Energy utilization of toad retina: A hydrogen-1 and phosphorus-31 NMR study
Brown, Darin Carlton
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https://hdl.handle.net/2142/23372
Description
Title
Energy utilization of toad retina: A hydrogen-1 and phosphorus-31 NMR study
Author(s)
Brown, Darin Carlton
Issue Date
1996
Doctoral Committee Chair(s)
Dawson, M. Joan
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Language
eng
Abstract
Vision is the most important of all human senses. Research concerning the physiology and biochemistry of components of the visual system can lead to advances in the prevention and treatment of diseases. Since the retina is part of the brain, studies of the retina can help provide information about the function of the central nervous system. Furthermore, the principles learned in retinal research concerning membrane protein interactions and second messengers (Ca$\sp{2+}$ and G-proteins) are generalizable to many other biological systems.
Magnetic resonance is a rapidly developing research and clinical tool that provides a unique opportunity to study living systems. Unlike most other biochemical or physiological techniques, nuclear magnetic resonance (NMR) can be performed on intact animals, tissues, or even on people. Phosphorus ($\sp{31}$P) NMR spectroscopy of the retina provides useful data about the control of biochemical reactions involving metabolites which contain phosphorus, such as how they are affected by light (Apte, 1993). Study of phosphorus-free metabolites, such as lactate, requires a different technique, namely proton ($\sp1$H) NMR spectroscopy. Combined, these techniques are especially powerful for the study of the relationship between biochemistry and physiology of isolated tissues, such as the retina.
Since $\sp1$H NMR spectra of the retina had not been previously recorded, the first goal of this research was to obtain a useful $\sp1$H spectrum under physiological conditions, and to identify the compounds that give rise to the signals observed. A necessary intermediate in this goal is to apply a method of solvent suppression, since the water proton concentration is more than a thousand-fold greater than the concentration of the substances of interest. For quantitative analysis, great care was taken to select appropriate methods for obtaining spectra. My selection of a water suppression pulse sequence that permits quantitation has not only formed the basis for this study, but has also suggested its use in other physiological preparations, including skeletal muscle (Shen et al., 1996).
After the procedure to maintain the retinas in the NMR probe had been established, and the corresponding $\sp1$H NMR spectrum obtained, there were three major questions I wished to answer. First, what metabolites are visible by $\sp1$H NMR spectroscopy of isolated retina, and is it possible to measure their concentrations, especially that of lactate? Secondly, what is the consequence of changing the concentrations of Ca$\sp{2+}$ ions in the perfusion solution on NMR-visible metabolites in the retina, especially lactate? Finally, are the results of altering Ca$\sp{2+}$ levels consistent with evidence obtained from $\sp{31}$P spectroscopy?
Motivation for these questions, as well as a brief review of the structure and function of the retina, is presented in chapter 1 of this paper. Chapter 2 summarizes the physics required for a basic understanding of NMR and the water suppression technique chosen for this research (along with a brief, general review of water suppression). The third chapter describes the materials and methods used, and the fourth summarizes the results of $\sp{31}$P and $\sp1$H NMR experiments, designed to answer the above questions.
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