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Author: Ashley

COVID-19: NAD+ deficiency

COVID-19: NAD+ deficiency may predispose the aged, obese and type2 diabetics to mortality through its effect on SIRT1 activity


The SARS-CoV-2 hyperinflammatory response is associated with high mortality. This hypothesis suggests that a deficiency of nicotinamide adenine dinucleotide (NAD+) may be the primary factor related to the SARS-Cov-2 disease spectrum and the risk for mortality, as subclinical nutritional deficiencies may be unmasked by any significant increase in oxidative stress.

NAD+ levels decline with age and are also reduced in conditions associated with oxidative stress as occurs with hypertension, diabetes and obesity. These groups have also been observed to have high mortality following infection with COVID-19. Further consumption of NAD+ in a pre-existent depleted state is more likely to cause progression to the hyperinflammatory stage of the disease through its limiting effects on the production of SIRT1.

This provides a unifying hypothesis as to why these groups are at high risk of mortality and suggests that nutritional support with NAD+ and SIRT1 activators, could minimise disease severity if administered prophylactically and or therapeutically. The significance of this, if proven, has far-reaching consequences in the management of COVID-19 especially in third world countries, where resources and finances are limited.


COVID-19 may be asymptomatic or manifest in 3 clinical phases, an initial upper respiratory tract infection, with a few patients thereafter progressing to a pneumonic phase, and an even smaller number to the hyperinflammatory phase which may be lethal [1]. The aim of any therapy would be to intervene at an early stage, either prophylactically or therapeutically, to prevent progression of the disease to a point where mechanical ventilation (MV) is required, or significant organ dysfunction occurs [2].

Risk factors for a poor outcome include older age, comorbidity (in particular diabetes, hypertension and cardiac disease), non-asthmatic respiratory disease, obesity, immunosuppression and male sex [2][3]. The independent associations of advancing age, male sex, chronic respiratory conditions (though not well controlled asthma), chronic cardiac and chronic neurological disease with in-hospital mortality, are in line with other international reports [4]. It is difficult however to determine why these conditions specifically are linked to mortality.

Docherty et al. report that severe SARS-CoV-2 infections are rare in those under 18 years of age, comprising only 1.4% of those admitted to hospital. Only 0.8% of those in this study were under 5 years of age, and this “J” shaped age distribution was starkly different from the “U” shaped distribution seen in seasonal influenza [5]. It has not been clear from observational studies however, why SARS-CoV-2 mostly spared children, but it has been speculated that it is due to differential expression of the ACE2 receptor in the developing lung [6]. Similarly, pregnancy has not been reported to be associated with mortality, in contrast to the situation with influenza [4][7].

While the general concept of an excessive or uncontrolled release of pro-inflammatory cytokines is well known, an actual definition of what a hyperinflammatory response or “Cytokine Storm” is, is lacking. Furthermore, there is a poor understanding of the molecular events that precipitate this response and the contribution it makes to pathogenesis. It is also not known what therapeutic strategies might be used to prevent this catastrophic progression of the disease or lessen its severity once initiated [8].

In this phase, there is an unbalanced and exacerbated inflammatory response with the release of inter alia, tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), as pro-inflammatory mediators together with interleukin 10 (IL-10) and interferon β as anti-inflammatory mediators. The complex interactions between TNF-α, the interleukins, chemokines and interferons in SARS-CoV-2 are currently poorly understood; however, they are associated with and related to a significant viraemia [9][10].

The fact that most international studies have indicated that certain risk factors were common suggested that a similar systemic abnormality might be present in those at high risk, predisposing to severe illness or mortality.

In this context, the nicotinamide adenine dinucleotide (NAD+)- and zinc-dependent molecule, the Silent Information Regulator 1 (SIRT1) represents a potential common thread in the aetiology of the hyperinflammatory response and increased mortality.

SARS-CoV-2 binding

SARS-CoV-2 targets and binds to the angiotensin-converting enzyme 2 receptor (ACE2R), a membrane-associated aminopeptidase also expressed in the vascular endothelium, renal and cardiovascular tissue, and small intestine, testis, and respiratory epithelia [11]. The ACE2R acts as the host cell receptor for the virus which binds via the spike protein on the viral capsid [12]. This stimulates clathrin-dependent endocytosis of both the ACE2R and virus, events that are essential for infectivity. This process induces ADAM 17 activity which reduces expression of ACE2 on the cell surface [13].

Nicotinamide adenine dinucleotide (NAD+)

NAD+ is a cofactor found in every cell of the body, and it is involved in multiple metabolic pathways. It is a fundamental housekeeping molecule that catalyses electron transfer in metabolic reduction–oxidation reactions, functioning as an electron shuttle in the production of adenosine triphosphate (ATP).

Increased age is a strong predictor of SARS-CoV-2-associated in-hospital mortality after adjusting for comorbidity [6]. Older patients have also been identified as having the lowest levels of NAD+ [14], while, conversely, those with the lowest risk, infants and children have the highest levels.

The decline in NAD+ levels with ageing is mainly dependent on CD38, a 45 kDa transmembrane molecule, encoded on chromosome 4. In leukocytes, it acts as a receptor in adhesion and signalling pathways [15]. CD38 expression is increased with insulin resistance, and may exacerbate the age-dependent decline of NAD+ [9]. NAD+ and NADP profoundly affect age-influencing factors such as oxidative stress and mitochondrial activity, and NAD+-dependent sirtuins also mediate the ageing process [10]. As humans age, antioxidant defence mechanisms such as glutathione production are also depleted and the associated increase in reactive oxidative species (ROS) causes all cells [16] to enter a state of pseudohypoxia in which the ratio of NAD+/NADP declines [17][18][19].

Oxidative stress also activates the NAD+-dependent enzyme, poly ADP ribose polymerase 1 (PARP1) [20]. Hyperactivity of PARP1 results in depletion of cellular NAD+ pools, leading to ATP deficiency, energy loss, and subsequent cell death. These processes have the potential to enhance the pro-inflammatory cascade.

NAD+ deficiency impairs SIRT1 function [21] and its successful activation. Whereas extreme niacin deficiency is associated with the development of pellagra, more subtle decreases occur in diabetes, ageing and hypertension with resultant attenuation of responsiveness to inflammatory stimuli [22].

Silent Information Regulator 1 (SIRT1)

Sirtuins are an ancient family of seven NAD+-dependent deacylase and mono-ADP-ribosyl transferase signalling proteins that are intrinsically involved in metabolic regulation and cellular homeostasis. Of particular interest is SIRT1, which downregulates ADAM 17 (A Disintegrin and Metalloproteinase Domain 17), also called TNF-α converting enzyme (TACE), by increasing expression of TIMP3 the gene that encodes for tissue metalloproteinase inhibitor 3 [23]. In so doing it decreases levels of TNF-α, IL-1b and IL-6. An increase in TNF-α causes SIRT1 to down-regulate ADAM 17, thereby controlling TNF-α formation in a negative feedback loop that secondarily influences IL-1b and IL-6 production, which are dependent on TNF-α [23].

SIRT1 is known to play a crucial role in obesity-associated metabolic diseases, cancer, ageing, cellular senescence, cardiac ageing and stress, prion-mediated neurodegeneration, inflammatory signalling in response to environmental stress, embryonal development of the heart, brain, spinal cord and dorsal root ganglia, and placental cell survival [24]. In its inactive or open state, it contains a Zn++ module and an NAD+ -binding site [25].

Whereas certain conditions such as ulcerative colitis, Crohn’s disease, short bowel syndrome, renal failure, alcoholism, and inadequate meat intake are specifically associated with zinc deficiency there is evidence that zinc intake among older adults might be marginal. An analysis of the Third National Health and Nutrition Examination Survey (NHANES III), 1988–1994 data found that 35–45% of adults aged 60 years or older had a zinc intake below estimated average requirements [26].

When NAD+ binds to the SIRT1 molecule in the presence of the Zn++ -binding module, it undergoes a structural change, enveloping the NAD+ molecule and causing it to be “closed” or activated [25]. The presence of both the Zn++ and the NAD+ moieties are imperative for its function.

SIRT1 downregulates ADAM17 and cytokine production

ADAM17 is a proteinase encoding gene. TNF-α and the cytokine receptor for IL-6 must be proteolytically cleaved in order to be systemically active, and ADAM17 provides this function. If ADAM17 expression is not downregulated by SIRT1, TNF-α and IL-6 are released, resulting in an uncontrolled hyperinflammatory response as may occur with COVID-19 [23][27][28][29][30]. SIRT 1, by inhibition of ADAM17 and thereby TNF-α and IL-6, performs an anti-inflammatory function [31][32][33][34][35][36][37][38].

If oxidative stress is severe, increased ADAM17 attempts to ameliorate tissue injury by converting active iron (Fe2+) to its inert form (Fe3+) which is stored in hepatocytes and macrophages and as ferritin by means of the Fenton reaction (Fe2+ + H2O2 → Fe3+ + HO + OH), (Fe3+ + H2O2 → Fe2+ + HO2  + H+). This also potentially transforms haemoglobin to methaemoglobin, reducing its capacity to bind to oxygen [39].Go to:

COVID-19 replication and SIRT1

SIRT1 not only controls and modifies the inflammatory response, but along with the Sirtuin family (SIRT1–7) is also a primary defence against DNA and RNA viral pathogens [40]. In some respiratory infections and cardiovascular conditions, SIRT1 promotes autophagy (the destruction of damaged or redundant cellular components occurring in vacuoles within the cell), and in so doing inhibits apoptosis and provides protection against hypoxic stress [37][38][39][40].

Upregulation of SIRT1 directly decreases viral replication and inhibits the activation of ADAM17, thereby decreasing TNF-α, IL-1b and IL-6. Conversely depletion of SIRT1 allows for increased viral replication with little or no inhibition of ADAM17 activity, causing uncontrolled increases in TNF-α, IL-6 and IL-1b. Whereas an increase in TNF-α would usually increase SIRT1 activity to downregulate ADAM17, in the presence of a deficiency of NAD+ or Zn++, this would not occur due to insufficient activation of SIRT1, causing an unchecked increase in TNF-α.

In both obesity and type 2 diabetes mellitus, intracellular NAD+ levels are decreased in multiple tissues, including adipose tissue, skeletal muscle, liver and the hypothalamus. [41] Furthermore, both conditions are characterised by low-grade inflammation associated with activation of both IL6 and TNF-α [42][43]. Obesity or type 2 diabetes mellitus would increase the risk for cytokine storm due to an inability to activate SIRT1.

SIRT1 maintains vascular endothelial function, preventing or reducing the potential for the metabolic syndrome, ischaemia–reperfusion injury and inflammation in obesity. With increasing age however, NAD+ levels and sirtuin activity decline and this is exacerbated by obesity and sedentary lifestyles [22]. SIRT1 is an effective inhibitor of oxidative stress in vascular endothelial cells (EC) [44] via various signalling pathways [45].

The endothelial glycocalyx (EG) is a web of membrane-bound glycoproteins on the luminal side of endothelial cells, associated with various glycosaminoglycans that cover the vascular endothelium [46]. The EG separates cellular blood components from the endothelium and maintains osmotic tension of the intravascular compartment [44][45].

Conditions causing damage to, and shedding or fragmentation of the EG, (as seen in SARSCoV-2 under severe oxidative stress induced by the hyperinflammatory response), exposes the endothelium, allowing adhesion, clumping and activation of platelets with degranulation and release of vasoactive substances. The EG has anticoagulant properties as it is a binding site for mediators such as heparin cofactor II, antithrombin, thrombomodulin and tissue factor pathway inhibitor (TFPI). Heparin cofactor II and dermatan sulphate inhibit thrombin, and antithrombin activity is enhanced when bound to heparan sulphate. Conversely, exposure of the endothelial cell surface protein, thrombomodulin, which contains a cofactor for thrombin, chondroitin sulphate, promotes coagulation via activation of tissue factor [46] as seen in SAR-CoV-2.

The EG is already compromised in systemic inflammatory states, such as diabetes, hyperglycaemia, surgery, trauma and sepsis [46]. Under conditions of more severe oxidative stress, as in the hyperinflammatory response, widespread damage may lead to its destruction, with the occurrence of capillary leak and oedema formation, accelerated inflammation, platelet aggregation, hypercoaguability and a loss of vascular responsiveness [47]. Inflammatory mediators that are implicated in this process are TNF-α, bradykinin, C-reactive protein and mast cell tryptase.

Given the above, it is possible that activation of SIRT1 may be a crucial factor in the prevention of the hyperinflammatory response and may be necessary for a successful defence against viral attack. Vulnerable patient groups would potentially be less likely or unable to ensure sufficient activation of SIRT1 due to low NAD+ levels or associated nutritional deficiencies including Zn++, and as such contribute to an inability to control viral replication and reduce the uncontrolled expression of pro-inflammatory cytokines.Go to:


The SARS-CoV-2 hyperinflammatory response is associated with high mortality. A deficiency of NAD+, in the context of an elevated CD38, may be the primary factor related to the SARS-Cov-2 disease spectrum and the risk of mortality, as subclinical nutritional deficiencies may be unmasked by any significant increase in oxidative stress.

NAD+ levels decline with age and are also reduced in conditions associated with oxidative stress as occurs with hypertension, diabetes and obesity. These same groups have also been observed to have high mortality following infection with COVID-19. Further consumption of NAD+ in a pre-existent depleted state is more likely to cause progression to the hyperinflammatory stage of the disease through its limiting effects on the production of SIRT1.

Given that activation of SIRT1 is dependent on the availability of NAD+ and zinc and that high levels of oxidative stress deplete NAD+, thereby decreasing SIRT1 activity, nutritional support with NAD+ precursors and SIRT1 activators, could minimise disease severity if administered prophylactically and or therapeutically. The significance of this hypothesis, if proven, has far-reaching consequences in the management of COVID-19 especially in third world countries, where resources and finances are limited.Go to:

Grants and support

NA.Go to:


We hypothesize that reduced Nicotinamide Adenine Dinucleotide (NAD+) levels with consequent deficient activity of the NAD+ dependent molecule SIRT1, which modulates cytokine production, may be the factor that predisposes the aged, obese, type 2 diabetics and other vulnerable groups to an increased mortality.Go to:

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Go to:


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Originally published here:

Rewinding the Clock

Treatment restores blood vessel growth, muscle vitality, boosts exercise endurance in aging animals  

By EKATERINA PESHEVA March 22, 2018 Research

We are as old as our arteries, the adage goes, so could reversing the aging of blood vessels hold the key to restoring youthful vitality?

The answer appears to be yes, at least in mice, according to a new study led by investigators at Harvard Medical School.

The research, published March 22 in Cell, identifies the key cellular mechanisms behind vascular aging and its effects on muscle health and has successfully reversed the process in animals.

The findings pinpoint a glitch in the normal crosstalk that occurs between muscles and blood vessels and keeps both tissues healthy.

Using the synthetic precursors of two molecules naturally present in the body, the scientists also managed to reverse blood vessel demise and muscle atrophy in aging mice, boosting their exercise endurance in the process.

The achievement, the team said, paves the way to identifying related therapies for humans.

“We’ve discovered a way to reverse vascular aging by boosting the presence of naturally occurring molecules in the body that augment the physiological response to exercise,” said study senior investigator David Sinclair, professor in the Department of Genetics at Harvard Medical School and co-director of the Paul F. Glenn Center for the Biology of Aging at Harvard Medical School.

“The approach stimulates blood vessel growth and boosts stamina and endurance in mice and sets the stage for therapies in humans to address the spectrum of diseases that arise from vascular aging,” added Sinclair, who is also a professor at the University of New South Wales School of Medical Sciences in Sydney, Australia.

The researchers caution that many promising treatments in mice don’t have the same effect in humans due to critical differences in biology. However, the results of the experiments were dramatic enough to prompt the research team to pursue experiments in humans. Clinical trials for safety are already under way, Sinclair said.

As old as our blood vessels

Sinclair and team set out to unravel the mechanisms behind one of biology’s inevitabilities: aging.

As we grow old, we become weak and frail. A constellation of physiological changes—some subtle, some dramatic—precipitate this inevitable decline. What exactly happens inside our cells to cause the biological shifts that lead to aging? It’s a question that has vexed Sinclair and team for years.

As we age, our tiniest blood vessels wither and die, causing reduced blood flow and compromised oxygenation of organs and tissues. Vascular aging is responsible for a constellation of disorders, such as cardiac and neurologic conditions, muscle loss, impaired wound healing and overall frailty, among others. Scientists have known that loss of blood flow to organs and tissues leads to the build-up of toxins and low oxygen levels. The so-called endothelial cells, which line blood vessels, are essential for the health and growth of blood vessels that supply oxygen-rich and nutrient-loaded blood to organs and tissues. But as these endothelial cells age, blood vessels atrophy, new blood vessels fail to form and blood flow to most parts of the body gradually diminishes. This dynamic is particularly striking in muscles, which are heavily vascularized and rely on robust blood supply to function.

Muscles begin to shrivel and grow weaker with age, a condition known as sarcopenia. The process can be slowed down with regular exercise, but gradually even exercise becomes less effective at holding off this weakening.

Sinclair and team wondered: What precisely curtails the blood flow and precipitates this unavoidable decline? Why does even exercise lose its protective power to sustain muscle vitality? Is this process reversible?

In a series of experiments, the team found that reduced blood flow develops as endothelial cells start to lose a critical protein known as sirtuin1, or SIRT1. Previous studies have shown that SIRT1 delays aging and extends life in yeast and mice.

SIRT1 loss is, in turn, precipitated by the loss of NAD+, a key regulator of protein interactions and DNA repair that was identified more than a century ago. Previous research by Sinclair and others has shown that NAD+, which also declines with age, boosts the activity of SIRT1.

A stimulating conversation

The study reveals that NAD+ and SIRT1 provide a critical interface that enables the conversation between endothelial cells in the walls of blood vessels and muscle cells.

Specifically, the experiments reveal that in young mouse muscle, SIRT1 signaling is activated and generates new capillaries, the tiniest blood vessels in the body that supply oxygen and nutrients to tissues and organs. However, as NAD+/SIRT1 activity diminishes over time, the study found, so does the blood flow, leaving muscle tissue nutrient-deprived and oxygen-starved.

Indeed, when researchers deleted SIRT1 in the endothelial cells of young mice, they observed markedly diminished capillary density and decreased number of capillaries, compared with mice that had intact SIRT1. Mice whose endothelial cells lacked SIRT1 had poor exercise tolerance, managing to run only half the distance covered by their SIRT1-intact peers. 

To determine SIRT1’s role in exercise-induced blood vessel growth, the researchers observed how SIRT1-deficient mice responded to exercise. After a month-long training regimen, the hind-leg muscles of SIRT1-deficient mice showed markedly diminished ability to form new blood vessels in response to exercise compared with same-age mice that had intact SIRT1 in their endothelial cells.

Exercise-induced blood vessel formation is known to occur in response to growth-stimulating proteins released by muscles under strain. SIRT1, however, appears to be the key messenger relaying growth-factor signaling from muscles to blood vessels, the study found. 

Experiments showed that endothelial cells lacking SIRT1 were desensitized to the growth-stimulating proteins released by exercised muscles.

“It’s as if these cells had grown deaf to the signals that muscles sent their way,” Sinclair said.

The observation, he added, explains why age-related loss of SIRT1 leads to muscle atrophy and blood vessel demise.

Since the experiments revealed the critical role of SIRT1 in exercise-induced blood vessel formation, the researchers wondered whether boosting SIRT1 levels would stimulate blood vessel growth and stave off muscle wasting.

Exercise in a pill?

The scientists set their sights on NAD+, a molecule conserved across many life forms, known to decline with age and previously shown to stimulate SIRT1 activity.

“We reasoned that declining NAD+ levels reduce SIRT1 activity and thus interfere with aging mice’s ability to grow new blood vessels,” said study first author Abhirup Das, who conducted the work as a post-doctoral fellow in Sinclair’s lab, currently a visiting scholar in genetics at Harvard Medical School and a post-doctoral research fellow at the University of South New Wales School of Medical Sciences.

To test this premise, scientists used a chemical compound called NMN, a NAD+ precursor, previously shown to play a role in repairing cellular DNA and maintaining cell vitality.

In lab dish experiments, endothelial cells from humans and mice treated with NMN showed enhanced growth capacity and reduced cell death.

Next, the team gave NMN over two months to a group of mice that were 20 months old—the rough equivalent of 70 in human years. NMN treatment restored the number of blood capillaries and capillary density to those seen in younger mice. Blood flow to the muscles also increased and was significantly higher than blood supply to the muscles seen in same-age mice that didn’t receive NMN.

The most striking effect, however, emerged in the aging mice’s ability to exercise. These animals showed between 56 and 80 percent greater exercise capacity, compared with untreated mice the study showed. The NMN-treated animals managed to run 430 meters, or about 1,400 feet, on average, compared with 240 meters, or 780 feet, on average, for their untreated peers.

To see whether the effects of NMN could be further augmented, the researchers added a second compound to the treatment regimen. The compound, sodium hydrosulfide (NaHS), is a precursor to hydrogen sulfide, which also boosts the activity of SIRT1.

A group of 32-month-old mice—the rough equivalent to 90 in human years—receiving the combo treatment for four weeks were able to run, on average, twice as long as untreated mice. In comparison, mice treated with NMN alone ran 1.6 times farther, on average, than untreated animals.

“These are really old mice so our finding that the combo treatment doubles their running capacity is nothing short of intriguing,” said study co-author James Mitchell, associate professor of genetics and complex diseases at the Harvard T. H. Chan School of Public Health.  Research led by Mitchell and published in the same issue of Cell also found sodium hydrosulfide to augment blood vessel formation in the muscles of mice.

Interestingly, the NMN treatment did not improve blood vessel density and exercise capacity in young sedentary mice. However, it did boost blood vessel formation and exercise capacity in young mice that had been exercising regularly for a month.

“This observation underscores the notion that age plays a critical role in the crosstalk between blood vessels and muscles and points to a loss of NAD+ and SIRT1 as the reason behind loss of exercise effectiveness after middle age,” Das said.

The researchers say their findings may pave the way to therapeutic advances that hold promise for the millions of older people for whom regular physical activity is not an option.

“Even if you’re an athlete, you eventually decline,” Sinclair said. “But there is another category of people—what about those who are in a wheelchair or those with otherwise reduced mobility?”

The team’s ultimate goal is to replicate the findings and, eventually, move toward developing small-molecule, NMN-based drugs that mimic the effects of exercise—enhanced blood flow and oxygenation of muscles and other tissues. Such therapies may even help with new vessel growth of organs that suffer tissue-damaging loss of blood supply and oxygen, a common scenario in heart attacks and ischemic strokes, the team said.

Neo-vascularization—the formation of new blood vessels—should be treated with caution, the researchers say, because increased blood supply could inadvertently fuel tumor growth.

“The last thing you want to do is provide extra blood and nourishment to a tumor if you already have one,” said study co-author Lindsay Wu, at the University of New South Wales School of Medical Sciences.

Sinclair and Wu point out that experiments done as part of the current study provide no evidence that treatment with NMN stimulated tumor development in animals treated with the compound.

Co-investigators included George Huang, Michael Bonkowski, Alban Longchamp, Catherine Li, Michael Schultz, Lynn-Jee Kim, Brenna Osborne, Sanket Joshi, Yuancheng Lu, Jose Humberto, Trevine-Villareal, Myung-Jin Kang, Tzong-tyng Hung, Brendan Lee, Eric Williams, Masaki Igarashi, James Mitchell, Nigel Turner, Zolt Arany and Leonard Guarente.

The work was supported by the Glenn Foundation for Medical Research (grants RO1 AG028730 and RO1 DK100263), and by the National Institutes of Health/National Heart, Lung and Blood Institute (grant RO1 HL094499).


Intravenous Glutathione Enhances Cycling Time Trial Performance

By William Misner Ph.D.


A healthy, fit 61-year old male endurance athlete received four (4 each) intravenous glutathione treatments consisting of 100 mg/cc, using 10cc (total 1000 milligrams) diluted total of 20cc normal saline solution slow IV-push delivery (15 minutes). The tests were recorded 36-days during a previously established aerobic cycling fitness on a common course. There were no changes in training or dietary protocols other than the administration of glutathione intravenously. The subject’s pre-treatment timed-trial was 57:30 (minutes:seconds). Performance improved progressively following each of 4 separate intravenous treatments to a peak performance of 52:21(minutes:seconds), representing a remarkable performance gain of 7.2%. The only variable to account for this performance gain was the intravenous glutathione treatments.


Whether antioxidant supplementation will enhance performance remains controversial. Exercise-induced changes in antioxidant scavengers and associated enzymes (e.g., glutathione, tocopherol, glutathione peroxidase) provide clues about demand imposed on the defense system. Exercise training is reported conclusively to result in an augmented antioxidant system including a reduction in lipid peroxidation. Supplementation with antioxidants appears to reduce lipid peroxidation but has not been shown to enhance exercise performance.

Endogenous glutathione, a tri-peptide referred to as “Reduced Glutathione”, is composed of the three amino acids: L-Cysteine, Glycine, and L-Glutamic Acid. Smaller peptides of 2 or 3 amino acids are absorbed without further reduction or delay, but there is some debate as to the effectiveness of taking peptides as opposed to free amino acids for enhancing endogenous glutathione stores. Most glutathione is found in the liver where it detoxifies harmful compounds later excreted in bile. Some glutathione is found in the red and white blood cells, the lungs, and the intestinal tract. The primary biological function of glutathione is to act as a nonenzymatic reducing agent to assist in cysteine thiol side chains in a reduced state on the surface of proteins.

Reduced glutathione is involved in the synthesis and repair of DNA, assists the recycling of vitamins C and E, blocks free radical damage, enhances the antioxidant activity of vitamin C, facilitates the transport of amino acids, and plays a critical role in detoxification. It is the base material for several other key antioxidant enzyme systems: glutathione-peroxidase, glutathione-reductase, and glutathione- transferase. Decline in glutathione concentrations in intracellular fluids correlate directly with indicators of longevity. Intravenous glutathione administration may provide the most direct and effective route for increasing intracellular glutathione levels. The glutathione (GSH) antioxidant system is foremost among the cellular protective mechanisms. Depletion of this small molecule is the common consequence of increased formation of reactive oxygen species during increased cellular activities. This phenomenon may occur in the lymphocytes during the development of an immune response or also in muscular cells during strenuous exercise. Time and transit of dietary oral GSH substrates may be a positive means of glutathione repletion. However, cysteine, found in milk proteins, is reported to be a crucial limiting amino acid for intracellular GSH4. Replenishing glutathione from dietary supplementation of Whey, N-Acetyl Cysteine, and Glutathione, are time- and dosedependant, but may further be inhibited by the influence of age, exercise stress, or environmental toxins. Bypassing gastric channels and hepatic intervention activity by intravenous loading protocol presents a model for resolving these deficits.

Exhaustive exercise depletes glutathione and simultaneously generates free radicals. This is evidenced by increases in lipid peroxidation, glutathione oxidation, and oxidative protein damage. It is well known that activity of cytosolic enzymes in blood plasma is increased as a result of exhaustive exercise.

Researchers accessed the antioxidant status and markers of oxidative damage in the members of the U.S. Men’s Alpine Ski Team during 10 days of intense training. Seven measures of antioxidant status were determined using Trolox equivalent antioxidant capacity, uric oxidase, alpha-tocopherol, total glutathione, cytosolic glutathione peroxidase, and superoxide dismutase. The results suggested that antioxidant status of elite alpine skiers declined during training. Further studies of blood values determined the importance of glutathione upon potentiating maximal oxygen carrying capacity. Higher glutathione levels influence red blood cell count, hematocrit, and hemoglobin. Conversely when red blood cells, hematocrit, and hemoglobin are reduced during anemia, low glutathione is implicated; the addition of GSH has been shown to resolve the aforementioned low blood markers.

Researchers reported the effects of reduced glutathione parenterally administered resolved the status in patients suffering from chronic renal failure and undergoing hemodialysis. Reduced glutathione and placebo were given for 120 days in a randomized double-blind fashion and the following measurements were performed: red blood cells reduced and oxidized glutathione, plasma reduced and oxidized glutathione, hematocrit, hemoglobin, reticulocytes, serum iron, transferrin, indirect bilirubin, urea, creatinine, calcium, phosphate, parathyroid hormone and alkaline phosphatase. In the treated group, during the supplementation period, there was an increase in the levels of red blood cells and plasma reduced glutathione, hematocrit and hemoglobin and a concomitant decrease in plasma oxidized glutathione and reticulocytes with a maximum effect on the 120th day of therapy. In the placebotreated group there were no significant variations of the parameters considered during the study period. When the therapy, on patients undergoing treatment, was terminated there was a drop in the analyzed parameters, which fell to pretreatment values at the subsequent controls. These findings indicate that reduced glutathione could represent a useful drug in the treatment and management of anemia in patients affected by chronic renal failure. Therefore, increased glutathione may enhance the blood oxygen carrying capacity and improve depleted glutathione stores, which hypothetically translates into enhanced performance outcome.


I hypothesized that performance gain would occur by increasing intravenous glutathione. The 61-year endurance athlete completed 1000 miles of aerobic cycling to establish base fitness, then completed a timed-trial over an 18.4 mile undulating hill course prior to receiving intravenous glutathione, 100 mg/cc, 10cc (total 1000 milligrams) dose diluted to a total volume of 20cc with normal saline by slow IVpush over a 15 minute time period. Subsequent treatments followed at 7-10 day intervals during 36 aerobic training days. Aerobic training levels were imposed as controls with the intent to maintain, not to increase performance outcome. Constant aerobic protocols do not contribute to enhanced time trial performance outcome. Constant aerobic cycling training did not exceed 75% maximum heart rate or maximum oxygen consumption, while diet and supplement protocols were also held constant throughout the test period. To evaluate the influence of IV-Glutathione on performance outcome, a cycling timed trial was pre-test recorded, then compared to a second time trial which was recorded between IV-Glutathione #1 and #2, then concluded after IV-Glutathione #3 & #4, with a comparison of a the final timedtrial recorded on the same 18.4 miles course, using the same bicycle, under similar conditions.

These results are shown in tables I, II, and III below:


Performance gain 7-10 days after treatment #1 was significant (+2.1%) with total performance gains from intravenous glutathione +7.2% over 36 days (57:30 to 52:21 minutes:seconds), averaging a remarkable +1.8% per treatment administered. The only variable to account for the remarkable improvement in performance was four glutathione injections. Measure of the efficiency of oxygen metabolization is closely tied to the mechanism of rapid glutathione repletion to resolve depleted levels. By increasing GSH serum levels, enhanced performance may result. Further research is needed with aging, fit, healthy subjects, measuring a variety of physiologic and metabolic endpoints to determine if the results of this case study prevail, and if so, the mechanism of any such benefits achieved.

Dr. Stanley B. Covert M.D., the Medical Director of High Road Clinic in Elk, Washington, (509) 292-2748 graciously provided expertise in glutathione pathway effects and medical administration of intravenous glutathione to this subject. Dr. Coverts work in resolving macular degeneration disorder using this slow push intravenous protocol is without equal.

The author was the subject of this open label case study. Dr. Covert was the supervising prescribing physician. The subject or the supervising physician reports no competing interests.


Emerging potential benefits of modulating NAD+ metabolism in cardiovascular disease


Nicotinamide adenine dinucleotide (NAD+) and related metabolites are central mediators of fuel oxidation and bioenergetics within cardiomyocytes. Additionally, NAD+ is required for the activity of multifunctional enzymes, including sirtuins and poly(ADP-ribose) polymerases that regulate posttranslational modifications, DNA damage responses, and Ca2+ signaling. Recent research has indicated that NAD+ participates in a multitude of processes dysregulated in cardiovascular diseases. Therefore, supplementation of NAD+ precursors, including nicotinamide riboside that boosts or repletes the NAD+ metabolome, may be cardioprotective. This review examines the molecular physiology and preclinical data with respect to NAD+ precursors in heart failure-related cardiac remodeling, ischemic-reperfusion injury, and arrhythmias. In addition, alternative NAD+-boosting strategies and potential systemic effects of NAD+ supplementation with implications on cardiovascular health and disease are surveyed.

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NAD+ Metabolism as an Emerging Therapeutic Target for Cardiovascular Diseases Associated With Sudden Cardiac Death


In addition to its central role in mediating oxidation reduction in fuel metabolism and bioenergetics, nicotinamide adenine dinucleotide (NAD+) has emerged as a vital co-substrate for a number of proteins involved in diverse cellular processes, including sirtuins, poly(ADP-ribose) polymerases and cyclic ADP-ribose synthetases. The connection with aging and age-associated diseases has led to a new wave of research in the cardiovascular field. Here, we review the basics of NAD+ homeostasis, the molecular physiology and new advances in ischemic-reperfusion injury, heart failure, and arrhythmias, all of which are associated with increased risks for sudden cardiac death. Finally, we summarize the progress of NAD+-boosting therapy in human cardiovascular diseases and the challenges for future studies.

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NAD+ and sirtuins in aging and disease


Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles’ heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide.

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