Objectives: In this study, we investigated dynamic thiol-disulfide homeostasis as a new indicator of oxidative stress in lung transplant recipients. In addition, we compared dynamic thiol-disulfide homeostasis parameters according to transplant indication and time after transplant.
Materials and Methods: This study had a single-center, observational, randomized design. In terms of transplant indications, lung transplant recipients were grouped as chronic obstructive pulmonary disease, interstitial lung disease, bronchiectasis, and other indications. To make comparisons based on time after transplant, lung transplant recipients were categorized into the following groups: >6 and ≤24 months, >24 and ≤48 months, >48 and ≤72 months, and >72 months. A fully automated spectrophotometric technique was used to measure dynamic thiol-disulfide homeostasis in fasting blood samples.
Results: Our study included 34 lung transplant recipients and 36 healthy volunteers. Native thiol (P = .005) and total thiol levels (P = .06) were lower in lung transplant recipients. Disulfide levels were similar. Disulfide-to-native thiol (P = .027) and disulfide-to-total thiol ratios (P = .027) were significantly higher in lung transplant recipients. Native thiol-to-total thiol ratios were lower in lung transplant recipients (P = .027). When we examined patients according to transplant indication, no statistically significant differences were found in dynamic thiol-disulfide homeostasis parameters, except for total thiol and disulfide levels. We also found no significant differences when we examined dynamic thiol-disulfide homeostasis parameters according to time after transplant.
Conclusions: Thiol-related antioxidant activity is significantly reduced after lung transplant, regardless of indication and transplant time. Ensuring oxidative balance in lung transplant recipients with an antioxidant supplement regimen can prevent damage from oxidative stress.
Key words : Oxidative status, Reactive oxygen species, Systemic inflammation
Lung transplant is a last resort treatment option for patients with end-stage lung diseases who are unresponsive to maximum medical therapy. The procedure significantly improves patients’ quality of life and survival rates. Despite advances in surgical techniques and immunosuppressant therapy, survival rates remain lower in lung transplant recipients than in other solid-organ transplant recipients.1 The causes of low survival rates among lung transplant recipients are primary graft dysfunction, acute rejection, and high mortality due to infections in the early period, as well as chronic lung allograft dysfunction (CLAD) in the late period. In addition to alloimmunologic damage, the vast epithelial surface of the lung and its constant contact with the external environment are risk factors for graft damage.2 Non-alloimmunologic factors such as toxic agent inhalation, infections, and gastro-esophageal reflux are thought to cause lung graft damage.1,2 These factors are related to the release of inflammatory mediators from various cells, such as epithelial cells, monocytes, macrophages, neutrophils, eosinophils, and dendritic cells. Increased reactive oxygen species (ROS) production and disrupted oxidative balance result from inflammatory mediators.3,4
Reactive oxygen species are unstable compounds with unpaired electrons that can initiate oxidation.5 Although ROS play an essential role in regulating many cellular and enzymatic reactions at low concentrations, they have a toxic effect on all cell elements, especially lipids, proteins, and nucleic acids at high concentrations.6,7 Damage from ROS can only be limited by oxidative balance.2 Physiologically produced oxidants exceed cells’ endogenous anti-oxidant capacity, and the disruption of intracellular redox homeostasis causes oxidative stress. Thiol is an organic compound that plays a critical role in protecting cells from oxidative stress.8 Thanks to its sulfhydryl content, thiols can eliminate ROS.9 The formation of reversible disulfide bonds is caused by thiol oxidation with oxidant molecules. When the oxidative stress condition is over, the disulfide bond structures formed can be reduced to thiol groups again, thus providing a dynamic thiol-disulfide balance.10 The relationship between oxidative stress and lung damage due to ischemia-reperfusion injury has been examined in many studies.11-16 However, there are few studies in the literature on long-term oxidative balance status in lung transplant recipients. In this study, we investigated dynamic thiol-disulfide homeostasis (TDH) as a new indicator of oxidative stress in lung transplant recipients. In addition, we compared dynamic TDH parameters according to the transplant indication and time after transplant.
Materials and Methods
Settings and study design
This single-center observational study was approved by our Institutional Ethics Committee (approval no. E1-21-1735). Written informed consent was obtained from all participants. All authors confirmed compliance with the World Medical Association Declaration of Helsinki on the ethical conduct of research involving human subjects.
We excluded recipients with single lung transplant, cystic fibrosis, active infections (bacterial, viral, or fungal), newly diagnosed acute cellular rejection, CLAD, acute or chronic kidney disease, heart failure, and liver dysfunction, as well as those who used antioxidants or herbal supplements. Lung transplant recipients with irreversible reduction (>20%), as measured by the best forced expiratory volume in 1 second, were not included in the study. As a result, 34 lung transplant recipients using the same immunosuppressant regimen consisting of a calcineurin inhibitor (tacrolimus), cell cycle inhibitor (mycophenolate mofetil), and low-dose systemic steroid (methylprednisolone) were included in the study. In addition, we also had a control group comprising 36 healthy volunteers randomized by age, sex, and body mass index (BMI; in kilograms divided by height in meters squared) and having no history of comorbidities or recent (within 1 month) trauma. No participants in the volunteer group were using antioxidants or herbal supplements, and none had a history of smoking more than 10 cigarette packages per year, inflammatory disease, autoimmune disease, diabetes mellitus, acute or chronic kidney disease, liver disease, cerebrovascular disease, or malignancy.
Lung transplant recipients were categorized according to their transplant indications and time after transplant. The dynamic TDH parameters were compared between the subgroups. Regarding the indications, the recipients were divided into 4 categories: chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD), bronchiectasis, and other indications. Two patients with lymphan-gioleiomyomatosis and 1 with histiocytosis X and Kartagener syndrome were included in the group of other indications. With regard to time after transplant, recipients were divided into the following 4 groups: >6 and ≤24 months, >24 and ≤48 months, >48 and ≤72 months, and >72 months posttransplant. Dynamic TDH parameters were compared
among the groups (Table 1). Age, sex, BMI, and laboratory results of all participants were noted. Time after transplant, actual pulmonary function test results, and 6-minute walk test results were also recorded.
Blood samples were obtained and placed into serum separator tubes after participants had fasted for 8 hours. The samples were centrifuged at 1600g for 10 minutes to obtain serum. Serum samples were stored at -80 °C until all results were analyzed together. Dynamic TDH parameters were measured with a fully automated spectrophotometric technique recently developed by Erel and Neselioglu.10 The technique is based on reducing dynamic disulfide bonds (-S-S-) to functional thiol groups (-SH). With the sodium borohydride (NABH4) used for this purpose, disulfide bonds are chemically reduced, and free and functional thiol groups (-SH) are formed. Sodium borohydride residues are removed with formaldehyde. Thus, the reduction of 5,5'-dithiobis-2-nitro benzoic acid (DTNB) and the reduction of disulfide bonds formed by the interaction of DTNB are prevented. We used DTNB to measure all thiol groups, both reduced and native. The total thiol content in the samples was measured using modified Ellman’s reagent. Half of the difference betweentotal thiol and native thiol gave the dynamic disulfide amount. After the native thiol, total thiol, and disulfide levels were determined, we calculated the disulfide-to-total thiol ratio, the native thiol-to-total thiol ratio, and the disulfide-to-native thiol
All analyses were performed using IBM SPSS Statistics for Windows, version 22.0 (SPSS Inc). Descriptive results are presented as frequencies and percentages for categorical variables and as means and standard deviations for numerical variables. Descriptive statistical methods (eg, mean, SD, median, frequency, and ratio) and Shapiro-Wilks test, histograms, and box plot charts were used to evaluate the distribution of the variables. Independent group comparisons were made with the Mann-Whitney U test and the t test according to the nonparametric and parametric distribution of the variables. Comparisons of dynamic TDH parameters in multiple groups were made with the Kruskal-Wallis test and one-way analysis of variance test. Post hoc tests were performed with the Dunn and Bonferroni tests. Spearman correlation test was used to analyze the correlation between TDH parameters and other parameters. All analyses were performed as 2-sided hypotheses with a 5% significance level and a 95% confidence interval. A 2-tail P < .05 was considered statistically significant.
The study population consisted of 70 participants: 34 lung transplant recipients (transplant group) and 36 healthy participants (control group). In the transplant group, the mean age was 49.3 ± 2.3 years; in the control group, the mean age was 47.1 ± 2.9 years. There were 8 women (23.5%) and 26 men (76.5%) in the transplant group and 10 women (27.7%) and 26 men (72.3%) in the control group. Demographic and laboratory findings are shown in Table 2. We observed no statistically significant differences between groups in age, sex, BMI, white blood cell (WBC), C-reactive protein (CRP) level, neutrophil-lymphocyte (N/L) ratio, and albumin level. However, total protein level was significantly higher in the control group (P = .001; Table 2).
Native thiol and total thiol levels were lower in the transplant group than in the control group (P = .005 and P = .06, respectively). There was no difference in disulfide levels between groups (P = .948). However, disulfide-to-native thiol and disulfide-to-total thiol ratios were higher (P = .027 and P = .027, respectively) and the native thiol-to-total thiol ratio was lower in the transplant group (P = 0.027). Figure 1 shows the comparative dynamic TDH parameters according to the transplant and control groups.
We compared indications for transplant (COPD, ILD, bronchiectasis, and others) in lung transplant recipients, including baseline characteristics, time after transplant, spirometric measurements, and 6-minute walking test results (Table 3). No statistically significant differences in demographic parameters were found between indications other than age. In post hoc tests, the mean age in the COPD group was similar to that of ILD. There was a statistically significant difference in age when we compared the COPD, bronchiectasis, and other groups (P < .001). We observed no significant difference in terms of dynamic TDH parameters, except for total thiol and disulfide levels. Our post hoc analysis revealed no difference between the COPD and ILD groups by total thiol and disulfide; however, there was a statistically significant difference between the COPD group and the bronchiectasis and the other indications groups. When we compared time after transplant, dynamic TDH parameters were similar, with no statistically significant difference (Table 3).
We performed Spearman’s correlation analyses for age, BMI, total protein, albumin, WBC, CRP, N/L ratio, disulfide, native thiol, total thiol, disulfide-to-native thiol ratio, disulfide-to-total thiol ratio, and native thiol-to-total thiol ratio in the lung transplant recipient group. No correlation was found between TDH parameters and BMI, WBC, CRP, N/L ratio, total protein, and albumin. We observed a moderate negative correlation between age and native thiol, total thiol, and disulfide (r = -040, -0.50, and -0.51, respectively; Figure 2).
To the best of our knowledge, the present study is the first in the literature to investigate dynamic TDH in lung transplant recipients. Native thiol and total thiol levels were significantly lower in our lung transplant recipients versus those shown in healthy controls. However, disulfide levels were similar, but disulfide-to-native thiol and disulfide-to-total thiol ratios were higher in lung transplant recipients. These results indicate that dynamic TDH is impaired in favor of disulfide, resulting in high thiol-related oxidative stress in lung transplant recipients.
Oxidative stress plays a role in the physiopathology of various lung diseases, such as asthma, COPD, lung fibrosis, cystic fibrosis, and lung cancer.16,17 It is also known that systemic inflammation, one of the most common complications in lung transplant recipients, is involved in the pathogenesis of cardiovascular diseases and malignancies.18,19 A number of studies have associated oxidative stress with varying degrees of CLAD in lung transplant recipients.2,20,21
Dynamic thiol-disulfide balance is essential in antioxidant defense and detoxification, apoptosis and enzyme activities, regulation of transcription, and cellular signal transduction mechanisms.9,10,22 Abnor-mal dynamic TDH is associated with many diseases, such as diabetes mellitus, cardiovascular diseases, kidney failure, asthma, COPD, and cancer.23-26 It has been shown that native thiol levels decrease linearly with the application of oxidation processes. Therefore, dynamic TDH measurements can be used to demonstrate the oxidative status in plasma clinically.10 In the present study, we used a recently developed automated measurement technique that provides a comprehensive assessment of dynamic TDH parameters. Before this novel technique, thiol and disulfide concentrations could be measured with low-molecular-weight compounds, such as cysteine, reduced glutathione, and oxidized glutathione, which constitute a small part of the thiol pool in the human body. The new measurement system can provide valuable information about the physiopathological and biochemical processes of diseases in lung transplant recipients.
There are a few reports about oxidative balance in lung transplant recipients. Williams and colleagues reported that the total thiol level was significantly lower in their lung transplant group of 19 recipients than in their control group.27 Similarly, we found lower total thiol levels in our lung transplant recipients. Additionally, their native thiol levels were also lower in their transplant group than in their control group. Furthermore, the recipients’ disulfide-to-total thiol and disulfide-to-native thiol ratios were higher than in the controls. These results indicate that oxidative balance is impaired in favor of oxidants, and the amount of antioxidants is decreased in lung transplant recipients. Lack of antioxidants may result from the decrease in antioxidants due to persistent inflammation and insufficient dietary intake of antioxidants. Further studies are necessary to elucidate the pathogenesis of persistent inflammation and antioxidant deficiency in lung transplant recipients.
With regard to transplant indications, total thiol and disulfide levels were lower in the COPD group than in the ILD, bronchiectasis, and other indication groups. However, there was no difference in terms of other dynamic TDH parameters. These results suggest that the dynamic TDH changes in favor of disulfide in lung transplant recipients regardless of the indications for lung transplant. Although total thiol levels were lower in the COPD group, we observed no difference between disulfide-to-total thiol, disulfide-to-native thiol, and native thiol-to-total thiol ratios. The higher mean age can explain the low amount of total thiol and disulfide in the COPD group. We also found moderate negative correlations between age and total thiol, native thiol, and disulfide in our study. Similarly, Ate? and colleagues reported a positive correlation between oxidative stress and age and found decreased native thiol levels with age.24 Babao?lu and colleagues indicated a negative relationship between age and total and native thiol.26 According to our results, thiol-related oxidative stress increases with age and dynamic TDH changes in favor of disulfide.
Various studies have shown increased oxidative stress in solid-organ transplant recipients.2,28-30 However, several authors investigating oxidative stress in lung transplant recipients have argued that oxidative stress occurs independently of the time after transplant.2,26 Similarly, when we categorized our lung transplant recipients according to time after transplant, we found that dynamic TDH was impaired in favor of disulfide, regardless of time. This result suggests persistent inflammation in lung transplant recipients.
Our study has 2 main limitations. The first one is the small sample size. The second is the absence of assessments regarding other oxidant and antioxidant markers.
Dynamic TDH changes in favor of disulfide after lung transplant, implying that thiol-related antioxidant activity is significantly reduced in lung transplant recipients due to oxidative stress. Determining dynamic TDH can provide valuable information to clarify the pathogenesis of diseases associated with lung transplant. Oxidative stress damage may be avoided by ensuring oxidative balance in the recipients. Further studies are needed to clarify the pathogenesis of oxidative stress and its effects on allografts in lung transplant recipients.
DOI : 10.6002/ect.2021.0360
From the 1Department of General Thoracic Surgery and Lung Transplantation, Ankara City Hospital, University of Health Sciences, Ankara; the 2Clinical Biochemistry Laboratory, Ministry of Health Ankara City Hospital, Ankara; and the 3Department of Biochemistry, Ankara Yıldırım Beyazıt University, Faculty of Medicine, Ankara, Turkey
Acknowledgements: The authors have not received any funding or grants in support of the presented research or for the preparation of this work and have no declarations of potential conflicts of interest.
Corresponding author: Muhammet Ali Beyoglu, Department of General Thoracic Surgery and Lung Transplantation, Ankara City Hospital, University of Health Sciences, MH2 binası, B1 katı, E2 bolumu, Universiteler Mahallesi 1604. Cadde No: 9, Çankaya, Ankara, Turkey
Table 1. Dynamic Thiol-Disulfide Homeostasis Parameters According to Posttransplant Time
Table 2. Baseline Demographics and Dynamic Thiol-Disulfide Homeostasis Parameters in Lung Transplant Recipient and Control Groups
Table 3. Baseline Characteristics and Dynamic Thiol-Disulfide Homeostasis Parameters of Lung Transplant Recipients According to Transplant Indication
Figure 1. Comparative Dynamic Thiol-Disulfide Homeostasis Parameters of Lung Transplant Recipient and Healthy Control Groups
Figure 2. Correlation of Age With Native Thiol, Total Thiol, and Disulfide Parameters in Lung Transplant Recipients