Oxidative-stress biomarkers in patients with pulmonary hypertension

Abstract Abstract

This controlled, prospective, nonrandomized clinical investigation has as its chief strength the fact that it was done in humans with active disease and apparently on fairly modest therapeutic regimens. The aim was to present the results of oxidative-stress biomarkers in humans suffering from pulmonary artery hypertension (PAH). Inflammation and oxidative stress are essential in PAH with increased lipid peroxidation and reduced antioxidant defenses. Twenty-four adult patients of both sexes, with a mean age of 21 years, were subdivided into 2 groups: a control group of 12 healthy, nonsmoking volunteers and a PAH group (PAHG) of 12 volunteers with PAH receiving outpatient treatment. Oxidative stress was evaluated by plasma activity of reduced glutathione (GSH); lipid peroxidation was expressed by malondialdehyde (MDA) and lipid hydroperoxide (ferrous oxidation of xylenol orange [FOX] assay); vitamin E was measured by high-performance liquid chromatography and tumor necrosis factor-α (TNF-α) by enzyme-linked immunosorbent assay. Statistical analyses showed significant differences for (1) the TNF-α measure, with highest values in PAHG patients; (2) the plasma GSH, with lowest values in PAHG patients; (3) vitamin E, with the lowest concentrations in PAHG patients; (4) MDA measure, with highest values in PAHG patients; and (5) the lipid hydroperoxide FOX measure, with highest values in PAHG patients. In conclusion, inflammation and oxidative stress are present in patients with PAH, as confirmed by increased lipid peroxidation, reduced GSH, and low concentrations of vitamin E.

Introduction

Vasoconstriction induced by oxidative stress is probably one of the most crucial factors in the early stages of pulmonary artery hypertension (PAH), and a reduction of endogenous NO bioavailability may contribute to its development. Early studies conducted in the mid-1990s suggested the beneficial effects of antioxidants on lipid peroxidation and the incidence of pulmonary hypertension syndrome in broilers.¹ A growing number of in vitro studies have reported beneficial effects of retinoids on cell migration, proliferation, apoptosis, matrix remodeling, fibrinolysis, coagulation, and inflammation, all of which are involved with vascular disease.² There is a rich literature on human PAH oxidative stress showing associations between increased oxidant-injury markers and poorer clinical outcomes. An increasing number of biomarkers have been described, which may assist the clinician in diagnosis and analysis of PAH severity and response to treatment.³

Besides NO, other markers of oxidative stress, such as plasma malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), reduced glutathione (GSH), vitamins E and A, and lipid hydroperoxide measured by ferrous oxidation of xylenol orange (FOX), are affected in PAH but still have not been widely explored. Therefore, this controlled, prospective, nonrandomized clinical investigation was carried out to evaluate oxidative stress through the measurement of plasma levels of these biomarkers in patients with idiopathic pulmonary or secondary hypertension. It was an observational study, and the biomarkers were randomly chosen. The aim was to present the results of the oxidative-stress biomarkers in humans suffering from PAH.

Patients and methods

Study population

The study was approved by the Ethics Committee of the Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo and was conducted at the Clinic of Pulmonary Hypertension and Interstitial Lung Diseases. In all patients, the diagnosis of PAH was confirmed prior to enrollment by right heart catheterization.

Twenty-four adult patients of both sexes, with a mean age of 21 years, were selected and subdivided into 2 groups: the control group (CG), 12 healthy volunteers who did not use any drugs or smoke, and the PAH group (PAHG), 12 patients receiving outpatient treatment for PAH. The PAHG has a notably lower average body mass index (22.91 ± 4.64 kg m⁻²) than the CG (30.42 ± 3.03 kg m⁻²). The level of PAH was assessed by echocardiography, which detected varying degrees of PAH (mean pulmonary artery pressure: 87.36 ± 21.61 mmHg). Concerning the etiology, PAH was idiopathic in 5 patients (41.67%), secondary to pulmonary thromboembolism in 5 patients (41.67%), and secondary to pulmonary blood overflow caused by ventricular septal defect in 2 patients (16.67%). No cases of pulmonary hypertension were due to collagen diseases. Five patients (41.67%) were in use of domiciliary oxygen, 7 (58.33%) were in use of sildenafil, 4 (33.33%) were in use of bosentan, and one (8.33%) used bosentan plus sildenafil.

Methods

The blood samples were frozen and stored at −70°C. Oxidative stress was evaluated by the levels of reduced GSH, and the final amount of lipid peroxidation was expressed by levels of MDA and lipid hydroperoxide (FOX assay). The MDA content was determined according to the method proposed by Gérard-Monnier et al.,⁴ with some adjustments. The FOX method was used for the determination of lipid hydroperoxides. Hydroperoxides oxidize Fe²⁺ to Fe³⁺, which reacts with xylenol orange to produce a chromophore that has an absorption maximum at 560 nm. This method can be used for the determination of hydroperoxides in aqueous and lipid phases. The reduced form of GSH was determined by the method of Sedlack and Lindsay.⁵ This method is based on the formation of the colored product (GSH-DTNB [5-5′-dithio-bis(2-nitrobenzoic acid)]), which is determined by a change in absorbance at 412 nm and expressed as mol/L blood serum. Serum levels of vitamin E were measured by high-performance liquid chromatography (Shimadzu model LC10A, Kyoto, Japan). TNF-α was measured with an enzyme-linked immunosorbent assay.

Statistical analysis

Data are expressed as median and the range between the 25% and 75% quartiles. Statistical analysis was carried out with nonparametric (Wilcoxon, Mann-Whitney) tests, and results were considered statistically significant when P < 0.05. The graphics were built with the software JMP 10 for Mac (SAS Institute, Cary, NC).

Results

A statistically significant difference (P = 0.0004) between the 2 groups was found when comparing the values for plasma MDA, with higher concentrations being found in the PAHG than in the CG, 0.82 (0.69–1.17) versus 0.18 (0.16–0.22) μmol/gram of protein (gP; Fig. 1). A statistically significant difference (P = 0.0179) between the 2 groups was found when comparing the values for plasma TNF-α, with the higher concentrations being found in the PAHG than in the CG, 54.48 (4.36–118.79) versus 1.29 (0.00–5.71) pg/mL (Fig. 2). A statistically significant difference (P = 0.0262) between the 2 groups was found when comparing the values for plasma vitamin E, with higher concentrations being found in the CG than in the PAHG, 13.41 (10.14–16.81) versus 8.48 (6.33–12.32) μmol/L (Fig. 3). A statistically significant difference (P = 0.0004) between the 2 groups was found when comparing the values for plasma GSH, with higher concentrations being found in the PAHG than in the CG, 173.88 (153.80–201.46) versus 0.10 (0.10–0.13) nmol/gP (Fig. 4). A statistically significant difference (P < 0.0001) between the 2 groups was found when comparing the values for plasma endoperoxides (FOX assay), with higher concentrations being found in the PAHG than in the CG, 2.97 (2.39–4.07) versus 0.70 (0.65–0.85) nmol/gP (Fig. 5).

Figure 1.

Values of plasma malondialdehyde (MDA) in absolute numbers in the control group (CG) and the pulmonary hypertension group (PAHG). Results are expressed as median and 25% and 75% quartiles; N = 12.

Figure 2.

Values of plasma tumor necrosis factor-α (TNF-α) in absolute numbers in the control group (CG) and the pulmonary hypertension group (PAHG). Results are expressed as median and 25% and 75% quartiles; N = 12.

Figure 3.

Values of plasma vitamin E in absolute numbers in the control group (CG) and the pulmonary hypertension group (PAHG). Results are expressed as median and 25% and 75% quartiles; N = 12.

Figure 4.

Values of plasma reduced glutathione (GSH) in absolute numbers in the control group (CG) and the pulmonary hypertension group (PAHG). Results are expressed as median and 25% and 75% quartiles; N = 12.

Figure 5.

Values of plasma lipid hydroperoxide (ferrous oxidation of xylenol orange [FOX] assay) in absolute numbers in the control group (CG) and the pulmonary hypertension group (PAHG). Results are expressed as median and 25% and 75% quartiles; N = 12.

Discussion

This study has shown that inflammation and oxidative stress play a vital role in the development and progression of pulmonary hypertension. This fact can be confirmed by the increased levels of circulating cytokines, such as TNF-α, increased lipid peroxidation, as shown by elevated plasma levels of MDA and lipid hydroperoxide (FOX assay), and decreased antioxidant defenses, as reflected in reduced plasma concentrations of GSH and vitamin E, in patients with PAH, relative to those in healthy controls. These findings can be explained by the reduction of endogenous antioxidants because oxidative stress causes an imbalance between the production of reactive oxygen species (ROSs) and the antioxidant enzyme system. This imbalance can impair vascular function via changes in lipid membranes, DNA, and proteins, thereby causing dysfunction and even cell death.^6,7

In addition, the superoxide anion is a potent inhibitor of NO, thereby contributing to endothelial dysfunction and increased vascular tone. Previously published studies suggest that endothelial dysfunction in PAH is not limited to the pulmonary vessels but also demands involvement of the systemic vasculature as a whole.^8,9 It is also known that MDA is a product of lipid peroxidation and reflects one of the multiple sources of ROSs. An increase in ROS levels in these patients may indicate the presence of extensive oxidative damage, particularly to endothelial cells.⁵ A high level of MDA shows that the antioxidant system is not able to prevent lipid peroxidation in these patients, which is clearly indicated by low levels of GSH and vitamin E. Thus, these results support the hypothesis that oxidative stress plays a critical role in the pathophysiology of PAH, causing endothelial damage and remodeling of blood vessels and contributing to progression of the disease.¹⁰ Measurement of MDA in biological samples is challenging. MDA is certainly well recognized as a product of lipid peroxidation. It is one of several hundred distinct and quantifiable products, and it is known to withstand secondary reactions with other biological macromolecules (e.g., adduction to DNA). Therefore, the use of an assay for MDA quantification more reliable than the classic TBARS (thiobarbituric acid reactive substances) assay would be appropriate. Technically, the conditions of the assay (heating to 45°C and treating with HCl) are still harsh enough to generate a false signal, especially if plasma samples are not delipidated before assay. In addition, the assay used at least has a significant potential to create a positive signal in the presence of hydroxyalkenals (e.g., 4-hydroxynonenal [4-HNE]), although these could also be argued to represent lipid peroxidation products. Lipid hydroperoxides present problems similar to, but more challenging than, those of MDA, since these are very short-lived species that are not particularly powerful markers of oxidant injury. In our study, the difference between the two groups in absolute values of lipid hydroperoxides was also more significant than the MDA difference. Therefore, based on the literature review, the investigations regarding lipid peroxidation data would consider more stable products of oxidative injury, such as the F2-isoprostanes or something else.^4,11

In the literature, there is agreement that inflammation is characteristic of the presence of PAH. In fact, it may develop in vascular remodeling and be part of the repair process, or it can be triggered by infections, drugs, or toxins. The production of inflammatory cytokines, among them TNF-α, contributes to the spread of the inflammatory process, leading to the production of growth factors and remodeling of the vascular bed.¹² Many studies have shown that circulating levels of cytokines, especially interleukin-6 (IL-6) and TNF-α, are increased in patients with idiopathic pulmonary hypertension; however, there also may be, to a lesser extent, increased levels of other cytokines, such as IL-2, IL-4, and IL-8.^12–15 One reason for the increased levels of plasma cytokines in patients with PAH may be related to oxidative stress present in these patients and the body’s efforts to regain vascular homeostasis through activation of the inflammatory cascade, which promotes excessive formation of ROSs and RNA and/or decreased antioxidant defenses.¹⁰ One study has linked increased plasma levels of inflammatory cytokines to the severity of disease, and the authors of one believe that they are predictive of response to optional therapies.⁹ Some studies show that patients with PAH who also have chronic obstructive pulmonary disease (COPD) and increased expression of cytokines demonstrated increased levels of vasoactive mediators, such as endothelin-1 (ET-1).^15,16 One possible explanation for this finding would be production of ET-1 by the airway epithelium due to exacerbation of the disease, which leads to increased protein expression of ET-1 by the messenger RNA of vessel endothelial cells.^15,16 There was no statistically significant difference between the plasma levels of IL-6 found in the 2 groups (P = 0.7287; results not shown); however, higher concentrations of IL-6 were seen in the CG than in the PAHG.

The findings of this study agree with those reported by Iqbal et al.¹⁷ and Wong et al.,¹⁸ where the values for GSH and α-tocopherol were extremely low compared to those in healthy controls. These data support the hypothesis that oxidative stress leads to mitochondrial dysfunction, with increased production of ROSs, and decreases the activity of antioxidant enzymes in PAH. These findings are in agreement with those of authors such as Cracowski et al.³ and Bowers et al.,⁸ who showed that there is decreased activity of antioxidant enzymes in the lungs of patients with severe PAH and that oxidative stress is present in these individuals, regardless of the PAH etiology. However, our study contradicts other investigations, for example, the report by Wu et al.,⁶ which showed that in patients with hypoxic PAH, there is increased protein expression of endothelial nitric oxide synthase and GSH. Liu and Thomas¹⁹ also noted increased expression of GSH in patients with PAH, highlighting its critical role in vascular homeostasis and endotoxic shock. Maintaining proper SH levels, turnover rates, and oxidation state are important for a number of critical cellular functions, and disruptions in these processes are observed in much human pathology. GSH deficiency manifests itself largely through an increased susceptibility to oxidative stress, and the resulting damage is thought to be a key step in the onset and progression of many disease states.

Joppa et al.²⁰ assessed the activity of antioxidant enzymes in patients with COPD and PAH and found that there are decreased levels of GSH in this group and that one possible explanation is the fact that these enzymes have a vital role in both pathology and pulmonary vascular physiology. Thus, they may alter the structure and function of pulmonary artery endothelial and smooth muscle cells. Moreover, ROSs act as vasoactive substances in the pulmonary circulation, and their production is balanced by the activity of antioxidant enzymes.

In conclusion, our study confirmed that the inflammatory process and oxidative stress are essential elements in PAH and are related to increased lipid peroxidation and reduced antioxidant defenses. However, this matter has been relatively little studied, and further investigations assessing oxidative-stress markers in patients with pulmonary hypertension are needed. The investigation has as its chief strength the fact that it was done in humans with active disease and apparently on fairly modest therapeutic regimens (e.g., reportedly, none of the patients received prostanoid treatment despite reported pulmonary artery pressures in the range of 85 mmHg for the PAH group).

Study limitations. The study is merely descriptive and does not provide insights into the potential clinical or mechanistic significance of the findings. It would be essential to consider correlations between the measured biomarkers and parameters of PAH severity, functional capacity, and prognosis. In addition, it would be helpful to stratify the data by PAH clinical group, but given the small number of patients and the heterogeneous nature of the PAH population, this analysis would be irrelevant. The lack of diet and nutritional status is another study limitation. Several of the measured outcomes (e.g., plasma GSH and vitamin E) could be influenced substantially by diet and nutritional status, independent of any differences in oxidative stress. This is essential to take into account because the PAHG had a notably lower average body mass index (22.91 ± 4.64 kg m⁻²) than the CG (30.42 ± 3.03 kg m⁻²), suggesting that there may, in fact, have been real differences in diet or nutritional status.

Source of Support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundacão de Apoio ao Ensino, Pesquisa e Assistência (FAEPA) do Hospital das Clínicas de Riberão Preto, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Conflict of Interest: None declared.