br Methods Approximately mL of whole blood was obtained

Methods Approximately 7.5mL of whole blood was obtained by venipuncture into citrate-coated tubes under vacuum. All whole blood samples were processed within 12h after the blood draw. Processing started by centrifuging the whole blood at 500g, and after removal of the buffy layer by aspiration, the ERYs were washed with physiological salt solution (PSS) prior to determination of hematocrit using a hematocrit centrifuge analyzer. Crude C-peptide was purified by high performance liquid chromatography; 100% purity was confirmed by mass spectrometry and used to prepare 15mL of a stock solution of approximately 8μM C-peptide in distilled and deionized water (concentration verified using a commercial ELISA kit). On the same day as an analysis of C-peptide binding, a working solution of C-peptide (800nM) was prepared by diluting 100μL of the 8μM stock solution to 1mL with distilled and deionized water (DDW). 20pmol of C-peptide (25μL of the 800nM solution) were added to approximately 900μL of PSS, followed by the immediate addition of an aliquot of the purified ERYs (~100μL, although amounts vary based on hematocrit of the packed, purified ERYs) to result in a 7% solution of ERYs in the final C-peptide containing sample. After 2h of incubation at 37°C, the sample was centrifuged at 500g. An aliquot of the supernatant above the packed ERYs, as well as all standards, was then diluted 1:50 in DDW and used as the sample in an ELISA for C-peptide. The amount of C-peptide remaining in the supernatant was measured, and by subtracting this buy pilocarpine hydrochloride amount from the 20pmol originally added to the sample, the amount bound to the ERYs was calculated. C-peptide standards were prepared in PSS, diluted, and measured in the same manner as the samples for quantitation, save for the addition of the ERYs. The average C-peptide±standard deviation is reported for all patient groups and statistical significance is reported for all groups using Student\'s t-test with associated p-values.
Results
Discussion C-peptide is secreted in equal amounts with insulin from pancreatic β-cell granules in vivo. Since its discovery in the late 1960’s, (Rubenstein et al., 1969) C-peptide has been regarded as a biologically inactive species once secreted from the β-cell granules (Luzi et al., 2007). In fact, other than facilitating the insulin production process, many believe C-peptide to be useful only as a biomarker for insulin production in patients with type 1 diabetes, due in large part to its longer half-life in the bloodstream (~30min) in comparison to insulin (<5min). Furthermore, no ERY C-peptide receptor has been identified, and the mechanism of ERY C-peptide uptake remains unknown. However, since the mid-1990’s, there have been numerous studies reporting beneficial effects of C-peptide replacement therapy to animals and humans with type 1 diabetes (Nordquist et al., 2008; Sima, 2003; Sima and Kamiya, 2004; Wahren et al., 2007). Many of these cellular and tissue effects involve improvements in blood flow (Forst et al., 2000; Forst and Kunt, 2004; Kunt et al., 1999). These studies reporting an improvement of overall blood flow inspired our group to investigate and report that C-peptide enhances the ability of ERYs to release adenosine triphosphate (ATP), a well-established stimuli of the potent vessel dilator and mediator of blood flow, nitric oxide (NO). During the course of our studies, we also reported that the ability of C-peptide to stimulate increased ERY-derived ATP also required zinc and serum albumin (Meyer et al., 2008; Liu et al., 2015). While C-peptide binds to the cell in the presence of albumin, there are no biological effects on the cell unless zinc is delivered along with the C-peptide (Liu et al., 2015). In fact, the ratio of C-peptide to zinc delivery to the cells is 1:1. Recently, others have shown enhanced effects of C-peptide if supplemented with zinc prior to in vivo delivery (Slinko et al., 2014).