We observed no differences in levels of plasma peroxynitrite in the saline-treated rats of all three weight groups, as reflected by insignificant differences of nitrotyrosine levels. However, with increases in BMI, a significant decrease in the ability to reduce tension through the endothelial-dependent vascular relaxation pathway was noted. The overweight rats had a significantly lower ability to reduce tension, even with a direct contribution of nitric oxide by SNP. Treatment with 10 mg/kg LPS induced a significant increase in the levels of nitrotyrosine in all weight groups as compared with saline-treated rats. We demonstrated that an increase in peroxynitrite leads to a decrease in the reduction of tension in the aortae in the underweight, normal weight and overweight rats.
Endothelial function is subject to alterations in biochemical factors, some of which are related to changes in body mass. Both endothelial function and plasma peroxynitrite are related to body mass [18, 19], and its effects on the levels of peroxynitrite were observed in the saline-treated rats. BMI significantly impacted peroxynitrite levels as measured by nitrotyrosine. The levels of nitrotyrosine were proportional to BMI: the higher the BMI, the higher the plasma nitrotyrosine. The elevated levels of peroxynitrite, as shown by higher nitrotyrosine levels, produced in the overweight rats, could be because of an increase in oxidative stress from the larger amount of adipose tissue . In other literature, lower body mass has also been associated with higher levels of peroxynitrite. This has been attributed to the damage that peroxynitrite causes to endothelium DNA [21, 22]. This was not so in our study, where the underweight rats had the lowest levels of nitrotyrosine.
We observed that in saline-treated rats, higher BMI was significantly associated with endothelial dysfunction: normal weight rats had significantly lower Rmax (121%) compared to underweight rats (Rmax = 137%) (P = 0.03). Further decrease in endothelial-dependent vascular relaxation in overweight rats was observed. Adipose tissue, a major source of iNOS , is observed in overweight. This may lead to an elevation in oxidative stress, as observed by the increase in peroxynitrite  which causes reduced endothelial-dependent vascular relaxation. Several literature have outlined that the higher levels of plasma peroxynitrite in overweight and underweight rats may be responsible for endothelial dysfunction in these weight groups [20,21,22]. In this study, acute changes in tension reduction among the different weight groups and varying peroxynitrite levels have been demonstrated in rat aortae.
To study only the effect of the endothelium, aortic ring response to endothelial-independent vascular relaxation was assessed using SNP. SNP is an exogenous agent that generates nitric oxide through nonenzymatic and enzymatic pathways in smooth muscle cells. It does not require a functional endothelium or NOS enzymes for its activity. Once infused, SNP interacts with oxyhaemoglobin, dissociating immediately and forming methaemoglobin while releasing cyanide and nitric oxide. Nitric oxide activates the enzyme guanylate cyclase found within the vascular smooth muscle. This results in increased intracellular concentrations of cyclic guanosine monophosphate, which inhibits calcium entry into vascular smooth muscle cells. The mechanism results in the increase of calcium uptake by the smooth endoplasmic reticulum to produce vasodilation . In the current study, the effectiveness of this pathway was noted by a significant change in Rmax after treatment with SNP.
Post SNP challenge, the underweight and normal weight rats showed insignificant differences in Rmax and logIC50 in the curves of saline-treated rats compared to those treated with LPS. This may be a demonstration of impairment of endothelial production and delivery of nitric oxide, rather than vascular smooth muscle sensitivity to nitric oxide. This is similar to other findings which have reported a decrease in endothelial dependant vascular relaxation, though not stratified by BMI [1, 15]. We observed that higher BMI was significantly associated with lower tension reduction propensity even when the pathway of direct nitric oxide donation (SNP mechanism) to the smooth muscle was used. This implies that there may be a more chronic reason for the increase in endothelial dysfunction in overweight rats. This gives an in-depth characterisation of the role of BMI in endothelial dysfunction via the increase in peroxynitrite levels. These findings are similar to those of O’Brien et al.  who also observed an impairment in endothelial-dependent and endothelial-independent vascular relaxation among obese rats.
We also observed the effect of increasing peroxynitrite levels (i.e., increased nitrotyrosine levels via LPS administration at 10 mg/kg) on endothelial function in all weight groups. When the characteristics of the dose-dependent curves of untreated rats and those treated with 10 mg/kg LPS were compared, a statistically significant reduction in endothelial-dependent ability to reduce tension was observed between saline-treated rats and those treated with the highest LPS dosage. In treatment with ACh, this reduction in endothelium-dependent vascular relaxation was noted by a significant decrease in Rmax. In a contracted aorta, the addition of ACh should typically result in vascular smooth muscle relaxation through the activation of protein kinase G [1, 32, 33], leading to dilation of the blood vessel. Increase in peroxynitrite causes significant endothelial dysfunction across the spectrum of body mass, as shown by reduced Rmax in all the weight groups. The diminished response to ACh supports the idea that increased levels of peroxynitrite cause a reduction in endothelium-dependent vascular relaxation. Others have reported that exposure of rat aortic rings to high concentrations of peroxynitrite caused a marked impairment of the endothelium-dependent vascular relaxation [15, 32]. Mihm et al.  found that brief incubations with relevant concentrations of nitrotyrosine elicited significant impairment of ACh-induced relaxation in a concentration-dependent manner.
There was no significant change in logIC50 in groups A and D of all the weight groups upon ACh treatment. Other literature has shown that increased peroxynitrite impairs tension reduction, as demonstrated by an increase in logIC50 . This increase was not statistically different.
Although there were significant differences in nitrotyrosine levels between rats treated with saline vs. those treated with the highest LPS dosage, more precise levels might have been recorded if nitrotyrosine levels were measured at a different time post-LPS treatment. Furthermore, we could have recorded more accurate levels of nitrotyrosine if chromatography/mass spectrometry were used instead of ELISA.