Date of Completion

2016

Document Type

Honors College Thesis

Department

Biology

Thesis Type

Honors College, College of Arts and Science Honors

First Advisor

Kalev Freeman

Second Advisor

Jim Vigoreaux

Third Advisor

George Wellman

Keywords

Oxidation-Reduction Potential, ORP, Redox, Trauma

Abstract

Oxidation-reduction potential (ORP) measurement has demonstrated oxidative stress in patients with severe illness/injury. A new ORP diagnostic platform using disposable sensors (Supplemental Figure 1) has been validated by comparison to mass spectrometry, but the response of ORP to experimental positive and negative control conditions has not been determined. Furthermore, optimal methods of sample handling for ORP measurement have not been studied. We sought to optimize ORP measurement in human plasma, under controlled conditions. We hypothesized that freeze-thawing of the sample, storage of the sample for up to one month, and the method of sample anticoagulation would influence ORP levels. Furthermore, we hypothesized that ORP can detect incremental experimental changes that either increase or decrease redox state. Finally, we hypothesized that trauma injuries cause increased levels of reactive oxygen species (ROS) in the circulating bloodstream, and therefore would reflect higher ORP signals in plasma measurements in both human and mouse subjects.

We enrolled healthy human volunteers in a prospective observational study and measured ORP in plasma prepared with heparin or citrate anticoagulants directly after blood draw and up to 28 days later. Additionally, we evaluated the platform’s ability to detect an exogenous increase and decrease in ORP by performing both positive and negative control titrations using six different concentrations of hydrogen peroxide (H2O2) and two different concentrations of ascorbic acid respectively. Lastly, we compared human trauma plasma samples in an uncontrolled emergency room setting with healthy human plasma and then mimicked this experiment in a more controlled mouse model experiment. We found that fresh plasma better retains the ORP signal as compared to freeze-thaw samples measured on subsequent days. We also found that the platform can detect exogenous, concentration-dependent oxidations with H2O2 and reductions with ascorbic acid. Furthermore, plasma prepared with heparin is more sensitive than using citrate anticoagulant when measuring ORP. Also, human trauma patients’ plasma (of varying degrees of trauma) samples were characterized by significantly higher ORP signals than healthy controls when measured in heparin anticoagulant, whereas mouse ORP signals did not change following a controlled, moderate traumatic brain injury. These data indicate that the diagnostic platform is capable of detecting exogenous increases and decreases in ORP signal from plasma samples with validity and is sensitive to different concentrations of positive and negative controls. We also show that plasma should be collected and centrifuged in heparin anticoagulant tubes and can be analyzed fresh or frozen for optimal results.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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