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European Journal of Heart Failure 2004 6(7):883-890; doi:10.1016/j.ejheart.2004.03.003
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© 2004 European Society of Cardiology

Elevated circulating levels of thioredoxin and stress in chronic heart failure

Andreas Jekella,1, Akter Hossaina, Urban Alehagenb, Ulf Dahlströmb and Anders Roséna,*

a Department of Biomedicine and Surgery, Division of Cell Biology, Linköping University SE-581 85 Linköping, Sweden
b Department of Medicine and Care, Division of Cardiology, Linköping University Linköping, Sweden

* Corresponding author. Tel.: +46-13-22-2794; Fax: +46-13-22-4314 E-mail address: Anders.Rosen{at}ibk.liu.se


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
Background: Chronic heart failure (CHF) is a complex syndrome, in which reactive oxygen species and inflammatory cytokines are important stressors that contribute to the pathogenesis.

Aim: We have studied physiological stress response parameters in CHF, in particular the redox-active regulator thioredoxin.

Subject: A case–control study was conducted including a consecutive sample of CHF patients (n=27) of NYHA class II and III; comparison control subjects (n=29) were recruited from an association for retired people.

Method: Baseline levels of Trx, lipid peroxides (oxidative stress), TNF and IL-6 cytokines, platelet-activation marker P-selectin, cortisol (as peripheral effector of HPA axis), and the potent antioxidant selenoprotein Trx-reductase were assessed.

Results: Mean (±S.E.M.) plasma levels of Trx were significantly higher in patients with CHF (32±3 ng/ml), than in the healthy subjects (12±3 ng/ml, P<0.0001). Trx levels increased in proportion to severity of disease (NYHA class III>NYHA class II) and degree of stress. Trx elevation correlated well with increased oxidative stress (lipid peroxides, P<0.0001), circulatory P-selectin (P<0.0001), morning level of free salivary cortisol (P=0.0002), and serum creatinine (P=0.0417), but not with pro-inflammatory cytokines TNF and IL-6.

Conclusion: Trx was strikingly elevated in heart failure cases compared with controls, signifying an adaptive stress response that is higher the more severe the disease.

Key Words: Thioredoxin • Oxidative stress • TNF • IL-6 • Inflammation • Chronic heart failure

Received August 22, 2003; Revised December 5, 2003; Accepted March 3, 2004


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
Chronic heart failure (CHF) is a complex neuro-hormonal and inflammatory syndrome [1]. Inflammatory cells (e.g. monocytes, neutrophils) contain NADPH-oxidases that produce large amounts of ROS upon stimulation with microbial antigens and cytokines (TNF/Fas receptor signalling) for example. These oxygen radicals (including NO) are necessary to combat infections, but serve also as specific signalling molecules under both physiological and pathophysiological conditions [2]. Excess ROS contribute to a condition of oxidative stress in CHF [3,4], in part caused by dampened antioxidant response systems essential to maintain homeostasis. In addition to ROS, proinflammatory cytokines such as TNF, IL-1, and IL-6 are overexpressed and released from the inflammatory cells in CHF [5,6]. ROS and cytokines have been shown to elicit apoptosis of cardiomyocytes [7,8], thus playing an important role in the progression and symptoms of CHF [5,6], i.e. cardiac cachexia, a serious complication of CHF leading to impaired survival [9]. Adaptation to stress (e.g. ROS, TNF, IL-6) evokes a series of neuroendocrine responses that activate the HPA axis and the sympathetic nervous system, striving at reestablishment of homeostasis in every peripheral tissue. Glucocorticoids serve as the major peripheral effector of the HPA axis, through the glucocorticoid receptor, and have multiple effects on immune cells and molecules including suppression of proinflammatory mediators [10].

Thioredoxin (Trx) is a multipotent protein and key regulator of cellular redox balance operating in synergy with Trx reductase and NADPH (the Trx system) [11]. Trx has gene regulatory activity of several transcription factors such as glucocorticoid receptor [12] and NF{kappa}-B via thiol-disulfide cystein control of DNA binding. Trx also controls in a fascinating redox-sensitive ‘on-off’ mechanism decisions for apoptotic or hypertrophic pathways [13]. We have previously reported growth stimulatory pathways of Trx and cytokines and cytokine receptors (IL-2R) [14]. Trx protects against H2O2 and TNF-mediated cytotoxicity, a pathway in which TNF receptor-binding generates ROS. In excess amounts, ROS is cytotoxic and activates in the cytoplasm the apoptosis signal-regulating kinase 1 (ASK-1) by releasing Trx that is structurally complexed to ASK-1 [15]. Trx is thus an exquisite redox-sensor. Trx has also direct antioxidant activity and reduces ROS through an interaction with the redox-center of Trx (-Cys-Gly-Pro-Cys-) [11]. In addition to its intracellular functions, Trx is released under physiological conditions of oxidative stress caused by a variety of stimuli such as mitogens and inflammatory signals [16], viral infections, such as HIV [17] and severe burns [18].

Thioredoxin reductase (Trx-reductase) is a redox-active selenoprotein that efficiently regenerates oxidized Trx to its reduced form in the presence of NADPH [19]. Trx-reductase requires selenium (selenocystein) for its activity. It is overexpressed and released upon oxidative stress and has recently been detected in plasma [20]. Circulating Trx-reductase is enzymatically active and may interact with plasma Trx [20]. It is of interest to note that selenium is essential for several antioxidant and anti-inflammatory proteins besides Trx-reductase, such as glutathione peroxidase, and that selenium deficiency is associated with a reversible dilated cardiomyopathy (Keshans's syndrome). Selenium supplementation may be protective against cardiovascular disease [21], as well as cancer, a condition in which several inflammatory mediators similarly are activated [22,23].

Since CHF is a disease with a poor prognosis despite optimal medical treatment [24]. An improved understanding of the underlying inflammatory mechanisms are important for design of novel therapy strategies. Anker and coworkers have formulated the hypothesis that altered gut permeability with bacterial translocation and endotoxin (LPS) release is triggering the immune activation [25]. This activation generates TNF, IL-1 and IL6, which all interplay at the level of the central nervous with psychological mental stressors (high demand/low control) to fire the HPA-axis [26]. CHF patients have elevated adrenocortical hormone release including cortisol levels [27]. Psychological, cardiovascular and metabolic parameters were recently found to correlate to free salivary cortisol stress recovery [28].

Based on two recent observations that reduction of oxidative stress may dampen inflammatory conditions [29] and that circulating Trx effectively suppresses inflammation by blocking chemotaxis of neutrophils [30], we were strongly impelled to investigate in this study whether circulating Trx in CHF patients were affected. We have analyzed the Trx and Trx-reductase redox-proteins in relation to biochemical stress and inflammatory markers and we discuss its potential role in the pathogenesis of cardiac dysfunction.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Statistical analysis
 4. Results
 5. Discussion
 References
 
2.1. Subjects
Twenty-seven male patients with CHF, NYHA functional class II–III, (74±0.9 years of age) (Table 1) and 29 healthy men of the same age group (75±1.0 years), as control subjects, were included in our study. The CHF patients were selected among consecutively attending patients at the Heart Failure Unit at Linköping University Hospital. The control subjects were healthy men in good physical condition, recruited from a section of an association for retired people with frequent sport activities such as walking, cycling and skiing on their program. In the medical examination including past medical history, the control subjects included in this study presented none of the clinical criteria of a cardiovascular risk profile. However, heredity for cardiovascular diseases did not exclude the persons in the control group to participate in the study. Of the 27 male patients included in the study, 19 had a history of ischemic heart disease (IHD), of which four had had myocardial infarction (MI), 7 dilated cardiomyopathy (DCM), and one hypertension (HT) (Table 1). They were medically well controlled and attended the Cardiology Unit on a regular basis. Exclusion criteria for both groups were conditions previously reported to have raised TNF, TNF-receptors and/or Trx levels, including smoking [31], diabetes mellitus [32], inflammatory diseases (inflammatory bowel diseases, rheumatoid arthritis or other rheumatic diagnosis) [33,34], cancer [35], obesity (BMI>34) [36], and active infection [17]. These conditions are also associated with oxidative stress [11]; and inclusion of patients with these conditions would unnecessarily confound the data interpretation in our heart failure study. All participants provided witnessed informed consent, and the ethics committee of Linköping University Hospital approved the study. ~25% of patients approached were included, 75% excluded/abstained.


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  		Table 1 Basal characteristics of patients with chronic heart failure

 
2.2. Sample collection
Venous blood and saliva samples were collected in the morning between 08:00 and 10:00 h. Prior to collection, the donor rested for 30 min in supine position. Blood was collected in tubes containing EDTA. For TNF and IL-6-sampling, the tubes were kept on ice. For Trx sampling, tubes were kept at room temperature and contained thrombolysis inhibitors 1 U/ml apyrase/salicylate. Trx-reductase sampling required pre-chilled tubes (0 °C) wrapped in aluminium foil containing a protease cocktail as previously described [20]. The Trx-reductase tubes were also used for thiobarbituric acid-reactive substances (TBARS) analysis. Samples were centrifuged at 1100xg for 15 min at 4 °C with the exception of Trx samples, which were centrifuged in room temperature. Directly after preparation, the plasma samples were aliquoted and stored at –70 °C for subsequent analyses.

For saliva collection, previously published procedures were followed [37], including special instructions for donors to refrain from eating, brushing of teeth and physical strain 60 min before collection. The mouth was rinsed with water 15 min before saliva collection using dental rolls (Salivettes, Sarstedt) held in mouth for 2 min, then centrifuged at 1100xg, for 2 min before aliquoting and freezing at –70 °C.

2.3. Enzyme immunoassays and oxidative stress determinations
Trx levels were determined in a sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (mAb) anti-Trx, clone 2G11 (BD Pharmingen), as a catcher Ab and biotinylated goat anti-Trx (IMCO Co, Stockholm) or biotinylated anti-Trx mAb 4H9 (BD Pharmingen) as an indicator Ab as previously described [35]. Trx-reductase was also analyzed in a sandwich ELISA using mAbs previously obtained in our laboratory [38]. Trx-reductase ELISA plates were always kept on ice and covered with aluminium foil during incubations in order to minimize proteolytic cleavage. IL-6 was determined by ELISA using a commercial set of mAbs obtained from Mabtech AB (Stockholm), according to the manufacturer's recommendations. TNF was analyzed by ELISA using matching antibody pairs (R&D System), using a fluorogenic substrate (4-MUP, Sigma Chemical Co). Reading was performed in a multi-label counter Wallac 1420 (Wallac Oy, Perkin ElmerTM). Lipid peroxides were determined by the TBARS assay (detecting TBA-malondialdehyde (MDA) complexes) as previously described [39]. Selected samples were separated in HPLC chromatography for quality determinations. Salivary cortisol was analyzed by a biotin-streptavidine immunoassay [37] in the laboratory of Dr C. Kirschbaum, Germany. P-selectin was determined by an ELISA employing mAbs from R&D Systems. For all assays used in the study, each sample was tested in triplicates in three separate experiments, for mean value determination, except for saliva samples, which were tested in triplicates once. Intra- and inter-assay coefficients of variation in the immunoassays were 9 and 11% for Trx; 5 and 6% for TNF; 9 and 5% for IL-6; 7 and 6% for Trx-reductase; 1.0 and 3% for TBARS. Lowest detectable level was 0.1 ng/ml for Trx; 0.1 ng/ml for Trx-reductase; 0.5 pg/ml for TNF; 1.25 pg/ml for IL-6; 0.01 µmol/l for TBARS, which was calculated from the background OD value +2 S.D.


   3. Statistical analysis
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
4. Results
 5. Discussion
 References
 
Statistical analysis of how continuous responses distribute differently across groups were evaluated by the Student's t-test and 1-way ANOVA analysis of variance. P values are two-tailed; all group data are expressed as means±S.E.M. Normal distribution was tested by evaluation of normal quantile plots. Deviating distributions were analyzed by the non-parametrical Wilcoxon signed rank test. Outlier analysis was performed with the Mahalonobis distance and Jackknife distance methods. Bivariate correlation analysis was performed with straight line fit using the least square regression.

Prospective power analysis was performed to determine minimal sample size of total study population. Estimates of group means, within-group variance (S.D.) and alpha levels were based on our previous studies on Trx in patients with burn damage [18]. Power analysis revealed a minimal sample size of n=55 for alpha 0.05. The patients were divided into two groups on the basis of their plasma level of Trx. The group with high level exceeded (by more than 2 S.D.) the mean level seen in the control group (i.e. levels ≥30 ng/ml). Patients whose level was below 30 ng/ml composed the group with low level of Trx. All data were evaluated by use of the statistical program JMP version 4.0.2 (SAS Institute Inc. Cary, NC, USA).


   4. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
5. Discussion
 References
 
The 27 patients with heart failure (basal characteristics, Table 1) had strikingly higher baseline plasma levels of Trx (32±3 ng/ml; mean±S.E.M.) than the 29 healthy subjects (12±3 ng/ml; P<0.0001) (Table 2). The Trx levels increased in proportion to the severity of the disease (Fig. 1), with a mean value of 37±6 ng Trx/ml in patients of NYHA functional class III, and 25±5 ng Trx/ml in NYHA class II patients vs. healthy subjects (P<0.0001, P=0.0024, respectively). Patients with high Trx-levels (≥30 ng/ml, i.e. >2 S.D. above the mean value of the control group) had a more advanced disease than the patients with low Trx values (<30 ng/ml), as evidenced by their increased values for serum creatinine (130 vs. 106 µmol/l, P=0.020). Serum creatinine increase is a marker of renal function defects, rising above normal range upon prolonged circulatory dysfunction such as found in CHF. Markers, previously reported to be associated with the disease [40,41] were also significantly raised in this study population compared to healthy subjects (Table 2): (i) Plasma lipid peroxides, indicator of oxidative stress: 12.5 vs. 9.5 µmol/l (P=0.0022), (ii) Plasma P-selectin, marker of platelet activation: 47 vs. 40 ng/ml (P=0.0454). In addition, body mass index was increased: 27.1 vs. 23.6 kg m–2 (P<0.0001). Bivariate correlation analysis was performed on the CHF and healthy donors’ biochemical and clinical parameter data by regression analysis (Fig. 2, Table 3). Trx was significantly associated with lipid peroxides (r=0.51, P<0.0001), P-selectin (r=0.51, P<0.0001), salivary cortisol (r=0.48, P=0.0002), and serum creatinine (r=0.39, P=0.0417). It is of interest to note that Trx-reductase significantly correlated with TNF and IL-6 (r=0.50, P<0.0001 and r=0.34, P=0.0100, respectively) and TNF correlated with IL-6 (r=0.82, P<0.0001).


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  		Table 2 Demographic data and biochemical markers in healthy subjects compared to heart failure patients. Significance levels

 


Figure 1
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  		Fig. 1 Thioredoxin levels in NYHA class II and class III patients, and in healthy subjects. Each circle represents the mean plasma Trx value of one donor. The mean value was derived from three separate experiments, each performed in triplicate. The diamonds show group mean values and the 95% confidence interval. P-values are two-tailed, derived from Anova/Student's t-test.

 


Figure 2
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  		Fig. 2 Relation between circulatory levels of thioredoxin and biochemical stress parameters. (a) Circulatory levels of thioredoxin correlated significantly with lipid peroxides determined in the TBARS assay (y=176+1.96x, n=56, r=0.52, P<0.0001). (b) Circulatory levels of thioredoxin correlated significantly with plasma P-selectin (y=35.7+0.33x, n=56, r=0.51, P<0.0001). (c) Circulatory levels of thioredoxin correlated significantly with morning levels of free salivary cortisol (y=12.3+0.31x, n=56, r=0.48, P=0.0002). (d) Circulatory levels of thioredoxin correlated significantly with serum creatinine (y=102+0.46x, n=27, r=0.39, P=0.0417). Open rings denote healthy subjects, closed rings denote CHF patients. Dashed lines denote 95% CI.

 


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  		Table 3 Correlation of thioredoxin with biochemical and clinical parameters

 
Body weight and body mass index in our 27 CHF male patients (Table 2) were higher than in healthy subjects (84 vs. 74 kg, and 27.1 vs. 23.6 kg m–2; P=0.0002 and P<0.0001, respectively). To gain further assurance that the high levels of Trx in patients were not due to increased body weight per se, the Trx concentration per kg body weight was calculated, and the mean values of the patient vs. healthy subjects were found to be significantly different (P=0.0003). Our patients were in NYHA class II to III, showing no signs of cachexia, which is a condition associated with advanced stage of chronic heart failure and also associated with high TNF and IL-6 levels [5,42]. In accordance, the TNF and IL-6 levels of patients and healthy subjects in this study did not differ significantly (Table 2), but there was an elevation trend in plasma TNF of NYHA class II and III patients (1.3 vs. 3.9 pg/ml, ns: P=0.068).


   5. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
References
 
The novel finding of this study is that severity of CHF (e.g. NYHA functional class) and stress response (e.g. raised lipid peroxides, free salivary cortisol, P-selectin, and serum creatinine), are associated with elevations in the redox-active protein Trx.

Firstly, the increase of circulatory baseline Trx concentrations, comparing the healthy subjects with the heart failure patients, was highly significant (Table 2). The outcome was greater than the expected estimates: 12 vs. 32 ng/ml (P<0.0001) for the healthy donors vs. CHF patients, respectively. The Trx increase was still evident after subdividing the patients, comparing healthy subjects (12 ng/ml; n=29) to NYHA class II patients (25 ng/ml; n=10; P=0.0024), and NYHA class III patients (37 ng/ml; n=17; P<0.0001). Mean age and age distribution in our study population was not significantly different from those of Swedish CHF patients in larger cohorts (UD and UA, personal communication). Our study population was restricted to males in order to obtain a homogenous population, reducing possible gender specific hormonal influences. We did not investigate patients with advanced disease in NYHA functional class IV. However, in the course of submitting our results, a parallel Japanese study, which supports our findings of Trx increase in CHF patients, was brought to our attention [43]. Trx level correlated positively with NYHA class III+IV, but negatively with left ventricular ejection fraction. The number of patients was, however, low and the patient profile differed compared to our study population in that patients outside inclusion criteria for chronic heart failure participated, e.g. dilated cardiomyopathy (n=5), acute coronary syndrome (n=7), stable angina (n=18), and control subjects (n=4).

Secondly, in order to approach an understanding of the mechanism behind the Trx increase, we determined the oxidative stress level by analysis of plasma lipid peroxides using the TBARS method. CHF patients revealed a significant increase of oxidative stress compared to the healthy subjects (P=0.0022). ROS originates from a chain of events as suggested in the prevailing neuroendocrine hypothesis, which includes an index event (initial injury), in which translocation of endotoxin (LPS) over the intestinal barrier occurs [25]. LPS induces the proinflammatory cytokines IL-1, IL-6 and TNF release in several cell types, which in turn activates NADPH-oxidase that produces large amounts of ROS. Several reports on chronic heart failure, show that oxidative stress is elevated [3,40]. Results in this study confirm previous reports and we found that the increase in oxidative stress was statistically significant (P=0.0022, Table 2) with correlation to Trx levels (P<0.0001) (Fig. 2).

Thirdly, based on the present knowledge that conditions of oxidative stress are generally well controlled under healthy physiological conditions, and that they are counteracted by a balanced induction of cellular antioxidants, including superoxide dismutase (SOD), catalase [44], and Trx-reductase [45], we determined the Trx-reductase levels in CHF and controls. Trx-reductase was previously discovered to reduce hydroperoxides, in particular lipid peroxides such as 15-S-HPETE that is associated with atherosclerosis and lipid peroxidation [46]. We found, however, in this study that the antioxidant Trx-reductase levels did not differ in the healthy subjects and patients (164 vs. 191 ng/ml; P=0.74, ns) (Table 2). This finding is in line with parallel observations that the antioxidants SOD and catalase were consumed in CHF patients [44], a phenomenon that may explain the non-elevated Trx-reductase levels in CHF patients. The apparent mismatch between Trx (highly elevated) and Trx-reductase (normal) may in addition be explained by a relative selenium deficiency in CHF patients [29] resulting in enzymatically inactivated Trx-reductase [47] (and glutathione peroxidase). Detailed knowledge on the ratio between reduced and oxidized forms and the mechanism for their shift (oscillation) requires further analysis. To tackle the question on how important is an optimal physiological selenium level for the function of antioxidants in particular (the Trx-system), and for the morbidity and mortality rates in general, we have initiated a long-term (4 years) intervention study. Selenium supplementation will be analyzed in a Swedish cohort (n=560) in a double-blind randomized, placebo-controlled study design. Cardiovascular protective effects of selenium supplementation in populations with low serum levels have been discussed recently by Rayman [21], but the mechanistic details are unknown.

A fourth point concerns the cellular origin of circulatory Trx in CHF patients. It is not known, but monocytes, platelets and the liver are possible sources. Monocytes contain high levels of both Trx and Trx-reductase released upon LPS/oxidative stress stimulation [20,48]. Platelets contain Trx, but not Trx-reductase [48,49], and in CHF patients there is a low grade of platelet activation ongoing, as suggested by the significantly higher plasma P-selectin values (P=0.0454; Table 2), thus enabling Trx release. In addition, Trx and P-selectin values correlated significantly (r=0.51, P<0.0001, Fig. 2). Trx is also secreted by the liver, as evidenced from previous reports in animal models, in which LPS injected mice up-regulated gene (mRNA) and protein expression for several antioxidants and acute phase reactants, including Trx, which was three-fold overexpressed [50]. The acute phase reactant C-reactive protein was reported in several studies to be raised in heart failure patients [51], supporting this point.

Comparison between healthy subjects and CHF patients shows a difference in BMI (23.6 vs. 27.1, P<0.0001), indicating that the healthy controls were somewhat supernormal with a lower BMI than expected for this particular age group. In anamnesis, the healthy subjects reported frequent physical activities including walking, cycling, and skiing. In recent studies physical training was found to enhance the expression of genes encoding antioxidative enzymes SOD and glutathione peroxidase, reduce oxidative stress and improve peak oxygen uptake [52]. The effect of exercise has also been studied in persons at risk of developing ischemic heart disease. The results showed that after a period of physical training, mononuclear blood cells had a reduced production of atherogenic cytokines (IL-1, TNF, IFN-{gamma}), and an increased level of atheroprotective cytokines, IL-4, IL-10 and TGF-β [53]. The results indicated that physical activity protects against ischemic heart disease. We expected that our healthy subjects would have lower IL-6 and TNF values compared with the CHF patient group, but we found no significant differences with regard to IL-6 and TNF, in spite of the clear differences in lipid peroxides, Trx and P-selectin (Table 2). Our study population consisted of patients of NYHA class II and class III, High IL-6 and TNF levels are, however, found preferentially in the more advanced stage of disease (NYHA class IV) [5,42].

Finally, we should consider the results presented in Fig. 2c and Table 3, that Trx and cortisol were significantly associated (P=0.0002). Elevated IL-1, IL-6 and TNF constitute potent physiological stressors that activate the HPA-axis, an activation that triggers cortisol release [26]. Cortisol will strongly influence the immunological balance; dampen T helper-1 responses (e.g. anti-viral, anti-tumor), which in turn will influence inflammatory condition [54]. These findings fit well with the endotoxin hypothesis forwarded by Anker et al. [25].

In conclusion, we observed that Trx, which is a biochemical stable protein of 12 kDa molecular weight (104 amino acids), was significantly increased in plasma of patients with CHF compared to healthy subjects. The increase was higher in patients with more advanced disease. The Trx elevation was also associated with elevated stress responses: salivary cortisol and lipid peroxides. Based on these findings, Trx may be used as a marker for disease development and monitoring.


   Acknowledgements
 
Special thanks to Kerstin Gustavsson and Ulf Axén, for technical support with sample collection and preparation, many thanks to Anita Söderberg, Anita Lönn and Ana María Barral for advanced technical support, and Gunilla Westermark for help with the TNF ELISA techniques. Many thanks also to Prof. Jan Ernerudh for critically reading the manuscript. This study was supported by funds from the Medical Faculty of Linköping University and the Swedish Cancer Association (no. 3171). GSD.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
1 A J and A H contributed equally to the study. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 

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