# Non-Enzymatic Formation of \(N\)-acetylated Amino Acid Conjugates in Urine
###### Abstract
It is believed that they play a crucial role in salvaging acetyl-CoA and enhancing the solubility of carboxylic acid intermediates for excretion in urine [18]. In aminoacidopathies, transferases catalyse the acetylation of amino acids to form \(N\)-acetylated amino acid (\(N\)-AcAA) conjugates [11; 19; 20; 21]. In contrast, in organic acidemias  
Figure 1: Overview of induced metabolites detected in urine of maple syrup urine disease (MSUD) cases by Dry [7]. The figure includes identification of unknown \(N\)-acylated amino acid (\(N\)-AAA) conjugates observed in three variant MSUD cases. Adapted from [PERSON] [7]. and fatty acid oxidation disorders GLYAT conjugates various acyl-CoA molecules, predominantly with GLY and other amino acids [7; 22; 23; 24; 25; 26; 27; 28; 29]. Aminoacidopathies thus exhibit elevated levels of \(N\)-AcAA conjugates corresponding to the increased amino acids, while organic acidemias exhibit elevated levels of \(N\)-acyl-Gly conjugates corresponding to the elevated acyl-CoA molecules. However, exceptions to this pattern include \(N\)-phenylacetyl-glutamine (-Gln) in phenylketonuria, \(N\)-isovaleryl-glutamate (-Glu), and -alanine (-Ala) in isovaleric acidemia, as they are products of GLYAT-mediated biotransformation [12; 22; 30]. Similarly, the presence of \(N\)-acylated BCAA conjugates in the above-mentioned variant MSUD cases deviates from the expected pattern [6; 7]. Previous studies by [PERSON] [7] and [PERSON] [6] have investigated and proposed hypotheses to elucidate the formation of these \(N\)-AAA conjugates in the mentioned cases. Although the biotransformation of \(N\)-acylated-CoAs with BCAAs by the GLYAT enzyme seemed a plausible explanation, the presence of \(N\)-AAA conjugates in MSUD cases was unexpected as \(N\)-acyl-glycine conjugates were not observed [7; 22]. Moreover, as can be seen in Figure 2, the enzyme defect in MSUD prevented or depleted the formation of apparent GLYAT substrates (\(N\)-isobutyryl-, \(N\)-isovaleryl-, and \(N\)-2-methylbutylryl-CoA) [6; 7]. [PERSON] [6] proposed a more likely hypothesis, suggesting the non-enzymatic formation of \(N\)-AAA conjugates from 2-keto acids and ammonia. This hypothesis built upon the mechanism proposed by [PERSON] et al. [31], which demonstrated that pyruvate and glyoxylic acid react with ammonium sulphate under specific conditions, resulting in a conjugate that yields amino acids upon hydrochloric acid (HCI) hydrolysis [31]. Figure 2: Summary of metabolomic obstructions caused by maple syrup urine disease (MSUD) indicating the prevention of typical GLYAT enzyme-substrate formation, acetyl-CoAs. The known enzymatic and non-enzymatic formations of \(N\)-AAA conjugates are also indicated. BCAA: branched-chain amino acid; BCKDH: branched-chain \(\alpha\)-keto acid dehydrogenase complex. However, the formation mechanism of this unique metabolomic profile and the clinical-biochemical effects of the hydantoin and \(N\)-AAA conjugates were deemed unclear. Understanding the origin of these constituents could provide insights into an individual's disorder/condition, shed light on unknown metabolic pathways, and troubleshoot potential issues with diagnostic methods. Studying variant metabolomic presentations is essential for: (1) further comparison of clinical-biochemical characteristics, (2) discovery of new diagnostic markers, (3) assessing responsiveness to treatment, (4) improving therapeutic approaches, and (5) expanding our knowledge of metabolic mechanisms, which may have implications beyond the specific disease context. Motivated by the confirmation of the alternative mechanism of \(N\)-AAA formation proposed by [PERSON] [7] and [PERSON] [6], this study aims to investigate and validate this mechanism. In this study, our aim was to investigate the formation of \(N\)-AAA conjugates and determine whether this process occurs enzymatically or non-enzymatically. To achieve this, we pursued several objectives. Firstly, we sought to determine the enantiomeric composition of \(N\)-AAA conjugates using chiral analysis.