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Clinical Chemistry 50: 1222-1228, 2004. First published May 6, 2004; 10.1373/clinchem.2004.031286
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(Clinical Chemistry. 2004;50:1222-1228.)
© 2004 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Measurement of N{epsilon}-(Carboxymethyl)lysine and N{epsilon}-(Carboxyethyl)lysine in Human Plasma Protein by Stable-Isotope-Dilution Tandem Mass Spectrometry

Tom Teerlink1,2,a, Rob Barto1, Herman J. ten Brink1 and Casper G. Schalkwijk1,2

1 Department of Clinical Chemistry and 2 Institute of Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands.

aAddress correspondence to this author at: Department of Clinical Chemistry, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Fax 31-204443895; e-mail t.teerlink{at}vumc.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 µmol/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) µmol/L (range, 2.1–3.4 µmol/L) and 0.82 (0.21) µmol/L (range, 0.5–1.2 µmol/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) µmol/L (P = 0.89) for CML, and 1.84 (0.39) vs 1.71 (0.42) µmol/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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glycation, or nonenzymatic glycosylation, is the nonenzymatic reaction of glucose and other reducing sugars with amino groups of proteins. The amino groups of the side chains of arginine and lysine are the primary targets for this type of posttranslational modification. Over time, the initial glycation products may undergo intramolecular rearrangements and oxidation reactions (glycoxidation) and ultimately transform into stable, so-called advanced glycation end products (AGEs).1 Likewise, peroxidation of lipids may lead to the formation of reactive carbonyl compounds that react with proteins, leading to the formation of advanced lipoxidation end products (ALEs). AGEs and ALEs play an important role in age-related pathologies, atherosclerosis, and diabetes [for a review, see Ref. (1)].

N{epsilon}-(Carboxymethyl)lysine (CML), one of the best-characterized of these compounds, can be regarded as either an AGE or an ALE because it can be formed on proteins by both glycoxidation and lipid peroxidation pathways (2). N{epsilon}-(Carboxyethyl)lysine (CEL) is a homolog of CML that is formed by the reaction of lysine residues in proteins with methylglyoxal as well as with triose phosphates and other sugars (3). CML and CEL are two major nonenzymatic chemical modifications on tissue proteins that can serve as biomarkers of oxidative stress resulting from sugar and lipid oxidation.

Antisera against well-defined AGEs such as CML are a valuable tool for assessing tissue distribution of AGEs by immunohistochemical techniques (4)(5). Immunoassays are often used for the quantification of AGEs, but for several reasons the use of antisera for quantitative immunoassays of protein-bound AGEs is questionable. One reason is that the specificity of the antibodies is often difficult to define with certainty and no monospecific antibodies are commercially available. Another reason is that proteins used to block nonspecific binding in immunoassays may also contain AGE epitopes and thus interact with the antibody. In addition, because of steric constraints, not all AGE epitopes on the protein may be available for interaction with the antibody. Finally, there is evidence for the presence in plasma of factors competing for the reaction between the anti-AGE antibody and its antigen. These factors include anti-AGE autoantibodies (6) and, possibly, complement (7). AGE immunoassays may thus yield only semiquantitative results. A better approach for the quantitative determination of specific AGE epitopes in proteins is to use a specific analytical technique for the analysis of these AGEs in protein hydrolysates. A major restriction of this approach is that not all AGE epitopes are stable during the harsh conditions used for protein hydrolysis. In these cases, the protein can be cleaved enzymatically. Fortunately, both CML and CEL are stable under the conditions used for acid protein hydrolysis.

CML and CEL have been analyzed by cation-exchange chromatography with postcolumn ninhydrin derivatization (8) and CML by reversed-phase HPLC with fluorescence detection after precolumn derivatization with o-phthaldialdehyde reagent (9). 6-Aminoquinolyl-N-hydroxysuccinimidyl-carbamate, a derivatization reagent that yields more stable derivatives, has been used for the analysis of early glycation products and AGEs, including CML and CEL, in human serum albumin (10)(11). These analytical techniques are suitable for the detection of AGEs in proteins after in vitro glycation, but the detection limits are usually too high for the quantification of endogenous AGEs in plasma proteins. Because of its very high selectivity, mass spectrometry is often used for the analysis of specific AGEs. Gas chromatography–mass spectrometry has been widely used for the analysis of CML and CEL in urine and protein hydrolysates (3)(12)(13)(14)(15). This analytical technique requires derivatization to increase the volatility of these highly polar compounds.

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is currently the method of choice for the analysis of nonvolatile compounds. One major advantage over gas chromatography–mass spectrometry is that usually no derivatization step is required. Most often, the analyte is separated from interfering compounds by reversed-phase chromatography. Because CML and CEL are very hydrophilic, they show little retention on reversed-phase columns. This may lead to coelution of these analytes with polar matrix components, which causes loss of sensitivity by reduced ionization as a result of ion-suppression effects (16). This problem can be overcome by increasing the retention by use of ion-pairing agents. The ion-pairing agents commonly used in chromatography are often incompatible with MS detection because these compounds are nonvolatile and severely reduce sensitivity. However, perfluorinated carboxylic acids are a class of volatile ion-pairing agents that has been found to be suitable for the analysis of amino acids by MS/MS in positive-ionization mode (17)(18)(19). We used this approach to develop a stable-isotope-dilution LC-MS/MS method for the simultaneous analysis of underivatized CML and CEL in plasma protein hydrolysates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
instrumentation
All LC-MS/MS analyses were performed on an API 3000 triple-quadrupole tandem mass spectrometer (Sciex-Applied Biosystems) interfaced with a TurboIonSpray source. The LC system consisted of a Series 200 autosampler, a Series 200 quaternary pump, and a Series 200 inline degasser (Perkin-Elmer). Mass spectra were acquired and processed with Analyst, Ver. 1.3.1, software (Sciex-Applied Biosystems). Mass spectrometry conditions were optimized by direct infusion of analytes into the mass spectrometer source with a syringe pump from Harvard Apparatus. For hydrolysis and evaporation of samples, we used a Reacti-Therm heating module combined with Reacti-Therm evaporator (Pierce).

materials
Nonafluoropentanoic acid (NFPA) was obtained from Sigma-Aldrich, trichloroacetic acid from Baker, boric acid and hydrochloric acid from Merck, and sodium borohydride from Fluka. All of these reagents were of analytical grade. HPLC-grade acetonitrile was from BDH. D,L-Lysine-4,4,5,5-2H4 · 2 HCl (99% 2H4) was from CDN Isotopes.

plasma samples
We used plasma samples from healthy volunteers (laboratory personnel) and dialysis patients participating in ongoing trials in the Department of Nephrology of the VU University Medical Center. The local ethics committee approved these trials, and all participants gave informed written consent.

synthesis and purification of cml, cel, and labeled analogs
Synthesis of CML, CEL, and their 2H4-labeled analogs was essentially as described for biocytin (20). Briefly, lysine (or 2H4-lysine) was converted to its copper complex to protect the {alpha}-amino group. This copper chelate was treated with 1.5 equivalents of 2-bromoacetic acid (for CML) or 2-bromopropionic acid (for CEL) in 1 mol/L NaOH for 3 days at room temperature. The pH was adjusted to 2 with HCl, copper was removed by treatment with hydrogen sulfide and filtration, and the resulting clear solution was dried under a stream of nitrogen. In addition to the desired product, the residue contained various amounts of unreacted lysine and dialkylated lysine, as determined by proton nuclear magnetic resonance.

CML, CEL, and their deuterated analogs were purified by preparative HPLC on a SymmetryPrep C18 column (7.8 x 300 mm; 7-µm particle size; Waters) with a mobile phase of 150 mL/L acetonitrile containing 5 mmol/L NFPA at a flow rate of 3.0 mL/min. Fractions (3-mL) were collected, and their purity was assessed by analytical LC-MS/MS as described below. Fractions in which the peak area of the analyte, identified by its retention time, accounted for >99% of total peak area were pooled and dried under reduced pressure. The isotopic purities of deuterated CML and CEL were 99.7% and 99.1%, respectively.

sample preparation
Plasma was centrifuged (20 000g for 5 min) to remove debris. A 25-µL portion of the supernatant was mixed with 100 µL of water in a 10-mL glass tube with a Teflon-lined screw-cap. After addition of 500 µL of 100 mmol/L sodium borohydride dissolved in 200 mmol/L borate buffer (pH 9.2), the samples were incubated for 2 h at room temperature. Proteins were then precipitated by addition of 2 mL of 200 g/L trichloroacetic acid and pelleted by centrifugation (2000g for 10 min). The supernatant was carefully removed by aspiration with a Pasteur pipette. The protein pellet was washed once by resuspension in 1 mL of 100 g/L trichloroacetic acid and centrifuged (2000g for 10 min). After removal of the supernatant as described above, 50 µL of a combined internal standard solution (containing 12.5 µmol/L 2H4-CML and 12.5 µmol/L 2H4-CEL dissolved in water) and 500 µL of 6 mol/L hydrochloric acid were added. Samples were hydrolyzed for 20 h at 110 °C in a heating block. The hydrolyzed samples were evaporated to dryness at 80 °C under a stream of nitrogen gas. The residue was dissolved in 500 µL of NFPA (5 mmol/L) and transferred to 1.5-mL Eppendorf vials. After centrifugation (20 000g for 10 min), 150 µL of the supernatant was transferred to an autosampler vial for LC-MS/MS analysis, and the remainder was stored at –20 °C.

calibrators
Stock solutions containing 0, 0.25, 1.0, 2.5, 10, and 25 µmol/L CML and CEL were prepared in water. The internal standard solution contained 12.5 µmol/L 2H4-CML and 12.5 µmol/L 2H4-CEL in water. Calibrators were prepared by mixing 100 µL of the respective stock solutions with 100 µL of the internal standard solution and 800 µL of a solution of 5 mmol/L NFPA in water.

lc-ms/ms conditions
Samples were analyzed at ambient temperature by reversed-phase HPLC on a Symmetry C18 column (3.9 x 150 mm; 5-µm particle size; Waters) with NFPA as the ion-pairing agent. Mobile phase A consisted of aqueous NFPA (5 mmol/L), and mobile phase B was acetonitrile. Compounds were eluted with a linear gradient from 10% to 25% B in 10 min. Thereafter, strongly retained compounds were removed from the column by a strong solvent wash: between 10 and 12 min, mobile phase B was increased to 80% and maintained at this percentage until 14 min. Between 14 and 16 min, the percentage of mobile phase B was reduced to initial conditions and maintained at this percentage for 5 min to equilibrate the column before the next injection at 21 min. The injection volume was 10 µL. The flow rate was 1 mL/min, and ~20% of the column effluent was introduced into the ion-spray source of the tandem mass spectrometer by means of a postcolumn T-piece. Analyses were performed in positive-ion mode. The ion-spray voltage was set at 4500 V, and the declustering and focusing potentials were set at 20 V and 240 V, respectively. The temperature of the turbo ion gas was 500 °C. Each mass transition was monitored for 150 ms with a 5-ms interchannel delay time, giving a cycle time of 930 ms. For both CML and CEL, the product ion at m/z 84.1 was used for quantification and the product ion at m/z 130.1 for confirmation. In each series, five calibration samples spanning the concentration ranges of 0–2.5 µmol/L for CML and 0–1.0 µmol/L for CEL were included. Calibration curves for CML and CEL, obtained by linear regression of a plot of the analyte/internal standard peak-area ratio vs analyte concentration, were used to calculate concentrations in plasma samples. Both low-and high-concentration quality-control samples were included in each analytical series.

statistics
Statistical analyses were performed with SPSS 10.0 for Windows. Significance of differences between groups was tested by the nonparametric Mann–Whitney test for independent samples. Spearman correlation coefficients were calculated to assess associations between variables. A two-tailed probability <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mass spectra
Product-ion mass spectra of the collision-induced decomposition of CML, CEL, and both deuterated internal standards are shown in Fig. 1 . The monoisotopic masses of CML and CEL are 204.11 and 218.12, respectively. The base peaks observed at m/z 205.1 and 219.1 in the spectra of CML and CEL, respectively, were thus consistent with the expected masses of the singly charged [M+H]+ species. Compared with the nondeuterated compounds, the masses of the protonated ions of the deuterated internal standards 2H4-CML and 2H4-CEL were shifted to a higher mass by 4 atomic mass units. The mass transitions used for multiple-reaction monitoring and the corresponding collision energies are listed in Table 1 , together with proposed neutral fragment losses associated with the formation of these product ions. The most intense product ion in the spectra of both CML and CEL was observed at m/z 84.1. After we optimized the intensity of this m/z 84.1 product ion by varying the collision energy, it was used for quantification. Likewise, the corresponding m/z 88.1 product ion of both internal standards was used for quantification. The less prominent product ion of m/z 130.1 in the spectra of both CML and CEL was used for confirmation of peak identity and purity in plasma samples.



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  		Figure 1. Product-ion mass spectra for calibrators and internal standard solutions.

Spectra were obtained by collision-induced fragmentation of the [M+H]+ precursor ions of m/z 205.1 for CML (A), m/z 209.1 for the internal standard 2H4-CML (B), m/z 219.1 for CEL (C), and m/z 223.1 for the internal standard 2H4-CEL (D). The spectra were recorded by direct infusion of 1 µmol/L solutions of the analytes into the source of the mass spectrometer at a flow rate of 20 µL/min.


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  		Table 1. Mass spectrometric settings for multiple-reaction monitoring of CML and CEL.

chromatography
During initial experiments, in which the analytes were separated by reversed-phase chromatography without ion-pairing agent, we observed a very low signal, probably attributable to ion suppression by early-eluting polar compounds. Increasing the retention times of the analytes by inclusion of the perfluorinated carboxylic acid NFPA as ion-pairing agent in the mobile phase solved this problem. Under these conditions, the peak areas of both internal standards did not differ between calibrators and plasma samples, indicating the absence of significant ion suppression. Typical mass chromatograms of a hydrolyzed plasma sample are shown in Fig. 2 . CML, CEL, and the internal standards eluted as symmetric peaks and were clearly resolved from interfering compounds. In general, the mass chromatographic traces of the CEL channel showed more additional peaks than were present in the CML channel, but no interference with the authentic CML and CEL peaks was ever observed. This was confirmed by the fact that the ratio of the m/z 84.1 and 130.1 product ions of the CML and CEL peaks in plasma hydrolysates was identical to the ratio observed for the peaks of the calibrators. Total analysis time, including a strong solvent wash to elute strongly retained compounds and reequilibration of the column, amounted to 21 min.



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  		Figure 2. LC/MS/MS spectra of a plasma sample obtained by multiple-reaction monitoring in positive-ion mode.

(Top to bottom), mass chromatograms of the following transitions: 205.1->84.1 for CML (A); 209.1->88.1 for the internal standard 2H4-CML (B); 219.1->84.1 for CEL (C); and 223.1->88.1 for the internal standard 2H4-CEL (D).

linearity and lower limit of quantification
After initial experiments revealed that acid hydrolysis of calibrator solutions did not affect the peak areas of CML and CEL, the calibrators were used without the hydrolysis step.

Calibration curves, obtained by linear regression of a plot of the analyte/internal standard peak-area ratio (y) vs analyte concentration (x), were linear over the range of concentrations tested (0–2.5 µmol/L for CML and 0–1.0 µmol/L for CEL), with correlation coefficients >0.999. The following regression lines were found: y = (9.41 (0.10)x – 0.028 (0.051) for CML; and y = 6.27 (0.19)x – 0.014 (0.091) for CEL.

The lower limits of quantification, at a signal-to-noise ratio of 10, were 0.2 pmol on column for both CML and CEL, corresponding to a concentration of 0.02 µmol/L.

recovery and precision
The recoveries of exogenous CML and CEL added to a plasma pool were determined at three concentrations (Table 2 ). The mean recoveries were 92% (CV, 2.2–4.3%) for CML and 98% (CV, 3.1–9.8%) for CEL. For quality control, plasma pools from healthy individuals (low-concentration) and hemodialysis patients (high-concentration), stored in aliquots at –20 °C, were included in each series of samples. The precision data (Table 3 ) showed that within-day CVs were <7.2% for CML and <8.2% for CEL. Between-day CVs were <8.5% for CML and <9.0% for CEL.


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  		Table 2. Accuracy data for the LC-MS/MS analysis of CEM and CEL.


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  		Table 3. Precision data for the LC-MS/MS analysis of CEM and CEL.

cml and cel in human plasma
The CML and CEL concentrations in hydrolysates of plasma proteins from healthy controls (n = 10), hemodialysis patients (n = 17), and peritoneal dialysis patients (n = 9) are shown in Fig. 3 . In healthy controls, mean (SD) CML and CEL concentrations were 2.80 (0.40) µmol/L and 0.82 (0.21) µmol/L, respectively. Compared with the healthy controls, both CML and CEL were significantly increased in both groups of dialysis patients (P <0.001 for both CML and CEL), without significant differences between patients on hemodialysis or peritoneal dialysis [7.26 (1.36) µmol/L vs 8.01 (3.80) µmol/L (P = 0.89) for CML, and 1.84 (0.39) µmol/L vs 1.71 (0.42) µmol/L (P = 0.53) for CEL]. In addition, CML and CEL concentrations did not differ between dialysis patients with and without diabetes mellitus [8.06 (3.36) µmol/L vs 7.18 (1.68) µmol/L (P = 0.62) for CML, and 1.78 (0.40) µmol/L vs 1.80 (0.41) µmol/L (P = 0.87) for CEL]. CML and CEL concentrations were not significantly correlated with each other or with serum creatinine in either the controls or the dialysis patients.



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  		Figure 3. CML (A) and CEL (B) concentrations in plasma of healthy controls, hemodialysis patients, and peritoneal dialysis patients.

Data are presented as the mean (SD; error bars).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The described MS/MS method is suitable for the simultaneous analysis of CML and CEL in hydrolysates of plasma proteins with high precision and accuracy. Sample pretreatment is straightforward and simple to perform on large numbers of samples. Hands-on time for a series of 50 samples (40 plasma samples and 10 calibrators and quality-control samples) is ~6 h, and the LC-MS/MS analyses can run unattended overnight. Because the method is very sensitive, only 25 µL of plasma is required. Before hydrolysis, a reduction step was performed to reduce early glycation products, such as fructoselysine, that might otherwise lead to formation of CML during heating in HCl (13). A major feature of the proposed method is that no derivatization of the analytes is needed, in contrast to gas chromatography–mass spectrometry methods, which require derivatization to increase the volatility of CML and CEL.

A common problem in the analysis of polar compounds by LC-MS is that they have very short retention times on reversed-phase columns, which may lead to loss of sensitivity as a result of coeluting polar contaminants that cause ion suppression. Because this effect may vary between samples, it can also lead to poor performance in terms of precision. To increase retention, polar compounds are often derivatized, but this complicates the sample pretreatment procedure, and excess reagent present in the samples may interfere with the chromatographic separation or cause ion suppression. Thornalley et al. (21) recently described a LC-MS/MS method for the quantitative screening of a large number of AGEs in enzymatic digests of cellular and plasma proteins. In this procedure, a porous graphite carbon column, which gives excellent retention of small polar compounds, was used. We used another approach to increase the retention of the underivatized analytes, i.e., addition of the volatile ion-pairing agent NFPA to the mobile phase. In addition, we used deuterium-labeled internal standards to compensate for residual ion-suppression effects. This stable-isotope-dilution procedure provided a robust LC-MS/MS method for the simultaneous analysis of CML and CEL. The robustness of the proposed method is illustrated by the fact that we have analyzed >1000 plasma samples with an acceptable long-term reproducibility without encountering any problems in terms of interference by unknown compounds or column deterioration.

The CML and CEL concentrations we measured in plasma hydrolysates from healthy controls are in reasonable agreement with data obtained by another LC-MS/MS technique that was reported recently (21). The authors of that study used enzymatic hydrolysis of plasma proteins to preserve AGEs, which are unstable under acid hydrolysis conditions. This may, by incomplete release of CML and CEL, possibly lead to slightly lower values compared with the results obtained in our study, in which acid hydrolysis was used. The two- to threefold increased concentrations of CML and CEL that we found in plasma proteins from dialysis patients and the lack of influence of dialysis mode, i.e., hemodialysis vs peritoneal dialysis, are in line with other reports (21)(22)(23)(24). The fact that we found no significant differences between CML and CEL concentrations in dialysis patients with or without diabetes confirms data on CML reported by Miyata et al. (23) and suggests that compared with nonenzymatic glycation, accumulation of reactive dicarbonyl intermediates and/or increased oxidative stress probably play a more important role in the generation of these compounds. This was corroborated by recent findings showing that oxidants generated by NADPH oxidase play a crucial role in CML formation (25).


   Acknowledgments
 
One of the authors (C.G.S.) was supported by a grant from the Diabetes Fonds Nederland. We thank Drs. S. Thorpe and J. Baynes (Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC), who kindly provided labeled CML and CEL in the initial phase of this study.


   Footnotes
 
1 Nonstandard abbreviations: AGE, advanced glycation end product; ALE, advanced lipoxidation end product; CEL, N{epsilon}-(carboxyethyl)lysine; CML, N{epsilon}-(carboxymethyl)lysine; LC-MS/MS, liquid chromatography–tandem mass spectrometry; and NFPA, nonafluoropentanoic acid.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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