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Rapid Formation of Advanced Glycation End Products by Intermediate Metabolites of Glycolytic Pathway and Polyol Pathway
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
228, 539–543 (1996)
1695
Rapid Formation of Advanced Glycation End Products by Intermediate Metabolites of Glycolytic Pathway and Polyol Pathway Yoji Hamada,1 Norie Araki,* Naoki Koh, Jiro Nakamura, Seikoh Horiuchi,* and Nigishi Hotta The Third Department of Internal Medicine, Nagoya University School of Medicine; and *The Department of Biochemistry, Kumamoto University School of Medicine Received October 11, 1996 To clarify roles of intermediate metabolites of the glycolytic pathway and the polyol pathway in nonenzymatic glycation under physiological conditions, we incubated bovine serum albumin with intermediates of both pathways in the micromolar range as well as with 20 mmol/l glucose, and observed the formation of advanced glycation end products (AGEs). We found that triose phosphates, glyceraldehyde, and a novel polyol pathway-related metabolite, fructose 3-phosphate along with its breakdown product, 3-deoxyglucosone were extremely potent glycating agents that at nearly physiological concentrations on incubation with albumin produced substantial amounts of AGEs as early as 24 hours, while 20 mmol/l glucose afforded trace amounts of AGEs after two week incubation. The results along with the previous evidence of the increased level of intermediates in diabetic states may suggest that the intermediate metabolites rather than glucose contribute to enhanced glycation in diabetic tissues, inspite of the much lower concentrations compared with glucose. q 1996 Academic Press, Inc.
Nonenzymatic glycation of biomolecules has been implicated in the etiology of diabetic complications (1). Although an elevated level of glucose has been thought to play a primary role via Maillard reaction in enhanced glycation and cross-linking in diabetic tissues (1), the nonenzymatic glycation is known to result from the action of various metabolites other than glucose. In addition to hexoses or pentoses that may be precursors of glycation products (2,3), some intermediate metabolites of the glycolytic pathway (4-7) and of the polyol pathway (6-10) have been shown to be potent glycating agents. Since diabetes induces, along with hyperglycemia, various metabolic alterations in the glycolytic and the polyol pathway metabolism (6, 11, 12), changes in the levels of intermediate metabolites seem to be potentially involved in enhanced glycation in diabetic tissues. It is of particular importance from the aspects of treatment of diabetic complications whether the intermediate metabolites significantly contribute to glycation in diabetic subjects, because such involvement of the metabolites may suggest a possibility that the normalization of intermediate metabolism by drugs, such as inhibitors of the polyol pathway, attenuate the glycation without correcting hyperglycemia. However, the much lower intracellular concentrations of these metabolites compared with glucose (6,11,13) raise the question about actual roles of the intermediates in glycation under physiological conditions. 1 Corresponding author: The Third Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan. Fax: /81-52-744-2213. Abbreviations: AGEs, advanced glycation end products; BSA, bovine serum albumin; PBS, phosphate bufferedsaline; G6-P, glucose 6-phosphate; F6-P, fructose 6-phosphate; F1,6-P, Fructose 1,6-diphosphate; DHAP, dihydroxyaceton phosphate; GA3-P, glyceraldehyde 3-phosphate; G3-P, glycerol 3-phosphate; GA, glyceraldehyde; F1-P, fructose 1-phosphate; F3-P, fructose 3-phosphate; 3-DG, 3-deoxyglucosone
539 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Vol. 228, No. 2, 1996
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The purpose of this study is to clarify whether any metabolites of the glycolytic pathway or the polyol pathway are potent enough to cause a significant glycation at physiological concentrations. We incubated BSA with various concentrations of intermediates of both pathways, including a novel polyol pathway-related metabolite, F3-P (9,14) and its possible breakdown product, 3-DG (9,15), and observed the formation of AGEs by enzyme-linked immunosorbent assay using monoclonal antibody raised against AGE-BSA as well as by measuring fluorescence at wavelengths specific to AGEs or pentosidine. MATERIALS AND METHODS Materials. F3-P was synthesized by the methods previously reported by Szwergold et al. (9). 3-DG was synthesized by the procedures described previously (16). Glucose and fructose were purchased from Katayama chemical (Osaka, Japan), F6-P and G6-P were from Nacalai Tesque (Kyoto, Japan), and avidin-biotin reagents were obtained from Vector (Burlingame, CA, USA). Other chemicals were purchased from Sigma (St. Louis, MO, USA). Ninety-six well immunoplates were obtained from Costar (Cambridge, MA, USA). Incubation of BSA with intermediate metabolites. Each metabolite at 50 mmol/l or 5 mmol/l were dissolved with BSA (10 mg/ml) in PBS, while D-Glucose was dissolved at concentrations of 5 mmol/l and 20 mmol/l. Solutions containing all intermediates together at 10, 20 and 50 mmol/l with 10 mg/ml BSA were also prepared. Each solution was sterilized by filtration using Millex-GS filter (Millipore, Bedford, MA, USA) and was dispensed into culture tubes under sterile condition. Each solution was then incubated at 37 7C for from 24 h to 14 days, and stored at 070 7C until analysis. Determination of AGEs. The amount of AGE was determined by a modification of competitive ELISA procedure previously described (17), using monoclonal antibody raised against AGE-BSA deriving from glucose (17). In brief, each well of an immunoplate was incubated with 0.1 ml of 0.5 mg/ml AGE-BSA in 50 mmol/l carbonate buffer (pH9.7) at room temperature for 2 h. The wells were washed three times with PBS containing 0.05 % (v/v) Tween 20 (Buffer A) and blocked with 0.25 ml of 0.5 % (w/v) gelatin in 5 mmol/l carbonate buffer (pH9.7) for 1 h. After washing the wells three times with buffer A, the pre-mixed solution of 50 ml each of sample and 0.1 mg/ml biotinylated anti-AGE antibody solution was placed in each well. The wells were incubated for 1 h at room temperature, washed again and then 0.1 ml of avidin-biotin horseradish peroxidase conjugate was added to each well. After incubation for 1 h at room temperature, the wells were washed, and then 0.1 ml of substrate solution was placed. Following incubation for 20-30 min at room temp in a dark place, absorbance at 405 nm was measured on a Bio-Rad 3550 UV microplatereader (Richmond, CA, USA). Examination of fluorescence. Fluorescence of each sample was determined at excitation/emission wavelengths of 370/440 nm as well as 335/385 nm using Simadzu RF-1500 spectrofluorophotometer (Kyoto, Japan). Ten mg/ml BSA solutions incubated without metabolites for the same period were used as controls.
RESULTS
Table 1 shows fluorescence specific to AGEs (Ex 370/ Em 440) and pentosidine (Ex 335/ Em 385) as well as AGE levels determined by ELISA in solutions containing BSA and each metabolite after 14 day incubation at 37 7C. Among the glycolytic intermediates, GA3-P, DHAP, and GA at 5 mmol/l on incubation with BSA produced high fluorescence at both wavelengths. F6-P and F1,6-P also caused an obvious fluorescence accumulation. Products from these metabolites and BSA reacted with antibody to glucose-derived AGE-BSA. The polyol pathway-related metabolites, F3-P and 3-DG also yielded definite fluorescence at both AGE- and pentosidine-wavelengths after 14 day incubation with BSA, while the reactivity of the solutions to the anti-AGE antibody was apparently lower compared with solutions of GA3P, DHAP, or GA. Small amounts of AGEs were produced from fructose and F1-P as well. GA3P, DHAP, GA, F1,6-P along with F3-P and 3-DG were able to modify BSA at a concentration of 50 mmol/l, that seems comparatively close to the physiological concentrations in diabetic states (6,11,13,18,19), causing fluorescence accumulation. Samples containing 50 mmol/l GA3-P, DHAP, or GA also showed a reactivity with the anti-AGE antibody. In contrast, incubation of BSA with 5 mmol/l glucose resulted in no detectable fluorescence and 20 mmol/l glucose produced only slight fluorescence at both wavelengths along with an insignificant reactivity in the ELISA system. 540
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Vol. 228, No. 2, 1996
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TABLE 1 Production of AGEs by Incubation of BSA with Intermediate Metabolites of the Glycolytic and the Polyol Pathways for 14 Days DFluorescence
Intermediates
Ex370/Em440
Ex335/Em385
AGEs (units/mg prot)
Glucose 5 mM 20 mM G6-P 5 mM 50 mM F6-P 5 mM 50 mM F1,6-P 5 mM 50 mM GA3-P 5 mM 50 mM DHAP 5 mM 50 mM G3-P 5 mM 50 mM GA 5 mM 50 mM Sorbitol 5 mM 50 mM Fructose 5 mM 50 mM F1-P 5 mM 50 mM F3-P 5 mM 50 mM 3-DG 5 mM 50 mM
ND 0.4 ND ND 2.9 ND 12.3 1.2 100.6 5.2 100.3 5.2 ND ND 108.8 6.7 ND ND 0.4 ND 1.9 ND 52.3 4.9 90.9 5.7
ND 0.6 0.1 ND 2.8 ND 50.1 8.7 168.3 5.7 286.0 5.7 ND ND 74.6 6.5 ND ND 1.1 ND 2.8 ND 127.1 5.8 123.6 8.5
ND ND ND ND 60 ND 10 ND 711 13 398 10 ND ND 370 13 ND ND 12 ND 11 ND 22 ND 49 ND
Fluorescence was expressed as differences of arbitrary units per mg proteins from control solution. One unit of AGEs was defined as the amount that showed reactivity to antibody equivalent to 1 ng of the glucose-derived AGE-BSA standard. ND, not detectable.
Figure 1 shows a time course of AGE-fluorescence and pentosidine-fluorescence by incubating BSA with a mixture of all metabolites at concentrations of 10 to 50 mmol/l as well as with 5 mmol/l or 20 mmol/l glucose. As can be seen fluorescence became detectable at both wavelengths after 24 h in samples containing 10, 20 and 50 mmol/l mixture of the intermediates and were rapidly increased in a concentration-dependent manner. In contrast, no fluorescence was detected in the 5 mmol/l or 20 mmol/l glucose solutions for 7 days and only a trace fluorescence accumulation resulted from 20 mmol/l glucose after 14 day incubation. DISCUSSION
We demonstrated in the present study that multiple intermediates in the glycolytic and the polyol pathways were capable of nonenzymatically modifying proteins. In particular, DHAP, GA3-P, GA, and a novel polyol pathway-related metabolite, F3-P along with its degradation product, 3-DG were highly reactive agents that in the micromolar range of concentrations formed more AGEs much faster than 20 mmol/l glucose. Glucose required the concentration of at least 100 mmol/l to produce 541
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Vol. 228, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Production of fluorescence at wavelengths specific to AGEs (A) or pentosidine (B) by incubating BSA with the mixture of 10 (n), 20 (s), and 50 (l) mmol/l intermediate metabolites as well as with 5 mmol/l (h) or 20 mmol/l (m) glucose. Fluorescence was expressed as differences of arbitrary units per mg proteins from control solutions.
detectable reactivity to the antibody to glucose-derived AGE-BSA after two week incubation (data are not shown). Although the concentration of each intermediate in human tissues largely varies among individuals and among investigations, the metabolites appear to range in concentration from a few mmol/l to less than 100 mmol/l (6,11,13,18,19). Thus it seems likely from the present results that the intermediate metabolites, rather than glucose, play tangible roles in the glycation of intracellular proteins, despite the fact that their concentrations are much lower than that of glucose. Among the intermediates, triose phosphates, F1,6-P, F3-P as well as fructose have been evidently shown to be increased in diabetic states (6,11,12,18-20). Given the fact that a physiological level of glucose, even in diabetic conditions, is much less potent than these intermediates to form glycation products, the enhanced glycation of proteins observed in tissues of diabetic subjects may be attributable to not a high level of glucose itself but the elevated levels of these intermediate metabolites induced by hyperglycemia or by diabetes. Of interest previous investigations have revealed that some glycolytic intermediates as well as polyol pathway metabolites are decreased by inhibitors of aldose reductase, the first enzyme of the polyol pathway (12,19). This may account, in part, for the suppressive effects of the drug on glycation as reported previously (21). Although we were unable to identify the forms of AGEs produced from each metabolite, a discrepancy in fluorescence and reactivity to the anti-AGE antibody seen among products may allow us to speculate that AGEs produced by these intermediates greatly vary in structure. One of the conceivable products from the metabolites may be pentosidine, a browning and fluorescent protein cross-link. Pentosidine is believed to derive from various hexoses or pentoses (3), while GA and DHAP may serve as potential precursors of pentosidine (22). The fragmentation of triose phosphates has also been revealed to form methylglyoxal, a reactive metabolite that modifies proteins, with formation of products similar to glucose-derived AGE (4,23). A product(s) from F3-P seems quite different from the glucose-derived AGE, considering the less immunoreactivity. Although mechanisms by which glycation products are formed under physiological circumstances are complex, the involvement of intermediate metabolism in the glycation appears undoubted and worth further investigation. 542
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Vol. 228, No. 2, 1996
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ACKNOWLEDGMENT This study was supported in part by a diabetes research grant from the Ministry of Health and Welfare of Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Brownlee, M., Vlassara, H., and Cerami, A. (1984) Ann. Intern. Med. 101, 527–537. Bunn, H. F., and Higgins, P. J. (1981) Science 213, 222–224. Grandhee, S. K., and Monnier, V. M. (1991) J. Biol. Chem. 266, 11649–11653. Phillips, S. A., and Thornalley, P. J. (1993) Eur. J. Biochem. 212, 101–105. Giardino, I., Edelstein, D., and Brownlee, M. (1994) J. Clin. Invest. 94, 110–117. Stevens, V. J., Vlassara, H., Abati, A., and Cerami, A. (1977) J. Biol. Chem. 252, 2998–3002. Kashimura, N., Morita, J., and Komano, T. (1979) Carbohyd. Res. 70, C3-C7. McPherson, J. D., Shilton, B. H., and Walton, D. J. (1988) Biochemistry 27, 1901–1907. Szwergold, B. S., Kappler, F., and Brown, T. R. (1990) Science 247, 451–454. Igaki, N., Sakai, M., Hata, H., Oimomi, M., Baba, S., and Kato, H. (1990) Clin. Chem. 36, 631–634. Travis, S. F., Morrison, A. D., Clements, R. S., Winegrad, A. I., and Oski, F. A. (1971) J. Clin. Invest. 50, 2104– 2112. Van den Enden, M. K., Nyengaard, J. R., Ostrow, E., Burgan, J. H., and Williamson, J. R. (1995) Invest. Ophthalmol. Vis. Sci. 36, 1675–1685. Minakami, S., Suzuki, C., Saito, T., and Yoshikawa, H. (1965) J. Biochem. 58, 543–550. Lal, S., Szwergold, B. S., Kappler, F., and Brown, T. (1993) J. Biol. Chem. 268, 7763–7767. Lal, S., Szwergold, B. S., Taylor, A. H., Randall, W. C., Kappler, F., Wells-Knecht, K., Baynes, J. W., and Brown, T. R. (1995) Arch. Biochem. Biophys. 318, 191–199. Khadem, H. E., Horton, D., Meshreki, M. H., and Nashed, M. A. (1971) Carbohyd. Res. 17, 183–192. Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329–7332. Petersen, A., Szwergold, B. S., Kappler, F., Weingarten, M., and Brown, T. R. (1990) J. Biol. Chem. 265, 17424– 17427. Hamada, Y., Odagaki, Y., Sakakibara, F., Naruse, K., Koh, N., and Hotta, N. (1995) Life Sci. 57, 23–29. Thurston, J. H., McDougal, D. B., Hauhart, R. E., and Schulz, D. W. (1995) Diabetes 44, 190–195. Sua´rez, G., Rajaram, R., Bhuyan, K. C., Oronsky, A. L., and Goidl, J. A. (1988) J. Clin. Invest. 82, 624–627. Dyer, D. G., Blackledge, J. A., Thorpe, S. R., and Baynes, J. W. (1991) J. Biol. Chem. 266, 11654–11660. Vander Jagt, D. L., Robinson, B., Taylor, K. K., and Hunsaker, L. A. (1992) J. Biol. Chem. 267, 4364–4369.
543
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6910$$$922
10-25-96 07:51:50
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228, 539–543 (1996)
1695
Rapid Formation of Advanced Glycation End Products by Intermediate Metabolites of Glycolytic Pathway and Polyol Pathway Yoji Hamada,1 Norie Araki,* Naoki Koh, Jiro Nakamura, Seikoh Horiuchi,* and Nigishi Hotta The Third Department of Internal Medicine, Nagoya University School of Medicine; and *The Department of Biochemistry, Kumamoto University School of Medicine Received October 11, 1996 To clarify roles of intermediate metabolites of the glycolytic pathway and the polyol pathway in nonenzymatic glycation under physiological conditions, we incubated bovine serum albumin with intermediates of both pathways in the micromolar range as well as with 20 mmol/l glucose, and observed the formation of advanced glycation end products (AGEs). We found that triose phosphates, glyceraldehyde, and a novel polyol pathway-related metabolite, fructose 3-phosphate along with its breakdown product, 3-deoxyglucosone were extremely potent glycating agents that at nearly physiological concentrations on incubation with albumin produced substantial amounts of AGEs as early as 24 hours, while 20 mmol/l glucose afforded trace amounts of AGEs after two week incubation. The results along with the previous evidence of the increased level of intermediates in diabetic states may suggest that the intermediate metabolites rather than glucose contribute to enhanced glycation in diabetic tissues, inspite of the much lower concentrations compared with glucose. q 1996 Academic Press, Inc.
Nonenzymatic glycation of biomolecules has been implicated in the etiology of diabetic complications (1). Although an elevated level of glucose has been thought to play a primary role via Maillard reaction in enhanced glycation and cross-linking in diabetic tissues (1), the nonenzymatic glycation is known to result from the action of various metabolites other than glucose. In addition to hexoses or pentoses that may be precursors of glycation products (2,3), some intermediate metabolites of the glycolytic pathway (4-7) and of the polyol pathway (6-10) have been shown to be potent glycating agents. Since diabetes induces, along with hyperglycemia, various metabolic alterations in the glycolytic and the polyol pathway metabolism (6, 11, 12), changes in the levels of intermediate metabolites seem to be potentially involved in enhanced glycation in diabetic tissues. It is of particular importance from the aspects of treatment of diabetic complications whether the intermediate metabolites significantly contribute to glycation in diabetic subjects, because such involvement of the metabolites may suggest a possibility that the normalization of intermediate metabolism by drugs, such as inhibitors of the polyol pathway, attenuate the glycation without correcting hyperglycemia. However, the much lower intracellular concentrations of these metabolites compared with glucose (6,11,13) raise the question about actual roles of the intermediates in glycation under physiological conditions. 1 Corresponding author: The Third Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan. Fax: /81-52-744-2213. Abbreviations: AGEs, advanced glycation end products; BSA, bovine serum albumin; PBS, phosphate bufferedsaline; G6-P, glucose 6-phosphate; F6-P, fructose 6-phosphate; F1,6-P, Fructose 1,6-diphosphate; DHAP, dihydroxyaceton phosphate; GA3-P, glyceraldehyde 3-phosphate; G3-P, glycerol 3-phosphate; GA, glyceraldehyde; F1-P, fructose 1-phosphate; F3-P, fructose 3-phosphate; 3-DG, 3-deoxyglucosone
539 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BBRC 5662
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Vol. 228, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The purpose of this study is to clarify whether any metabolites of the glycolytic pathway or the polyol pathway are potent enough to cause a significant glycation at physiological concentrations. We incubated BSA with various concentrations of intermediates of both pathways, including a novel polyol pathway-related metabolite, F3-P (9,14) and its possible breakdown product, 3-DG (9,15), and observed the formation of AGEs by enzyme-linked immunosorbent assay using monoclonal antibody raised against AGE-BSA as well as by measuring fluorescence at wavelengths specific to AGEs or pentosidine. MATERIALS AND METHODS Materials. F3-P was synthesized by the methods previously reported by Szwergold et al. (9). 3-DG was synthesized by the procedures described previously (16). Glucose and fructose were purchased from Katayama chemical (Osaka, Japan), F6-P and G6-P were from Nacalai Tesque (Kyoto, Japan), and avidin-biotin reagents were obtained from Vector (Burlingame, CA, USA). Other chemicals were purchased from Sigma (St. Louis, MO, USA). Ninety-six well immunoplates were obtained from Costar (Cambridge, MA, USA). Incubation of BSA with intermediate metabolites. Each metabolite at 50 mmol/l or 5 mmol/l were dissolved with BSA (10 mg/ml) in PBS, while D-Glucose was dissolved at concentrations of 5 mmol/l and 20 mmol/l. Solutions containing all intermediates together at 10, 20 and 50 mmol/l with 10 mg/ml BSA were also prepared. Each solution was sterilized by filtration using Millex-GS filter (Millipore, Bedford, MA, USA) and was dispensed into culture tubes under sterile condition. Each solution was then incubated at 37 7C for from 24 h to 14 days, and stored at 070 7C until analysis. Determination of AGEs. The amount of AGE was determined by a modification of competitive ELISA procedure previously described (17), using monoclonal antibody raised against AGE-BSA deriving from glucose (17). In brief, each well of an immunoplate was incubated with 0.1 ml of 0.5 mg/ml AGE-BSA in 50 mmol/l carbonate buffer (pH9.7) at room temperature for 2 h. The wells were washed three times with PBS containing 0.05 % (v/v) Tween 20 (Buffer A) and blocked with 0.25 ml of 0.5 % (w/v) gelatin in 5 mmol/l carbonate buffer (pH9.7) for 1 h. After washing the wells three times with buffer A, the pre-mixed solution of 50 ml each of sample and 0.1 mg/ml biotinylated anti-AGE antibody solution was placed in each well. The wells were incubated for 1 h at room temperature, washed again and then 0.1 ml of avidin-biotin horseradish peroxidase conjugate was added to each well. After incubation for 1 h at room temperature, the wells were washed, and then 0.1 ml of substrate solution was placed. Following incubation for 20-30 min at room temp in a dark place, absorbance at 405 nm was measured on a Bio-Rad 3550 UV microplatereader (Richmond, CA, USA). Examination of fluorescence. Fluorescence of each sample was determined at excitation/emission wavelengths of 370/440 nm as well as 335/385 nm using Simadzu RF-1500 spectrofluorophotometer (Kyoto, Japan). Ten mg/ml BSA solutions incubated without metabolites for the same period were used as controls.
RESULTS
Table 1 shows fluorescence specific to AGEs (Ex 370/ Em 440) and pentosidine (Ex 335/ Em 385) as well as AGE levels determined by ELISA in solutions containing BSA and each metabolite after 14 day incubation at 37 7C. Among the glycolytic intermediates, GA3-P, DHAP, and GA at 5 mmol/l on incubation with BSA produced high fluorescence at both wavelengths. F6-P and F1,6-P also caused an obvious fluorescence accumulation. Products from these metabolites and BSA reacted with antibody to glucose-derived AGE-BSA. The polyol pathway-related metabolites, F3-P and 3-DG also yielded definite fluorescence at both AGE- and pentosidine-wavelengths after 14 day incubation with BSA, while the reactivity of the solutions to the anti-AGE antibody was apparently lower compared with solutions of GA3P, DHAP, or GA. Small amounts of AGEs were produced from fructose and F1-P as well. GA3P, DHAP, GA, F1,6-P along with F3-P and 3-DG were able to modify BSA at a concentration of 50 mmol/l, that seems comparatively close to the physiological concentrations in diabetic states (6,11,13,18,19), causing fluorescence accumulation. Samples containing 50 mmol/l GA3-P, DHAP, or GA also showed a reactivity with the anti-AGE antibody. In contrast, incubation of BSA with 5 mmol/l glucose resulted in no detectable fluorescence and 20 mmol/l glucose produced only slight fluorescence at both wavelengths along with an insignificant reactivity in the ELISA system. 540
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TABLE 1 Production of AGEs by Incubation of BSA with Intermediate Metabolites of the Glycolytic and the Polyol Pathways for 14 Days DFluorescence
Intermediates
Ex370/Em440
Ex335/Em385
AGEs (units/mg prot)
Glucose 5 mM 20 mM G6-P 5 mM 50 mM F6-P 5 mM 50 mM F1,6-P 5 mM 50 mM GA3-P 5 mM 50 mM DHAP 5 mM 50 mM G3-P 5 mM 50 mM GA 5 mM 50 mM Sorbitol 5 mM 50 mM Fructose 5 mM 50 mM F1-P 5 mM 50 mM F3-P 5 mM 50 mM 3-DG 5 mM 50 mM
ND 0.4 ND ND 2.9 ND 12.3 1.2 100.6 5.2 100.3 5.2 ND ND 108.8 6.7 ND ND 0.4 ND 1.9 ND 52.3 4.9 90.9 5.7
ND 0.6 0.1 ND 2.8 ND 50.1 8.7 168.3 5.7 286.0 5.7 ND ND 74.6 6.5 ND ND 1.1 ND 2.8 ND 127.1 5.8 123.6 8.5
ND ND ND ND 60 ND 10 ND 711 13 398 10 ND ND 370 13 ND ND 12 ND 11 ND 22 ND 49 ND
Fluorescence was expressed as differences of arbitrary units per mg proteins from control solution. One unit of AGEs was defined as the amount that showed reactivity to antibody equivalent to 1 ng of the glucose-derived AGE-BSA standard. ND, not detectable.
Figure 1 shows a time course of AGE-fluorescence and pentosidine-fluorescence by incubating BSA with a mixture of all metabolites at concentrations of 10 to 50 mmol/l as well as with 5 mmol/l or 20 mmol/l glucose. As can be seen fluorescence became detectable at both wavelengths after 24 h in samples containing 10, 20 and 50 mmol/l mixture of the intermediates and were rapidly increased in a concentration-dependent manner. In contrast, no fluorescence was detected in the 5 mmol/l or 20 mmol/l glucose solutions for 7 days and only a trace fluorescence accumulation resulted from 20 mmol/l glucose after 14 day incubation. DISCUSSION
We demonstrated in the present study that multiple intermediates in the glycolytic and the polyol pathways were capable of nonenzymatically modifying proteins. In particular, DHAP, GA3-P, GA, and a novel polyol pathway-related metabolite, F3-P along with its degradation product, 3-DG were highly reactive agents that in the micromolar range of concentrations formed more AGEs much faster than 20 mmol/l glucose. Glucose required the concentration of at least 100 mmol/l to produce 541
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10-25-96 07:51:50
bbrcas
AP: BBRC
Vol. 228, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Production of fluorescence at wavelengths specific to AGEs (A) or pentosidine (B) by incubating BSA with the mixture of 10 (n), 20 (s), and 50 (l) mmol/l intermediate metabolites as well as with 5 mmol/l (h) or 20 mmol/l (m) glucose. Fluorescence was expressed as differences of arbitrary units per mg proteins from control solutions.
detectable reactivity to the antibody to glucose-derived AGE-BSA after two week incubation (data are not shown). Although the concentration of each intermediate in human tissues largely varies among individuals and among investigations, the metabolites appear to range in concentration from a few mmol/l to less than 100 mmol/l (6,11,13,18,19). Thus it seems likely from the present results that the intermediate metabolites, rather than glucose, play tangible roles in the glycation of intracellular proteins, despite the fact that their concentrations are much lower than that of glucose. Among the intermediates, triose phosphates, F1,6-P, F3-P as well as fructose have been evidently shown to be increased in diabetic states (6,11,12,18-20). Given the fact that a physiological level of glucose, even in diabetic conditions, is much less potent than these intermediates to form glycation products, the enhanced glycation of proteins observed in tissues of diabetic subjects may be attributable to not a high level of glucose itself but the elevated levels of these intermediate metabolites induced by hyperglycemia or by diabetes. Of interest previous investigations have revealed that some glycolytic intermediates as well as polyol pathway metabolites are decreased by inhibitors of aldose reductase, the first enzyme of the polyol pathway (12,19). This may account, in part, for the suppressive effects of the drug on glycation as reported previously (21). Although we were unable to identify the forms of AGEs produced from each metabolite, a discrepancy in fluorescence and reactivity to the anti-AGE antibody seen among products may allow us to speculate that AGEs produced by these intermediates greatly vary in structure. One of the conceivable products from the metabolites may be pentosidine, a browning and fluorescent protein cross-link. Pentosidine is believed to derive from various hexoses or pentoses (3), while GA and DHAP may serve as potential precursors of pentosidine (22). The fragmentation of triose phosphates has also been revealed to form methylglyoxal, a reactive metabolite that modifies proteins, with formation of products similar to glucose-derived AGE (4,23). A product(s) from F3-P seems quite different from the glucose-derived AGE, considering the less immunoreactivity. Although mechanisms by which glycation products are formed under physiological circumstances are complex, the involvement of intermediate metabolism in the glycation appears undoubted and worth further investigation. 542
AID
BBRC 5662
/
6910$$$922
10-25-96 07:51:50
bbrcas
AP: BBRC
Vol. 228, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENT This study was supported in part by a diabetes research grant from the Ministry of Health and Welfare of Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
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