New England Journal of Medicine
Vol. 349: 1605-1613 Oct. 23, 2003
Glutathione Peroxidase 1 Activity and Cardiovascular Events in Patients with Coronary Artery Disease
Stefan Blankenberg, M.D., Hans J. Rupprecht, M.D., Christoph Bickel, M.D., Michael Torzewski, M.D., Gerd Hafner, M.D., Laurence Tiret, Ph.D., Marek Smieja, M.D., Ph.D., François Cambien, M.D., Jürgen Meyer, M.D., Karl J. Lackner, M.D., for the AtheroGene Investigators
Background Cellular antioxidant enzymes such as glutathione peroxidase 1 and superoxide dismutase have a central role in the control of reactive oxygen species. In vitro data and studies in animal models suggest that these enzymes may protect against atherosclerosis, but little is known about their relevance to human disease.
Methods We conducted a prospective study among 636 patients with suspected coronary artery disease, with a median follow-up period of 4.7 years (maximum, 5.4) to assess the risk of cardiovascular events associated with base-line erythrocyte glutathione peroxidase 1 and superoxide dismutase activity.
Results Glutathione peroxidase 1 activity was among the strongest univariate predictors of the risk of cardiovascular events, whereas superoxide dismutase activity had no association with risk. The risk of cardiovascular events was inversely associated with increasing quartiles of glutathione peroxidase 1 activity (P for trend <0.001); patients in the highest quartile of glutathione peroxidase 1 activity had a hazard ratio of 0.29 (95 percent confidence interval, 0.15 to 0.58; P<0.001), as compared with those in the lowest quartile. Glutathione peroxidase 1 activity was affected by sex and smoking status but retained its predictive power in these subgroups. After adjustment for these and other cardiovascular risk factors, the inverse association between glutathione peroxidase 1 activity and cardiovascular events remained nearly unchanged.
Conclusions In patients with coronary artery disease, a low level of activity of red-cell glutathione peroxidase 1 is independently associated with an increased risk of cardiovascular events. Glutathione peroxidase 1 activity may have prognostic value in addition to that of traditional risk factors. Furthermore, increasing glutathione peroxidase 1 activity might lower the risk of cardiovascular events.
Ron Kennedy, M.D., received his certification from the American Board of Psychiatry and Neurology in 1975. Since that time, he has expanded his medical practice to cover a wide spectrum of health problems inclusive of cardiology.
Among the most interesting of recent findings from Western longevity researchers are those based on studies in Okinawa, a small island about 430 miles northeast of Taiwan and home of some of the world’s oldest and healthiest people. These findings are documented in The Okinawa Program, a massive scientific report that details why life expectancy on that small island is so long (Willcox 2001).
More centenarians live in Okinawa than any other place in the world: approximately 350% more than in the United States. The average life expectancy for Okinawan women is 86 years; for men, it is 78 years, a world record (Willcox 2001). It’s no accident: Researchers have found young-looking, clean arteries as well as healthy cholesterol, homocysteine, and blood pressure levels in these centenarians (Willcox 2001).
The Okinawan Diet: “The Antioxidant Network”
How do they do it? Perhaps the most important factor is diet. Green, leafy vegetables and high-protein foods such as fish are two staples of the Okinawan diet. These essential food groups contain an impressive array of prolongevity nutrients (Willcox 2001).
Nutrition specialists Dr. Lester Packer of the University of California, Berkeley, and Dr. Richard Passwater (1996) of Maryland’s Solgar Nutritional Research Center are calling those prolongevity nutrients the “antioxidant network.” This essential network—including vitamin groups, minerals, amino acids, and more—may be very important to longevity, as in-depth research suggests.
Amino Acids: The First Network Member
The amino acid glutathione often serves as a warrior in the body’s fight against free radicals and oxidative damage (Life Extension Foundation 1999). Glutathione is especially important to the cardiovascular system. Unfortunately, glutathione gets depleted by high levels of the compound homocysteine, which forms naturally during metabolism of another amino acid, methionine (Mayo Clinic 2002). Okinawans are able to control homocysteine levels through their low-fat, low-calorie diet (Willcox 2001).
One study, published in the American Heart Association journal Circulation, found that glutathione was able to produce a significant response in participants, especially in those people with coronary risk factors. These participants experienced an improvement in vascular function and arterial dilation (Kugiyama 1998). A report from the United Kingdom suggests that depleted glutathione levels are associated with an increased incidence of vascular cell damage. Take heart, however, as this “can be reduced when glutathione levels are restored” (Powell 2001).
Cysteine and glycine, found in foods such as fish, also are essential components of the antioxidant network (Willcox 2001). Studies are in their preliminary phases, but researchers from one prestigious East Coast medical school already have found that amino acids can be potent antioxidants. Examining the effects of these amino acids on bovine artery cells, the researchers found that cysteine and glycine are able to regulate oxidation, protect against dangerous free radicals, and boost intracellular levels of glutathione (Parinandi 1999). Researchers from the California Pacific Medical Center Research Institute have also shown that cysteine assists arterial flow and cardiovascular health in animal subjects (Holdefer 1994).
What Is Alpha-Lipoic Acid?
Once thought of as a vitamin, alpha-lipoic acid is a critical member of the antioxidant network. Many researchers tout alpha-lipoic acid as a “universal antioxidant” (Passwater 1996). It also is important to maintaining healthy levels of glutathione (Hultberg 2002).
According to Dr. Passwater (1996), alpha-lipoic acid has two primary functions: It metabolizes food, and it fights against oxidative damage. Indeed, an Italian study found that an antioxidant formula containing alpha-lipoic acid was able to promote cellular health and scavenge toxins in the bloodstream (Mosca 2002). One of the best sources of alpha-lipoic acid is high-protein foods such as fish, a mainstay of the Okinawan diet (Willcox 2001).
Vitamins and Minerals
Vitamins are likewise important antioxidant network members (Willcox 2001). A June 2002 report published in the renowned Journal of the American Medical Association provided solid affirmation of their power: Reviewing nearly 40 years of published studies, the researchers wrote of the many important benefits of several vitamin groups. “Inadequate intake of…vitamins,” they concluded, “has been linked to [many of the most prevalent health disorders in the world today]” (Fairfield 2002).
A placebo-controlled Indian study likewise gave a glowing notice for vitamins A, C, and E. Approximately 86% to 91% of the 175 subjects with cardiovascular problems saw benefits when administered these vitamins. The researchers concluded that “vitamins [A, C, and E] and fruits significantly decrease” low-density lipoprotein (“bad”) cholesterol levels and oxidation in people with cardiovascular problems (Singhal 2001).
Since Okinawans eat a diet rich in green, leafy plants, they also receive the benefits of selenium and zinc—additional components of the antioxidant network (Willcox 2001). These minerals also may reduce toxins and infections (Girodon 1997).
Maintaining Health and Longevity as Okinawans Do
Keeping optimal arterial function is an essential factor in maintaining health and longevity, and antioxidant supplementation may be one of the best ways to do so. But there is a wide array of available antioxidants, all with different specialties and benefits. Thankfully, researchers have discovered an antioxidant network that can maintain arterial health. Its dynamic formula allows you to combine the strengths of the many powerful antioxidants to reap the most fruitful rewards.
Fairfield, K.M. & Fletcher, R.H. (2002). “Vitamins for chronic disease prevention in adults: Scientific review.” JAMA; 287(23): 3116–26.
Girodon, F. et al. (1997). “Effect of micronutrient supplementation on infection in institutionalized elderly subjects: A controlled trial.” Annals of Nutrition & Metabolism; 41(2): 98–107.
Holdefer, M.M. et al. (1994). “Cardiotonic effects of reduced sulfhydryl amines after preservation of rabbit hearts.” Journal of Heart and Lung Transplantation; 13(1 Pt 1): 157–9.
Hultberg, B. et al. (2002). “Lipoic acid increases glutathione production and enhances the effect of mercury in human cell lines.” Toxicology; 175(1–3): 103–10.
Kugiyama, K. et al. (1998). “Intracoronary infusion of reduced glutathione improves endothelial vasomotor response to acetylcholine in human coronary circulation.” Circulation; 97(23): 2299–301.
Life Extension Foundation. (1999). “How selenium fights disease.” www.lef.org/prod_desc/seleniu1.htm.
Mosca, L. et al. (2002). “Modulation of apoptosis and improved redox metabolism with the use of a new antioxidant formula.” Biochemical Pharmacology; 63(7): 1305–14.
Parinandi, N.L. et al. (1999). “Phospholipase D activation in endothelial cells is redox sensitive.” Antioxidants & Redox Signalling; 1(2): 193–210.
Passwater, R.A. (1996). “Lipoic acid basics: An interview with Dr. Jim Clark.” Whole Foods; January: 1–5.
Powell, L.A. et al. (2001). “Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions.” Free Radical Biology & Medicine; 31(10): 1149–55.
Singhal, S. et al. (2001). “Comparison of antioxidant efficacy of vitamin E, vitamin C, vitamin A, and fruits in coronary heart disease: A controlled trial.” Journal of the Association of Physicians of India; 49: 327–31.
Willcox, B.J. et al. (2001). The Okinawa Program: How the World’s Longest-Lived People Achieve Everlasting Health—and How You Can, Too. New York, NY: Clarkson Potter.
A Healthy Heart (cardiovascular disease, cholesterol, atherosclerosis, stroke, etc.)
Lowering effect of dietary milk-whey protein
v. casein on plasma and liver cholesterol concentrations in rats
Zhang X, Beynen AC [Br J Nutr (1993 Jul) 70(1):139-46] The effect of dietary whey protein versus casein on plasma and liver cholesterol concentrations was investigated in female, weanling rats. Balanced, purified diets containing either whey protein or casein, or the amino acid mixtures simulating these proteins, were used. At the low dietary protein level, whey protein versus casein did not affect plasma total cholesterol, but lowered the concentration of liver cholesterol. At the high dietary-protein level, whey protein significantly lowered plasma and liver cholesterol and also plasma triacylglycerols. The hypocholesterolemic effect of whey protein was associated with a decrease in very-low-density-lipoprotein cholesterol. At the high dietary protein concentration, whey protein reduced the fecal excretion of bile acids when compared with casein. The effects of intact whey protein versus casein were not reproduced by the amino acid mixtures simulating these proteins. It is suggested tentatively that the cholesterol-lowering effect of whey protein in rats is caused by inhibition of hepatic cholesterol synthesis.
Serum Glutathione in Adolescent Males
Predicts Parental Coronary Heart Disease
John A. Morrison, PhD; Donald W. Jacobsen, PhD; Dennis L. Sprecher, MD; Killian Robinson, MD; Philip Khoury, MS; Stephen R. Daniels, MD, PhD [Circulation. 1999;100:2244] Traditional risk factors account for only half of the morbidity and mortality from coronary heart disease (CHD). There is substantial evidence that oxidative injury plays a major role in the atherosclerotic process. Thus, antioxidants may protect against development of atherosclerosis. Glutathione, an intracellular tripeptide with antioxidant properties, may be protective. This study found that low tGSH in adolescent boys is a significant independent predictor of parental CHD, in addition to elevated LDL cholesterol, low HDL cholesterol, and elevated total serum homocysteine concentrations.
Oxidant stress in the vasculature
Maytin M, Leopold J, Loscalzo J. [Curr Atheroscler Rep 1999 Sep;1(2):156-64] Vascular disease and vasomotor responses are largely influenced by oxidant stress. Numerous cellular antioxidant systems exist to defend against oxidant stress; glutathione and the enzymes superoxide dismutase and glutathione peroxidase are critical for maintaining the redox balance of the cell. However, the redox state is disrupted by certain vascular diseases. It appears that oxidant stress both promotes and is induced by diseases such as hypertension, atherosclerosis, and restenosis as well as by certain risk factors for coronary artery disease including hyperlipidemia, diabetes, and cigarette smoking. Once oxidant stress is invoked, characteristic pathophysiologic features ensue, namely adverse vessel reactivity, vascular smooth muscle cell proliferation, macrophage adhesion, platelet activation, and lipid peroxidation.
Erythrocyte susceptibility to lipid peroxidation in patients with coronary
Dincer Y, Akcay T, and others. [Acta Med Okayama 1999 Dec;53(6):259-64.] "Erythrocyte TBARS production was significantly higher in patients with coronary atherosclerosis than in the controls. On the other hand, the levels of plasma high-density lipoproteins, vitamin C, vitamin E and erythrocyte GSH were significantly lower, and the levels of plasma total cholesterol, triglycerides, low-density lipoproteins and TBARS were significantly higher in the patients with coronary atherosclerosis than in the controls. In conclusion, our results indicate that erythrocytes from patients with coronary atherosclerosis are more susceptible to oxidation than those of controls and that these patients have lowered antioxidant capacity as revealed by decreased plasma levels of vitamins C and E."
L-2-oxothiazolidine-4- carboxylic acid reverses endothelial dysfunction in
patients with coronary artery disease
Vita JA, Frei B, and others. [J Clin Invest 1998 Mar 15;101(6):1408-14.] "Cellular redox state...is a potential target for therapy in patients with coronary artery disease." These data suggest that augmenting cellular glutathione levels improves EDNO action in human atherosclerosis. Cellular redox state may be an important regulator of EDNO action, and is a potential target for therapy in patients with coronary artery disease.
Glutathione infusion enhances
coronary blood flow during oxidative stress
[Circulation 1998;97:2299-2301] Oxygen free radicals cause endothelial vasomotor dysfunction. Investigators found that reduced glutathione intravenously suppresses constriction of human coronary arteries in response to acetylcholine and increases the effect of nitroglycerin. Glutathione administration may be useful in patients with coronary artery disease, both as a result of improvement of endothelial dysfunction and augmentation of nitroglycerin-induced vasodilation and antiplatelet activity.
Serum glutathione in adolescent males predicts parental coronary heart disease
Morrison JA, Jacobsen DW, Sprecher DL, Robinson K, Khoury P, Daniels SR. [Circulation 1999 Nov 30;100(22):2244-7] There is substantial evidence that oxidative injury plays a major role in the atherosclerotic process. Thus, antioxidants may protect against development of atherosclerosis. Glutathione, an intracellular tripeptide with antioxidant properties, may be protective. This case-control study compared total serum glutathione (tGSH) in 81 adolescent male offspring of parents with premature CHD (ie, before 56 years of age) and 78 control male offspring of parents without known or suspected CHD. Case offspring had significantly lower tGSH than control offspring. Low tGSH in adolescent boys is a significant independent predictor of parental CHD, in addition to elevated LDL cholesterol, low HDL cholesterol, and elevated total serum homocysteine concentrations.
Glutathione reverses endothelial dysfunction and improves nitric oxide
Prasad A, Andrews NP, Padder FA, Husain M, Quyyumi AA. [J Am Coll Cardiol 1999 Aug;34(2):507-14] We investigated whether glutathione (GSH), a reduced thiol that modulates redox state and forms adducts of nitric oxide (NO), improves endothelium-dependent vasomotion and NO activity in atherosclerosis. Endothelial dysfunction and reduced NO activity are associated with atherosclerosis and its clinical manifestations such as unstable angina. Thiol supplementation with GSH selectively improves human endothelial dysfunction by enhancing NO activity.
Macrophage foam cell formation during early atherogenesis is determined by the
balance between pro-oxidants and anti-oxidants in arterial cells and blood
Aviram M. [Antioxid Redox Signal 1999 Winter;1(4):585-94] Atherosclerosis is a multifactorial disease, where more than one mechanism, along more than one step, contributes to macrophage cholesterol accumulation and foam cell formation, the hallmark of early atherogenesis. Intervention to inhibit LDL oxidation can affect the above additional LDL modifications. The balance between pro-oxidants and anti-oxidants in the LDL particle, as well as in arterial wall macrophages (such as NADPH oxidase vs. glutathione), determines the extent of LDL oxidation. Antioxidants can protect LDL from oxidation not only by their binding to the lipoprotein, but also following their accumulation in cells of the arterial wall.....the combination of antioxidants together with active paraoxonase decreases the formation of Ox-LDL and preserves PON1's ability to hydrolyze this atherogenic lipoprotein and hence, to attenuate atherosclerosis.
Hyperglycemia in diabetic rats reduces the glutathione content in the aortic
Tachi Y, Okuda Y, Bannai C, Bannai S, Shinohara M, Shimpuku H, Yamashita K, Ohura K. [Life Sci 2001 Jul 20;69(9):1039-47] The glutathione redox cycle plays a major role in scavenging hydrogen peroxide (H2O2) under physiological conditions. Recently, we demonstrated that a high glucose concentration in the culture medium reduced the level of H2O2 scavenging activity of human vascular smooth muscle cells (hVSMCs). We also showed that a high glucose concentration reduced the intracellular glutathione (GSH) content and the rate of uptake of cystine, which itself is a rate-limiting factor that maintains the GSH level. In the present study, we investigated whether the hyperglycemic condition in diabetic rats impairs the glutathione content in the aortic tissue in vivo. We demonstrated in vivo that the hyperglycemic condition in STZ-induced diabetic Wistar rats and OLETF rats reduced the GSH content in aortic tissue. This suggested reduced glutathione redox cycle function of aorta.
Effect of administration of fermented milk
containing whey protein concentrate to rats and healthy men on serum lipids and
Kawase M, Hashimoto H, Hosoda M, Morita H, Hosono A. [J Dairy Sci 2000 Feb;83(2):255-63] The effect of fermented milk supplemented with whey protein concentrate on the serum lipid level of rats was investigated. The serum total cholesterol level for the group fed fermented milk was significantly lower than that of the control group in rats. After 8 wk, the high density lipoprotein cholesterol level for the fermented milk group showed a significant rise after 4 wk , whereas that of the placebo group showed no change even after 4 wk. The triglyceride level for the fermented milk group lowered significantly after 4 wk, whereas that of the placebo group showed no change even after 4 wk. The atherogenic index [(total cholesterol - high density lipoprotein cholesterol)/high-density lipoprotein cholesterol] for the fermented milk group decreased significantly from 4.24 to 3.52. The systolic blood pressure lowered significantly by the intake of fermented milk. On the other hand, such effect was not observed in the placebo group. These results indicate potential of the development of fermented milk with multiple therapeutic effects.
Effect of milk protein and fat intake on blood pressure and the incidence of
cerebrovascular diseases in stroke-prone spontaneously hypertensive rats (SHRSP)
Ikeda K, Mochizuki S, Nara Y, Horie R, Yamori Y. [J Nutr Sci Vitaminol (Tokyo) 1987 Feb;33(1):31-6] The intake of two milk protein-rich diets containing casein and whey protein attenuated the development of severe hypertension in stroke-prone spontaneously hypertensive rats (SHRSP), and extended their life span in comparison with SHRSP on a regular stock diet. Milk fat-rich diet intake reduced the incidence of cerebrovascular disease in SHRSP without a significant fall in blood pressure. These results suggest that certain milk components have a preventive effect on hypertension and cerebrovascular disease in SHRSP.
Angiotensin I-converting enzyme inhibitory properties of whey protein digests:
concentration and characterization of active peptides
Pihlanto-Leppala A, Koskinen P, Piilola K, Tupasela T, Korhonen H. [J Dairy Res 2000 Feb;67(1):53-64] The aim of this study was to identify whey-derived peptides with angiotensin I-converting enzyme (ACE) inhibitory activity. The bovine whey proteins alpha-lactalbumin and beta-lactoglobulin were hydrolysed with pepsin, trypsin, chymotrypsin, pancreatin, elastase or carboxypeptidase alone and in combination. Whey protein digests gave a 50% inhibition (IC50) of ACE activity at concentration ranges within 345-1733 micrograms/ml. The IC50 values for the 1-30 kDa fractions ranged from 485 to 1134 micrograms/ml and for the < 1 kDa fraction from 109 to 837 mg/ml. Several ACE-inhibitory peptides were isolated from the hydrolysates by reversed-phase chromatography, and the potencies of the purified peptide fractions had IC50 values of 77-1062 microM. The ACE-inhibitory peptides identified were alpha-lactalbumin fractions (50-52), (99-108) and (104-108) and beta-lactoglobulin fractions (22-25), (32-40), (81-83), (94-100), (106-111) and (142-146).
Lactokinins: whey protein-derived ACE inhibitory peptides
FitzGerald RJ, Meisel H. [Nahrung 1999 Jun;43(3):165-7] Angiotensin-I-converting enzyme (ACE) has been classically associated with the renin-angiotensin system which regulates peripheral blood pressure. Peptides derived from the major whey proteins, i.e. alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in addition to bovine serum albumin (BSA), inhibit ACE. While they do not have the inhibitory potency of synthetic drugs commonly used in the treatment of hypertension, these naturally occurring peptides may represent nutraceutical /functional food ingredients for the prevention/treatment of high blood pressure. Studies with gastric and pancreatic proteinase digests of whey proteins indicate that enzyme specificity rather than extent of hydrolysis dictates the ACE inhibitory potency of whey hydrolysates.
Glutathione deficiencies exacerbate response to stroke
Phyllis G. Paterson, University of Saskatchewan - Saskatoon. Canada [Invited Symposium: The Therapeutic Potential of Phase II Enzyme Induction] The cascade of events responsible for the death of neural cells following a stroke include depletion of ATP, glutamate excitotoxicity, calcium overload, and production of strong oxidants that can overwhelm antioxidant defense. Glutathione (GSH) has a central role within the finely tuned network of antioxidant systems that can respond to the oxidative insult through its functions in peroxide scavenging via glutathione-S-transferase and the family of glutathione peroxidases, regeneration of alpha-tocopherol, and inhibition of NFkB which is required for the expression of pro-inflammatory genes. Our laboratory has used a nutritional approach to study the effects of GSH depletion in a rat model of stroke. We have found that a deficiency of sulfur amino acids used as a model of reduced cysteine supply for synthesis depresses GSH concentration in a number of brain regions. A second study demonstrated that acute sulfur amino acid deficiency exacerbates brain damage in a rat model of global hemispheric hypoxic ischemia. Approaches described in the literature for maintaining GSH under ischemia conditions have also been targeted towards increasing its synthesis. Administering a GSH ester immediately after an ischemic insult offered neuroprotection in one study as did the delivery of N-acetylcysteine, a compound that supports GSH synthesis by acting as a cysteine precursor. The series of studies reviewed suggests that GSH is an important determinant of the extent of secondary tissue damage in animal models of stroke. Strategies to enhance GSH in brain should be tested for their therapeutic efficacy in the human condition of stroke. (Funded by the Heart and Stroke Foundation of Saskatchewan)
Milk whey protein decreases oxygen free radical production in a murine model of chronic iron-overload cardiomyopathy
Bartfay WJ, Davis MT, Medves JM, Lugowski S. [Can J Cardiol. 2003 Sep;19(10):1163-8.]
Chronic iron overload is a major cause of organ failure worldwide, but its pathogenesis remains to be elucidated. Mice receiving iron treatments with whey supplementation had significantly lower concentrations of cytotoxic aldehydes and significantly higher cardiac levels of GPx and GSH activity than did iron-only treated mice. Additional basic research is warranted to examine the exact mechanisms by which milk whey protein protects the heart.
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