Viviana Cavalca

Age- and gender-related oxidative status determined in healthy subjects by means of OXY-SCORE, a potential new comprehensive index

Abstract Oxidative stress has been related to various diseases, gender and ageing, and has been measured by various markers. The authors developed a procedure to compute a global oxidative stress index (OXY-SCORE), reflecting both oxidative and antioxidant markers in healthy subjects. Its performance was tested in relation to age and gender and in coronary artery disease (CAD) patients. Eighty-two healthy subjects and 20 CAD patients were enrolled. Plasma free and total malondialdehyde (F- and T-MDA), glutathione disulphide/reduced form ratio (GSSG/GSH) and urine isoprostanes (iPF2α-III) levels were combined as oxidative damage markers (damage score). GSH, α- and γ-tocopherol (TH) levels, and individual antioxidant capacity were combined as antioxidant defence indexes (protection score). The OXY-SCORE was computed by subtracting the protection score from the damage score. Among single parameters, T-MDA and iPF2α-III significantly correlated with age; only GSH and both tocopherols correlated with male gender in healthy subjects. The OXY-SCORE was positively associated with age (p=0.004) and male gender (p=0.03). As expected, the OXY-SCORE was higher in CAD with a very significant p-value (<0.0001), after adjusting for age, gender and smoking. Combining different markers can potentially provide a powerful index in the evaluation of oxidative stress related to age, gender and CAD status.

Direct glutathione quantification in human blood by LC–MS/MS: comparison with HPLC with electrochemical detection

Glutathione plays a central role in the defence against oxidative damage and in signaling pathways. Upon oxidation the reduced glutathione (GSH) is transformed to glutathione disulfide (GSSG). The concentration of GSH and GSSG in whole blood samples and their ratios is useful indicator of the oxidative stress status and/or disease risk. Here we describe a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and we present the results of its comparison with a high-performance liquid-chromatographic method with electrochemical detection (HPLC-ECD). The method performed well in terms of validation parameters, i.e. linear range (0.01-50μM for both GSH and GSSG), precision (intra- and inter-day coefficients of variation were less than 10% for both GSH and GSSG), accuracy (bias% varied between -2.1 and 7.9% for both analytes), quantification limits (LLOQs were 0.5μM and 0.0625μM for GSH and GSSG respectively). Furthermore the LC-MS/MS method showed a good agreement with the HPLC-ECD assay. However, major benefits of LC-MS/MS are the improved selectivity, precision and accuracy, the higher sensitivity and the unaltered capacity of detection with time in contrast to ECD.

Glutathione, vitamin E and oxidative stress in coronary artery disease: relevance of age and gender

Background  Observational studies suggest that low levels of antioxidants are associated with high risk for coronary artery disease (CAD). We investigated whether the biomarkers of oxidative balance undergo the same modifications in all CAD patient groups, regardless of gender and age.

Analysis, physiological and clinical significance of 12-HETE: a neglected platelet-derived 12-lipoxygenase product.

While the importance of cyclooxygenase (COX) in platelet function has been amply elucidated, the identification of the role of 12-lipoxygenase (12-LOX) and of its stable metabolite, 12-hydroxyeicosatretraenoic acid (12-HETE), has not been clarified as yet. Many studies have analysed the implications of 12-LOX products in different pathological disorders but the information obtained from these works is controversial. Several analytical methods have been developed over the years to simultaneously detect eicosanoids, and specifically 12-HETE, in different biological matrices, essentially enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), high performance liquid chromatography (HPLC) and mass spectrometry coupled with both gas and liquid chromatography methods (GC- and LC-MS). This review is aimed at summarizing the up to now known physiological and clinical features of 12-HETE together with the analytical methods used for its determination, focusing on the critical issues regarding its measurement.

Nitric Oxide Synthetic Pathway in Red Blood Cells Is Impaired in Coronary Artery Disease

Background All the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls. Methods We determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[15N2]arginine conversion to L-[15N]citrulline respectively. Results Increased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity. Conclusion Our study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.

Simultaneous quantification of 8-iso-prostaglandin-F2α and 11-dehydro thromboxane B2 in human urine by liquid chromatography–tandem mass spectrometry

Both F(2)-isoprostanes (8-iso-PGF(2alpha)), a well-known marker of oxidative stress, and thromboxanes A(2) (TXA(2)) are involved in atherosclerosis through LDL oxidation and platelet activation. Different aspects of the pathology can be described by 8-iso-PGF(2alpha) and TXA(2) so it is important to determine both their concentrations to monitor the disease progression and/or therapy effects. We developed a simple and sensitive method based on liquid chromatography-tandem mass spectrometry, using electrospray ionization in negative-ion mode, for the simultaneous measurement of the concentration of 8-iso-PGF(2alpha) and 11-dehydro thromboxane B(2) (11-DH-TXB(2)), a TXA(2) metabolite. This method was applied to analyze urine samples collected overnight from 15 atherosclerotic patients, with documented carotid artery sclerosis (CAS), and from 20 controls. The detection limit was 0.097pg/microL for 8-iso-PGF(2alpha) and 0.375pg/microL for 11-DH-TXB(2), with a linear range of 0.78-25pg/microL; the inter- and intraday imprecision was <5% for both metabolites. These analytes were higher in CAS (P<0.005 vs controls) and were positively correlated in patients but not in controls, even after adjustment for age and gender (r=0.60; P=0.032). This highly sensitive, precise, and rapid method allows for the simultaneous determination of 8-iso-PGF(2alpha) and 11-DH-TXB(2) in human urine samples in order to evaluate oxidative stress and platelet aggregation.

8-hydroxy-2-deoxyguanosine levels and cardiovascular disease : a systematic review and meta-analysis of the literature

Abstract Significance: 8-Hydroxy-2-deoxyguanosine (8-OHdG) is generated after the repair of ROS-mediated DNA damages and, thus, is one of the most widely recognized biomarkers of oxidative damage of DNA because guanosine is the most oxidized among the DNA nucleobases. In several pathological conditions, high urinary levels of oxidized DNA-derived metabolites have been reported (e.g., cancer, atherosclerosis, hypertension, and diabetes). Recent Advances: Even if published studies have shown that DNA damage is significantly associated with the development of atherosclerosis, the exact role of this damage in the onset and progression of this pathology is not fully understood, and the association of oxidative damage to DNA with cardiovascular disease (CVD) still needs to be more extensively investigated. We performed a meta-analysis of the literature to investigate the association among 8-OHdG levels and CVD. Critical Issues: Fourteen studies (810 CVD patients and 1106 controls) were included in the analysis. We found that CVD patients showed higher 8-OHdG levels than controls (SMD: 1.04, 95%CI: 0.61, 1.47, p < 0.001, I2 = 94%, p < 0.001). The difference was confirmed both in studies in which 8-OHdG levels were assessed in urine (MD: 4.43, 95%CI: 1.71, 7.15, p = 0.001) and in blood samples (MD: 1.42, 95%CI: 0.64, 2.21, p = 0.0004). Meta-regression models showed that age, hypertension, and male gender significantly impacted on the difference in 8-OHdG levels among CVD patients and controls. Future Directions: 8-OHdG levels are higher in patients with CVD than in controls. However, larger prospective studies are needed to test 8-OHdG as a predictor of CVD. Antioxid. Redox Signal. 24, 548–555.

Effects of smoking regular or light cigarettes on brachial artery flow-mediated dilation

BACKGROUND AND PURPOSE To compare the effects of regular cigarettes (RCs) and light cigarettes (LCs) on brachial artery flow-mediated dilation (FMD) and sublingual glyceryl trinitrate-induced dilation (GTN), markers of endothelial dependant and independent function, respectively. METHODS 206 subjects (age 51.5 ± 12.8 yr, 122 men) had their smoking habits recorded and FMD and GTN measured by B-mode ultrasound. Cigarettes were categorized as RCs or LCs according to their content of tar, nicotine and CO. The chronic effect was assessed in current smokers of RCs (n = 85) or LCs (n = 53) and in never smokers (NS; n = 68). The acute effect was assessed in current smokers by measuring FMD before and 10-min after smoking a single regular (n = 29) or light (n = 51) cigarette. RESULTS FMD was significantly lower in consumers of RCs (6.26%, 95% C.I. 5.58, 6.94) or LCs (5.59%, 95% C.I. 4.74, 6.45) compared to NS (8.68%, 95% C.I. 7.92, 9.44) (both P < 0.0001), but did not differ (P > 0.05) when compared to each other. GTN was similar in the three groups. Analyses adjusted for clinical confounders and for markers involved in oxidative stress, arginine/nitric oxide pathway, and inflammation provided identical results. Smoking a single cigarette, either regular or light, reduced FMD (-0.88% and -1.17%, respectively, both P < 0.05), without significant difference between cigarette type. RCs and LCs produced analogous chronic and acute effects when FMD was calculated with respect to the last 60 s of the low-flow phase (FMD60s). CONCLUSIONS LCs impair endothelial-dependant vasodilation as much as RCs. Thus, smoking LCs cannot be considered an alternative to the only safe choice of a complete and permanent smoking cessation.

Circulating Levels of Dimethylarginines, Chronic Kidney Disease and Long-Term Clinical Outcome in Non-ST-Elevation Myocardial Infarction

Background Mechanisms linking chronic kidney disease (CKD) and adverse outcomes in acute coronary syndromes (ACS) are not fully understood. Among potential key players, reduced nitric oxide (NO) synthesis due to its endogenous inhibitors, asymmetric (ADMA) and symmetric (SDMA) dimethylarginine could be involved. We measured plasma concentration of arginine, ADMA and SDMA and investigated their relationship with CKD and long-term outcome in non-ST-elevation myocardial infarction (NSTEMI). Methodology/Principal Findings We prospectively measured arginine, ADMA, and SDMA at hospital admission in 104 NSTEMI patients. CKD was defined as an estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m2. We considered a primary end point of combined cardiac death and re-infarction at a median follow-up of 21 months. In CKD (n = 33) and no-CKD (n = 71) patients, arginine and ADMA were similar, whereas SDMA was significantly higher in CKD patients (0.65±0.23 vs. 0.42±0.12 µmol/L; P<0.0001). Twenty-four (23%) patients had an adverse cardiac event during follow-up: 12 (36%) were CKD and 12 (17%) no-CKD patients (P = 0.02). When study population was stratified according to arginine, ADMA and SDMA median values, only SDMA (median 0.46 µmol/L) was associated with the primary end-point (P = 0.0016). In models adjusted for age, hemoglobin and left ventricular ejection fraction, the hazard ratio (HR) for CKD and SDMA were high (HR 2.93, interquartile range [IQR] 1.15–7.53; P = 0.02 and HR 6.80, IQR 2.09–22.2; P = 0.001, respectively) but, after mutual adjustment, only SDMA remained significantly associated with the primary end point (HR 5.73, IQR 1.55–21.2; P = 0.009). Conclusions/Significance In NSTEMI patients, elevated SDMA plasma levels are associated with CKD and worse long-term prognosis.

Anesthetic Propofol Enhances Plasma -Tocopherol Levels in Patients Undergoing Cardiac Surgery

Background:Propofol (2,6-diisopropylphenol) is an anesthetic drug with antioxidant and antiinflammatory properties, documented both in vitro and in experimental models of ischemia–reperfusion injury and septic shock. These properties have been related to the similarity of its chemical structure to that of endogenous tocopherols, which are phenol-containing radical scavengers. This study evaluated the effects of propofol on &agr;- and &ggr;-tocopherol (&agr;- and &ggr;-T) levels and on selected markers of oxidant–antioxidant and inflammatory status in patients undergoing cardiac surgery. Methods:Patients were randomly assigned for anesthesia with either propofol (propofol group, n = 22) or sevoflurane (control group, n = 21). Plasma levels of &agr;- and &ggr;-T, individual antioxidant capacity, malondialdehyde, and interleukin 10 were measured before, during, and after anesthesia. In addition, levels of the proinflammatory prostaglandin E2 as a marker of cyclooxygenase-2 activity and those of interleukin 10 were measured in whole blood cultured with bacterial lipopolysaccharide. Results:&ggr;-T levels increased significantly during surgery in propofol group (P < 0.0001 vs. control group). By contrast, &agr;-T similarly decreased in both groups. Malondialdehyde and interleukin 10 increased markedly and individual antioxidant capacity decreased, without differences between groups. Prostaglandin E2 levels measured 24 h after anesthesia induction were significantly lower in the propofol than in the control group. In vitro studies highlighted the different capacity of &ggr;- and &agr;-T to impair prostaglandin E2 synthesis by human monocytes challenged with bacterial lipopolysaccharide. Conclusions:The antiinflammatory properties of propofol that may be linked to its effect on &ggr;-T levels could be relevant in controlling the inflammatory response that accompanies tissue injury during reperfusion.