzyme of GSH synthesis, i.e. g-glutamylcysteine synthetase thereby

dramatically decreasing GSH output by the liver . In a recent study in our laboratory, mice were injected i.p. with BSO (2 mmol/kg body wt) for 12 days along with 20 mM BSO in their drinking water. These treatment were found to deplete GSH content in liver, kidney, heart, and muscle to 23, 15, 10, and 7% of the normal values, respectively. The GSH-depleted mice and GSH adequate mice were then subjected to an acute bout of swimming to exhaustion. GSH content in the GSH adequate mice was decreased as a function of time in all tissues studied except quadriceps muscle which showed a slight increase. In the GSH depleted mice, exercise further lowered GSH levels in all tissues, accompanied with a higher MDA level than the GSH adequate mice. Furthermore, GPX activity was significantly elevated in the skeletal muscle of the GSH-depleted mice compared to the controls, whereas GST and GGT activity were significantly decreased with glutathione depletion. These alterations show that GSH-depletion can exacerbate the vulnerability of exercising muscle to reactive oxidants, while adaptations occur in related antioxidant system to compensate the loss of GSH. Since exercise decreases the reduced thiol pool in the body, including GSH, it might be beneficial to supplement thiols during acute or chronic exercise. Indeed, several compounds have been widely used in protecting ischemia-reperfusion caused myocardial oxidative damage and hyperoxia mediated lung injury, among these are N-acetylcysteine (NAC), GSH acetyl monoester and free GSH. Acute GSH injection i.p. at the dosages of 250-1000 mg/kg body wt has been reported to increase endurance time by 120-140% in swimming mice, although the level of oxidative stress was not determined in the study. NAC supplemented orally to human subjects for two days (total dose 2.4 g) was found to decrease GSSG levels provoked by an acute maximal exercise test. Regardless of the mechanisms in the mentioned studies, supplementation of exogenous thiol source may have potential merit in reducing oxidative stress during exercise. Conclusions Although the role of GSH in the biological systems has been studied extensively, its significance in relation with exercise physiology and sport medicine has not been fully appreciated until recently. From the available evidence accumulated in the past decade, it is now known that GSH is an important antioxidant protecting the cell against reactive oxygen species produced during aerobic exercise. The effectiveness of protection depends on both the concentration of tissue GSH and the capacity of the GSH-GSSG redox cycle under oxidative challenge. When there is an excessive production of hydroperoxide or when GSH content is

deprived in the cell, GSH/GSSG ratio will decrease markedly. This is indicative of an oxidative stress often accompanied with increased lipid peroxidation and disturbance of antioxidant enzyme systems. GSH is subject to constant turnover in the body. The liver exports GSH into the plasma whereas other organs and tissues take up GSH from the circulation. This interorgan transport facilitates the local needs of GSH to cope with a potential oxidative stress. There is evidence that skeletal muscle and heart increases its utilization of GSH during acute and chronic exercise, and that GSH efflux into plasma from the liver is enhanced. These regulatory mechanisms, however, can be interrupted by physiological, pharmacological, and nutritional interventions. Therefore, supplementation of GSH at appropriate levels and with proper means seems advisable in physically active individuals. References 1. Deneke, S. M. and B. L. Fanburg. Regulation of cellular glutathione. Am. J. Physiol. 257: L163-L173, 1989. 2. Gohil, K., Viguie, C., Stanley, W. C., Brooks, G. A., and L. Packer Blood glutathione oxidation during human exercise. J. Appl. Physiol. 64: 115-119, 1988 3. Hagen, T. M., G. T. Wierzbicka,, A. H. Sillau,, B. B. Bowman,, and D. P. Jones. Bioavailability of dietary glutathione: effect on plasma concentration. Am. J. Physiol. 259: G524-G529, 1990. 4. Ji, L. L. and R. G. Fu. and E. Mitchell. Glutathione and antioxidant enzyme in skeletal muscle: effect of fiber type and exercise intensity. J. Appl. Physiol. 73: 1854-1859, 1992. 5. Ji, L. L. and R. G. Fu. Responses of glutathione system and antioxidant enzymes to exhaustive exercise and hydroperoxide. Am. J. Physiol. 72: 549-554, 1992. 6. Kretzschmar, D., D. Muller,, J. Hubscher,, E. Marin,, W. and Klinger, (). Influences of aging, training and acute physical exercise on plasma glutathione and lipid peroxides in man Int. J. Sports Med. 12: 218-222, 1990. 7. Leeuwenbugh, C., R. Fiebig, R. Chandwaney, and L. L. Ji, (1994) Aging and exercise training in skeletal muscle: Response of glutathione and antioxidant enzyme systems. Am. J. Physiol. (Regulatory, integrative and Comparative Physiology) (in press) 8. Lew, H., S. Pyke,, and A. Quintanilha. Changes in the glutathione status of plasma, liver and muscle following exhaustive exercise in rats. FEBS, 185: 262-266, 1985. 9. Meister, A. Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy. Pharmac. Ther., 51, 155-194, 1991. 10. Pyke, S., H. Lew, and A. Quintanilha. Severe

depletion in liver glutathione during physical exercise. Biochem. Biophys. Res. Com. 139: 926-931, 1986. 11. Salminen, A., and V. Vihko. Endurance training reduces the susceptibility of mouse skeletal muscle to lipid peroxidation in vitro. Acta Physiol. Scand. 117, 109-113, 1983. 12. Sen, C. K., E. Marin, M. Kretzschmar, and O. Hanninen (1992). Skeletal muscle and liver glutathione homeostasis in response to training, exercise and immobilization. J. Appl. Physiol. 73:1265-1272. Figures Legends Figure 1. The g-Glutamyl-Cycle. AA, amino acids; GGT, g-glutamyltranspeptidase; GGC, g-glutamylcyclotransferase; 5-OP, oxoprolinase; GCS,g-glutamylcysteine synthetase; GS, glutathione synthetase; GSHT, glutathione transporter. Figure 2. Antioxidant function of glutathione. SOD, superoxide dismutase; GPX, glutathione peroxidase; GR, glutathione reductase; ICDH, isocitrate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; GST, glutathione S-transferase; GS., glutathione thiyl radical; X, compounds that react with GSH to form conjugates. Figure 3. Postulated outline of interorgan transport of glutathione. GS., glutathione thiyl radical; ROOH, hydrogen peroxide; ROH, alcohol; GGT g-glutamyltranspeptidase. http://www.sportsci.org/encyc/drafts/Glutathione.doc Glutathione Nature's Most Potent Ergogenic Aid By Paul Cribb B.H.Sci.HMS. Exercise Physiologist What if I told you science has already discovered natures most effective performance-enhancing, muscle building compound? What if I told you it is also physiologys most potent antioxidant, anti-aging, and anti-cancer compound? And what if I told you we have known about it for years? You have probably never heard of it because in supplement form it is useless to take. If youre too young to care about aging or decreasing cancer risks then think about this - research now demonstrates that without an abundant supply of this unique molecule in your body, your athletic performance plummets and you cannot build one ounce of muscle! This is a compound you dont have to buy. Even better, an array of studies demonstrate that if you provide your body with the correct nutritional material it will easily make tons of this precious stuff for you. Read carefully and Ill show you how to do it.

Glutathione - What is it? Research is confirming all these amazing properties and more about a little tri-peptide molecule called glutathione (GSH). In fact, over 300 published, peer reviewed journal articles have demonstrated that GSH is not only our body's premier antioxidant, it may well be the ever-elusive fountain of youth! Well, molecule of youth I suppose. Glutathione is found in virtually all plant and animal cells with highest concentrations in the liver, heart, eye lens, blood cells, and trained skeletal muscle. It must be synthesized within the cell because GSH cannot be transported into cells.1 GSH plays a central role in all antioxidant defense systems. It maintains a host of other anti-oxidants like vitamins C & E in their active form. Low cellular GSH levels are directly linked to various forms of cancer, Alzheimer's, Parkinson's and many other diseases normally associated with aging. In fact, low glutathione levels are so consistent with aging and degeneration they are used as cellular markers of health. Years ago studies showed that oral supplementation with the right nutritional material increased GSH levels. This reduced or eliminated various tumor/cancer growths and also dramatically improved the health of people with clinical illnesses. It has also been shown to increase the lifespan of healthy mice up to the equivalent of 33 human years! And now recent research has demonstrated that increasing GSH levels via precise supplementation may be the most potent ergogenic aid of all. A direct relationship between increasing GSH levels and enhancing exercise performance has recently been established. One well constructed study published in December last year demonstrated that healthy males and females taking an oral supplement designed to increase GSH levels not only increased these levels, it also significantly improved muscular performance in a variety of tests and the subjects grew muscle without performing any weight training! The researchers concluded that the enhanced performance was due directly to the increases in GSH and that the increased GSH levels decreased the amount of oxidative free radical damage that occurs during intense exercise. Antioxidants and Free Radicals - What Are They? In resting conditions oxygen content in arterial and venous blood of skeletal muscle is 20 and 15ml per 100ml of blood, respectively. Physical exercise increases the skeletal muscle arterio-venous oxygen difference by 3 fold and blood flow through tissue by 30 fold. As a result we may have up to a 100-fold increase in oxygen flux through active skeletal muscle during exercise. This unfortunately is a bad thing. Oxygen consumption and utilization involves a ton of

chemical reactions (the swapping /donating of electrons and molecule parts). As a result, oxygen particles "snap" or "fly off" and form highly reactive little buggers called "free radicals." These free radicals cause a heap of damage and destruction to our cells. A free radical is simply an oxygen molecule with an unpaired electron that desperately needs another electron to fill its outer shell. In fact, it is so desperate for this electron that it will rip an electron from any cell wall or lipid membrane surface. When this happens the free radical is happy or "quenched," but damage is done to the cell molecule that lost the electron and this disrupts its entire function. This process occurs a million times per second to every cell in your body 24 hours a day! Therefore you can imagine the kind of cellular damage Im talking about. However, things may not be as dramatic as that. Enter the anti-oxidant! An anti-oxidant is merely a molecule that easily donates its electron(s) to these free radicals neutralizing them into harmless molecules. Oxidative stress occurs when local antioxidant defenses are depleted. This depletion occurs because oxidants are being formed at a rate far greater than anti-oxidant systems can combat. This happens in skeletal muscle during intense exercise when oxidant/anti-oxidant balance shifts towards the pro-oxidant state. A mountain of research demonstrates intense or prolonged muscular exercise results in oxidative injury to lipids, proteins and DNA within skeletal muscle. The Role of GSH GSH is the center piece of all the bodys anti-oxidant defense systems. It not only directly neutralizes free radicals, it also donates its unique components to other anti-oxidant compounds like vitamins C & E1 and a host of antioxidant enzymes to maintain them in optimal working order. Low cellular levels of GSH mean all antioxidant defense systems will not work as well as they should. Over a prolonged period of time cells constantly bombarded by high levels of free radicals generated by such things as pollution, exercise, illness, junk-food, and other toxins do not function properly. The damage created at the DNA level impairs effective cell replication causing cells to die easier and quicker. This is the free radical theory of accelerated aging! The degenerative changes associated with aging are a result of the toxic effect of excessive free radical damage. Where as the data on age related changes in tissue when supplementing with vitamin E and other antioxidants are at best contradictory, the evidence demonstrating the importance of maintaining optimal GSH levels in your body is far more consistent. The

less GSH in your cells, the more poor your health and the quicker every cell in your body ages! Physical Exercise and Glutathione Levels If accelerated aging doesnt worry you, how about taking a look at what the latest reviews say about GSH levels and exercise performance. There is a direct association between exercise intensity and levels of oxidative stress. The harder you train the more free radical damage you produce and the more depleted your antioxidant defenses become, particularly muscle GSH levels. Studies on trained and untrained humans show the same thing - intense exercise devours our bodys stores of GSH. Condeming invivo studies with rats demonstrate exhaustive exercise depletes not only muscle GSH levels but also total body GSH pools (liver, muscle , blood, brain). Low GSH levels are disastrous to exercise performance. An array of studies demonstrate glutathione depleted bodies display only half the endurance of their counter parts. Muscles low in GSH also suffer far more oxidative damage. GSH deficiency weakens lipid phase and enzymatic antioxidants as well. Without optimal levels of GSH, free radical damage hits your muscles hard. Interestingly, humans and rats with high GSH levels maintain very favorable redox status in response to exercise induced oxidative stress. High levels of GSH in your body mean youll rip through your workouts and produce less damage to your cells in the process! This means faster and more muscle growth. A review of the literature shows that structured exercise training does get a resounding "thumbs up" in its ability to improve tissues defense against oxidative stress. This has also been linked to improved physical performance. Intelligent fitness programs that allow for optimal recuperation do produce a protective effect against free radical damage, and the more aerobically fit you are the more protection you have. This is one compelling reason bodybuilders should do aerobic work all year round. Dont worry, if you feed your body the correct ingredients and time your aerobics strategically, you wont lose muscle. However, I am also inclined to heed the words of Guvasto Bounous, the world's leading authority in GSH metabolism. In a recent report he concluded that the increased demand of the skeletal muscle imposed by frequent strenuous exercise deprives the immune cells of the capacity to replenish their cellular GSH levels - a prerequisite of optimal immune function. This diminished capacity to replenish GSH results in an undesirable competition between muscle and the immune system for sources of GSH. This easily creates a state of imbalance that quickly leads to prolonged poor performance. More serious repercussions are infections and illnesses such as Chronic Fatigue Syndrome.

Problems arise due to the fact that glutathione is utilized in such a vast array of physiological processes. It is directly involved in the immediate destruction of reactive oxygen compounds generated by illness and strenuous exercise. It detoxifies all foreign pollutants both dietary, such as alcohol, and environmental, such as carbon monoxide and heavy metals. It is an integral component of cell RNA and DNA proliferation1 and optimal immune response. It even protects from UV exposure (sunburn) and all other forms of radiation. However, it has been established that the necessary components needed to synthesize glutathione are extremely scarce in the fundamental diet. Clearly research shows that increasing your bodys GSH levels means less free radical damage and cellular destruction. Recovery is dramatically enhanced resulting in greater adaptation from training loads ultimately increasing physical performance. Increasing levels of GSH means optimal performance in every cell of every organ. This is the foundation of building a better, stronger, and more muscular body. GSH is utilized so extensively by the body yet only trace amounts of GSH precursors are found in regular food.1 Substantial GSH deficiencies are not uncommon among hard training individuals. Research has repeatedly shown that boosting GSH levels can be accomplished with the right supplementation. Does this sound intriguing? In the next article Ill show you how to increase your GSH levels naturally to power your performance, boost your immune function, build muscle, and keep you young. What more could you want? References: 1.Bounous G, Molson J. Competition for glutathione precursors between the immune system and the skeletal muscle: pathogenesis of chronic fatigue syndrome. Med Hypothesis 53;(4): 347-349 2.Bounous G et al. The influence of dietary whey protein on tissue glutathione and the diseases of aging. Clin Invest Med 12:343-9. 1989 3.Bounous G et al. Whey proteins as a food supplement in HIV-seropostive individuals. Clin.Invest. Med. Vol 16:3 p204-209. 1992. 4.Bounous G et al.The biological activity of undenatured dietary whey proteins: role of glutathione.Clin.Invest.Med. Vol 14:4.p296-309. 1991 5.Bounous G, G.Batist and P.Gold. Whey proteins in cancer prevention. Cancer Lett.57p91-94.1991 6.Blumburg JB, Meydani SN. Role of dietary antioxidants in aging. In Nutrition and Aging. Hutchinson MG, Munro HN (Eds). New York Academic Press, 85-97 1986. 7.Ebadi M, Srinivasan SK, Baxi MD. Oxidative stress

and antioxidant therapy in Parkinsons disease. Prog Neurobiol 48:1-19,1996 8.Jeandel C et al. Lipid peroxidation and free radical scavengers in Alzheimers diseaseGerontology 35:275-82 1989 9.Lands LC, Grey VL and Smountas AA. Effect of a cysteine donor on muscular performance.J Appl Physiol. 87 (4):1381-1385 1999 10.Lang CA, Naryshkin S, Schneider DL, Mills BJ et al. Low blood glutathione levels in healthy aging adults. J Lab Clin Med 120:720-5 1992 11.McIntosh GH et al. Dairy proteins protect against dimethylhydrazine-induced intestinal cancers in rats. J Nutr 125:809-16 1995 12.Powers SK, JI LL, Leeuwenburgh C. Exercise training-induced alterations in skeletal muscle antioxidant capacity:a brief review. Med. Sci. Sports Exerc. Vol31#7:987-997 1999. 13.Riederer P et al. Transition metals, ferritin, glutathione and ascorbic acid in Parkinsonian brains.J Neurochem. 52:515-20 1989 14.Sen CK. Glutathione homeostasis in response to exercise training and nutritional supplements. Molecular & Cellular Biochemistry. 196:31-42 1999 http://www.ast-ss.com/research/cribb/glutathione1-2-21-00.asp

I have been following whey protein research closely since it burst on to the commercial market about 10 years ago. Yet this amazing protein never ceases to astound me as science continues to reveal its new and exciting health benefits. Whey is already shown to be one of the best proteins for building muscle if correctly processed and this is very important. Most whey available on the store shelves is not correctly produced to yield the unique functional properties I'm about to explain. When whey protein is produced as a performance food it has the unique ability to boost glutathione levels, our most potent antioxidant. This is shown to directly increase athletic performance and increase lean muscle mass. Here are some of the latest findings you may not have known about whey. Whey protein supplements may help you cope better with stress. Increased brain serotonin levels improve the ability to cope with stress, while a decline in serotonin activity is associated with depression. Tryptophan is the amino acid that increases serotonin levels in the brain. Its transportation into the brain is dependent on other amino acids with a neutral charge. This influences tryptophan exchange via the ratio of tryptophan to the sum of other large neutral amino acids. A diet high in proteins that have high amounts of tryptophan may theoretically increase brain

serotonin levels. Twenty-nine (29) highly stress-vulnerable subjects and 29 other subjects participated in a double blind, placebo controlled study where all subjects were exposed to various experimental stresses after the intake of a whey protein (lactalbumin) or a casein supplement. Changes in mood, skin conductance, cortisol levels and plasma tryptophan/amino acid ratios were all assessed before and after the stress tests. The researchers chose to assess wheys lactabumin protein fraction as it possesses one of the highest concentrations of tryptophan of any known protein. The scientists were interested to see if a protein supplement could induce favorable brain serotonin levels. Results showed while the casein supplement produced little effect, the whey protein supplement increased the plasma tryptophan/amino acid ratio by 48%. In the stress vulnerable subjects the whey protein decreased cortisol levels and reduced depressive feelings and improved their ability to cope with the tasks presented. The scientists suggested the results obtained could only mean the whey protein supplement was able to increase brain serotonin levels. What does this mean to you? Although the word stress has many different meanings it does appear that serotonin levels in the brain influence mood and ability to cope with potentially stressful situations. A protein supplement that is high in lactalbumin has a natural ability to increase brain serotonin levels that produce beneficial effects in every day situations. So keep that whey protein shake handy to chug down next time you're stuck in a traffic jam or your boss calls you into his office. It may help you keep your cool and maintain a positive outlook! Wheys anti-cancer properties are twice as effective as soy. In an attempt to determine what protein possesses the best cancer fighting abilities, female rats were fed a diet containing either soy protein isolate, whey, or casein while the scientists attempted to induce tumors using the chemical 7,12-dimethylbenzanthracene. Also, the rats were mated with others fed the same protein to see if these protective effects could be passed on to the next generation. All rats grew well on these proteins. However, as the months went by, tumors developed in the casein and soy-fed rodents. The whey protein rats were virtually all clear. The whey and soy proved to be better than casein, while whey protein proved to be at least twice as effective as soy in reducing both tumor incidence and multiplicity. In many instances this protective effect of whey protein was able to be passed on to the second generation.

In another study published this year, lactoferrin (a whey protein fraction) was shown to inhibit colon carcinogenesis in male rats treated with another carcinogenic chemical azoxymethane.3 Most importantly these protective effects were demonstrated with easy to achieve, realistic amounts of the lactoferrin; about the same as contained in high quality whey oligopeptide formulations. Both a mere 2% and 0.2% of total protein intake proved highly effective in offering protection from the carcinogen. What this means to you? Wow, what a result in the first study! What a potent anticancer effect the whey possessed and the rats fed whey were actually able to pass on the protective effects of the whey to their offspring. These findings substantiate a series of studies performed with whey protein in the 80s and 90s that demonstrated wheys unique cancer fighting and prevention properties. With little doubt it appears that whey is a potent anticancer compound and should be your number 1 choice when it comes to protein selection for health whether you're male or female. Whey isolates are absorbed differently than other types of proteins. Researchers in Spain presented a rheokinetic study that examined the dynamic surface tension properties of whey isolate. Rheology and rheokinetics is the study of dispersion and flow of matter, in this case protein. Using constant temp (20o), pH (5) and ionic strength (0.05M) the results of the rheological parameters assessed show whey isolates possess different diffusion properties and interfacial unfolding actions to other proteins previously examined. However, these properties are related to the type of processing methods and concentrations used. Protein quality makes a difference. Choose your whey protein supplement carefully. Whey isolate is by far the safest bet in terms of effectiveness. While some companies put very misleading claims on the label and place a smidgen of whey isolate in their whey products, I know of only one that makes a 100% whey isolate - AST Sports Science. Conclusion Ask yourself, "Am I getting the results from my whey protein I should be getting?" Just because the label says "whey protein" does not mean you are getting the muscle building benefits of whey protein. There is a lot more to producing a whey protein supplement than just simply filtering away the fat. This may be fine for a "food grade" whey but not for for a performance protein supplement. This article points out just a few of the recent findings on the unique functional properties of whey protein and why it's not only important to use the

right whey protein, it's essential. If you're not, then you are robbing yourself of the real muscle building and health benefits the right whey protein possesses. Bargain hunting for whey protein is just like saying "Sorry muscles, this is all you get." References: 1.Am.J.of Clin.Nutr.71(6):1536-1544,2000. 2. Cancer Epidemiol Biomarkers Prev. 9(1):113-7, 2000. 3.Mutat Res 462(2-3):227-33,2000. 4. J.Agric Food Chem. 47(6):2241-8,1999. 5.J.Dairy Sci. 83(3):387-94,2000. ************************************************* Hepatitis and Glutathione ********************************************** Patricia A. L. Kongshavn, Ph.D Former Professor: Department of Medicine, McGill University, Montreal, Canada Infectious hepatitis is the most common of all serious infectious diseases in North America. Half a million Americans per year are estimated to contract the disease and, given the prevalence of a relatively new hepatitis virus C type, this number is likely to grow. Certain cases of infectious or toxic hepatitis may turn into chronic hepatitis, which poses a greater problem and may need treatment with steroids or interferon. Hepatologists know that glutathione plays a critical role in the liver. It is that organs most abundant antioxidant enzyme and glutathione concentrations are higher in the liver than in any other organ. This is because it is also the substrate for key detoxification processes in the liver. Glutathione neutralizes or conjugates the toxins, enabling them to be eliminated from the body through the gut or kidneys. Any impairment in this process allows buildup of toxins, leading to illness. Medical science has long known that glutathione deficiency invariably accompanies liver damage. Decreased liver production of glutathione is seen in alcoholic cirrhosis, sicknesses caused by exposure to hydrocarbons and other toxins, viral hepatitis, fatty livers and even aging individuals. Barbero et al. (1) described the systemic depletion of glutathione in

hepatitis C patients, suggesting that this deficiency could explain their resistance to interferon therapy. Ongoing research aims to raise glutathione levels in an attempt to improve liver function in patients with liver damage. Dentico et al. (2) showed that intravenous administration of high dose glutathione in patients with chronic steatosic liver disease significantly improved some liver function tests. Others such as Beloqui et al. (3) have seen benefit using the cysteine pro-drug N-acetylcysteine (NAC); by raising one groups glutathione levels with NAC, they showed that interferon therapy was enhanced. Watanabe et al. (4) treated hepatitis B and C patients with Immunocal for 12 weeks and found that plasma and lymphocyte glutathione concentrations increased to near control values in most of the patients by the fourth week. Immune parameters (NK cell activity and interleukin-2 levels) also improved, especially in the hepatitis B group. Immunocal is a safe, effective way to provide cysteine for the body to synthesize glutathione. It is proven to raise glutathione levels, is taken by mouth, and has no known side effects (unlike NAC). This is especially important in liver disease. References 1. Barbero G, Di Lorenzo G, Soldini M et al. Hepatic glutathione deficiency in chronic hepatitis C: quantitative evaluation in patients who are HIV positive and HIV negative and correlations with plasmatic and lymphocytic concentrations and with the activity of liver disease. Am J Gastroenterol 91:2569-73,1996 2. Dentico P, Volpe A, Buongiorno R et al. Glutathione in the treatment of fatty liver diseases. Recent Progr Med 86:290-293,1995 3. Beloqui O, Prieto J, Suarez M et al. N-acetylcysteine enhances the response to interferon-alpha in chronic hepatitis C: a pilot study. Journal of Interferon Research 13:279-282.1993 4. Watanabe, A, Higuchi K, Okada K et al. Third Department of Internal Medicine, Toyama Medical and Pharmacutical University, Toyama, Japan. Treatment of Chronic Hepatitis using whey protein (non-heated). Hepatology 241883,1996 Rev: 1001001 ******************************************************************** ******************************************************************** NAC for livers damaged by HCV TreatmentUpdate78 - Vol. 7, No. 8; June 1997 Sean Hosein

Background To protect cells from harmful chemical reactions, the body makes antioxidant enzymes using the molecule GSH (glutathione). Researchers in Italy have been studying the effect of hepatitis C virus (HCV) infection on the liver and the body's production of GSH. According to their results, people with chronic HCV have less than normal levels of GSH and may therefore benefit from supplements designed to boost GSH levels. Study details Doctors enrolled 156 adult subjects, 55 of whom were infected with both HIV and HCV, 50 others who had HCV infection alone and 51 healthy subjects with neither infection. Twenty-nine females and 127 males took part. Among the HIV-infected subjects, the average CD4+ cell count was 530 cells, while for those with HCV but not HIV, the figure was 720 cells. In the healthy group, the average was 750 cells. Results -- liver enzymes Levels of liver enzymes in the blood are often higher than normal in people with liver damage. Subjects with HCV in this study were no exception. The average level of the liver enzyme ALT was182 units/Litre (with or without HIV). In healthy subjects ALT levels averaged about 18 U/Litre. Results -- GSH On average, subjects with HIV and HCV had less than normal levels of GSH in their liver, blood and T cells, as did those with HCV alone. Moreover, subjects with HCV and low levels of GSH were more likely to have: * high levels of HCV * extensive liver damage The researchers think that levels of GSH in the liver could partly be responsible for the extent of liver damage caused by HCV. As well, T cells cannot function properly when their GSH levels are low. So researchers think that supplements of substances used to make GSH may be useful for people infected with HCV. To make GSH, the body needs the amino acid cysteine, which is found in eggs and dairy products including whey protein (sold as Immunocal / HMS 90). Another source of cysteine is the drug NAC (N-acetyl-cysteine) which is used in North America to treat livers damaged by high doses of Tylenol. As a general guide, several supplement programmes for PHAs suggest 1-2 grams/day of NAC. This drug is available on prescription in liquid form and is also available in capsules from some health food stores. REFERENCES: 1. Barbaro G, Di Lorenzo G, Soldini M, et al. Hepatic glutathione deficiency in chronic hepatitis C: quantitative evaluation in patients who are HIV positive and HIV negative and correlations with plasmatic and lymphocytic concentrations and with the

activity of liver disease. American Journal of Gastroenterology 1996;91(12):2569-2573. 970601 CATE7807 *************************************************************************** *************************************************************************** Hepatitis viral load correlates to glutathione levels. Abstract: Several recent scientific articles have found a direct correlation between Glutathione levels and viral activity for hepatitis B and C. When viral load increases, Glutathione decreases. Researchers from Germany report that adding NAC (N-acetyl cysteine) to HBV producing cells lines can reduce hepatitis viral load 50 fold. Glutathione is used by the liver to help break down toxins. Keywords: NEWSLETTER ARTICLE *Alternative Medicine Antigen Presentation *THERAPEUTIC USE Glutathione/*IMMUNOLOGY Hepatitis B/*IMMUNOLOGY Hepatitis C/*IMMUNOLOGY Human Viral Load 000730 A0070102 ************************************************************************************************************************ ******************************************** Glutathione Glutathione is hardly new; it is the cells primary antioxidant and is manufactured by the body. However, it is just now being recognized as perhaps the most important antioxidant. Glutathione is produced by the body from three amino acids found in foods: glutamic acid, cysteine, and glycine. It is found in every cell in the body. There are several million times more glutathione molecules in the cells than vitamin E. Glutathione is also found in the liver, where drugs, pollutants, alcohol, and other toxins are eliminated. In his book The Antioxidant Miracle, Lester Packer, Ph.D., perhaps the leading antioxidant researcher today, notes that glutathione plays many roles. It protects against damage that can lead to cancer and protects DNA, and is also an immune booster, as it increases the production of T cells-our primary disease-fighting cells. Glutathione also helps regulate the genes that cause chronic inflammation and lead to problems such as arthritis and autoimmune diseases. Glutathione is an essential part of the detoxification process-this is why it is found in large quantities in the liver. It has the ability to make a toxic compound water-soluble so it can be flushed out of the body via the kidneys. According to Packer, maintaining high levels of glutathione is critical for life-low glutathione levels are a marker for death at an early

age. Unfortunately, glutathione production by the body diminishes as we age, and glutathione supplements dont work. The key to getting enough glutathione is found in other supplements-such as alpha-lipoic acid and N-acetylcystine, which both spark glutathione production. Packer believes the best way to maintain healthy glutathione levels is through supplementing with alpha-lipoic acid. Alpha-lipoic acid Alpha-lipoic acid is a new antioxidant that may turn out to be one of the most important of all antioxidants. First of all, it is both water-soluble and fat-soluble. This means that it can access all parts of our cells, which have both water-soluble and fat-soluble components. This enhances its ability to destroy free radicals throughout the entire cell. Alpha-lipoic acid is especially powerful as an anti-aging substance. Aging can be described as a process that reduces the number of healthy cells in the body. The major factors in reducing healthy cells are free radical damage and glycation. Glycation is the process in which protein in our bodies reacts with excess blood sugar (glucose). This damage is as detrimental to our health as that caused by free radicals. Alpha-lipoic acid may keep blood sugar under control, which reduces glycation, and thus slow the aging process. Both free radicals and glycation affect the appearance of proteins found in the skin. Thus, a secondary benefit of alpha-lipoic acid (and all antioxidants) is that we look young for our age. Alpha-lipoic acid also works with other antioxidants to boost their levels. When you take alpha-lipoic acid, you also increase your levels of vitamins C and E, glutathione, and coenzyme Q10. According to Packer, alpha-lipoic acid can protect against stroke, heart disease, and cataracts; strengthen memory and prevent brain aging; and prevent and relieve complications from diabetes. N-acetylcysteine Although little-known, N-acetylcysteine (NAC) is a powerful antioxidant and a powerful tool in maintaining immunity. It has been used since the 1960s as a mucolytic; that is, a substance that breaks up mucus, especially in lung tissue, and has also been used for years in hospital emergency rooms to counteract acetaminophen poisoning. Acetaminophen is not all that NAC detoxifies. It also detoxifies such heavy metals as mercury, lead, and cadmium (J. Clin. Pharmacol. 13 (1973): 332-6), herbicides such as paraquat (Rev. Respir. Dis. 143, no. 4 part 2 (1991): A731), and some environmental pollutants. Clinical trials in Europe have indicated that NAC may also offer protection against the flu and flu-like

symptoms. Research into NAC also indicates that it may enhance the production of human T cells, an important part of the immune system.

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