INVESTIGATION OF PTERINS BY LIQUID CHROMATOGRAPHY/ELECTROCHEMISTRY

CRAIG EDWARD LUNTE, Purdue University

Abstract

The pterins are a family of nitrogenous heterocyclic compounds which serve as cofactors to several hydroxylase enzymes. Although a reduced pterin is required for the hydroxylation of several amino acids intimately involved in brain function, the exact role of pterins has not been well established. This lack of knowledge is due mostly to the absence of a suitable analytical methodology for the determination of pterins. The primary goal of the work embodied in this dissertation was to develop and demonstrate a methodology that would meet the requirements of a general method of pterin determination. In recent years, the electrochemical detector for liquid chromatography has been shown to be a powerful tool in biochemical studies. The development of a general analytical method for the determination of pterins required the use of a novel electrochemical detector, the dual-electrode detector. Therefore, development of electrochemical detection techniques made possible by the dual-electrode detector was also an aim of this work. With the use of dual-electrode liquid chromatography/electrochemistry an analytical methodology was developed which is capable of detection of both the oxidized and reduced forms of several pterin species in a variety of biological samples. Detection limits of less than a picomole were achieved for all species. In addition, response was linear over several orders of magnitude. This was the first method reported with all of these capabilities. The dual-electrode detector used in the parallel-adjacent configuration was shown to be advantageous in several ways. Both oxidizable and reducible compounds can be simultaneously detected. Voltammetric information can more readily be obtained for peak identification and peak purity assessment. Finally, easily oxidized compounds can be detected more selectively in the presence of hard to oxidize analytes. The general methodology for pterin determination was used to study several problems involving pterins. A method was developed to determine phenylalanine hydroxylase activity based on the oxidation of a tetrahydropterin. The fundamental electrochemistry of the pterins was also investigated. Finally, the tautomerization of quinonoid dihydropterin was studied. Liquid chromatography/electrochemistry was shown to have unique capabilities that made these investigations possible.

Degree

Ph.D.

Subject Area

Analytical chemistry

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