Specific and sensitive GC–MS analysis of hypusine, Nε-(4-amino-2-hydroxybutyl)lysine, a biomarker of hypusinated eukaryotic initiation factor eIF5A, and its application to the bi-ethnic ASOS study

Abstract

Hypusination is a unique two-step enzymatic post-translational modification of the Nε-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., Nε-(4-amino-2-hydroxybutyl)lysine. The method includes a two-step derivatization of Hyp: first esterification with 2 M HCl in CH3OH (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 CD3OD was used to prepare the internal standard. The major derivatization product was identified as the un-labelled (d0Me) and the deuterium-labelled methyl esters (d3Me) derivatives: d0Me-Hyp-(PFP)5 and d3Me-Hyp-(PFP)5, respectively. Negative-ion chemical ionization generated the most intense ions with m/z 811 for d0Me-Hyp-(PFP)5 and m/z 814 for the internal standard d3Me-Hyp-(PFP)5. 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.

Is the Novel Slot Blot a Useful Method for Quantification of Intracellular Advanced Glycation End-Products?

“…Although GC-MS was developed approximately 70 years ago, it is still used for the quantification of low-molecular weight compounds [10]. The target signal of the compounds and their degradation are detected as m/z, and the limitation of the range is 100-1000 Da [74]. Therefore, the molecular weights of the sample compounds must be less than 1000 Da.…”

“…Therefore, the molecular weights of the sample compounds must be less than 1000 Da. Furthermore, samples must be volatile when GC-MS identification is performed; the temperature conditions for detection are approximately 300-500 • C [10,74,75]. Although some types of AGEs have been detected and quantified using GC-MS, high molecular AGEs (e.g., CML-modified BSA [8], MGO-modified BSA [9], CML-modified type I collagen [71], and CML-modified HSA [71]) cannot be measured.…”

“…Although some types of AGEs have been detected and quantified using GC-MS, high molecular AGEs (e.g., CML-modified BSA [8], MGO-modified BSA [9], CML-modified type I collagen [71], and CML-modified HSA [71]) cannot be measured. Therefore, some researchers have performed hydrolysis of AGE-modified peptides or proteins and esterification of free AGEs (the low molecular compounds having the AGE structure) (Figure 6) [10,[74][75][76]. Esterification of the compounds produces the derived compounds, which must be volatile.…”

“…Esterification of the compounds produces the derived compounds, which must be volatile. A common condition of hydrolysis is treatment with 6 M hydrochloride, and esterification involves treatment with ethereal diazomethane [75] and 2 M hydrochloride/methanol [10,74]. If the condition of hydrolysis and esterification of free AGEs are successful, their retention time and mass spectrum must be previously analyzed for quantification.…”

Analysis of Crude, Diverse, and Multiple Advanced Glycation End-Product Patterns May Be Important and Beneficial

“…GC-MS has been used to perform absolute quantification of free-type AGEs [ 19 , 53 , 54 , 55 ]. However, AGE-modified proteins cannot be detected and analyzed because the conditions of GC-MS analysis (i) include a sample molecular weight range of 100–1000 Da, (ii) samples must be modified to their ester derivatives, (iii) samples should be heated and degraded at 250–500 °C, and (iv) the molecular weight of the target must be proven [ 19 , 56 ]. Therefore, AGE-modified proteins must be hydrolyzed to obtain free-type AGEs and undergo esterification to obtain free AGE-ester derivatives ( Figure 1 ) [ 19 ].…”