Identification and quantification of N ε-(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/production 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-epsilon-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. (C) 2004 Elsevier Inc. All rights. reserved.