Non-Enzymatic Formation of <i>N</i>-acetylated Amino Acid Conjugates in Urine
Unknown <i>N</i>-acylated amino acid (<i>N</i>-AAA) conjugates have been detected in maple syrup urine disease (MSUD) and other inborn errors of metabolism (IEMs). This study aimed to elucidate the mechanism behind the formation of urinary <i>N</i>-AAA conjugates....
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MDPI AG
2023-09-01
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| Series: | Applied Sciences |
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| Online Access: | https://www.mdpi.com/2076-3417/13/18/10002 |
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| author | Jano Jacobs Cornelia Gertina Catharina Elizabeth van Sittert Lodewyk Japie Mienie Marli Dercksen Monique Opperman Barend Christiaan Vorster |
| author_facet | Jano Jacobs Cornelia Gertina Catharina Elizabeth van Sittert Lodewyk Japie Mienie Marli Dercksen Monique Opperman Barend Christiaan Vorster |
| author_sort | Jano Jacobs |
| collection | DOAJ |
| description | Unknown <i>N</i>-acylated amino acid (<i>N</i>-AAA) conjugates have been detected in maple syrup urine disease (MSUD) and other inborn errors of metabolism (IEMs). This study aimed to elucidate the mechanism behind the formation of urinary <i>N</i>-AAA conjugates. Liquid–liquid extraction was employed to determine the enantiomeric composition of <i>N</i>-AAA conjugates, followed by liberation of conjugated amino acids through acid hydrolysis. Gas chromatography–mass spectrometry (GC–MS) was used to separate amino acid enantiomers. In vitro experiments were conducted to test the non-enzymatic formation of <i>N</i>-AAA conjugates from 2-keto acids and ammonia, with molecular modelling used to assess possible reaction mechanisms. Adequate amounts of <i>N</i>-AAA conjugates were obtained via organic acid extraction without concurrent extraction of native amino acids, and hydrolysis was complete without significant racemisation. GC–MS analysis successfully distinguished amino acid enantiomers, with some limitations observed for L-isoleucine and D-alloisoleucine. Furthermore, investigation of racemic <i>N</i>-AAA conjugates from an MSUD case confirmed its non-enzymatic origin. These findings highlight the value of employing chiral strategy and molecular modelling to investigate the origin of unknown constituents in biological samples. Additionally, these conjugates warrant further investigation as potential factors contributing to MSUD and other IEMs. |
| first_indexed | 2024-03-10T23:05:51Z |
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| institution | Directory Open Access Journal |
| issn | 2076-3417 |
| language | English |
| last_indexed | 2024-03-10T23:05:51Z |
| publishDate | 2023-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Applied Sciences |
| spelling | doaj.art-cfc3f0c9ea8b4ac7852ea03133b663d12023-11-19T09:21:39ZengMDPI AGApplied Sciences2076-34172023-09-0113181000210.3390/app131810002Non-Enzymatic Formation of <i>N</i>-acetylated Amino Acid Conjugates in UrineJano Jacobs0Cornelia Gertina Catharina Elizabeth van Sittert1Lodewyk Japie Mienie2Marli Dercksen3Monique Opperman4Barend Christiaan Vorster5Centre for Human Metabolomics, North-West University, Potchefstroom 2520, South AfricaLaboratory for Applied Molecular Modelling, Research Focus Area for Chemical Resource Beneficiation, North-West University, Potchefstroom 2520, South AfricaCentre for Human Metabolomics, North-West University, Potchefstroom 2520, South AfricaCentre for Human Metabolomics, North-West University, Potchefstroom 2520, South AfricaCentre for Human Metabolomics, North-West University, Potchefstroom 2520, South AfricaCentre for Human Metabolomics, North-West University, Potchefstroom 2520, South AfricaUnknown <i>N</i>-acylated amino acid (<i>N</i>-AAA) conjugates have been detected in maple syrup urine disease (MSUD) and other inborn errors of metabolism (IEMs). This study aimed to elucidate the mechanism behind the formation of urinary <i>N</i>-AAA conjugates. Liquid–liquid extraction was employed to determine the enantiomeric composition of <i>N</i>-AAA conjugates, followed by liberation of conjugated amino acids through acid hydrolysis. Gas chromatography–mass spectrometry (GC–MS) was used to separate amino acid enantiomers. In vitro experiments were conducted to test the non-enzymatic formation of <i>N</i>-AAA conjugates from 2-keto acids and ammonia, with molecular modelling used to assess possible reaction mechanisms. Adequate amounts of <i>N</i>-AAA conjugates were obtained via organic acid extraction without concurrent extraction of native amino acids, and hydrolysis was complete without significant racemisation. GC–MS analysis successfully distinguished amino acid enantiomers, with some limitations observed for L-isoleucine and D-alloisoleucine. Furthermore, investigation of racemic <i>N</i>-AAA conjugates from an MSUD case confirmed its non-enzymatic origin. These findings highlight the value of employing chiral strategy and molecular modelling to investigate the origin of unknown constituents in biological samples. Additionally, these conjugates warrant further investigation as potential factors contributing to MSUD and other IEMs.https://www.mdpi.com/2076-3417/13/18/10002<i>N</i>-acylated amino acid conjugatesnon-enzymatic reactionsmaple syrup urine diseaseinborn errors of metabolismracemic resolutiondensity functional theory (DFT) |
| spellingShingle | Jano Jacobs Cornelia Gertina Catharina Elizabeth van Sittert Lodewyk Japie Mienie Marli Dercksen Monique Opperman Barend Christiaan Vorster Non-Enzymatic Formation of <i>N</i>-acetylated Amino Acid Conjugates in Urine Applied Sciences <i>N</i>-acylated amino acid conjugates non-enzymatic reactions maple syrup urine disease inborn errors of metabolism racemic resolution density functional theory (DFT) |
| title | Non-Enzymatic Formation of <i>N</i>-acetylated Amino Acid Conjugates in Urine |
| title_full | Non-Enzymatic Formation of <i>N</i>-acetylated Amino Acid Conjugates in Urine |
| title_fullStr | Non-Enzymatic Formation of <i>N</i>-acetylated Amino Acid Conjugates in Urine |
| title_full_unstemmed | Non-Enzymatic Formation of <i>N</i>-acetylated Amino Acid Conjugates in Urine |
| title_short | Non-Enzymatic Formation of <i>N</i>-acetylated Amino Acid Conjugates in Urine |
| title_sort | non enzymatic formation of i n i acetylated amino acid conjugates in urine |
| topic | <i>N</i>-acylated amino acid conjugates non-enzymatic reactions maple syrup urine disease inborn errors of metabolism racemic resolution density functional theory (DFT) |
| url | https://www.mdpi.com/2076-3417/13/18/10002 |
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