42] Isotachophoretic analysis of porphyrin precursors: 5-aminolevulinic acid derivatives

Abstract

Publisher Summary This chapter describes the technique of isotachophoresis in separation and quantification of 5-Aminolevulinic acid (ALA) derivatives. 5-Aminolevulinic acid (ALA) and porphobilinogen (PBG) are important precursors in the biosynthesis of tetrapyrroles such as hemes, corrins, bilins, and chlorophylls and are found to be present widely in living tissues. The isolation and quantitative determination of ALA derivatives in urine and other tissues have always been complex and time consuming. This is mainly due to the necessity of prior separation of individual compounds before colorimetric determination because of the interference of other precursors and impurities. Isotachophoresis is an electrophoretic technique that can be used for the qualitative analysis of charged molecules in a discontinuous electrolyte system. Thus, the method can readily be applied for the simultaneous analysis of chemically analogous compounds. Isotachophoresis is carried out with a Model IP-2A equipped with a potential gradient detector. The isotachopherograms of ALA derivatives both in anion and cation analyses are shown. With respect to the separation of levulinic acid, a competitive inhibitor of PBG synthase (formerly ALA dehydratase, EC 4.2.1.24), complete resolution of ALA and levulinic acid (PU value = 0.109) is obtained in anion analysis, but separation of 4,5-dioxovaleric acid and levulinic acid is unsuccessful.