# Non-Enzymatic Formation of \(N\)-acetylated Amino Acid Conjugates in Urine
## 4 Discussion  
The metabolic significance of \(N\)-AAA conjugates is evident due to their involvement in various biological processes such as protein function [16], urea cycle regulation [15], myelin synthesis [17], biotransformation, medicine [44], and their association with certain IEMs. Enzymatic synthesis of \(N\)-AAA conjugates occurs through acyltransferases and their catabolism involves aminoacylases [16], predominantly yielding L-amino acid enantiomers [45]. Additionally, \(N\)-AAA conjugates have been observed in three variant MSUD cases [6;7], and the non-enzymatic synthesis of \(N\)-AAA conjugates has been proposed [31]. Enzymatic reactions exhibit high selectivity and are subject to stringent control mechanisms for maintaining homeostasis and targeted product synthesis [37]. In contrast, non-enzymatic reactions are unregulated, which can have supplementary or toxic implications for metabolism [37]. Therefore, it is important to differentiate between enzymatic and non-enzymatic reactions due to their varying impacts. The chiral analytical approach has been successful in distinguishing between enantiomers by exploiting the stereo-nonspecific nature of non-enzymatic reactions and the formation of racemic mixtures from prochiral substrates [46]. In our case study, the chiral analytical strategy effectively determined that the observed \(N\)-AAA conjugates consisted of both L- and D-enantiomers, indicating a non-enzymatic origin. This approach relied on specific steps: (1) urinary extraction of \(N\)-AAA conjugates without co-extraction of native BCAAs to avoid pseudo-enantiomeric excess, (2) hydrolysis of \(N\)-AAA conjugates without significant racemisation, and (3) conversion of extracted BCAAs to volatile diastereomers for separation via GC-MS without inducing racemisation during derivatisation. Through this qualitative approach, the enantiomeric composition of \(N\)-AAA conjugates could be determined, revealing their enzymatic or non-enzymatic origin. Whether this happens in vivo or in vitro merits further discussion following the results from molecular modelling. To synthesise \(N\)-AAA conjugates non-enzymatically, we incubated various 2-keto acids at pH < 2 and 37 \({}^{\circ}\)C. Although these conditions are unlikely to occur in vivo, they align with in vitro reaction conditions during sample preparation for organic acid analysis. However, the in vivo non-enzymatic formation of \(N\)-AAA conjugates cannot yet be ruled out, because [PERSON] et al. [31] synthesised \(N\)-Ac-Ala non-enzymatically at a temperature of 27 \({}^{\circ}\)C and a more acceptable pH of 4. While both our study and the work by [PERSON] et al. [31] achieved only low yields of \(N\)-AAA conjugates, the presence of these conjugates in an enzyme-free environment supports the occurrence of non-enzymatic reactions. Furthermore, the proposed reaction mechanism was confirmed, as evidenced by the predicted products being produced. It is important to note that this method is only valid when appropriate starting substrates are chosen, as non-enzymatic synthesis of \(N\)-AAA conjugates from acyl-chlorides and amino acids would not have provided meaningful results given that acyl-chlorides are not metabolites. In this study, molecular modelling successfully validated the proposed reaction mechanism and determined the energy requirements (E\({}_{\text{a}}\)). Although the calculated E\({}_{\text{a}}\) was relatively high compared to standard in vivo enzymatic reactions, it corresponded to the low yields observed during chemical synthesis. These results support the hypothesis of a slow in vivo reaction that may be facilitated by an unidentified catalyst or more appropriate reaction conditions, or they may indicate that the formation of \(N\)-AAA conjugates requires an acceptably low amount of energy during routine sample handling. The results neither confirm nor disprove the occurrence of non-enzymatic reactions in vivo or in vitro, but they support the hypothesis of non-enzymatic formation of \(N\)-AAA conjugates in urine.