The overarching hypothesis our group is pursuing is that gamma tocopherol is an important anti-inflammatory isoform of the antioxidant Vitamin E. This hypothesis is supported by preclinical studies by our group and others which show that gamma tocopherol has notable anti-inflammatory actions in animal models of airway allergy and innate immune response[18
]. The primary goals of the current study were to determine if one and two geltab doses of a gamma tocopherol-rich preparation were associated with changes in serum levels of α, δ and γ tocopherols as well as γ-CEHC, a primary metabolite of gamma tocopherol with anti-inflammatory action. We also assessed serum for levels of 5-nitro-γ-tocopherol, which is generated through the action of reactive nitrogen oxide species (RNOS) on γ-tocopherol and is thought to be a marker of RNOS stress in a number of diseases.
To assess the anti-inflammatory effect of γ-tocopherol, we examined serum cytokine levels, responsiveness of PBMCs recovered from volunteers before using supplements and after using 2 geltabs of gamma tocopherol enriched supplements for 1 week, and the effect of vitamin E isoforms on JNK and NFκB signaling in vitro using recovered PBMCs from allergic asthmatic volunteers. We also examined safety data from our volunteers with both doses of gamma tocopherol. There was no difference in symptom scores observed at baseline and after each dose of gamma tocopherol. There was no change in lung function or any of the systemic parameters we assessed during this study. These observations are an initial step in designing Phase I Proof of Concept studies to determine if gamma tocopherol has utility as an intervention for asthma and other respiratory tract and inflammatory diseases.
We were somewhat surprised that we did not observe some increase in circulating α-tocopherol levels following either dose of gamma tocopherol-rich supplementation. While our preparation had a relatively small amount of α-tocopherol present in our supplement preparation compared to other tocopherols, our daily doses of α-tocopherol were 61 and 122 mg/day (for the 1 and 2 geltab doses) and we would have expected to see an increase in these levels at 6 hours. This is especially true given the role that α-tocopherol transfer protein (αTTP) has in facilitating secretion of α-tocopherol from the liver into plasma [23
In contrast, we observed a notable increase in delta and gamma tocopehrol, though this appeared to drop significantly 24 hours after the first dose. With regard to γ tocopherol, these observations are not inconsistent with what has been previously reported, in which there is a relatively rapid increase in γ tocopherol levels at 12 hours with a gradual decline over 72 hours following consumption of a single dose of gamma tocopherol[26
]. We did find that the level of serum delta and gamma tocopherol tended to be higher six hours after the last dose was consumed for the initial dose in each eight day dosing schedule. While our phlebotomy schedule did not allow us to determine daily pharmacokinetics of these tocopherols, it seems likely that repeated dosing would gradually result in an increased level of gamma tocopherol following repeated dosing.
We also observed that there was a steady increase in γ-CEHC, a primary metabolite of γ-tocopherol, across the weekly dosing schedule for both the 1 geltab and 2 geltab test periods. The increases in γ-CEHC were gradual and consistent, and did not completely mimic the levels of γ-tocopherol. However, once consumption of gamma tocopherol stopped, there was a rapid decrease in circulating levels of gamma tocopherol, such that within one week after stopping supplementation (with either 1 or 2 geltabs), serum levels of gamma tocopherol returned to baseline levels. We found in both dosing regimens that there was a steady rise in gamma tocopherol which was maximal following the last dose of each dosing period. The higher (2 geltab) dose yielded the highest level of γ-CEHC. Of practical importance for our studies was the observation that circulating levels of δ tocopherol, γ tocopherol and γ CEHC all returned to baseline levels after stoping supplementation for one week, suggesting that it would be feasible to conduct relatively short phase I/II proof of concept studies examining the effect of gamma tocopherol supplementation on experimental exacerbation of lung disease in humans in a randomized,placebo controlled fashion.
Levels of γ-CEHC resulting from γ-tocopherol consumption may be very important as we have observed that γ-CEHC has been found to have substantial anti-inflammatory activity in animal studies and in vitro
, and likely accounts for a significant portion of the anti-inflammatory action of gamma tocopherol [17
]. γ-CEHC has anti-inflammatory activity as demonstrated using in vivo
rodent models of neutrophilic inflammation, as well as challenge of RAW cells (a macrophage cell line) with endotoxin[27
]. We did not find any notable change in any serum level of pro-inflammatory cytokines and chemokines. However, we found notably decreased IL-1β, IL-6, TNFα , MCP and MIP1α secretion from peripheral blood mononuclear cells following ex vivo
LPS challenge after volunteers had completed high dose supplementation when compared to PBMC responses at baseline.
To better appreciate mechanisms by which tocopherol species may exert anti-inflammatory responses, we performed in vitro studies with PBMCs recovered from a sub-set of allergic asthmatic volunteers. Here we found that γ-tocopherol, α-CEHC and γ-CEHC but not α-tocopherol, moderately inhibited LPS-induced IκBα degradation in recovered PBMCs after LPS stimulation. We also found that γ-tocopherol but not α-tocopherol moderately inhibited LPS-induced JNK phosphorylation, and α-CEHC and γ-CEHC differentially inhibited LPS-induced JNK phosphorylation. Taken together, these observations suggest that gamma (and alpha) tocopherol species inhibit NFκB activation by interfering with IκBα degradation. This would certainly account for decreased cytokine generation after tocopherol supplementation. We also found an inhibition of JNK activation, which would also result in decreased cytokine production.
We chose this approach for assessing anti-inflammatory actions of γT in the current study for several reasons. First, LPS is a standard innate immune stimulus which initiates a well characterized response in monocytic cells, with Il-1β, IL-6, and TNFα being important pro-inflammatory cytokines secreted in these responses[30
]. Second, environmentally encountered LPS (or endotoxin) is an important cause of exacerbation of asthma in occupational and domestic settings [31
]. Third, monocytic cells of the airway are crucial elements of the initial response to airborne irritants and exacerbants of asthma[31
]. Finally, we have developed an LPS inhalation challenge protocol which can be employed in normal volunteers, asthmatics, smokers and mild COPD patients to determine if gamma tocopherol inhibits endotoxin-induced responses in the airway [32
There are a number of reasons to hypothesize that gamma tocopherol would be a useful adjunct for treatment of airway diseases. First, as noted in the introduction, consumption of dietary vitamin E (which is predominantly gamma tocopherol) is associated with decreased occurrence of allergy and asthma [2
]. Secondly, exacerbation of asthma and other lung diseases is associated with activation of innate immune processes and exposure to oxidative stress, resulting from either innate inflammatory processes or exposure to environmental stressors [31
]. Amongst the most common environmental causes for exacerbation of lung disease is exposure to tobacco smoke hence studies examining the effect of smoking on tocopherol metabolism is relevant to the evaluation of gamma tocpoherol as an adjunct approach to decreasing asthma exacerbations [40
Cigarette smokers and nonsmokers exposed to cigarette smoke have significantly increased levels of gamma tocopherol despite having lower levels of other plasma antioxidants such as ascorbate and beta carotene[43
]. However, investigators using labeled alpha and gamma tocopherol to follow the pharmacokinetics of these agents found that that smokers have increased depletion of these isoforms of vitamin E compared to non-smokers [44
]. This may be due to increased metabolism of the tocopherols to their respective CEHC products. It is also possible that they are being directly modified by smoke-related oxidants. Exposure of γ tocopherol to tobacco smoke in vitro
results in production of 5 nitro γ-tocopherol, and smokers have double the level of 5 nitro γ-tocopherol than non-smokers [47
]. Production of 5 nitro γ-tocopherol likely protects cell membranes from reactive nitrogen stress[46
]. Increased levels of γ-tocopherol in smokers may allow for more production of γ-CEHC and serve as an antioxidant defense mechanism, both of which should reduce inflammation associated with smoke exposure.
Nitrosative stress is emerging and an important process in the pathophysiology of asthma[48
]. Exhaled nitric oxide (eNO), which derives primarily from inducible nitric oxide synthase (iNOS), has emerged as a marker of asthma severity, and is associated with increased airway eosinophilia. eNO is likely able to participate in nitrosative modification of airway and cell surface proteins. Acute endotoxin challenge has been shown enhance eNO in asthmatic, but not normal volunteers[52
], and children with asthma also have increased levels of 3-nitrotyrosine, a marker of nitrosative stress[48
]. It seems likely that increased consumption of γ tocopherol, which is a much more effective trap for reactive nitrogen species at its unmethylated C-5 position, may have specific advantages as an anti-asthma agent than other tocopherol isoforms, including the much more extensively studied α-tocopherol[15
]. Consistent with this observation is a report which demonstrates that γ tocopherol prevents protein nitration and ascorbate oxidation in rodents with zymosan-induced inflammation[28
We determined whether dosing with one and two geltab doses of a gamma tocopherol-rich preparation were associated with changes in serum 5-nitro-γ-tocopherol, and if in vitro
treatment with αT, γT, αCEHC or γ CEHC modified radical generation by PMA activated PBMCs. We observed that both supplement doses were associated with decreased serum 5-nitro-γ-tocopherol levels. We interpret these findings as a general decreased in RNOS generation, with decreased nitrosative stress. We also found that all tested tocopherol species inhibited ROS generation by PMA stimulated PBMCs. Whether this is due to direct scavenging of radicals by tocopherol species, γT's anti-inflammatory activity via iNOS down-regulation, or inhibition of cell signaling, which would also likely decrease NADPH oxidase and other oxidant generation systems present in PBMCs, these findings indicate that γ-tocopherol has potent antioxidant and anti-nitrosative activity. It is important to note that other oxidation products of tocopherols also elicit biological effects, and these include tocopheryl quinones and nitrite esters. Key oxidation products of αT include α-tocopherylquinone (αTQ), 5, 6-epoxy-α-tocopherylquinone (αTQE1) and 2,3-epoxy-α-tocopherylquinone (αTQE2). [56
has been demonstrated to have strong anti-oxidant properties and may protect against lipid oxidation [57
]. γ-tocopherylquinone (γ TQ), the quinone metabolite for γT, is associated with producing cytotoxic effects [58
] that are most likely due to its ability to give rise to Michael adducts which result in endoplasmic reticulum stress [59
]. In contrast to the negative cytotoxic effects, γTQ, particularly partially substituted ones, are also capable of functioning as beneficial biological antioxidants that can destroy multi-drug resistant cancer cells by inducing apoptosis [60
]. Nitrite esters are nitrosating agents and can potentially induce certain types of cancers such as gastrointestinal cancer [61
Given the effect of gamma tocopherol in inhibiting endotoxin induced inflammation in vitro
and in vivo
, as well as its effect on endotoxin induced responses of peripheral blood monocytes recovered from human volunteers after consumption of gamma tocopherol rich supplements, we propose to pursue studies examining the effect of gamma tocopherol on the response to inhaled endotoxin in normal and asthmatic volunteers. Endotoxin inhalation at doses that exceed 10,000 EU causes a neutrophilic inflammation in these volunteers[32
], and low level endotoxin which does not induce changes directly in the lung, have been shown to enhance response to allergen in allergic asthmatics after nasal[32
] and inhalational challenge [54
]. We have recently examined the effect of low doses of inhaled endotoxin on resident airway inflammatory cells of normal volunteers and asthmatics and found increased expression of mCD14, CD80 and CD86 on airway monocytes and macrophages, suggesting that innate immune stimulation enhances antigen presenting cell function and ability to response to airway irritants, events which likely play a role in asthma exacerbation [33
The results of the current study, coupled with our recent examination of the ability of gamma tocopherol to inhibit allergen and ozone induced eosinophilic airway inflammation in a rodent model of asthma, suggest that gamma tocopherol enriched preparations are an excellent candidate as a supplemental therapy to decreased asthma exacerbations and severity. Gamma tocopherol is a potent trap for reactive nitrogen species which is an important element of eosinophilic airway inflammation and is metabolized to the anti-inflammatory molecule γ-CEHC. To further determine the feasibility of using gamma tocopherol as an adjunct treatment for asthma, we are currently examining the effect of gamma tocopherol supplementation on allergen-induced inflammation following nasal allergen challenge in allergic volunteers. We have also initiated a double blinded placebo control crossover study of the effect of gamma tocopherol in decreasing inflammatory responses to inhaled endotoxin in both normal volunteers and asthmatics.