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OBJECTIVE To evaluate the predictive value of resting heart rate (RHR) for cardiac and total mortality in a large population of patients referred for coronary angiography with an extended follow-up, stratified in four subpopulations according to gender and age (50th percentile corresponding to 67 years).We studied 3559 subjects (2603 males, age: 66 ± 11 years, mean ± SD), obtaining patient data from the Institute electronic databank which saves demographic, clinical, instrumental and follow-up data of patients admitted to our department.During a mean follow-up period of 35 ± 25 months, 296 (8%) patients died; there were 173 (5%) cardiac deaths. In female patients irrespective of age, RHR (≥ 76 bpm, 75th percentile) did not appear predictive for cardiac death. In females, RHR was predictive for overall mortality after multivariate adjustment only in those aged ≥ 67 years (hazard ratio (HR) 1.7, 95% confidence interval (CI) 1-2.8, p ≤ 0.05). In male patients aged67 years, RHR remained as an independent predictive factor for overall mortality at the multivariate analysis (HR 2.5, 95% CI 1.5-4.2, p0.001), and as an independent predictor for both cardiac mortality (HR 1.8, 95% CI 1.2-2.7, p0.01) and total mortality (HR 1.6, 95% CI 1.2-2.3, p0.01) in male patients over 67 years.The current study suggests that the prognostic importance of RHR may differ according to the patient's gender and age, suggesting significant differences in cardiovascular physiopathology between female and male patients.

Myocardial tissue perfusion is not currently quantified in the clinical setting. Thus the aim of this paper is to review the quantitative information on myocardial perfusion provided by contrast echocardiography. In a circulatory model-without the capillary network interposed between injection and sampling point of contrast-the transit time of microbubbles (source of the echo contrast effect) is inversely related to absolute flow, thus providing accurate quantitation. A similar situation is represented by blood flow inside a vessel or a cardiac cavity, where, if the prerequisites for quantitation are respected, it is possible to measure blood flow by contrast echocardiography. In the coronary circulation, the transit time of contrast microbubbles varies according to their interaction with coronary microcirculation, and to the characteristics of contrast agents as flow tracers. Echo contrast agents with small microbubbles have been injected into the coronary branches of experimental animals, under both coronary autoregulation and maximal coronary dilation, providing good estimates of coronary blood flow. The accuracy of these measurements might improve when new contrast agents, with characteristics closer to those of a flow tracer, are available. If a tracer is injected before a bifurcation, and provided it mixes adequately, the amount of tracer distributed to each branch is proportional to the corresponding blood flow. A similar situation is encountered when an echo contrast agent is injected into the aortic root or into the left main coronary artery. Here, the ratio between myocardial signal intensity in the different perfusion territories reflects the corresponding ratio of blood flows. The validity of this approach has been previously demonstrated in experimental animals and validated in patients with coronary stenoses. The injection of contrast agents into the coronary circulation at baseline and under coronary hyperaemia has the potential for measuring coronary blood flow reserve. However, what is still unclear is whether contrast echo changes reflect changes in coronary blood flow (i.e. flow reserve), coronary blood volume (i.e. coronary recruitment) or both, and also whether they influence the different types of contrast agent. Finally, myocardial contrast echocardiography can provide information on the spatial distribution of myocardial perfusion, i.e. the presence, site and extent of perfused myocardium. Thus, in models where myocardial perfusion may be either present or absent, contrast echo can provide an accurate estimate of perfusion abnormalities.

Objectives. This study sought to assess the value of dipyridamole echocardiography in predicting reinfarction in patients evaluated early after uncomplicated acute myocardial infarction. Background. The identification of future nonfatal reinfarction seems an elusive target for physiologic testing. However, a large sample population is needed to detect minor differences in phenomena with a low event rate. Methods. We assessed the value of dipyridamole echocardiography in predicting reinfarction in 1,080 patients (mean [±SD] age 56 ± 9 years; 926 men, 154 women) evaluated early (10 ± 5 days) after uncomplicated acute myocardial infarction and followed up for 14 ± 10 months. Results. Submaximal studies due to limiting side effects occurred in 14 patients (1.3%); these test results were included in the analysis. Results of dipyridamole echocardiography were positive in 475 patients (44%). During follow-up, there were 50 reinfarctions: 45 nonfatal, 5 fatal (followed by cardiac death ≤4 days after reinfarction). Reinfarction (either nonfatal or fatal) occurred in 30 patients with positive and 20 with negative results (6.3% vs. 3.3%, p Conclusions. Dipyridamole echocardiographic positivity identifies patients evaluated early after uncomplicated acute myocardial infarction at higher risk of reinfarction, especially fatal reinfarction.

Uric acid (UA) is a potent endogenous antioxidant. However, high concentrations of this molecule have been associated with cardiovascular disease (CVD) and renal dysfunction, involving mechanisms that include oxidative stress, inflammatory processes, and endothelial injury. Experimental and in vitro results suggest that this biomarker behaves like other antioxidants, which can shift from the physiological antioxidant action to a pro-oxidizing effect according to their level and to microenvironment conditions. However, data on patients (general population or CAD cohorts) are controversial, so the debate on the role of hyperuricemia as a causative factor for CVD is still ongoing. Increasing evidence indicates UA as more meaningful to assess CVD in women, even though this aspect needs deeper investigation. It will be important to identify thresholds responsible for UA “biological shift” from protective to harmful effects in different pathological conditions, and according to possible gender-related differences. In any case, UA is a low-tech and inexpensive biomarker, generally performed at patient’s hospitalization and, therefore, easily accessible information for clinicians. For these reasons, UA might represent a useful additive tool as much as a CV risk marker. Thus, in view of available evidence, progressive UA elevation with levels higher than 6 mg/dL could be considered an “alarm” for increased CV risk.

Vitamin D (25-OHD) physiological functions have been expanded beyond traditional bone health, increasing the importance of its estimation in Laboratory Medicine, which renders validation of available methods mandatory.We evaluated some preanalytical and analytical aspects of 25-OHD determination and the effects of potentially confounding clinical variables by using the DiaSorin "LIAISON 25-OH Vitamin D TOTAL".25-OHD samples were extremely stable, at least in the short term, without requiring special transport or storage. Precision intervals (CV%) were: within run (7-11%) and total precision (8-11.5%). Mean (SD) recovery was 96 (2)%. The assay was linear on dilution. Comparison with radioimmunoassay (RIA) yielded acceptable correlation (Inter-rater agreement/kappa coefficient=0.94) and clinical equivalence in the interval from 6 to 55 ng/mL. The assay was evaluated on a general population (N=476, age: 60±14 years, 65 males). The status of 25-OHD resulted inversely related to parathyroid hormone levels (r=-0.21, p0.001), and aging (r=-0.17, p0.001), but not to sex. Levels of 25-OHD were found to be sufficient (≥30 ng/mL) only in 54 samples (12%). Marked seasonal 25-OHD variations were observed in 13 subjects (p0.05). Moreover, a marked seasonal fluctuation was seen in samples collected during the period of February 2010-October 2011 (p≤0.01). Lower 25-OHD concentration was observed in subjects with diabetes (19±9 vs 14±7 ng/mL, p0.01) and hypertension (20±9 vs 17±9 ng/mL, p0.01). Moreover, 25-OHD inversely correlated with BMI (r=-0.25, p0.001). Conversely, no difference in 25-OHD levels was observed between subjects due to smoking habits and dyslipidemia. In multiple logistic regression models, aging is the only significant independent risk factor for low 25-OHD levels (Odds ratio, 95% confidence intervals: 3.1, 1.3-7.3; p≤0.01).Results confirm the LIAISON 25-OHD assay as a useful tool for 25-OHD estimation in the clinical practice. Lack of vitamin D is common among Italian adults, and appears associated with several cardiovascular risk factors.

AIMS To evaluate the prognostic significance of oxidative stress on the rate of major adverse cardiovascular events (MACEs: cardiac and all-cause death, nonfatal myocardial infarction, coronary revascularization-PTCA/CABG) in CAD. METHODS We studied 97 angiographically proven CAD patients (78 males, age: 67±11 years, mean± SD). Reactive oxygen metabolites and total antioxidant status, assessed by commercially assays (d-ROMs and OXY-Adsorbent Test; Diacron, Grosseto, GR, Italy), were used to calculate the oxidant/antioxidant balance. Patient data were collected from the Institute's electronic databank, which saves demographic, clinical, instrumental and follow-up data of all patients admitted to our department. RESULTS Kaplan-Meier survival estimates showed a significantly worst outcome in patients presenting with elevated oxidative stress levels (>75th percentile, p

Background: Enhanced oxidative stress has been associated with atherosclerosis and coronary artery disease (CAD). However, the predictive value of circulating oxidative stress biomarkers for cardiovascular events (CE) in patients with CAD has remained poorly understood. Aim: To assess the prognostic signifi cance of reactive oxygen metabolites, estimated as index of oxidative stress in serum samples by means of a commercial kit (ROMs, Diacron, Italy) on the rate of mortality and major adverse CE (MACE) in CAD. Methods: A study of 93 consecutive patients with angiographically documented CAD (75 males, age: 68 ± 10 years, mean ± SD) was made during a mean follow-up of 66 months until the occurrence of one of the following CE: cardiac and all cause death, non-fatal myocardial infarction and coronary revascularization [percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass grafting (CABG)]. Patient data were retrospectively collected from the Institute ’ s electronic databank that saves demographic, clinical, instrumental and follow-up data of all patients admitted to our department. Results: The Kaplan-Meier survival estimates showed a signifi cantly worst outcome in patients presenting elevated ROM level ( > 75th percentile, corresponding to 481 AU) (log rank = 11, 7.5, 5.1; p < 0.001, p < 0.01, p < 0.05 for cardiac and all cause death and MACEs, respectively). In a multivariate Cox regression model, elevated oxidative stress remained a signifi cant predictor of cardiac and all cause death [hazard ratio (HR) 3.9, 95 % confi dence interval, 95 % (CI) 1.4 ‐ 11.1, p = 0.01; HR = 2.6, 95 % CI 1.1 ‐ 6.2, p = 0.02) and MACE (HR = 1.8, 95 % CI 1.1 ‐ 3.1, p = 0.03)]. Conclusions: The estimation of ROMs may represent an additional prognostic tool in the assessment of CE in CAD patients.

Objective To assess oxidative stress status in coronary artery disease (CAD) patients according to gender. Design Case-controlled, observational, retrospective study. Setting Clinical and research center. Patient(s) A total of 55 postmenopausal women and 108 men (mean age: 66 ± 9 years), including 72 patients with angiographically proven CAD (CAD(+), 19 women) and a group of 91 age-matched controls (CAD(−), 36 women). Intervention(s) None. Main Outcome Measure(s) Oxidant/antioxidant balance as a global index (oxidative index) obtained using two commercial assays (d-ROMs and OXY Adsorbent Test, respectively) for estimation of levels of reactive oxygen metabolites and total antioxidant status. Result(s) There was a statistically significant difference in oxidative stress status between men and women who were CAD(−) (−0.424 ± 1.3 vs. 0.64 ± 1.1 arbitrary units, respectively), but the CAD(+) women had oxidative stress levels almost three times those of the CAD(+) men (2.45 ± 2.5 vs. 0.9 ± 1.6 arbitrary units, respectively). After adjustment in the multivariate model, age and oxidative stress status in women and diabetes and age in men remained as statistically significant predictors of positive CAD findings. Conclusion(s) Oxidative stress status was a powerful predictor of CAD in women. This result may have important implications for the differences between sexes in CAD physiopathology.

BACKGROUND Magnitude and major causes of oxidative stress may be different between sexes, although limitedly addressed in clinical studies with controversial results. The present study aimed to determine whether any gender-related difference exists concerning oxidative stress in a population of 332 subjects of both sexes, in a wide age range, with and without cigarette smoking habit. METHODS The Oxidative-INDEX was calculated after evaluation of serum hydroperoxides (ROMs) and total antioxidant capacity (OXY) by means of commercial kits (d-ROMs and Oxy-adsorbent Tests, Diacron, Italy) subtracting the OXY standardized variable from the ROMs standardized variable. RESULTS The Oxidative-INDEX resulted higher in women with respect to men (p

Background:Carcinoembryonic antigen (CEA), one of the most widely used tumor markers, has been recently associated with carotid atherosclerosis. The aim of our study was to evaluate whether CEA concentrations have a role in coronary artery disease (CAD).Methods:Serum CEA concentrations were evaluated in 89 patients, including 50 patients with acute coronary syndrome (ACS) (Group I, 44 with acute myocardial infarction, six with unstable angina, 38 males, 65±2 years) and 39 patients with stable CAD (Group II, 33 males, 66±3 years). In addition, 33 subjects (16 males, 62±2 years) were also included as a control group (Group III).Results:ACS was significantly associated with increased mean CEA concentrations (3.1±0.3 vs. 1.75±0.1 and 1.7± 0.2 ng/mL in Groups I, II and III, respectively, pConclusions:CEA might represent a potential new candidate biomarker for the prediction of risk associated with ACS.Clin Chem Lab Med 2010;48:1339–43.

During the last few years, reliable and simple tests have been proposed to estimate oxidative stress in vivo. Many of them can be easily adapted to automated analyzers, permitting the simultaneous processing of a large number of samples in a greatly reduced time, avoiding manual sample and reagent handling, and reducing variability sources. In this chapter, description of protocols for the estimation of reactive oxygen metabolites and the antioxidant capacity (respectively the d-ROMs and OXY Adsorbent Test, Diacron, Grosseto, Italy) by using the clinical chemistry analyzer SYNCHRON, CX 9 PRO (Beckman Coulter, Brea, CA, USA) is reported as an example of such an automated procedure that can be applied in the clinical setting. Furthermore, a calculation to compute a global oxidative stress index (Oxidative-INDEX), reflecting both oxidative and antioxidant counterparts, and, therefore, a potentially more powerful parameter, is also described.

Oxidative stress plays a key role in the pathogenesis and development of atherosclerosis.To evaluate the relationship between a novel oxidative stress index (reflecting both oxidative and anti-oxidant counterparts) with traditional cardiovascular risk factors and C-reactive protein (CRP) in coronary artery disease (CAD).100 angiographically proven CAD and 70 control subjects (mean age: 65+/-10 years, 110 males), underwent a global cardiovascular risk assessment and serum CRP and oxidative stress estimation. The Oxidative-INDEX was calculated after automated evaluation of serum hydroperoxides and total anti-oxidant capacity (D-ROM and OXY-adsorbent Test, Diacron, Italy) subtracting the OXY standardized variable from the ROM standardized variable.The Oxidative-INDEX was higher in CAD with respect to control subjects (p0.001). A stepwise elevation in the Oxidative-INDEX levels was found depending on the number of affected vessels (p0.001). Oxidative stress was elevated according to the presence of diabetes (p0.001), smoking habit (p0.01), and hypercholesterolemia (p0.05). Oxidative-INDEX significantly correlated with aging (por = 0.05) and CRP (p0.001). The Oxidative-INDEX increased with the number of cardiovascular risk factors (p0.001). After adjustment for traditional CV risk factors, the multivariate logistic regression analysis indicated the Oxidative-INDEX concentration as an independent factor for CAD (odds ratio=1.4, confidence intervals=1.1-1.9, p0.05).Oxidative stress represents a shared molecular pathway in atherosclerotic-related conditions, and its estimation by the automated Oxidative-INDEX could represent a valuable tool and a promising target in the prevention, diagnosis and treatment of CAD in the clinical setting.

During the last few years, reliable and simple tests have been proposed to estimate oxidative stress in vivo. Many of them can be easily adapted to automated analyzers, permitting the simultaneous processing of a large number of samples in a greatly reduced time, avoiding manual sample and reagent handling, and reducing variability sources. In this chapter, description of protocols for the estimation of reactive oxygen metabolites and the antioxidant capacity (respectively the d-ROMs and OXY Adsorbent Test, Diacron, Grosseto, Italy) by using the clinical chemistry analyzer SYNCHRON, CX 9 PRO (Beckman Coulter, Brea, CA, USA) is reported as an example of such an automated procedure that can be applied in the clinical setting. Furthermore, a calculation to compute a global oxidative stress index (Oxidative-INDEX), reflecting both oxidative and antioxidant counterparts, and, therefore, a potentially more powerful parameter, is also described.

Adenosine modulates the immune system and inhibits inflammation via reduction of cytokine biosynthesis and neutrophil functions. Drugs able to prevent adenosine catabolism could represent an innovative strategy to treat inflammatory bowel disorders. In this study, the effects of 4-amino-2-(2-hydroxy-1-decyl)pyrazole[3,4-d]pyrimidine (APP; novel adenosine deaminase inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; standard adenosine deaminase inhibitor), and dexamethasone were tested in rats with colitis induced by 2,4-dinitrobenzenesulfonic acid (DNBS). DNBS-treated animals received APP (5, 15, or 45 micromol/kg), EHNA (10, 30, or 90 micromol/kg), or dexamethasone (0.25 micromol/kg) i.p. for 7 days starting 1 day before colitis induction. DNBS caused bowel inflammation associated with decrease in food intake and body weight. Animals treated with APP or EHNA, but not dexamethasone, displayed greater food intake and weight gain than inflamed rats. Colitis induced increment in spleen weight, which was counteracted by all test drugs. DNBS administration was followed by macroscopic and microscopic inflammatory colonic alterations, which were ameliorated by APP, EHNA, or dexamethasone. In DNBS-treated rats, colonic myeloperoxidase, malondialdehyde, and tumor necrosis factor (TNF)-alpha levels as well as plasma TNF-alpha and interleukin-6 were increased. All test drugs lowered these phlogistic indexes. Inflamed colonic tissues displayed an increment of inducible nitric-oxide synthase mRNA, which was unaffected by APP or EHNA, but reduced by dexamethasone. Cyclooxygenase-2 expression was unaffected by DNBS or test drugs. These findings indicate that 1) inhibition of adenosine deaminase results in a significant attenuation of intestinal inflammation and 2) the novel compound APP is more effective than EHNA in reducing systemic and intestinal inflammatory alterations.

Reactive oxygen species (ROS) play a major role in the pathogenesis of different chronic and degenerative diseases, including atherosclerosis. However, the lack of feasible and reliable methods limits the spread of oxidative stress estimation for routine application in clinical chemistry laboratories. We have recently evaluated the analytical characteristics of an automated test for the measurement of hydroperoxides (HPs) and its performance in determining oxidative stress levels in a general population. In this study we applied this method for the evaluation of oxidative stress in a cohort of patients with coronary artery disease (CAD).A total of 69 patients with angiographically verified CAD and 34 age- and sex-matched control subjects were enrolled in the study.HPs were higher in patients with CAD (p0.01), significantly increasing with disease severity (p0.01). HPs were also higher in subjects with diabetes, dyslipidemia or C-reactive protein1.5mg/L. A significant positive correlation was observed between glucose and HP levels. In a multivariate model, diabetes (odds ratio OR=3.5, 95% CI 1.2-10, p0.05) and CAD (OR=5.7, CI 1.1-28.5, p0.05) were independent determinants for the 75th HP percentile.The results obtained with this method largely reproduce those found using other oxidative stress biomarkers, but the method is faster, easy to perform and does not require skilled operators or complex instrumentation, and thus is a reliable procedure that might represent a feasible tool for oxidative stress estimation in the cardiovascular setting.

BACKGROUND The d-ROMs test for the evaluation of serum hydroperoxides (HP) is simple, reliable, and cheap. Furthermore, it can easily be adapted to automated analyzers. Changing from the manual to an automated procedure allows the simultaneous processing of a large number of samples in a greatly reduced time, avoiding manual handling of samples and reagents and reducing variability sources. METHODS This study was performed to adjust the manual procedure to a routine automated method in the clinical laboratory. We carried out the d-ROMs test in sera from 90 subjects of both sexes (34 men and 56 women) with age ranging from 20 to 80 years (mean 51+/-14 years). All subjects were free from acute or chronic inflammatory disease, immunological disease and history or evidence of malignancy. Subjects were not on vitamin and/or antioxidant therapies. RESULTS The detection limit of the assay was 40 AU. Linearity was observed up to 475 AU. The recovery ranged between 97% and 105%. Within- and between-run imprecision was

Objective To evaluate whether gender-related differences exist concerning oxidative stress levels in aged patients with coronary artery disease (CAD). Design Case-control. Setting Clinical and research center. Patient(s) Elderly subjects of both genders with or without CAD. Intervention(s) None. Main Outcome Measure(s) Serum hydroperoxides (HP) as index of oxidative stress levels. Result(s) The HP levels were comparable in aged control subjects of both genders (376 ± 20 arbitrary units [AU] in women, 333 ± 19 AU in men) but significantly increased in CAD (456 ± 15 AU) compared with all control subjects (357 ± 14 AU). Moreover, among CAD patients, the HP levels were higher in women than in men (536 ± 33 AU and 428 ± 15 AU, respectively). Multivariate analysis, in which CAD represented the dependent variable, indicated that dyslipidemia was independently associated with CAD in men (odds ratio [OR] 5.8), whereas HP >50th percentile represented the only strong independent risk factor for CAD in elderly women (OR 8.4). Conclusion(s) Differences in oxidative stress levels between elderly males and females might provide a biochemical basis for the epidemiologic differences in CAD, which might help to open new opportunities in the management of patients with cardiovascular disease from a gender point of view.

Background The administration of selective cyclooxygenase-2 inhibitors before surgery is regarded as an innovative option to manage postoperative pain. This study was designed to (1) examine the efficacy of preoperative cyclooxygenase-2 blockade on postoperative oral pain and (2) compare pain intensity with prostaglandin E2 (PGE2) production and cyclooxygenase isoform (cyclooxygenase-1, cyclooxygenase-2) messenger RNA (mRNA) expression at the surgical site during the postoperative period. Methods Sixty patients with impacted lower third molars were randomly allocated to three single-dose treatment groups--placebo, 50 mg rofecoxib, or 550 mg naproxen--1 h before extraction. Pain intensity was evaluated with categorical and visual analog scales every 30 min from 90 to 240 min after surgery. At these times, PGE2 production in the alveolar socket was also evaluated. Cyclooxygenase-1 and cyclooxygenase-2 mRNA expression was examined by reverse-transcription polymerase chain reaction in gingival specimens collected during tooth removal and 240 min after surgery. Results Pain intensity and PGE2 production in the placebo group increased throughout the observation period. Naproxen prevented pain and decreased PGE2 release at all time points. Rofecoxib reduced PGE2 production versus placebo from 150 min onward, while inducing analgesia through the whole observation period. mRNA assay in gingival specimens collected at tooth extraction revealed cyclooxygenase-1 expression, whereas cyclooxygenase 2 was undetectable. At the end of observation, cyclooxygenase-1 mRNA expression was unchanged, whereas cyclooxygenase-2 mRNA was significantly induced. Conclusions This study indicates that preoperative administration of a selective cyclooxygenase-2 inhibitor ensures effective control of postoperative pain. It is suggested that the selective blockade of inducible cyclooxygenase 2 at the surgical site does not entirely account for the analgesic action occurring in the postoperative period.

Although hormone replacement therapy (HRT) may offer considerable benefits for menopausal women, the potential cancer risk may limit its use. This work aimed at assessing whether HRT is able to induce DNA damage in postmenopausal women monitored by the micronucleus (MN) test, which provides a reliable biomarker of genotoxicity and cancer risk assessment. A group of 16 healthy women (non-smokers) in spontaneous menopause were given oral estradiol (2 mg oral micronized 17-beta estradiol daily) for 1 month, followed by a 30-day wash-out period and a transdermal treatment with 17-beta estradiol (1.5 mg gel daily) during 1 month. Oral intake of dihydrogesterone (10 mg/day for 12 days/month) was cyclically combined with oral or transdermal estradiol during the next 9 months. Venous blood samples were collected before the treatment, and after 1, 3, 6 and 12 months of therapy. Slides were scored blind and MN frequency was evaluated as number of micronuclei per 1000 binucleated cells. The baseline plasma levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) were simultaneously measured. The means of MN frequency were 18.2+/-1.6, 18.6+/-2.1, 14.8+/-1.5, 15.9+/-1.0 and 17.7+/-1.3 for samples collected before and at 1, 3, 6 and 12 months, respectively. The MN frequencies at every sampling time did not statistically differ from the basal values. In addition, no statistically significant associations between MN values and hormone levels of E2 and FSH were observed throughout the entire study. This study shows the absence of any significant increase of MN frequencies in women undergoing oral and/or transdermal HRT, sequentially monitored for up to 12 months of therapy.

Objective. It is well known that free radicals contribute to endothelial dysfunction and are involved in the pathogenesis and development of cardiovascular diseases, such as atherosclerosis. The aim of this study was to provide evidence for enhanced oxidative stress in coronary artery disease (CAD). Methods. Plasma levels of 8-isoprostane (8-epiPGF2α), marker of lipid peroxidation, were measured in 68 subjects (age: 60 ± 2 years, mean ± SEM). Subjects included 30 healthy control subjects and 38 patients with angiographically proven CAD. In addition, the total antioxidant power (PAO) was evaluated in a subgroup (40 subjects, 12 healthy and 28 CAD). Results. Levels of 8-epiPGF2α increased with the number of affected vessels (one- and multi-vessel disease versus control subjects, P

The inflammation hypothesis of atherosclerosis postulates that the crucial events involved in the initiation and progression of the lesion are represented mainly by inflammatory and fibroproliferative processes triggered by cytokines and growth factors.1 However, one of the most interesting developments in recent years has been the idea that infective agents may induce a pro-inflammatory effect and have a crucial role in atherothrombosis.1 In addition, at the beginning of the 1970s, the monoclonal hypothesis was first proposed, suggesting a potential role for viral infection in the atherosclerotic process.2 In particular, this theory proposed that a mutation or a viral agent may represent events able to transform a single smooth muscle cell into the progenitor of a proliferative clone, introducing the concept that the plaque may be considered a monoclonal benign neoplasm. Since then, many additional results have shown an association between infectious agents and the atherosclerotic process, although other studies have produced contrary results.1 Thus, the role of infection in the pathogenesis of this disease remains controversial. In particular, very few data on the relation …

Several methods to assess the total antioxidant capacity (TAC) are available. However, the final value of measured TAC in the sample depends on the procedure used in every specific assay. This makes crucial the comparison of different analytical methods. The aim of our study was to evaluate analytical characteristics and laboratory reliability of two different assays: the ferric-reducing ability (FRAP) assay and a new spectrophotometric test (OXY-adsorbent test, Diacron, Italy).Unselected outpatients referred to the Institute of Clinical Physiology were studied (n=187, 58 females, 129 males, mean age: 65±13 years). All blood samples were maintained on ice, centrifuged within 15 minutes after blood collection and then stored at −80°C until performance of assay procedures.OXY assay: The lower limit of sensitivity was 6 μmol HClO/ml. The assay was found to be linear up to 440 μmol HClO/ml (r=−0.99, pFRAP assay: The lower limit of sensitivity was 15 μmol/l. Linearity was observed up to 1000 μmol/l (r=−0.998, pFRAP vs. OXY: A significant direct relationship was observed when comparing FRAP with OXY levels in the whole population (r=0.22, pNeither of the methods are expensive and they are speedy and simple to perform. Values are reproducible and linearly correlated to the concentration of antioxidants present in the samples. For this reason, these methods may be considered practicable indicators of total antioxidant capacity, for routinely potential use in every laboratory and useful in all the studies concerning the evaluation of oxidative stress.

Background Cardiopulmonary bypass induces a systemic inflammatory response that may contribute to clinical morbidity. Gaseous nitric oxide at relatively low concentrations may elicit peripheral anti-inflammatory effects in addition to a reduction of pulmonary resistances. We examined the effects of 20 ppm of inhaled nitric oxide administered for 8 hours during and after cardiopulmonary bypass. Methods and results Twenty-nine consecutive patients undergoing aortic valve replacement combined with aortocoronary bypass were randomly allocated to either 20 ppm of inhaled nitric oxide (n = 14) or no additional inhalatory treatment (n = 15). Blood samples for total creatine kinase, creatine kinase MB fraction, and troponin I measurements were collected at 4, 12, 24, and 48 hours postsurgery. In addition, we collected perioperative blood samples for measurements of circulating nitric oxide by-products and brain natriuretic peptide. Soluble P-selectin was analyzed in blood samples withdrawn from the coronary sinus before and after aortic clamping. The area under the curve of creatine kinase MB fraction ( P = .03), total creatine kinase ( P = .04), and troponin I ( P = .04) levels were significantly decreased in the nitric oxide–treated patients. Moreover, in the same group we observed blunted P-selectin and brain natriuretic peptide release ( P = .01 and P = .02, respectively). Nitric oxide inhalation consistently enhanced nitric oxide metabolite levels ( P = .01). Conclusions Nitric oxide, when administered as a gas at low concentration, is able to blunt the release of markers of myocardial injury and to antagonize the left ventricular subclinical dysfunction during and immediately after cardiopulmonary bypass. The organ protection could be mediated, at least in part, by its anti-inflammatory properties.

The scientific assessment of diagnostic tests should not be based upon a small series of results published by the best academic institutions, but ought to require large scale, multicentre validation founded on grass roots institutions with real doctors, real patients and real problems. To this purpose, an international network of stress echo laboratories has been organized, and within a few years has collected data from thousands of studies using pharmacological stress echocardiography, performed with either dipyridamole (EPIC : Echo Persantine International Cooperative Study) or dobutamine (EDIC : Echo Dobutamine International Cooperative Study) stresses. In a widely deregulated field, all network laboratories have agreed : to pass a quality control examination on stress echo reading before entering the study ; to adopt an identical drug infusion protocol ; to code similarly the LV segments ; and to adopt a common scoring system for wall motion analysis. A minimum amount of historical, clinical, and-when available-stress electrocardiographic, angiographic and follow-up data have been collected on each patient, and disseminated in the various centres, facilitating the creation of an international stress echo language that will help, not only the production of high quality scientific data, but also the build up of a common stress echo lab, with a standardized way of making the studies, unifying the methods and terminology, and archiving data. To date, we have 50 echo laboratories from 15 nations across four continents (Europe, America, Asia and Africa) actively involved in this project. These data will ultimately fill the gap between the academic theory of journals and the pragmatic experience of daily life in a busy echocardiographic laboratory.

This study investigated the mechanisms involved in the protective actions exerted by lansoprazole against experimental gastric injury. Following the intraluminal injection of ethanol-HCl, the histomorphometric analysis of rat gastric sections demonstrated a pattern of mucosal lesions associated with a significant increase in the mucosal contents of malondialdehyde and 8-iso-prostaglandin F(2alpha) (indices of lipid peroxidation), as well as a decrease in the levels of mucosal sulfhydryl compounds, assayed as reduced glutathione (GSH). Pretreatment with lansoprazole 90 micromol/kg, given intraduodenally as single dose or once daily by intragastric route for 8 days, significantly prevented ethanol-HCl-induced gastric damage. The concomitant changes in the mucosal levels of malondialdehyde, 8-iso-prostaglandin F(2alpha) and GSH elicited by ethanol-HCl were also counteracted by lansoprazole. In separate experiments, performed on animals undergoing 2-h pylorus ligation, lansoprazole did not enhance the concentration of prostaglandin E(2), bicyclo-prostaglandin E(2), or nitric oxide (NO) metabolites into gastric juice. Western blot analysis revealed the expression of both type 1 and 2 cyclooxygenase (COX) isoforms in the gastric mucosa of pylorus-ligated rats. These expression patterns were not significantly modified by single-dose or repeated treatment with lansoprazole. Lansoprazole also exhibited direct antioxidant properties by reducing 8-iso-prostaglandin F(2alpha) generation in an in vitro system where human native low-density lipoproteins were subjected to oxidation upon exposure to CuSO(4). The present results suggest that the protective effects of lansoprazole can be ascribed to a reduction of gastric oxidative injury, resulting in an increased bioavailability of mucosal sulfhydryl compounds. It is also proposed that lansoprazole does not exert modulator effects on the gastric expression of COX isoforms as well as on the activity of NO pathways.

BACKGROUND It is well known that free radicals contribute to endothelial dysfunction and are involved in ageing and in the pathogenesis and development of many cardiovascular diseases, such as atherosclerosis. Measurement of F(2)-isoprostanes has emerged as probably the most reliable approach to assess oxidative stress status in vivo. In particular, 8-isoprostane (8-epiPGF(2alpha)) has been indicated as a marker of antioxidant deficiency and oxidative stress of potential relevance to assess human vascular diseases. DESIGN To provide evidence for enhanced oxidative stress in coronary artery disease (CAD). METHODS Plasma levels of 8-epiPGF(2alpha) (EIA, Cayman Chemicals, Ann Arbor, Michigan, USA) were measured in 51 patients (19 females, 32 males, age: 58.7+/-1.6 years, mean+/-SEM). Subjects included 13 healthy control subjects (group I), and 38 patients underwent coronary angiography; 11 patients without coronary artery atherosclerotic lesions (group II), and 27 with angiographically proven CAD (group III). RESULTS Plasma levels of 8-epiPGF(2alpha) were 123.2+/-9.5, 314.6+/-40 and 389.6+/-36.2 pg/ml in groups I, II and III respectively (P

Cardiovascular diseases and cancer are the main causes of death in developed countries. Mortality trends for these diseases suggest that they share common pathogenetic mechanisms. Glutathione S-transferase (GST) is a family of enzymes that detoxify reactive electrophiles, particularly present in tobacco smoke. Glutathione S-transferase null M1 and T1 (GSTM1 and GSTT1) genotypes have often been associated with increased risk of developing cancer. Our hypothesis was that the polymorphic GSTM1 and GSTT1 genes modulate the risk of smoking-coronary artery disease (CAD). We evaluated the distribution of GST genotypes in 430 angiographically defined patients (308 CAD and 122 non-CAD). The frequencies of GST null genotypes did not differ significantly between patients with CAD and without CAD. However, smokers with GSTM1 and GSTT1 null genotypes had a significantly higher risk of CAD than never-smokers with these genotypes present (OR 2.2 and 3.4 for smokers with null GSTM1 and GSTT1 genes, respectively). There was also evidence of multiple interaction between GSTM1 and GSTT1 deleted genotypes and smoking. In nonsmokers carrying both null genotypes the risk of CAD was 0.66. In smokers with both present genotypes the OR was 1.5 and was significantly increased in smokers with concurrent lack for GSTM1 and GSTT1 genes (OR=4.0). Moreover, smokers lacking GST genes had both more stenosed vessels and a higher Duke score than smokers expressing the genes. We also examined the levels of DNA damage in 66 men patients using the micronucleus test, a sensitive assay for evaluating chromosome damage. Micronucleus levels were higher in smokers with null genes than in smokers with present genes. These observations suggest that GST-null genotypes strengthen the effect of smoking on CAD risk by modulating the detoxification of genotoxic atherogens.

In the present study we assessed the effect of physical training on Laser Doppler skin flux (LDF) and nitric oxide (NO) release, before and after 3 min of brachial artery occlusion. To this end we performed laser Doppler measurements and the venous plasma assay of nitrite/nitrate (NOx) on 10 sedentary healthy subjects and 10 endurance athletes. The sedentary control subjects had lower basal and post reperfusion levels of NOx as compared to athletes (mean +/- SE: 27.8 +/- 3.5 vs. 33.2 +/- 3.4, 48.6 +/- 7.9 vs. 60.1 +/- 10.1 micromol/L; p < 0.05). LDF at baseline was not significantly different in the two groups (157.5 +/- 7.9 and 176.64 +/- 26.7 PU for sedentary subjects and athletes, respectively) while post ischemic LDF was significantly lower in nonathletic subjects than in athletes (209.9 +/- 13 and 343.8 +/- 21.3 PU, p < 0.001). In both groups the hyperaemic stimulus significantly increased LDF and NOx levels (p < 0.01 and p < 0.05, respectively). The flow reserve, estimated as peak/basal LDF, was significantly lower in control subjects than in athletes (1.34 +/- 0.2 and 2.32 +/- 0.9, respectively, p < 0.01). In athletes, as opposed to sedentary subjects, a direct correlation was found between plasma NOx concentration and LDF both in basal conditions (r = 0.92; p < 0.001), and during hyperaemia (r = 0.84; p < 0.01). In conclusion, compared to sedentary subjects, athletes had an enhanced nitric oxide release. Hyperaemia increased LDF and nitric oxide levels both in sedentary subjects and in athletes.

A common polymorphism at codon 72 (Arg72Pro) of the p53 gene, a gene which codes for a tumor-suppressor protein with both antiproliferative and pro-apoptotic actions, has recently been reported to be a risk factor for coronary luminal narrowing after angioplasty. However, the association of the polymorphism with coronary artery disease (CAD) risk has not been studied. We evaluated the distribution of the Arg72Pro genotype in 250 patients, 180 with angiographically documented CAD and 70 with normal coronary angiography, by using polymerase chain reaction amplification of patient DNA followed by restriction enzyme digestion. We also examined the association between the Arg72Pro genotype and chromosome damage in 82 male patients (60 CAD and 22 no-CAD) by the micronucleus (MN) test in human lymphocytes, a sensitive assay for chromosome breakage and aneuploidy. The frequencies of Pro/Pro, Pro/Arg, and Arg/Arg genotypes in CAD patients were not significantly different from those who were CAD-free (χ2 = 0.20, P = 0.90) and not significantly associated with the extent and severity of CAD. A significant increase in MN frequency was observed in relation to smoking status (8.4 ± 0.6, 11.9 ± 1 and 12.0 ± 1.6, for non smokers, ex-smokers and smokers, respectively; P = 0.02). Moreover, diabetic patients showed higher levels of MN than normal patients (13.5 ± 1.4 vs. 9.6 ± 0.5, P = 0.0025). Also, MN frequency was significantly higher in CAD patients than in no-CAD patients (11.2 ± 0.7 vs. 8.0 ± 0.9, P = 0.02) and increased with the number of affected vessels (9.3 ± 0.1, 12.2 ± 1.5 and 12.5 ± 1.3 for one-, two-, and three-vessel disease, respectively; P = 0.02). However, there were no associations between MN frequency and the Arg72Pro polymorphism. Although there appears to be an association between CAD and MN frequency, our results indicate that the Arg72Pro polymorphism does not have a significant impact on CAD or MN frequencies. Environ. Mol. Mutagen. 40:110–115, 2002. © 2002 Wiley-Liss, Inc.

The aim of this study was to evaluate the analytical performance and clinical usefulness of spectrophotometric assays for the measurement of the plasma levels of nitrite plus nitrate (NOx), and malondialdehyde (MDA), as an index of nitric oxide release and lipid peroxidation, respectively. We studied 30 healthy sedentary volunteers, 12 endurance athletes and 12 regular heavy smokers. The lower limit of quantification for plasma NOx concentration was 1 µmol/l, and linearity was observed from 1 to 40 µmol/l of NOx concentration. Variation in replicate samples within or between days was always below 5%. NOx levels were significantly higher in athletes compared to both control subjects and smokers (p

The purpose of this study was to detect myocardial perfusion defects as a result of coronary occlusion and myocardial reperfusion after thrombolysis with intravenous (i.v.) administration of the echo contrast agent BR1 (Bracco Research, Switzerland), which consists of microbubbles (median diameter 2.5 microm) containing sulfur exafluoride in a phospholipidic shell. To generate a coronary thrombosis, a copper coil was advanced into the left circumflex coronary artery in eight anesthetized dogs with opened chest cavities. Coronary occlusion occurred 18 +/- 10 minutes after the insertion of the coil and was documented both by an electromagnetic flow meter (as zero blood flow) and by radiolabeled microspheres (as myocardial perfusion defect). After 2 hours of occlusion, streptokinase was infused i.v.; reperfusion was documented by both the flow-meter and microspheres. Left ventricular cavity enhancement was apparent after all contrast injections. Peak cavity intensity did not increase with dose and was not affected by signal processing (suggesting signal saturation), whereas the duration of contrast effect significantly increased with the dose (from 26 +/- 16 to 147 +/- 74 seconds). Myocardial contrast intensity also increased after contrast (from 15 +/- 12 to 21 +/- 18 gray level/pixel, p0.001). Contrast echo detected myocardial perfusion defects (corresponding to 17% +/- 11% of LV cross-sectional area) in all the injections performed during coronary occlusion and detected myocardial reperfusion with a sensitivity of 50% versus microspheres. The extent of perfusion defects by contrast echo showed a good correlation with microspheres (r = 0.73). Myocardial reperfusion was not detected by changes in heart rate, aortic pressure, pulmonary arterial pressure, cardiac output, left ventricular fractional area change, or wall-motion score index. Hemodynamic parameters were not affected by contrast injections. Thus, the i.v. administration of BR1 allows us to accurately detect myocardial perfusion defects during coronary occlusion and, to a lesser extent, myocardial reperfusion after thrombolysis.

The aim of this study was to evaluate whether hydroperoxide levels, an index of oxidative stress, predict cardiac and total death in patients with cardiovascular disease. Serum hydroperoxide levels were measured in 157 consecutive inpatients, followed during a mean follow-up of 20+/-0.3 months. Elevated oxidative stress resulted in an independent predictors for cardiac death, suggesting that hydroperoxide evaluation could provide an adjunctive estimate in the evaluation of prognosis in the cardiovascular clinical setting.

F. Bamonti1, L. Vigna2, C. Novembrino1, R. De Giuseppe1, F. De Liso1, C. Vassalle3. 1Dip. Scienze Mediche, Universita degli Studi di Milano, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Fondazione IRCCS, Milano, Italy; 2Dip Medicina Preventiva Clinica e del Lavoro, U.O. Medicina del Lavoro I, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Fondazione IRCCS, Milano, Italy; 3Fondazione Toscana G. Monasterio, Istituto Fisiologia Clinica, CNR, Pisa, Italy E-mail: fabrizia.bamonti@unimi.it

losses of 0.6 to 1.2 g/kg per day have been reported. There is a marked reduction in key micronutrient levels in fevers (vitamin B2), tuberculosis (vitamins A, D, C and E, zinc and iron) and cancer (vitamin C, B2, B6, B12 and D, magnesium and zinc). Almost any nutritional deficiency can further compromise the immune functionality, impacting health outcome, quality of life and recovery from illness. It is therefore, essential to replace the nutritional losses sustained during illness as well as provide additional allowances for convalescence. Increased protein requirements of up to 2 g/day and micronutrient intakes to at least the recommended allowances or higher are indicated. Studies report positive effects of optimal nutritional support with significant increases in body weight, total lean mass and grip strength in TB subjects. Cancer subjects reported improved nutritional status, body weight, un-interrupted course of radiation (in case of subjects undergoing radiotherapy), reduced postoperative complications and improved quality of life as a result of nutrition intervention. Nutritional support should therefore be an integral part of the comprehensive care to optimise treatment for illness like fevers, TB and cancer.

Elite athletes show a high prevalence of symptoms and signs of asthma, but no study has assessed the acute effects of endurance exercise on airway cells in nonasthmatic athletes. We measured exhaled nitric oxide (NO) and collected samples of induced sputum after 3% NaCl aerosol administration for 20 min in nonasthmatic middle-aged amateur runners after the Fourth Palermo International Marathon and 6–9 wk later (habitual training period) at baseline. After the marathon, exhaled NO ( n = 9 subjects) was higher [27 ± 9 parts/billion (ppb)] than at baseline (12 ± 4 ppb; P < 0.0005). Polymorphonuclear neutrophil (PMN) counts in induced sputum were much higher in runners (91.2 ± 3.6% of total cells postmarathon and 78.7 ± 9.1% at baseline) than in sedentary control subjects (9.9 ± 5.9%; P < 0.001). Expression of L-selectin and CD11b/CD18 in sputum PMNs was lower after the race than at baseline and inversely related to the amount of exhaled NO ( r = −0.66 and −0.69, respectively; P < 0.05). Our data indicate that sputum PMNs are increased in nonasthmatic runners both after a marathon and at baseline and suggest that NO may modulate exercise-associated inflammatory airway changes.

AIM: This study investigated the mechanisms of protection afforded by the proton pump inhibitor lansoprazole against gastric injury induced by different non-steroidal anti-inflammatory drugs (NSAIDs) in rats. METHODS: Male Sprague-Dawley rats were orally treated with indomethacin (100 µmol/kg), diclofenac (60 µmol/kg), piroxicam (150 µmol/kg) or ketoprofen (150 µmol/kg). Thirty minutes before NSAIDs, animals were orally treated with lansoprazole 18 or 90 µmol/kg. Four hours after the end of treatments, the following parameters were assessed: gastric mucosal PGE2, malondialdehyde (MDA), myeloperoxidase (MPO) or non-proteic sulfhydryl compounds (GSH) levels; reverse transcription-polymerase chain reaction (RT-PCR) of mucosal COX-2 mRNA; gastric acid secretion in pylorus-ligated animals; in vitro effects of lansoprazole (1-300 µmol/L) on the oxidation of low density lipoproteins (LDLs) induced by copper sulphate. RESULTS: All NSAIDs elicited mucosal necrotic lesions which were associated with neutrophil infiltration and reduction of PGE2 levels. Increments of MPO and MDA contents, as well as a decrease in GSH levels were detected in the gastric mucosa of indomethacin- or piroxicam-treated animals. Indomethacin enhanced mucosal cyclooxygenase-2 expression, while not affecting cyclooxygenase-1. At the oral dose of 18 µmol/kg lansoprazole partly counteracted diclofenac-induced mucosal damage, whereas at 90 µmol/kg it markedly prevented injuries evoked by all test NSAIDs. Lansoprazole at 90 µmol/kg reversed also the effects of NSAIDs on MPO, MDA and GSH mucosal contents, without interfering with the decrease in PGE2 levels or indomethacin-induced cyclooxygenase-2 expression. However, both lansoprazole doses markedly inhibited acid secretion in pylorus-ligated rats. Lansoprazole concentration-dependently reduced the oxidation of LDLs in vitro. CONCLUSION: These results suggest that, besides the inhibition of acid secretion, lansoprazole protection against NSAID-induced gastric damage depends on a reduction in mucosal oxidative injury, which is also responsible for an increment of sulfhydryl radical bioavailability. It is also suggested that lansoprazole does not influence the down-regulation of gastric prostaglandin production associated with NSAID treatment.