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In Vitro Kinetic Studies of Formation of Antigenic Advanced Glycation End Products (AGEs)

1997; Elsevier BV; Volume: 272; Issue: 9 Linguagem: Inglês

10.1074/jbc.272.9.5430

1083-351X

A.Ashley Booth, Raja G. Khalifah, Parvin Todd, Billy G. Hudson,

Alcohol Consumption and Health Effects

Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products (“AGEs”) and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimer's disease, and normal aging. In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation. Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar. Of several derivatives of vitamins B1 and B6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed. Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates. Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products (“AGEs”) and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimer's disease, and normal aging. In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation. Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar. Of several derivatives of vitamins B1 and B6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed. Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates. INTRODUCTIONNonenzymatic protein glycation (glucosylation or glycosylation) by glucose is a complex cascade of condensations, rearrangements, fragmentations, and oxidative modifications that lead to poorly characterized heterogeneous products often collectively termed Advanced Glycation End Products or AGEs 1The abbreviations used are: AGEadvanced glycation end products (Maillard products)BSAbovine serum albuminRNasebovine pancreatic ribonuclease AELISAenzyme-linked immunosorbent assayCmLNε-carboxymethyllysine. (1Means G.E. Chang M.K. Diabetes. 1982; 31: 1-4Crossref Google Scholar, 2Harding J.J. Adv. Protein Chem. 1985; 37: 248-334Google Scholar, 3Baynes J.W. Diabetes. 1991; 40: 405-412Crossref PubMed Scopus (0) Google Scholar, 4Baynes J.W. Watkins N.G. Fisher C.I. Hull C.J. Patrick J.S. Ahmed M.U. Dunn J.A. Thorpe S.R. Monnier V. Baynes J.W. The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, Inc., New York1989: 43-67Google Scholar, 5Finot P.A. Aeschbacher H.U. Hurrell R.F. Liardon R.(eds) The Maillard Reaction in Food Processing, Human Nutrition and Physiology. Birkhauser Verlag, Basel1990Crossref Google Scholar, 6Ledl F. Finot P.A. Aeschbacher H.U. Hurrell R.F. Liardon R. The Maillard Reaction in Food Processing, Human Nutrition and Physiology. Birkhauser Verlag, Basel1990: 19-42Google Scholar, 7Ledl F. Schleicher E. Angew. Chem. Int. Ed. Engl. 1990; 29: 565-706Crossref Scopus (677) Google Scholar). These very slow Maillard reactions are believed to underlie the pathogenesis of diabetes (8Brownlee M. Cerami A. Annu. Rev. Biochem. 1981; 50: 385-431Crossref PubMed Scopus (350) Google Scholar, 9Nathan D.M. N. Engl. J. Med. 1993; 328: 1676-1685Crossref PubMed Scopus (1147) Google Scholar, 10McCance D.R. Dyer D.G. Dunn J.A. Bailie K.E. Thorpe S.R. Baynes J.W. Lyons T.J. J. Clin. Invest. 1993; 91: 2470-2478Crossref PubMed Scopus (398) Google Scholar, 11Vlassara H. Bucala R. Striker L. Lab. Invest. 1994; 70: 138-151PubMed Google Scholar, 12Reiser K.M. Proc. Soc. Exp. Biol. Med. 1991; 196: 17-29Crossref PubMed Scopus (138) Google Scholar, 13Reiser K. McCormick R.J. Rucker R.B. FASEB J. 1992; 6: 2439-2449Crossref PubMed Scopus (367) Google Scholar, 14Stevens V.J. Rouzer C.A. Monnier V.M. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2918-2922Crossref PubMed Scopus (415) Google Scholar, 15Ruderman N.B. Williamson J.R. Brownlee M. FASEB J. 1992; 6: 2905-2914Crossref PubMed Scopus (348) Google Scholar) and possibly neurodegenerative amyloidal diseases such as Alzheimer's (16Vitek M.P. Bhattacharya K. Glendening J.M. Stopa E. Vlassara H. Bucala R. Manogue K. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4766-4770Crossref PubMed Scopus (739) Google Scholar, 17Smith M.A. Taneda S. Richey P.L. Miyata S. Yan S. Stern D. Sayre L.M. Monnier V.M. Perry G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5710-5714Crossref PubMed Scopus (726) Google Scholar, 18Colaco C.A.L.S. Harrington C.R. Neuroreport. 1994; 5: 859-861Crossref PubMed Scopus (24) Google Scholar, 19Harrington C.R. Colaco C.A.L.S. Nature. 1994; 370: 247-248Crossref PubMed Scopus (63) Google Scholar, 20Kimura T. Takamatsu J. Araki N. Goto M. Kondo A. Miyakawa T. Horiuchi S. Neuroreport. 1995; 6: 866-868Crossref PubMed Scopus (56) Google Scholar, 21Smith M.A. Sayre L.M. Monnier V.M. Perry G. Trends Neurosci. 1995; 18: 172-176Abstract Full Text PDF PubMed Scopus (460) Google Scholar). Nonenzymatic glycation should normally always be occurring, although at a slower rate than in diabetes, and thus may contribute to the pathogenesis of aging (22Monnier V.M. Cerami A. Science. 1981; 211: 491-493Crossref PubMed Scopus (660) Google Scholar, 23Monnier V.M. Monnier V. Baynes J.W. The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, Inc., New York1989: 1-22Google Scholar, 24Monnier V.M. Sell D.R. Miyata S. Nagaraj R.H. Finot P.A. Aeschbacher H.U. Hurrell R.F. Liardon R. The Maillard Reaction in Food Processing, Human Nutrition and Physiology. Birkhauser Verlag, Basel1990: 393-414Google Scholar, 25Dyer D.G. Dunn J.A. Thorpe S.R. Bailie K.E. Lyons T.J. McCance D.R. Baynes J.W. J. Clin. Invest. 1993; 91: 2463-2469Crossref PubMed Scopus (637) Google Scholar). In vitro prepared serum proteins containing AGEs have also been shown to be toxic, immunogenic, and capable of triggering cellular injury responses after uptake by specific cellular receptors (26Vlassara H. Fuh H. Makita Z. Krungkrai S. Cerami A. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12043-12047Crossref PubMed Scopus (369) Google Scholar, 27Daniels B.S. Hauser E.B. Diabetes. 1992; 41: 1415-1421Crossref PubMed Scopus (37) Google Scholar, 28Brownlee M. Diabetes. 1994; 43: 836-841Crossref PubMed Scopus (633) Google Scholar, 29Cohen M.P. Hud E. Wu V.-Y. Kidney Int. 1994; 45: 1673-1679Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 30Brett J. Schmidt A.M. Yan S.D. Zou Y.S. Weidman E. Pinsky D. Nowygrod R. Neeper M. Przysiecki C. Shaw A. Migheli A. Stern D. Am. J. Pathol. 1993; 143: 1699-1712PubMed Google Scholar, 31Yan S.-D. Chen X. Schmidt A.-M. Brett J. Godman G. Zou Y.-S. Scott C.W. Caputo C. Frappier T. Smith M.A. Perry G. Yen S.-H. Stern D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7787-7791Crossref PubMed Scopus (524) Google Scholar). The discovery of chemical agents that can inhibit deleterious glycation reactions is potentially of great therapeutic benefit to all these pathologies, but no such pharmacological agents have entered clinical practice to date.Most kinetic studies of AGE inhibition so far have focused on observing the overall glycation of proteins in the presence of high, nonphysiological concentrations of reducing sugar. The elucidation of the mechanism of inhibition of any candidate AGE inhibitor under such conditions is an extremely difficult task due to the complexity of the glycation cascade (Scheme 1). For example, reactive AGE-forming intermediates can arise from oxidative reactions (“glycoxidation”) of free sugar or from initial Schiff base condensation products with protein amino groups, rather than just from the “classical” Amadori rearrangement products. Furthermore, an inhibitor could potentially act at more than one step of the glycation pathway, so that its contributions to each step would have to be evaluated. This is typified by the prominent AGE inhibitor aminoguanidine, or guanylhydrazine (Scheme 2), that can potentially react as a hydrazine with carbonyls of Amadori intermediates or can scavenge reactive dicarbonyls through its guanidinium moiety. Additionally, as a hydrazine it can block the reactive open chain carbonyl form of reducing sugars (32Khatami M. Suldan Z. David I. Li W. Rockey J.H. Life Sci. 1988; 43: 1725-1731Crossref PubMed Scopus (69) Google Scholar, 33Hirsch J. Petrakova E. Feather M.S. Barnes C.L. Carbohydr. Res. 1995; 267: 17-25Crossref PubMed Scopus (26) Google Scholar). Few studies have quantitatively measured the complete kinetics of protein glycation due to the slowness of the reactions with glucose, making it even more difficult to draw conclusions about inhibition. Limited studies have been carried out on small model Amadori compounds (34Smith P.R. Thornalley P.J. Eur. J. Biochem. 1992; 210: 729-739Crossref PubMed Scopus (111) Google Scholar, 35Glomb M.A. Monnier V.M. J. Biol. Chem. 1995; 270: 10017-10026Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 36Zyzak D.V. Richardson J.M. Thorpe S.R. Baynes J.W. Arch. Biochem. Biophys. 1995; 316: 547-554Crossref PubMed Scopus (102) Google Scholar) or on partially glycated protein intermediates (37Fu M.-X. Knecht K.J. Thorpe S.R. Baynes J.W. Diabetes. 1992; 41: 42-48Crossref PubMed Google Scholar, 38Wells-Knecht M.C. Thorpe S.R. Baynes J.W. Biochemistry. 1995; 34: 15134-15141Crossref PubMed Scopus (152) Google Scholar).Scheme 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We have recently reported a significant simplification in studying the kinetics and mechanism of protein glycation (39Khalifah R.G. Todd P. Booth A.A. Yang S.X. Mott J.D. Hudson B.G. Biochemistry. 1996; 35: 4645-4654Crossref PubMed Scopus (75) Google Scholar). Since glycation by glucose is usually a slow reaction (weeks and months), we have investigated glycation with the pentose ribose, a more reactive analogue of glucose (40Bunn H.F. Higgins P.J. Science. 1981; 213: 222-224Crossref PubMed Scopus (488) Google Scholar, 41Cervantes-Laurean D. Minter D.E. Jacobson E.L. Jacobson M.K. Biochemistry. 1993; 32: 1528-1534Crossref PubMed Scopus (101) Google Scholar). Those studies led to the unique preparation, kinetic characterization, and stabilization of a reactive glycation intermediate in ribonuclease A. After removing excess and reversibly bound ribose from this presumed Amadori intermediate (Scheme 1), it rapidly forms AGEs with a half-time for the exponential kinetics of about 10 h (39Khalifah R.G. Todd P. Booth A.A. Yang S.X. Mott J.D. Hudson B.G. Biochemistry. 1996; 35: 4645-4654Crossref PubMed Scopus (75) Google Scholar). This “interrupted glycation” method provides a new mechanism-based strategy for a better understanding of AGE inhibition by established inhibitors, such as aminoguanidine, through isolating post-Amadori pathways of AGE formation (cf. Scheme 1) and thus removing effects arising from glycoxidation of free sugar or Schiff base (Namiki pathway) (35Glomb M.A. Monnier V.M. J. Biol. Chem. 1995; 270: 10017-10026Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). More importantly, it opens the way to searching for novel inhibitors that specifically act on post-Amadori pathways of AGE formation. Baynes and co-workers (38Wells-Knecht M.C. Thorpe S.R. Baynes J.W. Biochemistry. 1995; 34: 15134-15141Crossref PubMed Scopus (152) Google Scholar) have recently emphasized the importance of Amadori products for the in vivo formation of AGEs. We note that exogenously administered Amadori and AGE proteins have been shown to produce diabetic-like glomerular sclerosis, basement membrane thickening, and albuminuria (42Vlassara H. Striker L.J. Teichberg S. Fuh H. Li Y.M. Steffes M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11704-11708Crossref PubMed Scopus (423) Google Scholar, 43Cohen M.P. Ziyadeh F.N. J. Am. Soc. Nephrol. 1996; 7: 183-190PubMed Google Scholar).We now report the successful use of the above approach to elucidate important aspects of the inhibition by aminoguanidine and to discover novel post-Amadori inhibitors of AGE formation. In particular, we examined several derivatives of vitamins B1 and B6 (Scheme 2) that were recently screened for possible AGE inhibition in the presence of high glucose (44Booth A.A. Khalifah R.G. Hudson B.G. Biochem. Biophys. Res. Commun. 1996; 220: 113-119Crossref PubMed Scopus (172) Google Scholar). They were chosen for study due to the chemistry of their participation as cofactors in many α-carbonyl reactions in carbohydrate metabolism, which could relate to α-carbonyls of Amadori intermediates. Two derivatives, pyridoxamine and thiamine pyrophosphate, proved to be unique post-Amadori inhibitors of AGE formation. In contrast, the classic AGE inhibitor aminoguanidine (45Brownlee M. Vlassara H. Kooney A. Ulrich P. Cerami A. Science. 1986; 232: 1629-1632Crossref PubMed Scopus (1077) Google Scholar) surprisingly showed little post-Amadori inhibition. We confirmed that our earlier inhibition results (44Booth A.A. Khalifah R.G. Hudson B.G. Biochem. Biophys. Res. Commun. 1996; 220: 113-119Crossref PubMed Scopus (172) Google Scholar) and the present ones are generally not specific for the proteins used, even though there are individual variations in rates of AGE formation and inhibition.RESULTSBefore presenting and then discussing the results, it may be useful to emphasize that there is no standard method of defining or following AGE formation and its inhibition. Quantitation of initial Schiff base condensation of labeled reducing sugars with protein amino groups provides little information on post-Amadori steps of AGE formation, since such steps may not lead to changes in the extent of labeling. Many studies of AGE formation have monitored the increase in blue fluorescence arising from “browning” glycation products. Such fluorescent changes can be produced by dicarbonyl or glycoxidation products that arise from free sugar, from the initial Schiff bases, and from Amadori and other intermediates (50Fuijimori E. Biochim. Biophys. Acta. 1989; 998: 105-110Crossref PubMed Scopus (66) Google Scholar, 51Wolff S.P. Dean R.T. Biochem. J. 1987; 245: 243-250Crossref PubMed Scopus (1137) Google Scholar, 52Hunt J.V. Dean R.T. Wolff S.P. Biochem. J. 1988; 256: 205-212Crossref PubMed Scopus (738) Google Scholar). Carboxymethyllysine (CmL), a notable AGE, can also arise from a variety of glycoxidation intermediates besides the Amadori product (35Glomb M.A. Monnier V.M. J. Biol. Chem. 1995; 270: 10017-10026Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 36Zyzak D.V. Richardson J.M. Thorpe S.R. Baynes J.W. Arch. Biochem. Biophys. 1995; 316: 547-554Crossref PubMed Scopus (102) Google Scholar, 53Wells-Knecht K.J. Zyzak D.V. Litchfield J.E. Thorpe S.R. Baynes J.W. Biochemistry. 1995; 34: 3702-3709Crossref PubMed Scopus (552) Google Scholar). The acid-stable fluorescent AGE pentosidine (54Sell D.R. Monnier V.M. J. Biol. Chem. 1989; 264: 21597-21602Abstract Full Text PDF PubMed Google Scholar, 55Grandhee S.K. Monnier V.M. J. Biol. Chem. 1991; 266: 11649-11653Abstract Full Text PDF PubMed Google Scholar, 56Dyer D.G. Blackledge J.A. Thorpe S.R. Baynes J.W. J. Biol. Chem. 1991; 266: 11654-11660Abstract Full Text PDF PubMed Google Scholar) can be utilized in principle, but the chemical work-up, protein hydrolysis, and high performance liquid chromatography separation required for each time point makes its use inconvenient for large sets of kinetics. In this work we chose to use sensitive ELISA techniques (46Nakayama H. Taneda S. Kuwajima S. Aoki S. Kuroda Y. Misawa K. Nakagawa S. Biochem. Biophys. Res. Commun. 1989; 162: 740-745Crossref PubMed Scopus (70) Google Scholar, 47Horiuchi S. Araki N. Morino Y. J. Biol. Chem. 1991; 266: 7329-7332Abstract Full Text PDF PubMed Google Scholar, 48Makita Z. Vlassara H. Cerami A. Bucala R. J. Biol. Chem. 1992; 267: 5133-5138Abstract Full Text PDF PubMed Google Scholar, 57Nakayama H. Taneda S. Mitsuhashi T. Kuwajima S. Aoki S. Kuroda Y. Misawa K. Yanagisawa K. Nakagawa S. J. Immunol. Methods. 1991; 140: 119-125Crossref PubMed Scopus (27) Google Scholar, 58Araki N. Ueno N. Chakrabarti B. Morino Y. Horiuchi S. J. Biol. Chem. 1992; 267: 10211-10214Abstract Full Text PDF PubMed Google Scholar) that utilize anti-AGE polyclonal antibodies developed against proteins typically glycated for 60-90 days with glucose. This approach recently proved highly suitable for detailed kinetics studies of the formation of antigenic AGE products and its inhibition (39Khalifah R.G. Todd P. Booth A.A. Yang S.X. Mott J.D. Hudson B.G. Biochemistry. 1996; 35: 4645-4654Crossref PubMed Scopus (75) Google Scholar, 44Booth A.A. Khalifah R.G. Hudson B.G. Biochem. Biophys. Res. Commun. 1996; 220: 113-119Crossref PubMed Scopus (172) Google Scholar).2The results in this section are presented as two series of inhibition experiments. In the first series, the indicated proteins are mixed with ribose to initiate glycation in the presence and absence of the inhibitors, and the formation of AGEs was monitored by ELISA. This assay is referred to as “overall glycation kinetics” or as “uninterrupted glycation.” In the second series of experiments, an interrupted glycation method was used to follow “post-Amadori kinetics” of AGE formation in absence of ribose. The proteins were first incubated at 37°C with ribose for 24 h during which Amadori intermediates (Scheme 1) accumulated (39Khalifah R.G. Todd P. Booth A.A. Yang S.X. Mott J.D. Hudson B.G. Biochemistry. 1996; 35: 4645-4654Crossref PubMed Scopus (75) Google Scholar). The excess and reversibly bound sugar was then removed by dialysis at 4°C, and AGE formation was initiated (the zero time) in the presence and absence of inhibitors by warming the solutions back to 37°C.Inhibition by Vitamin B6 Derivatives of the Overall Kinetics of AGE FormationThe inhibitory effects of the B1 and B6 vitamins on the kinetics of antigenic AGE formation were evaluated by polyclonal antibodies specific for AGEs. 3Polyclonal anti-AGE antibodies have proven to be a sensitive analytical tool for the study of AGE formation in vitro and in vivo, although the nature of the dominant antigenic AGE epitope of hapten remains in doubt. Baynes and co-workers (71Reddy S. Bichler J. Wells-Knecht K.J. Thorpe S.R. Baynes J.W. Biochemistry. 1995; 34: 10872-10878Crossref PubMed Scopus (461) Google Scholar) recently demonstrated that the protein glycoxidation product carboxymethyllysine (CmL) is a dominant antigen of their antibodies, and they were able to rationalize why earlier studies had failed to reveal strong ELISA reactivity with model carboxymethyllysine compounds (48Makita Z. Vlassara H. Cerami A. Bucala R. J. Biol. Chem. 1992; 267: 5133-5138Abstract Full Text PDF PubMed Google Scholar). Our preliminary characterization of our polyclonal antibodies reveals a strong, although variable, CML reactivity.5 Our initial inhibition studies were carried out on the glycation of bovine ribonuclease A (RNase) in the continuous presence of 0.05 M ribose. It has been shown elsewhere (39Khalifah R.G. Todd P. Booth A.A. Yang S.X. Mott J.D. Hudson B.G. Biochemistry. 1996; 35: 4645-4654Crossref PubMed Scopus (75) Google Scholar) that the rate of AGE formation is near-maximal at this concentration. Fig. 1 (control curves, filled rectangles) demonstrates that the formation of antigenic AGEs on RNase when incubated with 0.05 M ribose is relatively rapid, with a half-time of approximately 6 days under these temperature and buffer conditions. Pyridoxal 5′-phosphate (Fig. 1B) and pyridoxal (Fig. 1C) significantly inhibited the rate of AGE formation on RNase at concentrations of 50 and 15 mM. Surprisingly, pyridoxine, the alcohol form of vitamin B6 (Scheme 2), also moderately inhibited AGE formation on RNase (Fig. 1D). Of the B6 derivatives examined above, pyridoxamine at 50 mM provided the best inhibition of “final” levels of AGE formation on RNase over the 6-week period monitored (Fig. 1A).Fig. 1Effect of vitamin B6 derivatives on AGE formation during uninterrupted glycation of ribonuclease A by ribose. RNase (1 mg/ml) was incubated with 0.05 M ribose in the presence and absence of the various indicated derivatives in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 6 weeks. Aliquots were assayed by ELISA using R479 anti-AGE antibodies. Concentrations of the inhibitors were 0.5, 3.0, 15, and 50 mM. A, pyridoxamine (PM); B, pyridoxal-5′-phosphate (PLP); C, pyridoxal (PL); D, pyridoxine (PN).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inhibition by Vitamin B1 Derivatives of the Overall Kinetics of AGE FormationAll of the B1 vitamers inhibited antigenic AGE formation on RNase at high concentrations, but the inhibition appeared more complex than for the B6 derivatives (Fig. 2, A-C). In the case of thiamine pyrophosphate (Fig. 2A), both the rate of AGE formation and the final levels of AGE produced at the plateau appeared diminished by the compound. In the case of thiamine phosphate (Fig. 2B) and thiamine (Fig. 2C), there appeared little effect on the rate of AGE formation, but a substantial decrease in the final level of AGE formed in the presence of the highest concentration of inhibitor. In general, thiamine pyrophosphate demonstrated greater inhibition than the other two compounds at the lower concentrations examined.Fig. 2Effects of vitamin B1 derivatives and aminoguanidine on AGE formation during uninterrupted glycation of ribonuclease A by ribose. RNase (1 mg/ml) was incubated with 0.05 M ribose in the presence and absence of the various indicated derivatives in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 6 weeks. Aliquots were assayed by ELISA using R479 anti-AGE antibodies. Concentrations of the inhibitors were 0.5, 3.0, 15, and 50 mM. A, thiamine pyrophosphate (TPP); B, thiamine monophosphate (TP); C, thiamine (T); D, aminoguanidine (AG).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inhibition by Aminoguanidine of the Overall Kinetics of AGE FormationInhibition of AGE formation by aminoguanidine (Fig. 2D) was distinctly different from that seen by the B1 and B6 derivatives above. Increasing concentrations of aminoguanidine decreased the rate of AGE formation on RNase but did not reduce the final levels of AGE formed, so that the level of AGEs produced by the 6th week was almost identical to that of the control.Inhibition of the Overall Kinetics of AGE Formation in Serum Albumin and HemoglobinComparative studies were carried out with bovine serum albumin and human methemoglobin to determine whether the observed inhibition was protein-specific. The different derivatives of vitamin B6 (Fig. 3, A-D) and vitamin B1 (Fig. 4, A-C) exhibited similar inhibition trends when incubated with bovine serum albumin, with pyridoxamine and thiamine pyrophosphate being the most effective inhibitors in each of the respective families. Pyridoxine failed to inhibit AGE formation on BSA (Fig. 3D). Pyridoxal phosphate and pyridoxal (Fig. 3, B-C) mostly inhibited the rate of AGE formation but not the final levels. Pyridoxamine (Fig. 3A) again exhibited some inhibition at lower concentrations and at the highest concentration appeared to inhibit the final levels of AGE formation more effectively than the other B6 vitamers. In the case of the B1 vitamers, the overall extent of inhibition on BSA (Fig. 4, A-C) was less than that observed with RNase (Fig. 2, A-C). Higher concentrations of thiamine and thiamine monophosphate only slightly inhibited AGE formation on BSA (Fig. 4, B and C), whereas thiamine pyrophosphate inhibited the final levels of AGE formation without greatly affecting the rate of formation (Fig. 4A). Aminoguanidine again displayed the inhibition effects with BSA that it did with RNase (Fig. 4D), appearing to only slow the rate of AGE formation with lesser effects on decreasing the final levels of AGE.Fig. 3Effect of vitamin B6 derivatives on AGE formation during uninterrupted glycation of bovine serum albumin by ribose. BSA (10 mg/ml) was incubated with 0.05 M ribose in the presence and absence of the various indicated derivatives in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 6 weeks. Aliquots were assayed by ELISA using R618 anti-AGE antibodies. Concentrations of the inhibitors were 0.5, 3, 15, and 50 mM. A, pyridoxamine (PM); B, pyridoxal phosphate (PLP); C, pyridoxal (PL); D, pyridoxine (PN).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Effects of vitamin B1 derivatives and aminoguanidine on AGE formation during uninterrupted glycation of bovine serum albumin by ribose. BSA (10 mg/ml) was incubated with 0.05 M ribose in the presence and absence of the various indicated derivatives in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C for 6 weeks. Aliquots were assayed by ELISA using R618 anti-AGE antibodies. Concentrations of the inhibitors were 0.5, 3, 15, and 50 mM. A, thiamine pyrophosphate (TPP); B, thiamine monophosphate (TP); C, thiamine (T); D, aminoguanidine (AG).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The kinetics of AGE formation were also examined with human methemoglobin in the presence of the B6 and B1 vitamers and aminoguanidine. The absolute rates of AGE formation appeared higher with human methemoglobin than with the other two proteins,2 but the compounds revealed largely similar inhibition trends (data not shown). Of the vitamin B6 derivatives, pyridoxamine showed the greatest inhibition at concentrations of 3 mM and above when compared with pyridoxal phosphate, pyridoxal, and pyridoxine. In the case of the B1 compounds, the inhibitory effects were more similar to the BSA inhibition trends than to RNase. The inhibition was modest at the highest concentrations tested (50 mM), being nearly 30-50% for all three vitamers (data not shown). It was primarily manifest as a decrease in the final levels of AGE.Inhibition by Vitamin B6 Derivatives of the Kinetics of Post-Amadori Ribose AGE FormationIn the interrupted glycation assays for following post-Amadori AGE formation, kinetics were followed by incubating isolated Amadori intermediates of either RNase or BSA at 37°C in absence of free or reversibly bound ribose. Ribose sugar that was initially used to prepare the intermediates was removed b