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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:459-464 (2007)
© 2007 The Gerontological Society of America

Evaluation of Oxidative Stress and Effectiveness of Low-Dose Glucocorticoid Therapy on Exacerbation of Chronic Obstructive Pulmonary Disease

Fumio Komatsu, Hideki Kudoh and Yasuo Kagawa

The High Technology Research Center, Kagawa Nutrition University, Saitama, Japan.

Address correspondence to Fumio Komatsu, MD, Kagawa Nutrition University, 3-9-21 Chiyoda, Sakado, Saitama, 350-0288, Japan. E-mail: komatsu{at}eiyo.ac.jp


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Background. Chronic obstructive pulmonary disease (COPD) and its exacerbation are considered to be related to oxidative stress. We determined the levels of reactive oxygen metabolites (ROM) and a biological antioxidant potential (BAP) in blood before and after the exacerbation.

Methods. For these determinations, the Free Radical Analytical System 4 was used. Twenty-one male patients were divided into two groups.

Results. Group A patients (n = 11, for whom the disease was end-stage) showed high ROM levels and low BAP levels. Meanwhile, Group B patients (n = 10, for whom the disease was not end-stage but the exacerbation was repeated) exhibited variable ROM levels corresponding to the symptoms. Before the exacerbation, ROM levels increased. After administering high-dose glucocorticoids, the exacerbation disappeared and ROM levels decreased to the baseline. To suppress the relapse, the Group B patients were medicated with low-dose glucocorticoids by inhalation and systemic administration. Then, ROM levels did not increase or the relapse did not occur. BAP levels continued to be low, and were restored after a stable state was obtained.

Conclusion. Overproduction of ROM may precede exacerbation of COPD, and the low-dose glucocorticoid therapy may be effective to suppress its overproduction and to preserve COPD in a stable state.

CHRONIC obstructive pulmonary disease (COPD) is an obstructive airway disorder and shows slowly progressive pulmonary dysfunction. This disease is rare in middle-aged people but common in elderly people (1–3). Recently, the number of patients has been increasing. This disease is characterized by acute exacerbation, which often occurs after the common cold, acute bronchitis, and severe fatigue. The exacerbation means acute pulmonary insufficiency accompanied with serious dyspnea, hypoxemia, and cyanosis. Patients experience a decrease in quality of life, and nearly 50% of them are readmitted more than once in the following 6 months. Snow and colleagues (4) stated that a 180-day mortality rate in this disease was 33%. Therefore, the main treatment goal is reducing the exacerbation. For the therapy of exacerbation, high-dose glucocorticoids are often prescribed (5–7). However, this therapy is usually carried out only for a short period so as to avoid severe adverse effects such as diabetes mellitus and several infections. But, after discontinuing this therapy, the relapse soon occurs.

COPD is considered to be related to oxidative stress (1–3). Neutrophils and macrophages may infiltrate into lung tissue and play an important role in the production of reactive oxygen species (ROS) (8,9). ROS injure protein, lipids, and DNA and induce cell membrane destruction. The oxidative stress contributes to irreversible damage of both parenchyma and airway walls. The oxidative stress, in turn, results in alteration of the local immune response, which increases risk of infections.

Now, there have been few reports concerning the relationship of ROS production and exacerbation, because measurement of ROS and free radicals in routine laboratory tests is difficult owing to their biochemical characteristics. Recently, for measuring reactive oxygen metabolites (ROM) in blood, the d-ROM test has been developed. This test uses the Free Radical Analytical System (FRAS; Diacron, Grosseto, Italy) (10–12). The main component of ROM is hydroperoxide (11,12). Hydroperoxide causes cell death and tissue damage (13,14). Despite fair oxidant power, hydroperoxide in blood is relatively stable compared to the parent free radicals; therefore, the level can be adequately detected. By using this analyzer, a biological antioxidant potential (BAP) in blood can also be determined. The BAP test measures the capacity of blood to convert from Fe3+ reagent to Fe2+ (15,16). These tests have been recognized to be sensitive. In this study, we determined ROM and BAP levels in COPD patients, in particular, before and after the exacerbation. We medicated glucocorticoids at a high dosage for acute phase of exacerbation and at a low dosage for chronic phase to suppress the relapse. We describe the tendency of ROM and BAP levels in this disease and the effectiveness of low-dose glucocorticoids for suppressing the relapse.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Participants
COPD was diagnosed according to the COPD Guideline of the Japanese Respiratory Society in 2001. Decreasing of forced expiratory volume in one second (FEV1/FVC and FEV1) on a spirometry test and emphysematous changes on chest x-ray were important findings for the diagnosis. Smoking history, serious cough, and mucous sputum were also helpful. COPD staging was evaluated according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) Guidelines (17).

Twenty-one Japanese COPD patients were enrolled. For a comparison, Japanese healthy individuals (n = 220) and habitual heavy smokers (n = 24) were also enrolled. The protocol of this study was approved by the Ethics Committee of Kagawa Nutrition University (No. 178) according to the Declaration of Helsinki (Edinburgh, October 2000). The participants gave us their written informed consent before the study.

Measurement of ROM
To measure ROM levels, the d-ROM test was performed using the FRAS4. In brief, a 20 µL blood sample and 1 mL of buffered solution (R2 reagent of the kit, pH4.8) were mixed in a cuvette, and then 10 µL of chromogenic substrate (R1 reagent) was added to the cuvette. After mixing and centrifugation for 60 seconds, the cuvette was incubated in a thermostatic block for 5 minutes at 37°C. After that, the 505 nm absorbance was recorded. Measurements were expressed as Carr U. Reference values measured by the manufacturer (Diacron) are indicated as being from 250 and 300 Carr U; values higher than 300 Carr U suggest oxidative stress (10,11).

Measurement of BAP
To measure BAP levels, the BAP test was performed using the same analyzer. In brief, 50 µL of R2 reagent (ferric chloride) was added to the cuvette containing R1 reagent (thiocyanate derivative), and the absorbance was measured (to read and subtract the reagent blank value). Then, 10 µL of serum sample was added to the cuvette. After incubation for 5 minutes at 37°C, the 505 nm absorbance was recorded. The BAP levels were expressed as µmol/L. Reference values indicated by the manufacturer are shown to be higher than 2200 µmol/L; values lower than 2200 µmol/L suggest a reduced antioxidant capacity (15,16).

Measurements of Other Oxidative Stress Markers
We compared ROM and BAP levels with other oxidative stress markers that have been recognized to be reliable indicators. Peripheral blood was drawn following overnight fasting. Lipid peroxide (LPO) was measured by the Hemoglobin–Methylene Blue method (18). Malondialdehyde-modified low-density lipoprotein (MDA-LDL) was measured by using an enzyme-linked immunosorbent assay (ELISA) (19). Urinary 8-hydroxydeoxyguanosine (8-OHdG) was determined using the ELISA kit (20). Superoxide dismutase (SOD) activity was measured by the nitrite method (21).

Results were expressed as mean average ± standard deviation (M ± SD). For evaluation of the difference, Student's t test was used, and a p value <.05 was considered to be statistically significant.


   RESULTS
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
ROM and BAP Levels in Healthy Participants
Before the determination of ROM and BAP levels in COPD patients, we first determined them in healthy participants aged 20–98 years. In this study, smokers were not included. As shown in Figure 1, young participants (20–30 years) generally exhibited low ROM levels (around 300 Carr U), and the levels increased with age. Participants older than 70 years usually demonstrated high ROM levels. In Figure 2, BAP levels are shown. Most young participants exhibited high BAP levels (>2000 µmol/L), and the levels demonstrated a significant inverse correlation with age. This finding indicates that ROM and BAP levels change with age. Thus, evaluation of them in COPD patients should be performed by comparing the values with those in age-matched controls.


Figure 01
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  		Figure 1. Relationship of reactive oxygen metabolite (ROM) levels with ages in healthy participants. Y = young; O = old

 

Figure 02
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  		Figure 2. Relationship of biological antioxidant potential (BAP) levels with ages in healthy participants. Y = young; O = old

 
Because COPD is observed in men more than in women, ROM and BAP levels were compared between male and female healthy participants. As shown in Table 1, ROM levels showed no significant difference between men and women, whereas there was a significant difference in BAP levels. In participants older than 70 years, many women who were living in licensed nursing facilities for day care were included. They were served with nutritionally balanced diets by professional dieticians every day. Although they showed high ROM levels, their BAP levels did not always decrease compared to the participants aged 50–70 years. It is possible that the high antioxidant capacity of these women may be maintained by their desirable diets.


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  		Table 1. Comparison of ROM and BAP Levels Between Male and Female of Healthy Participants.

 
Because COPD often appears in habitual smokers more than in nonsmokers, ROM and BAP levels were compared between habitual heavy smokers and nonsmokers. The heavy smokers in this study were men and were not included in the above group of healthy participants, although they were healthy. They had smoked (>2 packs/d) for more than 5 years. As shown in Table 2, they demonstrated significant higher ROM levels than age-matched nonsmokers (controls; men from the above healthy participants shown in Figure 1), although there was no significant difference in BAP levels. This result suggests that habitual heavy smoking may affect oxidative stress.


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  		Table 2. ROM and BAP Levels in Habitual Heavy Smokers and COPD Patients.

 
ROM and BAP Levels in COPD Patients
COPD patients in this study were men (n = 21). All of them had smoked heavily (>2 packs/d) for 30–50 years until the diagnosis. They were divided into two groups: Group A and Group B.

Group A patients (n = 11) had suffered from persistent cough and shortness of breath for 5 years and had stayed in a hospital for 0.5–2 years. They showed hypoxemia and a decrease of FEV1 (FEV1 < 30% of predicted values, GOLD Guideline stage III) and mild emphysematous changes on chest x-ray. They needed continuous oxygen inhalation every day and could not leave the hospital. They were medicated with several drugs including short-acting and long-acting ß2-agonists, anticholinergic bronchodilators, and inhalant glucocorticoids (fluticasone, Flutide). The disease was end-stage for them, and four of them died within 1 year following cardiac or pulmonary insufficiency. As shown in Table 2, they demonstrated very high ROM levels and low BAP levels compared to age-matched healthy controls (men from the above group of healthy participants). We performed these tests repeatedly and obtained the same results consistently.

Group B patients (n = 10) were outpatients (30% < FEV1 < 60%, GOLD Guideline stage II) and had smoked for 30–40 years. The emphysematous findings of chest x-ray were slight or were not recognized. They could stay at home in a chronic stable state and did not need oxygen inhalation, but used long-acting ß2-agonists. The disease condition was not end-stage for them. However, eight Group B patients were admitted to a hospital. Although ROM levels were lower than those of Group A patients during the stable state, the levels increased at the exacerbation (Table 2).

Corticosteroid Therapy and Changes of ROM and BAP Levels
Group B patients showed severe symptoms such as hypoxemia, cyanosis, and general edema during the exacerbation, and FEV1 decreased below 30% of predicted values. These patients were treated with oxygen inhalation, short-acting ß2-agonists, and high-dose glucocorticoids (hydrocortisone 500 mg for 2 days + prednisolone 40 mg for 2 days then 20 mg for following 2 days). Within 1 or 2 weeks, they recovered from the serious symptoms. Hypoxemia, cyanosis, and general edema disappeared. FEV1 was restored to the levels of 30%–50%. ROM levels increased at the exacerbation and then returned to the baseline values along with the recovery (Table 2). BAP continued at low levels before and after the exacerbation. Transient hyperglycemia and urinary glucose were recognized in four patients during the high-dose glucocorticoid therapy.

The high-dose glucocorticoid therapy was discontinued after 4–5 days so as to avoid adverse effects. After that, the patients were medicated with long-acting ß2-agonists and Flutide (200 µg x 2/d) and spent 1–2 months without severe symptoms. However, after discontinuation of Flutide, the exacerbation relapsed within 2 months. Two patients died during the relapse, which was accompanied by further elevation of ROM levels. The patients were treated again with high-dose glucocorticoids as described above, and they then recovered. After repeating the relapse, we medicated the patients' low-dose glucocorticoids by inhalation (Flutide 100 µg x 1/d) and systemic administration (prednisolone 10 mg/d). After then, a stable state continued for over 1 year.

A typical case of repeating exacerbations is shown in Figure 3. The patient (S.N., 68-year-old man) had smoked heavily for 40 years. Dyspnea, persistent cough, mucous sputum, and shortness of breath appeared 5 years ago. Two years ago, he experienced a first exacerbation. After medication with high-dose glucocorticoids, he recovered from the symptoms. However, after 2 months, he experienced a second exacerbation. The same therapy was performed. After that, Flutide and prednisolone (10 mg/d) were prescribed. However, because his symptoms disappeared for 2 months, he stopped taking prednisolone. A third exacerbation soon appeared. He tried stopping again and experienced a fourth exacerbation. After he recovered from the last exacerbation, he continued taking the Flutide and prednisolone. After that, no relapse was observed for 1 year. As shown in Figure 3, ROM levels changed in correlation with the clinical symptoms, although BAP levels continued to be low. We determined other oxidative stress markers before and after the fourth exacerbation. As shown in Table 3, (i) LPO levels changed in parallel with ROM levels, (ii) changing of MDA-LDL levels appeared 3–5 days later than ROM levels, (iii) urinary 8-OHdG levels roughly correlated with ROM levels, and (iv) SOD activity changed within small ranges and did not always correlate with the symptoms. After a stable state was obtained, ROM levels stayed < 330 Carr U, and BAP levels > 1850 µmol/L.


Figure 03
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  		Figure 3. Changes of reactive oxygen metabolite (ROM) and biological antioxidant potential) BAP levels in Case (S.N.) before and after exacerbation. Discontinuation of glucocorticoids was associated with exacerbation. ROM levels changed corresponding to clinical symptoms. BAP levels restored after a stable state was obtained. (1), (2), and (3) = occurrences of dyspnea (short-acting inhaled ß2-agonist was necessary 2–3, 3–5, >5 times/d, respectively)

 

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  		Table 3. Determinants of Oxidative Stress Markers Before and After the Fourth Exacerbation in Case (S.N.).

 
Six patients have been taking this medication for more than 1 year, and none have reported any exacerbations, although FEV1 continued at the same levels of 30%–50% even after a stable state was obtained. No adverse effect has been observed. These findings suggest that the overproduction of ROM may precede the exacerbation, and the therapy using low-dose glucocorticoids (inhalation + systemic administration) may be effective to suppress the overproduction of ROM and maintain the condition of this disease in a chronic stable state.


   DISCUSSION
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
COPD is considered to be connected with oxidative stress. Cornelli and colleagues (8) stated that white cells (lymphocytes, neutrophils) as well as fibroblasts and endothelial cells are able to produce hydroxyl radicals and these produced free radicals cause a reactive phenomenon in the lung. Donma and Donma (3) stated that oxidant–antioxidant imbalance, like protease–antiprotease imbalance, is pathogenesis of smoking-induced COPD. In this study, we determined ROM and BAP levels in COPD patients and compared them with those in healthy participants. The manufacturer (Diacron) indicated that desirable ranges of ROM levels are 250–300 Carr U (10,11) and sufficient BAP levels are higher than 2200 µmol/L (15,16). But, in this study, ROM levels showed increases with age, and BAP levels demonstrated a negative correlation with age. COPD generally appears in older people; therefore, these levels in COPD patients should be evaluated by comparing them to those in age-matched controls. COPD patients demonstrated higher ROM level than the controls. With regard to gender difference, BAP levels were higher in women than in men. One of the reasons may be due to food styles. Japanese women follow a well regulated diet containing high levels of antioxidants, whereas Japanese men are not often able to maintain this diet. In this study, participants older than 70 years were enrolled. Many of them were women living in nursing facilities and were eating a well balanced diet. Their BAP levels were not always low compared with those of the participants aged 50–70 years. Their well balanced diets may have included foods containing antioxidants which are advantageous in preserving BAP levels. Effectiveness of antioxidants in COPD patients has been examined. Donma and Donma (3) described that supplementation of antioxidant vitamins was useful for improving this disease. Effect of N-acetylcysteine (NAC), a glutathione precursor, has also been tested. Anzalone and colleagues (22) and other authors (23,24) stated that NAC reduced the oxidative stress and risk of exacerbation, although Decramer and colleagues (25) did not find its effectiveness. Recently, Houstis and colleagues (26) reported that NAC stimulates the formation of endogenous glutathione, which scavenges H2O2 in cells. One of the reasons why COPD appears in men more than in women may be explained by low antioxidant capacity.

We compared ROM levels between heavy smokers and nonsmokers because COPD is said to be related to smoking. The result indicated that ROM levels in heavy smokers were higher than those in age-matched nonsmokers. Generally, smoking may induce oxidative stress (2,3). Smoking and its irritants enhance the infiltration of inflammatory cells into lung tissue. These cells produce interleukin-6, interleukin-8, tumor necrosis factor-{alpha}, and acute-phase proteins; furthermore, they induce an increase of intrapulmonary oxidant burden, in particular, an increase in the concentration of hydroperoxide (1,2).

In this study, we divided COPD patients into two groups. Group A patients (for whom the disease was end-stage) showed very high ROM levels and low BAP levels. This group showed a typical pattern of overt oxidative stress and reducing antioxidant capacity. Meanwhile, Group B patients (for whom the condition was not end-stage, but exacerbation repeated) demonstrated variable ROM levels. In chronic stable state, ROM levels were not always so high, but the levels markedly increased before the exacerbation. Patients were medicated with high-dose glucocorticoids, and then the severe symptoms were suppressed and ROM levels decreased to the baseline, although BAP levels were not restored until a stable state was obtained. These findings suggest that the overproduction of ROM precedes the deterioration of this disease. We compared ROM levels with other oxidative stress markers such as LPO, MDA-LDL, and urinary 8-OHdG concentrations because these markers are recognized as reliable indicators (27–29). In one case (S.N.), these markers showed roughly parallel changes with ROM levels. SOD activity was also determined. The changing ranges of SOD activity were small compared to those of BAP level. Thus, determination of BAP levels is preferable for evaluation of antioxidant status. Generally, the measurements of these markers are complicated. Meanwhile, d-ROM and BAP tests are simple, accurate, and reproducible. In particular, the d-ROM test may be useful in predicting the occurrence of exacerbation. Dekhuijzen and colleagues (30) reported that exhalation of hydrogen peroxide increased in patients with unstable COPD. With regard to the d-ROM test, if airway samples can adequately be mixed with the buffered solution of this kit, it may be possible to measure ROM levels in such samples, and its measurement may present meaningful information about the relationship between oxidative stress and clinical condition.

Group B patients repeated the exacerbations of this disease and high-dose glucocorticoids were used. However, after discontinuation of this therapy, the severe symptoms soon reoccurred. To suppress the relapse, we treated the patients with low-dose glucocorticoids even during the stable state. Concerning the effectiveness of glucocorticoids during the stable state, a controversy may occur because many authors reported that glucocorticoids did not show any effects on the chronic phase, although acute-phase symptoms were suppressed. Culpitt and colleagues (31) stated that steroids had no effect in stable COPD and did not redress the protease–antiprotease imbalance. Snow and colleagues (4) reviewed six randomized, placebo-controlled trials, and summarized that systemic glucocorticoids given for up to 2 weeks were helpful but no effect was observed after 2 weeks. Bach and colleagues (5), Niewoehner and colleagues (6), and Wood-Baker and colleagues (7) also stated mostly the same opinions. Usually, glucocorticoids have been used only for the short term, to avoid adverse effects. In contrast, Alsaeedi and colleagues (32) reported that long-term use of inhaled glucocorticoids (i.e., longer than 8 weeks) reduced airway inflammation, without significantly altering expiratory lung volumes.

In this study, low-dose glucocorticoids suppressed the relapse of exacerbation and overproduction of ROM. Previously, we had experienced difficulty in treatment of many patients with systemic lupus erythematosus (SLE). Early reduction of quantity of glucocorticoids invariably caused relapse of SLE. Slow reduction and continuous medication with low-dose glucocorticoids were effective for suppressing the relapse of SLE. We applied this therapy to COPD patients, although COPD is different from SLE. We speculate that glucocorticoids may suppress the infiltration of inflammatory cells into the lung, resulting in suppression of ROS overproduction. Total volumes of glucocorticoids used for the treatment could finally be reduced at small doses, and serious adverse effects (hyperglycemia and infections) were also avoided. High-unit Flutide therapy often induces infections such as candidiasis. Therefore, combination of low-unit inhalation (Flutide 50–100 µg/d) and low-dose systemic administration (prednisolone 5–10 mg/d) may be preferable. However, our data were obtained from a small number of patients, not enough for evaluation of clinical significance. Further study on many patients would be necessary.

Conclusion
COPD may intimately connect with oxidative stress. Overproduction of ROM may precede the exacerbation of this disease, and low-dose glucocorticoid therapy may be effective to suppress its overproduction and to preserve this disease in a stable state.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
We are grateful to Dr. Yoshinori Ui, the director of the Keijin-kai Daiichi Hospital, and Dr. Junnichi Morita, the director of Ryokueikai Hospital, for the management of this study. We thank all members of the Nursing Facility Mizuho-Mitaka and the Mizuho-Chouhu for their assistance in these determinations.


   Footnotes
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 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: Luigi Ferrucci, MD, PhD

Received March 5, 2006

Accepted July 25, 2006


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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