Metin Aytekin

Antioxidant effects of methionine, α-lipoic acid, N-acetylcysteine and homocysteine on lead-induced oxidative stress to erythrocytes in rats

Lead, widely used in industry, is a great environmental health problem. Many studies have examined its effects on the health of both humans and animals. Experimental studies have shown that sulphur-containing antioxidants have beneficial effects against the detrimental properties of lead. The present study was designed to investigate markers of oxidative stress (hemoglobin (Hb) in whole blood, malondialdehyde (MDA) in sera; superoxidase dismutase (SOD) and glutathione peroxidise (GSH-Px) in erythrocyte hemolysate and vitamins A and E in plasma) in rats given lead (2000ppm) with or without sulphur-containing antioxidants (l-methionine (Met) (100mg/kg/day), N-acetylcysteine (NAC) (800mg/kg/day), l-homocysteine (Hcy) (25mg/kg/day), lipoic acid (LA) (50mg/kg/day)) in their water for 5 weeks. In the lead group, Hb and plasma vitamin E levels were significantly lower whereas MDA levels were significantly higher compared to controls (p<0.05). Hb levels in lead-methionine and lead-LA groups were significantly higher than the lead group (p<0.01). MDA levels were reduced in all groups compared to the lead group (p<0.01). There was a decrease below control values in erythrocyte SOD (p<0.01) and GSH-Px (p<0.05) levels in the lead-LA group. Plasma vitamin A levels were significantly high in lead-methionine group compared to lead group (p<0.01). In conclusion, the data suggests that oxidative stress induced by lead is reduced by sulphur-containing compounds.

High levels of hyaluronan in idiopathic pulmonary arterial hypertension

Hyaluronan (HA), a large glycosaminoglycan found in the ECM, has major roles in lung and vascular biology and disease. However, its role in idiopathic pulmonary arterial hypertension (IPAH) is unknown. We hypothesized that HA metabolism is abnormal in IPAH. We measured the plasma levels of HA in IPAH and healthy individuals. We also evaluated HA synthesis and the expression of HA synthases and hyaluronidases in pulmonary artery smooth muscle cells (PASMCs) from explanted lungs. Plasma HA levels were markedly elevated in IPAH compared with controls [HA (ng/ml, mean +/- SD): IPAH 325 +/- 80, control 28 +/- 9; P = 0.02]. In vitro, unstimulated IPAH PASMCs produced high levels of HA compared with control cells [HA in supernatant (microg/ml, mean +/- SD): IPAH 12 +/- 2, controls 6 +/- 0.9; P = 0.04]. HA levels were also higher in IPAH PASMC lysates. The increased HA was biologically relevant as shown by tissue staining and increased HA-specific binding of mononuclear cells to IPAH compared with control PASMCs [number of bound cells x 10(4) (mean +/- SD): IPAH 9.5 +/- 3, control 3.0 +/- 1; P = 0.01]. This binding was abrogated by the addition of hyaluronidase. HA synthase-2 and hyaluronidase-2 were predominant in control and IPAH PASMCs. Interestingly, the expressions of HA synthase-2 and hyaluronidase-2 were approximately 2-fold lower in IPAH compared with controls [HA synthase-2 (relative expression mean +/- SE): IPAH 4.3 +/- 0.02, control 7.8 +/- 0.1; P = 0.0004; hyaluronidase-2 (relative expression mean +/- SE): IPAH 4.2 +/- 0.06, control 7.6 +/- 0.07; P = 0.008]. Thus patients with IPAH have higher circulating levels of HA, and PASMCs derived from IPAH lungs produce more HA compared with controls. This is associated with increased tissue levels and increased binding of inflammatory cells suggesting a role for HA in remodeling and inflammation in IPAH.

Plasma Levels of High-Density Lipoprotein Cholesterol and Outcomes in Pulmonary Arterial Hypertension

RATIONALE High-density lipoprotein cholesterol (HDL-C) promotes healthy vascular function, and it is decreased in insulin resistance. Insulin resistance predisposes to pulmonary vascular disease. OBJECTIVES We hypothesized that HDL-C is associated with clinical outcomes in pulmonary arterial hypertension (PAH). METHODS Plasma HDL-C concentrations were measured in 69 patients with PAH (age, 46.7 +/- 12.9 yr; female, 90%) and 229 control subjects (age, 57 +/- 13 yr; female, 48%). Clinical outcomes of interest included hospitalization for PAH, lung transplantation, and all-cause mortality. Survival and time to clinical worsening curves were derived by the Kaplan-Meier method. Cox regression modeling of outcome versus HDL-C with individual covariate adjustments was performed. MEASUREMENT AND MAIN RESULTS HDL-C was low in subjects with PAH compared with control subjects (median, interquartile range: PAH: 36, 29-40 mg/dl; control subjects: 49, 40-60 mg/dl; P < 0.001). An HDL-C level of 35 mg/dl discriminated survivors from nonsurvivors, with a sensitivity of 100% and specificity of 60%. After a median follow-up of 592 days, high HDL-C was associated with decreased mortality (hazard ratio for every 5-mg/dl increase in HDL-C, 0.643; 95% confidence interval, 0.504-0.822; P = 0.001) and less clinical worsening (hazard ratio for every 5-mg/dl increase in HDL-C, 0.798; 95% confidence interval, 0.663-0.960; P = 0.02). HDL-C remained a significant predictor of survival after adjusting for cardiovascular risk factors, C-reactive protein, indices of insulin resistance, and severity of PAH (all P < 0.05). CONCLUSIONS Low plasma HDL-C is associated with higher mortality and clinical worsening in PAH. This association does not appear to be explained by underlying cardiovascular risk factors, insulin resistance, or the severity of PAH.

The relationship between inflammation and slow coronary flow: increased red cell distribution width and serum uric acid levels

OBJECTIVES The underlying mechanism of slow coronary flow (SCF) has yet to be elucidated. Increased red cell distribution width (RDW) and uric acid level may be indicative of an underlying inflammatory state. We aimed to investigate RDW and serum uric acid levels in patients with normal coronary arteries and SCF without stenosis. STUDY DESIGN The study included 46 consecutive patients (25 males, 21 females; mean age 54 ± 11 years) with angiographically normal coronary arteries but having SCF in all three coronary arteries. The control group consisted of 40 patients (18 males, 22 females; mean age 54 ± 9 years) with angiographically normal coronary arteries without SCF. In both groups, RDW and serum uric acid levels were measured and compared. RESULTS In the SCF group, TIMI frame counts measured in the left anterior descending coronary artery, left circumflex coronary artery, and right coronary artery were significantly higher compared to the control group (p<0.05). Patients with SCF exhibited significantly higher RDW (13.4 ± 1.6% vs. 12.6 ± 1.2%, p=0.01) and serum uric acid levels (5.3 ± 1.6 mg/dl vs. 4.7 ± 1.3 mg/dl, p=0.01) compared to controls. In logistic regression analysis, uric acid [Exp(B)=1.612, 95% CI 0.206-5.35, p=0.021] and RDW [Exp(B)=1.496, 95% CI 0.403-4.72, p=0.030] were found as independent predictors of SCF. CONCLUSION Our findings show that patients with SCF have significantly increased RDW and serum uric acid levels. This may help throw more light on the pathophysiological basis of SCF.

Nitric oxide deficiency in pulmonary hypertension: Pathobiology and implications for therapy

Nitric oxide (NO) is a diffusible gas with diverse roles in human physiology and disease. Significant progress in the understanding of its biological effects has taken place in recent years. This has led to a better understanding of the pathobiology of pulmonary hypertension (PH) and the development of new therapies. This article provides an overview of the NO physiology and its role in the pathobiology of lung diseases, particularly PH. We also discuss current and emerging specific treatments that target NO signaling pathways in PH.

Sensitive cardiac troponin I predicts poor outcomes in pulmonary arterial hypertension

Circulating cardiac troponins are markers of myocardial injury. We sought to determine whether cardiac troponin I (cTnI), measured by a sensitive assay, is associated with disease severity and prognosis in pulmonary arterial hypertension (PAH). cTnI was measured in 68 patients with PAH diagnostic category 1 in a research-based sensitive immunoanalyser with a lower limit of detection of 0.008 ng·mL−1. The associations between cTnI and PAH severity and clinical outcomes were assessed using Chi-squared and Wilcoxon rank sum tests, Kaplan–Meier analysis and Cox regression models. cTnI was detected in 25% of patients. Patients with detectable cTnI had more advanced functional class symptoms, a shorter 6-min walk distance, more pericardial effusions, larger right atrial area, and higher B-type natriuretic peptide and C-reactive protein levels. 36-month transplant-free survival was 44% in patients with detectable cTnI versus 85% in those with undetectable cTnI. cTnI was associated with a 4.7-fold increased risk of death related to right ventricular failure or transplant (hazard ratio 4.74, 95% CI 1.89–11.89; p<0.001), even when adjusted individually for known parameters of PAH severity. Elevated plasma cTnI, even at subclinically detectable levels, is associated with more severe disease and worse outcomes in patients with PAH.

Abnormal platelet aggregation in idiopathic pulmonary arterial hypertension: role of nitric oxide

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease. Several processes are believed to lead to the fatal progressive pulmonary arterial narrowing seen in IPAH including vasoconstriction, cellular proliferation inflammation, vascular remodeling, abnormalities in the lung matrix, and in situ thrombosis. Nitric oxide (NO) produced by NO synthases (NOS) is a potent vasodilator and plays important roles in many other processes including platelet function. Reduced NO levels in patients with IPAH are known to contribute to the development of pulmonary hypertension and its complications. Platelet defects have been implied in IPAH, but original research supporting this hypothesis has been limited. Normal platelets are known to have NOS activity, but little is known about NOS expression and NO production by platelets in patients with IPAH. Here we characterized the phenotype of the platelets in IPAH and show a defect in their ability to be activated in vitro by thrombin receptor activating protein but not adenosine diphosphate. We also show that endothelial NOS (eNOS) levels in these platelets are reduced and demonstrate that NO is an important regulator of platelet function. Thus reduced levels of eNOS in platelets could impact their ability to regulate their own function appropriately.

The effect of adenotonsillectomy on right ventricle function and pulmonary artery pressure in children with adenotonsillar hypertrophy

OBJECTIVES Adenotonsillar hypertrophy (ATH) is the most common cause of upper airway obstruction in children. Severe upper airway obstruction may have an effect on chronic alveolar hypoventilation, which consequently may lead to right ventricle (RV) dysfunction induced by hypoxemic pulmonary vasoconstriction. The investigators aimed to study RV function and mean pulmonary artery pressure (mPAP) in patients with ATH who were undergoing adenotonsillectomy by using tissue Doppler echocardiography (TDE). METHODS The study examined 27 children with ATH who had a mean age of 8 ± 2 years. The subjects were comprised 17 (63%) males and 10 (37%) females. Hypertrophy of the tonsils was graded according to the Brodsky scale. Children having either grade 3 or 4 hypertrophied adenotonsils were recruited for the study. Adenotonsillectomy was performed on all subjects in the study group and echocardiographic examination was repeated 3 months postoperatively. RESULTS Tricuspid Em significantly increased after adenotonsillectomy (17.7 ± 3.6 vs. 19.1 ± 5.5, p=0.04). The RV myocardial performance index (MPI) and mPAP significantly decreased after adenotonsillectomy (RV MPI: 0.57 ± 0.13 vs. 0.40 ± 0.12, p<0.001 and mPAP (mmHg): 31 ± 9 vs. 25 ± 7, p=0.001). CONCLUSION The results of this study, evaluated with the results of previous studies, demonstrated that adenotonsillectomy improved RV performance and reduced mPAP in children with ATH.

Leptin levels predict survival in pulmonary arterial hypertension

Evidence suggests that leptin is involved in relevant processes in the cardiovascular system. Low serum leptin levels have been associated with increased cardiovascular events and mortality in patients with coronary artery, diabetes, or chronic kidney disease. We hypothesized that leptin is increased in pulmonary arterial hypertension (PAH) and provides prognostic information. We correlated leptin levels with clinical data and assessed its association with survival. Sixty-seven patients with PAH and 29 healthy controls were studied. Plasma leptin levels were nonlinearly associated with BMI. Leptin level <15 μg/l was associated with higher mortality in PAH patients, with an adjusted (age, gender, BMI, and smoking status) hazard ratio of 3.8 (95% CI: 1.3–11.2), P=0.016. Similarly, PAH patients with leptin/BMI ratio <0.5 μg * m2/kg * I had worse survival than those with a level >0.5 μg * m2/kg * I (P=0.046 by log-rank test). Two-year mortality in PAH patients was 24%. A receiver operating characteristic curve using leptin/BMI ratio as the test variable and 2-year mortality as the state variable showed an area under the curve of 0.74 (95% CI: 0.62–0.86). A leptin/BMI ratio cut-off of 0.6 had a high sensitivity (94%) and negative predictive value (96%) for predicting death of any cause at 2 years. In PAH, plasma leptin levels are directly associated with BMI. Lower leptin levels, when adjusted by BMI, are associated with an increased overall mortality and leptin/BMI ratio has high negative predictive value for mortality at 2 years.

NITRIC OXIDE AND ASTHMA SEVERITY: TOWARDS A BETTER UNDERSTANDING OF ASTHMA PHENOTYPES

Our ability to measure nitric oxide (NO) in exhaled breath has opened a new window on the lung that improved our understanding of asthma pathobiology (1–3). Standardization of the measurement of the fraction of exhaled NO (FENO) in breath (4), followed by several large clinical and population studies, has demonstrated that, when utilized in the appropriate clinical context, FENO is useful in asthma management and provides clinicians with a new non-invasive point-of-care test to monitor airway inflammation in asthma (5). Despite all these advances, the exact role of NO in the pathogenesis of asthma remains elusive. Whether NO is beneficial through its bronchodilator and antioxidant effects or harmful by inducing inflammation remains unclear (2, 6). Understanding the complex basic biology of NO in the lung will not only improve our understanding of asthma as a disease but will also enhance our ability to use FENO to manage asthma in the clinic. Endogenous NO is produced by nitric oxide synthases (NOS), including constitutive (neuronal, or type 1, and endothelial, or type 3) and inducible (type 2) enzymes, all isoforms of which are present in the lung (3). These enzymes utilize L-arginine and oxygen as substrates and require several cofactors including NADPH, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin (CaM) and tetrahyrobioptrein (BH4). Arginine is a semi-essential amino acid that is also used in protein and is a substrate for other enzymes like arginases (7). Arginases compete with NOSs for arginine as a substrate and convert it to ornithine and urea (7) (Figure). The arginase pathway may be responsible (at least in part) for a peculiar concept in nitric oxide metabolism known as the “L-arginine paradox”. It refers to the phenomenon that exogenous arginine causes NO-mediated effects despite the fact that NOS may already saturated with arginine and its activity should not be affected by increasing arginine concentration. This paradox is not fully understood but several theories have been put forth to explain it based on our current understanding of arginine and NO metabolism (7) including: the compartmentalization of arginine in the cytoplasm (extra cellular arginine may be preferentially utilized by NOS within this microenvironment); the inhibitory effects of L-citrulline (cells may need extra arginine to compete with citrulline), and competition from arginase for arginine (a substrate for both enzymes) making less arginine available for NO production by NOS (8). Figure A simplified schematic of the Arginine –Nitric oxide pathway(s). To add to the complexity of NO metabolism, NO is highly reactive and once produced it can rapidly lead to the formation of several nitric oxide related end products such as nitrothyrosine, S-nitrosothiols and nitrates (2). NO is rapidly consumed by reaction with superoxide (PNAS) which is produced in asthma either spontaneously or in response to external stimuli during an exacerbation (2). Thus, nitric oxide (NO) in exhaled breath (FENO) represents a final balance between all these competing pathways leading to NO production as well as the consumption reactions that follow (2). As a free radical that reacts with oxidants and antioxidants, nitric oxide (NO) in exhaled breath (FENO) also reflects the redox state of the airway(2). The dynamics of NO metabolism are further complicated during an asthmatic attack. Allergen challenge studies reveal multiple and sequential reactions that suggest a multifunctional role for NO in the airway (2, 9). NO rapidly consumes cytotoxic reactive oxygen species produced during the immediate asthmatic response and leads to the accumulation of less harmful reaction products. Nitrosylation reactions predominate during the late asthmatic response with accumulation of SNO, which have been proposed as safe reservoirs for removal of toxic NO derivatives. Thus, while NO may have some harmful effects in the airways as a free radical, a temporal sequence of NO participation in asthmatic airway chemical events suggests that it may also serve a protective role in the asthmatic response (2). This complexity in NO metabolism is why any FENO value needs to be taken within the clinical and biological contexts in order to be interpreted correctly (5, 10). The same complexity, however, may be the reason why FENO can be informative and useful in so many seemingly different settings. More recently, for example, it has been shown that despite the fact that FENO levels in severe and non-severe asthma were similar, when asthma is classified based on FENO levels, a distinct asthma phenotype emerged (11). Subclassification by FENO defines severe asthma phenotypes independent of current definitions for asthma severity. In fact, asthmatics who have high FENO levels share more characteristics as compared to asthmatics with low FENO levels regardless of asthma severity as it is currently defined (12). Asthmatics with high FENO are younger and diagnosed with asthma at a younger age. They are atopic and have more eosinophilic airway inflammation, more airway reactivity, more airflow limitation, and more hyperinflation. They also seem to be less aware of asthma symptoms. Within the severe asthma group of subjects, high FENO identifies a severe asthma phenotype that has the greatest eosinophilic airway inflammation, the most severe airflow limitation, and utilizes emergent care most often (11). It is in this context that the study by Yamamoto et al. in this issue of Clinical & Experimental Allergy adds important information to our understanding of the arginine/NO pathway and asthma severity (13). They determined the relationships of NOS expression/activation and arginase expression with asthma severity, FENO, nitrotyrosine, and eosinophilic inflammation. They confirmed that the functionality of the arginine NO pathway (measured by FENO and nitrotyrosine) was strongly related to NOSII (not arginase) levels. Interestingly, however, controlling NOSII mRNA for arginase 2 levels improved the identification of severe asthma. The investigators suggested that while NOSII expression is high in severe asthma, and may explain the high levels of FENO in these patients, factors controlling arginase expression significantly improve differentiation of severity. This study adds to the growing evidence of the importance and the complexity of the arginine-NO pathway in asthma. This accumulating knowledge will hopefully allow us to fine-tune or even revise altogether the way we grade asthma severity and define asthma phenotypes. Our classifications can be based more on the biology of the disease and biomarkers (like FENO) that reflect this biology and not only on the secondary clinical and physiologic manifestation.