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Original Research |
Discipline of Nutrition and Dietetics, University of Newcastle (L.G.W., M.L.G.), John Hunter Children’s Hospital, Newcastle
Department of Paediatrics (D.A.F., D.M.C.), John Hunter Children’s Hospital, Newcastle
Airway Research Centre, Department of Respiratory Medicine (P.G.G.), John Hunter Children’s Hospital, Newcastle
Department of Dietetics (C.E.C.), John Hunter Children’s Hospital, Newcastle
Department of Respiratory Medicine, New Children’s Hospital, Sydney (D.A.F.), New South Wales, AUSTRALIA
Address reprint requests to: Dr Manohar Garg, Head of Discipline of Nutrition and Dietetics, Faculty of Medicine & Health Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia. E-Mail: ndmg{at}medicine.newcastle.edu.au.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and antioxidant defenses, in relation to dietary intake, immune function and clinical status.
Methods: We measured total plasma concentrations of 8-iso-PGF2 and dietary antioxidants (vitamin E, vitamin C, ß-carotene), erythrocyte antioxidant enzyme activities (glutathione peroxidase and superoxide dismutase), lung function and dietary intake in 21 CF subjects and 21 healthy age- and gender-matched controls.
Results: Total plasma 8-iso-PGF2 concentration (median [quartile 1–quartile 3]) was significantly higher in CF subjects compared to controls (214 pg/mL (155–331) vs. 135 pg/mL (101–168), p=0.001). Neutrophil, monocyte and total white cell counts were elevated in the CF group and these correlated with 8-iso-PGF2 concentration. Despite similar dietary intake, lower plasma antioxidant concentrations were observed in the CF group (vitamin E, p < 0.001, vitamin C, p=0.004, ß-carotene, p=0.001). 8-iso-PGF2 correlated negatively with plasma vitamin E, C and ß-carotene concentrations.
Conclusion: Oxidative stress is increased in CF patients, despite normal dietary antioxidant intake. The immune response appears to be a key factor causing oxidative stress. Antioxidant intervention aimed at reducing oxidative stress in CF needs to be assessed.
Key words: cystic fibrosis, oxidative stress, antioxidants, isoprostanes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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is the most well-known isomer) is also a useful marker of oxidative stress in CF [9,10]. There are many factors that may contribute to increased oxidative stress in CF, as the disease combines increased production of ROS, with impaired antioxidant protection. CF causes intense and recurrent airway inflammation. The bacteria which colonize the lungs attract neutrophils which aim to destroy the invading pathogen by releasing high concentrations of ROS, some of which may leak into surrounding cells [11]. Free radical generation is also heightened in CF by an increase in metabolic rate which causes oxygen uptake to be 120% to 150% of normal [12], resulting in an increase in the quantity of ROS that are continuously generated as unwanted by-products of the respiration process. These factors lead to an increased oxidant burden, while, at the same time, patients have reduced antioxidant protection. Despite the administration of vitamin and pancreatic enzyme supplements to the 85% of CF patients with pancreatic insufficiency, residual steatorrhea and azotorrhea still occur [13], with the potential for ongoing malabsorption of fat soluble antioxidants, namely vitamin E and ß-carotene. The high fat, high energy diet which is recommended in CF may also be a cause of altered antioxidant protection. While increased dietary fat intake may increase levels of vitamin E, the intake of dietary antioxidants in foods such as fruits and vegetables (namely vitamin C and ß-carotene) may be limited. Furthermore, the loss of permeability of glutathione in the CFTR channel may result in the deficiency of glutathione in the airway surface liquid [14]. Consequently, many different and interacting pathways can potentially lead to increased oxidative stress in CF.
The aim of this study was to examine oxidative stress in CF by measuring total plasma 8-iso-PGF2 and antioxidant defenses in relation to dietary intake, immune function and clinical status.
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METHODS |
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Subject Characteristics
Routine pulmonary function tests were performed in all subjects using a spirometer (Medgraphics 1085D BreezeTM cardiorespiratory diagnostic software 1991, St Paul, MN), with established normal values [16]. Forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) were recorded and compared to predicted values. Height was measured with a Holtain, Crymych, Dyfed stadiometer. Weight was recorded using GEC/Avery digital scales, model number 824/890. Full blood counts were performed using a COULTER GEN-S analyser. For CF subjects other markers of respiratory status were recorded which included finger clubbing and chest auscultations (crackles or wheezing). Other characteristics recorded include genotype (
F508 zygosity), pancreatic sufficiency, current medications, presence of respiratory pathogens, history of antibiotic use and hospitalizations over the last two years for intravenous antibiotic treatment.
Vitamins and Mineral Analysis
Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) coated tubes and centrifuged at 3000 rpm, at 4°C for 10 minutes. Plasma was collected and frozen at -70°C within half an hour of blood collection. Plasma levels of vitamins A and E and ß-carotene were separated on a reverse phase high performance liquid chromatography (HPLC) column and measured using a program variable wavelength UV-visible detector [17]. Samples were thawed, mixed with ethanol to precipitate proteins, then hexane was added. After mixing again, samples were centrifuged and the hexane phase removed and injected into an HPLC column (lab-packed Whatman ODS 3 (5 micron) 300 x 3.5 mm ID), with a flow rate of 1 mL/min, run time of 20 minutes, at ambient temperature. At 0.01 minutes, vitamin A was measured at 310 nm, at 5.5 minutes, vitamin E was measured at 280 nm and at 9.0 minutes, ß-carotene was measured at 450 nm. Plasma vitamin C was separated on a reverse phase HPLC column and measured using an electrochemical detector [18]. Samples were mixed with trichloracetic acid to precipitate proteins, centrifuged and the supernatant injected into an HPLC column (lab-packed Whatman ODS 3 (5 micron) 150 x 3.5 mm ID), with a flow rate of 1 mL/min, run time of 15 minutes, at ambient temperature. Measurements were made with an amperometric (BAS) electrochemical detector with potential +0.6 volts versus Ag/AgCl reference electrode. Plasma levels of zinc, selenium and copper were analyzed by inductively coupled plasma—mass spectrometry (ICP-MS). Samples were diluted in an ammonium EDTA based diluent in a quantitative application. Platinum and rhodium were used as internal standards in the diluent. Calibration was by additions calibration in a pooled plasma base.
Total 8-iso-PGF2 Assay
Blood samples were collected in EDTA coated tubes, containing reduced glutathione (Sigma Chemical Company, St Louis, MI, USA) as an antioxidant [9]. The samples were centrifuged at 3000 rpm, at 4°C for 10 minutes. The plasma fraction was removed and stored at -70°C in tubes precoated with butylated hydroxytoluene (BHT) (Sigma) for isoprostane analysis. To an aliquot of plasma, a known amount of tritium-labeled thromboxane B2 (TxB2) (Amersham, Arlington Heights, IL) was added, to allow determination of recovery rate after purification procedure. Ethanol was added, the sample was chilled at 4°C, then centrifuged at 1500 g for 10 minutes to remove the precipitated proteins. The supernatant was decanted, an equal volume of 15% KOH was added and the resultant solution incubated at 40°C for 1 hour, to cleave any esterified isoprostane. The sample was diluted with H2O, then the pH was lowered with HCl to below 4.0. The sample was passed through a Sep-Pak C-18 reverse phase cartridge (Waters, Milford, MA), which had been activated by rinsing with methanol, then H2O. After passing the sample through, the cartridge was rinsed again with H2O, then hexane. Finally, the 8-iso-PGF2 was eluted with ethyl acetate containing 1% methanol. This solvent was evaporated using N2 and the sample reconstituted with assay buffer. Purified sample was added to aqueous biodegradable counting scintillant (Amersham) and counted using a liquid scintillation counter to determine recovery rates. A quantity of the remaining portion was analyzed with an 8-isoprostane enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Absorbance values were measured using a plate reader with a wavelength of 405 nm and the raw data corrected for recovery. The assay was validated by adding a series of known amounts of pure 8-iso-PGF2 standard to base volumes of purified plasma. The concentration of total 8-iso-PGF2 was determined using enzyme immunoassay (EIA). A high correlation (0.99) was obtained between the known amounts of 8-iso-PGF2 added and the concentration determined by EIA. The antiserum used in this assay has a 100% cross-reactivity with 8-iso-PGF2, 0.2% each with PGF2, PGF3, PGE1 and PGE2 and 0.1% with 6-keto-PGF1. The detection limit of the assay is 4 pg/mL. This kit has been used to measure 8-iso-PGF2 concentration in human plasma, BAL and other fluids [9,10,19].
GSHPx Enzyme Assay
Whole blood was collected into EDTA coated tubes and centrifuged at 8500 g at 4°C for 10 minutes. Plasma was discarded and cells washed with 10 volumes of cold buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM EDTA and 1 mM dithiothreitol). Samples were centrifuged again at 8500 g at 4°C for 10 minutes, and supernatant was discarded. Cells were then lysed by adding exactly 4 volumes of ice cold deionized water. After centrifuging again at 8500 g at 4°C for 10 minutes, supernatant was collected and stored at -70°C for analysis. Erythrocyte glutathione peroxidase (GSHPx) activities were measured using GPx-340 spectrophotometric assay kit (Bioxytech; OXIS International, Portland, OR), to obtain values in units per milliliter. The hemoglobin (Hb) concentration of the samples was also measured using Sigma Kit No. 525 for total hemoglobin (Sigma), to allow erythrocyte GSHPx activity to be expressed as units per gram of Hb.
Superoxide Dismutase (SOD) Enzyme Assay
Whole blood was collected into EDTA coated tubes and centrifuged at 3000 rpm at 4°C for 10 minutes. The erythrocyte pellet was separated and stored at -70°C before analysis. The erythrocyte pellet was thawed and resuspended in four volumes of ice-cold water and mixed thoroughly. Ice-cold extraction reagent (ethanol/chloroform, 62.5/37.5 v/v) was added to the erythrocyte suspension and mixed for 30 seconds. Samples were centrifuged at 3000 g and 4°C for 10 minutes. The upper phase was collected and stored at -70°C for analysis. Erythrocyte Zn/Cu-SOD activities were measured using SOD-525 spectrophotometric assay kit (Bioxytech; OXIS International), to obtain values in units per milliliter. The hemoglobin concentration of the samples was also measured using Sigma Kit No. 525 for total Hb, to allow erythrocyte Zn/Cu-SOD activity to be expressed as units per milligram of Hb.
Dietary Intake
Dietary intake was assessed using the 24 hour recall method [20]. Analysis of food records was conducted using the Diet/1 Nutrient Calculation Software, which is based on the 1992 Australian food tables and the composition of Australian manufactured foods (Goodhill, 1992). The mean intakes of energy, protein, fat, carbohydrates, fiber, vitamin A, ß-carotene, vitamin C, iron and zinc for each subject group were determined from these.
Statistical Analysis
Results were analyzed using Minitab Version 12 for Windows (Minitab Inc., State College, PA) [21]. Data were tested for normality using the Anderson-Darling test. Statistical comparisons were performed using the paired Student t test for normally distributed data and the Wilcoxon paired test for non-parametric data. The mean ±standard error are reported for normal data; for nonparametric data the median (quartile 1—quartile 3) are reported. Differences were considered significant when p < 0.05. Correlations between variables were studied by linear regression, with calculation of Pearson’s correlation coefficient for normal data and Spearman’s rank correlation coefficient for nonparametric data.
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RESULTS |
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F508 - 12/21 (57%), heterozygous F508 - 8/21 (38%) and other/other–1/21 (5%). Within the CF group, current medications included ß2 agonists, aerosol–15/21 (71%), inhaled corticosteroids–7/21 (33%), cromoglycate–3/21 (14%), ipratropium–5/21 (24%), DNase–4/21 (19%) and antibiotics–4/21 (19%). As expected, several markers of inflammation, including total white cell, neutrophil and monocyte counts, were significantly higher in the CF group than in the control group (Table 3).
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concentrations were elevated in the CF group compared to the control group (p < 0.001) (Fig. 1). Plasma 8-iso-PGF2 concentration correlated with total white cell, neutrophil and monocyte counts (Fig. 2). No correlation was observed between 8-iso-PGF2 concentrations and %FEV1, %FVC, height or weight. Plasma concentrations of the dietary antioxidant vitamins E, C and ß-carotene were significantly lower in the CF group than in the control group (Table 4). Plasma 8-iso-PGF2 correlated inversely with vitamin E, ß-carotene and vitamin C (Fig. 3). Plasma concentration of ß-carotene correlated inversely with total white cell, neutrophil and monocyte counts (Fig. 4). Similarly, plasma vitamin E correlated inversely with neutrophil and monocyte counts (Fig. 5). There was no significant difference in plasma concentrations of Se, Zn or Cu between the control and the CF groups. Similarly, there was no significant difference in the activity levels of the GSHPx or SOD enzymes in erythrocytes from the two groups.
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DISCUSSION |
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While there are many factors believed to contribute to raised oxidative stress, chronic bacterial colonization of the airways has been suggested as a major contributor to oxidative stress in CF [1,2,3,22,23]. In our study, significantly higher total white cell, neutrophil and monocyte counts support the presence of ongoing infection and inflammation in the CF group, even in the absence of severe pulmonary exacerbation. As the activity of the immune system is increased, the presence of increased quantities of ROS is anticipated.
Antioxidant defenses were deficient in the CF group, with low concentrations of the plasma antioxidants vitamin E, vitamin C and ß-carotene. The inverse correlations between ß-carotene and neutrophil, monocyte and white cell counts, as well as the inverse correlations between vitamin E and neutrophil and monocyte counts provides evidence that the production of ROS by the immune system (and resultant increased utilization of antioxidants) contributes strongly to the impaired antioxidant defenses. This evidence that ß-carotene is acting as an antioxidant in vivo is interesting in light of recent reports that ß-carotene supplementation is associated with adverse effects [24].
Reduced antioxidant defenses cannot be explained on the basis of dietary intake. Dietary analysis revealed no difference in the intake of vitamin A, vitamin C or ß-carotene between groups. While vitamin E intake is not available using the Diet1 software, it is assumed that the intake of this fat-soluble vitamin is also adequate, as food sources are similar to those of vitamin A and given the high fat intake of the CF group. Thus, the adoption of a high fat/high energy diet appears not to have altered the intake of dietary antioxidants. While previous studies have also reported low concentrations of plasma antioxidant vitamins [1,4–7,25–29], our data suggest that reduced dietary intake is not contributing to the low antioxidant defenses.
Malabsorption of fat soluble nutrients does not explain the antioxidant deficit. Another cross sectional sample of this same clinic population showed malabsorption to be generally well controlled (mean fecal fat excretion < 12%) [30]; thus, uptake of fat soluble vitamin E and ß-carotene should be adequate. Also, the correlation between dietary intake of ß-carotene and plasma ß-carotene concentration provides evidence that absorption has been normalized. Furthermore, the deficiency in water soluble vitamin C cannot be explained by fat malabsorption, and the correlation between dietary intake of vitamin C and plasma vitamin C concentration again suggests normal absorption.
As a result of impaired antioxidant protection, as well as the increased activity of the immune system, it is not surprising that 8-iso-PGF2 concentration is increased in the CF group, indicating increased levels of oxidative stress. The inverse correlations between 8-iso-PGF2 and vitamin E, vitamin C and ß-carotene provide evidence that the reduced antioxidant protection is contributing to elevated oxidative stress in the CF group. Similarly, the correlations between 8-iso-PGF2 and neutrophil, monocyte and total white cell counts provide direct evidence that the immune response to infection is contributing to elevated oxidative stress.
There was no correlation between plasma 8-iso-PGF2 and lung function tests. However, it has been suggested that, while diminished lung function may indicate previous lung damage, 8-iso-PGF2 concentration reflects the current pathological situation and may therefore reflect subsequent changes in lung function [19]. Thus, further studies examining changes in 8-iso-PGF2 and lung function over time are important. Additionally, isoprostanes have been shown to be potent vasoconstrictory agents in rat lungs [31]. While the situation in humans in vivo may be different, the full impact of the elevated isoprostane levels on pulmonary function remains to be established, and isoprostanes may prove to have an important biological role, as well as being an important in vivo marker of oxidative stress.
Activities of the Zn/Cu-SOD and GSHPx antioxidant enzymes were not reduced in the CF group in our study, nor were the respective cofactors of these enzymes (Zn, Cu and Se). Other studies have also shown normal erythrocyte levels of SOD, GSHPx, Se and/or Zn in CF patients [4,32]. However, some others have found reduced erythrocyte GSHPx activity levels [4]. This inconsistency suggests that enzyme activities may be poor indicators of oxidative stress. Unlike the dietary antioxidant vitamins, these enzymes are manufactured by the body. Thus it is possible that activity levels can be maintained or upregulated during times of increased demand, with a decline being only observed in the case of severe or prolonged nutrient deficiency or severely elevated oxidative stress.
The EIA methodology used in this study to measure 8-iso-PGF2 provides an inexpensive, accessible alternative to analysis by gas chromatography—mass spectrometry (GCMS). The values of total 8-iso-PGF2 we observed in normal plasma using the EIA method are similar to those obtained by Morrow et al. using GCMS [33]. As discussed by Morrow et al. [34], GCMS assay is expensive, labor intensive and uses technology that is not widely available. Thus, the use of specific immunoassays should expand research in this area [34]. Several studies using the EIA methodology have recently been reported [9,10,19].
Dietary data in this study was collected using the 24-hour recall method. While this method is reasonably well established for determining average intake levels of groups [20], the limitations of a method relying on subject recall and a short time frame are acknowledged. It is also acknowledged that analysis of diet records does not provide definitive information on antioxidant intake, which is also affected by factors such as food storage and processing. It should also be noted that the vitamin E concentrations for the control group are low, suggesting that this group may not be well nourished.
Although dietary antioxidant supplementation in CF patients will clearly not cure the disease, the evidence suggests that it may have a beneficial role. To date no study has shown an improvement in clinical status as a result of antioxidant supplementation in CF. Recent reports on dietary antioxidant supplementation normalizing MDA (a marker of lipid peroxidation) levels in plasma of children with CF [5,7,28] are provocative and highlight the relevance of using appropriate types and levels of antioxidant supplements in the treatment of CF patients.
In conclusion, this study has demonstrated increased total plasma 8-iso-PGF2 concentration, together with reduced antioxidant protection in CF, despite normal dietary intake. The data suggest that oxidative stress in these patients is strongly linked to the immune response to chronic inflammation and infection. Antioxidant supplementation in CF aimed at normalizing plasma 8-iso-PGF2 concentration may be an effective way of improving clinical outcome in CF.
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ACKNOWLEDGMENTS |
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Received August 29, 2000. Accepted February 9, 2001.
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