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Assay of early and advanced glycation adducts by enzymatic hydrolysis of proteins and HPLC of 6-aminoquinolylcarbonyl adducts
International Congress Series 1245 (2002) 279 – 283
Assay of early and advanced glycation adducts by enzymatic hydrolysis of proteins and HPLC of 6-aminoquinolylcarbonyl adducts Naila Ahmed, Paul J. Thornalley * Department of Biological Sciences, University of Essex, Central Campus, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK
Abstract A method for the assay of early and advanced glycation endproducts (AGEs) in proteins has been developed and validated. Protein samples were de-lipidified, hydrolysed enzymatically, the hydrolysate derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and AQC adducts analysed by HPLC with fluorimetric detection. Nq-Fructosyl-lysine and 12 AGEs were assayed. Amino acid analytes were also determined. HPLC analysis involved a custom ternary solvent system with custom acetonitrile and pH gradients. The limits of detection were 2 – 20 pmol and the interbatch coefficients of variation 4 – 29%—depending on the analyte. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Aminoquinolyl-N-hydroxysuccinimidyl carbamate; Glycation; Fructosyl-lysine; Advanced glycation endproducts; Hydroimidazolone
1. Introduction Glycation adducts found in proteins of physiological systems were a group of compounds of diverse structure and physiological function [1]. A comprehensive survey of the concentrations of advanced glycation endproducts (AGEs) in proteins was required
Abbreviations: AQC, aminoquinolyl-N-hydroxysuccinimidyl carbamate; AGEs, advanced glycation endproducts; AGEmin – HSA, human serum albumin minimally modified by glucose derived AGEs; HSA, human serum albumin; MGmin – HSA, human serum albumin minimally modified by methylglyoxal. * Corresponding author. Tel./fax: +44-1206-873-010. E-mail address: [email protected] (P.J. Thornalley). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 8 9 5 - 6
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N. Ahmed, P.J. Thornalley / International Congress Series 1245 (2002) 279–283
to associate particular AGEs with glycating agents and pathological conditions. We developed a procedure for the survey of a relatively wide range of AGEs in proteins using the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-based chromatographic assay [2]. Enzymatic hydrolysis procedures for the analysis of proteins in plasma and in haemoglobin were developed.
2. Materials and methods AQC and analytical glycation adduct standards (fructosyl-lysine and AGEs) were synthesised by modification of published methods [1,3,4] except structural isomers of hydroimidazolone AGEs were prepared by novel unambiguous synthesis—to be published elsewhere. Protein samples (500 Ag; 2 mg/ml) were extracted with 2 1 volume of watersaturated diethyl ether to remove lipids and the residual ether removed by centrifugal evaporation. The samples were washed three times with water by ultrafiltration (10 kDa cut-off membrane) and the protein concentration determined by the Bradford method. Protein samples (100 Ag) were diluted to 25 Al with water. Aliquots of 40 mM HCl (25 Al), pepsin solution (2 mg/ml in 20 mM HCl; 5 Al), and thymol solution (2 mg/ml in 20 mM HCl; 5 Al), were added and the samples were incubated at 37 jC for 24 h. The samples were neutralised and buffered at pH 7.4 by the addition of 25 Al of 0.5 M potassium phosphate buffer, pH 7.4, and 5 Al of 260 mM KOH. Pronase E solution (2 mg/ml in 10 mM KH2PO4, pH 7.4; 5 Al) was added and the samples were incubated at 37 jC for 24 h. Aminopeptidase solution (2 mg/ml in 10 mM KH2PO4, pH 7.4; 5 Al) and prolidase solution (2 mg/ml in 10 mM KH2PO4, pH 7.4; 5 Al) were added and the samples incubated at 37 jC for 48 h. This gave the final enzymatic hydrolysate. Aliquots of enzymatic hydrolysate (50 Al, equivalent to 50 Ag protein), internal standard (a-aminobutyric acid, 1 nmol; 10 Al), water (40 Al), derivatizing buffer (500 mM borate buffer, 400 AM DETAPAC, pH 8.8; 100 Al) and AQC (10 mM in acetonitrile; 200 Al) were mixed and incubated at 55 jC for 10 min. Calibration standards containing 0– 1000 pmol AGEs and 0– 2500 pmol fructosyl-lysine with 0 –20 nmol amino acids were derivatized similarly. The acetonitrile was then removed by centrifugal evaporation at room temperature and lyophilised to dryness. The sample was reconstituted in water (100 Al) and precipitated AQC degradation products removed by centrifugal filtration (0.2-Am pore). The filtrate was analysed for AGEs, fructosyl-lysine and amino acids by analytical reversed phase HPLC. Derivatized samples were analysed by reversed phase HPLC with a custom threesolvent gradient, adapted but significantly modified from that of van Wandelen and Cohen [2]. Changes were: (i) the extension of analysis time to 220 min; (ii) increased sample loading equivalent to ca. 220– 240 nmol of amino acids; and (iii) detection by absorbance spectrophotometry (amino acid analytes) and fluorescence (glycation adduct analytes). HPLC analysis was performed with a Waters HPLC system (717 plus autosampler with samples at 18 jC, 600 solvent delivery system, 474 fluorescence detector, 481 Lambda Max absorbance detector, with a two-channel data collection system). The columns were 3.9 150 mm, NOVAPAKk ODS, 4 Am, fitted with a 3.9 20 mm NOVAPAKk ODS guard column at 34 jC; the flow rate was 1 ml/min.
N. Ahmed, P.J. Thornalley / International Congress Series 1245 (2002) 279–283
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3. Results Derivatization of amino acids and glycation adducts with AQC and HPLC analysis of the AQC adducts gave an AGE assay technique of high sensitivity. The relatively mild derivatization technique [2] led to high recoveries of glycation adducts. Fructosyl-lysine, 12 AGEs and amino acid analytes were resolved and quantified—Figs. 1 and 2. Hydroimidazolone adducts of methylglyoxal (two epimeric pairs of two structural isomers, MGH1A, MG-H1B, MG-H2A, MG-H2B, peaks 12– 14 and 16), glyoxal (G-H, peak 9) and 3deoxyglucosone (3DG-H, peak 10), and two epimers of Nq-(1-carboxyethyl)-lysine (CELA, CELB, peaks 33 and 34) were detected for the first time. Two epimers of a further structural isomer of methylglyoxal hydroimidazolone, 2-amino-5-(2-amino-5hydro-5-methyl-4-imidazolon-3-yl)pentanoic acid (MG-H3), were detected but not quantified because of their instability under assay conditions. Arginine and threonine were unresolved but a second short chromatographic run with a slightly steeper acetonitrile gradient resolved these analytes. For the analysis of amino acids and AGEs in proteins glycated in vitro and in vivo by the AQC derivatization method, enzymatic hydrolysis was used since acid hydrolysis led to >90% loss in hydroimidazolone AGEs. The steps at pH
Fig. 1. Advanced glycation adducts detected in the AQC assay.
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N. Ahmed, P.J. Thornalley / International Congress Series 1245 (2002) 279–283
Fig. 2. Chromatographic assay of fructosyl-lysine, AGEs and amino acids by pre-column derivatization with AQC and (a) fluorescence detection and (b) absorbance detection. Chromatograms: calibration standard of fructosyl-lysine and AGEs (1 nmol) with amino acids (20 nmol). Key: peak number, retention time (min) and analyte: (1) 14.0, asp; (2) 17.3, ser; (3) 19.1, glu; (4) 22.1, gly; (5) 24.5, his; (6 and 7) 41.7, arg and thr; (8) 50.1, ala; (9) 61.0, G-H; (10) 66.5, 3DG-H; (11) 77.4, pro; (12) 82.8, MG – H1A; (13) 85.1, MG-H1B; (14) 86.8, MGH2A; (15) 89.3, IS (a-aminobutyric acid); (16) 94.1, MG-H2B and AQC degradation product; (17) 101.1, fructosyl-lysine; (18) 112.4, tyr; (19) 115.5, val; (20) 117.5, CML; (21) 122.6, met; (22) 125.3, DOLD; (23) 134.5, argpyrimidine; (24) 140.9, GOLD; (25) 155.3, ile; (26) 163.1, leu; (27) 166.1, MOLD; (28) 173.5, lys; (29) 175.6, pyrraline; (30) 188.4, phe; (31) 193.3, pentosidine; (32) 202.1, trp; (33) 205.6, CELA; (34) 206.5, CELB.
7.4 were performed under nitrogen or carbon monoxide (haemoglobin samples) to inhibit oxidative processes.
4. Discussion The AQC assay was particularly useful in the applications for the measurement of proteins glycated in vitro, to minimal and high extents, and this is the application where it may find most use. It was also useful for the resolution and detection of methylglyoxalderived hydroimidazolone structural isomers, MG-H1 and MG-H2. Structural isomerism of hydroimidazolones of methylglyoxal and glyoxal has been identified previously [5] or suspected [6]. Glycation adducts were investigated in human serum albumin minimally modified in vitro by methylglyoxal (MGmin – HSA) and minimally modified by glucose (AGEmin – HSA) under oxidative conditions [7]. Hydroimidazolone MG-H1 was the major methylglyoxal-derived protein adducts in MGmin – HSA. Fructosyl-lysine, glyoxalderived hydroimidazolone and Nq-carboxymethyl-lysine were major glycation adducts in AGEmin – HSA.
N. Ahmed, P.J. Thornalley / International Congress Series 1245 (2002) 279–283
283
References [1] P.J. Thornalley, Clinical significance of glycation, Clin. Lab. 45 (1999) 263 – 273. [2] C. van Wandelen, S.A. Cohen, Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxy-succinimidylcarbamate-derivatized amino acid mixtures, J. Chromatogr. 763 (1997) 11 – 22. [3] S.A. Cohen, D.P. Michaud, Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high performance liquid chromatography, Anal. Biochem. 211 (1993) 279 – 287. [4] P.J. Thornalley, A. Langborg, H.S. Minhas, Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose, Biochem. J. 344 (1999) 109 – 116. [5] T. Henle, A. Walter, R. Haebner, H. Klostermeryer, Detection and identification of a protein-bound imidazolone resulting from the reaction of arginine residues and methylglyoxal, Z. Lebensm.-Unters. Forsch. 199 (1994) 55 – 58. [6] T. Tajika, I. Bando, T. Furuta, N. Moriya, H. Koshino, M. Uramoto, Novel amino acid metabolite produced by Streptomyces sp.: taxonomy, isolation and structural elucidation, Biosci. Biotechnol. Biochem. 61 (1997) 1007 – 1010. [7] M.E. Westwood, O.K. Argirov, E.A. Abordo, P.J. Thornalley, Methylglyoxal-modified arginine residues—a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells, Biochim. Biophys. Acta 1356 (1997) 84 – 94.
Assay of early and advanced glycation adducts by enzymatic hydrolysis of proteins and HPLC of 6-aminoquinolylcarbonyl adducts Naila Ahmed, Paul J. Thornalley * Department of Biological Sciences, University of Essex, Central Campus, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK
Abstract A method for the assay of early and advanced glycation endproducts (AGEs) in proteins has been developed and validated. Protein samples were de-lipidified, hydrolysed enzymatically, the hydrolysate derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and AQC adducts analysed by HPLC with fluorimetric detection. Nq-Fructosyl-lysine and 12 AGEs were assayed. Amino acid analytes were also determined. HPLC analysis involved a custom ternary solvent system with custom acetonitrile and pH gradients. The limits of detection were 2 – 20 pmol and the interbatch coefficients of variation 4 – 29%—depending on the analyte. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Aminoquinolyl-N-hydroxysuccinimidyl carbamate; Glycation; Fructosyl-lysine; Advanced glycation endproducts; Hydroimidazolone
1. Introduction Glycation adducts found in proteins of physiological systems were a group of compounds of diverse structure and physiological function [1]. A comprehensive survey of the concentrations of advanced glycation endproducts (AGEs) in proteins was required
Abbreviations: AQC, aminoquinolyl-N-hydroxysuccinimidyl carbamate; AGEs, advanced glycation endproducts; AGEmin – HSA, human serum albumin minimally modified by glucose derived AGEs; HSA, human serum albumin; MGmin – HSA, human serum albumin minimally modified by methylglyoxal. * Corresponding author. Tel./fax: +44-1206-873-010. E-mail address: [email protected] (P.J. Thornalley). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 8 9 5 - 6
280
N. Ahmed, P.J. Thornalley / International Congress Series 1245 (2002) 279–283
to associate particular AGEs with glycating agents and pathological conditions. We developed a procedure for the survey of a relatively wide range of AGEs in proteins using the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-based chromatographic assay [2]. Enzymatic hydrolysis procedures for the analysis of proteins in plasma and in haemoglobin were developed.
2. Materials and methods AQC and analytical glycation adduct standards (fructosyl-lysine and AGEs) were synthesised by modification of published methods [1,3,4] except structural isomers of hydroimidazolone AGEs were prepared by novel unambiguous synthesis—to be published elsewhere. Protein samples (500 Ag; 2 mg/ml) were extracted with 2 1 volume of watersaturated diethyl ether to remove lipids and the residual ether removed by centrifugal evaporation. The samples were washed three times with water by ultrafiltration (10 kDa cut-off membrane) and the protein concentration determined by the Bradford method. Protein samples (100 Ag) were diluted to 25 Al with water. Aliquots of 40 mM HCl (25 Al), pepsin solution (2 mg/ml in 20 mM HCl; 5 Al), and thymol solution (2 mg/ml in 20 mM HCl; 5 Al), were added and the samples were incubated at 37 jC for 24 h. The samples were neutralised and buffered at pH 7.4 by the addition of 25 Al of 0.5 M potassium phosphate buffer, pH 7.4, and 5 Al of 260 mM KOH. Pronase E solution (2 mg/ml in 10 mM KH2PO4, pH 7.4; 5 Al) was added and the samples were incubated at 37 jC for 24 h. Aminopeptidase solution (2 mg/ml in 10 mM KH2PO4, pH 7.4; 5 Al) and prolidase solution (2 mg/ml in 10 mM KH2PO4, pH 7.4; 5 Al) were added and the samples incubated at 37 jC for 48 h. This gave the final enzymatic hydrolysate. Aliquots of enzymatic hydrolysate (50 Al, equivalent to 50 Ag protein), internal standard (a-aminobutyric acid, 1 nmol; 10 Al), water (40 Al), derivatizing buffer (500 mM borate buffer, 400 AM DETAPAC, pH 8.8; 100 Al) and AQC (10 mM in acetonitrile; 200 Al) were mixed and incubated at 55 jC for 10 min. Calibration standards containing 0– 1000 pmol AGEs and 0– 2500 pmol fructosyl-lysine with 0 –20 nmol amino acids were derivatized similarly. The acetonitrile was then removed by centrifugal evaporation at room temperature and lyophilised to dryness. The sample was reconstituted in water (100 Al) and precipitated AQC degradation products removed by centrifugal filtration (0.2-Am pore). The filtrate was analysed for AGEs, fructosyl-lysine and amino acids by analytical reversed phase HPLC. Derivatized samples were analysed by reversed phase HPLC with a custom threesolvent gradient, adapted but significantly modified from that of van Wandelen and Cohen [2]. Changes were: (i) the extension of analysis time to 220 min; (ii) increased sample loading equivalent to ca. 220– 240 nmol of amino acids; and (iii) detection by absorbance spectrophotometry (amino acid analytes) and fluorescence (glycation adduct analytes). HPLC analysis was performed with a Waters HPLC system (717 plus autosampler with samples at 18 jC, 600 solvent delivery system, 474 fluorescence detector, 481 Lambda Max absorbance detector, with a two-channel data collection system). The columns were 3.9 150 mm, NOVAPAKk ODS, 4 Am, fitted with a 3.9 20 mm NOVAPAKk ODS guard column at 34 jC; the flow rate was 1 ml/min.
N. Ahmed, P.J. Thornalley / International Congress Series 1245 (2002) 279–283
281
3. Results Derivatization of amino acids and glycation adducts with AQC and HPLC analysis of the AQC adducts gave an AGE assay technique of high sensitivity. The relatively mild derivatization technique [2] led to high recoveries of glycation adducts. Fructosyl-lysine, 12 AGEs and amino acid analytes were resolved and quantified—Figs. 1 and 2. Hydroimidazolone adducts of methylglyoxal (two epimeric pairs of two structural isomers, MGH1A, MG-H1B, MG-H2A, MG-H2B, peaks 12– 14 and 16), glyoxal (G-H, peak 9) and 3deoxyglucosone (3DG-H, peak 10), and two epimers of Nq-(1-carboxyethyl)-lysine (CELA, CELB, peaks 33 and 34) were detected for the first time. Two epimers of a further structural isomer of methylglyoxal hydroimidazolone, 2-amino-5-(2-amino-5hydro-5-methyl-4-imidazolon-3-yl)pentanoic acid (MG-H3), were detected but not quantified because of their instability under assay conditions. Arginine and threonine were unresolved but a second short chromatographic run with a slightly steeper acetonitrile gradient resolved these analytes. For the analysis of amino acids and AGEs in proteins glycated in vitro and in vivo by the AQC derivatization method, enzymatic hydrolysis was used since acid hydrolysis led to >90% loss in hydroimidazolone AGEs. The steps at pH
Fig. 1. Advanced glycation adducts detected in the AQC assay.
282
N. Ahmed, P.J. Thornalley / International Congress Series 1245 (2002) 279–283
Fig. 2. Chromatographic assay of fructosyl-lysine, AGEs and amino acids by pre-column derivatization with AQC and (a) fluorescence detection and (b) absorbance detection. Chromatograms: calibration standard of fructosyl-lysine and AGEs (1 nmol) with amino acids (20 nmol). Key: peak number, retention time (min) and analyte: (1) 14.0, asp; (2) 17.3, ser; (3) 19.1, glu; (4) 22.1, gly; (5) 24.5, his; (6 and 7) 41.7, arg and thr; (8) 50.1, ala; (9) 61.0, G-H; (10) 66.5, 3DG-H; (11) 77.4, pro; (12) 82.8, MG – H1A; (13) 85.1, MG-H1B; (14) 86.8, MGH2A; (15) 89.3, IS (a-aminobutyric acid); (16) 94.1, MG-H2B and AQC degradation product; (17) 101.1, fructosyl-lysine; (18) 112.4, tyr; (19) 115.5, val; (20) 117.5, CML; (21) 122.6, met; (22) 125.3, DOLD; (23) 134.5, argpyrimidine; (24) 140.9, GOLD; (25) 155.3, ile; (26) 163.1, leu; (27) 166.1, MOLD; (28) 173.5, lys; (29) 175.6, pyrraline; (30) 188.4, phe; (31) 193.3, pentosidine; (32) 202.1, trp; (33) 205.6, CELA; (34) 206.5, CELB.
7.4 were performed under nitrogen or carbon monoxide (haemoglobin samples) to inhibit oxidative processes.
4. Discussion The AQC assay was particularly useful in the applications for the measurement of proteins glycated in vitro, to minimal and high extents, and this is the application where it may find most use. It was also useful for the resolution and detection of methylglyoxalderived hydroimidazolone structural isomers, MG-H1 and MG-H2. Structural isomerism of hydroimidazolones of methylglyoxal and glyoxal has been identified previously [5] or suspected [6]. Glycation adducts were investigated in human serum albumin minimally modified in vitro by methylglyoxal (MGmin – HSA) and minimally modified by glucose (AGEmin – HSA) under oxidative conditions [7]. Hydroimidazolone MG-H1 was the major methylglyoxal-derived protein adducts in MGmin – HSA. Fructosyl-lysine, glyoxalderived hydroimidazolone and Nq-carboxymethyl-lysine were major glycation adducts in AGEmin – HSA.
N. Ahmed, P.J. Thornalley / International Congress Series 1245 (2002) 279–283
283
References [1] P.J. Thornalley, Clinical significance of glycation, Clin. Lab. 45 (1999) 263 – 273. [2] C. van Wandelen, S.A. Cohen, Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxy-succinimidylcarbamate-derivatized amino acid mixtures, J. Chromatogr. 763 (1997) 11 – 22. [3] S.A. Cohen, D.P. Michaud, Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high performance liquid chromatography, Anal. Biochem. 211 (1993) 279 – 287. [4] P.J. Thornalley, A. Langborg, H.S. Minhas, Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose, Biochem. J. 344 (1999) 109 – 116. [5] T. Henle, A. Walter, R. Haebner, H. Klostermeryer, Detection and identification of a protein-bound imidazolone resulting from the reaction of arginine residues and methylglyoxal, Z. Lebensm.-Unters. Forsch. 199 (1994) 55 – 58. [6] T. Tajika, I. Bando, T. Furuta, N. Moriya, H. Koshino, M. Uramoto, Novel amino acid metabolite produced by Streptomyces sp.: taxonomy, isolation and structural elucidation, Biosci. Biotechnol. Biochem. 61 (1997) 1007 – 1010. [7] M.E. Westwood, O.K. Argirov, E.A. Abordo, P.J. Thornalley, Methylglyoxal-modified arginine residues—a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells, Biochim. Biophys. Acta 1356 (1997) 84 – 94.