Glutathione: New Insights into Its Key Role in Regulation of Oxidative Stress and Age-Associated Inflammation
— Richard P. FeynmanBut Scientists, who ought to know,
Assure us that they must be so. . . .
Oh! let us never, never doubt
What nobody is sure about!
— Hilaire Belloc
From “The Microbe” (1912)
D&S comment: You can always try to get around the problem by calling everybody who disagrees with you a “skeptic” or “denier.” Oh! let us never, never doubt . . .
Before I came here, I was confused about this subject.
Having listened to your lecture, I am still confused.
But on a higher level.
— Enrico Fermi
Practical Do-It-Yourself Information—
Durk & Sandy’s Anti-Aging Regimen
If there were an anti-aging pyramid (like the food pyramid) for the elements of an anti-aging regimen, down at the base would be the antioxidant glutathione. As we discuss below (in the “Increasing Glutathione Levels” section of this article), supplementation with the amino acid cysteine is our own approach to increasing glutathione levels.
The importance of glutathione—a tripeptide comprised of glutamine, cysteine, and glycine (L-gamma-glutamyl-cysteinyl-glycine)—the major cell antioxidant, is well known, as are many of the effects (such as immune system depression) of cellular glutathione depletion. A new paper1 provides evidence of the decline in glutathione levels with aging (and the resulting increase in oxidative stress) as a major causative factor in the increased inflammatory activity with aging. (See below under “Decline in Glutathione Levels and Increased Inflammation with Aging.”)
Glutathione and Atherosclerosis
A recent paper2 reports that in the atherosclerosis-prone aortic arch of male ApoE-deficient mice, a commonly used model of atherosclerosis, glutathione depletion starts very early (10 weeks), as compared to age-matched wild-type controls. The glutathione depletion precedes the lipid peroxidation and detectable atherosclerotic lesions by several months. Moreover, the reduced synthesis of glutathione was associated with increased oxidative stress and reduced transcription and activity of the rate-limiting enzyme for glutathione synthesis, gamma-glutamylcysteine ligase, together with the glutathione-dependent antioxidant enzyme glutathione peroxidase. The authors conclude, “We suggest that glutathione deficiency is central to the failure of the intracellular antioxidant defenses and is causally implicated in the pathogenesis of atherosclerosis.”
Glutathione and Immune Function
It is well known that many bacterial and viral infections (e.g., herpes simplex, HIV, influenza) are preceded by sharp decreases in intracellular glutathione levels and that this results in oxidative stress and immune depression.3 Increasing glutathione levels (which, in this study, was done with supplemental glutathione) was shown to inhibit influenza infections in cultured Madin-Darby canine kidney cells or human small airway epithelial cells.3 The authors suggest that oxidative stress in vivo may enhance susceptibility to infection.
Increasing Glutathione Levels—Cysteine
Of the three amino acids comprising glutathione, cysteine is in the shortest supply; thus glutathione synthesis is limited by cysteine availability.4 A very recent paper4 reported on the effects of various amino acids on cellular glutathione levels. Cysteine was found to enhance glutathione (GSH) biosynthesis enzyme activity and increase cellular GSH levels. In fact, the researchers noted, “. . . supplementation with Cys [cysteine] would be a reasonable strategy for inducing de novo GSH synthesis.” We get our cysteine from our Root Food II™, a supplement we originally designed to support hair growth, which is highly dependent upon cysteine supplies. Each capsule contains 175 mg of cysteine (for comparison, the best food source is eggs, which contain an average of about 250 mg of cysteine per egg). Sandy takes 2 capsules four times a day (about the amount found in 5.6 eggs), while Durk takes 4 capsules four times a day (the equivalent of about 11 eggs, but without all the fat and cholesterol). Caution: To avoid the possibility of formation of cystine stones by oxidation of cysteine, cysteine should be taken with vitamin C in a ratio of 2:1 vitamin C to cysteine (as it is in our Root Food II).
Figure 2. Effects of amino acids on GSH content in H2O2-treated or nontreated Caco-2 cells. (A) The cells treated with amino acids (0–5 mM) for 2 h at 37 °C and then incubated with H2O2 (1 mM) for 6 h. (B) The cells treated with amino acids (0–5 mM) for 2 h at 37 °C. Control refers to cell cultures that were not treated with amino acids and not subjected to oxidative stress treatment by H2O2. #P < 0.05 compared with control cells. *P < 0.05 and **P < 0.01 compared with cells treated with H2O2 alone. Data are expressed as mean ± standard deviation of three experiments performed in triplicate.
We have two review papers5,6 by a scientist who proposes that a deficit in cysteine is a possible major causative factor of many aspects of aging and that “everybody is likely to have a cysteine deficiency sooner or later.” As he explains, clinical studies have shown that cysteine (as precursor to glutathione) decreased insulin responsiveness in the fasted state (you do not want insulin signaling unless in response to appropriate food intake), improved skeletal-muscle functions, decreased the body-fat/lean-mass ratio, decreased plasma levels of the inflammatory cytokine tumor necrosis factor-alpha, improved immune function, and increased plasma albumin levels. Moreover, “. . . the thiol/disulfide redox status shifts to more oxidative conditions in old age . . . As all these parameters degenerate with age, these findings suggest . . . that loss of youth, health, and quality of life may be partly explained by a deficit in cysteine and . . . that the dietary consumption of cysteine is generally suboptimal . . .”
As dysfunction of insulin signaling is an important aspect of aging, the results of another paper7 are of interest: here, the researchers found that dietary cysteine alleviates sucrose-induced oxidative stress and insulin resistance in rats fed a high-sucrose diet. They increased dietary cysteine intake with a supplement of cysteine-rich protein (an alpha-lactalbumin-rich whey concentrate) or N-acetylcysteine (a cysteine donor). The high-sucrose-diet-induced impairment in muscle insulin signaling (insulin resistance) was partially prevented by either the alpha-lactalbumin-rich whey supplement or the low-dose N-acetylcysteine supplement (10 g per kg of diet), while the high-dose N-acetylcysteine supplement (20 g per kg of diet) totally prevented the impairment.
Why Not Just Take Glutathione Supplements?
We don’t take glutathione to increase glutathione levels for the simple reason that it is a very costly way to deliver the amino acid cysteine to cells. To get cysteine from glutathione, the glutathione is first disassembled into its constitutive amino acids, and then the cell imports cysteine to manufacture glutathione. It is much less expensive to take cysteine itself rather than glutathione.
Why We Choose Cysteine Rather Than N-Acetylcysteine
N-acetylcysteine (NAC) is a potent nonphysiological (i.e., it is not found naturally in the body) precursor of GSH that increases GSH levels by donating cysteine. The problem with NAC is that, as reported in many papers, it is such a powerful antioxidant that it can inhibit reactive oxygen species (ROS) signaling that is a necessary part of many normal chemical pathways. Hence, we have been very reluctant to use it, at least as an everyday supplement. For example, one recent paper8 reported that NAC interfered with the ROS (reactive oxygen species) signaling pathway by which erythropoietin stimulates the creation of red blood cells. This was in a study of erythropoietin-induced differentiation of erythroid progenitors derived from mouse fetal liver. Treatment with another potent antioxidant, pyrrolidine dithiocarbamate (PDTC), also caused the attenuation of expression of TER119 (an erythroid-specific antigen). The authors conclude, “The results suggest reactive oxygen species are involved in Epo [erythropoietin]-mediated erythroid differentiation.” In fact, this may have happened to one of us (Sandy), who was taking N-acetylcysteine (but no longer does). Upon having her regular lab tests during her NAC supplementation period, she discovered that her red blood cell levels had declined to half of their previous amount.
Decline in Glutathione Levels and Increased Inflammation with Aging
A new paper1 reports on a potentially important link between declining glutathione levels and increased inflammation with aging. The authors discovered that glutathione is a suppressor of the activity of neutral sphingomyelinase-2 (NSMase-2), a plasma membrane enzyme whose increased activity, they found, is associated with the age-associated hyperresponsiveness to the proinflammatory cytokine IL-1beta. The 60–70% decrease in glutathione levels with aging in rat hepatocyte (liver cells) content resulted in increased NSMase-2 activity, which resulted in the age-associated increase in hyperresponsiveness to the proinflammatory cytokine IL-1beta. The researchers found that increasing GSH levels in the old rats to young rat levels with N-acetylcysteine inhibited the NSMase activity and restored normal response to IL-1beta. As we discuss below, GSH levels can be increased with cysteine supplements, which (as we will explain) we consider safer than N-acetylcysteine for use as a daily supplement.
NSMase-2 modulates cellular levels of ceramide, a substance that is an important regulator of cell growth and differentiation, apoptosis (programmed cell death), and inflammation. Ceramide accumulation, as well as increased sphingomyelinase activity, has been found in livers and brains of aged rodents.1 Interestingly, calorie-restricted rats had a higher GSH content in their hepatocytes and also had lower NSMase activity. In addition, calorie restriction prevented the inflammatory hyperresponsiveness seen in noncalorically restricted aged rats.
As the authors conclude, “. . . increased NSMase activity during aging may be causatively linked to the increased state of oxidative stress. Indications to that effect come from the fact that reduced glutathione [GSH] is a potent and reversible inhibitor of NSMase activity. Depletion of GSH in response to TNF-alpha [a proinflammatory cytokine] stimulation or hypoxia activates NSMase and increases ceramide content, whereas increases in cellular GSH levels prevent the hypoxia-induced generation of ceramide and apoptosis [programmed cell death].”
This paper, therefore, importantly identifies glutathione as a suppressor of increased NSMase-2-induced proinflammatory activity with aging and identifies NSMase-2 as “a link between increased oxidative stress and the onset of inflammation during aging.”
Other Ways to Increase Glutathione Levels—Docosahexaenoic Acid
A recent paper9 reports that docosahexaenoic acid (DHA) enhances the antioxidant response of human fibroblasts by upregulating gamma-GCL (gamma-cysteinyl ligase), the limiting enzyme in the synthesis of glutathione, and upregulating the enzyme glutathione reductase (which reduces GSSG, the oxidized form of GSH, back to GSH). The authors of this human-fibroblast cell-culture study reported finding that a 30 µmol/l concentration of DHA, considered a physiological level, induced a significant difference from controls for all biomarkers, including GSH, gamma-GCL, GR (glutathione reductase), and GST (glutathione S-transferase) activities. Our Omega-3 HeartFelt™ contains 1000 mg of a high-quality marine lipid concentrate (fish oils) per capsule that includes 120 mg of DHA as well as 180 mg of EPA (eicosapentaenoic acid). We each take two capsules with meals and another two capsules at bedtime.
Increasing Glutathione Levels—Certain Flavonoids
Another paper10 reported that onion extract, quercetin, kaempferol, and apigenin increased the activity of a transfected reporter gene for a GCS (gamma-glutamylcysteine synthetase) promoter subunit in a COS-1 cell culture study. They found that, as an earlier study, quercetin elevated the GSH level and the expression of both the regulatory and the catalytic subunit of GCS. In addition, they found that quercetin potently transactivates the GCSh promoter. You get quercetin if you take our Personal Radical Shield™. Each 3-capsule serving contains 32.5 mg, while the recommended daily dose (12 capsules total: 3 capsules taken with each meal and 3 capsules at bedtime) provides 130 mg of quercetin.
- Rutkute et al. Regulation of neutral sphingomyelinase-2 by GSH: a new insight to the role of oxidative stress in aging-associated inflammation. J Lipid Res48:2443-52 (2007).
- Biswas et al. Depressed glutathione synthesis precedes oxidative stress and atherogenesis in Apo-E-/- mice. Biochem Biophys Res Commun 338:1368-73 (2005).
- Cai et al. Inhibition of influenza infection by glutathione. Free Rad Biol Med34(7):928-36 (2003).
- Katayama and Mine. Antioxidative activity of amino acids on tissue oxidative stress in human intestinal epithelial cell model. J Agric Food Chem 55:8458-64 (2007).
- Wulf Droge. Oxidative stress and ageing: is ageing a cysteine deficiency syndrome? Phil Trans Roy Soc Lond B Biol Sci 360:2355-72 (2005).
- Wulf Droge. Oxidative aging and insulin receptor signaling. J Gerontol: Biol Sci60A(11):1378-85 (2005).
- Blouet et al. Dietary cysteine alleviates sucrose-induced oxidative stress and insulin resistance. Free Rad Biol Med 42:1089-97 (2007).
- Nagata et al. Antioxidant N-acetyl-L-cysteine inhibits erythropoietin-induced differentiation of erythroid progenitors derived from mouse fetal liver. Cell Biol Int 31:252-6 (2007).
- Arab et al. Docosahexaenoic acid enhances the antioxidant response of human fibroblasts by upregulating gamma-glutamyl-cysteinyl ligase and glutathione reductase. Brit J Nutr 95:18-26 (2006).
- Myhrstad et al. Flavonoids increase the intracellular glutathione level by transactivation of the gamma-glutamylcysteine synthetase catalytical subunit promoter. Free Rad Biol Med 32(5):386-93 (2002).