The Role of the Oxidative Stress Markers in Endotelial Dysfunction at Cardiovascular Disease Patients

GERMAINE SĂVOIU-BALINT, CLAUDIA BORZA, EMERIC TOTH, MIHAIELA ANDONI*, IOAN DEMETER*, SIMINA-AMELIA DEJICA, ANDREI NEAMTU, PETRU MATUSZ, VIRGIL CIOBANU University of Medicine and Pharmacy “Victor Babes” Timisoara, Faculty of Pharmacy, 2 Eftimie Murgu Sq., 300041 Timisoara, Romania University of Medicine and Pharmacy “Victor Babes” Timişoara, Faculty of Medicine, 2 Eftimie Murgu Sq., 300041 Timisoara, Romania


b.Measurement of serum glutathione
Serum glutathione was measured fluorometrically from fresh serum obtained by centrifugation of venous blood collected from healthy individuals and CVD individuals. The serum was then treated with 125 μL of 10% tricoloacetic acid and centrifuged (1900 g, 6 min). The supernatant was added to which phthalaldehyde (fluorescent agent) was added, pH 8.0, the absorbance being read on the excitation spectrofluorimeter: 350 nm; emission of 420 nm. Normal values of serum glutathione: 0.67-1.60 mEq / mL [11][12][13].

c.Determination of FMD
Assessment of endothelial function and endothelial dysfunction was performed by non-invasive measurement of flux-mediated vasodilation (FMD). By this method, vasomotor function can be quantified after nitric oxide dependent vasodilation. Furthermore, it allows the use of repeated measurements useful for assessing the effects of surgical or medicinal interventions that interfere with vascular function.
Measurement of FMD initially involves blood pressure measurement followed by longitudinal scanning of the brachial artery with arterial diathermy measurement (D1 -reference diameter.) Then the bladder of the strain gauge placed 50 mmHg over the predetermined blood pressure was swollen and held in position for 5 After 5 minutes the cuff is slowly decompressed to install the active throat. At one minute of active hyperemia, the longitudinal axis of the vessel, the vessel diameter D2, was measured [14][15][16].
FMD% was considered the procetual modification of the diameter obtained after the active hyperemia of the basal diameter: FMD was measured with an ultrasound (Aloka ProSound SSD 4000), under "a jeun" conditions, in a quiet and constant room. Patients were not allowed to smoke in the morning of the study, nor to consume drugs / vasoactive substances at least 12 hours prior to the experiments. The values taken in the study were the average of two consecutive determinations. Normal values:> 12% [17][18][19].

d.Determination of ox-LDL plasma concentration
The plasma concentration of ox-LDL cholesterol was measured by an immunoenzymatic method using solid-phase sandwich ELISA (Mercodia kit, Uppsala, Sweden)

e.Evaluation of vascular endothelium function through in vitro studies in organ bath
Experiments on vascular rings were performed in the presence of diclofenac (10 μmol/L) to avoid interference of arachidonic acid derivatives on endothelial function.
It has been shown that menadione produces endothelial dysfunction by increasing oxidative stress (by altering intracellular thiols), lowering intracellular glutathione and aATP, effects that produce cytotoxicity and loss of structural and functional cellular integrity and implicitly vascular endothelium dissorder with endothelial dysfunction

Results and discussions
In this study, we determined the value of FMD, as a marker of endothelial dysfunction, in the studied groups: patients with cardiovascular disease. healthy subjects as well as the determination of markers of oxidative stress: malondialdehyde and glutathione, but also ox-LDL-cholesterol (heavily atherogenic lipoprotein) to make a correlation between them in order to observe the existence of a direct link between endothelial dysfunction and oxidative stress patients. The results obtained are shown in Table 1. When comparing the FMD values from the BCV group we achieved a significant decrease of these vs. control (** p <0.01) (Fig. 1). When comparing MDA values from the BCV group, we achieved a significant increase of these vs. control (*** p <0.001) (Fig. 2).
When comparing the GS and ox-LDL values from BCV we obtained a decrease, respectively a significant increase of these vs. control (* p <0.05) (Fig. 3 and 4).  The results obtained have shown a significant decrease in FMD in patients with cardiovascular disease. This has also been described by other studies that consider FMD as the basis for assessing cardiovascular function and coronary risk (49). More importantly, DE evaluated through FMD can be considered a marker by which subjects with preclinical vascular disease can be identified.
When comparing ox-LDL values, a significantly higher value was seen in CVD patients, a result expected and described by many other studies that consider the increase in ox-LDL levels as an important step in the pathogenesis of atherosclerosis, a disease responsible for over 95% of cases of coronary artery disease (50).
When comparing the values of the oxidative stress parameters, we recorded an increase in serum malondialdehyde in CVD patients. Moreover, we also noticed a significant decrease in serum glutathione, an endogenous antioxidant in people with CVD. These results indicate the presence of significant oxidative stress in people with CVD.
To see exactly if the changes described above are responsible for the decrease in FMD or endothelial dysfunction, we sought to correlate the values obtained from the patients. Thus, in case of FMD correlation with serum malondialdehyde, we noticed the existence of a poor correlation for CVD patients. Moreover, in correlating the glutathione level we obtained a moderate correlation for the BCV group. In the case of the control group no correlations were obtained over the moderate ones. This indicates, in the present study, that the installation of oxidative stress produced by the decrease in antioxidant capacity is mostly responsible for endothelial dysfunction in people with CVD. Thus, we suggest that the decrease in antioxidant defense is the one that promotes the installation of an endothelial dysfunction, the latter being installed, then leads to an increase in the oxidative stress by increasing the production of SRO.

Conclusions
The present study demonstrates, by direct decrease of endothelium-mediated vasodilation accompanied / correlated with changes in important parameters of oxidative stress, the direct involvement of oxidative stress, mainly produced as an alteration of endothelial antioxidant capacity, in the production of direct effects on vascular endothelium, in the case of people with cardiovascular disease. Moreover, ox-LDL is negatively and significantly correlated with fluxmediated vasodilation, suggesting direct involvement of plasma lipoprotein levels (oxidized due to oxidative stress) on promoting endothelial dysfunction.
Incubation of menadione ring-shaped rings results in endothelial dysfunction by action on both endothelium dependent vasodilation and endothelium-independent vasodilation. Furthermore, selenium administration appears to lead to an improvement in endothelial function, on endothelial dysfunction model due to menadione.
Thus, we can suggest that endothelial diffusion occurs as a result of the decrease in endogenous antioxidant capacity and the consequent increase in oxidative stress, which leads to an increase in LDL-cholesterol oxidation and implicitly an increase in the risk of developing cardiovascular events. Administration of selenium could improve endothelial function by protecting the body from eventual cardiovascular events associated with endothelial dysfunction.