Measurement of Nepsilon-(carboxymethyl)lysine and Nepsilon-(carboxyethyl)lysine in human plasma protein by stable-isotope-dilution tandem mass spectrometry.

BACKGROUND

N(epsilon)-(Carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL) are two stable, nonenzymatic chemical modifications of protein lysine residues resulting from glycation and oxidation reactions. We developed a tandem mass spectrometric method for their simultaneous measurement in hydrolysates of plasma proteins.

METHODS

CML and CEL were liberated from plasma proteins by acid hydrolysis after addition of deuterated CML and CEL as internal standards. Chromatographic separation was performed by gradient-elution reversed-phase chromatography with a mobile phase containing 5 mmol/L nonafluoropentanoic acid as ion-pairing agent. Mass transitions of 205.1-->84.1 and 219.1-->84.1 for CML and CEL, respectively, and 209.1-->88.1 and 223.1-->88.1 for their respective internal standards were monitored in positive-ion mode.

RESULTS

CML and CEL were separated with baseline resolution with a total analysis time of 21 min. The lower limit of quantification was 0.02 micromol/L for both compounds. Mean recoveries from plasma samples to which CML and CEL had been added were 92% for CML and 98% for CEL. Within-day CVs were <7.2% for CML and <8.2% for CEL, and between-day CVs were <8.5% for CML and <9.0% for CEL. In healthy individuals (n = 10), mean (SD) plasma concentrations of CML and CEL were 2.80 (0.40) micromol/L (range, 2.1-3.4 micromol/L) and 0.82 (0.21) micromol/L (range, 0.5-1.2 micromol/L), respectively. In hemodialysis (n = 17) and peritoneal dialysis (n = 9) patients, plasma concentrations of CML and CEL were increased two- to threefold compared with controls, without significant differences between dialysis modes [7.26 (1.36) vs 8.01 (3.80) micromol/L (P = 0.89) for CML, and 1.84 (0.39) vs 1.71 (0.42) micromol/L (P = 0.53) for CEL].

CONCLUSIONS

This stable-isotope-dilution tandem mass spectrometry method is suitable for simultaneous analysis of CML and CEL in hydrolysates of plasma proteins. Its robustness makes it suitable for assessing the value of these compounds as biomarkers of oxidative stress resulting from sugar and lipid oxidation.