Superoxide anion production by neutrophils is associated with prevalent clinical manifestations in systemic lupus erythematosus

To determine the relation between neutrophil function and the clinical characteristics of systemic lupus erythematosus ( SLE), the superoxide anion (O(2)(-)) production by neutrophils, mediated by Fc gamma R and Fc gamma R/CR cooperation, was studied in 64 SLE patients classified according to their prevalent clinical manifestations. Three clinically distinct patterns were designated: ( 1) manifestations associated with the occurrence of cytotoxic antibodies ( SLE-I group); ( 2) manifestations associated with circulating immune complexes ( IC; SLE-II group), and ( 3) manifestations associated with IC and cytotoxic antibodies ( SLE-III group). O(2)(-) production was evaluated by a lucigenin-dependent chemiluminescent assay in neutrophils stimulated with ICIgG opsonized or not with complement. No difference in O(2)(-) production was observed when neutrophil responses from healthy controls were compared to the unclassified patients. However, when the SLE patient groups were considered, the following differences were observed: ( 1) SLE-I neutrophils showed lower O(2)(-) production mediated by the IgG receptor ( Fc gamma R) with the cooperation of complement receptors ( Fc gamma R/ CR) than observed in the SLE-II, SLE-III, and healthy groups; ( 2) neutrophils from the SLE-II group showed a decreased O(2)(-) production mediated by Fc gamma R/ CR compared to the SLE-III group, ( 3) SLE-III neutrophils produced more O(2)(-) than neutrophils from the SLE-II and control groups, and ( 4) CR showed inefficiency in mediating the O(2)(-) production by neutrophils from the SLE-I group. Comparative experiments on the kinetics of chemiluminescence ( CL; T(max) and CL(max)) disclosed differences only for the SLE- I group. Taken together, these results suggest that differences in oxidative metabolism of neutrophils mediated by Fc gamma R/ CR may reflect an acquired characteristic of disease associated with distinct clinical manifestations.

Assessment of Vascular Effects of Tamoxifen and Its Metabolites on the Rat Perfused Hindquarter Vascular Bed

Tamoxifen has been suggested to produce beneficial cardiovascular effects, although the mechanisms for these effects are not fully known. Moreover, although tamoxifen metabolites may exhibit 30-100 times higher potency than the parent drug, no previous study has compared the effects produced by tamoxifen and its metabolites on vascular function. Here, we assessed the vascular responses to acetylcholine and sodium nitroprusside on perfused hindquarter vascular bed of rats treated with tamoxifen or its main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen, and endoxifen) for 2 weeks. Plasma and whole-blood thiobarbituric acid reactive substances (TBARS) concentrations were determined using a fluorometric method. Plasma nitrite and NOx (nitrite + nitrate) concentrations were determined using an ozone-based chemiluminescence assay and Griess reaction, respectively. Treatment with tamoxifen reduced the responses to acetylcholine (pD(2) = 2.2 +/- 0.06 and 1.9 +/- 0.05 after vehicle and tamoxifen, respectively; P < 0.05), while its metabolites improved these responses (pD(2) = 2.5 +/- 0.04 after N-desmethyl-tamoxifen, 2.5 +/- 0.03 after 4-hydroxy-tamoxifen, and 2.6 +/- 0.08 after endoxifen; P < 0.01). Tamoxifen and its metabolites showed no effect on endothelial-independent responses to sodium nitroprusside (P > 0.05). While tamoxifen treatment resulted in significantly higher plasma and whole blood lipid peroxide levels (37% and 62%, respectively; both P < 0.05), its metabolites significantly decreased lipid peroxide levels (by approximately 50%; P < 0.05). While treatment with tamoxifen decreased the concentrations of markers of nitric oxide formation by approximately 50% (P < 0.05), tamoxifen metabolites had no effect on these parameters (P > 0.05). These results suggest that while tamoxifen produces detrimental effects, its metabolites produce counteracting beneficial effects on the vascular system and on nitric oxide/reactive oxygen species formation.

Enhanced concentrations of relevant markers of nitric oxide formation after exercise training in patients with metabolic syndrome

Metabolic syndrome (MetS) denotes a clustering of risk factors that may affect nitric oxide (NO) bioavailability and predispose to cardiovascular diseases, which are delayed by exercise training. However, no previous study has examined how MetS affects markers of NO formation, and whether exercise training increases NO formation in MetS patients. Here, we tested these two hypotheses. We studied 48 sedentary individuals: 20 healthy controls and 28 MetS patients. Eighteen MetS patients were subjected to a 3-month exercise training (E+group), while the remaining 10 MetS patients remained sedentary (E-group). The plasma concentrations of nitrite, cGMP, and ADMA (asymmetrical dimethylarginine: an endogenous nitric oxide synthase inhibitor), and the whole blood nitrite concentrations were determined at baseline and after exercise training using an ozone-based chemiluminescence assay, and commercial enzyme immunoassays. Thiobarbituric acid reactive species (TBA-RS) were measured in the plasma to assess oxidative stress using a fluorometric method. We found that, compared with healthy subjects, patients with MetS have lower concentrations of markers of NO formation, including whole blood nitrite, plasma nitrite, and plasma cGMP, and increased oxidative stress (all P < 0.05). Exercise training increased the concentrations of whole blood nitrite and cGMP, and decreased both oxidative stress and the circulating concentrations of ADMA (both P < 0.05). These findings show clinical evidence for lower endogenous NO formation in patients with MetS, and for improvements in NO formation associated with exercise training in MetS patients. (C) 2008 Elsevier Inc. All rights reserved.

Antihypertensive effects exerted by enalapril in mild to moderate hypertension are not associated with changes in the circulating levels of nitric oxide-related markers

The antihypertensive effects of angiotensin-converting enzyme inhibitors (ACEi) are explained, at least in part, by enhanced bradykinin-dependent nitric oxide (NO) formation and decreased angiotensin II-induced oxidative stress and vasoconstriction. We examined for the first time whether treatment with enalapril increases the plasma levels of markers of NO formation and decreases oxidative stress in mild to moderate hypertensive patients. Eighteen untreated hypertensive patients were treated with enalapril 10 mg/day (n = 10) or 20 mg/day (n = 8) for 60 days. Eighteen normotensive healthy controls were followed for the same period. Venous blood samples were collected at baseline and after 30/60 days of treatment with enalapril. Plasma NOx (nitrites + nitrates) concentrations were determined by using the Griess reaction. Plasma nitrite and whole blood nitrite concentrations were determined by using an ozone-based chemiluminescence assay. Plasma thiobarbituric acid-reactive species (TBARS) and 8-isoprostane concentrations were determined by a fluorimetric method and by ELISA, respectively. Treatment with enalapril decreased blood pressure in hypertensive patients. However, we found no significant changes in plasma NOx, nitrite, whole blood nitrite, and in the levels of markers of oxidative stress in both normotensive controls and hypertensive patients treated with enalapril. Our data show that enalapril 10-20 mg/day does not affect the concentrations of relevant markers of NO formation or markers of oxidative stress in mild to moderately hypertensive subjects, despite satisfactory blood pressure control. Our findings do not rule out the possibility that ACEi may produce such effects in more severely hypertensive patients treated with higher doses of ACEi.

Interethnic differences in ADMA concentrations and negative association with nitric oxide formation in preeclampsia

Background: Recent studies have suggested that impaired nitric oxide (NO) formation in preeclampsia may result from increased concentrations of an endogenous NO synthase inhibitor, the asymmetric dimethylarginine (ADMA). However, no previous study has examined whether a negative association exists between ADMA and nitrite concentrations in preeclampsia. Moreover, no previous study has compared ADMA and nitrite levels in black and white preeclamptic pregnant women. Methods: We measured plasma nitrite concentrations using an ozone-based chemiluminescence assay, and plasma ADMA levels using enzyme immunoassays in 94 pregnant (47 healthy pregnant: 16 blacks and 31 whites; and 47 preeclamptic: 14 blacks and 33 whites). Results: We found higher ADMA (2.199 +/- 0.016 mu mol/l vs. 2.112 +/- 0.012 mu mol/l; P < 0.0001) and lower plasma nitrite levels (102 +/- 7.1 nmol/l vs. 214.8 +/- 26.1 nmol/l; P<0.0001) in preeclamptic compared with healthy pregnant women. Black pregnant had higher ADMA levels than white pregnant women (P<0.05), both in preeclamptic (2.239 +/- 0.020 mu mol/l vs. 2.144 +/- 0.019 mu mol/l) and in healthy pregnant (2.172 +/- 0.025 mu mol/l vs. 2.077 +/- 0.018 mu mol/l). Conversely, we found no significant effects of ethnicity on the plasma nitrite levels, both in healthy pregnant and in preeclamptic women (P>0.05). We found a significant negative correlation (P<0.05) between these markers (r = 0.28; P<0.05). Conclusions: Our findings show higher ADMA and lower nitrite levels in preeclamptic compared with healthy pregnant, and the concentrations of these biomarkers are inversely associated. While ethnicity affected ADMA concentrations, no such effect was found with respect to nitrite levels. These results may have important implications for studies on NO biology and therapeutic approaches of preeclampsia. (C) 2010 Elsevier B.V. All rights reserved.

Alterations in cyclic GMP levels in preeclampsia may reflect increased B-type natriuretic peptide levels and not impaired nitric oxide activity

Objectives: We compared nitrite, B-type natriuretic peptide (BNP), and cGMP levels in preeclamptic with those found in healthy pregnant. Methods: We studied 21 healthy pregnant and 27 preeclamptic. Plasma cGMP and BNP levels were determined by ELISA. Nitrite levels were determined by chemiluminescence. Results: Higher cGMP and BNP, and lower nitrite levels were found in preeclamptic versus healthy pregnant Conclusions: Altered cGMP levels reflect increased BNP levels and not impaired nitric oxide activity in preeclampsia. (C) 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Increased circulating cell-free hemoglobin levels reduce nitric oxide bioavailability in preeclampsia

Contrasting with increased nitric oxide (NO) formation during healthy pregnancy, reduced NO bioavailability plays a role in preeclampsia. However, no study has examined whether increased NO consumption by enhanced circulating levels of cell-free hemoglobin plays a role in preeclampsia. We studied 82 pregnant women (38 healthy pregnant and 44 with preeclampsia). To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. Plasma ceruloplasmin concentrations and plasma NO consumption (pNOc) were assessed and plasma hemoglobin (pHb) concentrations were measured with a commercial immunoassay. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients (-48 and -39%, respectively; both P<0.05) compared with healthy pregnant women. Plasma samples from preeclamptic women consumed 63% more NO (P=0.003) and had 53% higher pHb and 10% higher ceruloplasmin levels than those found in healthy pregnant women (P<0.01). We found significant positive correlations between pHb and pNOc (r=0.61; P<0.0001), negative correlations between pNOc and whole blood or plasma nitrite concentrations (P=0.02; r=-0.32 and P=0.01: r=-0.34, respectively), and negative correlations between pHb and whole blood or plasma nitrite concentrations (P=0.03; r=-0.36 and P=0.01: r=-0.38, respectively). These findings suggest that increased pHb levels lead to increased NO consumption and lower NO bioavailability in preeclamptic compared with healthy pregnant women. (C) 2010 Elsevier Inc. All rights reserved.

Effects of eNOS polymorphisms on nitric oxide formation in healthy pregnancy and in pre-eclampsia

Pre-eclampsia (PE) is associated with decreased nitric oxide (NO) formation. However, no previous study has examined whether genetic variations in the endothelial NO synthase (eNOS) affect this alteration. We hypothesized that PE decreases NO formation depending on eNOS polymorphisms. We examined how three eNOS polymorphisms [T-786C, rs2070744; Glu298Asp, rs1799983; 27 bp variable number of tandem repeats (VNTR) in intron 4] affect plasma nitrite concentrations in 205 pregnant women [107 healthy pregnant (HP) and 98 PE]. Genotypes were determined and eNOS haplotypes were inferred using the PHASE 2.1 program. The plasma nitrite concentrations were determined using an ozone-based chemiluminescence assay. The Glu298Asp polymorphism had no effects on the plasma nitrite concentrations. Higher nitrite levels were found in HP women with the CC versus TT genotype for the T-786C polymorphism (277.9 +/- 19.5 versus 140.6 +/- 8.2 nM; P < 0.05). Lower nitrite levels were found in healthy women with the 4a4a versus 4b4b genotype for the VNTR polymorphism (95.1 +/- 3.3 versus 216.1 +/- 16.8 nM; P < 0.05). No effects of genotypes were found in PE women (all P > 0.05). The `C Glu b` haplotype was more frequent in the HP group than in the PE group (20 versus 5; P = 0.0044). This haplotype was associated with higher nitrite concentrations than the other haplotypes in healthy pregnancies (P < 0.05). No differences in nitrite concentrations were found among PE women with different eNOS haplotypes (P > 0.05). These findings indicate that eNOS polymorphisms affect endogenous NO formation in normal pregnancy, but not in PE, and that the `C Glu b` haplotype may protect against the development of PE by increasing endogenous NO formation.

Quercetin restores plasma nitrite and nitroso species levels in renovascular hypertension

Quercetin has antioxidants properties which may increase nitric oxide (NO) bioavailability. However, the effects of quercetin on NO status have been poorly studied. We evaluated whether quercetin improves the plasma levels of NO metabolites in two-kidney one-clip (2K1C) hypertensive rats and assessed its effect on endothelial function. Sham-operated and 2K1C rats were treated with quercetin (10 mg(-1) kg(-1) day(-1) by gavage) or vehicle for 3 weeks. Systolic blood pressure (SBP) was monitored weekly. Vascular responses to acetylcholine (Ach) and sodium nitroprusside (SNP) were assessed in hindquarter vascular bed. Plasma nitrate levels were assessed by Griess reagent and plasma nitrite and nitroso species (S, N-nitroso species) were assessed by ozone- based chemiluminescence. Aortic NADPH oxidase activity and superoxide production were evaluated. While quercetin had no effects in control normotensive rats (P > 0.05), it significantly reduced SBP in 2K1C rats (P < 0.05). At the end of treatment, plasma nitrate levels were similar in all experimental groups (P > 0.05). However, plasma nitrite and the nitroso species levels were significantly lower in 2K1C rats when compared with controls (P < 0.05). Quercetin treatment restored plasma nitrite and nitroso species levels to those found in the sham-vehicle group (P < 0.05). While quercetin treatment induced no significant changes in responses to SNP (P > 0.05), it restored the vascular responses to Ach. Quercetin significantly attenuated 2K1C-hypertension-induced increases in NADPH oxidase activity and vascular superoxide production (P < 0.05). These results suggest that the antihypertensive effects of quercetin were associated with increased NO formation and improved endothelial function, which probably result from its antioxidant effects.

Comparison of MB fraction of creatine kinase mass and troponin I serum levels with necropsy findings in acute myocardial infarction

Serum levels of troponin and heart-related fraction of creatine kinase (CK-MB) mass are used as diagnostic and prognostic criteria in myocardial infarction, but the relation between those levels and-the necropsy-determined size of necrosis has not been tested in human beings. In this retrospective study, 1-cm-thick transverse sections of the ventricles were cut from the base to the apex in the necropsy hearts of 27 patients aged 47 to 86 years (mean 66, median 69; 19 men). Total and necrotic areas were measured using a computer-linked image analysis system. The weights of the necrotic areas were also calculated. The correlations of the areas and weights of necrotic myocardium with the highest serum values of CK-MB mass and troponin 1, which had been quantified during life by chemiluminescence immunoassays, were verified by Pearson`s test; results were considered significant at p <= 50.05. Significant correlations were detected between CK-MB mass peak and infarct size (r = 0.63, p < 0.01) and weight (r = 0.69, p < 0.01) and between CK-MB mass and highest troponin level (r = 0.73, p < 0.01); however, the correlations between highest troponin level and myocardial infarct size (r = 0.31, p = 0.11) and weight (r = 0.35, p = 0.07) were small and nonsignificant. In conclusion, despite the well-established role of serum levels of troponin as a diagnostic tool for myocardial infarction, their highest values showed poor correlations with the extent of infarct. In contrast, the highest serum level of CK-MB mass was well correlated with myocardial infarct size. (c) 2008 Elsevier Inc. All rights reserved.

Daily variations in human plasma fluoride concentrations

This study investigated the variations in human plasma fluoride concentrations ([F]) and sought to determine the causes. Five subjects (27-33 years old) received a low-F diet during the 5 days of the study. Plasma samples and urine were collected every 3 h from 8 a.m. to 8 p.m. F, PTH, Ca and P were analyzed with the electrode, by chemiluminescence, AAS and colorimetry, respectively. A trend for the plasma [F] was found. The peak [F], 0.55 +/- 0.11 mu mol L(-1), occurred at 11 a.m. and the lowest [F], 0.50 +/- 0.06 mu mol L(-1) occurred between 5 and 8 p.m. Plasma [F] were positively correlated with urinary F excretion rates and with serum PTH levels, but not with the Ca or P levels. Serum PTH levels were positively correlated with urinary F excretion rates and negatively correlated with plasma Ca. The results suggest that the renal system seems to control the daily fluctuations in plasma [F]. (c) 2008 Elsevier B.V. All rights reserved.

Inverse relationship between markers of nitric oxide formation and plasma matrix metalloproteinase-9 levels in healthy volunteers

Background: Nitric oxide (NO) is a major regulator of cardiovascular homeostasis and has anti-atherogenic properties. Reduced NO formation is associated with endothelial dysfunction and with cardiovascular risk factors. Although NO downregulates the expression and activity of the pro-atherogenic enzyme matrix metalloproteinase-9 (MMP-9), no previous clinical study has examined whether endogenous NO formation is inversely associated with the circulating levels of pro-MMP-9, which are associated with cardiovascular events. We examined this hypothesis in 175 healthy male subjects who were non-smokers. Methods: To assess NO bioavailability, the plasma concentrations of nitrite, nitrate, and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Pro-MMP-9 and pro-MMP-2 levels were measured in plasma samples by gelatin zymography. Results: We found significant negative correlations between pro-MMP-9 levels and plasma nitrite (P=0.035, rs=-0.159), nitrate (P=0.040, rs=-0.158), and cGMP (P=0.011, rs=-0.189) concentrations. However, no significant correlations were found between pro-MMP-2 levels and the plasma concentrations of markers of NO bioavailability (all P>0.05). Conclusions: There is an inverse relationship between markers of NO formation and plasma MMP-9 levels. This finding may shed some light on the possible mechanisms involved in the increased cardiovascular risk of apparently healthy subjects with low NO bioavailability or high circulating levels of pro-MMP-9. (C) 2008 Elsevier B.V. All rights reserved.

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial. species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique. (c) 2007 Elsevier GmbH. All rights reserved.

Iron deficiency anaemia and blood lead concentrations in Brazilian children

This study investigated the relationship between iron deficiency/iron deficiency anaemia, assessed by several parameters, and blood lead concentration in children. This cross-sectional study involved 384 Brazilian children, aged 2-11 years, who lived near a lead-manipulating industry. Complete blood counts were obtained by an automated cell counter. Serum iron, total iron binding capacity (TIBC) and ferritin were determined respectively, by colorimetric, turbidimetric methods and chemiluminescence. Blood lead was measured by atomic absorption spectrophotometry. The impact of several parameters for assessment of iron status (haemoglobin, serum iron, TIBC, transferrin saturation, ferritin, red cell indices and red cell distribution width) and variables (gender, age, mother`s education, income, body mass index, iron intake, and distance from home to lead-manipulating industry) on blood lead concentration was determined by multiple linear regression. There were significant negative associations between blood lead and the distance from home to the lead-manipulating industry (P < 0.001), Hb (P = 0.019), and ferritin (P=0.023) (R(2)=0.14). Based on these results, further epidemiological studies are necessary to investigate the impact of interventions like iron supplementation or fortification, as an attempt to decrease blood lead in children. (C) 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

The impact of maternal cortisol concentrations on child arterial elasticity

Epidemiological studies suggest that glucocorticoid excess in the fetus may contribute to the pathophysiology of cardiovascular diseases in adulthood. However, the impact of maternal glucocorticoid on the cardiovascular system of the offspring has not been much explored in studies involving humans, especially in childhood. The objective of this study was to assess the influence of maternal cortisol concentrations on child arterial elasticity. One hundred and thirty pregnant women followed from 1997 to 2000, and respective children 5-7 years of age followed from 2004 to 2006 were included in the study. Maternal cortisol was determined in saliva by an enzyme immunoassay utilizing the mean concentration of nine samples of saliva. Arterial elasticity was assessed by the large artery elasticity index (LAEI; the capacitive elasticity of large arteries) by recording radial artery pulse wave, utilizing the equipment HDI/PulseWave CR-2000 Cardiovascular Profiling System (R). The nutritional status of the children was determined by the body mass index (BMI). Insulin concentration was assessed by chemiluminescence, and insulin resistance by the homeostasis model assessment. Blood glucose, total cholesterol and fractions (LDL-c and HDL-c) and triglyceride concentrations were determined by automated enzymatic methods. The association between maternal cortisol and child arterial elasticity was assessed by multivariate linear regression analysis. There was a statistically significant association between maternal cortisol and LAEI (P=0.02), controlling for birth weight, age, BMI and HDL-c of the children. This study suggests that exposure to higher glucocorticoid concentrations in the prenatal period is associated to lower arterial elasticity in childhood, an earlier cardiovascular risk marker.