Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany.
Medical Research Centre, University Hospital, University Duisburg-Essen, Hufelandstrasse 55, 45128, Essen, Germany.
Amino Acids. 2022 Jul;54(7):1083-1099. doi: 10.1007/s00726-022-03142-8. Epub 2022 Mar 3.
Hypusination is a unique two-step enzymatic post-translational modification of the N-amino group of lysine-50 of the eukaryotic initiation factor 5A (eIF5A). We developed a specific and sensitive gas chromatography-mass spectrometry (GC-MS) method for the measurement of biological hypusine (Hyp), i.e., N-(4-amino-2-hydroxybutyl)lysine. The method includes a two-step derivatization of Hyp: first esterification with 2 M HCl in CHOH (60 min, 80 °C) to the methyl ester (Me) and then acylation with penta-fluoro-propionic (PFP) anhydride in ethyl acetate (30 min, 65 °C). Esterification with 2 M HCl in CDOD was used to prepare the internal standard. The major derivatization product was identified as the un-labelled (dMe) and the deuterium-labelled methyl esters (dMe) derivatives: dMe-Hyp-(PFP) and dMe-Hyp-(PFP), respectively. Negative-ion chemical ionization generated the most intense ions with m/z 811 for dMe-Hyp-(PFP) and m/z 814 for the internal standard dMe-Hyp-(PFP). Selected-ion monitoring of m/z 811 and m/z 814 was used in quantitative analyses. Free Hyp was found in spot urine samples (10 µL) of two healthy subjects at 0.60 µM (0.29 µmol Hyp/mmol creatinine) in the female and 1.80 µM (0.19 µmol Hyp/mmol creatinine) in the male subject. The mean accuracy of the method in these urine samples spiked with 1-5 µM Hyp was 91-94%. The limit of detection (LOD) of the method is 1.4 fmol Hyp. The method was applied to measure the urinary excretion rates of Hyp in healthy black (n = 38, age 7.8 ± 0.7 years) and white (n = 41, age 7.7 ± 1.0 years) boys of the Arterial Stiffness in Offspring Study (ASOS). The Hyp concentrations were 3.55 [2.68-5.31] µM (range 0.54-9.84 µM) in the black boys and 3.87 [2.95-5.06] µM (range 1.0-11.7 µM) in the white boys (P = 0.64). The creatinine-corrected excretion rates were 0.25 [0.20-0.29] µmol/mmol (range 0.11-0.36 µmol/mmol) in the black boys and 0.26 [0.21-0.30] µmol/mmol (range 0.10-0.45 µmol/mmol) in the white boys (P = 0.82). These results suggest that there is no ethnic-related difference in the ASOS population in the eIF5A modification. Remarkable differences were found between black and white boys with respect to correlations of urinary Hyp with amino acids and advanced glycation end-products of Lys, Arg and Cys. Deoxyhypusine, formally the direct precursor of Hyp, seems not to be excreted in the urine by healthy subjects.
Hypusination 是真核起始因子 5A（eIF5A）赖氨酸-50 的 N-氨基的独特两步酶促翻译后修饰。我们开发了一种用于测量生物 Hypusine（Hyp）的特异性和灵敏性气相色谱-质谱（GC-MS）方法，即 N-（4-氨基-2-羟基丁基）赖氨酸。该方法包括 Hyp 的两步衍生化：首先用 2 M HCl 在 CHOH 中酯化（60 分钟，80°C）成甲酯（Me），然后用五氟丙酸酐在乙酸乙酯中酰化（30 分钟，65°C）。用 2 M HCl 在 CDOD 中酯化用于制备内标。主要的衍生化产物被鉴定为未标记（dMe）和氘标记的甲酯（dMe）衍生物：dMe-Hyp-（PFP）和 dMe-Hyp-（PFP），分别。负离子化学电离产生最强烈的离子，m/z 811 用于 dMe-Hyp-（PFP），m/z 814 用于内标 dMe-Hyp-（PFP）。m/z 811 和 m/z 814 的选择离子监测用于定量分析。在两名健康受试者的尿液样本（10 μL）中发现了游离 Hyp，女性受试者为 0.60 μM（0.29 μmol Hyp/mmol 肌酐），男性受试者为 1.80 μM（0.19 μmol Hyp/mmol 肌酐）。该方法在这些尿液样本中加入 1-5 μM Hyp 的平均准确度为 91-94%。该方法的检测限（LOD）为 1.4 fmol Hyp。该方法用于测量动脉硬度后代研究（ASOS）中健康的黑人（n = 38，年龄 7.8 ± 0.7 岁）和白人（n = 41，年龄 7.7 ± 1.0 岁）男孩的 Hyp 排泄率。黑人男孩的 Hyp 浓度为 3.55 [2.68-5.31] μM（范围 0.54-9.84 μM），白人男孩的 Hyp 浓度为 3.87 [2.95-5.06] μM（范围 1.0-11.7 μM）（P = 0.64）。黑人男孩的肌酐校正排泄率为 0.25 [0.20-0.29] μmol/mmol（范围 0.11-0.36 μmol/mmol），白人男孩的肌酐校正排泄率为 0.26 [0.21-0.30] μmol/mmol（范围 0.10-0.45 μmol/mmol）（P = 0.82）。这些结果表明，ASOS 人群中 eIF5A 修饰没有种族相关的差异。黑人男孩和白人男孩之间的 Hyp 与氨基酸和赖氨酸、精氨酸和半胱氨酸的高级糖基化终产物的相关性存在显著差异。脱氧 Hypusine，正式地说是 Hyp 的直接前体，似乎没有被健康受试者排泄在尿液中。
