Absolute Concentration Determination of Phosphorus Metabolites in the Langendorff-Perfused Rabbit Heart by Phosphorus-31 Nuclear Magnetic Resonance (Quantification, Metabolism, Intact Tissue, Nmr)
Gard, Janice Koles
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https://hdl.handle.net/2142/70272
Description
Title
Absolute Concentration Determination of Phosphorus Metabolites in the Langendorff-Perfused Rabbit Heart by Phosphorus-31 Nuclear Magnetic Resonance (Quantification, Metabolism, Intact Tissue, Nmr)
Author(s)
Gard, Janice Koles
Issue Date
1984
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, Medical
Abstract
The concentrations of mobile high energy phosphorus metabolites and intracellular pH of Langendorff-perfused rabbit heart have been determined under control and reduced flow conditions. Absolute concentration determination was accomplished by Lorentzian lineshape analysis after development of hexachlorocyclotriphosphazene as an external intensity standard. Hearts were demonstrated to be bichemically and physiologically competent during control perfusion periods and compromised during reduced flow conditions by independent hemodynamic and metabolic measurements coincident with the NMR experiment.
Reduction in perfusate flow from 20 mL/min to 5.0 mL/min (25% flow) or 2.5 mL/min (12.5% flow) demonstrated a fall in phosphocreatine and adenosine triphosphate concentrations, a rise in cytosolic inorganic phosphate concentrations, and drops in pH. Subsequent recovery upon reflow was observed. Utilizing a creatine kinase equilibrium model, cytosolic ADP concentrations were derived, but not detected by differences in the integrated areas of the gamma and beta phosphate resonances of ATP. The derived values for the free concentration of ADP were very close to the reported values of the Michaelis constant for respiratory stimulation, implicating a regulatory role for this molecule in cellular respiration. Strong evidence that the creating kinase reaction was in equilibrium in the 25% flow study was seen. The NMR observable correlated closely with myocardial performance and biochemical indices of metabolic function, and supported the use of phosphocreatine as an indicator of current metabolic integrity.
In a separate way of HEPES Krebs-Henseleit perfused hearts, significant protection of myocardium during reduced flow periods was seen. In addition, ventricular function appeared to be dissociated from the pH. Therefore, the loss of ventricular function could not be explained in terms of metabolic acidosis.
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