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Title: Amino acid oxidation
Author: Beckett, Philip Ronald
ISNI:       0000 0001 3453 4383
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1989
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The aim of the study was to determine the efficiency with which the carbon skeletons of essential amino acids are utilised by the pig when they are limiting in its diet. This requires the oxidation rate of an amino acid to be measured accurately using a tracer labelled with an isotope of carbon or hydrogen. The use of carbon labelled tracers requires the animal to be enclosed in a sealed chamber for the collection of CO2; to avoid this the use of [3H]-labelled tracers was investigated. The oxidation rates of [14C]-labelled tracers when the tracee was limiting and the animal's nitrogen intake was 2 g/kg0.75.d, showed that leucine was used with an efficiency of 0.88; for phenylalanine and histidine the efficiency was over 0.99. When the animal's nitrogen intake was reduced to 0.23 gN/kg0.75.d, and the limiting amino acid was removed entirely from the diet, the oxidation rates did not change significantly. The oxidation rates of [3H]amino acids were very variable compared to the [14C]amino acids and the techniques used were critically analysed. Three reasons were identified for the differences between the [14C]- and [3H]tracers. These were the measurement of tritiated water, the purity of the infused tracers and the position of the isotope in the labelled amino acid. The first two were solved by the adaption of techniques to the high accuracy required. The third reason was a problem of isotope becoming incorporated into synthetic pathways via exchange reactions. No amino acids were available labelled with a [3H] atom which would not be incorporated; therefore it was necessary to synthesise one. L-[33H]valine was chosen because a suitable precursor for its synthesis, L-[2,3-3H]valine was available and because L-[1-14C]valine was also available for comparison The L-[2,3-3H]valine was acetylated to produce N-acetyl-D, L-[3-3H]valine which was then hydrolysed by the stereospecific enzyme acylase I to yield N-acetyl-D-[3-3H]valine and L-[3-3H]valine. The products were separated using a cation exchange column and the L-[3-3H]valine was purified by paper chromatography. Contamination with D-[3-3H]valine was measured using D-amino acid oxidase and L-[2,3-3H]valine using L-amino acid oxidase. The oxidation rates of L-[1-14D]valine and L-[3-3H]valine were compared in the rat and the oxidation rate of L-[3-3H]valine was significantly higher. The experiment was repeated using L-[3-3H]valine synthesised by Amersham International plc. using the same method; the structure was established by NMR. In this case both tracers were oxidised at the same rate. The difference in the first experiment was therefore probably due to slight contamination of L-[3-3H]valine with L-[2,3-3H]valine, the tritium in position 2 being removed by reversible transamination of valine, i.e., not only during its oxidation. Finally, the two tracers were compared in the pig. It was found that the valine flux rates calculated from the two tracers were in closer agreement when a correction was made for infused D-labelled valine. Using the techniques developed throughout this work, L-[1-14C]valine and L-[3-3H]valine measured the same rate of valine oxidation. L-3-3H]valine was a more precise tracer of valine oxidation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry