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Development of Analytical Methods to Assist with the Purification & Characterization of Novel Endogenous Cardiotonic Steroids Extracted from Sus domesticas Skeletal Muscle

Stiner, Cory A.
http://orcid.org/0000-0002-9288-6181
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1522318918009782

Abstract Details

2018, PhD, University of Cincinnati, Arts and Sciences: Chemistry.
A new analytical method was designed to quantify the activity of the Na+, K+ ATPase in skeletal muscle and red blood cells based on ICP-MS-MS. This new approach will aid in determining the physiological effects of novel endogenous cardiotonic steroids extracted from pig skeletal muscle. The Na+, K+ ATPase is a vital enzyme in all eukaryotic cells. The Na+, K+ ATPase is also the receptor for cardiotonic steroids, a class of compounds that are used in the treatment of heart failure as well as many other medical conditions. Endogenous cardiotonic steroids are present at low levels in tissues and blood, and these compounds play a significant role in blood pressure regulation and cardiac function. The presence of uncharacterized cardiotonic steroids in tissues presents a relevant area for research. The first part of my dissertation focuses on metal ion transport by the Na+, K+ ATPase in cells which were quantified by ICP-MS-MS. The activity of the Na+, K+ ATPase in mouse skeletal muscles and human red blood cells was measured based on the amount of Rb, as a K analog and K present upon quantification by ICP-MS-MS. The previous method used to quantify Rb and measure Na+, K+ ATPase activity in tissues and cells required the use of radioactive materials. The instrument used for the previous method is called a scintillation counter which can detect and measure the ionizing radiation from 86Rb. The new approach using ICP-MS-MS does not require the use of radioactive 86Rb. Instead, natural abundance 85Rb and 87Rb can be used to quantify Rb within the cells to determine the activity of the Na+, K+ ATPase. This new method helps to study physiological processes that are metal-dependent and can be used for various cell types and conditions. The second part of my dissertation focuses on ICP-MS-MS analysis of micro-sized biological samples with heteroatoms as an internal tag for mass-free quantification of selected elements. ICP-MS-MS has been used for elemental analysis at trace and ultra-trace levels in various research areas. In biomedical research, the application of ICP-MS-MS has been limited by the sample volumes obtained. A new approach was developed which allowed for mass-free quantification of selected elements in biological samples that have sub-microgram mass, processed into low microliter volumes. The biological samples used were H. capsulatum, B. dermatitidis yeast, RAW macrophages, and skeletal myofiber cells. The third part of my dissertation focuses on the isolation and purification of novel endogenous cardiotonic steroids from pig skeletal muscle. Cardiotonic steroids are a class of compounds that can be extracted from various living organisms. It is known that these compounds can bind to the Na+, K+ ATPase and inhibit ion transportation across the plasma membrane. A variety of techniques were used to refine these endogenous cardiotonic steroids from pig skeletal muscle. Throughout the purification process, multiple assays are conducted to ensure that the compound is present in each phase of the purification process. The results have shown that these endogenous compounds are very active and have a high affinity for the Na+, K+ ATPase. The fourth portion of my dissertation focuses on the physiological effects of novel endogenous cardiotonic steroids extracted from pig skeletal muscle tissue. Various assays were used such as the ATPase assay, red blood cell assay, and the reversibility red blood cell assay. These assays help to determine if the unknown compounds have inhibitory effects on the Na+, K+ ATPase, if they work in higher order mammals, and if they can be easily unbound from the enzyme. The final portion of my dissertation focuses on the characterization of novel endogenous cardiotonic steroids extracted from pig skeletal muscle.
Julio Landero Figueroa, Ph.D. (Committee Chair)
Edward Merino, Ph.D. (Committee Chair)
Judith Heiny, Ph.D. (Committee Member)
Pearl Tsang, Ph.D. (Committee Member)
174 p.
Analytical Chemistry
Inductively Coupled Plasma Mass Spectrometry; High Performance Liquid Chromatography; Cardiotonic Steroids; Na, K-ATPase; Metal Analysis; Metal Ion Flux

Recommended Citations

Citations

  • Stiner, C. A. (2018). Development of Analytical Methods to Assist with the Purification & Characterization of Novel Endogenous Cardiotonic Steroids Extracted from Sus domesticas Skeletal Muscle [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1522318918009782

    APA Style (7th edition)

  • Stiner, Cory. Development of Analytical Methods to Assist with the Purification & Characterization of Novel Endogenous Cardiotonic Steroids Extracted from Sus domesticas Skeletal Muscle. 2018. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1522318918009782.

    MLA Style (8th edition)

  • Stiner, Cory. "Development of Analytical Methods to Assist with the Purification & Characterization of Novel Endogenous Cardiotonic Steroids Extracted from Sus domesticas Skeletal Muscle." Doctoral dissertation, University of Cincinnati, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1522318918009782

    Chicago Manual of Style (17th edition)

ucin1522318918009782
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