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Determination of Glycated Nucleobases in Human Urine by a New Monoclonal Antibody Specific for N2-Carboxyethyl-2′-deoxyguanosine Marc Schneider,† Gerlinde Thoss,† Christa Hu¨bner-Parajsz,‡ Rose Kientsch-Engel,‡ Peter Stahl,‡ and Monika Pischetsrieder*,† Institute of Pharmacy and Food Chemistry, Friedrich-Alexander-University, Schuhstrasse 19, D-91052 Erlangen, Germany, and Roche Diagnostics GmbH, D-82372 Penzberg, Germany Received March 2, 2004
Sugars and sugar degradation products react in vivo readily with proteins (glycation) resulting in the formation of a heterogeneous group of reaction products, which are called advanced glycation end products (AGEs). AGEs notably change the structure and function of proteins so that extended protein-AGE formation is linked to complications such as nephropathy, atherosclerosis, and cataract. DNA can be glycated in vitro in a similar way as proteins, and the two diastereomers of N2-carboxyethyl-2′-deoxyguanosine (CEdGA,B) were identified as major DNA AGEs. It was postulated that DNA AGEs play an important role in aging, diabetes, and uremia. However, at the moment, sensitive methods to measure the extent and impact of DNA AGEs in vivo do not exist. In this study, we developed a monoclonal antibody, which recognized CEdGA,B with high affinity and specificity (MAb M-5.1.6). The I50 value for CEdGA,B was 2.1 ng/mL, whereas other modified nuclueobases and AGE proteins showed negligible crossreactivity. Unmodified 2′-deoxyguanosine was only weakly recognized with an I50 value > 600 000 ng/mL, which is the limit of solubility. MAb M-5.1.6 was then used to measure the urinary excretion of AGE-modified nucleobases in a competitive enzyme-linked immunosorbent assay. The recovery of CEdGA,B from human urine was between 87.4 and 99.7% with coefficients of variations between 8.0 and 22.2%. The detection limit was 0.06 ng/mL, and the determination limit was 0.15 ng/mL with a linear range between 0.3 and 100 ng/mL. CEdG equivalents were analyzed in urine samples from 121 healthy volunteers, and concentrations between 1.2 and 117 ng CEdG equiv/mg creatinine were detected.
Introduction Sugars (e.g., glucose) and sugar degradation products (e.g., glyceraldehyde, methylglyoxal) are reactive compounds, which readily bind to proteins (glycation). As a result, a heterogeneous group of reaction products is formed, which are summarized by the term advanced glycation end products (AGEs) (1). Sugars and sugar degradation products are endogenously formed in vivo, but they can also derive from exogenous sources, such as nutrition or smoking. In vivo, AGEs have been detected on extra- (2) and intracellular (3) proteins, where they greatly change protein structure (4) and function (5). In a healthy organism, AGE formation is rather slow, but a remarkable accumulation has been observed on proteins with long half-lives, such as lens crystalline (6) or skin collagen (7). In diabetes and uremia, two diseases that are linked to elevated concentrations of sugars or sugar degradation products, glycation reactions are severely accelerated. As a consequence, high levels of AGE modifications can be detected even on proteins with shorter half-lives, such as hemoglobin (8) or serum proteins (9). * To whom correspondence should be addressed. Tel: ++49-91318524102. Fax: ++49-9131-8522587. E-mail: pischetsrieder@lmchemie. uni-erlangen.de. † Friedrich-Alexander-University. ‡ Roche Diagnostics GmbH.
When DNA is reacted with sugars in vitro at physiological temperatures, the formation of analogous DNAbound AGEs can be observed (10). Furthermore, it was shown that glycation of DNA in vitro leads to depurination (11), single strand breaks (12), and mutations, such as insertions, deletions (13), and transposition (14). Therefore, it was postulated that sugars and sugar degradation products are an important source of endogenous and exogenous noxae, which continuously induce DNA lesions (15). As a consequence, they could contribute to the well-established loss of genomic integrity (16), which occurs during aging and which may contribute to age-related complications. However, up to now, fast and sensitive methods to assess the extent of DNA glycation in vivo do not exist. The two diastereomers of N2-carboxyethyl-2′-deoxyguanosine (CEdGA,B) (17) were identified as main DNA AGEs, which are formed upon the reaction of DNA with various sugars in vitro (10) (Figure 1). The corresponding derivatives of N2-carboxyethylguanosine (CEGA,B) and N2-carboxyethylguanine (CEguanine) are formed during the glycation of guanosine or guanine, respectively (17, 18). A polyclonal antibody was developed, which specifically recognizes CEdGA,B and which was used to monitor DNA AGE formation in vitro by ELISA (10). However, the assay was not sensitive enough to record DNA glycation in living cells or in vivo. Sensitive chromatographic methods to measure glycated
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Figure 1. Formation of CEdGA,B from sugars and sugar degradation products.
nucleobases in human specimen, for example, by LC/MS, have not been described. Here, we report the development of a monoclonal antibody, which binds with high affinity and specificity to CEdGA,B and which can be used to measure AGE-modified nucleobases in human urine.
Materials and Methods Apparatus. A microplate washer Columbus (Tecan Group Ltd., Maennedorf, Germany), a BioRad microplate reader 550 (BioRad Laboratories, Hercules, United States), and a fluorescence activated cell sorter, FACStar plus (Becton Dickinson, Heidelberg, Germany) were used. Preparation of CEGA,B and CEdGA,B. The two diastereomers of CEGA,B were obtained from guanosine and dihydroxyacetone (12) and further purified by preparative HPLC on a C18 reversed phase column (19). The products were eluted with methanol:water:50 mM ammoniumformiat buffer, pH 4 (6:84: 10). The two diastereomers of CEdG were synthesized according to the literature (17). The absolute configuration of the diastereomers was not determined, and the assignment (A,B) was made randomly. Preparation of the Immunogen and the Coating Agent. The immunogen was prepared by coupling CEGA,B to keyhole limpet hemocyanine (KLH) carrier protein according to the procedure of Erlanger and Beiser (20). Twenty milligrams of CEGA,B was stirred at room temperature for 20 min in 1 mL of an aqueous 0.2 M NaIO4 solution. The reaction was stopped by the addition of 4 µL of ethylene glycol. Twenty milligrams of KLH (Sigma, Taufkirchen, Germany) was solved in 2 mL of coupling buffer (31 mM sodium phosphate buffer, 460 mM NaCl, and 41 mM saccharose) and was then added dropwise to the CEGA,B solution. The pH was adjusted to 9.5 with 2 M Na2CO3 solution. After the mixture was stirred for 45 min, 500 µL of sodium cyanoborhydride solution (120 mg/mL in water) was added to the CEguanine-protein complex and was kept at 4 °C overnight. To avoid foam formation, 20 µL of n-octanol was dropped to the reaction mixture and then the pH was adjusted to 6 with formic acid. The modified protein was finally dialyzed three times against 2.5 L of coupling buffer. The modification rate was calculated from the UV absorbance and the protein concentration (BioRad Laboratories, Hercules) and was about 190 mol CEguanine/mol KLH, assuming a molecular weight of 1 700 000 for KLH. The immunogen, a fine suspension in coupling buffer, was stored at -22 °C. The same method was applied to the synthesis of the conjugate of CEG and bovine serum albumin (CEguanine-BSA), which was used for coating the microtiter plates for ELISA. CEguanine-BSA was dialyzed against water, lyophilyzed, and stored at -22 °C. The modification rate was 10 mol CEguanine/ mol BSA. Preparation of the Competitors. N2-Carboxymethyl-guanosine (CMG) was prepared as described in the literature (21). N-carboxyethyllysine-BSA (CEL-BSA) was synthesized by incubating 176 mg of BSA at 37 °C for 24 h with 15 mg of pyruvic acid and 20 mg of NaCNBH3 in 10 mL of 0.2 M sodium phosphate buffer, pH 7.8. After incubation, the protein was
Schneider et al. dialyzed extensively against PBS. Guanine-BSA was prepared in the same way as described in detail for CEguanine-BSA, except for the use of guanosine instead of CEGA,B. Guanosine and 2′-deoxyguanosine monohydrate were purchased by Fluka (Taufkirchen, Germany), and N2-methylguanosine, N2-N2-dimethylguanosine, N2-9-diacetylguanine, 6-Omethylguanine, and 8-hydroxy-2′-deoxyguanosine were obtained from Sigma-Aldrich. Immunization and Development of Monoclonal Antibodies (MAbs). Ten female Balb/c mice were immunized with the CEguanine-KLH conjugate. For priming, 100 µg of immunogen in 250 µL of a 1:1 mixture of isotonic NaCl solution and complete Freund’s adjuvant was injected intraperitoneally into the mice. Six, 10, and 14 weeks after the first immunization, the procedure was repeated with incomplete Freund’s adjuvant. Four weeks later, blood samples were taken retroorbitally and assayed by direct ELISA for antibody activity as described below. Six months later, the mice showing the highest titers were first boosted by intraperitoneal injection of 250 µg of CEguanine-KLH and the following 2 days with 200 µg, and at the fourth day, 100 µg of immunogen in PBS was injected intravenously. The next day, the mice were sacrificed and the spleens were excised. The spleen cells were fused with P3 × 63-Ag8.653 myeloma cells using polyethylenglycol (22). The hybridomas were cultured in hypoxanthin-azaserin (Hybri-Max, Sigma) selection medium containing interleukin-6 as a growth factor. About 2 weeks later, culture media were screened for anti-CEguanine specific MAbs by direct ELISA. Hybridoma cultures, which yielded an absorption of 2.0 or more, were expanded and cryopreserved. Selected cultures were cloned by using a fluorescence-activated cell sorter. The clones were also cryopreserved. Characterization of Selected MAb Producing Clones. Specificity and affinity of the MAbs were determined by competitive ELISA. The MAb isotype was determined by an immunoassay using a commercial kit (IsoStrip-Kit, Roche Diagnostics Corporation, Indianapolis, IN). In addition, the specific MAb productivity of the clones was measured by ELISA for quantitation of mouse immunoglobulins in hybridoma culture media and ascites fluid (Roche Diagnostics GmbH, Mannheim, Germany). Direct ELISA for the Screening of Mouse Sera and MAb Secreting Hybridomas. Ninety-six well microtiter plates (Maxisorb from Nalgene Nunc International, Rochester, NY) were coated with 100 µL/well of a CEguanine-BSA solution (0.2 µg/mL BSA conjugate in 0.2 M sodium carbonate buffer, pH 9.7) at 4 °C overnight. The plates were washed with washing buffer [1 mM KH2PO4, 7 mM K2HPO4, 15 mM NaCl, 0.02 mM potassium sorbate, and 0.05% Tween (v/v)] twice after each step. Nonspecific binding to the plate was minimized by blocking the wells for 1.5 h with 150 µL of 3% skim milk powder in water. The mouse sera were added in a dilution of 1:6000 in diluting buffer (0.2% BSA and 0.05% Tween-20 in PBS). For the hybridoma supernatants, a dilution of 1:500 was necessary. After 1 h of incubation at room temperature, 100 µL of antimouse IgG horseradish peroxidase conjugated Fab fragments [from sheep, 0.06 U/mL (Chemicon International Ltd., Hofheim, Germany) in 0.1% BSA in PBS] was added and the plates were incubated for 45 min. After the plates were washed three times with washing buffer, 100 µL of ABTS (2,2′-azino-di[3-ethylbenzthiazoline sulfonate]) solution (Roche Diagnostics GmbH) was added. The absorbance was measured at 405 nm. Competitive ELISA for Carboxyethyldeoxyguanosine (CEdG). The competitive ELISA was applied to the immunochemical characterization of selected MAbs and to the detection and quantitation of glycated nucleobases in human urine samples. Competitive ELISA was performed similar to the direct ELISA with the following differences. For characterization of the selected MAbs, the hybridoma culture media were diluted in diluting buffer as required and mixed with the competitors. Labeling was performed with anti-mouse IgG horseradish peroxidase conjugate (Chemicon International Ltd.). After the
ELISA for DNA-AGEs in Human Urine media were washed three times, antibody binding was detected using 100 µL of tetramethylbenzidine solution. The reaction was stopped after 30 min by adding 25 µL of 2 N sulfuric acid, and the absorbance was measured at 450 nm. The I50 (concentration of competitor producing 50% inhibition of MAb binding to the coating complex) of selected compounds was determined. For the determination of glycated nucleobases in human urine samples, the hybridoma culture medium of clone M-5.1.6 was diluted 1:340 and then mixed with an equal volume of the prepared urine samples. A 100 µL amount of the sample-MAb mixture was added to a well after coating and blocking. Concentrations of CE-modified nucleobases amounts were calculated from a calibration curve using CEdGA,B (17) as the standard. The results obtained from the urine samples are expressed as ng CEdG equivalents/mg creatinine. Preparation of Human Urine Samples. Spontaneous urine samples were obtained from 121 healthy individuals (1961 years old). The creatinine concentration of each urine sample was determined by a colorimetric or enzymatic test using commercial creatinine kits (Sigma Diagnostics, St. Louis, MO, or Roche Diagnostics GmbH). All urine samples were stored at -20 °C before analysis. Before the test, 1 mL of the urine samples was first vortexed for 30 s and then diluted with an equal volume of bidistilled water. After centrifugation at 2000g for 10 min, concentrations of CE-modified nucleobases were determined in the supernatants by competitive ELISA as described before. Validation of the Competitive ELISA for the Determination of CEdG Equivalents in Human Urine. For determination of the recovery and precision, human urine samples were spiked with increasing concentrations of CEdGA,B as indicated. The reading of the unspiked urine was subtracted as a blank. The recovery was calculated as: measured concentration/spiked concentration (mean; n ) 12 for each concentration). The precision was determined under intraassay conditions and was the mean of two independent experiments (n ) 2 × 6). It is shown as the standard deviation and coefficient of variation. According to the blank method, the detection limit was calculated as y (blank) + 3.3 × SD (blank) and the determination limit was determined as y (blank) + 10 × SD (blank). The linear range was taken from nine independent standard curves after logit-log transformation. A representative standard curve is shown in Figure 4.
Results Hapten Synthesis and Conjugation. It was the goal of this study to generate a monoclonal antibody, which binds with maximal affinity to CEdGA,B, whereas unglycated nucleobases, modified nucleobases, which are formed by other reactions, and glycated proteins should have minimal cross-reactivity. Therefore, we coupled the hapten via the sugar moiety to the carrier protein, KLH, according to the method of Erlanger and Beiser (20). KLH was chosen because of its high immunogenicity and its absence from human biological samples. A mixture of the two diastereomers of CEGA,B, which can be prepared in high yields from guanosine and dihydroxyacetone, was used as the starting material. After oxidative cleavage of the ribose, coupling was achieved by reductive alkylation of the protein. The resulting conjugate, CEguanine-KLH (Figure 2), was used for the immunization of 10 mice. Hybridoma Generation and Preliminary Evaluation of the Hybridoma Culture Supernatants. One hundred fifty-one hybridoma cultures, produced from two fusions, secreted antibodies with high affinity to the coated complex CEguanine-BSA (OD > 1.2). Fifteen supernatants of these cultures showed strong antibody binding to CEguanine-BSA in the direct ELISA at a
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Figure 2. Structure of the CEguanine-KLH conjugate, which was used for immunization. Table 1. Binding Specificity of MAb M-5.1.6 Characterized in the Competitive CEdG ELISA
a
compound
I50 (ng/mL)
CEguanine-BSA CEdGA,B CEdGA CEdGB CEGA CEGB CEguanine CMG guanine-BSA guanosine 2′-deoxyguanosine CEL-BSA N2-methylguanosine N2-9-diacetylguanine N2-N2-dimethylguanosine 6-O-methylguanine 8-hydroxy-2′-deoxyguanosine
284 2.1 (6.1 pmol/mL) 63 (186 pmol/mL) 1.5 (4.4 pmol/mL) 82 (231 pmol/mL) 4 (11.3 pmol/mL) 13 (58.2 pmol/mL) 55 (161 pmol/mL) >700 000a >250 000a >600 000a bld bld bld bld bld bld
Max solubility, bld ) below the limit of detection.
dilution of 1:500 but no cross-reactivity to protein-bound CEL-BSA or protein-bound guanine-BSA and were selected for evaluation by competitive ELISA. CEL-BSA was chosen for primary evaluation, because it is crucial that the antibody does not cross-react with glycated proteins. CEL is the protein-bound AGE, which has the most structural similarities to CEdG (Figure 3). The supernatants of the CEguanine specific hybridomas were titrated for their affinity to CEguanine-BSA, CEL-BSA, guanine-BSA, CEguanine, CEG, and CEdG. Six of the tested hybridomas that showed highest affinity to carboxyethylguanine derivates but no cross-reactivity to structurally related substances were then cloned by using a fluorescence-activated cell sorter. Three clones of each primary culture with the highest affinity to CEguanineBSA were expanded, and the specific MAb productivity was determined. Finally, the clone 5.1.6, which produced MAbs showing the highest affinity and selectivity for CEdGA,B, was used for further experiments. Characterization of MAb M-5.1.6. The isotype of the MAb produced by hybridoma M-5.1.6 was IgG1,κ. The 24 h productive capacity was determined by quantitative immunoassay to be 15 µg IgG/106 cells per day. The binding specificity of M-5.1.6 was examined by competitive ELISA, and the highest affinity was detected for CEdGA,B followed by CEG and CEguanine (Figure 4). In contrast, unmodified nucleobases guanosine and 2′-deoxyguanosine as well as the guanine derivates N2-methylguanosine, N2-N2-dimethylguanosine, N2-9-diacetylguanine, 6-O-methylguanine, and 8-hydroxy-2′deoxyguanosine showed only low or no detectable crossreactivity. The I50 values are displayed in Table 1. The competitors were chosen either because of their structural similarities (Figure 3) or because of their particular relevance in vivo. The antibody cross-reacted weakly
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Figure 3. Structures of selected compounds tested for cross-reactivity with MAb M-5.1.6.
Figure 4. ELISA competition curve for CEdGA,B (-2-), CEdGA (-9-), and CEdGB (-]-) using MAb M-5.1.6.
with 2′-deoxyguanosine and guanosine, but the competitor concentration resulting in 50% inhibition was over the limit of solubility and at least more than 250 000 and 600 000-fold higher than the I50 concentration of CEdGA,B. Assuming a physiological concentration of 70 ng/mL guanosine, 2 ng/mL 2′-deoxyguanosine, and 195 ng/mL guanine in human urine (23) and a 250 000fold lower affinity, the calculated response of unmodified nucleobases in the CEdG ELISA is far below the detection limit. Assay Validation. MAb M-5.1.6 was then used in a competitive ELISA to measure glycated nucleobases in human urine samples. First, the assay conditions were optimized for maximal sensitivity, precision, accuracy, and robustness for the matrix urine. Under the optimized conditions, the ELISA assay was validated (according to the requirements of DIN EN ISO/IEC 17025:2000 and the European Community’s decision 93/256/EEC). The detection limit was 0.06 ng/mL, and the determination limit was 0.15 ng/mL with a linear range between 0.3 and 100 ng/mL for CEdGA,B. For the determination of recovery and precision, human urine samples were spiked
Figure 5. Concentrations of CEdG equivalents in urine samples from healthy volunteers (n ) 121, ages between 19 and 61 years) by competitive CEdG ELISA using MAb M-5.1.6. Table 2. Accuracy and Intraassay Precision of the CEdG ELISA as Determined for Human Urine Samples Spiked with CEdGA,B concentration of CEdGA,B added (ng/mL)
recovery (%)
precision (coefficient of variation) (%)
1.25 2.5 5.0 10.0
87.4 99.7 97.2 95.4
22.2 16.1 8.0 9.6
with four different concentrations of CEdGA,B (Table 2). Interassay variability resulted in a coefficient of variation of 11.3% for a urine sample, which was measured 10 times by three different persons at different days. Quantification of CEdG Equivalents in Human Urine. The concentrations of glycated nucleobases were determined in urine samples of 121 healthy volunteers (19-61 years old) by the newly developed competitive ELISA. The concentrations ranged from 1.2 to 117 ng
ELISA for DNA-AGEs in Human Urine
CEdG equiv/mg creatinine (Figure 5) and did not change with the age of the probands.
Discussion This publication describes the development of the monoclonal anti-CEdGA,B antibody MAb M-5.1.6 and its application in a competitive ELISA to detect glycated nucleobases in human urine. The antibody recognizes several glycated guanine derivatives with high affinity, so that the most important AGE-modified nucleobases can be determined in a single assay. Because sensitive chromatographic methods are not yet available, it is not clear yet, which DNA AGEs predominate in vivo. The binding affinity to CEdGA,B was more than 600 000-fold higher than the affinity to the parent compound 2′deoxyguanosine. Therefore, a detectable signal from physiological concentrations of unmodified nucleobases is not expected. Most notably, affinity of the antibody to related structures such as 8-hydroxy-2′-deoxyguanosine or other modified DNA bases was not observed. Even more important is the absence of cross-reactivity with AGE proteins, mainly CEL-modified proteins, because AGE proteins and AGE peptides are present in most biological samples and tissues (24, 25). However, there is a significant difference in the affinity of the antibody to both diastereomers of CEdG. In case of patient-topatient variations in the elimination of the diastereomers, over- or underestimation of the CEdG concentration in some samples could occur. Therefore, the results are given in CEdG equivalents. In vitro, sugars and sugar degradation products modify DNA readily under the formation of CEdG adducts, resulting in gross alteration of DNA structure and function (10, 12-14). Thus, it was hypothesized that DNA is also glycated in vivo and that this process is an important factor in aging and also in diseases that are related to increased carbonyl stress, such as diabetes and uremia (15, 26). However, up to now, fast, reliable, and sensitive methods have been missing to evaluate the role of DNA AGEs in the human organism under physiological and pathological conditions. In this study, we used a newly developed monoclonal antibody against CEdGA,B to analyze glycated nucleobases in human urine samples. In all samples, glycated nucleobases were determined in a concentration range between 1.2 and 117 ng CEdG equiv/mg creatinine. Thus, for the first time, we obtained strong evidence that DNA is glycated in vivo. Immunoaffinity chromatography using antibodies against modified nucleobases coupled with HPLC-DAD or LC-MS/MS is a very sensitive method, which allows very specific and reliable identification of defined DNA adducts. However, the procedure is time-consuming and thus not applicable for the screening of a large number of samples. Furthermore, a stable isotope-labeled standard is strongly recommended for reliable quantification when biological samples are purified by immunoaffinity chromatography (27). The newly developed ELISA method has several advantages. It is time-saving, sensitive, accurate, and reproducible in our assay system using human urine samples. In addition, the sample preparation is reduced to a dilution and a clarification step without further purification, and urine is a matrix that is easily available and that reflects DNA damage in vivo (28). Analysis of urinary excretion of glycated nucleobases of 121 healthy individuals (19-61 years old) with no
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history of cancer or diabetes showed the efficiency of the assay to test a large number of urine samples. Further investigations are now required to determine if urinary excretion of AGE nucleobases is a valid marker for the attack of DNA by sugars and sugar degradation products. In summary, glycation reactions may be one of several exogenous and endogenous factors that chronically assault the structure of the genome. A noninvasive technique is the method of choice to perform studies on the effects of various conditions that induce DNA damage as a result of glycation or glycoxidation reactions. Urine from human subjects is easy to access and to handle as compared to other biological fluids. The analytical potential of the newly developed ELISA technique for the identification and quantitation of glycated nucleobases in urine can be used to improve our understanding about the role of DNA AGE in aging and disease.
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TX049929D
