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.

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.

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.

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.

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.

Nr1d1, an important circadian pathway regulatory gene, is suppressed by cigarette smoke in murine lungs.

Nuclear receptor subfamily 1, group D member 1 (Nr1d1), also known as Rev-erb-alpha, belongs to the family of "orphan receptors" and functions as a member of clock gene family. In addition to being an important member of clock circuitry, Nr1d1, also regulates cell proliferation, lipid metabolism, and inflammation and is also touted as a tumor suppressor. Our focus on Nr1d1 was stimulated by data from a genome-wide search for mRNA correlates of cigarette smoke (CS) sensitive--whole smoke (WS) and filtered smoke (FS)--lung transcriptomes in tumor-resistant C57BL6 and tumor-susceptible AJ mice strains. Differential analysis of approximately 15,000 genes using Affymetrix 430A 2.0 high-density oligonucleotide arrays identified modulation of genes related to circadian pathways by CS in lungs of both mouse strains. Nr1d1 expression was downregulated by both WS and FS irrespective of mouse strain as compared to respective air-breathing controls. WS was more effective than FS on decreasing Nr1d1 expression. The present data suggest that transcriptional regulation of Nr1d1 by CS may affect circadian rhythmicity and thus may play a complementary role in CS-induced lung respiratory tract pathobiology and/or lung tumorigenesis.

Effects of dietary carotenoids on mouse lung genomic profiles and their modulatory effects on short-term cigarette smoke exposures.

Male C57BL/6 mice were fed diets supplemented with either beta-carotene (BC) or lycopene (LY) that were formulated for human consumption. Four weeks of dietary supplementations results in plasma and lung carotenoid (CAR) concentrations that approximated the levels detected in humans. Bioactivity of the CARs was determined by assaying their effects on the activity of the lung transcriptome (~8,500 mRNAs). Both CARs activated the cytochrome P450 1A1 gene but only BC induced the retinol dehydrogenase gene. The contrasting effects of the two CARs on the lung transcriptome were further uncovered in mice exposed to cigarette smoke (CS) for 3 days; only LY activated ~50 genes detected in the lungs of CS-exposed mice. These genes encoded inflammatory-immune proteins. Our data suggest that mice offer a viable in vivo model for studying bioactivities of dietary CARs and their modulatory effects on lung genomic expression in both health and after exposure to CS toxicants.

Mice lacking alpha-tocopherol transfer protein gene have severe alpha-tocopherol deficiency in multiple regions of the central nervous system.

Ataxia with vitamin E deficiency is caused by mutations in alpha-tocopherol transfer protein (alpha-TTP) gene and it can be experimentally generated in mice by alpha-TTP gene inactivation (alpha-TTP-KO). This study compared alpha-tocopherol (alpha-T) concentrations of five brain regions and of four peripheral organs from 5 months old, male and female, wild-type (WT) and alpha-TTP-KO mice. All brain regions of female WT mice contained significantly higher alpha-T than those from WT males. alpha-T concentration in the cerebellum was significantly lower than that in other brain regions of WT mice. These sex and regional differences in brain alpha-T concentrations do not appear to be determined by alpha-TTP expression which was undetectable in all brain regions. All the brain regions of alpha-TTP-KO mice were severely depleted in alpha-T. The concentration of another endogenous antioxidant, total glutathione, was unaffected by gender but was decreased slightly but significantly in most brain regions of alpha-TTP-KO mice. The results show that both gender and the hepatic alpha-TTP, but not brain alpha-TTP gene expression are important in determining alpha-T concentrations within the brain. Interestingly, functional abnormality (ataxia) develops only very late in alpha-TTP-KO mice in spite of the severe alpha-tocopherol deficiency in the brain starting at an early age.

Lung vitamin E transport processes are affected by both age and environmental oxidants in mice.

Despite the physiological importance of alpha-tocopherol (AT), the molecular mechanisms involved in maintaining cellular and tissue tocopherol levels remain to be fully characterized. Scavenger receptor B1 (SRB1), one of a large family of scavenger receptors, has been shown to facilitate AT transfer from HDL to peripheral tissues via apo A-1-mediated processes and to be important in the delivery of AT to the lung cells. In the present studies the effects of age and two environmental oxidants ozone (O(3)) (0.25 ppm 6 h/day) and cigarette smoke (CS) (60 mg/m(3) 6 h/day) for 4 days on selected aspects of AT transport in murine lung tissues were assessed. While AT levels were 25% higher (p<0.05) and 15% lower (p<0.05) in plasma and lung tissue, respectively, in aged versus young mice, acute environmental exposure to O(3) or CS at the doses used had no effect. Gene expression levels, determined by RT-PCR of AT transport protein (ATTP), SRB1, CD36, ATP binding cassette 3 (ABCA3) and ABCA1 and protein levels, determined by Western blots for SRB1, ATTP and ABCA1 were assessed. Aged mouse lung showed a lower levels of ATTP, ABCA3 and SRB1 and a higher level CD36 and ABCA1. Acute exposure to either O(3) or CS induced declines in ATTP and SRB1 in both aged and young mice lung. CD36 increased in both young and aged mice lung upon exposure to O(3) and CS. These findings suggest that both age and environmental oxidant exposure affect pathways related to lung AT homeostasis and do so in a way that favors declines in lung AT. However, given the approach taken, the effects cannot be traced to changes in these pathways or AT content in any specific lung associated cell type and thus highlight the need for further follow-up studies looking at specific lung associated cell types.

Antioxidants in cystic fibrosis. Conclusions from the CF antioxidant workshop, Bethesda, Maryland, November 11-12, 2003.

Although great strides are being made in the care of individuals with cystic fibrosis (CF), this condition remains the most common fatal hereditary disease in North America. Numerous links exist between progression of CF lung disease and oxidative stress. The defect in CF is the loss of function of the transmembrane conductance regulator (CFTR) protein; recent evidence that CFTR expression and function are modulated by oxidative stress suggests that the loss may result in a poor adaptive response to oxidants. Pancreatic insufficiency in CF also increases susceptibility to deficiencies in lipophilic antioxidants. Finally the airway infection and inflammatory processes in the CF lung are potential sources of oxidants that can affect normal airway physiology and contribute to the mechanisms causing characteristic changes associated with bronchiectasis and loss of lung function. These multiple abnormalities in the oxidant/antioxidant balance raise several possibilities for therapeutic interventions that must be carefully assessed.

Does gamma-tocopherol play a role in the primary prevention of heart disease and cancer? A review.

Vitamin E consists of a group of eight isomers, four tocopherols (alpha-, beta-, gamma-, delta-tocopherol) and four tocotrienols (alpha-, beta-, gamma-, delta-tocotrienol). While extensive literature has been published on the potential health benefits of alpha-tocopherol, little is known about gamma-tocopherol, the major form of vitamin E in food in the U.S. gamma-tocopherol has recently received more research attention based on findings from in vitro and animal studies indicating that it has potent anti-inflammatory and antioxidant properties. Based on these recent studies, it is important to investigate the possible health benefits of gamma-tocopherol in humans. In this article, we review publications on dietary gamma-tocopherol intake, plasma gamma-tocopherol levels, cardiovascular disease and cancer risk in humans.

Regulation of the alpha-tocopherol transfer protein in mice: lack of response to dietary vitamin E or oxidative stress.

The alpha-tocopherol transfer protein (TTP) plays an important role in the regulation of plasma alpha-tocopherol concentrations. We hypothesized that hepatic TTP levels would be modulated by dietary vitamin E supplementation and/or by oxidative stress. Mice were fed either a High E (1150 mg RRR-alpha-tocopheryl acetate/kg diet) or a Low E (11.5 mg/kg diet) diet for 2 wk. High E increased plasma and liver alpha-tocopherol concentrations approximately 8- and 40-fold, respectively, compared with Low E-fed mice, whereas hepatic TTP increased approximately 20%. Hepatic TTP concentrations were unaffected by fasting (24 h) in mice fed either diet. To induce oxidative stress, chow-fed mice were exposed for 3 d to environmental tobacco smoke (ETS) for 6 h/d (total suspended particulate, 57.4 +/- 1.8 mg/m3). ETS exposure, while resulting in pulmonary and systemic oxidative stress, had no effect on hepatic alpha-tocopherol concentrations or hepatic TTP. Overall, changes in hepatic TTP concentrations were minimal in response to dietary vitamin E levels or ETS-related oxidative stress. Thus, hepatic TTP concentrations may be at sufficient levels such that they are unaffected by either modulations of dietary vitamin E or by the conditions of environmentally related oxidative stress used in the present studies.