Non-enzymatic formation of N-acylated amino acid conjugates in urine

Abstract

Comprehensive metabolic profiling is routinely utilised for the diagnosis of inborn errors of metabolism (IEMs). Observed disease-specific constituents are mostly due to induced secondary pathways, resulting in the build-up of these metabolites, which may contribute to a phenotypic presentation. There are however cases in which no enzymatic pathway is identified as a contributing factor. This is true for some N-acylated amino acid (N-AAA) conjugates, reported in isolated cases of maple syrup urine disease (MSUD). The aim of this study was to identify a potential mechanism for the formation of N-AAA conjugates, identified in urine of South African MSUD patients. Emphasis was placed on the general stereo-nonspecific nature of non-enzymatic reactions yielding racemic mixtures. A strategic approach was subsequently employed in which the enantiomeric composition of N-AAA conjugates was determined, to establish the origin of these compounds. Several applications were utilised to identify the enantiomeric composition of the N-AAA conjugates. These included (1) a liquid-liquid extraction of N-AAA conjugates, followed by acid-hydrolysis to liberate the conjugated amino acids, (2) the separation of amino acid enantiomers by chiral derivatisation via gas chromatography-mass spectrometry (GC-MS) and (3) molecular modelling to assess the reaction mechanism for the non-enzymatic formation from 2-keto acids and ammonia. The organic acid extraction method yielded adequate amounts of N-AAA conjugates without concomitant extraction of native amino acids. Hydrolysis was complete without significant hydrolysis-induced racemisation. Amino acid enantiomers were distinguishable through GC-MS analysis with limitations noted in L-isoleucine and D-allo-isoleucine. After standardisation of the methods, this chiral strategy was employed to investigate an available MSUD case, which was found to contain racemic N-AAA conjugates. From the results, it was deduced that the N-AAA conjugates were indeed from non-enzymatic origin. The findings also illustrate the usefulness of a chiral strategy and molecular modelling in investigating the origin of unknown constituents in biological samples. These conjugates can now be studied as a potential disease contributing factor in MSUD and other IEMs.