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6-25-1991 Formation of Pentosidine during Nonenzymatic Browning of Proteins by Glucose Daniel G. Dyer
James A. Blackledge
Suzanne R. Thorpe
John W. Baynes University of South Carolina - Columbia, [email protected]
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Publication Info Published in Journal of Biological Chemistry, Volume 266, Issue 18, 1991, pages 11654-11660. This research was originally published in the Journal of Biological Chemistry. Dyer DG, Blackledge JA, Thorpe SR, Baynes JW. Formation of Pentosidine during Nonenzymatic Browning of Proteins by Glucose. Journal of Biological Chemistry. 1991, 266:11654-11660. © the American Society for Biochemistry and Molecular Biology.
This Article is brought to you by the Chemistry and Biochemistry, Department of at Scholar Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. THEJOURNAL OF BIOLOGICALCHEMISTRY Vol. 266, No. 18,, Issue of June 25, pp. 11654-11660,1991 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Formation of Pentosidine during NonenzymaticBrowning of Proteins by Glucose IDENTIFICATION OF GLUCOSE AND OTHER CARBOHYDRATES AS POSSIBLEPRECURSORS OF PENTOSIDINE IN VIVO*
(Received for publication, January 14, 1991)
Daniel G.Dyer$, James A. Blackledge$, Suzanne R. Thorpet, and John W. Baynes$gT From the $Department of Chemistry and §School of Medicine, University of South Carolina, Columbia, South Carolina 29208
A fluorescent compound has been detected in proteins of the Maillard reaction and to evaluate their possible role in browned during Maillard reactions with glucose in the development of complications. Fluorescent products have vitro and shown to be identical to pentosidine, a pen- provided a focus for this work since fluorescence increases in tose-derived fluorescent cross-link formed between ar- proteins during browning reactions in vitro (8, 91, and the ginine and lysine residues in collagen (Sell, D. R., and fluorescence of long-lived proteins, such as lens crystallins Monnier, V. M. (1989) J. Biol. Chem. 264, 21597- and tissue collagens, increases with age and at anaccelerated 2 1602). Pentosidine was the major fluorophore formed rate in diabetes (10). Increased collagen-linked fluorescence during nonenzymatic browning of ribonuclease and is also correlated with the appearance of complications in lysozyme by glucose, but accounted for <1% of non- diabetes (11). disulfide cross-links in protein dimers formed during In 1989, Sell and Monnier isolated and characterized the the reaction. Pentosidine was formed in greatest yields fluorescent cross-link, pentosidine (Fig. l), in human colla- in reactions of pentoses with lysine and arginine in gens andother tissue proteins.Pentosidine was found to model systems but was alsoformed from glucose, fruc- accumulate in tissue collagens with age and at anaccelerated tose, ascorbate, Amadoricompounds, 3-deoxygluco- rate in diabetic patients with renal complications (13). Since sone, and other sugars. Pentosidine was not formed it was readily formed in syntheticreactions between pentoses, from peroxidized polyunsaturated fatty acids or ma- arginine, and lysine, Sell and Monnier (12) proposed that this londialdehyde. Its formation from carbohydrates was compound was also formed spontaneously by reaction be- inhibited under nitrogen or anaerobic conditions and tween pentoses and proteins in vivo. At the same time our by aminoguanidine,an inhibitor of advanced glycation laboratory had identified a fluorescent compound, named and browning reactions. Pentosidine was detected in Maillard Fluorescent Product 1 (MFP-l),’ which was formed human lens proteins, where its concentration increased in browning reactions of glucose with model proteins in vitro gradually with age,but it did not exceed trace concen- and was also observed to accumulate in tissue proteins with trations (55 Fmol/mol lysine), even inthe 80-year-old lens. Although its precise carbohydrate source in vivo age and in diabetes (14-16). Because of their similar chro- is uncertain and it is present in only trace concentra- matographic properties, absorbance maxima (328 nm) and tions in tissue proteins, pentosidine appears to be a fluorescence spectra (excitation and emission maxima of 328 useful biomarker for assessing cumulative damage to and 378 nm, respectively), we considered that MFP-1 and proteins by nonenzymatic browning reactions with pentosidine might be related compounds, perhaps differing by carbohydrates. a single carbon atom, i.e. containing six and five carbohydrate- derived carbons, respectively. In thisreport we show, however, that MFP-1 andpentosidine are the same compound and that pentosidine is formed by reaction of proteins with glucose, pentose, and numerous other carbohydrates and Maillard Glycation (nonenzymatic glycosylation) and Maillard (non- reaction products under aerobic conditions in vitro (Fig. 1). enzymatic browning) reactions of proteins by reducing sugars We also present evidence that pentosidine accounts for 4% are thought to contribute to the aging and cross-linking of of the total cross-links formed in proteins during browning tissueproteins (1-3). Acceleration of these reactions as a reactions with glucose in vitro. Although its precise carbohy- result of hyperglycemia in diabetes and the consequent in- drate source is uncertainand it is present in only trace crease in structural and functional modification of long-lived concentrations in tissue proteins, the results presented below proteins are implicated in the pathogenesis of the chronic indicate that pentosidine should be useful as a biomarker for complications of diabetes (4). Since the extent of glycation of assessing cumulative damage to proteins by nonenzymatic protein is relatively constant with age (5-7), a major challenge browning reactions with carbohydrates. in research on the Maillard reaction in vivo is to identify specific products formed in tissue proteins during laterstages MATERIALS AND METHODS AND RESULTS* DISCUSSION * This work was supported by Research Grant DK-19971 from the National Institute of Diabetes and Digestive and Kidney Diseases The Origin of Pentosidine in Tissue Proteins-The glyca- and National Science Foundation Grant CHE-89-04942. The costs of publication of this article were defrayed in part by the payment of The abbreviations used are: MFP-1, Maillard fluorescent product page charges. This article must therefore be hereby marked “aduer- 1, shown in this paper to be pentosidine; CML, N“(carboxy- tisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate methy1)lysine; CMhL, N“(carboxymethy1)hydroxylysine; FL, fruc- this fact. toselysine; HPLC, high performance liquid chromatography. 8 To whom correspondence should be addressed. Tel.: 803-777- ’’ Portions of this paper (including “Materials and Methods,” “Re- 7272; Fax: 803-777-9521. sults,’’ Figures 2-8 and Tables I-IV) are presented in miniprint at
11654 Formation of Pentosidine during Nonenzymatic Browning of Proteins 11655 Lysine as might be expected if ribose were derived directly from CARBOHYDRATE glucose or indirectly from Amadori adducts. The resolution of this question may be complicated since pentoses could be Aldose, KBlO(iB Arglnine IHl Dicarbonyl Sugar Dicarbonyl ( HzJa (CHxJa a major source of the age-dependent increase in pentosidine Amadori Compound Amadori 'i I in non-diabetic tissue, while the incremental increase in pen- CH tosidine in diabetic tissue could be attributed to glucose. Mechanism of Formation of Pentosidine-The requirement PENTOSIDINE for oxygen in the formation of pentosidine from glucose, ribose, ascorbate, and Amadori compounds provides evidence FIG. 1. Generalized pathway for formation of pentosidine. that oxidation is required at some stage in the formation of In addition to arginine and lysine, this scheme includes several possible carbohydrate precursors of pentosidine identified in this pentosidine. The requirement for the Amadori rearrangement study, Oxygen is required forthe formation of pentosidine from these is less certain.Pentosidine is formed from Amadori com- precursors. pounds, such as 1-deoxy-1-morpholino-D-fructose(Table IV) and Ne-formyl-W-fructoselysine (data not shown), and from tion hypothesis proposes that the pathogenesis of long-term 3-deoxyglucosone (Table 11) which is formed on re- complications in diabetes is a consequence of chemical, struc- arrangement and dissociation of Amadori adducts (33). How- tural, and functional modifications of proteins resulting from ever, kinetic experiments, such as in Fig. 6, show at most a non-enzymatic reactions with glucose. The glucose or Mail- short lag-phase in the formation of pentosidine from glucose. lard theory of aging (1, 3) also treats aging as a natural Thus, oxidative fragmentation of Schiff base adducts or consequence of cumulative chemical modification of proteins eneaminol intermediates (34) cannot be excluded, since these via the Maillard reaction and considers diabetic complications are formed rapidly from sugars and amines and may also be to be, in part, an expression of accelerated age-related pa- formed from Amadori compounds by reversal of the Amadori thology resulting from hyperglycemia. The chemical modifi- rearrangement (19). Numerous oxidation and cleavage prod- cations and damage to proteins and other biomolecules are ucts are known to be formed on oxidation of carbohydrates thought to be produced during the late or browning stages of (35), but only trace amounts of the critical intermediate(s) the Maillard reaction, however, evidence for age-dependent would be required for formation of pentosidine at the yields accumulation of Maillard products in tissue proteins is still obtained in our experiments. Among the likely candidates for limited to only three compounds: (i) N"(carboxymethy1)lysine pentosidine precursors are dicarbonyl sugars (35), which (CML) in lens proteins(5) and skin collagen (7), (ii) N" should react readily with arginine (36). Pentoses and5-carbon (carboxymethy1)hydroxylysine(CMhL) in skin collagen (7), dicarbonyl sugars may also be intermediates in the formation and (iii) pentosidine in lens proteins (Fig. 8) and collagens of pentosidine from 6-carbon and smaller precursors. The 5- (12). Levels of CML, CMhL, and pentosidine are also in- carbon sugars could be formed from larger sugars by oxidative creased in diabetic, compared to non-diabetic skin collagen fragmentation (35) or from other sugars by condensation and/ (12),3 consistent with the hypothesized role of the Maillard or reverse aldol reactions. reaction in the accelerated aging of proteins in diabetes. Significance of Pentosidine in Tissue Proteins-Our esti- There is increasing uncertainty about the origin(s) of Mail- mate of the pentosidine content of skin collagen (up to 7 pmol lard products in tissue proteins. CML and CMhL, which were pentosidine/mg collagen at 70 years of age) appears to be originally observed as products of oxidative cleavage of glu- about 5-fold lower than reported by Sell and Monnier (-32 cose-derived Amadori compounds in vitro (19), may also be pmol/mg at age 70) (12, 13), but there is essential agreement derived from reactions of proteins with ascorbate (31), aswell that pentosidine is present at only trace concentrations. Our as glucose (19), ribose, threose, and probably other sugars in measurements indicate approximately 2 mmol pentosidine/ vivo (31). Thus, while CML and CMhL can be measured as mol triple-stranded collagen and 25pmol pentosidine/mol indicators of carbohydrate-derived damage, the exact identity lens crystallin (assuming crystallin M, = 20,000) at age 70, of the carbohydrate source remains uncertain. Similarly, while with at most a 2-fold increase in diabete~.~We thinkit Sell and Monnier (12, 13) characterized pentosidine asa unlikely that this level of pentosidine would have a major pentose-derived fluorophore in proteins, the results of the impact oncollagen or lens proteinstructure, function, or present study suggest that glucose itself, or Amadori adducts turnover. However, even in studies on the browning of lyso- of glucose to protein, can also be sources of pentosidine, thus zyme (Fig. 2) or RNase (Fig. 6) by glucose in vitro, when the explaining the increase in pentosidine in tissue proteins in proteins are extensively modified by glucose, pentosidine ac- diabetes. However, the origin of pentosidine from ascorbate, counts for only a small fraction of the total modification of fructose, pentose, and lower sugars cannot be rigorously ex- lysine residues in the protein and less than 1%of the cross- cluded, either during the normal aging process or in diabetes, links in protein dimers formed during the glycation reaction and glucose may be only one of many sources of Maillard (Figs. 6 and 7). At the same time, the kinetics of pentosidine reaction damage to tissue proteins. formation in the proteins during glycation reactions (Fig. 6) Measurements of the pentoses in non-diabetic serum indi- reflect the accumulation of more extensive chemical modifi- cate that they are present in micromolar concentrations (re- cations (loss of lysine residues, formation of CML and flu- viewed in Ref. 12), but, considering the relative reactivity of orescent products, cross-linking of the protein) and damage glucose and pentoses in vitro (Table 11), this would still be to the protein. Although pentosidine does not discriminate sufficient to support a significant role for pentoses in the between browning derived from various sugars, it does appear formation of pentosidine in uiuo. However, there is no evi- to be specific for carbohydrates since it is not formed on dence that pentose concentrations are increased in diabetes, incubation of lysine and arginine with malondialdehyde (Table 11) or other products of oxidation of fatty acids. Thus, the end of this paper. Miniprint is easily read with the aid of a despite its low concentration intissues, pentosidine should be standard magnifying glass. Full size photocopies are included in the useful as aunique biomarker of carbohydrate-dependent dam- microfilm edition of the Journal that isavailable from Waverly Press. age to protein via the Maillard reaction. "D. G. Dyer, J. A. Blackledge, S. R. Thorpe, D. R. McCance, T. J. Lyons, and J. W. Baynes, manuscript in preparation. Acknowledgments-We wish to thank Dr. Vincent Monnier for 11656 Formation of Pentosidineduring Nonenzymatic Browning of Proteins helpful discussions and for providing experimental information from D. R., Lyons, T. J., and Baynes, J. W. (1990) in The Maillard his laboratory prior to publication. We also thank Dr. A. Ronald Reaction in Food Processing, Human Nutrition and Physiology Garber for NMR spectroscopy analyses and Dr. Michael D. Walla for (Finot, P. A., Aeschbacher, H. U., Hurrell, R. F., and Liardon, mass spectrometry analyses. R., eds) pp. 379-384, Birkhauser Verlag, Basel, Switzerland 17. Richmond, M. L., Barfuss, D. L., Hare, B. R., Gray, J. I., and REFERENCES Stine, C. M. (1982) J. Dairy Sci. 65, 1394-1400 18. Watkins, N. G., Thorpe, S. and Baynes, J. W. (1985) J. Biol. 1. Cerami, A. (1985) J. Am. Geriatr. SOC.33, 626-634 R., Chem. 260, 10629-10636 2. Baynes, J. W., and Monnier, V. M. (eds) (1989) The Maillard 19. Ahmed, M. U., Thorpe, S. R., and Baynes, J. W. (1986) J. Biol. Reaction in Aging, Diabetes and Nutrition, Alan R. Liss, New Chem. 26 1,4889-4894 York 20. Watkins, N. G., Neglia-Fisher, C. I., Dyer, D. G., Thorpe, S. R., 3. Monnier, V. M. (1990) J. Gerontol. Biol. 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U., Murtiashaw, M. H., Richardson, J. 13. Sell, D. R., and Monnier V. M. (1990) J. Clin. Znuest. 85, 380- M., Walla, M. D., Thorpe, S. R., and Baynes, J. W. (1990) 384 Biochemistry 29, 10964-10970 14. Baynes, J. W., Dunn, J. A., Dyer, D. G., Knecht, K. J., Ahmed, 32. Heath, H. (1962) Erp. Eye Res. 1,362-367 M. U., Thorpe, S. R. (1990) in Glycated Proteins in Diabetes 33. Ledl, F., and Schleicher, E. (1990) Angew. Chem. Int. Ed. Engl. Mellitus (Ryall, R. G., ed) pp. 221-236, Adelaide University 29,565-594 Press, Adelaide, Australia 34. Hayashi T.,Mase S., and Namiki, M. (1985) Agric. Biol. Chem. 15. Lyons, T. J., Silvestri, G., Dyer, D. G., Dum, J.A., and Baynes, 49,3131-3137 J. W. (1990) in Glycated Proteins in Diabetes Mellitus (Ryall, 35. Schuchmann, M. N., and von Sonntag, C. (1977) J. Chem. SOC. R.G., ed) pp. 265-270, Adelaide University Press, Adelaide, Perkin 11, 1958-1963 Australia 36. Means, G. E, and Feeney, R. E. (1971) Chemical Modification of 16. Dunn, J. A,, Dyer, D. 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FORMATION OF PENTOSIDINE DURINQ NONENIYMTIC BROWNINGOF PROTEINS BY GLUCOSE: IDENTIFICATION OF GLUCOSE AND OTHER CARBOHYDRATES A8 POSSIBLE PRECURSORSOF PENTOSIDINE IN VNO
by
Daniel G. Dyer. James A. Blackledge, Suzanne R. Thorpe. and John W. Baynes
MTERIALS AND METHODS of pentosidlne and measurement of the pentosidine content of tissue proteins. Chromatography Was performed on a Varian 5500 liquid chromatograph using C-18 laterials - Protease (type XIV, Pronase E), leucine aminopeptidase (type silica co1Ymn5. a Supelcosil LC-118 column (250 x 1.6 mm, 5 pm; Supelco) for IV-s). hen egg white lysozyme (grade I) and bovine pancreatic ribonuclease A analytical separations and quantitative analyses, and a Hi-Pore RP-318 column (Type XII-A), were obtained from Sigma Chemical CO., Bnd o-chymotrypsin from (250 x 10 mm. 5pm; Blo-Rad) for preparative isolations; flow rates were I and Cooper Biomedical. [l"'C], [6"'C] and [U"*C]Glucose were purchased from 1.5 ml/rnin, respectively. Two different gradient systems were used to optimize AmericanRadiolabeled Chemicals and purified by chromatography on an Aminex HPX- various separations, one cantainlng 0.1% trifluoroacetic acid (TFA), and the 87C carbohydrate column (Ca"-form; Bio-Rad) using deionized water as eluent other, 0.1% heptafluorobutyric acid (HFBA) a16 organic modifier in each solvent. (17,18). When radioactive glucose was diluted with nan-radioactive glucose in In both cases Solvent A was HiO and Solvent 8 was 50% acetonitrile. The solvent experiments. the specific radioactivity of the glucose war based on the glucose gradients were: for the TFA system. 0 ain=lOOI A, 15 mi"= 9% 8. 25 mln=9% 8.
concentration measured using a glucose oxidase-peroxidase kit (Aaresco). N" 70 mi" = 60% 8. 80 min = 100% Bi and for the HFBA system. 0 min n 10% 8. 10 ain farmyl-N'-fructoselysine and N"(carboxymethy1)lysine (CML) were prepared as = 18% 8, 60 ain = 18% 8, 65 min = 100% B. In both Cases pentosidine eluted near described previously (19). N'-hexitollysines, a mixture of mannitol- and the end of an iEocratiC region of the gradient, 25 min in TFI (see Figure 2) and glucitol-lysine, were prepared by reduction Of N'-fonnyl-N'-fructorelysine With 53 min in HFBii (see Figure 3). and was detected by its fluorescence at EX/Em = NaBH', followed by acid hydrolysis to remove the formyl group (19). All Other 3281378 nm, using either a Cilford Fluoro-IV or Shirnadzu RF-5000 spectro reagents were obtained fron Sigma Chemical Co,, unless otherwise indicated. fluorometer, equipped with a continuous flow cell. Peak areas Were integratr HPLC Anoilyaea - Amino acid analysis was performed by ion exchange HPLC using a ~aselineChromatography Wortstation (Dynamic Solutions). Yslng a pH and salt gradient (NaC1-Na citrate) on a Waters HPLC System equipped SyIltheeIs and puantitation of Radioaotive Pentollidin. - N"Acety1-[G-'HI- with a post-column reactor for measuring amino acids as their o-phthaldialdehyde arginine) was prepared by adding 25 moles arginine in 200'pl acetic acid to 250 derivatives (18-20). Reversed-phase HPLC (UP-HPLC) was used for purification pci [G9HIarginine (ICN Blomedicals), followed by 200 p1 (-2.5 mmol) acetlc Formation of Pentosidine during Nonenzymatic Browning of Proteins 11657
anhydride. The reaction was quenched after 5 ninby the addition of 1.5 ml Of 8p.EtlO.OOpY - NMR Spectra Yere recorded On a BrUker "500 spectrometer water, and the "at== and acetic acid removed by rotary evaporation. The running at 500 and 125 MHz for proton and "C-NUR, respectively. High recovery of LT-acetyl-[G-'H]arginine was >95% by amino acidanalysis. N"AcetY1- resolution fast atom bombardment mass spectrometry was performed on L VG-7OSQ [4,5"H]lyaine was prepared from [4,5-'H)lysine (ICN Biomedicals). Briefly, spectrometer. radioactive lysine (400 pci) vas dried under a stream Of N1 and mixed with 40 urn01 lysine, 300 pl (-4 mrnol) acetic anhydride. and 100 111 acetic acid at 25'C. RESULTS The mixture was vortexed and quenched after 45 seconds by the addition of 2 ml of water. N'-acetyllysina and lysinewere separated on the aminoacid analyrer, D.t.Etion of P.ntoaidin. im Br0m.d Lysosy.. - Lysozyme WZ.B used as D model and aliquots of eluant fractions counted for radioactivity. The reaction protein in preliminary studies on the farmation Of fluorescent products during yielded N"acetyl1yeine (27%). N'-acetyllySine (3%). N.N"diaCetyllySine 120%) Drowning reactions with glucose. After incubation Of lysozyme with 0.25 M and lysine (501). Theunreacted lysins Was recoverad and desalted by glucose in 0.2 u phosphate buffer, pH 7.4, for 28 days at 37-C, thefluorescence chromatography on a 2 ml c~lumnof DOWBX-50-H' using 2 N NH'OH as eluant. spectrum Of the dialyzed.browned protein shoved emissionand excitation maxima recycled through the acetylationand purification procedure,aind Pooled with the at approximatsly 325 and 400 nm, respectively. The identification Of specific urn01 N'-acetyl-(4.5-'Hlly.ine (48% original product to yield 19.2 recovered glucose-derived flwrophores formed in the browning reactionat physiolqical yield). The specific activity Of the radioactive N.-acetyl amino acids WBQ pH and tenperature was complicated by the fact that acid hydrolysis Of the determined experimentally by amino acid analysis with On-line flow-thrOUgh browned protein produced about a 10-fold increase in flYOresCenCe. TO avoid the radioactivity detection (Flo-One\Bata; Radiomatic Instruments). production Of artifacts during hydrolysis. the browned protein was reduced and Radioactive pentosidine, labeled with either arginine, lysineor glucose. S-carboxynethylated, then digested sequentially with chymotrypsin,pronase and was prepared by including the radioactive compoundin reaction mixtures (500 p1 aminopeptidase, as described in "Materials and Uethods". The fluoreroence of mU total volume) containing 100 each glucosa, N'-acetylarginina and N*- the protein vas not siqnificantly increased ( hydrolysate of browned RNase A, 8 protein which doer not contain tryptophan (data not shown). Syntb.sIs of P.ntosIQIn. from QlYCOS~- The flUOrOphOrB isolated from browned lysozyme andlens proteins had fluorescence propertiee similar to those Of pentosidine, the pentare-derived fluorescent compound describedby Sell and Hannier (12). i.e.. an absorbance maximum at 328 nm. fluorescence maxima at 3281378 nm, and a minimum in fluorescence intensity at pH 9. We prepared pentosidine fromN'-acetylarginine, V-acetyllysine and ribose. using conditions similar to those described bySell and NOnnier (12) (see legend to Figure3). The glucose-derived fluorophore was prepared using the same conditions. but Substituting glucose for ribosein the reaction mixture. Since pentosidinewas originally described in tissue collagens (12). we a160 compared the RP-HPLC chromatographic behavior Of the fluorescent product recovered in hydrolysates Of skin collagen with those prepared synthetically fromglucoBe or ribose. AS shown in Figure3, the glucose-derived fluorophore andpentosidine had identical chromatographic properties (also using the TFA solvent system and various qradiontmodifications), Which, Coupled vith the Similarities in absorbance and flvorercencepropertier. strongly Suggestedthatthe glucose-derived fluorophore was, in fact, pentosidine. The yield Of the fluorophore from ribose reaction mixtures Was typically 500-1000 fold greater than that fromglucose (legend to Figure 3, and Table11, discussed below). To exclude possible riboseor pentose contaminants in theg1ucose used for the protein browning and Synthetic reaction mixtures. the pentose content ofglucoae the was determined by measuringglucose and pentoses as their BlditOl acetates (25) by selected ion monitoring gas chromatography-mass SpectOmetry. These analyses yieldedless than 10 ppm ribose or pentose contaminationin the glucose preparation, a level 100 foldlower than Aromatic Aiiphalk that required to explain the yield Of pentosidine from ribose contaminants. . 1.2 IA I I IIIII ~igure4. proton ana 'lc ~ll~*p.ctra or pantoaidin.. Pentosidine (1.2 mg) vas synthesizedfrom glucose. N*-acetylarginine and V-acetyllysine,and purified as described in "naterials and Methods". NMR spectrawere recorded in deuterium oxide (1 ml for '*C, and 2 nl for proton). The proton assignments in the upper frame are based on those of Sell and Nannier (12). The "C-NUR spectrum in the lower frame was resolvedinto unprotonated IO), singly protonated (1) and doubly protonated (2) carbons by INEPT analysis. At higher resolution. the tall peak at53 ppm was a150 resolved into two separate peaks, one Of which was separated in singly and doubly protonated Carbons by INEPT analysis. The presence Of three unprotonated and three singly protonated aromatic Carbons is consistent with the proposed structure of pentosidine. The singly protonated o-carbons of the amino acids resonate53 ppm at and theseven 0 10 20 30 40 50 60 backbone Carbons 01 the amino acids at 22-53 ppm, Time (rnin) Figure 3. Identification or p.ato~ia1r.e in buman skin collagan (AI ana In ..action* or QUEOI~ (81 or rib... LC) with IP-.oetyl.rginin. ana r-.c.tyl- lysin.. Sampler Were analyzed using the HFBA gradient system with the Shimadzu RF-5000 SpeCtTOfluarophotometer. A, Pentosidine in skin collagen sample (500 Syntbeoim of PentosIdIn. fromRaaioaotIv. Pr.cursor'. - Pentosidine prepared uq) from a 20 year old donor. B and C, GlU~ose or ribose (100 mN) were from N'-acetyl-[G-'H].rginine and N.-acetyl-[(,5-'H]lysine yieldedsimilar incubated with 100 mn Ne-acetylarginine and N'-acetyllysine in 0.2 M phosphate specific activities (molaminoacid/fluores.encaareaunit; Table I), consistent buffer, pH 9, for 6 hOUFs at 6OPC. The samples were reduced with NaBH, and With incorporation of equimolar amounts of arginine and lysins per mol deacetylated by heating in 6 N HC1 for 2 hr at 100°C. Thevolume injected from pantoridine. As shovnin Figure 5 and summarized in Table pentoatidinsI, could the glu~osereaction (c) was equivalent to 1000 times thevolume injected from also be labeled with[u-"c] and [6-"c] and [l-"C]qlucore. The consistent.-lot the ribose reaction (8). lover specific activity obtainad with [u-"C]glucosa, compared to amino acids (Table I), is consistent withloss of 1 carbon from Uniformly-labeled glucose duringformation of pentosidine. (The theoretical specific activity for The experimental data Suggested that pentosidinewas formed from g1ucose. pentosidine synthesized from [U-l4C]glucose would 3.8be nmol/lo' fluorescence with loss of one carbon in the process. but itvas conceivable that theglUCOse area unit, i.e., 83% that of pentosidine labeled with amino acids.) One and ribose products were different, but structurally related compounds which possible route to formationof pentosidine from glucose would be the formation were not separable by RP-HPtC analysis. For thisreason we decided to proceed of a +carbon intermediate by oxidative elimination Of C-1 from glucooe. with the preparative synthesis and characterization glucose Of the fluorophore, HOY~VBT,the comparable specific radioactivity obtainedfmm [U-"C] and [6-"CI as described in "Materials and Methods". Analysis of the product by 'H NplR glucose and the decreased specific activity Obtained with [6-"Clglucasa, (Figure 4A) indicated that the glucose-derived lluorophore was, infact, compared to amino acidlabels (Table I), suggests that some pentosidine may be pentosidine (Figure 1). The "C NMR spectrum in Figure 48 confirmed the formed by pathways involving elimination Of C-6,or rearrangement andcleavage presence Of the6 aromatic carbons in theimidaP0[4,5-b]pyridinillm ring. INEPT Of the glucose chain. Partial elimination Of c-6 is also supported by the analysis (26) also confirmed the presence Of 3 quaternary and 3 singly experiment in Figure 5c. in which pentosidine is labeled, albeit to lower protonated aromatic Carbons in the compound, consistent with the Structural Specificradioactivity. with [I-"C]qlucose. Althoughthe uniformity Or aSIignment Of Sell and Hannier (12). High resolution faetatom bombardmentmass specificity of labeling of the various glucose preparations used in these SpeCtrQSCOpy also yielded m/z = 379.2089 Compared to the calculated mass of experiments vas not confirmed experimentally. the[l-"c]glucose was synthesized 379.2094 (1.3 ppm error) for pentosidine, CI,H2,Na0,, These results established by the cyanohydrin procedure(27). SO that significant labeling at other than that the glucose-derived fluorescent compound we whichhad originally identified C-1 is unlikely. Thus, the labeling Of pentosidine by (1-l4C]glucOsa indicates a5 MFP-1 (16-16) Vas, in fact. pentosidine, and indicated that pentosidine could that as much as 259 Of pentosidine formation from glucose may result from be formed lrom glucose. fragmentations not involving theloso Of the C-l carbon. Formation of Pentosidine during Nonenzymatic Browningof Proteins 11659 Although measurable amount5 of pentosidineare detected in all of the reactions conducted invacuo, the presenceOf trace amounts of oxygen cannotbe rigorously excluded. It is also possible, however, that the sugars could be oxidized anaerobically by intermolecular redox reactions. Notably, as Shown near the bottom Of Table 11, among the conpounds tested, nalondialdehyde,a product of peroxidation of Unsaturated lipids. did not form pentosidine. We were also unable to detect pentasidine in reactions which included linoleic, orlinolenic 2000 arachidonic acids peroxidized by treatment With H202 in the presence Of ferrous iron (data not Shown). 1000 TABLE I1 0 The formation of pentosidine from various compounds and the effects Of anaerobic incubation conditione. compounds were incubated with 100 mM N"-acetylarginine, 100 mM N*-acetyl- lysine and 200 mM phosphate. pH 9.0, at 60'C for 6 hr, under air or Vacuum. Pentosidine Was quantitated by reversed phase HPLCas described in "Materials and Methods" and legend to Figure3. C om p ou nd Pentosidine Compound Pentosidine % of Air' 8 of Ribose' (air1 (YICuYmD) "inOl/rnl ,lmOl/U,l D"G1"CC.S. 0.11 0.01 9.0 0.2 n-Giucose + 2 mn DTPA 0.08 0.007 8.8 0.2 D-FrUCtOSB 0.37 0.01 1.0 0.8 1500 D-Galactose 0.67 0.02 3.5 1.4 L -A s co rb i c Acid 0.06L-AscorbicAcid 0.002 3.9 0.1 NDO _d 1000 D-GluconicAcid 3-DeoxyglYc050ne' 0.18 1.4 D-Ribose 48.0' 1.5 3.2 100.0 500 O-Ribose + 2 mM DTPA 55.6 1.6 2.9 116.0 n -x y lo se 22.1 n-xylose 1.1 4.8 46.0 0 D-Erythrose 4.7 0.31 7.0 9.1 D-Threose 1.1 0.08 7.3 2.2 51 52 53 54 55 56 DL-Glyceraldehyde 3.1 0.14 4.5 6.4 Time (min) Dihydroxyacetone 12.6 0.51 4.2 16.2 ~igure 5. Characterization of radioactivepentosiaine by RP-HPLc. NDMalondialdehyde PentOsldine was Synthesized from radioactive prec~rsors and purified as F o rm a ld eh y de ND Formaldehyde described in "Materials andMethods'q. This figure shows chromatograms obtained in the final purification stepby RP-HPLC using the HFBA gradient system. The a Expressed as a percentage of the yield from parallel the incubation underax. specific radioactivity of tha various preparations is described in TableI. Expressed as a percentage of the yield from the ribose incubation underair. ' ND, not detected; < 0.4 pmol/ml. -, not determined. The concentration of the amino acids sugarand in this experiment was 25 mM: TABLE I the 8 of rlbose is based on a parallel incubation with 25 mn ribose. I The 8 yield Of pentosidine based on ribose is 0.051. Summary of the synthesis and quantitation of radioactive pentosidine. 11101 R a di o ch e mi ca l specific Radioactivity specific Radiochemical Inhibition Of pentoridine formation by siminoguanidine - AG has been identified as a potent inhibitor of the browning, cros51inking and development offluorescence of collagen in vivo (28) and in vitro (9). TableI11 illustrates that AG also lnhlblts the formation of pentosidine from hexose and pentose prec~rsorsin -3 dase-dependent fashion. Notably, AC Was effective at concentrations significantly lower than that of the startingsugar, consistent with itsrole in trapping eithera product of oxidationOf the sugar or a sugar- aminoacld adduct formed during the Maillard reaction. The experiments summarized in Table IV suggest that pentosldlne prec~rsors mayalso be formed by decomposition of Amadori adducts to protein in biological systems, since pentosidine is farmed fromthe commerclally available Amadori Compound. l-deoxy- Mean i SD. l-morpholinO-D-fructose in the presence N'-acetylarginineOf and N"-acetyllysine ' Expressed as nmol Of radiochemical per 10' area units as determined from the at PH 7.e and 37OC. In this case, too. AG was an effective inhibitor Of integration Of the pentosidine peak from the HPLC Chromatogram. Quantitatlon pentosidine formation. of fluorescence and radioactivitywere as described in "Materials and Methods" and Figure 5. TABLE I11 - Formation of Pantosidine from Other Carb0hYdrat.s Table I1 summarizes The effect of aminoquanidine (AG) on the formation of pentosidine the results ofa number of experiments in Which varioussugars were evaluated from glucose and ribose. as possible precursors Of pentosidine. These experiments indicate that while pentoses are the most efficientsource Of pentosidine, other compounds present An aqueous solution of 100 mM each sugar. N'-acetylarginine and N*-acetyl- in biological systems mayalso be p~ecursors of pentosidine, including hexoses lysine and 200 mW phosphate. pH 9.0, was heated a< 60'C for 6 hr in the absence (both aldosms and ketoses), ascorbicac'id, 3-deoXYgluCOSone and simplerEYgarL. or presence of varying concentrations of aminoguandins (AC). Qumtitation of Such as tetrose* and trioses. In each case, the pentosidine was identified by pentosidine was as described in "Materials and Methods" and legend to Figure1. itsRP-HPLC elution position, its fluorescence maxima and decrease in fluorescence at pH9, compared topH 2 (12). The yield from non- of pentosidine Pentosidineconditions % Inhibition' pentose sugars was typically lessthan 108 of that fromthe pentoses,but in all cases the presence of significant amounts (>lo ppnl of pentose was excluded by analysis for pentosesas their alditol acetates(251 by gas chromatography-mass spectrometry. The fact that pentosidine is formed fromEL variety such ofsuqars 0 suggests that a Common intermediate my be formed by a variety of pathways. 40 Aldol condensation reactions must be involved in the formationof pentosidine invitro from sugars with less than 5 carbons. although the biological 71 significance of this pathway is questionable becauseOf the low concentrations 84 of tetrose and triosesugars in vivo. In all cases the yield of pentosidinewas significantly decreased under v.acuum, indicating an essential role for oxygen Ribose control 39300 0 in the oxidation, and possibly the oxidative fragmentation. Ofsugars the or of Ribose + 1 AG 38500 2 some precursor to pentosidine. Surprisingly. the transition metal chelator, Ribose + 10 mM AG 28700 27 diethylenetri~ininepentaacetic acid (DTPAI, which is often used to inhibit autoxidative reactions Ofsugars, was not an efficient inhibitor of pentosidine Ribose + 20 mM AC 18400 53 formation Under air, although formationof CML was Dore than 90% inhibited. In fact. DTPA consistently stimulated the formation of pentosidine from ribose. 11660 Formation of Pentosidine during Nonenzymatic Browning of Proteins TABLE IV As shown in Figure 6B, pentosidine, like CUL, appeared early in the reaction and folloved a kinatic OOurse similar to thatof CML. HoVever. even The effects of various reagents on the formtion or pentosidine from the at the end of 5 months. pentosidine accounted for only a mall fraction Of the Amodori rearrangement product, I-deoxy-I-morpholino"-fr"=t~~~(DMFJ. total modification of lysine residues in the protein. i.e.. (0.01 mol pentosidine/mol RNase, or <0.11 of the 10 lysine residues in RNase. TOtal Aqueous solutions containing 10 mn DMF in 200 mu phosphate buffer, pH 7.4, fluorescence and pentosidine in the protein increased in parallel. but the were incubated at 37.C for 14 days in the absence Or presence of various additives (N'-acetylarginine, AcAeq; K-acetyllysine, AcLyS; aminoguanidine, fractional contribution of pantosidine to the totalfluoreecensa at 128/178nn AG). Quantitation of pentosidine was as described in "Materials and Methods" declined from -901 of the totalfluor'oscencs at 1 day. to -501 after 14 days and and lagend to figure 3. 301 at 140 days Of incubation. Although these results indicate that other flUOrOphOres are formed during the c0UI-s~Of the reaction and COntribUte to the overall fluorescence of theprotein. there Were no Significant differenCBP in Additive Pantosidine Remarks either the fluorescence maxima or the 3-dimensional fluorescence Surface plots of the protein glyoated tor 28 days "8. 140 days(data not shown). pmol/ml fractionation of the browned RNase by gel exclusion chromatography On sephadax G-75 (Phsrmacia), shown in Figure 1, revealed that fluoresicancs was not None ND. significantly enriched in dimer compared to monomer. The pentosidins content ACArg ND Of the peak fractions vas also Similar, -0.005 mol pentosidinejmol RNa.. monomer AELys ND (see also figure 68). indioating that pentosidine aCcOUnts for I 1000.0 xin.tio. and Pmauot. oc a1yoatic.n ma Browning or Pnotein in vitm - Because lysozyme gradually precipitates from solution during glycation reactions, we used RNase as a model protein for studying the kinetics of modification Of lysine residues, formation of pantosidina and development Of f1uoreBcence during long-term browning reactions. Glycltion of RNase increased rapidly during the first iew weeks Of reaction (Figure 6A1, reaching a steady state of approximately 1.8 mol glucosajmol protein for the remainder of the 5 month period. CNL, formed on Oxidative olewage of the Amadori compound, fruotosalysina, also appeared early in the reaction and continued to aCEYmUlllte (figure 611). exceeding the concentration of frustosalysina after about2 months. The kinetics Of disappearance Of lysine (Figure 6A). measured by amino acid analysis, were consistent with the cumulative rate of formation of fructoselysine and CUL, although there was an increasing discrepancy of up to 2 no1 lysine modified (unrecovered during amino acid analysis) per mol RNase. starting from the first week of incubation. At the conclusion of the experiment, fruotoselyeine and CUL accounted for -4 out of 10 mol lysine/nol RNase. and -101 of the totalloss of lysine residues in the protein, as measured bV amino acid anLlVsis,.. althouqh .. the Dosaible reqenaration Of lysine from SDme products during acid hydrolysis cannot be excluded. -l PlUOteaCeIIo. and Pantosidin. in ByUn TisSY.s - The changes occurring during glycatian and brovninq of protein in vitro (Fiqure 6) show interesting parallels to proces~esWhich occur naturally in tisaua proteins. ThUB, in the model System, the level of theAmadori adduct, fructoselysine, reaches a ateady state, while the level of theoxidation product, CML, increases gr&dually vith time (figure 6). similarly, levels or fructoselysine are relatively constant with age in both lens protein (5.6,9) and skin collagen (7.9). vhila level8 Of $4 CIlL increase gradually with age in both tissues (5.1.9). The COnEBntratiOn Of - pentosidine in lens proteins, shown in Figure 8, a160 increases qrsdually vith age. consistent with the accumulation Of pentoeidine in proteins in vitro E (Figure 6). and mirroring the aFcUnUlatiOn of pentoaidine in tissue 5011agenS Y (12). The approximate 4-fold increase in pentosidine in lens protein between E2 20 and 80 yeam of age agrees well vith the relative increase in pentosidine in dura matter sollagan through the Same age range (12). Pentobidine accounted for modification of t0.01: of lysine residues in lens proteins in the Oldest sample shown in Figure 9, i.e., or -25 pol pentoridinajmol lens crystallin. thus it clearly does not have a major role in the age-dependent aggregation and Crosslinking of lens Droteina. 0 7 0 0 50 100 150 Time(days) Figure 6. Pozutio~01 bromiag and oxidation produotm duzimg qlyaation or W.... RNase A (20 ngjml) vas glycated as described in "Materials and 0 Methods". Aliquots were removed at various times, reduced with NOiBH', and hydrolyzed in 6 N HC1. I,Lysine modification (open squares), hexitallysines (open triangles) and CML (closed triangles) were measured by amino acid analysis. 8. Pentosidina (open circles) was measured by RP-HPLC, as described in "Materials and Methode". A separate aliquot wee dialyzed at 4.C against eeveral changes of 0.5 M acetic acid. diluted to 250 !Jq/ml vith 0.5 M acetic acid. pH 2, and the fluorescence measured at EX = 328nm and Em = 378 nm (closed circles). Note the 1000-fold difference in vertical scales in frames A and 8.