Viviana Cavalca

The effects of the oral glucose tolerance test (OGTT) on red blood cells (RBCs) have not been thoroughly investigated, although it is known that the ingestion of 75 g of glucose during OGTT results in a systemic state of inflammation and oxidative stress. Therefore, we evaluated the effect of OGTT on oxidative stress and L-arginine/Nitric Oxide (L-Arg/NO) metabolic pathway in RBCs obtained from patients with prediabetes. Blood samples were collected from all participants before (T0) and at 10 (T1), 20 (T2), 30 (T3), 60 (T4), 90 (T5), 120 (T6), 150 (T7), and 180 (T8) minutes after glucose loading. Results showed a significant increase in oxidative stress status characterized by a rise in the GSSG/GSH ratio at T4 and T6 that increased in parallel with a reduction of NO production in RBCs. In addition, in this time frame, increased exposure of phosphatidylserine on RBCs membrane was observed. These metabolic modifications were rescued at T8, together with an increase in activated RBC NO synthase expression. These findings provide a possible explanation of the phenomena occurring after glucose loading and suggest that, even in the early stages of diabetes, it may be important to avoid acute variations in glycemia in order to prevent diabetic complications.

Clinical data indicate that low circulating l-homoarginine (HArg) concentrations are associated with cardiovascular (CV) disease, CV mortality, and all-cause mortality. A high number of LC-based analytical methods for the quantification of HArg, in combination with the l-arginine (Arg)-related pathway metabolites, have been reported. However, these methods usually consider a limited panel of analytes. Thus, in order to achieve a comprehensive picture of the Arg metabolism, we described an improved targeted metabolomic approach based on a multiple reaction monitoring (MRM) mass spectrometry method for the simultaneous quantification of the Arg/nitric oxide (NO) pathway metabolites. This methodology was then employed to quantify the plasma concentrations of these analytes in a cohort of individuals with different grades/types of coronary artery disease (CAD) in order to increase knowledge about the role of HArg and its associated metabolites in the CV field. Our results showed that the MRM method here implemented is suitable for the simultaneous assessment of a wide panel of amino acids involved in the Arg/NO metabolic pathway in plasma samples from patients with CV disease. Further, our findings highlighted an impairment of the Arg/NO metabolic pathway, and suggest a sex-dependent regulation of this metabolic route.

Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily (od), low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase (COX)-1 inhibition. We performed a multicenter, double-blind trial to investigate the efficacy of three aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2-dependent vascular thromboresistance. Two-hundred-forty-five patients on chronic od low-dose aspirin were randomized (1:1:1) to receive 100 mg aspirin od, twice-daily, bid), or three-times daily (tid) for 2 weeks. Serum thromboxane B₂ (sTXB₂), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate endpoints of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated. Evaluable patients assigned to the bid and tid regimens showed substantially reduced inter-individual variability and lower median values of sTXB₂: 19.3[9.7-40], 4 [2.1-6.7], and 2.5[1.4-5.65] ng/ml in the od (n=85), bid (n=79) and tid (n=79) arms, respectively. Urinary PGIM was comparable in the three arms. Urinary TXM was significantly reduced by 35% in both experimental arms. Patients in the tid arm reported a higher abdominal discomfort score. In conclusion, the currently recommended aspirin regimen of 75-100 od for cardiovascular prophylaxis appears largely inadequate in reducing platelet activation in the vast majority of ET patients. The antiplatelet response to low-dose aspirin can be markedly improved by shortening the dosing interval to 12 hours, with no improvement by further reducing it. (EudraCT 2016-002885-30)

As no studies before now have thoroughly investigated the risk associated with the exposure to low-dose ionizing radiations in patients undergoing catheter ablation (CA), we aimed to evaluate the oxidative and DNA damage in 59 CA patients (10 of whom received N-acetylcysteine (NAC) before the procedure). A burst of oxidized/reduced glutathione ratio was observed 3 hours after procedure that was diminished by NAC administration. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) concentrations, index of oxidative DNA damage, showed a peak 24 hours after CA. A positive correlation between 8-OHdG peak and fluoroscopy time and a negative correlation among 8-OHdG decrease (from the peak to 48 hours after CA) and all procedure parameters were found. Furthermore, DNA tail percentages (which reflect the extent and the number of DNA strand breaks) positively correlated with 8-OHdG concentrations. This study evaluates for the first time the kinetic of oxidative damage in patients undergoing CA procedure. Our findings raise the question of whether 8-OHdG can be used as a circulating biomarker of DNA oxidative damage induced by low-dose ionizing radiations in different clinical settings. Antioxid. Redox Signal. 28, 1137–1143.

The serum TXB₂ (sTXB₂) assay reflects the pharmacodynamics of platelet inhibition by low-dose aspirin. However, different studies reported variable sTXB₂ values. sTXB₂ assay requires whole blood incubation at 37 °C as a condition for optimal thrombin generation, arachidonic acid release and its metabolism by platelet cyclooxygenase-1 to form TXA₂. Access to 37 °C incubation may be variably delayed, and different methods to quantitate sTXB₂ may contribute to variable results between different Centers. We investigated whether delaying 37 °C incubation and/or analytical issues affect sTXB₂ concentrations, biasing the assessment of aspirin responsiveness. Sixty-eight samples from 54 volunteers, on- and off-aspirin, were incubated at 37 °C immediately after sampling (reference sample) or after 5, 10, 15, 20, 30 or 60 minutes at room temperature (RT); 8 samples remained at RT 60 minutes, without subsequent incubation; 314 sera were measured by enzyme immunoassay (EIA) and liquid chromatography-tandem mass-spectrometry (LC/MS-MS) methods. sTXB₂ concentrations decreased exponentially as a function of the delay before 37 °C incubation, ranging from 94 ± 11 % at 5 minutes to 23 ± 22 % of the reference sample after 60 minutes at RT. There was high agreement between EIA and LC/MS-MS. Moreover, we simulated the influence of a 15– or 30-minute delayed incubation on 300 sTXB2 measurements from previously-studied, aspirin-treated patients. Delayed incubation reduced the percentage of aspirin ‘non-responders’ by 22 % to 52 %, depending on the response threshold. In conclusion, a variable delay in the 37 °C incubation of blood samples may affect the assessment of platelet cyclooxygenase-1 inhibition by aspirin and confound the characterization of the determinants of aspirin responsiveness.

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, I ² = 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.

Platelet activation is persistently enhanced, and its inhibition by low-dose aspirin is impaired in type 2 diabetes mellitus. We investigated in vivo thromboxane (TX) and prostacyclin (PGI2) biosynthesis and their determinants, as well as aspirin responsiveness, in young adult subjects with type 1 diabetes mellitus (T1DM) without overt cardiovascular disease and stable glycemic control. The biosynthesis of TXA2 was persistently increased in subjects with T1DM versus matched healthy subjects, with females showing higher urinary TX metabolite (TXM) excretion than male subjects with T1DM. Microalbuminuria and urinary 8-iso-PGF2α, an index of in vivo oxidative stress, independently predicted TXM excretion in T1DM. No homeostatic increase in PGI2 biosynthesis was detected. Platelet COX-1 suppression by low-dose aspirin and the kinetics of its recovery after drug withdrawal were similar in patients and control subjects and were unaffected by glucose variability. We conclude that patients with T1DM and stable glycemic control display enhanced platelet activation correlating with female sex and microvascular and oxidative damages. Moreover, aspirin responsiveness is unimpaired in T1DM, suggesting that the metabolic disturbance per se is unrelated to altered pharmacodynamics. The efficacy and safety of low-dose aspirin in T1DM warrant further clinical investigation.