Alpha-Tocopherol from People to Plants Is an Essential Cog in the Metabolic Machinery.

Significance: Protection from oxygen, a diradical, became a necessity with the evolution of photosynthetic organisms about 2.7 billion years. α-Tocopherol plays an essential, protective role in organisms from plants to people. An overview of human conditions that result in severe vitamin E (α-tocopherol) deficiency is provided. Recent Advances: α-Tocopherol has a critical role in the oxygen protection system by stopping lipid peroxidation, its induced damage, and cellular death by ferroptosis. Recent findings in bacteria and plants support the concept of why lipid peroxidation is so dangerous to life and why the family of tocochromanols are essential for aerobic organisms and for plants. Critical Issues: The hypothesis that prevention of the propagation of lipid peroxidation is the basis for the α-tocopherol requirement in vertebrates is proposed and further that its absence dysregulates energy metabolism, one-carbon metabolism, and thiol homeostasis. By recruiting intermediate metabolites from adjacent pathways to sustain effective lipid hydroperoxide elimination, α-tocopherol function is linked not only to NADPH metabolism and its formation through the pentose phosphate pathway via glucose metabolism but also to sulfur-containing amino acid metabolism and to one-carbon metabolism. Future Directions: Evidence from humans, animals, and plants supports the hypothesis, but future studies are needed to assess the genetic sensors that detect lipid peroxidation and cause the ensuing metabolic dysregulation. Antioxid. Redox Signal. 38, 775-791.

α-Tocopherol Pharmacokinetics in Adults with Cystic Fibrosis: Benefits of Supplemental Vitamin C Administration.

BACKGROUND: Numerous abnormalities in cystic fibrosis (CF) could influence tocopherol absorption, transportation, storage, metabolism and excretion. We hypothesized that the oxidative distress due to inflammation in CF increases vitamin E utilization, which could be positively influenced by supplemental vitamin C administration. METHODS: Immediately before and after receiving vitamin C (500 mg) twice daily for 3.5 weeks, adult CF patients (n = 6) with moderately advanced respiratory tract (RT) disease consumed a standardized breakfast with 30% fat and a capsule containing 50 mg each hexadeuterium (d6)-α- and dideuterium (d2)-γ-tocopheryl acetates. Blood samples were taken frequently up to 72 h; plasma tocopherol pharmacokinetics were determined. During both trials, d6-α- and d2-γ-tocopherols were similarly absorbed and reached similar maximal plasma concentrations ~18-20 h. As predicted, during vitamin C supplementation, the rates of plasma d6-α-tocopherol decline were significantly slower. CONCLUSIONS: The vitamin C-induced decrease in the plasma disappearance rate of α-tocopherol suggests that vitamin C recycled α-tocopherol, thereby augmenting its concentrations. We conclude that some attention should be paid to plasma ascorbic acid concentrations in CF patients, particularly to those individuals with more advanced RT inflammatory disease and including those with severe exacerbations.

Alpha-1 antitrypsin deficiency and recombinant protein sources with focus on plant sources: Updates, challenges and perspectives.

Alpha-1 antitrypsin deficiency (A1ATD) is an autosomal recessive disease characterized by low plasma levels of A1AT, a serine protease inhibitor representing the most abundant circulating antiprotease normally present at plasma levels of 1-2 g/L. The dominant clinical manifestations include predispositions to early onset emphysema due to protease/antiprotease imbalance in distal lung parenchyma and liver disease largely due to unsecreted polymerized accumulations of misfolded mutant A1AT within the endoplasmic reticulum of hepatocytes. Since 1987, the only FDA licensed specific therapy for the emphysema component has been infusions of A1AT purified from pooled human plasma at the 2020 cost of up to US $200,000/year with the risk of intermittent shortages. In the past three decades various, potentially less expensive, recombinant forms of human A1AT have reached early stages of development, one of which is just reaching the stage of human clinical trials. The focus of this review is to update strategies for the treatment of the pulmonary component of A1ATD with some focus on perspectives for therapeutic production and regulatory approval of a recombinant product from plants. We review other competitive technologies for treating the lung disease manifestations of A1ATD, highlight strategies for the generation of data potentially helpful for securing FDA Investigational New Drug (IND) approval and present challenges in the selection of clinical trial strategies required for FDA licensing of a New Drug Approval (NDA) for this disease.

Quantitative assessment of cyanide in cystic fibrosis sputum and its oxidative catabolism by hypochlorous acid.

RATIONALE: Cystic fibrosis (CF) patients are known to produce cyanide (CN-) although challenges exist in determinations of total levels, the precise bioactive levels, and specificity of its production by CF microflora, especially P. aeruginosa. Our objective was to measure total CN- levels in CF sputa by a simple and novel technique in P. aeruginosa positive and negative adult patients, to review respiratory tract (RT) mechanisms for the production and degradation of CN-, and to interrogate sputa for post-translational protein modification by CN- metabolites. METHODS: Sputa CN- concentrations were determined by using a commercially available CN- electrode, measuring levels before and after addition of cobinamide, a compound with extremely high affinity for CN-. Detection of protein carbamoylation was measured by Western blot. MEASUREMENTS AND MAIN RESULTS: The commercial CN- electrode was found to overestimate CN- levels in CF sputum in a highly variable manner; cobinamide addition rectified this analytical issue. Although P. aeruginosa positive patients tended to have higher total CN- values, no significant differences in CN- levels were found between positive and negative sputa. The inflammatory oxidant hypochlorous acid (HOCl) was shown to rapidly decompose CN-, forming cyanogen chloride (CNCl) and the carbamoylating species cyanate (NCO-). Carbamoylated proteins were found in CF sputa, analogous to reported findings in asthma. CONCLUSIONS: Our studies indicate that CN- is a transient species in the inflamed CF airway due to multiple biosynthetic and metabolic processes. Stable metabolites of CN-, such as cyanate, or carbamoylated proteins, may be suitable biomarkers of overall CN- production in CF airways.

An oxidation-resistant, recombinant alpha-1 antitrypsin produced in Nicotiana benthamiana.

Proteases and reactive oxygen species (ROS) have long been implicated in playing key roles in host tissue injury at sites of inflammation dominated by macrophage activations and/or neutrophil infiltrations. Imbalances between proteases/antiproteases and ROS/antioxidants are recognized to contribute to amplification of inflammatory-based host tissue injury. This has been especially well-documented in such respiratory tract diseases as chronic obstructive pulmonary disease, cystic fibrosis, and acute respiratory distress syndrome. Inflammation-related protease/ROS disequilibria are further confounded by recognition that proteases can increase ROS by several different mechanisms and that ROS can inactivate proteases. The major human antiprotease, alpha-1 antitrypsin (AAT), is dramatically inactivated by ROS. AAT deficiency is the most prevalent genetic predisposing factor leading to emphysema, a condition treated by replacement infusions of plasma-derived AAT (hAAT) at a cost of up to $200,000 per year per patient. An updated method for production of a plant-made recombinant AAT (prAAT) engineered for enhanced oxidation resistance compared to hAAT is presented. Plant-made recombinant AAT shows comparable antiprotease activity to hAAT, and retains full activity under oxidative conditions that would deactivate hAAT. Additionally, we show that prAAT has similar effectiveness in preventing neutrophil elastase-induced cell death in an in vitro human bronchial epithelial cell culture model. We conclude that prAAT is potentially a "biobetter" AAT product that could be made available to individuals with a wide spectrum of inflammatory disorders characterized by overly aggressive neutrophilic infiltrations.

The Diagnosis and Management of Alpha-1 Antitrypsin Deficiency in the Adult.

Background: The diagnosis and clinical management of adults with alpha-1 antitrypsin deficiency (AATD) have been the subject of ongoing debate, ever since the publication of the first American Thoracic Society guideline statement in 1989.1 In 2003, the "American Thoracic Society (ATS)/European Respiratory Society (ERS) Statement: Standards for the Diagnosis and Management of Individuals with Alpha-1 Antitrypsin Deficiency" made a series of evidence-based recommendations, including a strong recommendation for broad-based diagnostic testing of all symptomatic adults with chronic obstructive pulmonary disease (COPD).2 Even so, AATD remains widely under-recognized. To update the 2003 systematic review and clinical guidance, the Alpha-1 Foundation sponsored a committee of experts to examine all relevant, recent literature in order to provide concise recommendations for the diagnosis and management of individuals with AATD. Purpose: To provide recommendations for: (1) the performance and interpretation of diagnostic testing for AATD, and (2) the current management of adults with AATD and its associated medical conditions. Methods: A systematic review addressing the most pressing questions asked by clinicians (clinician-centric) was performed to identify citations related to AATD that were published since the 2003 comprehensive review, specifically evaluating publications between January 2002 and December 2014. Important, more recent publications were solicited from the writing committee members as well. The combined comprehensive literature reviews of the 2003 document and this current review comprise the evidence upon which the committee's conclusions and recommendations are based. Results: Recommendations for the diagnosis and management of AATD were formulated by the committee. Conclusions: The major recommendations continue to endorse and reinforce the importance of testing for AATD in all adults with symptomatic fixed airflow obstruction, whether clinically labeled as COPD or asthma. Individuals with unexplained bronchiectasis or liver disease also should be tested. Family testing of first-degree relatives is currently the most efficient detection technique. In general, individuals with AATD and emphysema, bronchiectasis, and/or liver disease should be managed according to usual guidelines for these clinical conditions. In countries where intravenous augmentation therapy with purified pooled human plasma-derived alpha-1 antitrypsin is available, recent evidence now provides strong support for its use in appropriate individuals with lung disease due to AATD.

Inhibition of myeloperoxidase: evaluation of 2H-indazoles and 1H-indazolones.

Myeloperoxidase (MPO) produces hypohalous acids as a key component of the innate immune response; however, release of these acids extracellularly results in inflammatory cell and tissue damage. The two-step, one-pot Davis-Beirut reaction was used to synthesize a library of 2H-indazoles and 1H-indazolones as putative inhibitors of MPO. A structure-activity relationship study was undertaken wherein compounds were evaluated utilizing taurine-chloramine and MPO-mediated H2O2 consumption assays. Docking studies as well as toxicophore and Lipinski analyses were performed. Fourteen compounds were found to be potent inhibitors with IC50 values <1µM, suggesting these compounds could be considered as potential modulators of pro-oxidative tissue injury pertubated by the inflammatory MPO/H2O2/HOCl/HOBr system.

Inflammation, oxidative stress, and cardiovascular disease risk factors in adults with cystic fibrosis.

Cystic fibrosis (CF) represents one of a number of localized lung and non-lung diseases with an intense chronic inflammatory component associated with evidence of systemic oxidative stress. Many of these chronic inflammatory diseases are accompanied by an array of atherosclerotic processes and cardiovascular disease (CVD), another condition strongly related to inflammation and oxidative stress. As a consequence of a dramatic increase in long-lived patients with CF in recent decades, the specter of CVD must be considered in these patients who are now reaching middle age and beyond. Buttressed by recent data documenting that CF patients exhibit evidence of endothelial dysfunction, a recognized precursor of atherosclerosis and CVD, the spectrum of risk factors for CVD in CF is reviewed here. Epidemiological data further characterizing the presence and extent of atherogenic processes in CF patients would seem important to obtain. Such studies should further inform and offer mechanistic insights into how other chronic inflammatory diseases potentiate the processes leading to CVDs.

Evaluation of long-term vitamin E insufficiency or excess on bone mass, density, and microarchitecture in rodents.

High dietary α-tocopherol levels reportedly result in osteopenia in growing rats, whereas α-tocopherol deficiency in α-tocopherol transfer protein-knockout (α-TTP-KO) mice results in increased cancellous bone mass. Because osteoporosis is a disease associated primarily with aging, we hypothesized that age-related bone loss would be attenuated in α-TTP-KO mice. Cancellous and cortical bone mass and microarchitecture were assessed using dual-energy X-ray absorptiometry and micro-computed tomography in 2-year-old α-TTP-KO and wild-type (WT) male and female mice fed dl-α-tocopherol acetate. In contrast to our expectations, differences in cancellous bone were not detected between WT and α-TTP-KO mice of either gender, and α-TTP-KO males had lower (p<0.05) cortical bone mass than WT males. We therefore evaluated bone mass, density, and microarchitecture in proximal femur of skeletally mature (8.5-month-old) male Sprague-Dawley rats fed diets containing low (15 IU/kg diet), adequate (75 IU/kg diet), or high (500 IU/kg diet) dl-α-tocopherol acetate for 13 weeks. Low dietary α-tocopherol did not increase bone mass. Furthermore, no reductions in cancellous or cortical bone mass were detected with high dietary α-tocopherol. Failure to detect increased bone mass in aged α-TTP-KO mice or bone changes in skeletally mature rats fed either low or high levels of α-tocopherol does not support the hypothesis that α-tocopherol has a negative impact on bone mass, density, or microarchitecture in rodents.

Omics approaches in cystic fibrosis research: a focus on oxylipin profiling in airway secretions.

Cystic fibrosis (CF) is associated with abnormal lipid metabolism, intense respiratory tract (RT) infection, and inflammation, eventually resulting in lung tissue destruction and respiratory failure. The CF RT inflammatory milieu, as reflected by airway secretions, includes a complex array of inflammatory mediators, bacterial products, and host secretions. It is dominated by neutrophils and their proteolytic and oxidative products and includes a wide spectrum of bioactive lipids produced by both host and presumably microbial metabolic pathways. The fairly recent advent of "omics" technologies has greatly increased capabilities of further interrogating this easily obtainable RT compartment that represents the apical culture media of the underlying RT epithelial cells. This paper discusses issues related to the study of CF omics with a focus on the profiling of CF RT oxylipins. Challenges in their identification/quantitation in RT fluids, their pathways of origin, and their potential utility for understanding CF RT inflammatory and oxidative processes are highlighted. Finally, the utility of oxylipin metabolic profiling in directing optimal therapeutic approaches and determining the efficacy of various interventions is discussed.

Metabolomic profiling of regulatory lipid mediators in sputum from adult cystic fibrosis patients.

Retained respiratory tract (RT) secretions, infection, and exuberant inflammatory responses are core abnormalities in cystic fibrosis (CF) lung disease. Factors contributing to the destructive CF airway inflammatory processes remain incompletely characterized. The pro-oxidative inflammatory CF RT milieu is known to contain enzymatically and nonenzymatically produced regulatory lipid mediators, a panel of structurally defined oxidized metabolites of polyunsaturated fatty acids known to play a role in pathology related to inflammation. Using an extraction protocol that maximizes recoveries of sputum-spiked deuterated standards, coupled with an LC/MS/MS detection system, this study presents a metabolomic method to assess a broad spectrum of regulatory lipid mediators in freshly obtained sputum from CF patients. A broad range of both proinflammatory and anti-inflammatory lipid mediators was detected, including PGE2, PGD2, TXB2, LTB4, 6-trans-LTB4, 20-OH-LTB4, 20-COOH-LTB4, 20-HETE, 15-HETE, 11-HETE, 12-HETE, 8-HETE, 9-HETE, 5-HETE, EpETrEs, diols, resolvin E1, 15-deoxy-PGJ2, and LXA4. The vast majority of these oxylipins have not been reported previously in CF RT secretions. Whereas direct associations of individual proinflammatory lipid mediators with compromised lung function (FEV-1) were observed, the relationships were not robust. However, multiple statistical analyses revealed that the regulatory lipid mediators profile taken in aggregate proved to have a stronger association with lung function in relatively stable outpatient adult CF patients. Our data reveal a relative paucity of the anti-inflammatory lipid mediator lipoxin A4 in CF sputum. Patients displaying detectable levels of the anti-inflammatory lipid mediator resolvin E1 demonstrated a better lung function compared to those patients with undetectable levels. Our data suggest that comprehensive metabolomic profiling of regulatory lipid mediators in CF sputum should contribute to a better understanding of the molecular mechanisms underlying CF RT inflammatory pathobiology. Further studies are required to determine the extent to which nutritional or pharmacological interventions alter the regulatory lipid mediators profile of the CF RT and the impact of potential modulations of RT regulatory lipid mediators on the clinical progression of CF lung disease.

A high-fat diet containing whole walnuts (Juglans regia) reduces tumour size and growth along with plasma insulin-like growth factor 1 in the transgenic adenocarcinoma of the mouse prostate model.

Prostate cancer (PCa) has been linked to fat intake, but the effects of both different dietary fat levels and types remain inconsistent and incompletely characterised. The effects on PCa in the transgenic adenocarcinoma of the mouse prostate (TRAMP) cancer model of an elevated fat (20 % of energy as fat) diet containing 155 g of whole walnuts were compared to those of an elevated fat (20 % of energy as soyabean oil) diet with matched macronutrients, tocopherols as well as a low-fat (8 % of energy as soyabean oil) diet. Mice, starting at 8 weeks of age, consumed one of the three different diets ad libitum; and prostates, livers and blood were obtained after 9, 18 or 24 weeks of feeding. No differences were observed in whole animal growth rates in either high-fat (HF) diet group, but prostate tumour weight and growth rate were reduced in the walnut diet group. Walnut diet group prostate weight, plasma insulin-like growth factor 1, resistin and LDL were lower at 18 weeks, while no statistically significant prostate weight differences by diet were seen at 9 or 24 weeks. Multiple metabolites in the livers differed by diet at 9 and 18 weeks. The walnut diet's beneficial effects probably represent the effects of whole walnuts' multiple constituents and not via a specific fatty acid or tocopherols. Moreover, as the two HF diets had dissimilar effects on prostate tumour growth rate and size, and yet had the same total fat and tocopherol composition and content, this suggests that these are not strongly linked to PCa growth.

Evaluation of thiol-based antioxidant therapeutics in cystic fibrosis sputum: Focus on myeloperoxidase.

Neutrophil-dependent reactions catalysed by myeloperoxidase (MPO) are thought to play important roles in the pulmonary pathobiology of cystic fibrosis (CF). Aerosolized thiol antioxidants such as glutathione (GSH) and N-acetylcysteine (NAC) are currently being utilized as therapeutics to modify CF respiratory tract oxidative processes. This study hypothesized that MPO in CF airway lining fluids may be a target of such therapeutics. MPO activity in sputum from 21 adult CF patients was found to be inversely associated with lung function (FEV(1)). In contrast, systemic inflammation (assessed by plasma C-reactive protein) was not correlated with lung function. Ex vivo studies revealed that GSH and NAC effectively scavenged N-chloramines in sputum and inhibited sputum MPO activity with potency exquisitely dependent upon MPO activity levels. Detailed kinetic analyses revealed that NAC and GSH inhibit MPO by distinct mechanisms. Activation of the key pro-inflammatory transcription factor NF-κB in cultured HBE1 cells was inhibited by GSH. The findings reveal that MPO activity and its reactive products represent useful predictors of the doses of inhaled thiol antioxidants required to ameliorate airway oxidative stress and inflammation in CF patients and provide mechanistic insight into the antioxidative/anti-inflammatory mechanisms of action of GSH and NAC when administered into the CF lung.

Cigarette smoke exposure causes changes in Scavenger Receptor B1 level and distribution in lung cells.

Scavenger Receptor B1 has been shown to play a prominent role in the uptake and delivery of vitamin E from HDL and is likely involved in regulating vitamin E in the lung. We have previously demonstrated that lung Scavenger Receptor B1 levels (protein and mRNA) are modulated by cigarette smoke in mice and this was accompanied by changes in lung vitamin E. To further characterize the molecular mechanism(s) involved in this process, human alveolar epithelial cells were exposed to cigarette smoke and Scavenger Receptor B1 cellular levels and distribution were assessed. Results demonstrated that Scavenger Receptor B1 localizes in patches on the cellular membrane and in the per nuclear area of control cells. Upon cigarette smoke exposure, Scavenger Receptor B1 first translocated to the cell surface (within the first 12h of exposure) and then cell levels (protein and mRNA levels) decreased significantly at 24h. This decline was accompanied by increased Scavenger Receptor B1 ubiquitination which may explain the decrease in the protein levels. Cigarette smoke induced changes in both sub-cellular redistribution and ubiquitination of Scavenger Receptor B1 together with our previous in vivo data provides evidence that cigarette smoke exposure may alter lung's ability to control its tocopherol levels.

Dietary alpha-tocopherol and neuromuscular health: search for optimal dose and molecular mechanisms continues!

Rodents fed alpha-tocopherol (alphaT)-depleted diets develop neuromuscular deficits. Unequivocal role of alphaT in the prevention of these deficits is confounded by possible neurotoxic oxidant products generated, ex vivo in alphaT-depleted diets. The discovery that large doses of alphaT could ameliorate neuromuscular deficits, attributed to very low serum alphaT caused by mutations in either the microsomal triglyceride transfer protein or the alphaT-transfer protein (alphaTTP), underscores the necessity of alphaT for neuromuscular health in humans. The discovery of human alphaTTP provided physiological relevance to biochemical data from rodents documenting alphaT-binding transfer protein, expressed exclusively in liver. The cloning of alphaTTP gene and the creation of alphaTTP-knockout mice allowed to achieve severe systemic alphaT deficiency in brain and muscles, possibly at birth, eliminating the possible confounding effects of ex vivo-generated oxidant products in vitamin E-stripped diets. alphaTTP-knockout mice have proven useful models to discover alphaT-regulated phenotypes and molecular actions of alphaT in vivo. The results suggest that antioxidant and non-antioxidant actions of alphaT in vivo may not be mutually exclusive. These studies also suggest that low levels of dietary alphaT can achieve in excess of nanomolar alphaT levels in tissues and maintain normal neuromuscular functions. This is consistent with biochemical and crystallographic data of alpha-TTP and of other alphaT-binding proteins that have dissociation constants in nanomolar range. Molecular mechanisms that cause a long delay for the development of deficiency symptoms remain enigmatic. It is likely that alphaT is metabolically stable in post-mitotic neurons and myocytes and, if it undergoes redox-cycling in vivo, a large repertoire of alphaT-regenerating systems maintains its biological activity before it is totally depleted.

Myeloperoxidase-dependent oxidative metabolism of nitric oxide in the cystic fibrosis airway.

BACKGROUND: Decreased expired nitric oxide (eNO) is commonly observed in cystic fibrosis (CF) patients and is usually explained by dysregulation of NO synthase (NOS) isoforms in respiratory tract epithelium. Later stages of this disease are accompanied by intense airway infiltration of phagocytes with high NOS activity, abundant levels of the hemoprotein myeloperoxidase (MPO) and significant production of significant reactive oxygen species. METHODS: This study characterizes the contribution of the high airway levels of MPO to decreased eNO levels in adult CF patients. NO metabolites (NO(x)) and MPO levels in fresh sputum of control and adult CF patients were determined and related to measurements of eNO and to in vitro consumption of NO in CF sputum. RESULTS: Despite essentially equal levels of NO(x) in sputum, eNO was 2- to 3-fold lower in CF patients compared to healthy controls. In CF patients, eNO levels were negatively associated with sputum peroxidase activity. In vivo correlations were confirmed by ex vivo studies of NO consumption by MPO in CF sputum. Immunodepletion studies confirmed MPO as the major heme peroxidase in CF sputum contributing to the hydrogen peroxide (H(2)O(2))-dependent consumption of NO. CONCLUSIONS: In CF airways MPO acts as a phagocyte-derived NO oxidase that diminishes NO bioavailability at airway surfaces, possibly identifying this peroxidase as a potential target for therapeutic intervention.

Modulation of ozone-sensitive genes in alpha-tocopherol transfer protein null mice.

Alpha-tocopherol transfer protein (ATTP) null mice (ATTP-/-) have a systemic alpha-tocopherol (AT) deficiency, with their lung AT levels being < 10% of those in AT-replete ATTP(+/+) mice when fed a standard rodent chow diet. ATTP(+/+) and ATTP(-/-) mice (4 wk old male mice, n = 16 per group) were fed a standard diet (35 IU AT/kg diet) for 8-12 wk, exposed 6 h/day for 3 days to either to O(3) (0.5 ppm) or filtered air, then sacrificed. No significant differences in plasma or lung AT concentrations were observed in response to this level of O(3) exposure. Lung genomic responses of the lungs to O(3) were determined using Affymetrix 430A 2.0 arrays containing over 22,600 probe sets representing 14,000 well-characterized mouse genes. As compared with filtered air exposure, O(3) exposure resulted in 99 genes being differentially expressed in ATTP(-/-) mice, as compared to 52 differentially expressed genes in ATTP(+/+) mice. The data revealed an O(3)-induced upregulation of genes related to cell proliferation/DNA repair and inflammatory-immune responses in both ATTP(+/+) and ATTP(-/-) mice, with the expression of 22 genes being common to both, whereas 30 and 77 genes were unique to ATTP(+/+) and ATTP(-/-) mice, respectively. The expressions of O(3) sensitive genes-Timp1, Areg, Birc5 and Tnc-were seen to be further modulated by AT status. The present study reveals AT modulation of adaptive response of lung genome to O(3) exposure.