Elucidating the Structure of N¹-Acetylisoputreanine: A Novel Polyamine Catabolite in Human Urine

Abstract

An untargeted metabolomics approach was utilized to determine urinary metabolites that could serve as small-molecule biomarkers for treatment response to standard tuberculosis treatment. However, the majority of metabolites that most accurately distinguished patient samples at the time of diagnosis from those at 1 month after the start of therapy lacked structural identification. The detection of unknown metabolite structures is a well-known limitation of untargeted metabolomics and underscores a need for continued elucidation of novel metabolite structures. In this study, we sought to define the structure of a urine metabolite with an experimentally determined mass of 202.1326 Da, classified as molecular feature (MF) 202.1326. A hypothesized structure of N1-acetylisoputreanine was developed for MF 202.1326 using in silico tools and liquid chromatography–tandem mass spectrometry (LC–MS/MS). In the absence of a commercial standard, synthetic N1-acetylisoputreanine was generated using enzymatic and chemical syntheses, and LC–MS/MS was used to confirm the structure of MF 202.1326 as N1-acetylisoputreanine, a proposed terminal polyamine catabolite that had not been previously detected in biological samples. Further analysis demonstrated that N1-acetylisoputreanine and an alternative form of this metabolite, N1-acetylisoputreanine-γ-lactam, are both present in human urine and are likely end-products of polyamine metabolism.

Software Tools and Approaches for Compound Identification of LC-MS/MS Data in Metabolomics

“…A clear benefit of LC-MS/MS approaches is the limited amount of material needed, in comparison to LC-MS/MS-NMR methods. A recent report annotated N 1 -acetylisoputreanine and N 1 -acetylisoputreanine-gamma-lactam by metabolic profiling and used custom synthesis to confirm the commercially unavailable metabolite [ 180 ]. Another approach used multiple-stage tandem mass spectrometry (MS 4 ) and custom synthesis to identify and confirm N , N , N -trimethyl-l-alanyl-l-proline betaine in human plasma.…”

Gender-Related Differences on Polyamine Metabolome in Liquid Biopsies by a Simple and Sensitive Two-Step Liquid-Liquid Extraction and LC-MS/MS

“…In addition, the chromatographic behavior of AcDAP (6.98 min) and AcCAD (7.54 min) with respect to 1,3-DAP (12.84 min) and CAD (13.22 min) was consistent with the behaviors of AcPUT (7.93 min) and PUT (13.81 min), for which we had commercial standards. The other tentatively identified compound was N1-AcIsoPUTR, which is an end-product of polyamine metabolism first described in urine by Fitzgerald et al [41]. The expected dansylated precursor ion was observed with an error lower than 5 ppm, and the main fragment from the loss of dansylated GABA at 100.0753 m/z [M + H-Dns-C4H9NO2(GABA)] was also detected with an error <5 ppm ( Figure 2).…”

“…The other tentatively identified compound was N1-AcIsoPUTR, which is an end-product of polyamine metabolism first described in urine by Fitzgerald et al [41]. The expected dansylated precursor ion was observed with an error lower than 5 ppm, and the main fragment from the loss of dansylated GABA at 100.0753 m/z [M + H-Dns-C 4 H 9 NO 2 (GABA)] was also detected with an error <5 ppm (Figure 2).…”

“…The expected dansylated precursor ion was observed with an error lower than 5 ppm, and the main fragment from the loss of dansylated GABA at 100.0753 m/z [M + H-Dns-C 4 H 9 NO 2 (GABA)] was also detected with an error <5 ppm (Figure 2). The product ion of 126.0906 m/z has also been described by Fitzgerald et al [41]. was an Agilent 6550 QTOF, and the chromatographic conditions were the same as those used in the targeted approach.…”

“…Here, this extraction procedure was compared with different previously described extraction protocols, all of them combined with dansyl chloride derivatization of polyamines. Moreover, we included several acetylated and diacetylated polyamines and other compounds that were recently described as catabolites of polyamine metabolism [41]. Finally, we applied the developed method to a small trial to validate the method usefulness in a clinical environment.…”