©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Protein Modification by Glyoxal and Glycolaldehyde, Reactive Intermediates of the Maillard Reaction (*)

Marcus A. Glomb (§) , Vincent M. Monnier

From the (1) Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The role of glyoxal and glycolaldehyde in protein cross-linking and N-(carboxymethyl)lysine (CML) formation during Maillard reaction under physiological conditions was investigated. Incubation of bovine serum albumin with these reagents lead to rapid formation of C-2-imine cross-links and CML. Initial CML formation rate from glyoxal was not dependent on oxidation, suggesting an intramolecular Cannizzaro reaction. CML formation from glucose/lysine or Amadori product of both was strongly dependent on oxidation. Blocking of Amadori product by boric acid totally suppressed CML formation from Amadori product, but only by 37% in the glucose/lysine system. Trapping of glyoxal with aminoguanidine hardly suppressed CML formation from Amadori product, whereas it blocked 50% of CML production in the glucose/lysine system. While these results would support a significant role for glucose autoxidation in CML formation, the addition of lysine to a glucose/aminoguanidine incubation system catalyzed glyoxal-triazine formation 7-fold, thereby strongly suggesting that glucose autoxidation is not a factor for glyoxal-mediated CML formation. Based on these results, it can be estimated that approximately 50% of the CML forming in a glucose/lysine system originates from oxidation of Amadori product, and 40-50% originates from a pre-Amadori stage largely independent from glucose autoxidation. This step may be related to the so-called Namiki pathway of the Maillard reaction.


INTRODUCTION

The reaction between reducing sugars and amino structures in amino acids or proteins (also called Maillard reaction) has been shown to proceed in living systems and to correlate with age and severity of diabetes (1) . Unlike organic syntheses, it does not result in one or few well defined products but proceeds through complex reaction pathways resulting in a large number of structures (2) . After the initial formation of a Schiff base adduct between the carbonyl and the amine moeity, the aldimine rearranges to a more stable ketoamine or Amadori product. Enolization, dehydration, cyclization, fragmentation, and oxidation reactions form reactive intermediates that ultimately lead to stable end products.

Although enormous effort was devoted to elucidate the nature of these protein modifications, so far only two structures besides the Amadori product have been successfully established in vivo, pentosidine and N-carboxymethyllysine (CML)()(3, 4) . A third structure, pyrraline, was found in tissue matrix by immunochemical and chromatographic methods (5, 6, 7) , but its existence in vivo is controversial (8) .

The slow progress in elucidating the structure of the major Maillard cross-link in proteins together with the finding of a C-2-glucose fragmentation product in form of CML, suggested to us that the pathway proposed by Namiki (9) may play an important role in CML formation and cross-linking. In this pathway, glyoxal and glycolaldehyde were detected as Maillard fragmentation products in heated model systems of sugars and alkylamines (10, 11) . According to Namiki, the Schiff base adduct 1 undergoes retro aldol condensation at C-2-C-3 to yield fragment 2 (Fig. 1). Compound 2 is the Schiff base adduct of the amine with glycolaldehyde, which on one hand could hydrolyze to release glycolaldehyde or rearrange to form aldoamine 3. Condensation with a second molecule 3 was proposed to result in pyrazine 4, which has been shown to be easily oxidized and fragmented to give, for example, di-Schiff base adduct 5 or to release glyoxal (12) . Condensation with a second amine compound would yield imine 7. Thus, the formation of glyoxal and glycolaldehyde in this pathway are linked together, and both, upon reaction with amines, would result in common structures. Both molecules are highly reactive intermediates and have been reported to modify and cross-link proteins, although structures were only proposed (13, 14, 15) .


Figure 1: Reaction scheme for the formation of CML and imine cross-links during the Maillard reaction.



Based on these preliminary observations and the finding that carboxymethylation of amino acids can occur with glyoxal in heated reaction mixtures (16, 17) , we hypothesize that a mechanism involving glyoxal/glycolaldehyde could contribute to CML formation and protein cross-linking under physiological conditions. This hypothesis departs from the previous notion that CML originates only from the Amadori product (18, 19) .

In the first part of this study, we investigated the relationship between glyoxal/glycolaldehyde and presumed C-2-imine cross-links 5/ 7 in proteins incubated with these carbonyl compounds and various sugars. In the second part, we studied the mechanism of CML formation from these reagents. In addition, we evaluated the contribution of glyoxal versus oxidative cleavage of the Amadori product on CML formation and the relative importance of the ``Namiki pathway'' during incubations with reducing sugars.


EXPERIMENTAL PROCEDURES

Reagents Reagents of highest quality available were obtained from Sigma, Fisher, and Aldrich, unless otherwise indicated. Spectra H and C nuclear magnetic resonance (NMR) spectra (MeSi (CDCl), 3-(trimethylsilyl)propionic-2,2,3,3- d-acid (DO) as internal standards) were recorded with a Varian 300 MHz spectrometer Gemini-300 (Varian Associates, Inc. Palo Alto, CA). High resolution fast atom bombardment mass spectral data were obtained at Michigan State University Mass Spectrometry Facility, and high resolution mass spectra were obtained with a Kratos MS 25 RFA dual beam, double focussing, magnetic sector mass spectometer (direct probe insertion, EI 20 eV). High Performance Liquid Chromatography HPLC was performed on a Waters gradient system (Waters Chromatography Division, Milford, MA) equipped with two model 510 pumps and a model 470 fluorescence detector. Amino acids were derivatized postcolumn with o-phthaldialdehyde (Aldrich) (20) . System 1 for detection of CML consisted of water (eluent A) and 70% methanol in water (eluent B), both with 0.01 M heptafluorobutyric acid (Aldrich), C18 column (0.4 25 cm, 5 µm, VYDAC 218TP54, Hesperia, CA), flow rate 1 ml/min, gradient 2% B for 20 min and then in 5 min to 100% B. System 2 for detection of CML and glucitolyl-lysine was as follows: column as system 1, with 5% propanol (eluent A) and 60% propanol in water (eluent B), both with 3 g of SDS (Fluka)/liter, 1 g of monobasic sodium phosphate monohydrate/liter, and adjusted to pH 2.8 with phosphoric acid, gradient 15% B 22% B in 30 min 40% B in 20 min 100% B in 5 min and flow rate 1 ml/min. System 3 for detection of 6 was as follows: column, flow and eluents like system 1, gradient 15% B 40% B in 25 min 50% B in 22 min 100% B in 3 min. System 4 for detection of 6 was as follows: column and flow like system 2, eluent A 5% propanol and eluent B 60% propanol in water, both with 3 g of SDS/liter, 1.5 g of monobasic sodium phosphate monohydrate/liter and adjusted to pH 7 with potassium hydroxide, gradient 15% B 25% B in 30 min 35% B in 5 min 100% B in 5 min. System 5 for preparative isolation of 6 was as follows: eluents like system 1, C18 column (2.2 25 cm, 10 µm, VYDAC 218TP1022), gradient 15% B 50% B in 50 min 100% B in 25 min and flow 8 ml/min. System 6 for preparative isolation of CML and 9 was as follows: eluents, column and flow like system 5, gradient 2% B for 40 min then to 100% B in 5 min. System 7 for detection of 9 was as follows: column, flow, and eluents like system 2, gradient 20% B 28% B in 40 min 100% B in 5 min. Coupled Gas Chromatography-Mass Spectrometry GC/MS was performed on a Hewlett Packard 5890 series II chromatograph (Wilmington, DE), quartz capillary column (25 m, inner diameter 0.2 mm, Ultra 5 (Hewlett Packard), 0.33 µm, He, 12 psi, 26.26 cm/s, constant flow program on), injection port 270 °C, interface 280 °C, temperature program 100 °C 200 °C/5 °Cminto 200 °C 270 °C/10 °Cminto 10 min isothermal 270 °C; connected to Hewlett Packard 5971 series mass selective detector, EI and positive CH-CI mode. Chromatography Silica gel 60 FMerck 5554 (EM Separations, Gibbstown, NJ) was used for thin-layer chromatography (TLC) and silica gel (63-200 µm) (Alltech, Deerfield, IL) was used for column chromatography. Chromatography solvents were all ACS grade. From the individual chromatographic fractions, solvents were evaporated under reduced pressure. Synthetic Procedures

Ethylenedihexylamine

A solution of 0.5 g (4.2 mmol) of 2,3-dihydroxy-1,4-dioxane (Fluka) and 1.25 g (12.5 mmol) of hexylamine (Fisher) in 5 ml of anhydrous methanol was stirred at 25 °C for 30 min, 380 mg of sodium borohydride (Fluka) was added, and after 15 min of stirring, the solvents were evaporated to dryness. The residue was taken up in 1 N NaOH and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and the solvents were evaporated. The residue was dissolved in 10 ml of anhydrous pyridine, and 5 ml of trifluoroacetic anhydride (Aldrich) were slowly added at 0 °C. The solution was stirred for 5 min at 0 °C and then for 55 min at ambient temperature. The solvents were evaporated to dryness, and the resulting brown oil was purified by column chromatography (eluent CHCl). Fractions containing N, N`-di-(trifluoroacetyl)ethylenedihexylamine ( R0.56, TLC (same solvent)) were combined and evaporated to dryness (300 mg (17%), colorless oil, GC t23.21 min). CI-GC/MS, m/ z 421 (M + 1;100) 307 (10) 224 (13) 210 (3) 198 (6) 154 (5) . To remove the trifluoroacetyl groups, 300 mg of N, N`-di(trifluoroacetyl)ethylenedihexylamine was dissolved in 0.5% potassium hydroxide solution in methanol and stirred for 45 min at 80 °C. TLC (solvent as above) showed complete absence of trifluoroacetyl derivative. Solvents were evaporated and the residue was taken up in 1 N NaOH and extracted with diethylether. The combined organic layers were dried over anhydrous sodium sulfate, and solvents were evaporated to yield ethylenedihexylamine (150 mg (overall 16%) of colorless oil, TLC R0.49 (solvent butanol/water/acetic acid, 25:5:3), GC t19.02 min). CI-GC/MS, m/ z 229 (M + 1;100) 227 (91) 157 (40) 128 (51) 114 (53) . H NMR(CDCl), (ppm) 0.88 (t, 6H, J = 6.9 Hz) 1.28 (m, 12H) 1.48 (m, 4H) 2.59 (t, 4H, J = 7.5 Hz) 2.71 (s, 4H).

2,15-Diamino-7,10-diaza-hexadecane-1,16-dioic acid 6

A suspension of 0.25 g (2.1 mmol) of 2,3-dihydroxy-1,4-dioxane, 1.28 g (5.2 mmol) of N- t-Boc-lysine (Bachem, Torrance, CA) and three potassium hydroxide pellets in 5 ml of anhydrous methanol was stirred for 1 h at room temperature. Following addition of 190 mg (5 mmol) of sodium borohydride, the solution was stirred for another 15 min, and the pH was then adjusted to 7 with 1 N HCl. TLC (solvent butanol/water/acetic acid, 25:5:3) revealed a new spot at R0.10. Eluents were evaporated to dryness, protecting groups were removed by treatment with 3 N HCl for 30 min, and the solution was then freeze dried. The lyophilisate was taken up in water and subjected to preparative HPLC system 5. Fractions containing 6 were collected and freeze dried (65 mg (10%), colorless amorphous hygroscopic powder, HPLC system 5, t63 min; system 3, t47.5 min; system 4, t30.3 min; GC ( 6 as its trifluoroacetylmethylester derivative) t32.81 min). EI(70 eV)-GC/MS, m/ z 378 (M-352;24) 356 (9) 319 (13) 309 (35) 305 (54) 194 (29) 180 (100) 178 (48) 152 (15) 140 (98) 126 (19) 114 (9) 96 (10) 84 (10) 67 (40) . H NMR(DO), (ppm) 1.38 (m, 4H), 1.62 (p, 4H, J = 7.7 Hz) 1.80 (m, 4H), 2.99 (t, 4H, J = 7.7 Hz), 3.28 (s, 4H), 3.81 (t, 2H, J = 6.3 Hz). C NMR(D2O), (ppm) 23.85, 27.56, 31.85, 45.39, 50.17, 55.68, 175.26. High resolution fast atom bombardment mass spectrometry, 319.4217 calculated 319.4233 for C14 H31 N4 O4. Elementary analysis showed that material obtained was the 64 heptafluorobutyrate salt.

N-(Carboxymethyl)lysine

246 mg (1 mmol) of N- t-Boc-lysine and 186 mg (1 mmol) of iodoacetic acid (Aldrich) were dissolved in 10 ml of phosphate buffer, pH 10, and stirred overnight at ambient temperature. The solution was freeze dried and purified by column chromatography (solvent methanol/ethyl acetate, 2:1). Fractions containing material with R0.69 (TLC/solvent methanol) were evaporated, taken up in 3 N HCl, and stirred for 60 min at room temperature. Solvents were removed by freeze drying, and the lyophilisate was subjected to preparative HPLC system 6. Fractions containing CML were combined and freeze dried (45 mg (22%), colorless amorphous powder, HPLC system 6 t22.1 min, system 1 t9.4 min, system 2 t26.3 min, GC (CML as its trifluoroacetylmethylester derivative) t22.03 min, spectroscopic data compliant with the literature (4) , elementary analysis showed that the material obtained was the CML2 heptafluorobutyrate salt).

N-(Glucitolyl)lysine (reduced Amadori product of glucose and lysine)

A solution of 0.6 g (3.3 mmol) of glucose (Sigma), 0.82 g (3.3 mmol) of N- t-Boc-lysine, and 0.21 g (3.3 mmol) of sodium cyanoborohydride (Aldrich) in 8.5 ml of anhydrous methanol was stirred overnight, 2 g of silica gel was added, solvents were evaporated, and the resulting powder was fractionated by column chromatography (eluent methanol/ethyl acetate, 1:2). Fractions containing N- t-Boc- N-(glucitolyl)lysine ( R0.12, TLC, eluent butanol/water/acetic acid, 25:5:3) were evaporated. The residue was taken up in 3 N HCl, stirred for 60 min at room temperature, and freeze dried. N-(glucitolyl)lysine was obtained as colorless crystals (125 mg (12%), HPLC system 2 t39.1 min, spectroscopic data compliant with the literature (21) ).

N-(2-Hydroxyethyl)lysine 9

273 mg (1.1 mmol) of N- t-Boc-lysine, 66 mg (1.1 mmol) of glycolaldehyde, and 69 mg (1.1 mmol) of sodium cyanoborohydride were dissolved in 7 ml of anhydrous methanol and stirred overnight. Solvents were evaporated, and the resulting oil was applied to column chromatography (eluent methanol/ethyl acetate, 1.75:1) to remove inorganic salts. Fractions containing material with R0.37 (TLC solvent n-butyl alcohol/water/acetic acid, 25:5:3) were combined, eluents were removed, and the residue was taken up in 3 N HCl and stirred for 60 min at room temperature. Solvents were removed by freeze drying, and the lyophilisate was subjected to preparative HPLC system 6. Fractions with 9 were combined and freeze dried. (75 mg (35%), colorless amorphous powder, HPLC system 6 t37 min, system 1 t15.5 min, system 7 t37.1 min, GC ( 9 as its trifluoroacetylmethylester derivative) t20.31 min, elementary analysis showed that the material obtained was the HEL2 heptafluorobutyrate salt). EI(70 eV)-GC/MS, m/ z 460 (M-32;14) 433 (6) 395 (6) 379 (9) 346 (12) 319 (50) 266 (35) 180 (59) 141 (100) 69 (50) . High resolution mass spectrometry, m/ z 492.0948 (M°), calculated 492.0943 for CHFNO, m/ z 460.0636, calculated 460.0591 for CHFNO, m/ z 266.0254, calculated 266.0256 for CHFNO.

N-(2-Hydroxy-1-D-ethyl) ( 9`) and N-(2-hydroxy-1,2- d-ethyl)lysine ( 9") were synthesized like nonlabeled 9 but with the use of sodium cyanoborodeuteride ( 9`) or sodium cyanoborodeuteride (Aldrich) and glyoxal ( 9"). EI(70 eV)-GC/MS, 9` (trifluoroacetylmethylester derivative), m/ z 461 (M°-32;14) 434 (5) 396 (5) 380 (8) 347 (9) 320 (59) 267 (45) 180 (63) 142 (100) 69 (71) . EI(70 eV)-GC/MS 9" (trifluoroacetylmethylester derivative), m/ z 462 (M°-32;11) 435 (4) 397 (5) 381 (6) 348 (10) 321 (48) 268 (31) 180 (47) 143 (100) 69 (51) .

Radioactive Labeled N-(1-Deoxyfructos-1-yl)propylamine

Unlabeled glucose was added to 250 µCi of 1-C- and 6-C-labeled glucose (Amersham Corp.) to adjust the specific activity to 10.8 mCi/mmol. After complete removal of water, the residue was taken up in 50 µl of anhydrous methanol and 150 µl of a solution of 4.2 µl of propylamine/100 µl of methanol, and the solution was heated for 45 min at 65 °C in a closed system. Solvents were evaporated, 100 µl of a solution of 2.94 mg of oxalic acid2HO/100 µl of methanol were added, and the mixture was heated for 15 min at 65 °C in a closed system. The methanol was removed, 0.5 ml of 1 N NaOH were added, and the solution was extracted 5 times with methylenechloride. After neutralization with 1 N HCl, the aqueous layer was freeze dried, and the lyophilisate was taken up in 400 µl of water and applied to column chromatography (Dowex 50W-X4-400, Aldrich, 3 cm height in Pasteur pipette). To remove unreacted glucose, the column was first washed with 6 ml of water; the Amadori product was then eluted with 0.2 N pyridine formate, pH 5.5, and proper fractions were combined (control by TLC (eluent n-butyl alcohol/water/acetic acid, 25:5:3, R0.16) and radioactivity TLC scanner (Berthold, Germany)) and freeze dried.

N- t-Boc-N-(1-deoxyfructos-1-yl)lysine and N- t-Boc- N-(1-deoxyribulos-1-yl)lysine were synthesized mainly according to Ref. 22. N-(1-Deoxyfructos-1-yl)propylamine and 3-amino-1,2,4-triazine (GC t8.40 min (triazine as its trimethylsilyl derivative)) according to Refs. 23 and 24, respectively. Incubations All incubations were conducted at 37 °C in a shaker incubator after sterile filtration (0.2-µm filter, Gelman Science, Ann Arbor, MI), followed by reduction prior to hydrolysis. Deaerated conditions were achieved by the presence of 1 mM phytic acid (Sigma), 1 mM diethylenetriaminepentaacetic acid (Sigma), and gassing for 3 min with argon. Phosphate-buffered saline (pH 7.4, unless otherwise indicated) was used in all glyoxal/glycolaldehyde experiments; in all other cases, 0.2 M phosphate buffer, pH 7.4, was used. Samples were reduced for 1 h at room temperature in the presence of a 1.25-fold (glyoxal/glycolaldehyde) and a 5.3-fold (all other) molar excess of sodium borohydride to the carbonyl compounds used. In protein incubations, BSA (Sigma) concentration was 0.1 mM. After reduction, the protein was precipitated and washed twice with trichloroacetic acid (Sigma) (10, 5, and 5%). The resulting precipitate was dried and subjected to acid hydrolysis (6 N HCl) for 20 h at 110 °C under argon. For incubations with radioactive labeled sugars (specific activity, 10.8 mCi/mmol) concentrations of 77.3 µCi/500 µl were used. Analytical Procedures Imines 5 and 7 were monitored as their common reduction product 6. For HPLC analysis, hydrolysates were evaporated in a Savant Speed-Vac concentrator (Hicksville, NY) and taken up in water. 9 was determined with HPLC system 7 after material had been collected from HPLC system 1 ( t13.5-18.0 min) without postcolumn derivatization. CML from incubations with radioactive labeled sugars was first collected from HPLC system 1 without postcolumn detection ( t6-11.5 min) and then quantified on HPLC system 2 by counting the radioactivity of proper fractions (LS6000, Beckman, Fullerton, CA) or postcolumn derivatization.

For GC/MS analysis of amino acids, proper fractions were collected from HPLC without postcolumn derivatization (HPLC system 5 for 6, HPLC system 6 for CML and 9, 9`, 9"), solvents were removed to absolute dryness, and material obtained was derivatized as trifluoroacetylmethylester derivatives. First, up to 10 mg of material was dissolved in 1 ml of 0.1 M thionylchloride (Fluka) solution in anhydrous methanol and heated for 1 h at 110 °C. Solvents were evaporated to absolute dryness. The residue was taken up in 300 µl of pyridine and 100 µl of trifluoroacetic anhydride (Aldrich), and the reaction mixture was heated for 10 min at 65 °C. After complete removal of solvents, the residue was dissolved in ethyl acetate and passed over a short silica gel column (eluent ethyl acetate). Proper fractions were combined and subjected to GC/MS.

3-Amino-1,2,4-triazine was extracted from incubations containing aminoguanidine with ethyl acetate. Solvents were evaporated after drying the combined organic layers over anhydrous sodium sulfate. The residue was derivatized using a 1:1 (v/v) ratio of pyridine and N, O-bistrimethyl silylacetamide (Fluka), and the reaction mixture was analyzed with GC/MS.

Amadori product concentration in incubations with N- t-Boc-lysine was determined prior to acid hydrolysis after reduction and removal of the protecting groups by 3 N HCl as N-(glucitolyl)lysine. Solvents were removed in a Savant Speed-Vac concentrator, and the residue was taken up in water and subjected to HPLC system 2. SDS-Polyacrylamide Gel Electrophoresis For estimation of the extent of cross-linking mediated by glyoxal/glycolaldehyde at various time points, RNase (0.5 mM, Sigma) incubations were reduced, dialyzed overnight at 4 °C against phosphate-buffered saline, pH 7.4, and analyzed by SDS-gel electrophoresis on a 12% acrylamide gel. The amounts of RNase loaded onto the gel was 20 µg in each lane. The gel was stained with Coomassie Blue for 1 h and destained in a mixture of 40% methanol and 10% acetic acid in water.


RESULTS

Because of the instability of the C-2-imines 5 and 7 (Fig. 1) during acid hydrolysis, their common reduced form 6, 2,15-diamino-7,10-diaza-hexadecane-1,16-dioic acid, was synthesized from N- t-Boc-lysine and 2,3-dihydroxy-1,4-dioxane and purified by preparative HPLC. The structure was unequivocally established from its spectroscopical data as described under ``Experimental Procedures.'' To optimize conditions, the synthesis of the hexylamine derivative of 6 was studied first. Best conditions were obtained using 2,3-dihydroxy-1,4-dioxane as a water-free glyoxal substitute (25) , strong alkaline reaction conditions, and subsequent reduction with sodium borohydride rather than the use of commercially available glyoxal/water solutions under neutral conditions in the presence of sodium cyanoborohydride. Product formation was assessed by coupled GC/MS.

Structure 6 was found to be stable under the conditions used for acid hydrolysis of proteins. To confirm the nature of 6 in hydrolysates, two different HPLC systems with o-phthaldialdehyde-postcolumn derivatization and fluorescence detection were developed, and the absence of the corresponding peak in nonreduced samples was confirmed throughout all investigations. Additionally, in selected cases, the material was collected by HPLC, derivatized, and applied to GC/MS.

In BSA incubations, the formation of diimine 5 monitored after reduction to 6 was dependent on the glyoxal concentration and began to level off above 10-20 mM (Fig. 2 A). We chose 20 mM to investigate cross-link structures 5 and 7 in BSA incubated with glyoxal or glycolaldehyde, respectively. Time course experiments ranging from 5 min to 170 h showed that the formation quickly reached a maximum at about 0.5 h followed by degradation (Fig. 2 B). Remarkably, in the case of glycolaldehyde, the initial peak was much smaller, but the steady level reached later was higher than with glyoxal. These findings were correlated with the rate of cross-linking using SDS-polyacrylamide gel electrophoresis after dialysis of the reduced and unreduced reaction mixtures (Fig. 2 B, inset). Reduced and unreduced samples showed no visible difference (data not shown). Cross-linking increased with time. With glyoxal, the trimer became faintly visible at 20 mM, and more intramolecular modifications could be observed in contrast to glycolaldehyde that had significant intermolecular reactions.


Figure 2: A, effect of glyoxal concentration on the formation of C-2-imine 5 in BSA incubations. B, time-dependent formation of C-2-imines 5 and 7 in BSA incubations with 20 mM glyoxal () and 20 mM glycolaldehyde (▾). B, inset, formation of polymers during incubation of RNase with 20 mM glyoxal ( 8/5 h, 7/170 h), 200 mM glyoxal ( 6/5 h, 5/170 h), and 20 mM glycolaldehyde ( 4/5 h, 3/170 h), monitored by SDS-polyacrylamide gel electrophoresis. Lane 1 shows low molecular mass standard (14.4-97.4 kDa), and lane 2 shows control incubation (170 h) without carbonyl compounds added.



C-2-imines 5/ 7 were also detected in other sugar-BSA incubations (). The rate of formation was time-dependent and roughly followed the reactivity of the sugar. Glucose protein mixtures showed no cross-link formation under the same conditions (detection limit of this experiment, 0.1 mmol/mol of lysine). In a detailed time course experiment, the cross-links formed progressively during incubation with ribose until a peak was reached at about 120 h with 0.28 mmol/mol of lysine followed by degradation. Retention times and mass spectra of derivatized collected HPLC material from glyoxal/glycolaldehyde/ribose incubation mixtures were identical to the data of the synthesized authentic product 6 (Fig. 3).


Figure 3: Identification of C-2-imines 5 and 7 as their reduction product 6 in BSA incubations with 20 mM glyoxal (B), 20 mM glycolaldehyde ( C) and 200 mM ribose ( D). Mass spectra obtained after derivatization are identical to the one of synthesized authentic amino acid 6 (A).



The data presented so far suggested that a significant amount of 5 and 7 was further degraded into one or several more stable products. One known product, which contains a C-2 fragment of the original sugar, is CML. Incubations of BSA with both glyoxal and glycolaldehyde revealed indeed large quantities of CML formed (Fig. 4). The identity of CML detected by HPLC was confirmed after collection and derivatization by GC/MS (Fig. 5). For comparison, authentic CML was synthesized. Deaerated conditions and presence of transition metal ion chelator altered the rate of formation from both carbonyl compounds.


Figure 4: Time-dependent formation of CML in BSA incubations with () 20 mM glyoxal and (▾) 20 mM glycolaldehyde. Full symbols indicate aerated conditions; open symbols indicate deaerated conditions. Inset shows same experiment up to 5 h.




Figure 5: Identification of CML formed in BSA incubations with 20 mM glyoxal ( B) and 20 mM glycolaldehyde ( C) by GC/MS. Mass spectra obtained after derivatization are identical to the one of authentic CML ( A).



However, the role of oxidation in CML formation from glyoxal and glycolaldehyde is complex since the initial formation rate of CML from glyoxal is not dependent on oxygen, whereas it is totally suppressed under anaerobic conditions for glycolaldehyde (Fig. 4, inset). This suggests that CML formation from glyoxal can occur through an intramolecular Cannizzaro reaction without oxidation (Fig. 6, intermediate 8). In support of this possibility, pH studies showed that CML synthesis from glyoxal was favored by alkaline conditions, pH 9, but totally suppressed at pH 5 (data not shown).


Figure 6: Proposed mechanism for formation of CML from glyoxal and glycolaldehyde.



Whereas oxidation of Amadori-rearranged glycolaldehyde adduct 3 (Fig. 6) is a necessary step for initial CML formation, the finding that CML formation during long term incubation of BSA with glycolaldehyde progresses in spite of deaerated conditions suggests a possible intermolecular Cannizzaro reaction of aldoamine 3 into acid CML and alcohol N-(2-hydroxyethyl)lysine 9. In order to clarify the significance of this pathway, authentic 9 was synthesized and detected in high levels upon trapping the Schiff base 2 with sodium cyanoborohydride. On the other hand, only small levels were recovered without reducing reagent ( i.e. 4.5 compared with 29.2 mmol/mol of lysine of CML at 170 h). Since even lower levels were detected under deaerated conditions (2.0 mmol/mol of lysine), intermolecular Cannizzaro reaction is excluded as a significant source of CML from glycolaldehyde.

Additional experiments with 5 mM aminoguanidine as a trapping reagent specific for -dicarbonyl compounds (24) showed that whereas only 1.16% (aeration) and 0.95% (deaeration) of glycolaldehyde was transformed into glyoxal, as measured as by the corresponding triazine, as much as 37% CML formation was suppressed. The formation of an aromatic ring structure represents such gain of stability that not only free glyoxal will react, but hidden glyoxal in Schiff bases like 8 or aminals (26) will also react. Thus, detection of the glyoxal-triazine and significant inhibition of CML formation suggests glyoxal or glyoxal derivatives 8 as major intermediates for CML synthesis in glycolaldehyde incubations.

The data so far implicate unequivocally glyoxal and glycolaldehyde in CML formation and raise the question of the mechanisms by which CML forms from higher sugars, as demonstrated in and by others (19, 27) . In our own experiments (), CML formation rates in BSA incubations generally followed the anomerization rates of the sugar except for D-ribose, which was a potent generator of CML.

The key question concerning CML synthesis from higher sugars is whether formation rates are primarily dependent on sugar autoxidation or Schiff base fragmentation as proposed by Wolff (28) and Namiki (10) , respectively, or whether the major source stems from oxidative breakdown of the Amadori product of the sugar (19) . Clarification of this question is problematic since all molecules are simultaneously present in sugar-amine incubation systems.

As previously reported (18) , CML formation from glucose and N- t-Boc-lysine was strongly dependent on presence of oxygen and free metals (Fig. 7). Aminoguanidine and boric acid were used as tools to dissect the relative importance of Amadori product versus glucose as a precursor of CML in incubations with N- t-Boc-lysine. Only when the reaction was started from glucose itself did aminoguanidine have an important impact, as evidenced by almost 50% reduction in CML formation. In presence of Amadori product, incubations with aminoguanidine showed almost no effect up to 100 h, although the absolute amount of inhibition after 170 h was larger compared with glucose experiments. Formation of the triazine derived from glyoxal and aminoguanidine measured by GC/MS was strictly dependent on oxidation (). After 170 h, the Amadori product produced about 4 times more glyoxal than the sugar. In addition, 7-fold catalytic effect of N- t-Boc-lysine on glyoxal formation as the amine component in glucose systems is evident. In contrast to aminoguanidine, 10 times excess boric acid completely inhibit CML formation from the Amadori compound, but only by about 37% when the reaction was initiated by glucose. This suggests an additional mechanism, which is not dependent on the Amadori product. Indeed, whereas in presence of Amadori product, only as much as 40% degradation within 170 h yielded 2.8 mmol of glyoxal triazine/mol of lysine (), only 0.7% Amadori product were actually detected in the glucose/ N- t-Boc-lysine system after the same time (100% refers to the initial Amadori concentration in Amadori product only incubations).


Figure 7: Time dependent formation of CML in incubations of ( A) glucose (42.2 mM) and N- t-Boc-lysine (42.2 mM) ( B) Amadori product of glucose and N- t-Boc-lysine. Incubations were conducted under (▾) aerated, aerated in presence of aminoguanidine (5 mM, ), and in the presence of boric acid (420 mM, ), and () deaerated conditions.



Two different approaches were utilized to establish the formation of glyoxal and glycolaldehyde in incubations of higher sugars without the use of trapping reagent. First, the formation of N-(2-hydroxyethyl)lysine 9 in reduced incubations of glucose and ribose was confirmed reaching concentrations of 0.1 and 7.5 mmol/mol of lysine, respectively, after 170 h. In the case of ribose, the nature of the original C-2 carbonyl intermediates could be elucidated by reducing with sodium borodeuterate. Isotopic distribution of characteristic fragments in the mass spectrum, as 461 for N-(2-hydroxy-1- d-ethyl)lysine 9` and 462 for N-(2-hydroxy-1, 2- d-ethyl)lysine 9`', revealed a ratio of 4:5 of 9` to 9" through comparison with the spectra of authentic synthesized compounds 9, 9`, and 9". Signals at 461 and 462 represent loss of methanol, which is a common fragmentation pattern for methylesters. Second, in incubations of the Amadori product (0.5 mM) of glucose and propylamine with N- t-Boc-lysine (42 mM), the formation of CML was confirmed and found to increase with time (0.47 mmol/mol of lysine at 170 h).

Investigations into the origin of carbon atoms involved in CML formation utilizing C-1 and C-6 radiolabeled sugars revealed surprising findings. By comparison of CML measured by HPLC using radioactivity with total CML using postcolumn derivatization 74% (5 days)/68% (10 days) of CML was attributed to the C-1 and 29%/31% to the C-6 portion of N-(1-deoxyfructos-1-yl)propylamine. Starting the incubation with glucose only 38% (5 days)/32% (10 days) for the C-1 and 22%/36% for the C-6 region were recovered, suggesting thereby that about 30% of CML is stemming from the C-2-C-5 region of the original carbon backbone.


DISCUSSION

The purpose of this study was to clarify the mechanism of formation of structures incorporating a C-2 fragment of the original sugar during the Maillard reaction of reducing sugars and proteins in vivo. Clarification of this question is important because the major advanced Maillard product found in vivo so far is CML. Its levels increase with age in human skin (29) and in the presence of diabetes, and the levels can be correlated with severity of diabetic complications (30) . In addition, CML was detected in human lens, where it is thought to originate from ascorbate (4, 27) . Previous mechanistic studies by Dunn et al. (19) suggested that CML forms from the Amadori product of glucose or threose through metal-catalyzed oxidative fragmentation.

The data in this study show unequivocally that, under physiological conditions at 37 °C and pH 7.4, glyoxal and glycolaldehyde are immediate precursors in the formation of CML and that they are involved in formation of C-2-imine cross-link 5, 7 (Fig. 1) and other more important, but yet unknown, structures. Formation of CML and 5/ 7 during Maillard reaction from higher sugars like glucose is much more complex because all three potential sources, i.e. the free sugar, the Schiff base adduct, and the Amadori product, are present together as soon as the reaction proceeds. Additionally, our findings raise questions of the importance of glyoxal and glycolaldehyde in the reaction pathways leading to CML and cross-linking.

When the reaction was initiated from the Amadori product of glucose, very large quantities of CML were formed that were almost totally stalled by deaerated conditions (Fig. 7 B). The fact, that aminoguanidine had no suppressive effect until 100 h strongly favors a mechanism involving direct oxidative cleavage of the Amadori product. In contrast, when starting the reaction from glucose in the presence of N- t-Boc-lysine, 50% inhibition of CML formation by aminoguanidine indicates approximately a 50% participation of the C-2 intermediates glyoxal/glycolaldehyde and 50% of CML originating from the Amadori product via oxidative cleavage, which is not influenced by aminoguanidine.

The key question therefore is what is the major source of glyoxal/glycoladehyde production leading to CML. Fig. 7B shows at 170 h that only 15% of total CML formation from the Amadori product can be attributed to C-2 intermediates. Indeed, the amount of glyoxal-triazine generated in the presence of 42 mM Amadori product was only 2.8 mmol/mol of initial sugar concentration. Thus, less than 0.3% of the original Amadori product was transformed into glyoxal (). As the Amadori product concentration in a glucose/ N- t-Boc-lysine system reaches only 0.7% of 42 mM, this source contributes only minor quantities CML by C-2 intermediates. Based on Fig. 7 B, it can be estimated at approximately 7%. This leaves 43% of total CML originating from other sources via glyoxal/glycolaldehyde (Fig. 8).


Figure 8: Formation of CML from various sugars during the Maillard reaction. Contribution of oxidative cleavage versus glyoxal is calculated on data in Fig. 7.



Utilizing the data on 3-amino-1,2,4-triazine formation, glucose autoxidation, as proposed by Wolff et al. (28) , can be excluded as a significant source of glyoxal generation (). We recovered small quantities of the triazine from glucose incubated in absence of amine. However, about 7 times higher levels were formed when the identical experiment was carried out in the presence of N- t-Boc-lysine.

Thus, in an incubation system starting from glucose in the presence of amines, the major pathway leading to glyoxal is not autoxidation of glucose or degradation of the Amadori product but degradation of an amine-assisted oxidative step prior to formation of the Amadori product, presumably the Schiff base adduct (Fig. 8).

Incubations conducted in the presence of boric acid support the findings with aminoguanidine. Boric acid forms a stable chelating complex with the coplanar cis diol groups of the furanoic structure of 1-deoxyfructos-1-yl-amines (31) and thus totally blocks CML formation from Amadori products via oxidative cleavage and glyoxal/glycolaldehyde (Fig. 7 B). Based on the experiments with aminoguanidine, this should result in a decrease of 57% CML synthesis in glucoseamine systems. The actual decrease of 37% was found to be smaller and is probably due to accelerated CML synthesis linked to increased saltconcentrations (18) .

Since aminoguanidine itself is an amine compound, it could compete with the target amine of the system used and contribute to the generation of reactive intermediates. The possibility of generating glyoxal/glycolaldehyde solely by this reaction pathway was discounted based on the detection of CML in incubations of the Amadori product of propylamine and glucose with N- t-Boc-lysine. The Amadori product will primarily form carboxymethylated propylamine via oxidative cleavage and glyoxal generation, but part of the glyoxal will also react with lysine to give carboxymethylated lysine or CML (Fig. 8).

Other independent evidence for glyoxal and glycolaldehyde generation from higher sugars was gained from the abundance of imine-intermediates 2 and 3 as glycolaldehyde and 8 as glyoxal derivatives (Figs. 1 and 6) in incubation mixtures with proteins. After reduction, 9 as their common product was detected in incubations with glucose and in much higher levels also with ribose. Use of a deuterated reduction reagent made it possible to distinguish between the N-(2-hydroxy-1- d-ethyl)lysine 9` for reduced 2 and 3 and N-(2-hydroxy-1, 2- d-ethyl)lysine 9" for reduced 8 in ribose incubations. This experiment unequivocally proves the existence of glycolaldehyde and glyoxal as their imine derivatives in incubations under physiological conditions and therefore emphasizes their role as potent contributors of CML formation from higher sugars.

Based on these considerations, and the finding that Amadori products are present in significant concentrations in tissues, the source of CML formation in vivo is expected to follow the sequence of precursor reactivity: Amadori product (via oxidative cleavage) > Schiff base adduct (via glyoxal/glycolaldehyde) >>> glucose (via autoxidation). Indeed, when using the data of CML and glyoxal-triazine formation to calculate the contribution for CML formation under severe diabetic conditions, i.e. 30 mM glucose and 2 mM Amadori product (32) , most would originate from the Amadori product via oxidative cleavage, but glyoxal from Amadori product and glucose-amine interaction would also account for 17% (I).

The fact that addition of a heterologous Amadori product to N- t-Boc-lysine induced CML formation led to investigations about the origin of the C-2 fragments with radiolabeled sugars. Incubating glucose with protein, about 30% of total CML incorporate the C-1 and about 30% incorporate the C-6 region, representing a C-2-C-3 and C-4-C-5 split of the sugar backbone. This indicates that significant amounts of C-2 fragments originate in between C-1 and C-6 and are therefore not accounted for by radioactivity. About 70% originate from the C-1 and about 30% from the C-6 part, when CML was induced by labeled Amadori product of glucose and propylamine. In this case, only glyoxal/glycolaldehyde transfer to the -amino lysine residues can generate CML. Thus, other reaction pathways in addition to the one proposed by Namiki contribute to CML formation. These may not be in contradiction with the importance of the Schiff base as a major contributor to C-2 fragmentation as evidenced above, because activation of the sugar by the imine bond may result in enolization through the whole molecule and facilitated fragmentation in multiple sites (2) .

Hayashi et al. (10) reported the formation of glyoxalalkyldiimines in heated reaction systems. The data shown in this paper establish the formation of analogue lysine derivative diimine 5 and also imine 7 (Fig. 1) under physiological conditions. Both structures could be detected in protein incubations with their immediate precursors glyoxal and glycolaldehyde, as well as from various sugars. Although the C-2-imine cross-links are formed in vitro, they are not stable and, as such, of minor importance as permanent cross-links. For this reason, no attempt was made to distinguish between 5 and 7. The concentrations formed were very small compared with other modifications like CML and were strictly dependent on the amount of glyoxal and glycolaldehyde present in the system. Since the expected steady-state levels of both carbonyl compounds formed in living systems are extremely low, and neither 5 nor 7 accumulate, this cross-link may therefore be present only in trace amounts in vivo and not contribute to protein cross-linking during aging or diabetes. Thus, it is not surprising that preliminary analysis of human skin and aorta tissue and serum samples showed no evidence for the formation of 5 and 7.

In summary, the generation of glyoxal and glycolaldehyde from higher sugars under physiological conditions and the tight relationship between these carbonyl compounds and CML formation became apparent in this study. In addition the in vitro data of protein cross-linking by us and others (33) provide new insights in the possible nature and mechanisms of sugar-mediated protein modification in vivo. Mechanistic studies in vivo are now necessary to probe the significance of the proposed pathway for CML formation and cross-linking. It is conceivable that some answers may come from in vivo determination of the type of triazines formed during the aminoguanidine therapy (34) .

  
Table: C-2-imine/CML formed in various sugar-BSA incubations


  
Table: 3-Amino-1,2,4-triazine formed after 170 h in various sugar/amine-incubations (all 42.2 mM) in the presence of 5 mM aminoguanidine


  
Table: Calculated CML and glyoxal formation after 170 h of incubation mimicking severe diabetic conditions, based on results presented in Table II and Fig. 7



FOOTNOTES

*
This work was supported by grants from the National Eye Institute (EY 07099) and the National Institute on Aging (AG 05601). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Fellowship of the Deutsche Forschungsgemeinschaft, Germany. To whom correspondence should be addressed: Inst. of Pathology, Case Western Reserve University, 2085 Adelbert Rd., Cleveland, Ohio 44106. Tel.: 216-368-6613; Fax: 216-844-1810.

The abbreviations used are: CML, N-(carboxymethyl)lysine; GC/MS, coupled gas chromatography-mass spectrometry; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; N-(glucitolyl)lysine, reduced Amadori product of glucose and lysine; TLC, thin-layer chromatography; t-Boc, t-butoxycarbonyl.


ACKNOWLEDGEMENTS

We thank Dr. Douglas Gage at the Michigan State University Mass Spectrometry Facility for performing high resolution fast atom bombardment spectral analysis. This facility is supported, in part, by a grant (DRR-00480) from the Biotechnology Resources Branch, Division of Research Resources, National Institutes of Health.


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