Increased hydrogen peroxide in the exhaled breath of uraemic patients unaffected by haemodialysis

Jacek Rysz1, Marek Kasielski2, Joanna Apanasiewicz3, Maciej Król3, Andrzej Woznicki3, Marek Luciak1 and Dariusz Nowak3

1Department of Internal Medicine and Dialysotherapy Medical University of Lodz, Lodz, 2Practical Clinical Training Centre, Medical University of Lodz, Lodz and 3Department of Experimental and Clinical Physiology, Institute of Physiology and Biochemistry, Medical University of Lodz, Lodz, Poland

Correspondence and offprint requests to: Dariusz Nowak, Department of Experimental and Clinical Physiology, Institute of Physiology and Biochemistry, Medical University of Lodz, Mazowiecka Str. 6/8, 92-215 Lodz, Poland. Email: dnowak{at}zdn.am.lodz.pl



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Uraemia is accompanied by conditions favouring the rise of H2O2 activity in body fluids. This results from the increased release of H2O2 by polymorphonuclear leukocytes and decreased plasma glutathione peroxidase activity. The purpose of this study was to determine if patients on chronic haemodialysis (HD) exhale more H2O2 than healthy individuals, and if dialysis affects breath H2O2 content.

Methods. We studied 29 chronic HD patients (mean age 49 ± 11 years) and 40 healthy persons (mean age 44 ± 9 years). H2O2, which is volatile, was measured fluorimetrically with the homovanillic acid method in the exhaled breath condensate (EBC) of the study cohort. EBC was collected immediately before and after the HD session and also at 20 and 60 min of HD treatment (n = 14) and once in controls. Peak expiratory flow (PEF), white blood cell (WBC) count, PaO2 and circulatory cyclic guanosine monophosphate (cGMP), Il-6 and Il-8 concentrations were measured concomitantly. Finally, H2O2 diffusion through the dialyser cuprophane membrane was determined in an in vitro experiment.

Results. At baseline, EBC H2O2 concentration was 22 times higher in HD patients than in controls (2.92 ± 4.64 vs 0.16 ± 0.13 µM, P < 0.001). Although the maximum decrease in PEF (431 ± 52 vs 398 ± 56 l/min, P < 0.01) and WBC count (6.72 ± 1.02 vs 3.82 ± 1.51 x 103/µl, P < 0.01) occurred at 20 min after the start of HD, no significant changes in breath H2O2 levels were noted throughout the session. Plasma IL-6 and IL-8 levels remained unchanged whereas cGMP rose 1.3 times at 60 min (P < 0.01). In vitro, H2O2 rapidly diffused through the cuprophane membrane.

Conclusion. Chronic HD patients exhale more H2O2 than healthy subjects. Although no change of breath H2O2 concentration was observed during HD, as H2O2 easily diffuses through the dialyser membrane, it is not possible to rule out that HD stimulates H2O2 generation.

Keywords: Exhaled breath condensate; haemodialysis; hydrogen peroxide; uraemia



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Polymorphonuclear leukocytes (PMN) in patients with chronic renal insufficiency are primed for enhanced oxidative bursts and produce more reactive oxygen species (ROS), including H2O2, than in healthy individuals [1,2]. The activity of glutathione peroxidase, one of the main antioxidant enzymes responsible for H2O2 decomposition, is suppressed in uraemic patients [3]. Therefore, uraemia is accompanied by conditions that favour increased production of H2O2 and impair its decomposition into water. Although studies examining the effect of haemodialysis (HD) on ROS production by PMN have yielded conflicting results—with this function being reported as suppressed, normal and increased [46], the transient pulmonary leuko-sequestration during HD [7] may result in an increased ROS overload in lungs. Moreover, HD-induced leuko-sequestration is accompanied by decreased pulmonary diffusing capacity [8], which suggests the occurrence of an interstitial pulmonary inflammatory response. As plasma contains detectable concentrations of H2O2 [9], it is possible that part of the H2O2 released from PMN attached to the pulmonary endothelium (under conditions of decreased glutathione peroxidase activity) may diffuse into the fluid lining the epithelium of the lower airways and from there exhaled into air. In this study we wanted to determine H2O2 concentrations in exhaled breath condensate (EBC) collected from uraemic patients just before (0 min), during (at 20 and 60 min) and after (at 240 min) HD and compare them with levels found in healthy controls.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Study population
The study involved 29 (HIV- and HBV-negative) patients with end-stage renal disease who had been on regular maintenance HD from 0.5 to 12 years (mean 5.6 ± 2.4 years) and 40 non-smoking healthy individuals as controls (Table 1). No subject had suffered from any infectious disease during at least the 3 months preceding the study. The controls were not on any medications, and their routine physical examinations disclosed no abnormalities. None of the uraemic patients had diabetes, symptomatic hyperparathyroidism or symptoms of aluminium toxicity. All medications were stopped starting 2 months prior to the study (including vitamins with antioxidant properties and blood transfusions), but recombinant human erythropoietin continued to be administered in doses necessary to maintain haematocrit between 30 and 35% (n = 24), as did the combinations of beta-blockers and angiotensin-converting enzyme inhibitors needed to control hypertension (n = 14).


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Table 1. Patient characteristics

 
All subjects enrolled in the study gave informed consent, and the study protocol was approved by Ethics Committee of the Medical University of Lodz.

Collection of expired breath condensate
EBC was collected before and after the 4-h HD sessions—(using AK–100 GAMBRO, Lund Sweden), with single-use cuprophan dialyser (Clirens C–101 Terumo Corp, Tokyo Japan)—as described previously [10], with some modifications. Briefly, the collecting device consisted of a mouthpiece with a saliva trap connected to a glass Liebig tube cooler (cooling and collecting tube 55 cm long, internal diameter 10 mm, external jacket diameter 36 mm; Labmed, Lodz, Poland, cat. no. 6010). The tube was cooled with ethanol pumped through the closed circuit and its temperature was kept at -9°C with Multi Temp III (Pharmacia Biotech). This temperature was the lowest that allowed collecting liquid EBC in the sterile plastic tube covered with ice (Sarstedt, capacity 13 ml, internal diameter 14 mm) and mounted at the base of Liebig tube cooler. Subjects were asked to exhale through the mouthpiece and inhale with the mouthpiece removed, for 20 min as described previously [10]. Aliquots of 5 ml of EBC were collected and were stored at -80°C for at most 7 days pending H2O2 measurement. In 14 patients, EBC was also collected at 20 and 60 min of HD and the time of collection was shortened to 7 min. The patients started to breathe out into a collection device 3.5 min before the chosen time-point and finished the EBC collection procedure 7 min later.

Measurement of H2O2 in expired breath condensate
H2O2 was measured following the procedure of Ruch et al. [11] with some modifications [10]. Briefly, 600 µl of EBC was mixed with 600 µl of horseradish peroxidase solution (1 U/ml) containing 100 µM homovanillic acid and incubated for 60 min at 37°C. Then, the sample was mixed with 150 µl 0.1 M glycine–NaOH buffer (pH 12.0), 25 mM of EDTA was added and, after specimen excitation at 312 nm, emission at 420 nm was measured using a Perkin Elmer Luminescence Spectrometer LS-50B. The samples used to control the assay received deionized water instead of EBC. Readings were converted into micromoles using the regression equation Y = 0.012 (X - Xo) - 0.007 (where Y = micromoles of H2O2 per litre of EBC; X = intensity of emission at 420 nm expressed in arbitrary units; Xo = intensity of emission of the reference sample that received deionized water). The lower limit of H2O2 detection was 0.083 µM [10]. The intra-assay variability did not exceed 2% for standard H2O2 solutions ranging from 0.1 to 0.5 µM. Of the EBC specimens with detectable H2O2 levels, four obtained from the controls and four from uraemic patients were pre-incubated with 10 µl of aqueous catalase solution containing 20 U of enzyme activity (catalase from Aspergillus niger 6600 U/mg protein; Sigma, St Louis, MO, USA) or 10 µl of distilled water for 30 min at 37°C, after which the concentration of H2O2 was measured again.

H2O2 diffusion through cuprophane membrane
Two independent closed circuits working in the counterflow system were mounted in the capillary cuprophane membrane dialyser (Diacap CE 1600, B.Braun, Melsungen AG, Germany). The first circuit (blood circuit) was filled with 250 ml of 0.9% NaCl and the second one with 430 ml of dialysing fluid (140 mEq/l Na+, 2.5 mEq/l K+, 3.0 mEq/l Ca2+, 1.0 mEq/l Mg2+, 35 mEq/l HCO3-, 111.5 mEq Cl-, 3.68 mM acetic acid). Samples of the dialysis fluid (950 µl) leaving the dialyser were collected just before and every 1 min for 8 min after the addition of 427 µl of 35.12 mM H2O2 solution in 0.9% NaCl into the blood circuit (just beyond the dialyser) and placed immediately into a luminometer to measure H2O2. The pressures in both circuits were equal, and the fluid flow was started simultaneously with the first specimen collection and maintained at 130 ml/min with a peristaltic pump.

Measurement of H2O2 in dialysis fluid
The concentration of H2O2 was determined by measuring H2O2 horseradish peroxidase-dependent luminol chemiluminescence [12]. Briefly, 950 µl aliquots of the dialysing fluid were pre-incubated in a 1251 Luminometer (Bio-Orbit® Oy, Turku, Finland) for 5 min at 37°C and then the baseline chemiluminescence was recorded continously for 1 min. Afterwards, 50 µl of 1 µM luminol solution in 0.07 M phosphate buffer (pH 8.4) containing horseradish peroxidase (4 U/ml) was automatically injected, and the total light emmission (integrated chemiluminescence) was counted for a further 2 min. The results were converted into micromoles using the calibration curve obtained with 20 increasing concentrations of H2O2 ranging from 0.1 to 50 µM. The method sensitivity was 0.25 µM H2O2.

Other measurements
Spirometry was performed with a Lungtest 1000 spirometer (MES S.c., Kraków, Poland) according to American Thoracic Society standards [13] the day before EBC collection.

Peak expiratory flow (PEF) was measured with a mini-Wright peak flow meter (with the patient sitting) at 0, 20, 60 and 240 min of HD [14]. At the same points of time, arterial blood was drawn to measure: haematocrit; white blood cells (WBC); arterial oxygen pressure (PaO2); plasma concentrations of cyclic guanosine monophosphate (cGMP) (Cyclic GMP Enzyme Immunoassay Kit, Cayman Chemical, sensitivity 0.9 pmol/ml), Il-6 and Il-8 (Quantikine TM Human Interleukin Immunoassay D6050 and D8050, R&D Systems, USA, sensitivity 0.7 and 0.9 pg/ml). WBC and PaO2 were determined in the Diagnostic Laboratory of Medical University Teaching Hospital of Lodz. PEF measurements were repeated during the second and third consecutive HD sessions with the inhalation of one puff of salbutamol (0.1 mg) or ipratropium bromide (0.02 mg) just before and 10 min prior to the initiation of HD, respectively. The values of plasma concentrations tested during and at the end of HD were corrected for changes in haematocrit.

Statistical analysis
Data are expressed as mean ± SD. Median and quartile ranges (QR) were also calculated for H2O2 results. For readings below the methods sensitivity, H2O2 concentrations in EBC were assumed to be 0 nM. The differences between the groups were computed with the Mann–Whitney U test. Variables changing over time during HD were analyzed with ANOVA or Friedman ANOVA followed by the Student and Wilcoxon matched pairs tests according to the sample distribution. Correlation coefficients were calculated with the Pearson test or Spearman test. A P value < 0.05 was considered significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients with end-stage renal disease who are on regular HD exhaled 22 times more H2O2 (2.92 ± 4.64 vs 0.16 ± 0.13 µM, P < 0.001) (median = 0.63, QR = 2.00 vs median = 0.14, QR = 0.13) than healthy non-smokers (Figure 1). H2O2 levels noted in these patients at the end (240 min) of HD (2.74 ± 4.78 µM, median = 0.91, QR = 2.10) also were higher than in healthy individuals (17 times, P < 0.001). Although in healthy controls cigarette smoking elevated EBC H2O2 levels [10], there were no significant differences in H2O2 exhalation between currently smoking and non-smoking uraemic patients (2.72 ± 4.71 µM, n = 6 vs 2.91 ± 4.86 µM, n = 23, P > 0.05). In accordance with the results of previous studies [7,14], HD caused transient decreases in WBC and PEF (Table 2). The lowest PEF and WBC values were observed at 20 min of HD; however, these were not accompanied by significant changes in H2O2 exhalation (Figure 2). The time course of H2O2 exhalation in 14 uraemic patients was as follows: 2.03 ± 3.85, 1.24 ± 1.65, 1.85 ± 3.79 and 2.40 ± 5.00 µM at 0, 20, 60 and 240 min of HD (P > 0.05), respectively. The addition of catalase to the EBC specimens of uraemic patients and healthy persons who had previously revealed high H2O2 exhalation completely abolished homovanillic acid oxidation (Table 3). This indicates that our method for H2O2 determination is specific and other compounds, such as reactive oxygen species, do not contribute to high H2O2 readings especially in our uraemic patients.



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Fig. 1. H2O2 levels in EBC of 40 healthy controls (A) and of 29 uraemic patients just before (B) and after (C) HD session. *P < 0.001 vs healthy subjects. No significant changes were found in H2O2 exhalation before and after the HD session.

 

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Table 2. Changes of selected clinical parameters during HD

 


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Fig. 2. Time course of H2O2 exhalation in 14 uraemic patients during HD. EBC for H2O2 determination was collected just before (0 min) and at 20, 60 and 240 min (end) of HD. No significant changes were found from baseline (0 min).

 

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Table 3. Effect of catalase on hydrogen peroxide concentration in EBC obtained from four healthy persons and four uraemic patients

 
In vitro, H2O2 diffused rapidly through the dialyser cuprophane membrane (Figure 3), reaching an equilibrium concentration of ~22 µM in the dialysis fluid within 3 min from H2O2 injection into the blood circuit. This concentration was nearly equal to that calculated (22 µM) based on the amount of H2O2 added and the total fluid volume of both circuits. This suggests that under our experimental conditions H2O2 did not react with the dialyser membrane.



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Fig. 3. H2O2 diffusion through the cuprophane membrane. 427 µl of 35.12 mM H2O2 solution in 0.9% NaCl were added into blood circuit of the cuprophane capillary dialyser (Diacap CE 1600, B.Braun, Melsungen AG, Germany) filled with 250 ml 0.9% NaCl. Specimens of dialysis fluid (950 µl) were collected from the second circuit (volume 430 ml) just before (0 min) and after H2O2 addition. The fluid flow in both circuits was 130 ml/min. H2O2 was measured with the chemiluminescence method [12].

 
Inhalation of salbutamol or ipratropium bromide just before and 10 min prior HD completely prevented PEF decrease. No significant alterations of circulatory Il-6 and Il-8 levels in response to HD were noted. Only the mean plasma concentration of cGMP, the vasodilatory second messenger of NO, increased 1.3 times (P < 0.01) at 60 min of HD (Table 2). No significant correlations were found between exhaled H2O2 and spirometric parameters, the cause of chronic renal insufficiency, duration of HD treatment, plasma Il-6, Il-8 or cGMP (data not shown). There was no association between H2O2 levels and treatment with beta-blockers and angiotensin-converting enzyme inhibitors (data not shown).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Uraemic patients regularly on HD were found to exhale many times more H2O2 than healthy persons. To our knowledge this is the first report that shows increased exhalation of H2O2 by patients with end-stage renal disease. Exhaled H2O2 may originate in pulmonary tissue (e.g. alveolar macrophages, type II pneumocytes) [10], circulating phagocytes or both. HD patients with chronic renal insufficiency had elevated levels of the plasma protein 3-chlorotyrozine, formed from tyrozine residues by the myeloperoxidase–H2O2–Cl- system as a consequence of polymorphonuclear leukocyte activation and H2O2 release into circulation [15]. This supports the hypothesis that circulating phagocytes could be a source of exhaled H2O2 in uraemics on HD.

Plasma glutathione peroxidase activity is decreased in uraemic patients [3]. In addition, HD patients have an increased activity of plasma superoxide dismutase, which catalyses dismutation of superoxide radical into H2O2 [16]. These may promote elevated H2O2 levels in body fluids, including that lining the epithelium of airways, and consequently increased H2O2 exhalation by uraemic patients.

HD is believed to induce systemic oxidative stress [17]; moreover, it causes sequestration in pulmonary microvasculature [7] of activated PMN, producers of more ROS [18]. This is accompanied by the impairment of respiration [8,14] and increased nitric oxide exhalation [19]. Although, our patients revealed transient leukopenia and PEF decrease, we surprisingly did not observe a rise in EBC H2O2 levels during HD. Possible explanations for this are: (i) an increase in plasma glutathione peroxidase activity after HD [3] and consequently higher H2O2 decomposition in pulmonary microvasculature; (ii) H2O2 diffusion through the dialyser membrane into the dialysing fluid; (iii) increased systemic and airway NO production in response to HD [19,20]. NO may react with superoxide radicals, precursors of H2O2, thus limiting its formation. The rise in the plasma concentration of cGMP (the second messenger of NO) during HD accords with this hypothesis.

Our in vitro experiments showed rapid H2O2 diffusion through the dialyser cuprophane membrane. This seems to be responsible for the transient (although not significant) decrease of exhaled H2O2 during HD. Moreover, it is not possible to exclude that H2O2 diffusion abolishes the stimulatory effect of HD on the production by circulating phagocytes of ROS [6]—thus, the insignificant rise in EBC H2O2 levels noted in response to HD.

On the other hand, a possibility that cannot be excluded is that the systemic and airway inflammatory response to HD was too small to induce a rise in H2O2 exhalation. Patients in our study had no significant changes in plasma Il-6 and Il-8 concentrations; and any mild, transient decrease of PEF in response to HD was easily prevented by single doses of bronchodilators.

In conclusion, we have found that uraemic patients on long-term, regular HD exhale many times more H2O2 than healthy subjects. However, H2O2 exhalation did not change significantly during or as a result of HD. Further prospective studies with uraemic patients starting HD are necessary to identify the sources of exhaled H2O2 and to explain precisely the effect of HD on EBC H2O2 levels and its significance with respect to lung function.



   Acknowledgments
 
The study was supported by grant 503-104-4 from the Medical University of Lodz.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 20. 8.02
Accepted in revised form: 6. 8.04