The act of nitric oxide (NO) is multifaced. It can be as a messenger for either a pro-oxidant or an antioxidant in biological systems . All organic nitrates undergo biotransformation releasing nitric oxide (NO) . Chronic exposure of nitrates induces oxidative stress and exerts disastrous pathophysiological effects including oxidative stress, inflammation, autoimmune disease and cancer . This may be directly due to potential liability of mediating DNA damage or indirectly through production of reactive nitrogen species (RNS) free radicals. NO has short half-life and widely formed through the vascular endothelium, resulting in direct relief of the smooth muscles of the vascular tissues . Serum level of both Nitates and Nitrites were used to estimate the level of NO due to its very short half-life . We chose the rat model since rats are more similar to human physiology over mice model, making them better models for the studying pathological conditions in pre-clinical trials [37, 38]. Moreover, high sucrose/fat diet (HSF) induces hypertension rats; called spontaneously hypertensive rats (SHR) which are extensively used as an in vivo model for essential hypertension and cardiovascular disease . This SHR rat model has elevated basal myocardial NO content which may be due to an increase in the protein-bound of dinitrosyl nonheme iron complexes which liberates the NO to the peripheral circulation that helps in treating the hypertensive state. Moreover, it has been previously published that SHR rats showed increased NO synthase activity (NOS) III in the cardiac and aortic endothelia. Both of these two enzymes helps in regulating the vasoreactivity in the SHR rats .
Our data go along with the previously published data for using antioxidant for treating the nitrate tolerance and they gave similar clinical data (34, 38-39). Meanwhile, the authors stressed on the oxidative stress markers. Here, we studied a very important stress marker; nrf2, has a great role in the antioxidant metabolism pathway and has important role against reactive oxygen species producing the cellular injury in the myocardium muscles . This gives a molecular explanation of the previously published results and quantitatively determined the dose of vitamin C that can be used clinically. Moreover, we studied the activity of pro-inflammatory mediator gene; NF-kB. Our results showed another mechanism for the protective effect of the vitamin c against NO deleterious effect on the myocardium tissue. Besides, our data on the apoptosis marker; caspase-3, gives the an explanation for the end result of the protective effect of vitamin c on the myocardium tissue.
SOD, GSH, CAT and MDA are the first line defense mechanisms against oxidative stress. They give an indication about the stressful conditions inside the cells . It has been previously reported a significant decrease in the activity of SOD, GSH and CAT, and a significant elevated level of plasma MDA . The suggested mechanism is that this may be an indirect effect of the significant increased NO plasma level or directly due to the significant elevated level of ROS and RNS. Meanwhile, the previous study  has not proved the tightly bound to that hypothesized subcellular event. In our study, we proved these suggestions through finding the dose-dependent response according to the plasma NO levels. Yet, vitamin C; a free radical scavenger, restored the change of these first line defense mechanisms against oxidative stress. We measured the cellular levels of pro-inflammatory cytokines marker (NF-κB), apoptosis markers (caspase-3) and mitochondrial oxidative stress marker (nrf2) in myocardium muscles. nrf2 is a suppressive mitochondrial oxidative stress marker. If this marker is increased, this indicates an increased oxidative stress conditions inside the cell. We found that ISMN elevated the cellular activity of nrf2 in a concentration-dependent manner. Moreover, ISMN reduced the cellular activity of both pro-inflammatory cytokines marker (NF-κB) and apoptosis markers (caspase-3) in a dose-dependent manner. Vitamin C succeeded to restore the cellular levels of the three markers in all tested concentrations of ISMN. We expected that the vasodilatory effect of ISMN will remain in chronic administration of ISMN due to the restoring of the endothelial function of myocardium and no need to establish the NO free period. This nitrate free period for patients that may cause elevation of the risk of the cardiovascular events during such nitrate-free periods, specifically in the early light phase.
Reduction of the plasma hemoglobin level and increase in the level of both methemoglobin and carboxyhemoglobin may be the consequences of the liberated nitric oxide and nitrate ions during isosorbide mononitrate metabolism which can oxidize hemoglobin to methemoglobin and carboxyhemoglobin [39, 40]. Increased methemoglobin level can cause congenital enzymatic defects, variation in hemoglobin molecule [41, 42]. Methemoglobin does not bind to oxygen efficiently causing a reduction in the oxygen carrying capacity of the blood and reduction in saturated oxygen and oxygen content this decrease is significant in high doses of nitrates . Vitamin C protects the blood from the oxidant effect of nitric oxide (antioxidant effect) and induces decreasing of level of met-hemoglobin concentration . This reduction in methemoglobin levels induced by vitamin C concluded that erythrocyte alone had a negligible ability to reduce methemoglobin in the absence of exogenous ascorbate. Ascorbic acid preserves the Hb in a reduced ferrous redox state [43, 44].
It has been previously reported that carboxyhemoglobin showed a significant increase with high doses of organic nitrates only . Our results showed that low doses of ISMN can significantly increase the plasma level of MET-Hb. This may be due to the impairment in the antioxidant enzymes defense system of the erythrocyte which may cause an elevation of the abnormal hemoglobin derivatives as carboxyhemoglobin . An elevation in carboxyhemoglobin levels were noticed in all ISNN treated groups in a concentration-dependent manner. However, following vitamin C administration, a significant reduction of the carboxyhemoglobin was obtained in all treated groups. A reduction in hematocrit, RBC, and WBC may be a result of hemoglobin reduction and oxidative stress induced by nitric oxide [46, 47].
The present study showed a significant decrease in both serum iron level and serum total iron binding capacity (TIBC) in all ISMN treated groups in a concentration-dependent manner. This result can be explained by the increased serum nitric oxide by the increased ISMN dose and by the induced oxidative stress decreasing the serum iron and total iron binding capacity (TIBC) . It has been reported that iron deficiency anemia increases through NO production, and elevated NO concentrations in iron deficiency anemia. This effect can be revered by iron supplementation to regain its normal levels . In this study an improvement of the serum iron level and total iron binding capacity (TIBC) was observed by the effect of vitamin C. This improvement may be due to the beneficial effect of vitamin C to enhance iron absorption [47, 48]. In our study all groups treated with ISMN showed a significant increase in serum nitric oxide concentration. It has been previously recorded that vitamin C significantly decrease nitric oxide concentration .
Vitamin C can decrease the accumulation of superoxide and peroxynitrite by directly scavenging superoxide . Vitamin C safeguards against oxidative stress that induces pathological vasoconstriction and destruction of the endothelial barrier through blocking tetrahydrobiopterin (BH4) oxidation, the cofactor of the endothelial nitric oxide synthase (eNOS), thereby inhibiting endothelial nitric oxide (eNO) depletion and eNOS uncoupling. Ascorbate blocks inducible nitric oxide synthase (iNOS) preventing abundant production of NO .