The GSNO Connection

Supplementing L-arginine to Augment Formation of S-nitrosoglutathione (GSNO) To Enhance the Benefits of Supplemental GSH in the Management of Cystic Fibrosis Pathology

by Patricia M. Chapman, August 2005

One of the fundamental factors present in the pathology of Cystic Fibrosis (CF) is the progressive systemic deficit of extracellular reduced Glutathione (GSH) whereby the production of adequate levels of extracellular GSH is eventually outpaced by the build-up of toxins (1) leading to oxidative stress. GSH has been recognized as one of the body’s primary antioxidants, and is vital for a properly functioning immune system. In the absence of adequate levels of GSH, whether due to lack of production or lack of transport, oxidative stress eventually leads to complications both primary and secondary in nature. GSH deficiency causes damage to individual cells as a result of oxidative stress and increased lipid peroxidation (2, 9). Immune cells, which are able to efflux GSH, may also become depleted of GSH in CF patients, leading to accelerated cell death and inefficient bacterial defense (2).

Secondary complications of GSH deficiency manifest in damage to organs such as the lungs, pancreas, and liver (1). In the CF lung secondary complications of GSH deficiency include, but are not limited to, degradation of lung surfactant, increased adherence hence colonization of bacteria, impaired ciliary beat function, and pulmonary fibrosis. Moreover, exuberant inflammation is simultaneously incapable of eradicating pathogens, perhaps due to a lack of Nitric Oxide as a result of L-arginine deficiency. Gastrointestinal complications of long term GSH deficiency include, but also are not limited to, pancreatic insufficiency, inflammation of the gut, decreased absorption of nutrients, and liver fibrosis (1).

The pathology associated with GSH deficiency has been shown to correlate almost precisely with the pathology of CF (1). Therefore, augmenting GSH as early as possible in the CF diagnosis may be considered first and foremost of greatest importance in the effort to slow, halt and even reverse the effects of oxidative stress in CF. However, while in older CF patients symptoms of oxidative stress are typically immediately alleviated by GSH supplementation, secondary complications of GSH deficiency will usually not respond to GSH augmentation alone (1). Therefore, given the implications arising from secondary complications of systemic GSH deficiency, it is useful to ask how long term oxidative stress may govern the level of relief experienced by CF patients who supplement GSH, and how GSH augmentation can be enhanced to bring the greatest benefit possible in the management of progressed CF pathology.

The following discussion is based on studies demonstrating the benefits of augmenting GSNO in the CF patient by supplementing the amino acid L-arginine with an emphasis on increasing host defense against bacterial colonization, as well as the demonstrated benefits of oral supplementation of L-arginine to alleviate complications in the GI tract commonly present in CF.

The CF Lung and Bacteria
Bacterial colonization of the CF lung is one of the most familiar characteristics of the CF pathology. Typically, airway colonization begins with infection caused by Staphylococcus Aureus (SA) or Haemophilus Influenzae (6). Pseudomonas Aeruginosa (PA) is an opportunistic pathogen that colonizes the already compromised CF lung and eventually becomes the dominant pathogen with as many as 80% of CF patients culturing PA (3) within the first few years of life.

PA is a denitrifying pathogen consuming airway NO and causing increased levels of Ammonia (NH4+), a byproduct of NO metabolism (6, 21, 22). Increased levels of NH4+ contribute to the affinity for bacterial colonization in the CF lung and are regarded as a marker for more severe lung damage. Moreover, PA will commonly mutate from non-mucoid to mucoid by forming a protective lining that increases the resistance of PA to antibiotic treatments (4). The process of mutation to mucoid from non-mucoid is facilitated by Hydrogen Peroxide (H2O2), which is increased in CF patients as a result of GSH deficiency (1, 3, 4, 9). Once PA becomes mucoid the most that is generally expected of traditional allopathic anti-Pseudomonas treatment is a reduction in the colonization, but never eradication of the bacteria (4, 12).

GSH Deficit in the Pathology of Cystic Fibrosis
GSH is a water-soluble tripeptide formed inside the cells from the three amino acids glycine, glutamine, and cysteine. GSH is found everywhere throughout the body, both inside and outside cells (1), however, mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein causes GSH to be trapped inside the cells of the CF patient. This malfunction of the CFTR protein leads to systemic deficit of extracellular GSH (1) in CF patients.

GSH deficiency is responsible for exaggerated bronchial inflammation, greater viscosity of mucus, and increased lipid peroxidation causing damage to cells (1). GSH deficiency is also responsible for chronic inflammation of the gut, reduced bile flow, and decreased absorption of nutrients (1). Secondary complications of GSH deficiency include oxidant damage to the liver and pancreas, onset of diabetes mellitus, cachexia (tissue wasting), rheumatoid arthritis, peripheral nerve damage, and myocardial injury (1).

Nitric Oxide Deficiency in Cystic Fibrosis

Nitric Oxide (NO) is a free radical gas produced inside and outside the cells (3). The amino acid L-arginine is a substrate for the production of NO and is believed to be deficient in CF patients, either resulting from issues of mal-absorption or lack of bioavailability. The deficiency of L-arginine would therefore account for the lack of production of NO in the CF patient (7).

Nitric Oxide is important for its role in cell signaling, facilitating the building of muscle tissue, and the uptake of nutrients and glucose by muscle tissue where it is used for energy (1, 8). Additionally, NO is responsible for smooth muscle relaxation and regulating ciliary beat function (1). NO is also responsible for increased conductance of the CFTR protein, enhancing chloride transport while down-regulating sodium absorption (1, 3, 6, 7) resulting in increased hydration of endobronchial secretions, facilitating elimination of bacteria from the lungs (1,7,19). NO may also decrease neutrophil accumulation in the lung (20, 21).

Finally, NO is known to act as a bactericidal agent against two of the most prevalent pathogens common in the pathology of Cystic Fibrosis, Pseudomonas Aeruginosa (PA) and Staphylococcus Aerus (SA) (1, 18). The bactericidal effects of NO work first by reducing adherence of bacteria in the CF lung, and second by disrupting microbial functions by interacting with critical membranes and cellular thiols in bacteria (23, 24, 25). As adherence is the initial step in establishing infection, augmenting NO very early in CF would in theory have therapeutic effects (3).

GSNO Deficit in the Pathology of Cystic Fibrosis
S-nitrosoglutathione (GSNO) is formed when GSH and NO bind together (1). One of the roles of GSNO is that of holding in reserve GSH and NO (1). GSNO is a critical component in the availability of NO because it is the vehicle by which NO is transported within in the body (1, 7, 26, and 27). However, adequate levels of both GSH and NO must be available to facilitate formation of GSNO and the transport of NO to areas where it is needed most, such as the lungs to provide for defense against pathogens (1, 8).

GSNO by way of its breakdown increases expired NO (10) and provides two important mediums for CF lung defense: GSH which reduces Hydrogen Peroxide (H2O2) to water (1, 4) thereby limiting the mutation of PA from non-mucoid to mucoid; and NO which is responsible for increased conductance of the CFTR protein (1, 5, 6), and which is also recognized as a bactericidal agent against SA and PA (2).

Low levels of GSNO in CF airways would seem to account for the affinity of the CF lung to colonize SA and PA since a lack of NO is recognized for increased adherence of bacteria in the CF lung (1, 3, 5, 7). It is reasonable to suggest, therefore, that normalizing airway GSNO in CF will increase host defense against these common CF pathogens.

Augmenting Production of NO and GSNO in Cystic Fibrosis Airways
The formation of nitrosothiols is known to occur upon inhalation of NO gas (8). Additionally, the safety of augmenting GSH and NO using nebulized forms of each has been demonstrated in vivo (15). In a study conducted by Solomons, Cotton and R Dubois on the effects of L-arginine in CF supplementing the NO donor L-arginine in CF patients resulted in increased airway clearance and weight gain (10) and improvements in vital capacity, believed to be a result of increased mucolytic action (9).

While the half life of NO is 3 to 7 seconds (7, 11) the half-life of GSNO is approximately 5.5 hours. Supplementing L-arginine as a substrate for the production of NO without first correcting the GSH deficit cannot be expected to result in the same long-term positive effects of augmenting GSNO. Of primary importance, however, in augmenting GSNO is the necessity to first address the systemic deficit of GSH which exists in progressed CF pathology.

Augmenting airway GSNO using a nebulized form of GSNO is also well tolerated, improves gas exchange in CF patients (11) and results in elevated levels of exhaled NO. A study by Gaston, et al whereby GSNO was augmented in the CF airways demonstrated that expired NO is immediately increased by GSNO augmentation in the CF airways and remains above baseline for approximately 30 minutes (11). However, lack of stability as well as the cost of GSNO, which is approximately ten times the cost of L-arginine, are inhibiting factors to directly augmenting GSNO.

Recognizing the necessary positive effects of augmenting GSNO in CF and the impracticality of augmenting GSNO directly, other methods of augmenting GSNO in the CF patient must be considered, with markers for measuring benefit being increased levels of exhaled NO, as well as reduction, and possible eradication, of bacterial colonization of PA and SA, especially if treatment begins very early in the disease (4).

Supplementing Oral L-arginine in Cystic Fibrosis

In the GI tract L-arginine as a substrate for NO production protects against injury of the gastric mucosa. reduces intestinal inflammation, and is involved in pancreatic secretion (5), all of which are significant characteristics of CF.

In the study conducted by Solomons, et al, it was revealed that a small percentage of orally administered L-arginine makes its way into the airways (9). This would suggest that oral administration of L-arginine produces some airway NO, however, to what extent this production is beneficial would depend on exactly how much NO is produced and if GSH is available to form GSNO. In Solomon's study oral supplementation of L-arginine was administered to 22 CF patients. Results included weight gain, increased fat absorption, and decreased abdominal pain (9), suggesting that pancreatic insufficiency is caused at least in part by a deficiency of L-arginine. The significance of the results in weight gain, fat absorption and decreased abdominal pain may be considered markers for measuring the benefit of augmenting NO via oral administration of L-arginine.

The following protocol demonstrates a theoretically feasibility, practicality, and cost efficient method for augmenting GSNO in CF using a buffered L-arginine for oral and inhaled administration specifically designed for use with GSH supplementation.

Protocol for Augmenting GSNO with Inhaled GSH and Inhaled L-arginine
Based on studies indicating GSNO is formed on inhalation of NO gas, assuming GSH levels are adequate, the formation of GSNO may, in theory, also be achieved by administering the NO donor L-arginine as a buffered, nebulized form immediately after administration of inhaled buffered reduced L-glutathione.

A pharmaceutical grade, buffered L-glutathione suitable for inhalation is currently available through one manufacturer, Theranaturals. The GSH dosing protocol designed by Dr. Clark Bishop is available at

The protocol for the inhaled buffered L-arginine based on Dr. Solomons study consists of the following measurements for mixing the inhaled L-arginine solution: 0.9g of L-arginine free base and 15g of L-arginine hydrochloride dissolved in 300ml of distilled or purified water. The mixture should have a pH of 7.4 - 7.6 and is refrigerated. In Solomon's study, the mixture was inhaled four times a day for thirty minutes.

However, it is worth considering that smaller doses of buffered L-arginine for inhalation would be effective if this treatment were preceded by inhaled buffered L-glutathione. The resulting formation of GSNO will make NO available over the course of several hours. Additionally, it is recommended that the inhaled buffered L-arginine solution be mixed in batches equal to a one day supply, discarding the unused portion after 24 hours. Therefore, the following measurements for mixing are recommended: 2.5g of L-arginine hydrochloride, .15g of L-arginine free-base, and 50cc of boiled distilled or purified water, refrigerate mixture.

Protocol for Supplementing Oral L-arginine with Oral GSH
The protocol for augmenting GSNO orally requires an adequate level of extracellular GSH to facilitate adduct of the NO produced via oral administration of L-arginine. Oral GSH takes approximately 30 minutes to reach peak blood plasma levels. Since NO has a half-life of only 3 to 7 seconds, a wait period of 30 minutes is suggested after administering oral GSH prior to administering oral L-arginine.

Dr. Solomons study observed L-arginine supplemented in doses upward of 1 gram per kg of body weight up to 25 grams daily, administered in equal doses over the course of 24-hours. Although L-arginine is very well tolerated at high doses with no known toxicity, it may be worthwhile to determine the effectiveness of smaller doses of L-arginine when combined with Glutamine and/or Leucine to remedy weight loss (cachexia)*, which is a secondary complication of GSH deficiency (13, 14).

It is noteworthy that since a small percentage of L-arginine taken orally will find its way to the bronchial fluids (9) it may be suggested that oral supplementation of L-arginine will also count for a small percentage of formation of airway NO which, in turn may increase the formation of GSNO in the presence of adequate levels of airway GSH*.

Evidence suggests the amino acid, L-arginine, is deficient in CF patients, leading to decreased production of NO which, in turn causes a deficit of GSNO. Based on the vast number of studies indicating that L-arginine is a safe donor of NO to form GSNO, supplementing L-arginine may prove to be an effective method for enhancing the benefit of GSH augmentation in CF. It has been well established that the formation of GSNO is inhibited in CF airways. As well, the importance of GSNO in CF has also been firmly established.

Augmenting GSNO in CF airways has been achieved and demonstrated to be well tolerated and safe. Additionally, oral administration of GSH and L-arginine has also been demonstrated as both safe and beneficial. Since for cost reasons it is not practical to supplement GSNO directly, supplementing L-arginine to increase production of NO in the CF patient as part of a regimen that includes GSH augmentation would also be expected to enhance formation of GSNO.

While further empirical work is necessary to confirm these hypotheses, evidence strongly suggests the critical role of GSNO in CF, as well as the feasibility of supplementing both GSH and NO to augment production of GSNO in the CF patient.

Footnotes: *The author has administered 3g of L-arginine in a 100lb child in even doses over 24 hour periods for approximately 4 months without achieving weight gain, however obvious indicators of increased airway NO have been present. Secondary complications of cachexia have existed in the child for approximately 4 years. Research indicates that Leucine, Isoleucine and Valine in a ratio of 2:1:1 by oral supplementation have shown positive results in tissues in catabolic states (14). Additionally, Glutamine deficiency has been observed in cases of respiratory stress (14). Therefore, it may be beneficial in cases of secondary complications of GSH deficiency involving cachexia (tissue wasting) to orally supplement the amino acids L-arginine, Leucine, Isoleucine, Valine, and Glutamine along with an exercise program specific to the needs of a CF patient as a corrective measure toward increasing weight, muscle mass and increased bone density in the CF patient.

Acknowledgement: This work is dedicated to Jesse David Cornett, born March 14, 1990, and David-John Heidmann, born July 21, 1980. I would like to express my sincere gratitude to Valerie M. Hudson for her encouragement and support in this work, as well as for her research in GSH and Cystic Fibrosis, without which this paper would not have been accomplished.

CF, Cystic Fibrosis; H202, Hydrogen Peroxide; NH4+, Ammonia; NO, Nitric Oxide; GSH, reduced Glutathione; PA, Pseudomonas Aeruginosa; SA, Staphylococcus Aureus


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