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Clinical Chemistry 47: 1249-1255, 2001;
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(Clinical Chemistry. 2001;47:1249-1255.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Immunochemical Assay of Hemoglobin with N{epsilon}-(Carboxymethyl)lysine at Lysine 66 of the ß Chain

Hisahiko Iwamoto1a, Yoshihiro Motomiya2, Keisuke Miura1, Masayo Morisawa2, Yoshimichi Yoshimura1 and Ikuro Maruyama3

1 R&D for Diagnostics, A&T Corporation, 2023-1, Endo, Fujisawa-City, Kanagawa 252-0816, Japan.
2 Suiyukai Clinic, 676-1, Kuzumoto-chou, Kashihara-City, Nara 634-0007, Japan.
3 Department of Laboratory Medicine, Kagoshima University School of Medicine, Kagoshima 890-0075, Japan.


aAuthor for correspondence. Fax 81-466-86-8676; e-mail iwamoto{at}alice.aandt.co.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: N{epsilon}-(Carboxymethyl)lysine (CML), a well-characterized and major advanced glycation end product structure, is produced via a Maillard reaction by nonenzymatic glycation and/or oxidation. Although few of the carboxymethylation sites of lysine residues on proteins have been identified, it is known that the possible lysine glycation site in hemoglobin (Hb) is Lys-66 on the ß chain. We aimed to develop an assay for the Hb with a CML (CML-Hb) site specific to Lys-66 on the Hb ß chain and to determine whether the lysine residue at that site is carboxymethylated.

Methods: Ala-His-Gly-Lys-Lys(CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys, the peptide derived from the ß chain of human Hb, was synthesized as an immunogen, and a monoclonal antibody against the peptide was prepared. A latex immunoassay method was established using the antibody on an automatic analyzer. In this study, 20 samples from healthy subjects and 80 samples from nondiabetic patients undergoing hemodialysis (HD) were analyzed.

Results: The latex immunoassay method using the antibody correlated significantly with the ELISA method using the antibody (r = 0.95; P <0.001). Between healthy subjects (n = 20) and nondiabetic HD patients (n = 80), a significant difference was seen in circulating CML-Hb (525 ± 76 vs 778 ± 137 pmol CML/mg of Hb; P <0.0001).

Conclusion: The latex method for the CML-Hb site specific to Lys-66 on the ß chain can measure large numbers of samples on an automatic analyzer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nonenzymatic glycation and lipoxidation may play roles in the pathogenesis of diabetic complications (1)(2)(3)(4)(5), end-stage renal disease (6), and the aging process (7)(8). Advanced glycation end products (AGEs) 1 are well-known results of these reactions. AGEs are a heterogeneous group of irreversibly modified adducts and products. Because of their diversity, the precise chemical structures of most AGEs have yet to be elucidated, although several, such as N{epsilon}-(carboxymethyl)lysine (CML) (9), pentosidine (10), crossline (11), imidazolone (12), and pyrraline (13), have been structurally identified.

CML, a well-characterized and major AGE structure, is known to be a product of both glycation and oxidation reactions (14), and it is known that CMLs are found on hemoglobin (Hb) (15), albumin (16), and collagen (17) in vivo. In Hb, the N-terminal valine is known to react with glucose, and it has been reported by Cai and Hurst (18) that N-(carboxymethyl)valine is generated from the glycated valine by the same process as CML. On the other hand, although it has become clear that the amino group of Lys-66 on the ß chain in Hb is glycated in vivo (19), the carboxymethylation sites of the amino group within the side-chain of lysine residues on Hb have not been identified.

In this study, we raised a specific monoclonal antibody against carboxymethylated peptide (CM peptide) analogous to the amino acid sequence of the carboxymethylated ß-chain of human Hb at Lys-66 and established an assay system specific for CML-Hb carboxymethylated at Lys-66 on the ß chain in circulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
synthesis of cm peptide as a immunogen
All commercial chemicals and keyhole limpet hemocyanin (KLH) were purchased from Wako Chemical Co. The Hb peptide with the appropriate sequence for carboxymethylation was synthesized on an automated machine (Model 430; Applied Biosystems) using solid-phase techniques. Briefly, Boc-Ala-His(Tos)-Gly-Lys(Z)Lys(Fmoc)-Val-Leu-Gly-Ala-Phe-Ser(Bzl)-Cys(MeBzl), where Boc is N-tert-butoxycarbonyl, Tos is p-toluenesulfonyl, Z is benzyloxycarbonyl, Fmoc is 9-fluorenylmethoxycarbonyl, Bzl is benzoyl, and MeBzl is methylbenzoyl, was synthesized as the peptide, and Fmoc on the peptide was deprotected in dimethylformamide containing 200 mL/L piperidine at room temperature for 15 min. After removal of the piperidine, 1 mg of the synthesized peptide was carboxymethylated by reaction for 12 h at 0 °C in dichloromethane containing 0.25 mol/L glyoxylic acid and 1 mg of sodium cyanoborohydride as described previously (20). Subsequently, the peptide was treated with HF containing p-thiocresol, m-cresol, dimethyl sulfide, and anisole for 1.5 h at 0 °C to remove the other protective group and was purified by reversed-phase chromatography using an ODS column (TSKgel ODS-80Ts; 2 x 20 cm; TOSOH). Separation was achieved after 30 min of a linear gradient of buffer A (1 g/L trifluoroacetic acid) to buffer B (1 g/L trifluoroacetic acid in 700 mL/L acetonitrile) at 7 mL/min.

The peptide was coupled to KLH by m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as described previously (21). Briefly, MBS in dimethylformamide was added to a final concentration of 5 g/L in 100 mmol/L sodium phosphate buffer (pH 7.2) containing the peptide and was incubated at room temperature for 30 min. Sufficient KLH dissolved in sodium phosphate buffer was then added to the buffer containing the peptide and incubated for 3 h at room temperature with stirring. Finally, the KLH-coupled peptide was dialyzed against sodium phosphate buffer to remove the noncoupled CM peptide and MBS and was subsequently injected into mice.

enzymatic digestion of cm peptide by lysyl endopeptidase
Lysyl endopeptidase (2 mg; Wako) was added to 20 mmol/L glycine-NaOH (pH 9.0) buffer containing 1 mg of CM peptide and incubated at 37 °C for 3 h.

mass analysis of the synthesized cm peptide
Enzymatic digestion of CM peptide yielded a sample that was dilute and contained lysyl endopeptidase. The digested CM peptide was purified on a C18 reversed-phase microcolumn (Sep-Pak C-18; Waters Co.) and then freeze-dried before matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. Using {alpha}-cyano-4-hydroxycinnamic acid (Sigma) as the MALDI matrix, we prepared the CM peptide for analysis by mixing 10 µL of matrix solution with 3 µL of the analyte on the probe. The CM peptide layer was formed by drying the solutions in a stream of cold air. The mass spectrometer used in the experiments was a home-built linear TOF instrument equipped with a nitrogen laser at 337 nm (LASERMAT; Finnigan MAT). Only positively charged ions were analyzed, and 16 single-shot spectra were accumulated. All further processing, including spectrum calibration, was performed using the software package provided by the manufacturer.

amino acid analysis
CM peptide was hydrolyzed in 6 mol/L HCl for 20 h at 110 °C. The hydrolyzed sample was then dried and subjected to amino acid analysis.

Amino acid analysis was performed using an amino acid analyzer (Model L-8500; Hitachi Co.) with the ninhydrin method described previously (22). The ninhydrin reagent and buffer solution for the Model L-8500 and the calibration solution were purchased from Wako. The concentration of each amino acid in the CM peptide was calculated based on the peak area of each amino acid of unknown concentration compared with the amino acid calibration solution.

preparation of monoclonal anti-cml antibody
Monoclonal antibody was prepared according to the method of Galfre and Milstein (23) with modifications.

Briefly, Balb/c mice received subcutaneous injections of 0.5 mg of KLH-coupled CM peptide in 500 mL/L Freund’s complete adjuvant. After the first immunization, mice received four booster injections of 0.5 mg of KLH-coupled CM peptide in 500 mL/L Freund’s incomplete adjuvant every 2 weeks. The titers to CM peptide in the sera of immunized mice were determined by ELISA. The splenic lymphocytes from the immunized mouse were fused to myeloma P3U1 cells in the presence of polyethylene glycol. Hybrid cells were cultured in medium containing aminopterin, hypoxanthine, and thymidine (HAT medium) with 100 mL/L fetal calf serum. The cell lines were selected and then injected into Balb/c mice to produce ascites fluid. Antibody was precipitated from cell-free ascites fluid by ammonium sulfate (40% saturation). Precipitated materials were dissolved in 50 mL of 10 mmol/L Tris-HCl (pH 8.0), dialyzed against the same buffer, and further purified by DEAE-cellulose chromatography.

chemical modification of proteins
Carboxymethylated human serum albumin (CML-HSA) was prepared as described previously (15)(17). In brief, HSA (fraction V, 1 mg; Sigma) was incubated in 1 mL of 0.1 mol/L borate buffer (pH 9.0) containing 0.25 mol/L glyoxylic acid and 1 mg of sodium cyanoborohydride for 12 h at 0 °C. Carboxymethylated Hb (CML-Hb) was also prepared from Hb (fraction V, 1 mg; Sigma) in the same manner.

direct elisa
Each well was incubated at 37 °C for 20 min with 0.05 mL of the above CM peptide or non-CM peptide in 50 mmol/L carbonate buffer (pH 9.7) and washed three times with phosphate-buffered saline (pH 7.4) containing 0.5 mL/L Tween 20 (washing buffer). Each well was then blocked at 37 °C for 1 h with 0.3 mL of Block Ace (Dainippon Pharmaceutical) and diluted four times with 50 mmol/L carbonate buffer. Each well was washed three times with washing buffer and incubated at 37 °C for 1 h with 0.05 mL of anti-CML monoclonal antibody (5 g/L). Wells were washed three times with washing buffer and incubated at 37 °C for 1 h with 0.05 mL of biotinylated anti-mouse IgG antibody and reacted with avidin-biotin-alkaline phosphatase complexes (vectastain ABC reagent set; Vector), followed by reaction with p-nitrophenyl phosphate (Bio-Rad). The reaction was terminated by 0.4 mol/L NaOH, and the absorbance at 405 nm was read by an EIA reader (Model 2550; Bio-Rad).

binding assay of the antibodies against cm peptide or cm proteins
For investigation of the specificity of monoclonal anti-CML antibody, a BIAcore system (BIAcore 2000; Pharmacia Biosensor AB) was used. The CM or non-CM peptide was immobilized on a CM5 (dextran matrix) chip of the BIAcore apparatus. The CM or non-CM proteins were also immobilized in the same manner. The measurements were performed at an antibody concentration of 50 mg/L with a continuous flow of 200 µL/min.

latex immunoassay
After dialysis of the purified antibody (0.3 g/L) against 0.05 mol/L glycine-NaOH buffer (pH 8.2), 1-mL aliquots were incubated with 1 mL of 10 g/L latex suspension at 37 °C for 1 h. Two milliliters of 0.05 mol/L glycine-NaOH buffer (pH 8.2) containing 5 g/L bovine serum albumin was added, and the mixture was incubated at 37 °C for 2 h. The latex particles were pelleted by centrifugation (16 000g for 50 min), washed with 0.05 mol/L glycine-NaOH buffer (pH 8.2), and resuspended in 3.35 mL of 0.1 mol/L Tris-HCl buffer (pH 8.2) containing 0.1 mol/L NaCl and 1 g/L NaN3. The antibody-coated latex solution (R2) was stored at 4 °C.

In the CML-Hb assay, additional buffer (R1; 0.1 mol/L Tris-HCl buffer (pH 8.2) containing 0.1 mol/L NaCl and 1 g/L NaN3) was prepared and used to dilute R2 to suppress nonspecific binding between protein(s) or lipid(s) in samples and the antibody-coated latex. Whole blood was collected from a healthy volunteer, the concentration of CML-Hb was measured by amino acid analysis, and the material was used as a calibrator. Calibrator and samples were finally prepared by diluting 10 µL of whole blood with 10 mL of 1.5 g/L sodium dodecyl sulfate. We mixed 220 µL of R1 and 5 µL of sample and preincubated the mixture for 4 min at 37 °C in the reaction cell. We then added 100 µL of R2, measured the change in absorbance in each sample and calibrator, and calculated the concentration of the sample. Finally, the CML-Hb concentration was corrected for the Hb concentration in each sample.

patients
Healthy subjects (n = 20) and nondiabetic patients undergoing maintenance hemodialysis (HD; n = 80) were enrolled in this study. The mean age of the healthy subjects was 30 years (age range, 20–50 years). The mean age of the nondiabetic patients undergoing maintenance HD was 40 years (age range, 20–70 years). The length of HD treatment ranged from 3 to 258 months (mean ± SD, 128.6 ± 63.2 months).

statistical analysis
Data are expressed as the mean ± SD. The Mann-Whitney test was used to compare the two groups. Correlation was determined by Pearson’s correlation. P <0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
identification of synthesized cm peptide
To confirm whether the synthesized CM peptide was Ala-His-Gly-Lys-Lys (CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys, as derived from the amino acid sequence of the ß chain in human Hb, mass spectrometric and amino acid analyses were performed.

The resulting mass spectrum of the CM peptide is shown in Fig. 1A . The CM peptide was identified by this analysis as one peak at Mr 1275, which was consistent with the calculated molecular weight of CM peptide (Mr 1276). A commercial renin substrate (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser; Mr 1759; Sigma) was used as an internal standard for the mass analysis. Mass spectrometric analysis of CM peptide digested by lysyl endopeptidase was also performed. The CM peptide was cleaved into two peptide fragments, Ala-His-Gly-Lys and Lys (CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys, by this enzymatic digestion. One of the CM peptide fragments gave a peak at Mr 882, but the peak for the other fragment disappeared against the background and could not be identified (Fig. 1B ). The molecular weight obtained for the digested CM peptide corresponded with the molecular weight calculated for the digested CM peptide. This result suggested that modification took place at the fifth lysine from the NH2 terminus, but not at the fourth lysine. The peaks at m/z 376, 378, 439, and 441 were derived from the {alpha}-cyano-4-hydroxycinnamic acid used as the MALDI matrix because those peaks appeared when the matrix without CM peptide was analyzed (data not shown).



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  		Figure 1. MALDI mass spectra of synthesized CM peptide [Ala-His-Gly-Lys-Lys(CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys; A] and the CM peptide digested by lysyl endopeptidase [Lys(CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys; B] using {alpha}-cyano-4-hydroxycinnamic acid matrix.

Renin substrate was used as an internal standard.

The mole ratios of the amino acid residues in the CM peptide were confirmed by amino acid analysis. The result is shown in Table 1 . The mole ratio of each amino acid was determined by amino acid analysis and was found to be consistent with the theoretical mole ratio. Because one of the two lysines in the CM peptide is carboxymethylated, the theoretical lysine mole ratio may be 1.0. Therefore, the synthesized CM peptide was identified as Ala-His-Gly-Lys-Lys(CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys, where Lys(CM) is the carboxymethylated lysine.


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  		Table 1. Mole ratios of the amino acid constituents of the CM peptide.

specificity of anti-cml antibody
The specificity of the anti-CML monoclonal antibody was assessed using the direct ELISA method, and the antibody was found to be specific for CM peptide, in which the {epsilon}-amino group on Lys-66 in the peptide derived from human Hb ß chain was carboxymethylated, but not for non-CM peptide (Ala-His-Gly-Lys-Lys-Val-Leu-Gly-Ala-Phe-Ser; Fig. 2 ).



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  		Figure 2. Direct ELISA using anti-CML monoclonal antibody.

Curves represent the reaction of the antibody to CM peptide ({diamondsuit}) or non-CM peptide (). Experimental details are described in Materials and Methods.

Similarly, the specificity and affinity of the antibody to the CM peptide were analyzed by on the BIAcore after the CM peptide was coupled to a Biosensor chip (Fig. 3A ). The CM peptide was immobilized by the thiol-coupling method using the cysteine at the end of the COOH terminus. The antibody exhibited an off-rate of 8.35 x 10-5/s and was found to have an on-rate of 4.58 x 105 L · mol-1 · s-1. By dividing the measured off-rate by the on-rate, a kD of 1.82 x 10-10 mol/L was determined for the antibody. The non-CM peptide did not react with the antibody to any degree, and this result was similar to the result obtained by the ELISA method. In addition, as shown in Fig. 3B , although the antibody bound strongly to CML-Hb, it did not bind to Hb, CML-HSA, or HSA.



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  		Figure 3. Kinetics of the binding of monoclonal anti-CML antibody to CM or non-CM peptide (A) and specificity of binding of monoclonal anti-CML antibody to various carboxymethylated proteins or noncarboxymethylated proteins (B).

(A), the antibody was applied to a sensor chip to which CM or non-CM peptide was immobilized. (B), the antibody was applied to a sensor chip to which CML-Hb, nonmodified Hb, CML-HSA, or nonmodified HSA was immobilized. See Materials and Methods for experimental details.

calibration curve for elisa method
Assays were performed with 0–500 µg/L CM peptide as the calibrator. As shown in Fig. 2Up , the calibration curve was prepared by measuring the absorbance at 405 nm.

The concentration of CML-Hb was measured in the same manner. The concentration of CML-Hb in blood from a healthy volunteer was ~500 pmol CML/mg of Hb when calculated from the calibration curve (data not shown).

correlation between elisa method and latex immunoassay method
To test whether CML-Hb could be measured conveniently by the latex immunoassay method, we examined the correlation between the measurements obtained by ELISA and those obtained by the latex immunoassay method, using blood from healthy volunteers and nondiabetic HD patients (n = 10; Fig. 4 ). Our results for the correlation between the measurements obtained by ELISA and those obtained by the latex immunoassay method were considered preliminary because no more than 10 points were studied, but a strong correlation could be found between both methods (r = 0.95; P <0.001). This result supports the latex immunoassay method as a suitable method for CML-Hb.



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  		Figure 4. Correlation between CML-Hb in blood from controls and diabetics measured by ELISA and latex immunoassay.

detection limit and imprecision
The detection limit of the latex immunoassay method, defined as the minimum concentration of CML-Hb that differed from 0 at the 95% confidence level, was 50 pmol CML/mg of Hb. The imprecision of the method was estimated by measuring two blood samples with high (1000 pmol CML/mg of Hb) and normal (500 pmol CML/mg of Hb) concentrations of CML-Hb. The intraassay CVs (n = 10) of samples with high and normal concentrations of CML-Hb were 3.1% and 2.4%, respectively, whereas the interassay CVs (n = 5) were 3.5% and 2.9%, respectively.

circulating cml-hb concentrations
There was a significant difference in circulating CML-Hb concentrations between the healthy control subjects and the group of nondiabetic HD patients (P <0.0001; Fig. 5 ). CML-Hb concentrations were 430–650 pmol CML/mg of Hb (525 ± 76 pmol CML/mg of Hb) in the 20 healthy control subjects. On the other hand, CML-Hb concentrations were 500-1210 pmol CML/mg of Hb (778 ± 137 pmol CML/mg Hb) in the 80 nondiabetic HD patients.



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  		Figure 5. Comparative study of circulating concentrations of CML-Hb in healthy volunteers and nondiabetic HD patients.

A significant difference was found between the two groups (P <0.0001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Carboxymethylated proteins have been recognized as one of the predominant AGE proteins in vivo. Until the present report, however, which of the lysine residues on various proteins underwent carboxymethylation had not been identified.

On the other hand, the presence of glycohemoglobin has been widely confirmed in vivo. In general, the N-terminal valine has been considered the most likely to react with glucose, and N-(carboxymethyl)valine was reported as an advanced product of glycated valine by Cai and Hurst (18). In addition, it is known that fructoselysine, formed by reaction of {epsilon}-amino groups on lysine residues in proteins with reducing sugars, is converted to CML by oxidative cleavage between C-2 and C-3 of the carbohydrate chain (9). After the amino group at the NH2 terminus of the ß chain of Hb, the amino group of Lys-66 on the ß chain is the site most likely to undergo glycation in vivo (19). Therefore, the glycated structures of lysine residues in Hb are thought to be carboxymethylated as well.

We raised a specific antibody against CM peptide with an amino acid sequence similar to that of the ß chain carboxymethylated at Lys-66. The molecular structure of the CM peptide synthesized as an immunogen was identified by mass analysis, and the site specificity of carboxymethylation at Lys-66 was confirmed by selective endopeptidase digestion of the Lys-Lys(CM) bond. The results indicated that the antibody against CM peptide does not recognize the CML, regardless of surrounding peptide sequence, but that it recognizes the specific CM peptide sequence because the antibody bound to CML-Hb but not Hb, CML-HSA, or HSA.

Compared with ELISA, the latex immunoassay is generally less difficult and more available for mass screening. Taking this into account, we established an assay system for CML-Hb based on a latex immunoassay method using our monoclonal antibody.

Our assay system detected the presence of circulating CML-Hb in healthy subjects. In addition, significantly increased CML-Hb concentrations as high as 1.5-fold higher than those of controls were found in HD patients, which was consistent with early results obtained by the immunological dot-blotting method (14). It is thought that acceleration of oxidative stress may be one source of the higher CML-Hb concentrations in HD patients.

With the increasing interest in AGEs in the life sciences, we believe that our method may provide valuable information as a surrogate marker of oxidative stress in various clinical setting, e.g., the aging process and dialysis.

Because 1 mg of Hb corresponds to 15.5 nmol of Hb, our result of 525 pmol CML/mg of Hb in healthy subjects indicates that 3.4% of Hb is modified by carboxymethylation of Lys-66 on one of two ß chains. Hb is composed of two {alpha} and two ß chains. Taking this into account, the percentage mentioned above might be overestimated because Lys-66 could be carboxymethylated in both of the ß chains in vivo. If the lysines at position 66 on both of ß chains are carboxymethylated, the percentage should be lowered by half.

In conclusion, we established a latex immunoassay for measurement of the CML-Hb site specific to Lys-66 on the ß chain. The assay can be adapted to the automated analysis of large numbers of samples. This method is valuable as a new tool for investigating the carboxymethylated modification of Lys-66 on the ß chain in human Hb in vivo.


   Footnotes
 
1 Nonstandard abbreviations: AGE, advanced glycation end product; CML, N{epsilon}-(carboxymethyl)lysine; Hb, hemoglobin; CM peptide, carboxymethylated peptide; KLH, keyhole limpet hemocyanin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; HSA, human serum albumin; CML-Hb, hemoglobin containing a N{epsilon}-(carboxymethyl)lysine; and HD, hemodialysis.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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U. Friess, A. Beck, E. Kohne, R. Lehmann, S. Koch, H.-U. Haring, R.-M. Schmuelling, and E. Schleicher
Novel Hemoglobin Variant [{beta}66(E10) Lys->Asn], with Decreased Oxygen Affinity, Causes Falsely Low Hemoglobin A1c Values by HPLC
Clin. Chem., August 1, 2003; 49(8): 1412 - 1415.
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