Identification and quantification of N(epsilon)-(Hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry.
The identification and quantification of N(epsilon)-(hexanoyl)lysine (N(epsilon)-HEL), which was found from the reactions between lipid hydroperoxide and lysine, from human urine was examined using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The N(epsilon)-HEL in the partially purified urine fraction was identified using LC/MS/MS by several approaches including precursor/product ion scans. The peak found by the multiple-reaction monitoring (MRM) of the collision-induced fragmentation of N(epsilon)-HEL was clearly observed in urine, and the elution position coincided with the synthetic standard N(epsilon)-HEL. The product, estimated N(epsilon)-HEL, was absorbed by a specific antibody to N(epsilon)-HEL. Moreover, N(alpha)-HEL, one of the plausible hexanoyl adducts from the reaction between the N(alpha) moiety of L-lysine and the peroxidized lipid, was hardly detected in urine samples, suggesting that the origin of the N(epsilon)-HEL is the peroxidized lipid-modified proteins but not artificial hexanoylated L-lysine. Using the MRM technique, the amount of urinary N(epsilon)-HEL from the control subjects (observed healthy) was estimated to be 1.58 +/- 0.23 mumol/mol of creatinine. A comparative study of the urinary N(epsilon)-HEL with an oxidative stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, showed a high correlation (r = 0.844) between the two biomarkers. Furthermore, the quantification of N(epsilon)-HEL in the control and diabetic urines revealed that the urinary N(epsilon)-HEL from diabetic subjects (3.21 +/- 0.65 mumol/mol of creatinine) was significantly higher than that from the control subjects.
Kato
Yoji
Y
School of Human Science and Environment, University of Hyogo, Himeji 670-0092, Japan. yojikato@shse.u-hyogo.ac.jp
Yoshida
Akihiro
A
Naito
Michitaka
M
Kawai
Yoshichika
Y
Tsuji
Kentaro
K
Kitamura
Masayasu
M
Kitamoto
Noritoshi
N
Osawa
Toshihiko
T
D000906:Antibodies / Q000276:immunology; D015415:Biomarkers / Q000652:urine; D002853:Chromatography, Liquid; D003920:Diabetes Mellitus / Q000175:diagnosis* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000276:immunology / Q000652:urine*; D013058:Mass Spectrometry; D018384:Oxidative Stress
D000906:Antibodies; D015415:Biomarkers; C120589:N(epsilon)-hexanoyllysine; D008239:Lysine