A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism |
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| Authors: | Shunsuke Moriya Kaori Iwasaki Keijiro Samejima Koichi Takao Kohfuku Kohda Kyoko Hiramatsu Masao Kawakita |
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| Affiliation: | 1. Department of Molecular Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506, Japan;2. Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan;3. Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo, Tokyo 202-8585, Japan |
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| Abstract: | An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N1-acetylspermidine (N1AcSpd), N8-acetylspermidine (N8AcSpd), N1-acetylspermine, N1,N8-diacetylspermidine, and N1,N12-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N1AcSpd and N8AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with 13C2-N1AcSpd and 13C2-N8AcSpd which have the 13C2-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N1-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N1-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-15N3]-N1-acetylspermine and [1,4,8-15N3]spermidine (15N3-Spd), respectively; for SMO, [1,4,8,12-15N4]spermine and 15N3-Spd, respectively; and for SSAT, 15N3-Spd and [1,4,8-15N3]-N1-acetylspermidine, respectively. |
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| Keywords: | SSAT, spermidine/spermine N1-acetyltransferase APAO, N1-acetylpolyamine oxidase SMO, spermine oxidase N1AcSpd, N1-acetylspermidine N8AcSpd, N8-acetylspermidine N1AcSpm, N1-acetylspermine DiAcSpd, N1,N8-diacetylspermidine DiAcSpm, N1,N12-diacetylspermine AcSpd, a mixture of N1AcSpd and N8AcSpd 15N2-Put, [1,4-15N2]putrescine 13C2,15N2-Put, [1,4-13C2,1,4-15N2]putrescine 15N3-Spd, [1,4,8-15N3]spermidine 15N2,D4-Spd, N-(3-aminopropyl)-1,4-15N2-butanediamine-1,1,4,4-d4 15N4-Spm, [1,4,9,12-15N4]spermine 15N4,D4-Spm, N,N&prime -(3-aminopropyl-15N)-1,4-15N2-butanediamine-1,1,4,4-d4 15N3-N1AcSpd, [1,4,8-15N3]-N1-acetylspermidine 15N3-DiAcSpd, [1,4,8-15N3]-N1,N8-diacetylspermidine 15N4-DiAcSpm, [1,4,9,12-15N4]-N1,N12-diacetylspermine 13C2-N8AcSpd, [acetyl-1,2-13C2]-N8-acetylspermidine 13C2-N1AcSpd, [acetyl-1,2-13C2]-N1-acetylspermidine 13C3,15N3-N1AcSpd, [acetyl-2-13C,5,8-13C2,1,4,8-15N3]-N1-acetylspermidine 15N3-N1AcSpm, [4,9,12-15N3]-N1-acetylspermine HFB, heptafluorobutylic RSD, relative standard deviation MDL72527, an inhibitor of APAO |
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