Graduate Thesis Or Dissertation
 

The determination of pyridoxal phosphate by the measurement of ¹⁴Co₂ enzymatically released from tyrosine-1-¹⁴C

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  • The increasing knowledge of the significance of vitamin B₆ in human metabolic processes has emphasized the importance of developing reliable means of assessing nutritional status with respect to this vitamin. One possible approach to this end is the measurement of pyridoxal phosphate in blood or plasma, particularly since this phosphorylated component is the most common metabolically active form. Although some methods of measurement have been proposed, none has proved completely satisfactory. This research was undertaken in an effort to find an improved procedure for determining pyridoxal phosphate, which might be used for evaluation of vitamin B₆ nutrition. The earliest method for the determination of pyridoxal-5-phosphate was a manometric procedure which involved the measurement of CO₂ released from tyrosine by the action of tyrosine decarboxylase. Since pyridoxal phosphate is the coenzyme of tyrosine decarboxylase, the rate of the reaction is proportional to the amount of this form of vitamin B₆ which is present. This method has been adapted for use with whole blood, plasma, and leukocytes. More recently, the sensitivity and precision of the method have been increased by incorporating radioactive tracer techniques. Tyrosine-U-¹⁴C was used as the substrate and the radioactive tyramine and tyrosine were measured after separation by paper chromatography. The investigations which led to the development of a procedure parallel to that above, but employing tyrosine-1-¹⁴C as the substrate, are described in this report. The enzymatically released ¹⁴CO₂ is measured by liquid scintillation techniques. This procedure is less complicated than any of the others, involving simple apparatus and reaction steps. It presupposes, however, the availability of some type of liquid scintillation counter. Moreover, the procedure is extremely sensitive, approximately equalling the sensitivity of microbiological methods. Included in this report are descriptions of the apparatus which was developed, and the tests which were made to establish optimum levels and ratios of reactants, as well as optimum conditions for the reaction and for detecting the ¹⁴C-labeled carbon dioxide. Preliminary tests were made applying this procedure to the assay of whole blood or plasma, thereby demonstrating that it can provide a feasible assay method. In addition, there are some recommended steps for future work which would be of value in correlating the use of this procedure with other means of assessing vitamin B₆ nutritional status.
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