Tanize do Espirito Santo Faulin

Lipid Peroxidation Is Associated with the Severity of Periodontal Disease and Local Inflammatory Markers in Patients with Type 2 Diabetes

CONTEXT Periodontitis is the most common lytic disease of bone and is recognized as a common complication of diabetes. Lipid peroxidation (LPO) is increased in diabetes and may be related to modulation of the inflammatory response. LPO levels in patients with diabetes and periodontal disease have not been evaluated. OBJECTIVE The aim of this study was to evaluate the levels of LPO and its correlation with periodontal status and inflammatory cytokines in type 2 diabetic and nondiabetic patients. DESIGN AND SETTING This is a cross-sectional study involving Brazilian patients recruited at the State University of São Paulo. PATIENTS The sample comprised 120 patients divided into four groups based upon diabetic and dyslipidemic status: poorly controlled diabetics with dyslipidemia, well-controlled diabetics with dyslipidemia, normoglycemic individuals with dyslipidemia, and healthy individuals. MAIN OUTCOME MEASURES Blood analyses were carried out for fasting plasma glucose, glycated hemoglobin, and lipid profile. Periodontal examinations were performed, and gingival crevicular fluid was collected. LPO levels were evaluated by measuring oxidized low-density lipoprotein (ELISA) and malondialdehyde (HPLC). Cytokines were evaluated by the multiplex bead technique. RESULTS LPO evaluated by malondialdehyde in plasma and gingival crevicular fluid was significantly increased in diabetes groups. Significant correlations between LPO markers and periodontal parameters indicate a direct relationship between these levels and the severity of inflammation and secretion of inflammatory cytokines, particularly in diabetic patients. CONCLUSION These findings suggest an important association for LPO with the severity of the local inflammatory response to bacteria and the susceptibility to periodontal disease in diabetic patients.

Validation of a novel ELISA for measurement of electronegative low-density lipoprotein

Background: Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. LDL(-) is present in blood plasma of healthy subjects and at higher concentrations in diseases with high cardiovascular risk, such as familial hypercholesterolemia or diabetes. Methods: We developed and validated a sandwich ELISA for LDL(-) in human plasma using two monoclonal antibodies against LDL(-) that do not bind to native LDL, extensively copper-oxidized LDL or malondialdehyde-modified LDL. The characteristics of assay performance, such as limits of detection and quantification, accuracy, inter- and intra-assay precision were evaluated. The linearity, interferences and stability tests were also performed. Results: The calibration range of the assay is 0.625-20.0 mU/L at 1:2000 sample dilution. ELISA validation showed intra- and inter-assay precision and recovery within the required limits for immunoassays. The limits of detection and quantification were 0.423 mU/L and 0.517 mU/L LDL(-), respectively. The intra- and inter-assay coefficient of variation ranged from 9.5% to 11.5% and from 11.3% to 18.9%, respectively. Recovery of LDL(-) ranged from 92.8% to 105.1%. Conclusions: This ELISA represents a very practical tool for measuring LDL(-) in human blood for widespread research and clinical sample use.BACKGROUND Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. LDL(-) is present in blood plasma of healthy subjects and at higher concentrations in diseases with high cardiovascular risk, such as familial hypercholesterolemia or diabetes. METHODS We developed and validated a sandwich ELISA for LDL(-) in human plasma using two monoclonal antibodies against LDL(-) that do not bind to native LDL, extensively copper-oxidized LDL or malondialdehyde-modified LDL. The characteristics of assay performance, such as limits of detection and quantification, accuracy, inter- and intra-assay precision were evaluated. The linearity, interferences and stability tests were also performed. RESULTS The calibration range of the assay is 0.625-20.0 mU/L at 1:2000 sample dilution. ELISA validation showed intra- and inter-assay precision and recovery within the required limits for immunoassays. The limits of detection and quantification were 0.423 mU/L and 0.517 mU/L LDL(-), respectively. The intra- and inter-assay coefficient of variation ranged from 9.5% to 11.5% and from 11.3% to 18.9%, respectively. Recovery of LDL(-) ranged from 92.8% to 105.1%. CONCLUSIONS This ELISA represents a very practical tool for measuring LDL(-) in human blood for widespread research and clinical sample use.

Development of immunoassays for anti-electronegative LDL autoantibodies and immune complexes

BACKGROUND Electronegative low-density lipoprotein (LDL-) promotes atherosclerosis through inflammatory and immunologic mechanisms that lead to the production of anti-LDL(-) autoantibodies and to the subsequent formation of immune complexes (IC) and macrophage foam cells. We described the development and validation of an ELISA for the quantification of free anti-LDL(-) autoantibodies and an ELISA for the quantification of IC consisting of LDL(-)-bound IgG in human plasma. METHODS LDL(-) purified from human plasma, and anti-LDL(-) monoclonal antibody Fab fragments were adsorbed onto ELISA plates to capture anti-LDL(-) autoantibodies and IC-LDL(-), respectively. The performance characteristics of both ELISAs, including the limits of detection and quantification, accuracy and inter- and intra-assay precision were evaluated. Linearity, interference and stability tests were also performed. RESULTS The calibration range of the anti-LDL(-) assay was 0.004-0.125 mU/l and plasma demonstrated a dilutional linearity when diluted 1:100, 1:200, 1:400 and 1:800. The calibration range of the IC-LDL(-) assay was 0.06-4 U/l, and plasma demonstrated a dilutional linearity when diluted 1:12.5, 1:25, 1:50 and 1:100. Both ELISAs showed intra- and inter-assay precision and recovery within the required limits for immunoassays. CONCLUSION These ELISAs can be used in clinical studies and for the biochemical investigation of atherosclerosis. In addition, they will enable the comprehensive evaluation of the importance of bound or free autoantibodies against LDL(-) in this disease.

Violet ZnSe/ZnS as an alternative to green CdSe/ZnS in nanocrystal-fluorescent protein FRET systems.

Fluorescent proteins from the green fluorescent protein family strongly interact with CdSe/ZnS and ZnSe/ZnS nanocrystals at neutral pH. Green emitting CdSe/ZnS nanocrystals and red emitting fluorescent protein dTomato constitute a 72% efficiency FRET system with the largest alteration of the overall photoluminescence profile, following complex formation, observed so far. The substitution of ZnSe/ZnS for CdSe/ZnS nanocrystals as energy donors enabled the use of a green fluorescent protein, GFP5, as energy acceptor. Violet emitting ZnSe/ZnS nanocrystals and green GFP5 constitute a system with 43% FRET efficiency and an unusually strong sensitized emission. ZnSe/ZnS-GFP5 provides a cadmium-free, high-contrast FRET system that covers only the high-energy part of the visible spectrum, leaving room for simultaneous use of the yellow and red color channels. Anisotropic fluorescence measurements confirmed the depolarization of GFP5 sensitized emission.

Increased electronegative LDL and decreased antibodies against electronegative LDL levels correlate with inflammatory markers and adhesion molecules in hemodialysed patients.

BACKGROUND Chronic Kidney Disease (CKD) patients present high levels of electronegative LDL (LDL-) that can modulate the expression of molecules involved in inflammation and it is closely linked to atherosclerosis. We investigated the association between LDL(-) and inflammatory markers in patients undergoing hemodialysis (HD). METHODS Forty-seven HD patients from a private clinic in Rio de Janeiro, Brazil were studied and compared with 20 age matched healthy individuals. Serum LDL(-) and anti-LDL(-) autoantibody levels were measured by ELISA; TNF-α, IL-6, VCAM-1 and ICAM-1 were determined by a multiplex assay kit. RESULTS HD patients presented higher IL-6 and TNF-α concentrations (4.1 ± 1.6 and 5.5 ± 2.1 pg/ml, respectively) than healthy subjects (2.6 ± 0.2 and 2.4 ± 1.1 pg/ml, respectively) (p=0.0001). In addition, they presented higher VCAM-1 and ICAM-1 levels and, LDL(-) concentrations were also increased (0.18 ± 0.12 U/l) when compared to healthy individuals (0.10 ± 0.08 U/l) (p<0.02). In contrast, the anti-LDL(-) autoantibody levels were lower in HD patients (0.02 ± 0.01 mg/l) than in healthy subjects (0.05 ± 0.03 mg/l) (p<0.001). There was a positive correlation between LDL(-) and IL-6 (r=0.25, p=0.004) and ICAM-1 (r=0.36; p=0.003). There was also a negative correlation between anti-LDL(-) autoantibodies and TNF-α (r=-0.37; p=0.003) and VCAM-1 (r=-0.50; p=0.0001). CONCLUSIONS The association between LDL(-) and inflammation and the lower levels of anti-LDL(-) autoantibodies are important risk factors related to atherosclerosis in CKD.

Reduced Plasma Zinc Levels, Lipid Peroxidation, and Inflammation Biomarkers Levels in Hemodialysis Patients: Implications to Cardiovascular Mortality

Despite the fact that low plasma zinc (Zn) levels play important roles in the oxidative stress, the relationships between lipid peroxidation and inflammation biomarkers with low plasma Zn levels have not been investigated in chronic kidney disease (CKD) patients. The aim of this study was to evaluate the Zn plasma levels, electronegative LDL [LDL(–)] levels, and inflammation markers as predictors of cardiovascular (CV) mortality in hemodialysis (HD) patients. Forty-five HD patients (28 men, 54.2 ± 12.7 years, 62.2 ± 51.4 months on dialysis and BMI 24.3 ± 4.1 kg/m2) were studied and compared to 20 healthy individuals (9 men, 51.6 ± 15.6 years, BMI 25.2 ± 3.9 kg/m2) and followed for 24 months to investigate the risks for CV mortality. LDL(–) levels were measured by ELISA, plasma Zn levels by atomic absorption spectrophotometry, C-reactive protein (CRP) level by immunoturbidimetric method, and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) levels by a multiplex assay kit. HD patients presented low plasma Zn levels (54.9 ± 16.1 μg/dL) and high-LDL(–) (0.18 ± 0.12 U/L) and TNF-α (5.5 ± 2.2 pg/mL) levels when compared to healthy subjects (78.8 ± 9.4μ g/dL, 0.10 ± 0.08U/L, 2.4 ± 1.1 pg/mL, respectively, p < 0.05). Zn plasma levels were negatively correlated to TNF-α (r = –0.49; p = 0.0001) and LDL(–) (r = –0.33; p = 0.008). During the 2 years, 24.4% of the patients died, all due to CV disease. Analysis by the Cox model showed that high CRP, TNF-α, IL-6 levels, and long duration of HD were significant predictors of mortality. In conclusion, reduced Zn levels were associated with lipid peroxidation and inflammation, and we confirm here in a Brazilian cohort of HD patients that inflammation markers are strong predictors of CV death.

Diabetes subdiagnosticado e necrose miocárdica: preditores de hiperglicemia no infarto do miocárdio

BACKGROUND Hyperglycemia in the acute phase of myocardial infarction is an important prognostic factor. However, its pathophysiology is not fully understood. OBJECTIVE To analyze simultaneously the correlation between hyperglycemia and biochemical markers related to stress, glucose and lipid metabolism, coagulation, inflammation, and myocardial necrosis. METHODS Eighty patients with acute myocardial infarction were prospectively included. The following parameters were analyzed: blood glucose; stress hormones (cortisol and norepinephrine); glucose metabolism factors [glycated hemoglobin (HbA1c); insulin]; lipoproteins (total cholesterol, LDL, HDL, minimally modified electronegative LDL, and adiponectin); glycerides (triglycerides, VLDL and fatty acids); coagulation factors (factor VII, fibrinogen, plasminogen activator inhibitor-1); inflammation (high-sensitivity C reactive protein); and myocardial necrosis (CK-MB and troponin). Continuous variables were converted into degrees of relevance using fuzzy logic. RESULTS Significant correlation was observed between hyperglycemia and glucose metabolism (p < 0.001), lipoproteins (p = 0.03), and necrosis factors (p = 0.03). In the multivariate analysis, only glucose metabolism (OR = 4.3; CI = 2.1-68.9; and p < 0.001) and myocardial necrosis (OR = 22.5; CI = 2-253; and p = 0.012) showed independent and significant correlation. For the analysis of the influence of history of diabetes mellitus, a regression model including only patients without diabetes mellitus was developed, and the results did not change. Finally, in the model adjusted for age, gender, and clinical variables (history of diabetes mellitus, hypertension and dyslipidemia), three variables maintained a significant and independent association with hyperglycemia: glucose metabolism (OR = 24.1; CI = 4.8-122.1; and p < 0.001), myocardial necrosis (OR = 21.9; CI = 1.3-360.9; and p = 0.03), and history of DM (OR = 27; CI = 3.7-195.7; and p = 0.001). CONCLUSION Glucose metabolism and myocardial necrosis markers were the best predictors of hyperglycemia in patients with acute myocardial infarction.

Role of electronegative LDL and its associated antibodies in the pathogenesis of atherosclerosis

Abstract Atherosclerotic vascular disease has a wide range of clinical and pathological manifestations; nevertheless, inflammation is common in all stages of the disease. Electronegative LDL, or LDL(-), is a minimally modified form of LDL with distinct biological activities that not appear in native LDL. LDL(-) can elicit several inflammatory responses, including the induction of chemotactic or proinflammatory proteins by endothelial cells and macrophages, which contribute to atherogenesis. LDL(-) promotes atherosclerosis through inflammatory and immunologic mechanisms that lead to the production of autoantibodies and to the subsequent formation of immune complexes and macrophage foam cells. This article reviews the involvement and contribution of LDL(-) to the pathogenesis of atherosclerosis.

Avaliação da aterosclerose subclínica e de níveis plasmáticos de LDL minimamente modificada em pacientes com espondilite anquilosante e sua correlação com a atividade da doença

INTRODUCTION Accelerated atherosclerosis has been shown in some autoimmune diseases, mainly in Systemic Lupus Erythematosus and Rheumatoid Arthritis. Although high prevalence of corticosteroids use may be a confounding factor due to their detrimental effects on several risk factors, systemic inflammation per se is supposed to play an important role in atherogenesis in these patients. METHODS We have evaluated sub-clinical atherosclerosis and plasma levels of circulating electronegative LDL, which represents the fraction of LDL that is minimally modified, in patients with ankylosing spondylitis (AS). Fourteen patients who fulfilled the modified New York criteria for AS were compared with 13 paired controls. Carotid intimal-media thickness (IMT) was assessed by ultrasonography bilaterally in common carotid artery, internal carotid artery and in the bifurcation. Groups were homogeneous regarding cardiovascular risk factors. Only a single patient in AS group was in use of corticosteroid. RESULTS The presence of active inflammation was demonstrated by elevated BASDAI and higher CRP levels and in patients versus controls (12.36 vs. 3.45 mg/dl, P = 0.002). No difference was found in carotid IMT between both groups, in any site of artery. Averaged IMT (6 measurements, at 3 pre-specified sites bilaterally) was 0.72 ± 0.28 in AS group and 0.70 ± 0.45 mm in controls (P = 0.91). Minimally modified LDL did not differ significantly either between patients and controls (14.03 ± 17.40 vs. 13.21 ± 10.21; P = 0.88). CONCLUSIONS Patients with AS did not show increased carotid IMT in comparison to controls. In the same way, circulating plasma levels of LDL (-), did not differ significantly in both groups.

The hypolipidemic and pleiotropic effects of rosuvastatin are not enhanced by its association with zinc and selenium supplementation in coronary artery disease patients: a double blind randomized controlled study.

Objective Statins treatment may modify the levels of zinc and selenium, minerals that can improve vascular function and reduce oxidative damage and inflammation in atherosclerotic patients. This study aimed to evaluate the effects of rosuvastatin, alone or associated with zinc and selenium supplementation, on lipid profile, antioxidant enzymes and mineral status in coronary artery disease patients. Material and Methods A double-blind randomized clinical trial was performed in which patients (n = 76) were treated with 10 mg rosuvastatin over 4 months associated or not with zinc (30 mg/d) and selenium (150 μg/d) supplementation. The following parameters were analyzed before and after the intervention: anthropometric measurements, lipid profile, high sensitivity C-reactive protein (hs-CRP), electronegative low density lipoprotein (LDL(-)) concentrations, activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), zinc and selenium concentrations in blood plasma and erythocytes. Significance was determined using an α of 5% (two-tailed). Results We found that rosuvastatin therapy was efficient in reducing total cholesterol, LDL-cholesterol, non-HDL cholesterol, triglycerides, and hs-CRP independently of mineral supplementation. Neither treatment was associated with significant changes in LDL(-). Similarly, the antioxidant enzymes GPx and SOD activity were unchanged by treatments. Neither treatment was associated with significant differences in concentrations of zinc or selenium in blood plasma and erythocytes of studied groups. Conclusion Rosuvastatin treatment did not affect zinc and selenium levels in coronary artery disease patients. The zinc and selenium supplementation at doses used in this study did not change lipid profile or SOD and GPx activity in patients receiving rosuvastatin. Further studies should be focused on testing alternative doses and supplements in different populations to contribute for a consensus on the ideal choice of antioxidants to be used as possible complementary therapies in atherosclerotic patients. Trial Registration ClinicalTrials.gov NCT01547377