Identification and quantification of N-(Hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry

The identification and quantification of N-(hexanoyl)lysine (N -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 -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 -HEL was clearly observed in urine, and the elution position coincided with the synthetic standard N -HEL. The product, estimated N -HEL, was absorbed by a specific antibody to N -HEL. Moreover, Nα-HEL, one of the plausible hexanoyl adducts from the reaction between the Nα moiety of L-lysine and the peroxidized lipid, was hardly detected in urine samples, suggesting that the origin of the N -HEL is the peroxidized lipid-modified proteins but not artificial hexanoylated L-lysine. Using the MRM technique, the amount of urinary N -HEL from the control subjects (observed healthy) was estimated to be 1.58 ± 0.23 μmol/mol of creatinine. A comparative study of the urinary N -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 -HEL in the control and diabetic urines revealed that the urinary N -HEL from diabetic subjects (3.21 ± 0.65 μmol/mol of creatinine) was significantly higher than that from the control subjects.