Vitamin C

status by protecting polyunsaturated fatty acid from oxidative degradation, as their supple‐

Levels of plasma carotenoids such as β-carotene, β-cryptoxanthin, and total lycopene are significantly lowered in CF patients and this has been associated with higher susceptibility to lipid peroxidation. Rust et al. [14] examined the effect of long-term oral β-carotene supple‐ mentation in patients with CF. Patients of the CF supplementation group received 1 mg βcarotene/kg body weight/day (maximally 50 mg β-carotene/day). During high-dose treatment, a significant decrease in the MDA level and a correction of total antioxidative capacity was

Renner et al. reported distinct clinical benefits from high-dose (1 mg/kg body weight/ day, maximum 50 mg/day) supplements. Their patients required significantly fewer antibiotics during the phase of high-dose β-carotene supplementation and showed a decrease in pulmo‐ nary exacerbations [15]. Lepage et al. reported that the two-month supplementation of CF patients with 4.42 mg β-carotene, three times per day, led to the normalization of increased

At the same time, toxicity issues have been raised for supplementation with water- miscible vitamin A formulations in CF patients, which may increase serum retinol and possible risk of CF-associated liver and bone complications [17]. However, β-carotene supplementation seems to be safe since it does not affect serum concentrations of other carotenoides and retinol [14]. Recent studies have analysed the use and safety of a new CF polyvitamin (AquADEKs ®), which comprises almost 90% vitamin A in retinol form. Patients on AquADEKs ® maintained a high level of serum β-carotene, but serum retinol was not above the normal levels. β-carotene levels were associated with lung function and better nutritional status [18,19], while lipid

α-tocopherol acts as a membrane antioxidant closely associated with polyunsaturated fatty acids. Vitamin E's antioxidative properties might be helpful in reducing the negative effects of free radicals. Current recommended supplementation of vitamin E in CF patients only includes α-tocopherol. Supplementation with high levels of α-tocopherol alone may result in further imbalances in CF patients: such supplementation has been shown to deplete γtocopherol in the blood and tissues [21]. Papas et al. [16] evaluated vitamin E supplementation with mixed tocopherols. The increase in the blood of levels of γ-tocopherol may be particularly important for CF patients due to its function as a scavenger of reactive nitrogen species and its synergistic effects with α-tocopherol [22]. Cystic fibrosis is characterized by neutrophildominated airway inflammation. Activated neutrophils release oxidants, proteases, and cytokines, further sustaining and increasing the inflammatory response and causing direct injury to the lungs. Improved antioxidant capacity with γ-tocopherol, especially if present in the lungs, could potentially decrease oxidant-mediated damage and limit the cytokinemediated neutrophil recruitment. It has also been reported that reduced serum levels of vitamin E are associated with an increased rate of pulmonary exacerbations in CF [23].

MDA level and increased plasma β-carotene from 0.08 ± 0.03 to 3.99 ± 0.92 μM [16].

peroxidation markers were not affected [20].

Vitamin E (α-tocopherol)

mentation augments levels of plasma polyunsaturated fatty acid.

Carotenoids

76 Cystic Fibrosis in the Light of New Research

observed.

Vitamin C is present in the respiratory lining fluid of human lungs, and local deficit occurs during oxidative stress. Experimental findings confirm that vitamin C induced the openings of CFTR Cl− channels without a detectable increase in intracellular cyclic AMP levels. Vitamin C instilled into the nasal epithelium of human subjects effectively activates Cl− transport, too.

The pool of vitamin C in the respiratory tract represents a potential nutraceutical and phar‐ maceutical target for the complementary treatment of oxidative stress in patients with CF [24]. In a study by Winklhofer-Roob et al. [25] on 122 children and young adults with CF, ascorbicacid concentrations decreased with age, and low vitamin C levels were associated with the highest indexes of inflammation, so the authors concluded that ascorbic acid could interact with an inflammation-amplifying circle of activation of alveolar neutrophils and macrophages.

Water-soluble vitamins seem to be well absorbed by patients with CF, but there is documented evidence of poor dietary intake. A supplement of at least 50–100 mg vitamin C/day should be prescribed for patients with an unbalanced diet, or if there is evidence of deficiency.

### Selenium

Dietary intake of selenium is inversely related to inflammatory markers such as sialic acid and triacylglycerol [26]. Moreover, a possible role of selenium in the modulation of serum com‐ plement 3, which may be an early marker of metabolic syndrome manifestations, has also been documented.

Wood et al. carried out an eight-week, double-blind, randomized intervention trial, providing two groups of patients with low- and high-dose vitamin supplements (500 μg vitamin A and 10 mg vitamin E vs. 500 μg vitamin A, 25 mg β-carotene, 200 mg vitamin E, 300 mg vitamin C and 90 μg selenium). They demonstrated significant improvement of clinical indicators after treatment. Increased serum β-carotene, selenium, and fatty-acid concentrations were linked to improved lung function [27].

Consensus regarding supplementation of antioxidants in CF to include selenium is yet to be established. Two studies have attempted to demonstrate successfully how the administration of 2.8 μg/kg/day and 90 μg/day of selenium can decrease oxidative stress in CF [28].

### **d. Glutathione and n-acetylcysteine**

The discovery that CF is associated with significantly diminished efflux of reduced glutathione (GSH) from most cells in the body [29] offers a new perspective on the pathophysiology of this disease. GSH plays several important roles; among the most important are the following: 1) primary water-soluble antioxidant; 2) mucolytic capable of cleaving disulphide bonds; and 3) regulator of immune-system function [30].

The relationship between redox ratio (GSH:GSSG) and total glutathione (GSH+GSSG) and the initiation of inflammation is well established [29,30]. GSH is also an important component of the epithelial lining fluid of the intestines, helping to keep intestinal mucus thin, serving to defend the intestinal system against reactive oxygen species, and keeping inflammation in check under normal circumstances [31].

In a recent placebo-controlled, randomized, double-blinded, clinical trial in 44 paediatric CF patients aged between 18 months and 10 years [32], treatment with oral glutathione (65 mg/kg/day) increased weight and BMI z score and improved measures of gut inflammation (faecal calprotectin) over the course of six months, without adverse side effects. The authors therefore concluded that Oral GSH might primarily be beneficial in those children with more severe inflammation of the gut, and suggested that early intervention with oral glutathione in young CF children with growth failure could forestall decline in pulmonary function in later years.

Several studies have investigated the potential therapeutic role of inhaled GSH in patients with CF. Three short-term clinical trials, including a placebo-controlled one, have shown the tolerability and efficacy of inhaled GSH on pulmonary function in these subjects [33-34]. A recent 12-month randomized single-blind placebo-controlled trial demonstrated the efficacy of inhaled GSH (600 mg twice daily) on lung function in CF adults [35]. Three months of therapy with inhaled GSH resulted in a statistically significant improvement in percentagepredicted FEV1, measured as a pre-post difference from baseline values, when compared to the placebo, which persisted at six and nine but not at 12 months. A reduced compliance with therapy in adult patients could explain the decrease in FEV1 values registered in the last visit. The best improvements in functional parameters were registered in the subgroup of patients with moderate lung disease (FEV1 below 81%). These results are in concordance with those reported by Griese et al. [36], who showed a significant increase of FEV1 absolute values (but not when expressed as percentage-predicted) from the baseline after three months of GSH therapy.

N-acetylcysteine (NAC), a well-known cysteine donor for the synthesis of glutathione, has been used in different diseases to treat GSH deficiency [37]. High-dose oral NAC has been shown to increase neutrophil GSH levels, decrease airway neutrophil recruitment and reduce neutrophilic release of airway elastase in CF patients [38]. Skov et al. demonstrated that highdose oral NAC (1200 mg x 2/day for 30 days) in CF patients with chronic *P. Aeruginosa* infection decreased the level of oxidized vitamin C [39].

Indications of a positive effect of NAC treatment on the lung function of a subgroup of CF patients have previously been published [40]. Recently, a placebo-controlled randomized clinical trial (70 CF patients) was conducted in the USA to study the effect of oral NAC on lung inflammation (ClinicalTrials.gov Identifier: NCT00809094). Oral NAC was administered in a dose of 1800 mg/day divided into two dosages over a period of 24 weeks and the effects on the sputum levels of human neutrophil elastase (HNE) were assessed as a primary end-point. While no statistical significant difference was found between the two groups with regard to the primary end-point, an improvement in the predicted FEV1% was observed in the NACtreated group.

A recent Cochrane review on the use of thiol derivatives, such as NAC, did not find sufficient evidence to recommend the use of these compounds in the management of CF lung disease, but concluded that further studies were warranted [41].

γ-Glutamylcysteine ethyl ester (GCEE) is another potentially interesting GSH pro-drug, which has proved to have some efficacy in the amelioration of oxidative stress, e.g., in experimental myocardial infarction [42] and central-nervous-system conditions (see, e.g., [43]). However, GCEE has not been investigated in CF yet.

New CFTR modulation therapies are being designed to correct the function of the defective protein (CFTR) made by the CF gene, allowing chloride and sodium to move properly in and out of cells lining the lungs and other organs. N6022 is a new injectable compound that modulates the function of the defective CFTR protein and decreases inflammation in the lung. N6022 is the first of a new class of compounds that increase levels of an important signalling molecule in the body called S-nitrosoglutathione, or GSNO. These novel compounds have been shown in preliminary results (Phase 1b trial) to increase the amount of CFTR that reaches the cell membrane and to stabilize CFTR so that its function can be improved.
