Control of airway inflammation is the cornerstone of asthma management. The aim of the present pilot study was to assess the effects of a leukotriene receptor antagonist (LTRA) added to a basic treatment of inhaled corticosteroids (ICS) and long-acting beta-agonist (LABA) on airway hyperresponsiveness, inflammation, and quality of life in well-controlled patients with asthma.Seventeen patients  with well-controlled asthma presenting airway hyperresponsiveness to mannitol and methacholine challenge were given add-on montelukast on a stable ICS + LABA for 4 weeks. Quality of life and selected parameters of airway inflammation were measured at baseline and at study end. ).1 , cumulated higher dose of mannitol  and methacholine  in the provocation test, lower airway reactivity with mannitol and methacholine (response dose ratio (RDR) –50.0%, p = 0.024 and –44.3%, p = 0.006, respectively), and improved airway sensitivity to mannitol and methacholine . Changes in inflammation parameters  were consistent with these findings. Asthma-related quality of life improved significantly in all domains and overall . The main limitation was the absence of a control group.Adding montelukast to ICS + LABA resulted in an increase in mean FEVThe consistency of the changes in airway hyperresponsiveness and inflammation as well as in quality of life observed with an add-on therapy with montelukast in well-controlled asthma patients during 4 weeks suggests that residual inflammation may represent an area for further improvement of asthma control to be explored in adequately powered randomized controlled trials. Thus, control of airway inflammation is considered the cornerstone of asthma management1. Inhaled corticosteroids (ICS) are effective anti-inflammatory agents widely used for improving asthma control1. However, ICS may not suppress all components of airway inflammation3. Oral leukotriene receptor antagonists (LTRAs) have been shown to possess anti-inflammatory properties complementary to ICS5 and may reach small airways not easily accessed by inhaled formulations6. LTRAs have also been shown to attenuate airway hyperresponsiveness to both specific and non-specific stimuli8 and, as an add-on therapy to ICS, to improve asthma control9–12. When added to ICS, LTRAs may primarily have anti-eosinophilic effects14 and/or modulate airway hyperresponsiveness16.Asthma is a chronic inflammatory disorder of the airways in which chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing1 from baseline . Airway reactivity is characterized by the response dose ratio (RDR), i.e. the final % fall in FEV1 divided by the total cumulative dose needed to induce that % fall in FEV117. The assessment of airway reactivity by challenge tests is well-established and recommended in the Global Initiative for Asthma (GINA) guidelines for establishing the diagnosis of asthma since 200717.Airway sensitivity to a challenge test is expressed as the cumulated provocative dose (in mg) of either methacholine or mannitol that induces a predefined and substance-specific percentage  of the fall in FEVThe aim of the present pilot study was to evaluate the effects of add-on montelukast to standardized ICS + LABA therapy on airway hyperresponsiveness, selected markers of airway inflammation, and quality of life in a cohort of already clinically well-controlled patients with asthma.All patients were recruited at the outpatient clinic of Allergology and Clinical Immunology of the University Hospital of Bern, Switzerland. Seventeen adult patients older than 18 years and younger than 65 years of age with clinically well-controlled asthma under a fixed combination of budesonide/formoterol  were included after having signed an informed consent form. Clinically well-controlled asthma was defined as the absence of symptoms and exacerbations during the 8 weeks prior to inclusion. In addition, documented airway hyperresponsiveness to mannitol and methacholine as well as current non-smoker status were required for inclusion. Smokers, women planning a pregnancy or pregnant, and patients with any significant illness that could interfere with the feasibility or the results of the provocation tests, such as acute asthma or severe cardiovascular disease, were not included.At baseline, all patients underwent a mannitol provocation test and the fraction of exhaled nitric oxide (FeNO) was measured. If positive, and at least 2 days later, a methacholine provocation test and repeated measurement of FeNO were performed. For standardization all patients were switched to the same dose-equivalent ICS + LABA therapy in a fixed combination with budesonide/formoterol (200/6 µgbid). After a 2-week run-in period on this therapy, 10 mg montelukast was added for a 4 weeks’ treatment duration (interim visit). All patients included attended the final visit in which the provocation tests were repeated.1 was assessed by using a MasterScreen Pneumo spirometer  with filter and according to ATS recommendations18. The best of three values FEV1 repeatable to within 100 ml were recorded and the percentage of predicted values19 were calculated. Mannitol challenge tests were carried out using the protocol described by Anderson et al.20. Mannitol was administered as a dry powder in capsule form, inhaled from a RS01 device . An empty capsule was used as a control at baseline. The test was started with 5 mg and increased doubling the doses up to 160 mg. In case of no response at the 160 mg dose, this dose was repeated up to a maximum cumulative dose of 635 mg. FEV1 was measured 60 seconds after delivery of each dose. The challenge was stopped if FEV1 fell by 15% or more, or when the maximum dose had been administered. The dose causing a 15% fall in FEV1 (PD15 FEV1) was read by interpolation on the plotted dose–response curve. Methacholine testing was carried out according to the protocol of the SAPALDIA Study21 which was identical to the protocol of the European Community Respiratory Health Survey (ECRHS) “method 2 short-protocol”23. The provocation test started with inhalation of saline diluent, and the maximum post-diluent FEV1 recorded 2 minutes later was the control value. All solutions of methacholine were prepared by the pharmacy of University Hospital Bern. Methacholine was delivered using a Mefar MB3 dosimeter  set to deliver the aerosol over a period of 1 second. FEV1 was recorded 2 minutes later and in the absence of a 20% fall in FEV1 from baseline the next dose was given. Methacholine was inhaled with quadrupling doses until a fall in FEV1 greater than 20% from the control value was observed or the maximum cumulative dose of 5.515 mg was reached. The dose causing a 20% fall in FEV1 (PD20 FEV1) was read by interpolation on the plotted dose–response curve. Patients were asked to refrain from short-acting beta-2-agonist for 8 hours, from the combined inhaled therapy (formoterol and budesonide) for 24 hours and from montelukast for 4 days prior to bronchial challenge testing.FEV24 by using an NIOX nitric oxide analyzer with computed biofeedback software by Aerocrine AB, Solna, Sweden.Exhaled nitric oxide was measured with the same equipment throughout the study according to ATS guidelines25. Each item was rated by the patient on a numerical scale ranging from 1 (extreme limitations) to 7 .Asthma-related quality of life (AQLQ) was assessed by applying the self-administered Juniper questionnaire at the baseline, interim and final visit. This validated questionnaire encompasses 32 items clustered in four domains: asthma symptoms, limitations in daily physical activities, emotional function, and exposure to environmental stimuliBlood samples were drawn at the baseline and final visit before any challenge tests, for determination of blood eosinophils and eosinophil cationic protein serum levels .1 in the mannitol test, PD20 FEV1 in the methacholine test, the RDR for mannitol and methacholine, the asthma related quality of life, FeNO, blood eosinophils and eosinophil cationic protein concentrations between baseline and the final visit. Descriptive statistical analysis methods were used. No PD15 or PD20 value was assigned to subjects who did not achieve PD15 or PD20. As all parameters were normally distributed, means, standard deviations and corresponding 95% confidence intervals were calculated. For mean PD15 FEV1, PD20 FEV1, RDR, and FeNO the base 10 antilog (the antilog of the mean of log-transformed data corresponds to the geometric mean) was calculated as previously suggested27. Calculations of statistical significance  and corresponding power for a 5% significance level were performed by Student’s t-test using StatsDirect software version 2.7.7, Altrincham, Cheshire, UK.Predefined parameters of interest were the changes in PD15 FEVThe study was approved by the ethics committee of the University Hospital of Bern (KEK-BE 240/06), Switzerland. The study is registered with ClinicalTrials.gov (NCT01725360).1 (3.35 L) and Tiffeneau index  were consistent with patients with normal lung function, as required by the inclusion criteria. While mean blood eosinophil counts were within normal range, mean serum ECP levels were slightly above. Asthma-related quality of life was high at baseline, scoring 5.3 on a numerical scale up to 7.Seventeen patients , with a mean age of 32 (±11) years were included with baseline characteristics shown in 1 values , significantly higher mean dose of mannitol that could be administered during the provocation test , lower sensitivity to mannitol as suggested by decreasing PD15 FEV1 values , and lower bronchial reactivity as suggested by a significantly lower maximal percent decrease in FEV1  and by a decreasing RDR . Fourteen out of 17 patients had no PD15 to mannitol at the end of the study. As shown in 1, higher cumulated methacholine dose, lower maximal % decrease in FEV1, improved airway sensitivity) and to a significantly decreased airway reactivity . Nine out of 17 patients had no PD20 to methacholine at the end of the study.As given in Changes of blood eosinophil counts, serum ECPs, and FeNO were consistent with the results obtained by the provocation tests. Differences between baseline and final visit did not reach statistical significance; all numerical trends were consistently in favor of a possible modest improvement of airway inflammation .p < 0.001). In addition, 11 patients (65%) experienced a clinically relevant improvement in asthma-related quality of life (>0.5 improvement in score).Asthma-related quality of life assessed by the AQLQ improved significantly in all four domains covered by the Juniper questionnaire . The oveIn this pilot study with 17 well-controlled asthma patients, short-term treatment with montelukast added to a basis therapy with ICS + LABA for 4 weeks improved all parameters of airway hyperresponsiveness and inflammation in a consistent manner, suggesting that, even in clinically well-controlled asthmatics, residual inflammation amenable to therapeutic intervention may persist.28. Indirect challenge tests may be a closer reflection of active airway inflammation than direct challenge tests30. While direct challenge tests (such as the methacholine test) act directly on the airway smooth muscle, indirect challenge tests (such as the mannitol test) act by increasing the osmolarity of the airway surface liquid which leads to the release of mediators from inflammatory cells  and of neuropeptides from sensory nerves. Ultimately, in subjects with bronchial hyperresponsiveness this cascade of events leads to airway narrowing and reduction of FEV1. Thus, as most stimuli that provoke an attack of asthma in daily life do so indirectly, indirect tests may be considered more specific for asthma17. The present study showed improvements in airway hyperresponsiveness when using an indirect challenge test with mannitol. A positive test to mannitol was shown to correlate with asthma severity, asthma worsening during stepwise reduction of ICS, asthma symptoms, and asthma airway inflammation consistent with the presence of inflammatory cells such as eosinophils or their mediators such as prostaglandins, leukotrienes, and histamine31–33. Earlier observations suggested that leukotriene receptor antagonists play a key role in sustaining but not in initiating the airway response to mannitol35, which is consistent with our observations. On the other hand, a negative result may be due to an insufficient number of inflammatory cells (as it may occur in a patient on ICS), insufficient concentration of mediators or lack of airway hyperresponsiveness to mediators (as it may occur in nonasthmatic patients with eosinophilic bronchitis)36. Keeping this limitation in mind, the mannitol test is useful to confirm diagnosis of a currently active asthma in untreated or of insufficiently controlled asthma in treated patients36. In the present study, montelukast added to ICS + LABA over 4 weeks significantly improved bronchial reactivity and lead to a significant increase of the cumulated dose of mannitol administered. On the other hand, montelukast had no effect on airway sensitivity assessed by the mannitol PD15. Thus, the improvements reported in the present study suggest that montelukast might possibly contribute to additionally decrease airway hyperresponsiveness when added to a fixed combination of ICS + LABA even in already well-controlled patients. Bronchial provocation testing with methacholine is generally considered sensitive, i.e. useful to rule out the presence of asthma in a person not taking ICS37. However, 27% of adults reporting physician diagnosed asthma, generally with normal or near normal spirometry, were shown to have a negative methacholine challenge test38. On the other hand, a positive methacholine provocation test does not necessarily rule in the diagnosis of asthma31 so that airway hyperresponsiveness has been described in patients with allergic rhinitis39 and with chronic obstructive pulmonary disease40. Sensitivity to the test is expressed as the PD20, i.e. the cumulated provocative dose of methacholine (in mg) inducing a 20% fall in FEV1. Reactivity is characterized by the response–dose ratio (RDR), i.e. the final % fall in FEV1 divided by the total cumulative dose needed to induce that % fall in FEV141. The latter was first suggested as a simple index of non-specific airway reactivity summarizing the dose–response curve by O’Connor et al.42 and applied to the findings of the Swiss SAPALDIA study to establish reference values for methacholine reactivity43. In the present study, airway reactivity assessed by the methacholine challenge significantly improved after 4 weeks of add-on therapy with montelukast compared to baseline, which is consistent with previous findings in patients with asthma44–46. However, airway sensitivity only numerically improved by up to 48% without reaching statistical significance. These observations support previously reported additional protection against excessive airway narrowing provided by montelukast added to a stable dose ICS in a 12 weeks’ placebo controlled study in patients with controlled asthma. In this study, montelukast significantly improved the FEV1 decline and increased PD20 FEV1 vs. placebo, thus providing additional protection to ICS with regard to excessive airway narrowing47.Airway hyperresponsiveness is a hallmark of asthma that can be assessed by provocation tests using direct and/or indirect stimuli48 and the number of eosinophils in the blood or the sputum, may be of interest to assess the level of inflammation underlying the current asthma status, guide treatment choice, and monitor treatment response49. Eosinophils present in increased numbers in airways release proteins that may damage airway epithelial cells. They also play a role in the release of growth factors and airway remodeling50. Cysteinyl leukotrienes are potent proinflammatory mediators mainly derived from eosinophils and mast cells. They are the only mediator whose inhibition has been associated with an improvement in lung function and asthma symptoms51. Furthermore, there is increasing evidence for the involvement of cysteinyl leukotrienes in the pathophysiology of airway hyperresponsiveness and, subsequently, in airway remodeling52–54. Thus, the trend in numerical decrease of blood eosinophils observed in the present study is consistent with previously published findings showing that montelukast decreases the number of eosinophils and mast cells in airway tissues55 and the blood of asthma patients56.Although their contribution to asthma management remains unclear, non-invasive markers of airway inflammation, such as the levels of exhaled nitric oxide57. Although normal ranges are still to be defined58, NO can be measured in the exhaled air with non-invasive standardized methods59 allowing for longitudinal monitoring. Exhaled NO can be considered a surrogate marker of airway inflammation which increases with loss of asthma control61 and decreases with improving inflammation control62. In our study, exhaled NO levels decreased markedly and early (2 weeks) after initiation of the add-on therapy with montelukast, which is consistent with improved control of airway inflammation, especially when taken together with the effects on the other non-invasive markers of inflammation. It is well-known that ICS dose-tapering in well-controlled patients may lead to loss of asthma control. Airway hyperresponsiveness to mannitol was identified as a predictor of asthma exacerbations after ICS dose reduction32 so that responsiveness to mannitol can be used to predict risk of exacerbation during back titration of steroids63. On the other hand, airway hyperresponsiveness correlates with noninvasive markers of airway inflammation such as exhaled nitric oxide and sputum eosinophils in steroid-naive asthmatics only33. Thus the incremental response of montelukast added to ICS + LABA observed in the present study may suggest that montelukast contributes to improved asthma control via improved control of airway inflammation which is consistent with earlier reports indicating that leukotriene receptor antagonists oppose systemic mediators of inflammation which are not controlled by or not accessible to inhaled corticosteroids64.Nitric oxide (NO) is produced by many cells, including eosinophils. NO can act as a dilator of bronchial and vascular smooth muscle, a neurotransmitter and an immune response mediator65–67. In the present study, the majorities of patients included were women, had no symptoms, and were non-smokers. A potential confounder was the presence or absence of allergic rhinitis which was not assessed at inclusion. A future study should aim at also characterizing such determinants of quality of life in asthmatics not covered by the Juniper questionnaire, e.g. for a stratified analysis. Keeping in mind that the present study had no control group, but given the fact that patients were well-controlled at inclusion, this suggests that montelukast may contribute to improve quality of life in asthmatics by still unknown mechanisms beyond inflammation control and lung function improvement.Quality of life improvements observed in the present study were statistically significant in all domains and clinically relevant in two-thirds of the patients. The determinants of quality of life in asthma are not fully understood. Female sex, the presence of respiratory symptoms, being a current or former smoker, and the presence of an associated allergic rhinitis were independently associated with poorer quality of life in asthmaticsThis pilot study has several limitations. First, this study had no control group such that reported statistical significances relate to differences versus baseline. A future study should ideally include a placebo control group. Furthermore, only patients with well-controlled asthma on ICS and LABA were included. A future study may want to explore the effects of montelukast added to ICS alone and use patients on ICS alone or on ICS plus LABA as a control group. The number of patients included in this pilot study was too small for assessing airway sensitivity but deemed sufficient for assessing airway reactivity. The present results should allow for adequate power calculations, depending on the primary endpoint chosen. Finally, all comparisons were made versus baseline, that is before the 2-week run-in on a standardized ICS + LABA regimen. In a future study, it would be advisable to re-assess the primary outcomes following run-in.In this small cohort of 17 patients consistent trends across all tested dimensions of airway hyperresponsiveness and inflammation suggest that in an adequately powered study, the add-on effects of montelukast added to a basis therapy with ICS + LABA may contribute to improved asthma control. The present results may contribute to facilitate power and thus sample size calculations in future clinical trials aimed at exploring more in depth the effects of montelukast on airway inflammation and hyperresponsiveness.Montelukast added to a fixed combination of ICS + LABA in well-controlled asthma patients was shown to numerically improve airway hyperresponsiveness and selected parameters of airway inflammation. While statistical significance was not reached in this pilot study, the consistency of the reported numerical trends across all parameters of interest deserves further research in adequately designed and powered randomized controlled trials.


We aimed to investigate peripheral blood eosinophil chemotaxis, generation of spontaneous reactive oxygen species (ROS), and apoptosis in patients with allergic asthma after bronchial allergen challenge.A total of 18 patients with allergic asthma (AA), 14 with allergic rhinitis (AR), and 10 healthy subjects (HS) underwent bronchial challenge with a specific allergen extract. Eosinophils from peripheral blood were isolated 24 h before as well as 7 and 24 h after bronchial allergen challenge. Chemotaxis, spontaneous ROS production in eosinophils, and apoptosis were analyzed by flow cytometry. Serum and induced sputum IL-5 levels were measured by ELISA; the cell count in sputum was analyzed by the May-Grünwald-Giemsa method.P < 0.05). Meanwhile, eosinophil chemotaxis and ROS generation markedly increased in the patients with AA 7 h and 24 h after challenge compared with other groups and baseline values (P < 0.05). The percentage of apoptotic eosinophils in the patients with AA decreased at 7 h as well as 24 h after challenge when compared with other groups and the baseline values (P < 0.05). There was a significant correlation between the migrated peripheral blood eosinophil count and the sputum eosinophil count  and the sputum IL-5 level  at 24 h after bronchial challenge only in the patients with AA. Furthermore, the percentage of peripheral blood apoptotic eosinophils significantly correlated with eosinophil count in sputum , and ROS production correlated with the serum IL-5 levels .Before bronchial allergen challenge, peripheral blood eosinophil chemotaxis, spontaneous ROS production was enhanced and eosinophil apoptosis was reduced in the patients with AA as compared with AR patients and HS (During allergen-induced late-phase airway inflammation, peripheral blood eosinophils demonstrated further alterations of their functional activity manifested by enhanced spontaneous ROS production, increased chemotaxis, and diminished apoptosis in patients with AA. Asthma is an inflammatory disorder of the airways involving T‐cells, mast cells, and eosinophils . The toxIL-5 plays a key role in eosinophil proliferation, differentiation, maturation, migration to tissue sites and survival, as well as prevention of eosinophil apoptosis ,7. EosinIn the absence of any inflammatory survival-prolonging factors, eosinophils die by apoptosis in a few days, but in inflamed airways, eosinophils survival is thought to be prolonged due to the surrounding proinflammatory factors such as IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor ,17. TherThere is no doubt that eosinophils are important cells which participate in allergic airway inflammation. Meanwhile, associations between eosinophil infiltration in the airways and peripheral blood eosinophil chemotaxis, ROS production, and apoptosis have not been completely elucidated yet. Therefore, the regulation of blood eosinophil activity in asthmatic patients especially after allergen challenge needs to be investigated.We hypothesized that evaluating peripheral blood eosinophils functional activity  during allergen-induced late-phase airway inflammation is important for understanding the pathogenesis of eosinophilic inflammation in the airways.ClinicalTrials.gov with identifier NCT02214303.A total of 42 nonsmoking adults (13 men and 29 women) were examined: 18 patients with intermittent or mild-to-moderate persistent allergic asthma, defined according to the GINA criteria , 14 patiDermatophagoides pteronyssinus (D. pteronyssinus), birch pollen, or mixture of 5 grasses. All the patients were not using inhaled, nasal, or oral steroids at least 1 month before visits; short-acting β2 agonists, at least 12 h; long-acting β2 agonists, at least 48 h prior the lung function test, and antihistamines and antileukotrienes, 7 days before the skin prick test and the lung function test. None of the patients had a history of smoking. Baseline forced expiratory volume in one second (FEV1) was more than 70% of the predicted value in all patients. All the healthy subjects were nonsmokers, without symptoms of asthma or rhinitis, with normal findings of spirometry, and all showed negative results of the skin prick test.Patients with allergic asthma and rhinitis had a clinical history of the disease for ≥1 year, current symptoms, and positive results of skin prick test (≥3 mm) with the following allergens: D. pteronyssinus, D. farinae, cat and dog dander, mixture of pollen of 5 grasses, birch pollen, mugwort, Alternaria, Aspergillus, and Cladosporium. Histamine hydrochloride (10 mg/mL) was used for a positive control. Skin testing was read 15 min after application. The results of the skin prick test were considered positive if the mean wheal diameter was ≥ 3 mm  vs. 0.16 × 109/L [0.04–0.88] and 0.13 × 109/L [0.04–0.94], P <0.05). At 24 hours after bronchial challenge, the peripheral blood eosinophil count was significantly increased in the patients with allergic asthma compared with the baseline values and the healthy subjects . Meanwhile, at the baseline as well as 7 h and 24 h after bronchial challenge, the total eosinophil count in the sputum was significantly higher in the patients with allergic asthma than those with allergic rhinitis and the healthy subjects (P < 0.05) . Bronchial allergen challenge had no impact on eosinophil count in healthy subjects.The eosinophil count in the peripheral blood 24 h before bronchial allergen challenge was significantly higher in the patients with allergic asthma compared with the patients with allergic rhinitis and healthy subjects (0.32 × 10P < 0.05). At 7 h and 24 h after bronchial challenge, eosinophil chemotaxis was significantly enhanced in the patients with allergic asthma compared with the baseline values, and it was greater than in the patients with allergic rhinitis and the healthy subjects  had an impact on peripheral blood eosinophil chemotaxis in all the studied groups, but the highest concentration had the greatest effect. At the baseline, peripheral blood eosinophil chemotaxis after the stimulation with 1000 ng/mL of eotaxin was higher in the patients with allergic asthma compared with those with allergic rhinitis and the healthy subjects (P < 0.05). At 7 h and 24 h after bronchial challenge, ROS generation was significantly greater in the patients with allergic asthma compared with other groups and the baseline values  .Before bronchial allergen challenge, the patients with allergic asthma had a significantly lower percentage of apoptotic peripheral blood eosinophils than those with allergic rhinitis and the healthy subjects  Figure . FurtherThere was a significant increase in the induced sputum IL-5 levels in the patients with allergic asthma and those with allergic rhinitis compared with the healthy subjects at 24 h before bronchial challenge  of eotaxin. Our results showed that before bronchial allergen challenge, peripheral blood eosinophil chemotaxis stimulated with the highest eotaxin concentration was greatest in the patients with allergic asthma as compared with other groups. These data suggest that eosinophils in the peripheral blood of individuals with allergic asthma are already primed and more sensitive to a chemokine.It is known that eosinophils are recruited to sites of inflammation by released chemotactic agents. Eotaxin, a CC chemokine, is one of the strongest stimulator of eosinophil chemotaxis . It alsoin vivo injection of eotaxin into the skin of mice and rhesus monkeys showed local accumulation of eosinophils, and the kinetics of allergen-induced production of eotaxin is paralleled by eosinophil accumulation in a guinea-pig model of allergic airways [Experiments with  airways ,26.We found that 7 h and 24 h after bronchial allergen challenge, eosinophil chemotaxis was significantly greater in the patients with allergic asthma than other groups and also compared with the baseline values. This shows that not only allergen challenge stimulates the functional activity of eosinophils, but response to eotaxin confirms the undeniable importance of this chemokine. Moreover, at 24 h after bronchial challenge, the increased eosinophil count in the sputum significantly correlated with the increased migrated eosinophil count in the peripheral blood of asthmatic patients. That can reflect the hallmark characteristic of allergic asthma by infiltration of eosinophils to the airway.The inflammatory cells recruited to the asthmatic airways are exceptionally capable of producing ROS, resulting in abnormal physiologic function of DNA, proteins, and lipids that clinically can augment bronchial hyperresponsiveness and inflammation . HoweverOur study showed that spontaneous ROS production in peripheral blood eosinophils was significantly greater in the patients with allergic asthma than those with allergic rhinitis and the healthy subjects. Moreover, at 7 h and 24 h after bronchial allergen challenge, ROS generation in peripheral blood eosinophils was significantly greater in the patients with allergic asthma compared with the baseline values and other groups.The upregulation of ROS production by blood eosinophils in allergic patients has been previously documented in several studies ,28, and P < 0.05). These findings suggest that enhanced ROS generation can activate a number of redox-sensitive signaling cascades, stimulate production-interaction of proinflammatory cytokines, and promote inflammation [Moreover, it was demonstrated that a low concentration of IL-5 enhanced chemokine-primed ROS production by eosinophils . Consequammation .As it has been reported that IL-5 can enhance eosinophil migration by the upregulation of adhesion molecules on eosinophils , we analThe accumulation of eosinophils in the asthmatic lungs is a complex process, which involves their maturation in and release from the bone marrow, adhesion and transmigration through the post-capillary endothelium, and then their chemotaxis to and activation/degranulation at inflammatory foci ,31. In tFinally, we evaluated relationships between eosinophil counts in the sputum and eosinophil viability after bronchi al allergen challenge, which could represent a link between eosinophilic influx to the airways possibly caused by delayed peripheral blood eosinophil apoptosis in allergen-induced late-phase airway inflammation.Significant correlations were observed only 24 h after bronchial challenge and only in the patients with allergic asthma. The lower percentage of peripheral blood eosinophils was negatively correlated with the increased eosinophil count in the sputum. It might be that diminished apoptotic potential represents a mechanism that promotes resolution of eosinophilic inflammation in asthma.In conclusion, this study has shown that allergic asthma patients demonstrates enhanced spontaneous ROS production, increased chemotaxis, and diminished apoptosis of peripheral blood eosinophils, and these alterations are more pronounced during allergen-induced late-phase airway inflammation.


Background: Hypertension is commonly found in patients with Diabetic Kidney Disease (DKD). Microalbuminuria is the first clinical sign of involvement of kidneys in patients with type 2 diabetes. Uncontrolled hypertension induces a higher risk of cardiovascular events, including death, increasing proteinuria and progression to kidney disease.Objectives: To determine the correlation between microalbuminuria and hypertension and their association with other risk factors in type 2 diabetic patients.Methods: One hundred and thirteen type 2 diabetic patients attending the diabetic clinic of Shaikh Zayed Postgraduate Medical Institute, Lahore, Pakistan were screened for microalbuminuria and raised blood pressure. The study was conducted from November 2012 to June 2013.Results: Patients were divided into two groups. Group 1, those with normoalbuminuria (n=63) and Group 2, those having microalbuminuria (n=50). Group 2 patients showed higher blood pressure values as compared to Group 1. The results were statistically significant and showed poor glycemic control as a contributing risk factor.Conclusion: The study concluded that there is high frequency of hypertension among type 2 diabetics but still much higher among those having microalbuminuria. So, early recognition of renal dysfunction through detection of microalbuminuria and to start treatment without any delay will confer future protection from end stage renal disease as well as hypertension and its complications in type 2 diabetic patients. At the time of diagnosis of type 2 Diabetes Mellitus, hypertension is found in approximately 70-80% of patients. Still blood pressure rises further in those patients who subsequently develop diabetic nephropathy.2Diabetes Mellitus remains a tremendous challenge to public health worldwide. Diabetes and Hypertension are common diseases that coexist at a greater frequency than chance alone would predict. Hypertension in the diabetic individual markedly increases the risk and accelerates the course of cardiac disease, peripheral vascular disease, stroke, retinopathy, and nephropathy.3 It also accounts for a third of all patients requiring renal replacement therapies, which are prohibitively expensive and not widely available in our country due to cost and lack of expertise. The development of diabetic nephropathy is characterized by a progressive increase in the excretion of protein, particularly albumin, an early and continuing rise in systolic blood pressure, and a late decline in glomerular filtration rate, leading eventually to end stage renal failure. Systolic blood pressure may be particularly important and in the UKPDS (United Kingdom Prospective Diabetes Study), higher blood pressure was associated with a higher risk of macro vascular and micro vascular disease.4Diabetic nephropathy is the leading cause of end stage renal disease (ESRD) worldwide.Hypertension and Microalbuminuria commonly coexist. The mechanism is controversial but is thought to be a renal manifestation of generalized vascular endothelial dysfunction and strongly linked with increased cardiovascular risk. It is well appreciated both that coexisting hypertension exacerbates diabetic nephropathy and that diabetic nephropathy somehow results in a markedly increased risk of hypertension.5 Screening for the earliest stages of renal damage and aggressively controlling blood pressure can help prevent more severe renal involvement. Blood pressure control is at least as important as glucose control, especially after the onset of renal damage.6The American Diabetes Association recommends that patients with type 2 diabetes be tested for albuminuria at the time of initial diabetes diagnosis and yearly thereafter.The present study was initiated to see the correlation between microalbuminuria and hypertension in type 2 diabetics and its association with other risk factors.A total of one hundred and thirteen type 2 diabetic patients who attended the diabetic outdoor clinic of Shaikh Zayed Postgraduate Medical Institute (SZPGMI), Lahore, Pakistan were included in the study.The study was conducted over a period of eight months. Patient’s age ranges from 30-70 years. Informed consent was obtained from each patient. A structured questionnaire regarding the demographic data such as age, gender, height, body weight while wearing light weight clothing, without shoes and family history of DM.Blood pressure, smoking habit were recorded for each patient. Diabetic patients suffering from any other medical problem were excluded from the study. Co-morbidities like chronic heart disease, renal and liver diseases as evidenced by either history of ischemic heart disease or alteration in ECG; history of renal disease or disturbed BUN creatinine; history of liver disease such as hepatitis B or C positive or disturbed liver function tests were excluded from the study.2). Blood pressure was measured with a suitable mercury sphygmomanometer. Readings were taken after the patients had been resting comfortably, back supported in the sitting or supine position, for at least 5 minutes and at least 30 minutes after smoking or coffee ingestion, by a physician or a trained nurse. Blood pressure was measured two times at 5 minutes interval. The 1st and the 5th Korotokoff’s sound were used to determine the systolic (SBP) and diastolic (DBP) measurements respectively. The second blood pressure measurement was used as the blood pressure for the individual. Mean arterial pressure (MAP) was calculated as systolic + (2xdiastolic) pressure.BMI was calculated as weight (Kg) divided by height  ≥140mmhg and/ or diastolic blood pressure (DBP) of ≥90mmhg or use of anti-hypertensive medications.HbA1c was assessed by mean of three consecutive Hba1c levels, either <6.5% or ≥6.5%. Those patient were included who had no evidence of proteinuria in urinalysis and without abnormal serum BUN and creatinine.Morning mid-stream urine sample, negative for proteinuria by Albustix was used to calculate micro albumin: creatinine ratio in mg/g. Micro albumin was carried out using ELISA assay developed at NHRC and validated at NETRIA UK, whereas creatinine was done by calorimetric methods using Fortress kit USA. If the ratio was <30mg/g the patient was normoalbuminuric, ratios between 30-300mg/g were indicative of microalbuminuria and above 300mg/g revealed macroalbuminuria.Subjects were excluded from the study if they came to the clinic after vigorous exercise, had any serious illness such as history of heart failure, UTI or were known patients of nephropathy.Statistical analysis was done using SPSS version 20. Descriptive statistics were compared for continuous variables, and percentages for categorical variables. Association between outcome variables and independent variables were sought using T-Test.Patients were divided into two groups as assessed by microalbuminuria calculated as micro albumin: creatinine ratio in mg/g.Group 1: (n=63) were those patients with micro albumin: creatinine ratio <30mg/g.Group 2: (n=50) were those patients with micro albumin: creatinine ratio between 30-300mg/g.There were 16 males and 47 females in group 1 with a mean age of 49.89 ±9.34 in years while in group 2 there were 23 males and 27 females with a mean age of 48.4 ±9.35 in years.Baseline Characteristics of two groups:Positive family history of DM was in 63.49% in group 1 and 78.6% in group 2 respectively. Mean BMI in group 1 was 28.90 ±4.17 while in group 2 it was 28.61 ±4.09. These results showed no significant statistical difference . Mean HbThe two groups were also assessed for difference of blood pressure that included systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP). Group 2 showed mean systolic pressure (MSP) higher than group 1 as evidenced by p<0.001 that is statistically significant. Similarly group 2 showed mean diastolic pressure (MDP) and mean arterial pressure (MAP) higher than group 1 as evidenced by p<0.001 that is again statistically significant. These results showed significant statistical difference . Group 2 patients showed higher blood pressure values as compared to group 1 patients.8 This current global epidemic is associated with an increase of cardiovascular diseases that primarily accounts for the increase in morbidity and mortality seen in patients with diabetes.9,10 The high prevalence of microvascular complications of diabetes such as diabetic nephropathy means that the number of patients with end stage renal disease due to diabetes will also increase dramatically.11 Hence diabetes especially type 2 diabetes is becoming the main reason for patients to start renal replacement therapy.12,13Over the past decades, there has been a significant worldwide increase in the incidence of diabetes mellitus.4United Kingdom prospective diabetes study (UKPDS) a well-known study on diabetes mellitus showed that the presence of hypertension is a risk factor for microalbuminuria and retinopathy and that reducing the incidence of chronic complications was significantly associated with the amplitude of systolic blood pressure decrease, the lowest risk corresponding to a systolic blood pressure below 120mmHg.14,15 In general microalbuminuria is a sensitive marker for damage induced by diabetes.16The risk factors associated with microalbuminuria were found to be poor glycemic control and hypertension. It is now widely appreciated that the excretion of even small amounts of albumin in the urine may portend serious future events, such as elevation of systemic arterial pressure, cardiovascular disease and progressive renal dysfunction.6 Hypertension is unfortunately also very common in patients with diabetes. Hypertension occurs in 50% of patients with Diabetes and results in a sevenfold increase in mortality.17 Concomitant nephropathy in patients with diabetes and hypertension results in a 37-fold increase in mortality. Hypertension is probably both a cause and an effect of diabetic nephropathy.18The combination of hypertension and diabetes is an especially dangerous clinical situation, both for risk of macro vascular and microvascualr complications of diabetes and for diabetes related and overall mortality.19 Similarly Arkedani et al. and Varghese et al. reported a good statistical correlation between the prevalence of microalbuminuria and the diastolic blood pressure.20,21 Pasko et al found that microalbuminuric patients had higher systolic and diastolic blood pressure, suggesting that systolic blood pressure is a significant risk factor for diabetic nephropathy.22 Svenson et al. showed that high blood pressure increased the risk of developing signs of nephropathy.23 Our study also found a significant statistical correlation between hypertension and microalbuminuria and this finding was also strongly associated with poor glycemic control.Studies have shown an association between microalbuminuria and high levels of blood pressure. Ahmedani et al. showed microalbuminuria positive group had a higher systolic and diastolic blood pressure compared to microalbuminuria negative group (p<0.001) which has been observed by others.17,24 Higher values of Systolic blood pressure and diastolic blood pressure in microalbuminuric than in normoalbuminuric patients suggests that hypertension is associated with microalbuminuria.25Hypertension can cause microalbuminuria and hypertensive nephropathy that can accelerate the progression of diabetic nephropathy. Thus hypertension is probably both a cause and an effect of diabetic nephropathy. Epidemiological studies have shown that identifying and monitoring patients with microalbuminuria is important because its treatment can prevent or postpone overt nephropathy.High prevalence of microalbuminuria in diabetic patients and its positive association with blood pressure suggests that screening for microalbuminuria is essential for intervention and prevention of further complications like end stage renal disease and cardiovascular disease. Once overt nephropathy is present, progression cannot be halted, only slowed. It is much more effective to screen for early nephropathy with sensitive tests for microalbuminuria and to prevent or halt the earliest stages of damage by vigorous control of hypertension and hyperglycemia.FI, AT, AA: Conceived, designed, and did statistical analysis and editing of manuscriptAA, MJA: Did data collection and manuscript writing.ZI: Did review and final approval of manuscript.


Hypertension is a premier risk factor for cardiovascular disease which can be recognized if sought and treated effectively. Effective management of high blood pressure is possible when the magnitude of the problem is identified. So, a cross-sectional community based survey among 1,239 respondents aged ≥30 years was designed to estimate the prevalence and the sociodemographic correlates of hypertension among adults aged ≥30 years. Data was collected by personal interviews, followed by anthropometric and blood pressure measurements. Analysis was done using Statistical Package for the Social Sciences (SPSS) version 11.5. The prevalence of hypertension was 43.3%, with the prevalence being more among males (51.6%) as compared to females (38.9%). Of the total prevalence 23.1% (287) were known cases, and 20.2% (250) were newly detected cases. Based on the seventh report of the Joint National Committee (JNC VII) on high blood pressure, prehypertension was noted among 38.7%. Advancing age, male gender, current diabetic status, central obesity, overweight and obesity as defined by body mass index, and family history of hypertension were identified as significant correlates for hypertension by multivariate logistic regression. Hypertension (HTN) is an enormous health problem and is one of the biggest health challenges in the 21st century. Although the condition is common, readily detectable, and easily treatable, it is usually asymptomatic and often leads to lethal complications if left untreated . In IndiIndia, being a culturally and socially diverse nation, differences would be noted in the region-wise prevalence of hypertension, but research regarding the same is inadequate in coastal Karnataka. This inadequacy necessitated us to conduct this study with the objective of assessing the prevalence of hypertension and study the sociodemographic correlates of hypertension. A cross-sectional community-based survey was conducted among individuals of either sex, aged 30 years and above. The study was carried out in the field practice area of the Department of Community Medicine, Kasturba Medical College, Manipal in coastal Karnataka, a Southern state of India. The field practice area covers a population of 45,587 individuals living in 7,164 families spread out in 11 villages. The population in these villages is homogeneous in terms of occupation, socioeconomic status, and food habits.Institutional ethical clearance was obtained prior to initiation of the study. Study population included all men and women aged 30 years and above. Pregnant or lactating women up to 12 weeks after partum were excluded from the study, due to possible variations in blood pressure during this period.Considering a prevalence of 14% for rural adults, with an allowable error of 15% and 95% confidence level, the sample size estimated was 1,092. A nonresponse rate of 20% required a sample of 1,310 to be studied. The detailed methodology of selection of subjects has been described earlier [During house visits, the objectives of the study were explained to the eligible household members, and data was collected by personal interviews using a predesigned questionnaire. The questionnaire included details on sociodemographic variables, past/family history of hypertension, and physical activity status . Socioec2). Waist circumference was measured to the nearest 0.1 cm at the midpoint between costal margin and iliac crest using a nonstretchable measuring tape. Hip circumference was measured at the level of the greater trochanters (widest portion of the hip) to the nearest 0.1 cm by a measuring tape. Waist-hip ratio was calculated as the ratio of waist circumference over hip circumference [Weight was recorded using a standard weighing scale  kept on a firm horizontal surface. Weight was recorded to the nearest 500 gm. Height was recorded using a measuring tape to the nearest 1 cm. Subjects were requested to stand upright, without shoes, with their back against the wall, heels together and looking forward. Body mass index (BMI) was calculated using the formula of weight (kg)/height  with adult size cuff was used. The first appearance of (phase 1 of Korotkoff sounds) sound was used to define systolic blood pressure (SBP). The disappearance of sound (phase 5) was used to define diastolic blood pressure (DBP). Two readings were taken five minutes apart, and the average of the two readings was taken as the final blood pressure reading. A person was considered to be a hypertensive if he/she was an already diagnosed case of hypertension and/or on treatment or had current SBP ≥ 140 mm of Hg and/or DBP ≥ 90 mm of Hg  .Blood sugar estimation was done for all the subjects using a glucometer. A person was considered to be a diabetic if he/she was an already diagnosed case of diabetes and/or on treatment or had current fasting capillary blood glucose ≥110 mg/dL  [Individuals with either a parent or a sibling (brother or sister) having hypertension were considered to have a positive family history. Eligible subjects unavailable during the first house visit were approached on another preinformed date as per their convenience. Even after two such visits if the subject was noncompliant, then he/she was considered as a nonrespondent.P < .05 as statistically significant. The odds ratios (ORs) of the statistically significant variables detected in the univariate analysis and their 95% confidence intervals were calculated. A multiple logistic regression analysis was carried out to obtain adjusted odds ratios for the variables. All the variables having a P < .2 in the univariate analysis were included in the multiple logistic regression analysis, with hypertension as a dichotomous outcome and age, sex, socioeconomic status, physical activity, positive family history of hypertension, current diabetic status, BMI, and central obesity as independent variables. Variables with significant adjusted odds ratios (P < .05) were considered to be independently associated with hypertension.All statistical analyses were performed using Statistical Package for Social Sciences (SPSS) version 11.5. Prevalence and risk factors of hypertension are presented as percentages. The association between hypertension and sociodemographic variables, diabetes, obesity, physical activity, and family history of hypertension was assessed by comparing the prevalence of hypertension in individuals with and without these risk factors. The chi-square test was used to analyze the differences, considering a The baseline characteristics of the study subjects are shown in P value  <  .001).The prevalence of hypertension was 43.3% of which 20.2% were previously undiagnosed cases. A higher prevalence was noted among males (51.6%) as compared to females (38.9%), as shown in χtrend2 = 113.93, P < .001, df = 1).The blood pressure recording of the study subjects classified according to JNC VII criteria with respect to age has been described in Advancing age, male gender, current diabetic status, overweight and obesity defined by BMI, and central obesity identified in the univariate analysis were also found to have significant association with hypertension in multivariate analysis, as shown in The World Health Report 2002 identified high blood pressure (BP) as one of the five important risk factors for noncommunicable diseases worldwide. It is estimated that elevated BP alone causes about 50% of cardiovascular disease (CVD) worldwide. It is important to emphasize that while 10–30% of adults worldwide suffer from high BP as currently defined by the JNC VII report, an additional 50%–60% could improve their prognosis if they had lower BP . A downwThe prevalence of hypertension was found to be high in the present study (43.3%), as compared to other reported literature, but was comparable to that of Kerala, which is similar to our coastal study area with respect to diet, occupation, and high literacy levels , 15. CroA community-based study has the inherent limitation of resource constraints in terms of manpower, which is acceptable to the population. So, blood pressure measurements were taken during a single visit and repeated measurements on different occasions and different settings could not be done. But, estimation of blood pressure was done by a single trained investigator in order to have a uniform pattern of blood pressure measurement. The authors do agree that there was a poor representation of males, and an attempt to quantify life style changes was not made in the present study. Although we accept these shortcomings, it is also true that this study was planned to quantify the problem of hypertension in the community, so that future interventions could be planned based on the existing level of the risk factor in the study population. There were no earlier data on prevalence of hypertension, in the area, which necessitated us to conduct this study. The methodology was fairly rigorous and the sample size was also sufficiently large. The high prevalence of hypertension, noted in the study, necessitates the need to plan future community-based studies in the same region with an additional objective to quantify the lifestyle factors responsible for the same. Stress, staying away from home due to being overboard on the boats for fishing purposes, odd hours of work with changed sleep pattern, consumption of salted fish, less physical activity predisposing to obesity, and probable indulgence in usage of tobacco and alcohol to beat loneliness could be some of the potential reasons for the high prevalence of hypertension noted in the area, as speculated by the authors. Central obesity was found to be present in a substantial proportion of the population, which points towards the need for lifestyle modifications in order to reduce this risk factor. Research into the underlying mechanisms may have policy implications to address the problem of hypertension.A significant number of individuals were identified to be in the prehypertension category, stressing the need to initiate screening strategies at an earlier age and promote opportunistic screening for hypertension during routine health care visits, so that major health gains can be made through the implementation of primary prevention strategies.


Hypertension affects about 10 % of pregnancies and is responsible for both maternal and neonatal devastating complications such as eclampsia, HELLP syndrome, prematurity and maternal and neonatal death. The resolution of the disease occurs in the first twelve weeks postpartum. The behavior of blood pressure and occurrence of very high blood pressure episodes among women with severe preeclampsia is related to remodeling of the dynamics of body fluids with consequent increase in intravascular volume. The persistence of hypertension in critical levels results in increased puerperal morbidity.To evaluate the effectiveness of furosemide in accelerating blood pressure recovery among women with severe preeclampsia.A triple-masked placebo controlled clinical trial, will be conducted including 120 postpartum women with severe preeclampsia, after eclampsia prophylaxis with magnesium sulfate and with adequate diuresis. Women with chronic hypertension and users of diuretics will be deleted. Informed consent will be obtained from all participants. Patients will be randomized to receive furosemide  or placebo. The variables are systolic and diastolic blood pressure, frequency of hypertensive crises, need for maintenance of antihypertensive therapy, number of antihypertensive agents used to control blood pressure, urine output, length of hospital stay, adverse effects and maternal complications. This study was approved by the Research Ethics Committee in humans of our institution. All participants will be duly informed about the aims of the project and will be included only if they agree to participate voluntarily, signing the informed consent.NCT02163655. .This study was registered on Clinical Trials.gov under the number  P values for all tests will be two tailed at a 5 % level of significance.The characteristics of the participants in each group will be compared with Student’s http://moosenose.com/EBCalculator.htm]; in case of adverse effects the number needed to harm (NNH) and its 95 % confidence will also be calculated.Risk ratios and their 95 % confidence intervals will be calculated as a measure of relative risk. The number needed to treat (NNT) and its 95 % confidence interval will be calculated for the outcomes in which a beneficial effect of using furosemide is achieved, using the EBM calculator [The researchers will maintain a record of problems occurred during the study and any doubt should be solved with the Steering Committee.https://clinicaltrials.gov/ct2/show/NCT02163655).The original protocol of this research proposal has already obtained approval of the local Institutional Review Board from the coordinating center , and of the National Committee for Ethics in Research (CONEP) of the Brazilian Ministry of Health, under the number 3957. The protocol also was published in the Clinical Trials Register under the number NCT02163655. (Patients will only be included if they agree to participate and sign the informed consent. All principles related to research in human beings established by the Brazilian National Health Council according to the Declaration of Helsinki will be followed. The confidentiality on women’s data and medical care will be ensured regardless of whether they participate in the study or not.Treatment of patients with preeclampsia and eclampsia still represents a challenge in the postpartum period, since the pressure levels may continue to rise, which increases morbidity, mortality and length of hospital stay. There is a rationale for the use of furosemide for the purpose to reduce these complications since this drug eliminates excess intravascular volume resulting from the mobilization of liquid which was previously in the interstitial and extravascular space. However, evidence from randomized clinical trials is scarce and insufficient for a recommendation to incorporate the use of furosemide in daily clinical practice.Furosemide, a loop diuretic, is a cheap drug, safe, widely available and considered level 1 for use during breastfeeding, especially if used for shorter periods. Long-term treatment may inhibit lactation but is not the case of this proposed therapy, using low dose of only five days after birth. If it is demonstrated its effectiveness in reducing blood pressure levels, the need for antihypertensive drugs and shortening of hospitalization, significant benefits can be obtained for women with pre-eclampsia and the health system. With the reduction in blood pressure and the elimination of excess intravascular, the incidence of acute pulmonary edema and other diseases resulting from fluid overload can be decreased. The reduced need for antihypertensive drugs, beyond avoiding side effects for women, can along with the reduction of hospital days, lead to substantial savings for the health system.


Baroreflex failure syndrome is a rare disorder which causes labile blood pressure, headache, flushing, diaphoresis and emotional lability. It is caused by history of trauma or radiotherapy in the cervical legion, bilateral carotid-body tumor or resection of glossopharyngeal nerve. We experienced a case of hemodialysis patient who had difficulty in controlling blood pressure during dialysis because of his baroreflex failure syndrome and successfully controlled his blood pressure by adjusting dialysate temperature.We report a case of a 68-year-old CKD5 patient who had difficulty in hemodialysis treatment because of severe fluctuations in blood pressure with hypertensive attacks and hypotensive episodes which caused him a severe discomfort. His dialysis treatment was started in 2010 and since that time baroreflex failure syndrome has been suspected because of his clinical manifestations and history of radiotherapy in the cervical region for his lingual cancer in 1994. Baroreflex failure syndrome is diagnosed by symptoms and cold stressor test. We performed a cold stressor test on an experimental baroreflex failure syndrome mouse and induced a significant elevation of blood pressure. From this experimental finding of model mouse, we changed the patients dialysate temperature between 34-38° according to his change in blood pressure though 80–240 mmHg. From this attempt, his blood pressure was successfully controlled between 100–180 mmHg and he was able to continue hemodialysis without any discomfort.In our case, environmental stimulation such as temperature change modified the patients fluctuating blood pressure. Change of dialysate temperature could be an option for controlling the unstable blood pressure due to baroreflex failure syndrome. The arterial baroreflex which involves multiple components of the baroreflex arc, prevents excessive fluctuations of arterial blood pressure.The signals by distention of the vessel wall are sent from baroreceptors in each carotid sinus to the brain stem via the glossopharyngeal nerve . The information from other baroreceptors in the aortic arch and the great vessels of the thorax are sent through the vagus nerve  to the brain stem. Thoracic blood volume change detected by low-pressure receptors is also sent by the vagus nerve to the brain stem.According to these mentioned mechanism of baroreflex, any abnormalities in the vascular baroreceptors, the glossopharyngeal or vagus nerves, or the brain stem could cause baroreflex failure .In clinical setting, baroreflex failure is often caused by denervation of carotid body tumor resection, carotid artery surgery, neck irradiation and neck trauma. The frequent symptoms are labile hypertension and hypotension, often with headache, diaphoresis, face flashing and emotional instability.We experienced a hemodialysis case with labile blood pressure which fluctuated through 80 mmHg to 240 mmHg during dialysis Figure .Figure 1Although it was hard to diagnose, we differentiated our case from autonomic failure, which is common in chronic hemodialysis patients, by the hyperactive result to cold pressure stimuli. This is because sympathetic efferents to the vasculature are intact in baroreflex failure and patients exhibit a normal or even an increased pressor response to cold-pressure test .In general, the use of low-temperature dialysate is recommended to decrease the frequency and intensity of symptomatic hypotension. From this evidence, we considered applying this method to stabilize the patients’ labile blood pressure.This is a case report of baroreflex failure hemodialysis patient in whom dialysate temperature adjustment was an effective treatment for labile blood pressure during hemodialysis caused by baroreflex failure syndrome.A 68-year-old Danish male presented with multiple episodes of lightheadedness and the feeling of “passing out” while he was getting into his bed or when food was administered to his gastric fistula tube. And he also had sudden onset of hypertensive attacks several times a day. His past medical history was significant for chronic kidney disease and hypertension since he was 30-years-old. The patient was diagnosed having lingual cancer for which he received external radiation therapy (RT) at 51-year-old. Ten years after RT, the patient started having dysphagia and had several episodes of aspiration pneumonia. Modified barium swallow showed he was having silent aspiration and Percutaneous Endoscopic Gastrostomy (PEG) was created. Although the patient didn’t have any oral intake, he still repeated pneumonia and eventually he underwent tracheotomy. At the same time, his renal function declined and hemodialysis was started three times a week. Since then he has been suffering labile blood pressure.During dialysis, interdialytic weight increase was controlled between 1.5–1.7 kg (2-3% of body weight) and ultrafiltration rate was about 6.0 – 6.3 ml/hr/kg. The patients’ sodium concentration was mostly controlled between 140 to 142 mEq/L and diffusive sodium balance during dialysis was negative. Although this stable weight and sodium control, blood pressure fluctuated through 80 mmHg to 240 mmHg especially during dialysis and was difficult to control with medications .From his current symptom of labile blood pressure with stable heart rate and medical history of RT and brachial vein stenosis, we diagnosed him having Baroreflex Failure.The patient’s labile blood pressure during hemodialysis was treated with dialysate temperature change. We decreased dialysate temperature to 34° when patient’s blood pressure was low and increased it to 37° when pressure was getting high. From this trial, his blood pressure was stabilized between 120–180 mmHg which was ranging through 80–240 mmHg before the temperature adjustment . The dialysate sodium level was 140 mEq/L and the patients’ pre dialysis plasma sodium level was mostly maintained between 140 mEq/L to 142 mEq/L. . According to the lab results during dialysis, the diffusive sodium balance during dialysis was negative and the ultrafiltration rate was 6.30 ml/hr/kg. The transition of lab results during convectional dialysis without temperature adjustment is shown below (Table From these results, the patients hemodynamic instability during dialysis was not thought to be caused by volume overload nor sodium imbalance.In the process of making the diagnosis, we noticed that the patients’ heart rate did not change and was stable compared to his blood pressure instability. From his past medical history of RT and unusual blood pressure elevation in cold pressor test, we diagnosed him having baroreflex failure.The key for diagnosing this case was his medical history of RT in the cervical region. Among several causes of baroreflex failure, a history of irradiation around the cervical region is one of the main reasons . Since tBy the facts that Carotid Ultrasonography showed iso-echoic plaques and he had a brachial vein stenosis which was cured by PTA, it was compatible to our thought that the fibrosis of the artery is main reason to cause his baroreflex failure after radiation therapy.The recommended medication for baroreflex failure is antihypertensive and/or vasopressor agents to prevent labile change in blood pressure . Our patThe 2005 K/DOQI guidelines  recommenIn our case the diffuse sodium balance was negative and ultrafiltration rate was 6.30 ml/hr/kg and 6.16 ml/hr/kg in convectional dialysis Figure . From thFrom this negative diffuse sodium balance and ultrafiltration rate similarity between convectional and temperature adjusting dialysis, we presume that the blood pressure was successfully controlled through peripheral temperature stimulation in temperature adjusting dialysis.From our case, we considered baroreflex failure patient have more susceptibility to temperature change than usual hemodialysis patient and temperature adjusting dialysis is an effective way to control blood pressure in these patients.Baroreflex failure occurs as a chronic complication for neck irradiation therapy. Patients who have baroreflex failure often suffer from their unstable blood pressure. Baroreflex failure dialysis patient who had an extreme change in blood pressure during dialysis was treated successfully by adjusting the dialysate temperature according to the labile blood pressure during hemodialysis.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Objectives. There are many studies regarding unnecessary polysomnography (PSG) when obstructive sleep apnea syndrome (OSAS) is suspected. In order to reduce unnecessary PSG, this study aims to predict the apnea-hypopnea index (AHI) via simple clinical data for patients who complain of OSAS symptoms. Method. Demographic, anthropometric, physical examination and laboratory data of a total of 390 patients  who were subject to diagnostic PSG were obtained and evaluated retrospectively. The relationship between these data and the PSG results was analyzed. A multivariate linear regression analysis was performed step by step to identify independent AHI predictors. Results. Useful parameters were found in this analysis in terms of body mass index (BMI), waist circumference (WC), neck circumference (NC), oxygen saturation measured by pulse oximetry (SpO2), and tonsil size (TS) to predict the AHI. The formula derived from these parameters was the predicted AHI = (0.797 × BMI) + (2.286 × NC) − (1.272 × SpO2) + (5.114 × TS) + (0.314 × WC). Conclusion. This study showed a strong correlation between AHI score and indicators of obesity. This formula, in terms of predicting the AHI for patients who complain about snoring, witnessed apneas, and excessive daytime sleepiness, may be used to predict OSAS prior to PSG and prevent unnecessary PSG. OSAS is one of the most significant health problems in middle-aged people and leads to daytime sleepiness and cognitive deficiencies as well as many systemic diseases. It is a highly prevalent disorder, and at least 4% of middle-aged males and 2% of middle-aged females are estimated to be affected . DiagnosIn this observational study, we retrospectively evaluated 390 consecutive, unselected subjects who were referred to the sleep laboratory of a university hospital to evaluate presumed sleep-disordered breathing and who had undergone PSG. Each individual included in this study was referred to sleep laboratory by otolaryngology department after detailed ear, nose, and throat examination which was performed by the same specialist. Demographic data , anthropometric measurements , and medical history were evaluated.2). Neck circumference was measured in centimeters at the level of the cricothyroid membrane. Waist circumference was measured in centimeters at the level above the iliac crest. Hip circumference was measured in centimeters while patients were standing still and their feet were fairly close and at the level of the point where the maximum circumference over the buttocks was measured.BMI, the most commonly used method to measure obesity, was calculated by dividing weight in kilograms by the square of height in meters . Scores higher than 10 were considered to be sleepiness. Pulmonary function tests (including spirometry and flow volume curves), chest X-rays, pulse oximeters in polyclinics to evaluate peripheral oxygen saturation , arterial blood gas analysis, and a full-night in-laboratory PSG were performed on all subjects.Detailed otolaryngologic physical examinations were performed by the same specialist. Parameters obtained in these examinations were included in statistical evaluations and included the following: protrusion and retrusion of the mandibles, tonsil sizes, the relationship between the soft palate and the neutral position of the tongue, the tongue-base size, nasal septum deviation, the inferior turbinate size, the endoscopic nasal cavity, and nasopharynx examinations using a 0° rigid endoscope, endoscopic larynx, and hypopharynx examinations using a 70° rigid endoscope .Mandibular retrognathism was evaluated in accordance with the position of the pogonion when the virtual imaginary line between the vermilion line and the chin on a patient sitting in a Frankfort horizontal position is taken into consideration. The examinations of the oral cavity started with an inspection of the relative position of the hard and of the soft palate to the tongue inside the mouth, without protrusion using the modified Mallampati index (MMI), ranging from Class I to Class IV, with Class I representing the highest visibility  and Class IV representing the lowest level of visibility  of the posterior oropharynx . The tonAll subjects underwent a full overnight in-laboratory diagnostic PSG . Electroencephalography electrodes were positioned according to the international 10–20 system. PSG consisted of monitoring of sleep by electroencephalography, electrooculography, electromyography, airflow, and respiratory muscle effort and included measures of electrocardiographic rhythm and blood oxygen saturation. Thoracoabdominal plethysmograph, oronasal temperature thermistor, and nasal-cannula pressure transducer system were used to identify apneas and hypopneas. Transcutaneous finger pulse oximeter was used to measure oxygen saturation. Sleep was recorded and scored according to the standard method . BesidesU test was performed to evaluate the statistical difference between AHI values of men and women. Following this test it was not a statistically significant difference between men and women according to AHI values (P = 0.190). Therefore different models were not developed regarding the gender. Prior to the correlation analysis, we identified whether these variables provided parametric assumptions. We found in the results of the Kolmogorov-Smirnov test that variables did not demonstrate normal distribution. Spearman's rank order correlation analysis was performed because variables did not provide normal distribution as required. Following this analysis, variables that were identified to have had a correlation with the AHI were integrated into the regression analysis. Each variable was integrated into the regression in order to identify how these variables as a whole affected the AHI via a step-by-step, multiple linear regression analysis. When the P value was <0.05, it was regarded as statistically significant.SPSS version 17.0 was used for statistical analysis. First, the correlation between the AHI and all of the variables included in the study was analyzed in order to identify variables to be used to predict the AHI. Mann-Whitney The demographic data , anthropometric measurements , and medical history variables are shown in Variables that were integrated into the regression analysis after we found that they were correlated with the AHI in Spearman's rho test were the BMI, NC, WC, hip circumference (HC), smoking rate in packages/year, force vital capacity (FVC), forced expiratory volume ratio in one second (FEV1%), FEV1/FVC ratio, Pao2, PaCO2, ESS score, oxygen saturation level via a pulse oximeter, the Epworth Scale score, and tonsil size. Correlation coefficients of these variables in nonparametric Spearman's rank order correlation analysis are shown in R2 = 0.682. Therefore, the following model was developed to predict the AHI: AHI prediction = (0.797 × BMI) + (2.286 × NC) − (1.272 × SpO2) + (5.114 × TS) + (0.314 × WC). The most significant variables in the regression model developed to the AHI prediction via a step-by-step multilinear regression analysis are shown in Thereafter, each variable was integrated into the regression analysis in order to identify how these variables as a whole affected the AHI via a step-by-step multiple linear regression analysis. In the seventh phase, the explanatory power of the model reached its peak: F = 155.348, P < 0.05). According to this formula, 68.2% of the variation in the AHI could be explained via these variables, while 32.8% could be attributed to other variables. Bland Altman plot comparing real AHI values obtained from PSG and the predicted AHI values obtained from the prediction formula is shown in We found this model to be statistically significant  in the upper airway (UAW) in patients suspected of OSAS. The UAW is assumed to be narrower and/or more prone to collapse multilevelly in OSAS patients. The relationship between anatomic abnormalities and the severity of OSAS has not been well established in previous studies because UAW abnormalities are not the only causative factors . In thisThere is no consensus regarding the effect of nasal airway blockage on OSAS pathogenesis and the severity of the disease. While some authors argued that the nasal passage affects OSAS development and is related to AHI, other authors claimed the converse . In thisThe Mallampati index, which was defined by anesthesiologists in 1985 to identify difficult intubation, was later suggested as an MMI to be used in predicting OSAS . This inIn many studies, pulmonary function tests were not included in the data to detect OSAS predictors. Herer et al. evaluated 102 obese patients by clinical features, pulmonary function tests, arterial blood gas tensions, and oximetry for prediction of OSAS prior to PSG and stated that none of these data is sufficient to prove the existence of OSAS . In a stHowever, such formulas include different variables within different populations, whose differences may be a significant limitation of this study. Therefore, this method may not be sufficient since a screening technique and its validity need to be further tested in other series and would need to include more patients. It is our next aim to design a prospective study to evaluate the predictability of the formula obtained from this study.As a result, a mathematical model created via data obtained through simple office procedures to predict OSAS may reduce redundant PSG and result in more rapid diagnoses and treatment processes. The potential advantage of this approach is its ease of use, its cost effectiveness, its applicable nature in office environments, and its contribution to more rapid decisions. Validity of this prediction formula needs to be further tested in other prospective researches. It is not possible to make a clear clinical statement about this formula yet. Validated prediction models, which will be created via collective studies by more sleep centers and with more groups of patients, are needed because no single, sufficient model has yet been described.


P=0.039), whereas C-reactive protein increased significantly . From body composition analysis, a significant impairment of fat-free mass percentage at the end of the study was demonstrated , probably related to total body water increase. The muscular mass, body cell mass and body cell mass index are significantly decreased after 6 weeks of LDP (T2). The phase angle is significantly reduced at the end of the study compared with basal values . This study indicated that LPD slows down the progression of kidney disease but worsens patients' nutritional state.Chronic kidney disease (CKD) is becoming increasingly widespread in the world. Slowing its progression means to prevent uremic complications and improve quality of life of patients. Currently, a low-protein diet (LPD) is one of the tools most used in renal conservative therapy but a possible risk connected to LPD is protein-energy wasting. The aim of this study is evaluate the possible correlation between LPD and malnutrition onset. We enrolled 41 CKD patients, stages IIIb/IV according to K-DIGO guidelines, who followed for 6 weeks a diet with controlled protein intake . Our patients showed a significant decrease of serum albumin values after 6 weeks of LDP (T2) compared with baseline values (T0) ( CKD is more frequent among older individuals and subjects with hypertension and diabetes, suggesting that CKD needs to have a central part in future public health planning.2 In 2007, average inpatient cost for the first month of dialysis in the US was $9846 per Medicare member and $22 841 per employer group health plan member.3 According to the United States Renal Data System database, in 2010, the total expenses per patient for hemodialysis was $87 561 compared with $66 751 for PD.3 Epidemiological data collected in Italy suggest that the prevalence of CKD in male subjects is 8.1% and in female subjects is 7.8%; these values are lower in comparison with European and non-European countries, probably because of the Mediterranean-style food.4–7 Thus, slowing the progression of CKD is necessary not only to improve the quality of life of uremic patients but also to reduce the significant impact that this disease has on public health costs.Chronic kidney disease (CKD) represents a major problem of public health and its evolution in end-stage renal disease requires renal replacement therapy  or transplantation). Worldwide, CKD incidence is high.8 During the 1960s, for the first time, Giovannetti and Maggiore10 Despite the obvious benefits of protein restriction  several studies have demonstrated that especially very LPDs could cause deterioration in the nutritional status of CKD patients.10 Menon and coworkers11 compared LPD with very LPD supplemented with keto acids and amino acids and demonstrated that both diets had no effects on the progression of kidney disease but increased the long-term risk of death.In the last 100 years, CKD patient management of restriction of dietary daily protein intake, has a central role.12 A 10-year cohort study in 206 hemodialysis patients showed that serum albumin level was a stronger predictor of mortality, more than inflammatory markers or intima-media thickness of the common carotid artery.13 In CKD population, surrogates of over-nutrition, for instance obesity or hyperlipidemia, seem to be associated with increased survival; this phenomenon is now acknowledged as 'reverse epidemiology'.14In CKD patients, protein-energy wasting is a frequent condition, characterized by low serum levels of albumin or transthyretin, sarcopenia and weight loss. Protein-energy wasting is strongly related to mortality in CKD patients.The aim of this study is to evaluate whether 6 weeks of low-protein diet has impact on body composition, inflammatory markers and progression of CKD in nephropathic patients (stage IIIb/IV according to K-DIGO guidelines).Furthermore, we measured for the first time erythrocyte glutathione S-transferase (e-GST), a well-characterized cellular detoxifying enzyme, and erythrocyte catalase (e-CAT), a protecting enzyme against oxidative stress in CKD patients enrolled in LPD protocol.15–17In previous studies, e-GST was already considered a potential biomarker of blood toxicity and adequacy of the dialytic treatments in nephropathic patients.All the enrolled 41 subjects completed the study, and all their data were eligible for the analysis. In P=0.039). At the same time-points, total proteins were significantly lower at T1 (3 weeks of LPD) and T2 (6 weeks of LPD) with respect to T0 (P<0.0001) .Moreover, we observed a significant decrease of serum albumin values after 6 weeks of LPD (T2) compared with baseline values (T0) (<0.0001) . Inverse18Regarding lipid profile , we observed changes statistically significant only for total cholesterol and low-density lipoprotein cholesterol; in particular, we measured a reduction at T1 for both parameters , as prevIn addition, we found a significant lowering of albuminuria and azoturia in every time-point . The sysversus T2; P=0.0489). These data are probably because of the increase of total body water observed.In versus T2 and T1 versus T2  and at T2 (P=0.04) showing that LPD aggravated the malnourishment of CKD patients  to baseline value . Interesersus T2 . These ppatients .versus T2; P=0.0001, and T1 versus T2; P=0.0015); this parameter is a negative prognostic factor for survival as previously demonstrated in the literature.22The phase angle (PA) is significantly reduced at the end of the study compared with basal values . Therefore, the reduced protein intake for the first 3 weeks showed no changes in the serum concentration of albumin.25In this study, we confirmed the benefits of this nutritional intervention in decreasing nitrogenous wastes and improving estimated glomerular filtration rate (GFR), as previous demonstrated.12 In all time-points of our study, we observed a significant reduction of BMI.Traditionally, reduced serum albumin and decreased BMI are among the primary indicators of protein-energy malnutrition in uremic patients.27 In this context, in addition to a deterioration of the nutritional status of the patients, we also found, after 6 weeks of LPD, a significant increase in inflammation parameters, evaluated by measuring CRP.The progressive malnutrition observed in CKD patients is associated with some markers of a chronic inflammation state that characterized the malnutrition-inflammation-atherosclerosis syndrome, inducing a vicious cycle where malnutrition, inflammation and atherosclerosis coexist.per se is related to skeletal mass loss, causing the condition called ‘uremic sarcopenia’.28–30 The etiology of uremic sarcopenia is not well understood, although several factors  are involved.Our data suggest that LPD triggers a constant catabolic phase, which causes loss of protein from skeletal muscle. In fact, we observed a significant reduction of muscular mass at the end of the diet treatment. CKD 22 A substantial and significant decrease of PA was showed during patients' diet treatment. The PA is used as an indicator of cellular density; a low PA is associated to the damage of cell’s membrane and their impaired function. Actually, in literature, PA is negatively related to survival in several pathological conditions such as cancer, uremia and HIV infection.31–33 The PA is an independent predictor of survival in hemodialysis patients.34 An interesting study on PD patients showed that PA could be considered as an independent prognosis marker for survival and clinical improvement: the PD patients with PA>6.0° had higher survival compared with those with PA values lower than 6.0°.21 A PA reduction can reflect an increase in the ratio between extracellular and intracellular water or a decrease in BCM.21Another index linked to malnourishment state, assessed by bio-impedance analysis, is the PA.35 In addition to BCM, lean tissue consists also in ECM. ECM/BCM ratio, which directly reflects the proportions between intracellular and extracellular space, is one of the most sensitive index of malnutrition.20 Avram et al.20 had demonstrated that an increased ECM/BCM ratio was considered a negative independent predictor index of survival in PD patients. Malnutrition features, characterized by a pronounced depletion of BCM, with a greater ECM/BCM ratio due to the lack of functional muscle mass, was observed, for example, in HIV patients and reported as an early marker of malnutrition.36In the CKD patients, the assessment of the fluid state such as total body water and extracellular water and the evaluation of BCM is crucial to verify the nutritional state. BCM represents the metabolically active cellular fraction of the body and, for this reason, it could be considered as a potentially sensitive indicator of lean tissue loss.37 It was demonstrated that arterial hypertension and CKD are closely associated. In fact, BP typically arises when renal function is compromised, and induces the progression of kidney disease.38 Experimental animal models have shown that arterial hypertension causes kidney damage and decreases the kidney's ability to remove sodium.39 High dietary salt intake not only exacerbates arterial hypertension in nephropathic patients, but also has the potential to directly aggravate kidney dysfunction. In a Cochrane database systemic review, it was demonstrated that on reducing salt intake in CKD patients, there was a significant decrease of systolic and diastolic blood pressure.40In this study, we also observed a significant decrease of systolic blood pressure (BP), related with the restriction of the sodium in the diet, according to guidelines maintaining sodium intake <2 g/day.41 This finding confirms a persistent blood toxicity status during the LPD even if a weak renal function improvement was achieved.Interestingly, e-GST and e-CAT levels remained stable during the period of the study. This is a strong indication that toxins and oxidative stress inducers did not increase during the LPD. The level of e-GST in all patients before the diet was high and corresponded to the one found previously in other CKD stage IV patients.In this study, we confirmed, coherently to the literature, that the LPD induces an improvement of the renal function, but at the same time, it contributes of the state of malnutrition usually observed in CKD patients.The time limit of LPD did not allow us to evaluate a significant reduction of muscle mass, which is one of the pathognomonic criteria of uremic sarcopenia. Therefore, additional and prolonged studies will be needed to corroborate our hypothesis.A total of 41 patients , aged 73±14 years, were recruited from the Department of Internal Medicine, Hypertension and Nephrology Unit, ‘‘Tor Vergata’’ University Hospital and Haemodyalisis and Nutrition Service, ‘Nuova Clinica Annunziatella’, Rome  according to the level of kidney function .37The selected patients had a mean GFR of 23±1 ml/min corresponding to stages IIIb/IV according to K-DIGO guidelines.42 Controls were carried out at baseline (T0), after 3 weeks (T1) and after 6 weeks (T2). All the subjects provided a signed consensus at the enrollment, according to a protocol previously approved by the ‘Tor Vergata’ University Medical Ethical Committee, Rome, Italy.The primary causes of renal failure were: chronic glomerulonephritis (32%), nephroangiosclerosis (58%), chronic pyelonephritis and interstitial nephropathy (2%), autosomal dominant polycystic kidney disease (2%) and other causes (~5%) . Exclusi11 The daily energy intake is aimed at 30/35 kcal per kilogram ideal body weight per day.42 From the time of enrollment (T0) to the next 6 weeks, patients followed a diet with controlled protein intake  and low-phosphorus intake (8–12 mg/g of protein). The restriction of potassium intake varies according to blood values, usually about 8–17 mg/kg/day, sodium intake <2 g/day and calcium intake<2 g/day.In CKD, nutritional therapy has the aim to reduce intake of proteins, phosphorus, potassium and sodium, preserving an adequate energy intake. In CKD patients, the energy ingested is a crucial aspect of diet treatment. In fact, neutral or positive nitrogen balance normally requires adequate energy intake and poor energy intake may directly result in protein wasting.The macronutrient composition of the diet therapy was: carbohydrates, 50–60% kcal/day; proteins, 14% kcal/day; total fat, 30% kcal/day , 15–20 g of fiber, folic acid 200–300 mcg. In the diet plan, we have used ‘non proteic’  carbohydrates in order to replace cereal foods  with similar products but with a low fat content and constantly controlled protein, sodium, potassium, tyrosine and phenylalanine. They also include inulin, a soluble dietary fiber. No alcoholic beverages were allowed.The plan for each subject was obtained from a dietetic software package .43 Height (m) was measured using a stadiometer to the nearest 0.1 cm (Invernizzi), BMI was calculated according to Quetelet Index ).After 12-h overnight fast, anthropometric measurements were performed on subjects in underwear without clothes and shoes. According to standard methods, the body weight (kg) was measured to the nearest 0.01 kg, using an accurate balance scale .Resistance, reactance, impedance and PA at 50 kHz frequency were measured using bioelectrical impedance analysis .The total body analysis allows the assessment of nutritional status and body fluids according to a bi-compartmental model. Bioelectrical impedance analysis is a safe, low-cost, noninvasive, rapid method for the assessment of body composition, displaying a great potential if used for epidemiological and clinical studies. Measurements were taken on left side of the body, with injection and sensor electrodes placed on the hand and foot in reference position.44Total body water, intracellular water, extracellular water, BCM, fat mass and fat-free mass are estimated using the manufacturer’s equations.45The estimated GFR was calculated using the CKD epidemiology collaboration (CKD-EPI) formula.Systolic and diastolic blood pressures were registered during each visit and defined as the average of two measurements 1 min apart, with 5 min rest before the first measurement.via venipuncture from the antecubital vein. All materials were immediately placed on ice and plasma was separated by centrifugation at 1600×g for 10 min at 4 °C. We measured serum lipid profile that included plasma total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein-cholesterol and triglyceride concentrations; for kidney function, we measured creatinine, azotemia and albumin, CRP, erythrocyte sedimentation rate, GST, e-CAT activity, uric acid and serum and urinary electrolytes .Early morning blood samples were taken from each patient for biochemical screening test after a 12-h overnight fasting. Blood samples were collected into sterile tubes containing ethylenediaminetetraacetic acid , For determination of C-reactive protein, a nephelometric assay was used .The lipid profile that included total cholesterol (TC), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglyceride was determined through standard enzymatic colorimetric techniques , according to the manufacturer’s procedures, with reagents provided by the same company.All other parameters were analyzed according to standard techniques by the accredited Clinical Chemical Laboratories of the ‘Tor Vergata’ Policlinico (PTV) Rome, Italy.2O2 and all other reagents were purchased from Sigma–Aldrich  and used without further purification.Glutathione, 1-chloro-2,4-dinitrobenzene, ethylenediaminetetraacetic acid, Hμl) of whole blood was diluted in 25 volumes (1 ml) of bi-distilled water causing red blood cell hemolysis. After 2 min, 0.1 ml were diluted to a final volume of 1 ml containing 1 mM glutathione, 1 mM 1-chloro-2,4-dinitrobenzene in 0.1 M potassium phosphate buffer, pH 6.5 according to the standard procedure of Habig and coworkers.46 Results were expressed as enzyme units (U) per gram of hemoglobin (Hb) (U/gHb): one unit represents the amount of enzyme that catalyzes the conjugation of 1 micromole of glutathione to 1-chloro-2,4-dinitrobenzene in 1 min at 37 °C.41e-GST activity was determined with a spectrophotometric assay at 340 nm (37 °C), using an Uvikon 941 Plus spectrophotometer . Briefly, one volume  per gram of Hb (U/gHb): one unit represents the amount of enzyme that catalyzes the decomposition of 1 micromole of H2O2 in 1 min at 25 °C.e-CAT activity was determined with a spectrophotometric assay at 240 nm (25 °C), using a Kontron Uvikon 941 Plus spectrophotometer (Kontron Instruments). One volume of 5 t-test and Mann–Whitney test, as indicated The minimal level of significance of the differences was fixed at P≤0.05. Statistical analysis was performed using computer software packaged .Data are represented as means±standard error of the mean (S.E.M.). All continuous variables were checked for normality using Kolmogorov–Smirnov test. Differences between baselines and final values were tested used paired samples