The research presented here shows that zinc is delivered to the red blood cell (RBC) and investigates the effects of this in multiple sclerosis. A review of MS, as well as prior research investigating RBC-derived adenosine triphosphate (ATP) and the stimulation of nitric oxide (NO) are presented here. This dissertation hypothesizes that the increase in RBC-derived ATP seen in MS patients may be the result of increased zinc levels and leads to increased levels of NO, a molecule known to increase the permeability of the blood brain barrier (BBB), which is a precursor to lesion formation. C-peptide, a biologically active byproduct in the formation of insulin, also increases the release of ATP from RBCs, but only when bound to Zn2+. Evidence is presented here showing that C-peptide can deliver Zn2+ to RBCs. When bound to C-peptide, 2.54 ± 0.23 pmol of Zn2+ were delivered to RBCs, compared to 0.09 ± 0.21 pmol when Zn2+ alone was introduced RBCs. The significance of this in diabetes mellitus will be discussed.Because of these findings, C-peptide was used to deliver Zn2+ to the RBCs of MS patients. Significantly more Zn2+ is delivered to the RBCs of MS patients, at a value of 3.61 ± 0.22 pmol, than to those of healthy controls, at a value of 2.26 ± 0.24 pmol. Additionally, the basal level of Zn2+ in the RBCs of MS patients and those of healthy controls was measured. The RBCs of MS patients were found to have 41.8 ± 1.7 μg of Zn2+/g Hb where the RBCs of healthy controls only had 32.9 ± 2.2 μg Zn2+/g Hb.To further the research into the increase ATP release from the RBCs of MS patients, this was measured in a flow system that mimics the shear stress experienced by the RBCs in vivo. It was found that the RBCs of MS patients release significantly more ATP, at a value of 344.7 ± 46.8 nM, than those of healthy controls, at a value of 132.1 ± 14.1 nM. Glybenclamide, an inhibitor of ATP release from the RBC, decreased this value to 65.3 ± 11.6 nM in the RBCs of MS patients, showing that this is indeed the result of increase release of ATP, as opposed to cell lysis. The glucose uptake into these cells that may be leading to the increased ATP release is also discussed.Finally, reports have shown that estrogens have a protective effect in MS. Here, the effect of estradiol and estriol on the RBC-derived ATP was measured. Estradiol and estriol reduced the ATP release from healthy RBCs to 74 ± 4% and 70 ± 11% that of healthy controls, respectively. Through the use of a microfluidic device, the effects of estradiol on RBC-derived ATP and the subsequent endothelial cell NO production were investigated. When these RBCs were incubated estradiol, the NO production from the endothelial cells was attenuated to a value that was only 59 ± 7% of RBCs in the absence of estradiol. The ability of estrogens to decrease the ATP release from RBCs and subsequently the NO production of endothelial cells has major implications in the treatment of MS.
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