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* From the Departments of Clinical Chemistry (Drs. Lases, Gerritsen, and Haas) and Pulmonary Diseases (Drs. Duurkens), St. Antonius Hospital, Nieuwegein, The Netherlands.
Correspondence to: Vincent A.M. Duurkens, MD; Department of Pulmonary Diseases, St. Antonius Hospital, PO Box 2500, NL-3430 EM Nieuwegein, The Netherlands;
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Design: A prospective study.
Setting: A specialized thoracic surgical unit in a large referral hospital.
Patients: Twenty-eight patients with lung carcinoma undergoing thoracotomy.
Measurements: Exhaled H2O2 concentrations in breath condensate were measured by spectrophotometry, while malondialdehyde (MDA) levels in urine samples collected every 24 h were measured by reversed-phase, ion-pair high-performance liquid chromatography using ultraviolet detection.
Results: Our results show increased H2O2 and MDA levels in lobectomy patients compared with pneumonectomy patients. A strong correlation was found between the levels of H2O2 and MDA.
Conclusion: The present data support the hypothesis that oxidative stress may occur following pulmonary resection.
Key Words: hydrogen peroxide • lobectomy • malondialdehyde • oxidative stress • pneumonectomy • predictive value of tests • pulmonary edema
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of the present study was to investigate whether oxidative stress may occur following pulmonary resection and to evaluate whether parameters of oxidative stress might be of value in the assessment of the diagnosis, course, and prognosis of postoperative complications, like PPE.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Collection of Expired Breath Condensate and
H2O2 Measurement
The patients’ samples of exhaled breath condensate were
collected at 8:00 AM on the day of surgery, 30 min after
surgery, and 1 day after surgery. The samples of healthy volunteers
only were collected at 8:00 AM. Before collection, patients
and control subjects rinsed their mouths with 0.2% chlorhexidine
gluconate (Zeneca; Ridderkerk, The Netherlands) to exclude the effect
of mouth flora. Subsequently, the participants breathed through a face
mask with a two-way valve. The expired air was conducted through a
connection with a "cold finger" collecting system that was
connected to an 8-mL tube. In this way, approximately 1 to 4 mL of
breath condensate was collected within 30 min with tidal breathing.
After surgery, the collection system was connected to the expiratory
port of the ventilator, since all patients were intubated. The samples
were immediately frozen at -70°C.
H2O2 measurements were
performed within 1 week after sample collection, since preliminary data
(results not shown) demonstrated that
H2O2 concentrations had not
changed during this period. The method described by Gallati and
Pracht14
was applied. In this method, 100 µL 420
µmol/L 3,3',5,5'-tetramethylbenzidine (Aldrich; Milwaukee, WI) were
dissolved in 0.42 mol/L citrate buffer (pH, 3.8) (sodium citrate;
Merck; Darmstadt, Germany; citric acid; Sigma; St. Louis, MO) and 10
µL 52.5 U/mL of horseradish peroxidase (Sigma) were added to 200 µL
condensate and to mixtures of 190 µL condensate and 10 µL 14,000
U/mL catalase (Sigma). Catalase was used to prove the specificity of
the H2O2 measurement. The
reaction proceeded for 30 min at room temperature. Subsequently, the
mixture was acidified to pH 1 with 10 µL of 95 to 97% sulfuric acid
(Baker; Deventer, The Netherlands). The reaction product was measured
spectrophotometrically at 450 nm using an automated microplate reader
(Titertek Twinreader type 380; Flow Laboratories Amstelstad B.V.;
Zwanenburg, The Netherlands). The absorbance was directly proportional
to the H2O2 concentration
in the range of 0 to 10 µmol/L. All samples were measured in
duplicate; mean values were used for subsequent analysis.
Collection of Urine and MDA Measurement
The patients’ samples of urine were collected on the day
before, the day of, and the day after surgery. The urine samples
collected every 24 h were collected in a container without any
addition. MDA measurements were performed on the day of collection,
since preliminary data (results not shown) demonstrated that MDA levels
were changed during a 24-h storage time. Reversed-phase, ion-pair
high-performance liquid chromatography using ultraviolet detection at
254 nm was used for the measurement.13
Aliquots of a urine
specimen, filtered through a sterile 0.2-µm filter (Acrodisc;
Gelman Sciences; Ann Arbor, MI), were analyzed using a high-performance
liquid chromatography system (LKB-HPLC; Pharmacia Biotech AB;
Uppsala, Sweden) and were separated on a 150 x 4.6-mm 3-µm packing
column (Supelcosil LC-IST HPLC; Supelco Inc; Bellefonte, PA) using a
gradient elution. Absorbance was monitored, and data were collected
with a databox (500 series, model 2600; Perkin-Elmer; Norwalk, CT)
using computer software (Nelson Analytical Chromatography Software,
revision 5.0, 1987; Perkin-Elmer). The following gradient was used for
the chromatographic separation: 15 min in 100% of buffer A (10 mmol/L
tetrabutylammoniumhydroxide [Sigma]/10 mmol/L
KH2PO4 [Merck]; pH, 7.0);
and 15 min in up to 100% of buffer B (2.8 mmol/L
tetrabutylammoniumhydroxide /100 mmol/L
KH2PO4; pH, 5.5), and the
solution was held here for an additional 6 min. The initial conditions
were restored after 9 min by washing with buffer A.
A single assay was completed in 45 min. MDA concentrations were calculated by the measurement of peak-area ratios of the sample and the standard (1,1,3,3-tetraethoxypropane, 20 µmol/L; Sigma). The reproducibility of the method, expressed as the intrarun coefficient of variation, was 1.2% and the detection limit (blank value + 3 SDs) was 0.2 µmol/L. This method had a linearity up to 20 µmol/L. The MDA excretion is expressed as the ratio of the urinary creatinine.
Statistical Analysis
The measured values were nonnormally distributed. Thus, all
statistical analyses followed the nonparametric approach. Consequently,
all values are presented as median and the 95% confidence interval
(CI). Comparisons within the lobectomy and pneumonectomy groups were
made using the Wilcoxon signed rank test. The Mann-Whitney U
test was used for comparisons between the groups. Statistical
significance was assumed at p < 0.05. Spearman rank correlation
tests were performed to detect a correlation between the concentration
of exhaled H2O2 in breath
condensate and the MDA excretion in urine samples collected every
24 h.15
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A strong correlation (r = 0.79) was found between the levels of H2O2 and MDA (n = 18) in patients who underwent thoracotomy (Fig 3 ). In 20 cases the concentration of H2O2 in breath condensates was below the detection limit of 0.14 µmol/L.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Before discussing the implications of our findings, we should address some possible limitations of our methodology. First, it was not possible to obtain all data points in all patients. Among particular missing values were those belonging to the time point 1 day after surgery. In our statistical analysis, we corrected for these missing data because fewer values lead to a broadening of the 95% CIs. Second, we cannot exclude the possibility that mechanical ventilation affects H2O2 and MDA levels due to intubation. However, all patients were intubated and receiving mechanical ventilation for the same time period. Therefore, any possible effect of intubation on H2O2 and MDA levels may be assumed to be constant in both groups. Consequently, the observed differences are valid. Besides, intubation is unavoidable in these patients.
Our results show that in lobectomy patients the concentrations of exhaled H2O2 and MDA excretion were higher after surgery than in the pneumonectomy patients. This might be explained by the fact that in patients undergoing lobectomy the lobes have to be separated by the surgeon, which means much more manipulation than in a patient undergoing pneumonectomy. Therefore, lobectomy seems to be more stressful. Besides, the remaining lobe seems more prone to oxidative stress than an empty cavity after pneumonectomy.
Recently, Williams et al18 found changes in several markers of oxidative stress (protein thiol, protein carbonyl, and myeloperoxidase levels) following lung resection. They expected that the lobectomy patient group would be subjected to a greater degree of oxidative stress than the pneumonectomy group, but they were not able to conclude this from their results. A possible explanation might be that the parameters, which were measured in plasma, do not reflect the degree of oxidative stress in the lung. In this study, we used direct measurement of oxidative stress in the lung by exhaled H2O2. This method should be able to distinguish between lobectomy and pneumonectomy, assuming the higher stress in lobectomy.
Only one patient developed PPE in our pilot study. This could be expected because the frequency of PPE following pneumonectomy is 4%. No other postoperative complications were encountered. Nine pneumonectomy patients showed the same downward trend after surgery, while the PPE patient displayed significantly elevated levels of H2O2 and MDA in comparison with the other pneumonectomy patients. Because only one patient in our study developed postoperative complications, it is possible only to indicate a trend.
The fact that the increases in levels of H2O2 and MDA were similar suggests that a relationship between these two parameters for oxidative stress might exist. Indeed, we found a significant correlation between the levels of H2O2 and MDA in thoracotomy patients. This correlation between two different classified and measured parameters in different patients strengthens the hypothesis that oxidative stress may occur following pulmonary resection.
In conclusion, the results of the present study support the hypothesis that oxidative stress may occur following lung resection. Lobectomy patients showed higher H2O2 and MDA levels than pneumonectomy patients. In one PPE patient, both H2O2 and MDA levels increased steadily in comparison with the other pneumonectomy patients. Large-scale prospective studies are indicated to explore the potential predictive value of these types of markers.
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Acknowledgements |
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Footnotes |
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Received for publication September 10, 1999. Accepted for publication November 10, 1999.
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