Oxidative deamination of lysine residue in plasma protein of diabetic rats Novel mechanism via the Maillard reaction Mitsugu Akagawa, Takeshi Sasaki and Kyozo Suyama Department of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Japan The levels of a-aminoadipic-d-semialdehyde residue, the oxidative deamination product of lysine residue, in plasma protein from streptozotocin-induced diabetic rats were evaluated. a-Aminoadipic-d-semialdehyde was converted to a bisphenol derivative by acid hydrolysis in the presence of phenol, and determined by high performance liquid chro- matography. Analysis of plasma proteins revealed three timeshigherlevelsofa-aminoadipic-d-semialdehyde in dia- betic subjects compared with normal controls. Furthermore, we explored the oxidative deamination via the Maillard reaction and demonstrated that the lysine residue of bovine serum albumin is oxidatively deaminated during the incu- bation with various carbohydrates in the presence of Cu 2+ at a physiological pH and temperature. This experiment showed that 3-deoxyglucosone and methylglyoxal are the most efficient oxidants of the lysine residue. When the reaction was initiated from glucose, a significant amount of a-aminoadipic-d-semialdehyde was also formed in the presence of Cu 2+ . The reaction was significantly inhibited by deoxygenation, catalase, and a hydroxyl radical scavenger. The mechanism we propose for the oxidative deamination is the Strecker-type reaction and the reactive oxygen species- mediated oxidation. Based on these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residue via the Maillard reaction. Keywords: Maillard reaction; glycation; reactive oxygen species; a-aminoadipic-d-semialdehyde; oxidative deamina- tion. The Maillard reaction (nonenzymatic glycation) is thought to contribute to the pathogenesis of diabetic complications and the ageing process. The first step in this reaction is the formation of a Schiff base between a reducing sugar and an amino group in proteins, followed by an Amadori rear- rangement to yield a relatively stable ketoamine adduct. Subsequently, the adducts (Amadori products) are further degraded to form a variety of structurally diverse com- pounds known as advanced glycation end products (AGEs) which frequently have chromophores, fluorophores, and protein cross-links [1]. Recent researches have demonstrated that the formation and accumulation of AGEs in plasma and tissue are associated with aging [1–5] and the long-term complications of diabetes [1,6–10]. In the process of the Maillard reaction, several reactive a-dicarbonyl compounds are found in vitro and in vivo. 3-Deoxyglucosone (3-DG) is produced from the multiple dehydration in the early stage Maillard reaction and by the fragmentation of fructose 3 phosphate [11,12]. Methylgly- oxal (MG) is mainly formed by amine-catalyzed sugar fragmentation reactions and by spontaneous decomposition of triose phosphate intermediates in glycolysis [12]. Glyoxal (GO) is formed by the spontaneous oxidative degradation of glucose, the degradation of glycated proteins, and lipid peroxidation [12]. In addition, increased levels of 3-DG, MG, and GO are found in blood from diabetic patients and streptozotocin (STZ)-induced diabetic rats [11–14]. In the advanced stages of the Maillard reaction, these a-dicarbonyl compounds irreversibly modify lysine and arginine residues in proteins at physiological conditions, leading to the formation of various AGEs in vitro, which are also identified in vivo [1,5,7–10,15,16]. Therefore, a-dicarbonyl compounds have been recognized as the major intermediates and precursors in AGEs formation in vivo. Recently it has been proposed that a-dicarbonyl stress is among the major factors in the pathogenesis of diabetic complications [1,7,11,16]. The oxidative degradation of a-amino acid by a-dicar- bonyls is known as the so-called Strecker degradation in food science. In the Strecker degradation [17,18], a number of carbohydrate-derived a-dicarbonyls as well as glucose are able to degrade a-amino acids at high temperatures, thus generating an aldehyde with one carbon atom less than a-amino acids. On the other hand, o-quinone compounds, which have an a-dicarbonyl group, are known to catalyze the oxidative deamination of primary amines to form the corresponding aldehydes under physiological conditions Correspondence to K. Suyama, Department of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981–8555, Japan. Fax: +81 22 717 8820, Tel.: +81 22 717 8818, E-mail: suyama@bios.tohoku.ac.jp Abbreviations: ACPP, 1-amino-1-carboxy-5,5-bis-p-hydroxyphenyl- pentane; AGE, advanced glycation end-product; 3-DG, 3-deoxy- glucosone; LTQ, lysine tyrosylquinone; MG, methylglyoxal; GO,glyoxal;STZ,streptozotocin. Enzyme: lysyl oxidase (EC 1.4.3.13). (Received 14 May 2002, revised 4 September 2002, accepted 9 September 2002) Eur. J. Biochem. 269, 5451–5458 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03243.x [19]. Lysyl oxidase (EC 1.4.3.13), a copper-containing amine oxidase, catalyzes the oxidative deamination of the e-amino group of lysine residue of elastin and collagen, which are connective tissue proteins, to form a-aminoadi- pic-d-semialdehyde [20]. Lysyl oxidase has a lysine tyrosyl- quinone (LTQ) cofactor consisting of an amino-o-quinone skeleton at the active site [21,22], and the LTQ cofactor itself catalyzes the amine oxidation [23]. The a-dicarbonyls derived from the Maillard reaction exist as free compounds in vivo and thus potentially may serve as adventitious oxidants of amines possibly including protein lysine residues and, in the course of the oxidative deamination, generate a-aminoadipic-d-semialdehyde. On the other hand, it was recently found that a-aminoadipic-d-semialdehyde is produced from protein lysine residue by the attack of oxygen-derived free radicals [24–26]. In addition, the aldehyde residue has been identified in serum albumin collected from some mammalian species [24]. The Maillard reaction can give rise to oxygen free radicals in the presence of O 2 and transition metal ions [27– 30] and the formation of some AGEs has been shown to require oxygen free radicals [30–32]. Therefore, the oxygen free radicals derived from the Maillard reaction in vivo may also serve as adventitious oxidants of lysine residues. a-Aminoadipic-d-semialdehyde residue is known as a pre- cursor of cross-links in elastin and collagen [21–23], thus implying the formation of cross-links, i.e. AGEs. In the present study, we measured the a-aminoadipic-d- semialdehyde in plasma protein. Analysis of rat plasma proteins by RP-HPLC revealed significantly higher levels of a-aminoadipic-d-semialdehyde residues in STZ-induced diabetic rats compared with normal controls. Furthermore, we explored the oxidative-deamination reaction via the Maillard reaction and demonstrated the occurrence of the oxidative deamination of the lysine residues in BSA via the Maillard reaction at a physiological pH and tempera- ture. Based on these findings, we propose a novel mechan- ism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residue via Maillard reaction. MATERIALS AND METHODS Materials Methanol was of HPLC grade from Nacalai Tesque Co., Kyoto, Japan. D -Ribose and catalase from bovine liver were from Tokyo Kasei Co, Tokyo, Japan. STZ was from Sigma Chemical Co, St. Louis, MO. Biuret reagent was from Wako Pure Chemical Industries Co, Osaka, Japan. 3-DG was from Dojindo Laboratories Co, Kumamoto, Japan. All other chemicals were from Nacalai Tesque Co. Diabetic model rats Animal experiments were carried out according to a protocol approved by the Animal Care Committee of Tohoku University. Male Wistar rats (8 weeks old, Japan SLC Co., Shizuoka, Japan), weighing 180–200 g, were used for the disease model study. Experimental diabetes was induced by a single i.p. dose of STZ (55 mgÆkg )1 ). STZ was dissolved in 0.1 M citrate buffer (pH 4.5). The animals were fasted overnight prior to STZ administration. Two weeks after STZ administration, all animals with plasma glucose level > 400 mgÆdL )1 were considered diabetic and were included in the study. Plasma glucose levels were measured using the commercial kit, Glucose CII Test Wako (Wako Pure Chemical Industries Co). Control animals received 0.1 M citrate buffer (pH 4.5). During the experiment the rats were housed in groups of two or three per cage. Tap water and pelleted standard diet Laboratory MR (Nihon Nosan Kogyo Co, Yokohama, Japan) were available ad libitum. The rats were housed in temperature- (23 ± 1 °C), humi- dity- (55 ± 5%), and light- (8.00–20.00 h) controlled room. Plasma protein After 14 days of administration, blood was drawn from the abdominal aorta of rats under light anesthesia with diethyl ether and placed into heparinized tubes. Plasma was immediately prepared by centrifugation at 1500 g for 20 min. The concentration of protein in the plasma samples was measured by the Biuret reaction using BSA as reference protein. Each plasma sample (500 lL) in a Pyrex test tube with a Teflon-lined screw cap was treated with 2 mL of cold 10% (w/v) trichloroacetic acid. All subsequent steps were performed in these tubes, and all samples were kept on ice during processing. After 5 min, the mixture was centrifuged at 2000 g for 30 min, and the resulting pellet of precipitated protein was separated. The pellet was washed with 2 mL of cold 5% (w/v) trichloroacetic acid. Then the resulting protein was hydrolyzed for RP-HPLC analysis as described below. Detection of a-aminoadipic-d-semialdehyde by RP-HPLC a-Aminoadipic-d-semialdehyde was derivatized to a bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxy- phenylpentane (ACPP), and determined by a modification of the previous method [33,34] as follows. The protein in a Pyrex test tube with a Teflon-lined screw cap was hydrolyzed in a conventional manner for 48 h at 110 °C with 4 mL of 6 M HCl containing 3% (v/v) phenol. The hydrolysate was extracted twice with 2.0 mL of diethyl ether, and the water layer was dried by rotary evaporation in vacuo followed by reconstitution in 500 lL of distilled water. A Sep–Pak plus C 18 environmental cartridge (Waters Co, Milford, MA, USA) was used for prepurifi- cation as follows. The Sep–Pak cartridge was flushed with methanol (10 mL) and then distilled water (10 mL), and the sample (500 lL) was put onto the cartridge. After the cartridge was washed with 20.0 mL of distilled water, ACPP was eluted with 4.0 mL of distilled water/methanol (1 : 1, v/v). The eluate was evaporated to dryness in vacuo, and then reconstituted in 500 lL of distilled water. After filtration with a poly(vinylidene difluoride) (PVDF) syringe filter (0.45 lm pore size, Whatman Co, Clifton, NJ), 40-lL portion of it was injected into an HPLC apparatus with a C-18 reversed phase column (COSMOSIL 5C 18 -AR-II, 250 · 4.6 mm, Nacalai Tesque Co). The RP-HPLC ana- lysis was performed on a Perkin Elmer Liquid Chroma- tograph Integral 4000 system (Norwalk, CT, USA). Solvent A was 10% methanol, and solvent B was methanol. The solvents were degassed by sonication and then continuously bubbled with a slow stream of helium during chromatography. The column was equilibrated with 5452 M. Akagawa et al.(Eur. J. Biochem. 269) Ó FEBS 2002 solvent A. The elution started with a linear gradient from 0 to 15% B in 20 min. Then solvent B was increased to 95% over the following 15 min. Finally, the eluting solvent was changed linearly to 100% A over a period of 20 min. The column was reequilibrated for 15 min with solvent A before the next run was started. The column oven was maintained at 40 °C. ACPP was eluted at 15.4 min using a flow rate of 1.0 mLÆmin )1 . Quantification of ACPP was performed by calculating the peak area of the HPLC absorbance profile (at 278 nm) of purified ACPP and comparing it with those of samples. Selective reduction of a-aminoadipic-d-semialdehyde in plasma protein Human plasma from a nondiabetic patient was dialyzed for 24 h at 4 °C against phosphate buffered saline. For reduction with sodium borohydride (NaBH 4 ), plasma sample (500 lL) in a Pyrex test tube with a Teflon-lined screw cap was diluted with 3.0 mL of 0.1 M sodium borate buffer (pH 9.0) followed by the addition of NaBH 4 (25 mg, 0.66 mmol). For reduction with sodium cyanoborohydride (NaBH 3 CN), plasma sample (500 lL) in a Pyrex test tube with a Teflon-lined screw cap was diluted with 3.0 mL of 0.1 M sodium phosphate buffer (pH 6.0) followed by the addition of NaBH 3 CN (50 mg, 0.80 mmol). The mixture was incubated for 24 h at 37 °C with shaking in the dark. After incubation, each plasma sample was treated with 5 mL of acetonitrile. The mixture was centrifuged at 2000 g for 30 min, and the resulting pellet of precipitated protein was separated. The pellet was washed with 5 mL of acetonitrile and 2 mL of cold 5% (w/v) trichloroacetic acid. Then the resulting protein was hydrolyzed for RP-HPLC analysis as described above. Incubation of BSA with carbohydrates General procedure. Reaction mixtures (2.0 mL) in a Pyrex test tube with a Teflon-lined screw cap contained 10.0 mgÆmL )1 BSA, 5 l M CuSO 4 , each carbohydrate, and 50 m M sodium phosphate buffer (pH 7.4). In some experi- ments, we added dimethylsulfoxide (50 m M ) or catalase (200 UÆmL )1 ) to the mixture. Carbohydrate concentrations are given in the respective legends to figures and tables. The reaction mixtures containing 10 lL of toluene were incu- bated at 37 °C with shaking in the dark. After incubation, the mixture was treated with 2 mL of cold 10% (w/v) trichloroacetic acid in ice bath. After 5 min, the mixture was centrifuged at 2000 g for 30 min, and the resulting pellet of precipitated protein was separated. The pellet was washed with 2 mL of cold 5% (w/v) trichloroacetic acid. Then the resulting protein was hydrolyzed for RP-HPLC analysis as described above. Incubation under nitrogen. The test tube was tightly fitted with a silicone rubber cap. The tube was evacuated and then filled with N 2 gas through a hypodermic needle. After another hypodermic needle was inserted in the tube to serve as an outlet port, gas was passed through the incubation mixture for 10 min and charged until the pressure of 0.05 MPa inside the tube was reached. Then the reaction mixture was incubated at 37 °C for 3 weeks with shaking in the dark. Statistical analysis The significance of changes in the experimental variables measured was assessed by Student’s t-test. We considered a change with a P-value < 0.05 statistically significant. The STATVIEW program (StatView J-4.5, Abacus Concepts, Berkeley, CA) was used for the analysis. RESULTS Detection of a-aminoadipic-d-semialdehyde in rat plasma protein Rat plasma protein from STZ-induced diabetic and control subjects was investigated for the presence of a-aminoadipic- d-semialdehyde. Protein hydrolysis with 6 M HCl contain- ing 3% phenol converts a-aminoadipic-d-semialdehyde to a bisphenol derivative (ACPP) which is a condensation product of one a-aminoadipic-d-semialdehyde residue and two phenol molecules by Baeyer’s reaction [33,34]. After hydrolysis, ACPP was measured by RP-HPLC using a linear gradient solvent system. Figure 1 shows the HPLC chromatograms of the ACPP standard and the hydrolysate of diabetic rat plasma protein followed by detection at 278 nm with a diode array detector. ACPP was eluted in a t R of 15.4 min (Fig. 1A) and observed in the hydrolyzate of diabetic rat plasma protein (Fig. 1B). We identified the peak as ACPP either by cochromatography with an authentic standard or by comparing UV spectra with the standard using a diode array detector (data not shown). The concentration of a-aminoadipic-d-semialdehyde in rat plasma protein from STZ-induced diabetic and control subjects was determined using the RP-HPLC analytical Fig. 1. Determination of a-aminoadipic-d-semialdehyde in diabetic rat plasma protein by RP-HPLC. After the protein was hydrolyzed with 6 M HCl containing 3% phenol at 110 °C for 48 h, ACPP, a bisphenol derivative of a-aminoadipic-d-semialdehyde, was measured by RP- HPLC with detection at 278 nm. (A) Chromatogram of ACPP standard. (B) Chromatogram of hydrolyzate of plasma protein from diabetic rat. Details are shown in the Experimental procedures section. Ó FEBS 2002 Oxidative deamination via the Maillard reaction (Eur. J. Biochem. 269) 5453 procedure (Fig. 2). The mean ± SD of a-aminoadipic- d-semialdehyde concentration was 3.21 ± 0.88 nmolÆmg )1 proteinindiabetic(n ¼ 7) and 0.99 ± 0.29 nmolÆmg )1 protein in control subjects (n ¼ 10). The 3.2-fold increase in a-aminoadipic-d-semialdehyde was statistically significant by Student’s t-test (P <0.001). Selective reduction of plasma protein We examined whether the a-aminoadipic-d-semialdehyde residue exists as aldehyde, or Schiff base, or a mixture of the two structures in vivo by selective reduction. Human plasma was reduced, and then a-aminoadipic-d-semialdehyde was analyzed by RP-HPLC. Figure 3 shows HPLC chromato- grams of the plasma protein (A) and the NaBH 4 -reduced plasma protein (B). The ACPP peak is abolished by the reduction with NaBH 4 . On the other hand, reduction of plasma protein with NaBH 3 CN, which is a selective reductant toward Schiff base at pH 6–7 [35], only resulted in a 5% decrease in the a-aminoadipic-d-semialdehyde peak (Fig. 3C). This result indicates that the a-aminoadipic-d- semialdehyde residue exists primarily as the free aldehyde in vivo. Oxidative deamination of lysine residue in BSA by carbohydrates The oxidative deamination of the e-amino groups of lysine residue via the Maillard reaction was assessed by the reaction of BSA with glucose. BSA (10.0 mgÆmL )1 )was incubated with 100 m M glucose in 50 m M sodium phos- phate buffer (pH 7.4) in the presence and absence of 5 l M Cu 2+ at 37 °C. After glucose-incubated BSA was hydro- lyzed with 6 M HCl containing 3% phenol, ACPP was measured by RP-HPLC. Figure 4A shows the formation of a-aminoadipic-d-semialdehyde with the incubation of BSA with glucose. Native BSA also contained 0.06 nmolÆmg )1 protein of a-aminoadipic-d-semialdehyde but the content remained constant during the incubation without carbohy- drates. The incubation with glucose in the absence of Cu 2+ did not increase a-aminoadipic-d-semialdehyde content (Fig. 4A). As shown in Fig. 4A, in the presence of 5 l M Cu 2+ , a significant amount of a-aminoadipic-d-semialde- hyde was produced by the reaction with glucose. There was a time-dependent increase in the concentration of a-aminoadipic-d-semialdehyde throughout the incubation period (3 weeks). We also evaluated various carbohydrates as possible oxidants of the lysine residue. The formation of a-amino- adipic-d-semialdehyde in BSA after incubation with various sugars for 3 weeks in the presence of Cu 2+ is summarized in Table 1. In the case of aldose, pentoses were more effective oxidants than hexoses. A marked increase was observed with an ascorbic acid/Cu 2+ system that generates reactive oxygen species. This result is consistent with a recent report by Stadtman et al. [26]. Furthermore, a significant increase was found with a low concentration (1.0 m M )ofMGand Fig. 3. Selective reduction of a-aminoadipic-d-semialdehyde residue in plasma protein. Plasma protein was reduced with NaBH 4 or NaBH 3 CN as described in the Experimental procedures section. After the protein was hydrolyzed with 6 M HCl containing 3% phenol at 110 °C for 48 h, ACPP was measured by RP-HPLC with detection at 278 nm. (A) Intact plasma protein. (B) NaBH 4 -reduced plasma pro- tein. (C) NaBH 3 CN-reduced plasma protein. Fig. 2. a-Aminoadipic-d-semialdehyde levels in rat plasma protein de- rived from STZ-induced diabetic and control subjects. After plasma protein was hydrolyzed with 6 M HCl containing 3% phenol at 110 °C for 48 h, ACPP, a bisphenol derivative of a-aminoadipic-d-semialde- hyde, was measured by RP-HPLC as described in the Experimental Procedures section. Mean ± SD was 3.21 ± 0.88 and 0.99 ± 0.29 nmolÆmg )1 protein in STZ-induced diabetic and controls, respectively; P < 0.0001 by Student’s t-test. 5454 M. Akagawa et al.(Eur. J. Biochem. 269) Ó FEBS 2002 3-DG but not GO. Figure 4B,C shows the formation of a-aminoadipic-d-semialdehyde in BSA by MG and 3-DG, respectively, with the advance of time. BSA (10 mgÆmL )1 ) was incubated in 50 m M phosphate buffer with 1.0 m M of each a-dicarbonyl under a physiological pH and tempera- ture (pH 7.4, 37 °C). As shown in Fig. 4B, in the presence of Cu 2+ , a significant amount of aldehyde was produced by the reaction with 3-DG but not in the absence of Cu 2+ .MG oxidatively deaminated the lysine residue in the presence and absence of Cu 2+ (Fig. 4C). The oxidation was appar- ently stimulated by the addition of Cu 2+ . Effect of scavengers on the oxidative deamination of BSA The presence of oxygen plays an important role in the Maillard reaction [36], and, actually, oxygen is required for the formation of some AGEs [30,37]. To assess for the participation of oxygen in the reaction, BSA was incubated with glucose, 3-DG, and MG in the presence of Cu 2+ under nitrogen atmosphere. Indeed, the reaction under a nitrogen atmosphere almost completely inhibited the oxidative deamination by glucose, clearly illustrating the involvement of oxygen in the oxidative deamination (Table 2). In addition, the deoxygenation caused a significant decrease in the production of a-aminoadipic-d-semialdehyde by 3-DG and MG. Further, we investigated the participation of reactive oxygen species in the reaction. BSA and Cu 2+ were incubatedwithglucose,3-DG,andMGinthepresenceof catalase and dimethylsulfoxide, which is a hydroxyl radical scavenger. As shown in Table 2, addition of catalase (100 UÆmL )1 ) markedly inhibited the formation of a-aminoadipic-d-semialdehyde by glucose and significantly inhibited it by 3-DG and MG. The oxidation of BSA by glucose, 3-DG, and MG was also significantly inhibited in the presence of 50 m M dimethylsulfoxide. These results suggest that the hydroxyl radical is produced by the Maillard reaction and is responsible for the a-aminoadi- pic-d-semialdehyde formation. DISCUSSION In the present study, we identified the a-aminoadipic- d-semialdehyde residue in rat plasma protein and demon- strated that the a-aminoadipic-d-semialdehyde level in STZ-induced diabetic rat plasma is significantly higher than Fig. 4. Time course of oxidative deamination of BSA by glucose, 3-DG, and MG. BSA (10 mgÆmL )1 ) was incubated with 100 m M glucose (A), 1.0 m M 3-DG (B), or 1.0 m M MG(C)in50m M phosphate buffer (pH 7.4) in the presence or absence of 5 l M Cu 2+ at 37 °C. After the reaction was terminated, a-aminoadipic-d-semialdehyde was measured by RP-HPLC. Table 1. Formation of a-aminoadipic-d-semialdehyde by incubation of BSA with various carbohydrates. BSA (10 mgÆmL )1 )wasincubated with 5 l M Cu 2+ and each indicated carbohydrate in 50 m M sodium phosphate buffer (pH 7.4) at 37 °Cfor3weeks.a-Aminoadipic-d- semialdehyde was quantitated by RP-HPLC as described in Experi- mental procedures. Carbohydrate Concentration (m M ) a-Aminoadipic-d-semialdehyde (nmolÆmg protein )1 ) None — 0.06 D -Glucose 50 0.13 D -Galactose 50 0.21 D -Fructose 50 0.15 D -Ribose 50 1.60 D -Xylose 50 0.72 L -Ascorbic acid 50 2.04 3-Deoxyglucosone 1 0.26 Methylglyoxal 1 0.32 Glyoxal 1 0.07 Ó FEBS 2002 Oxidative deamination via the Maillard reaction (Eur. J. Biochem. 269) 5455 that in normal rat plasma. Analysis of selectively reduced plasma protein suggested that the a-aminoadipic-d-semial- dehyde residue exists primarily as the free aldehyde form in vivo. Furthermore, we explored the oxidative-deamination reaction via the Maillard reaction, and demonstrated the occurrence of the oxidation of the lysine residue of BSA in the incubation with various carbohydrates in the presence of Cu 2+ at a physiological pH and temperature. This experi- ment showed that 3-DG and MG are the most efficient oxidant of the lysine residue. When the reaction was initiated from glucose, a significant amount of a-aminoad- ipic-d-semialdehyde was also formed in the presence of Cu 2+ . We have also determined the effects of oxygen and scavenger on the oxidative deamination. The formation of a-aminoadipic-d-semialdehyde by glucose, 3-DG, and MG was inhibited by deoxygenation, catalase, and dimethylsulf- oxide. From these results we propose the Strecker-type reaction by a-dicarbonyls and the reactive oxygen species- mediated oxidation for the oxidative deamination mechan- ism via the Maillard reaction. The proposed mechanism of the formation of a-aminoadipic-d-semialdehyde from the lysine residue by the Strecker-type reaction is summarized in Fig. 5. The formation of a-dicarbonyls is induced through the autoxidation of glucose and the degradation of Amadori products or Schiff base adduct by metal ion-catalysis [12]. Subsequently, the resulting MG and 3-DG could condense with protein lysine residues to form the Schiff base adduct, an iminoketone (I). Then the e-proton of the lysine moiety would be abstracted by basic media, and the enolization might give an iminoenaminol (II). In the electron transfer process, it is assumed that Cu 2+ serves as the electron-pair acceptor and stabilized II through the formation of a coordination complex because the requirement of Cu 2+ was observed in model studies. Finally, spontaneous hydrolysis of II can lead to the release of an enaminol (III) and the formation of a-aminoadipic-d-semialdehyde (IV). The mechanism of the Strecker-type reaction is not accompanied by decarboxylation and is consistent with the o-quinone- mediated mechanism previously proposed [23]. Although the incubation of BSA with GO produced a very small amount of a-aminoadipic-d-semialdehyde, GO may prefer- entially form stable inter- and intramolecular cross-links or carboxymethyllysine during the incubation with BSA because of its high reactivity [38,39]. Another possible pathway of a-aminoadipic-d-semialdehyde formation via the Maillard reaction is reactive oxygen species-mediated oxidative deamination. The fact that the aerobic glycation of proteins in the presence of transition metals is accom- panied by radical-generating reactions supports this possi- bility [27]. Firstly, protein-bound products of the Amadori reaction may subsequently degrade, in a transition metal- catalyzed process, to yield H 2 O 2 , reactive oxidants and further protein-reactive aldehydes [28]. The production of Table 2. Effect of O 2 and scavengers on the formation of a-aminoadipic-d-semialdehyde in BSA by glucose, 3-DG, and MG. BSA (10 mgÆmL )1 )was incubatedwith5l M Cu 2+ and each of indicated carbohydrate in 50 m M sodium phosphate buffer (pH 7.4) at 37 °Cfor3 weeks.a-Aminoadipic-d- semialdehyde was quantitated by RP-HPLC as described in Experimental procedures. The values are shown as mean ± SEM (n ¼ 3). Native BSA contained 0.06 ± 0.01 nmolÆmg )1 protein of a-aminoadipic-d-semialdehyde. a-Aminoadipic-d-semialdehyde (nmolÆmg protein )1 ) Condition Glucose (50 m M ) 3-DG (1 m M )MG(1m M ) None 0.25 ± 0.03 0.26 ± 0.03 0.32 ± 0.02 N 2 a 0.07 ± 0.02 0.20 ± 0.03 0.24 ± 0.04 Catalase (200 UÆmL )1 ) 0.13 ± 0.03 0.17 ± 0.02 0.23 ± 0.06 Dimethylsulfoxide (50 m M ) 0.09 ± 0.02 0.12 ± 0.01 0.22 ± 0.05 a Incubation under N 2 atmosphere. Fig. 5. Proposed mechanism of oxidative deamination of lysine residue by the Strecker-type reaction. 5456 M. Akagawa et al.(Eur. J. Biochem. 269) Ó FEBS 2002 oxidants by the oxidation of glucose-protein adducts has been termed glycoxidation. Furthermore, 3-DG, MG, and GO also produce superoxides during the reaction with lysine and arginine [29]. In this study, catalase and dimethylsulfoxide significantly inhibited the oxidative deamination by glucose, 3-DG, and MG, indicating the participation of hydroxyl radicals. Dependency of the oxidative deamination on both oxygen and Cu 2+ is also consistent with a metal ion-catalyzed mechanism for the production of hydroxyl radicals, probably through the intermediary of superoxide and H 2 O 2 . Recently it has been demonstrated that lysine residue is oxidatively deaminated to a-aminoadipic-d-semialdehyde residue by reactive oxy- gen species [24–26]. In addition, we have found that various primary amines are converted to the corresponding alde- hydes in the presence of H 2 O 2 and transition metal ions, and the oxidation is effectively prevented by catalase and dimethylsulfoxide [25]. Therefore, the hydroxyl radical generated by the Fenton-type reaction is also likely to contribute to the oxidative deamination via the Maillard reaction. Based on these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residues via the Maillard reaction. Our proposed mechan- ism of oxidation may also be the case in vivo.Infact, increased levels of 3-DG, MG, and GO are found in blood from diabetic patients and STZ-induced diabetic rats [11– 14]. These a-dicarbonyls are likely to react with the lysine residue to form Schiff base adducts in the first step of protein modification in vivo. Therefore, the Strecker-type oxidative deamination may contribute to the formation of a-amino- adipic-d-semialdehyde in diabetes. Oxidative stress has the potential for causing gross oxidative damage to biological molecules, which is mainly induced by reactive oxygen species, and is apparently responsible for diabetic complica- tions [40]. Diabetes is associated with increased free radical formation and malondialdehyde, which is the most exten- sively studied marker in lipid peroxidation. Recently, a-aminoadipic-d-semialdehyde is thought to be a biomarker of oxidative damage to proteins in vivo because a-aminoad- ipic-d-semialdehyde originates from the lysine residue by the attack of oxygen-derived free radicals [24,26]. When oxida- tive stress is induced in rats by treatment with tert-butyl hydroperoxide, which induces oxidative stress as a conse- quence of the production of reactive oxygen species, this aldehyde is found to be significantly higher compared with control rats [24]. Furthermore, the content of a-aminoadipic- d-semialdehyde in plasma proteins shows a positive corre- lation with the rat’s age. Therefore, the formation of oxygen free radicals during the Maillard reaction is also a possible source of the formation of a-aminoadipic-d-semialdehyde in diabetes. It has been observed that blood Cu 2+ levels are higher than normal in diabetic individuals, although it is not clear whether this is caused by an increase in ceruloplasmin or an increase in the pool of copper associated with albumin or low molecular weight chelates [27]. In diabetes, copper may play a significant role in the oxidation of lysine residues in plasma. The potential pathobiological role for a-amino- adipic-d-semialdehyde residue in diabetes is still speculative. The conversion of lysine residues to aldehydes might reflect changes in protein conformation as a result of the continu- ously decreasing loss of positive charge, and then the protein will be inactivated. Lysyl oxidase, a copper-containing amine oxidase, catalyzes the oxidative deamination of certain lysine residues in elastin and collagen to form a-aminoadipic- d-semialdehyde, which participates in cross-linking reactions in these connective tissue proteins [20]. Once generated, a-aminoadipic-d-semialdehydes condense with each other via aldol condensation or with lysine residue via Schiff base formation to form various inter- and intramolecular cross- links spontaneously [41–46]. Therefore, a-aminoadipic- d-semialdehyde residues may be candidates of a precursor for the formation of protein cross-links, i.e. AGEs, in diabetes although a-aminoadipic-d-semialdehyde derived cross-links are not found in plasma protein. The protein cross-linking leads to increasing resistance to removal by proteolytic means as well as impeding function. 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Oxidative deamination of lysine residue in BSA by carbohydrates The oxidative deamination of the e-amino groups of lysine residue via the Maillard reaction was assessed by the reaction of. explored the oxidative- deamination reaction via the Maillard reaction and demonstrated the occurrence of the oxidative deamination of the lysine residues in BSA via the Maillard reaction at a physiological. propose a novel mechanism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residue via the Maillard reaction. Keywords: Maillard reaction; glycation;