Mechanism of formation of the Maillard protein cross-link pentosidine. Glucose, fructose, and ascorbate as pentosidine precursors

Pentosidine is a recently discovered fluorescent protein cross-link from human extracellular matrix that involves lysyl and arginyl residues in an imidazo (4, 5b) pyridinium ring. Pentosidine could be synthesized in vitro by the reaction of ribose, lysine, and arginine. The potential biological significance of the molecule prompted us to investigate its mechanism of formation from D-ribose and key Maillard intermediates, as well as from other potential precursor sugars. The yield of pentosidine from N alpha-t-Boc-lysine, N alpha-t-Boc-arginine, and D-ribose was highest at pH 9.0 and 65 degrees C, but was unaffected by reactant ratios at alkaline pH suggesting an important role for base catalysis. Ribated Boc-lysine on incubation with N alpha-t-Boc-arginine afforded a fluorescent compound with UV, fluorescence, 1H NMR, and MS properties identical with those from native or synthetic pentosidine. 3-Deoxypentosone, however, was not a major pentosidine precursor. Pentosidine became slowly detectable in bovine serum albumin incubated with 0.25 M and 1.0 M glucose and reached, at 30 days, 13.2 and 17 pmol/mg bovine serum albumin, respectively. Spectroscopical properties of glucose-derived pentosidine were identical with those from ribose-derived pentosidine. Pentosidine formed from glucated Boc-lysine with N alpha-t-Boc-arginine in higher yields than from glucose under standard conditions. Fructose, and unexpectedly ascorbate, also formed pentosidine in similar yields as glucose. The discovery that pentosidine can form not only from pentoses but also from hexoses and ascorbate raises major new questions concerning biochemical pathways of the Maillard reaction in vivo.     

Formation of pentosidine during nonenzymatic browning of proteins by glucose. Identification of glucose and other carbohydrates as possible precursors of pentosidine in vivo

A fluorescent compound has been detected in proteins browned during Maillard reactions with glucose in vitro and shown to be identical to pentosidine, a pentose-derived fluorescent cross-link formed between arginine and lysine residues in collagen (Sell, D. R., and Monnier, V. M. (1989) J. Biol. Chem. 264, 21597-21602). Pentosidine was the major fluorophore formed during nonenzymatic browning of ribonuclease and lysozyme by glucose, but accounted for less than 1% of non-disulfide cross-links in protein dimers formed during the reaction. Pentosidine was formed in greatest yields in reactions of pentoses with lysine and arginine in model systems but was also formed from glucose, fructose, ascorbate, Amadori compounds, 3-deoxyglucosone, and other sugars. Pentosidine was not formed from peroxidized polyunsaturated fatty acids or malondialdehyde. Its formation from carbohydrates was inhibited under nitrogen or anaerobic conditions and by aminoguanidine, an inhibitor of advanced glycation and browning reactions. Pentosidine was detected in human lens proteins, where its concentration increased gradually with age, but it did not exceed trace concentrations (less than or equal to 5 mumol/mol lysine), even in the 80-year-old lens. Although its precise carbohydrate source in vivo is uncertain and it is present in only trace concentrations in tissue proteins, pentosidine appears to be a useful biomarker for assessing cumulative damage to proteins by nonenzymatic browning reactions with carbohydrates.     

Isolation, purification and characterization of histidino-threosidine, a novel Maillard reaction protein crosslink from threose, lysine and histidine

We isolated a novel acid-labile yellow chromophore from the incubation of lysine, histidine and d-threose and identified its chemical structure by one and two-dimensional NMR spectroscopy combined with LC-tandem mass spectrometry. This new cross-link exhibits a UV absorbance maximum at 305 nm and a molecular mass of 451 Da. The proposed structure is 2-amino-5-(3-((4-(2-amino-2-carboxyethyl)-1H-imidazol-1-yl)methyl)-4-(1,2-dihydroxyethyl)-2-formyl-1H-pyrrol-1-yl)pentatonic acid, a cross-link between lysine and histidine with addition of two threose molecules. It was in part deduced and confirmed through synthesis of the analogous compound from n-butylamine, imidazole and d-threose. We assigned the compound the trivial name histidino-threosidine. Systemic incubation revealed that histidino-threosidine can be formed in low amounts from fructose, glyceraldehyde, methylglyoxal, glycolaldehyde, ascorbic acid, and dehydroascorbic acid, but at a much higher yield with degradation products of ascorbic acid, i.e. threose, erythrose, and erythrulose. Bovine lens protein incubated with 10 and 50 mM threose for two weeks yielded 560 and 2840 pmol/mg histidino-threosidine. Histidino-threosidine is to our knowledge the first Maillard reaction product known to involve histidine in a crosslink.     

Structure elucidation of a novel yellow chromophore from human lens protein

We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and (1)H, (13)C, and two-dimensional NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and fluorescence at 410 nm when excited at 343 nm. Analysis of the purified compound by reversed-phase HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues, and a five-carbon ring. Because this cross-link contains two lysine residues and a dihydropyridinium ring, we assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests were made using a high-performance liquid chromatograph equipped with a diode array detector. These measurements revealed a significant enhancement of K2P in cataract lens proteins (613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of WS protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of water-soluble (WS) protein). These data provide chemical evidence for increased protein cross-linking during aging and cataract development in vivo. This new cross-link may serve as a quantitatively more significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.     

Glyceraldehyde-derived advanced glycation end-products having pyrrolopyridinium-based crosslinks

Reducing sugars and reactive aldehydes, such as glyceraldehyde, non-enzymatically react with amino or guanidino groups of proteins to form advanced glycation end-products (AGEs) by the Maillard reaction that involves Schiff base formation followed by Amadori rearrangement. AGEs are found relatively in abundance in the human eye and to accumulate at a higher rate in diseases that impair vision such as cataract, diabetic retinopathy or age-related macular degeneration. We identified two novel AGEs of pyrrolopyridinium lysine dimer derived from glyceraldehyde, PPG1 and PPG2, in the Maillard reaction of N^α-acetyl-l-lysine with glyceraldehyde under physiological conditions. Having fluorophores similar to that of vesperlysine A, which was isolated from the human lens, PPGs were found to act as photosensitizers producing singlet oxygen in response to blue light irradiation. Moreover, PPG2 interacts with receptor for AGE (RAGE) in vitro with a higher binding affinity than GLAP, a well-known ligand of the receptor. We also proposed a pathway to form PPGs and discussed how they would be formed in vitro. As glyceraldehyde-derived AGEs have been studied extensively in connection with various hyperglycemia-related diseases, further studies will be required to find PPGs in vivo such as in the lens or other tissues.     

Amides are novel protein modifications formed by physiological sugars

The Maillard reaction, or nonenzymatic browning, proceeds in vivo, and the resulting protein modifications (advanced glycation end products) have been associated with various pathologies. Despite intensive research only very few structures have been established in vivo. We report here for the first time N(6)-[2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl]lysine (GOLA) and N(6)-glycoloyllysine (GALA) as prototypes for novel amide protein modifications produced by reducing sugars. Their identity was confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with N(alpha)-t-butoxycarbonyl-lysine showed that glyoxal and glycolaldehyde are immediate precursors, and reaction pathways are directly linked to N(epsilon)-carboxymethyllysine via glyoxal-imine structures. GOLA, the amide cross-link, and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), the imidazolium cross-link, share a common intermediate. The ratio of GOLA to GOLD is greater when glyoxal levels are low at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose and lysine depends directly upon oxidation. With the advanced glycation end product inhibitors aminoguanidine and pyridoxamine we were able to dissect oxidative fragmentation of the Amadori product as a second mechanism of GOLA formation exactly coinciding with N(epsilon)-carboxymethyllysine synthesis. In contrast, the formation of GALA appears to depend solely upon glyoxal-imines. After enzymatic hydrolysis GOLA was found at 66 pmol/mg of brunescent lens protein. This suggests amide protein modifications as important markers of pathophysiological processes.     

The reaction of alpha-crystallin with the cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) demonstrates close proximity of the C termini of alphaA and alphaB in the native assembly

The chaperone-like activity of human lens alpha-crystallin in inhibiting the aggregation of denatured proteins suggests a role for alpha-crystallin in cataract prevention. Although a variety of techniques have generated structural information relevant to its chaperone-like activity, the size and heterogeneity of alpha-crystallin have prevented determination of its crystal structure. Even though synthetic cross-linkers have provided considerable information about protein structures, they have not previously been used to study the proximity and orientation of subunits within human alpha-crystallin. Cross-linkers provide structural insight into proteins by binding the side chains of amino acids within close proximity. To identify the cross-linked residues, the modified protein is digested and the resulting peptides are analyzed by mass spectrometry. Analysis of products from the reaction of alpha-crystallin with 3,3'dithiobis(sulfosuccinimidyl propionate), DTSSP, identified several modifications to both alphaA and alphaB. The most structurally informative of these modifications was a cross-link between lysine 166 of alphaA and lysine 175 of alphaB. This cross-link provides experimental evidence supporting theoretical structural models that place the C termini of alphaA and alphaB within close proximity in the native aggregate.     

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.     

Detection of nonenzymatic browning products in the human lens

The nonenzymatic browning or Maillard reaction is an aging process in stored foods. The initial stage of this reaction, nonenzymatic glycosylation, has been shown to occur in the human lens. The possible occurrence of further steps of the Maillard reaction involving lysine residues and glucose has been investigated. A lipid-free protein extract from a pool of human cataractous lenses was reduced, alkylated, and digested with pronase. The digest was reduced with [³H]borohydride, acid hydrolyzed and fractionated by Sephadex G-15 chromatography. The fractions eluting ahead of ?-1-deoxyglucitolyllysine were pooled and separated with an amino acid analyzer. Four fluorescent, yellow, and radioactive peaks were obtained. One of these, which co-eluted with tyrosine, was isolated, acetylated, and further analyzed by reverse phase chromatography using HPLC. Two new peaks were separated which co-chromatographed with lysine derivatives isolated from the nonenzymatic browning reaction of α-tert-butyloxycarbonyllysine with glucose. Control experiments showed that they were not artifacts due to acid hydrolysis of ?-glucitolyllysine. These results suggest that dehydration and rearrangement of the Amadori product, ?-fructosyllysine, has occured in vivo, thus leading to the formation of at least two nonenzymatic browning products.     

Glycation of lysine-containing dipeptides.

Protein glycation through Maillard reaction (MR) is a fundamental reaction both in foods and in the human body. The first step of the reaction is the formation of Amadori product (AP) that is converted into intermediate and advanced MR products during reaction development. Although the MR is not an enzymatic reaction, a certain degree of specificity in the glycation site has been observed. In the present study, we have monitored the glycation of different lysine-containing dipeptides to evaluate the influence on the NH(2) reactivity of the neighboring amino acid.Lysine dipeptides were reacted with glucose, galactose, lactose and maltose. The formation and identification of glycated compounds were monitored by mass spectrometry (MALDI-TOF and ESI-MS/MS) and by HPLC of their Fmoc derivatives. MS/MS analysis showed that the glucose APs formed on dipeptides have a characteristic fragmentation pattern: the fragment at [M - 84](+) due to the formation of pyrylium and furylium ion is mainly present in the monoglucosylated form, while the [M - 162](+) and the [M - 324](+) are more evident in the fragmentation pattern of the diglucosylated forms.The nature of the vicinal amino acids strongly affects lysine reactivity towards the different carbohydrates: the presence of hydrophobic residues such as Ile, Leu, Phe strongly increases lysine reactivity. Contrasting results were obtained with basic residues. The Lys-Arg dipeptide was among the most reactive while the Lys-Lys was not.     

Generation of intramolecular and intermolecular sulfenamides, sulfinamides, and sulfonamides by hypochlorous acid: a potential pathway for oxidative cross-linking of low-density lipoprotein by myeloperoxidase.

Oxidized low-density lipoprotein (LDL) is implicated in atherogenesis, and human atherosclerotic lesions contain LDL oxidized by myeloperoxidase, a heme protein secreted by activated phagocytes. Using hydrogen peroxide (H(2)O(2)), myeloperoxidase generates hypochlorous acid (HOCl), a powerful oxidant. We now demonstrate that HOCl produces sulfenamides, sulfinamides, and sulfonamides in model peptides, which suggests a potential mechanism for LDL oxidation and cross-linking. When we exposed the synthetic peptide PFKCG to HOCl, the peptide's thiol residue reacted rapidly, generating a near-quantitative yield of products. Tandem mass spectrometric analysis identified the products as the sulfenamide, sulfinamide, and sulfonamide, all formed by intramolecular cross-linking of the peptide's thiol and lysine residues. An intramolecular sulfinamide was also observed after the peptide PFRCG was exposed to HOCl, indicating that the guanidine group of arginine can also form a sulfur-nitrogen cross-link. The synthetic peptide PFVCG, which contains a free thiol residue but lacks nucleophilic amino acid side chains, formed an intermolecular sulfonamide when exposed to HOCl. Tandem mass spectrometric analysis of the dimer revealed that the free N-terminal amino group of one PFVCG molecule cross-linked with the thiol residue of another. This peptide also formed intermolecular sulfonamide cross-links with N(alpha)-acetyllysine after exposure to HOCl, demonstrating that the epsilon-amino group of a lysine residue can undergo a similar reaction. Moreover, human neutrophils used the myeloperoxidase-H(2)O(2) system to generate sulfinamides in model peptides containing lysine or arginine residues. Collectively, our observations raise the possibility that HOCl generated by myeloperoxidase contributes to intramolecular and intermolecular protein cross-linking in the artery wall. Myeloperoxidase might also use this mechanism to form sulfur-nitrogen cross-links in other inflammatory conditions.     

2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses

Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the nonproteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nalpha-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nalpha-t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties.     

Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen

A stable nonreducible trifunctional cross-linking amino acid has been isolated from mature bovine skin collagen fibrils. Previous cross-link peptide isolations and amino acid analyses indicate the compound has properties identical with those of hydroxyaldolhistidine. Its newly proposed structure was verified using fast atom bombardment mass spectrometry, and 1H and 13C nuclear magnetic resonance. The data indicated that the cross-link consists of the prosthetic groups from one residue each of histidine, hydroxylysine, and lysine. The 1H and 13C nuclear magnetic resonance data indicated that imidazole C-2 of histidine is linked to C-6 of norleucine (epsilon-deaminated lysine residue) which in turn is linked to the C-6 amino group of hydroxylysine. Based on the trivial names for other cross-linking residues found in collagen and elastin it was given the name histidinohydroxylysinonorleucine. In vitro incubation studies for up to 24 weeks, in aqueous solution at physiological pH and ionic strength, using 6-month-old bovine embryo skin demonstrated a one-to-one stoichiometric relationship between the disappearance of the labile reducible bifunctional cross-link dehydrohydroxylysinonorleucine and the appearance of histidinohydroxylsinonorleucine. These results can partially explain the previously observed disappearance of dehydrohydroxylysinonorleucine with chronological age.     

Fluorescent-labeled poly(ethylene glycol) lipid conjugates with distal cationic headgroups

The synthesis of a new class of fluorescent cationic poly(ethylene glycol) lipid conjugates (CPLs) is described. These lipids consist of a hydrophobic distearoyl-phosphatidylethanolamine (DSPE) anchor coupled to a highly fluorescent N(epsilon)-dansyl lysine moiety, which is attached to a hydrophilic poly(ethylene glycol) (PEG) spacer that is linked to a cationic headgroup made of lysine residues. Introduction of the dansyl moiety allows rapid and accurate quantification of CPLs within lipid bilayers using fluorescence techniques. The synthetic scheme is straightforward, using repeated amino-carboxyl coupling reaction steps, with purification by precipitation. A series of dansylated CPLs was synthesized with zero, one, three, and seven lysine residues located at the distal end of the PEG chain, giving rise to CPLs with one, two, four, and eight distal positive charges, respectively. The structures of the CPLs were confirmed by (1)H NMR spectroscopy and chemical analysis. CPLs provide a means of introducing positive charge to a bilayer that is localized some distance from the membrane surface, and are of particular interest for nonviral gene delivery applications. The usefulness of CPLs is demonstrated by the enhanced in vitro cellular binding and uptake of liposomes containing CPL(4).     

Creatine plays a direct role as a protein modifier in the formation of a novel advanced glycation end product

Pentosidine, a cross-link structure between lysine and arginine residues, is one of the major advanced glycation end products (AGE). It is formed by the reaction of ribose with lysine and arginine. The pentosidine concentration produced by in vitro incubation of plasma obtained from uremic patients was reported to be higher than in normal plasma, indicating that uremic plasma contains an enhancer(s) for pentosidine formation [Miyata, T., Ueda, Y., Yamada, Y., Izuhara, Y., Wada, T., Jadoul, M., Saito, A., Kurokawa, K., and Strihou, C.Y. (1998) J. Am. Soc. Nephrol. 9, 2349-2356]. Since our preliminary study using a monoclonal anti-pentosidine antibody identified creatine as the most effective enhancer, the purpose of the present study was to clarify the mechanism by which creatine contributes to pentosidine formation. Lysine was incubated with ribose in the presence of creatine and analyzed by reverse phase high performance liquid chromatography. A novel fluorescent peak (lambda(ex/em) = 335/385 nm) was detected at 8 min, under conditions at which the authentic pentosidine (lysine was incubated with ribose in the presence of arginine under identical conditions) eluted at 12 min. Structural analyses of this compound revealed a pentosidine-like structure in which the arginine residue was replaced by creatine. This novel AGE-structure, named here creatine-derived pentosidine (C-pentosidine), was detected in the plasma of patients on hemodialysis. These results indicate that creatine increases the formation of C-pentosidine but not authentic pentosidine. This study indicates that creatine plays a direct role as a protein modifier in C-pentosidine formation, although the clinical significance of C-pentosidine is still unknown.     

Increase in three alpha,beta-dicarbonyl compound levels in human uremic plasma: specific in vivo determination of intermediates in advanced Maillard reaction

Methylglyoxal (MGO), glypxal (GO) and 3-deoxyglucosone (3-DG) are reactive alpha,beta-dicarbonyl intermediates in advanced Maillard reaction, which form advanced glycation and oxidation end products (AGEs) by reaction with both lysine and arginine residues in protein. We measured these three dicarbonyl compound levels in human plasma to estimate the relationship between accumulation of alpha, beta-dicarbonyl compounds and AGE formation reactions in uremia and diabetes in human plasma by a highly selective and specific assay, electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS). We show that 3-DG and MGO levels are significantly higher in uremia and diabetes compared with age-matched healthy controls. Only the GO level in uremic plasma is significantly higher compared to diabetes and healthy controls. In both diabetic and uremic patients, these dicarbonyl compounds promote AGE accumulation in vivo, and especially in uremic patients, increased accumulation of GO could result from accelerating oxidative stress.     

Advanced glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid: consequences for the lens

Upon aging, the lens accumulates brown fluorophores, mainly derived from the Maillard reaction between vitamin C oxidation products and crystallins lysine residues. At the same time, the concentration of UVA filters decreases, allowing some radiation to be absorbed by lenticular advanced glycation endproducts (AGEs). This paper quantifies the photosensitizing activity of AGEs at various oxygen pressures, and compares it to that of lenticular riboflavin (RF). Solutions containing the sensitizer and the substrates tryptophan (Trp) and ascorbate (AH(-)) were irradiated at 365 nm. We show that the AGEs-photosensitized Trp oxidation rate increases with AGEs concentration and is optimal at 5% oxygen, the pressure in the lens. By contrast, for AH(-), the photooxidation rate increases with oxygen concentration. Despite the higher quantum yield of RF-depending reactions, its low concentration as compared to that of AGEs in aging lenses induces significantly higher Trp and AH(-) photodegradation rates with AGEs than with RF. As ascorbate is more rapidly photodegraded than Trp, the antioxidant competitively protects Trp from oxidation up to 1 mM, although not absolutely. We conclude that in the aging lens, AH(-) exerts a strong UVA protecting activity, but does not impede some Trp residue to be photodegraded proportionally to the AGEs concentration.     

Isolation and identification of 4-hydroxy- and 4-oxoretinoic acid. In vitro metabolites of all-trans-retinoic acid in hamster trachea and liver.

Incubation of [3H]retinoic acid in the presence of hamster liver 10000g supernatant produces several metabolites that are more polar than the parent compound. Two of these metabolites are identical with synthetic all-trans-4-hydroxyretinoic acid and all-trans-4-oxoretinoic acid both in ultraviolet absorption and mass spectral characteristics and in migration rates on two different reverse-phase high-pressure liquid chromatographic systems. The metabolites produced in a cell-free liver incubation reaction also migrate on a high-pressure liquid chromatography column together with metabolites isolated from a tracheal organ culture system. Both the metabolites and the synthetic standards show less biological activity than the parent all-trans-retinoic acid in a tracheal organ culture assay.     

LC-MS display of the total modified amino acids in cataract lens proteins and in lens proteins glycated by ascorbic acid in vitro

We previously reported chromatographic evidence supporting the similarity of yellow chromophores isolated from aged human lens proteins, early brunescent cataract lens proteins and calf lens proteins ascorbylated in vitro [Cheng, R. et al. Biochimica et Biophysica Acta 1537, 14-26, 2001]. In this paper, new evidence supporting the chemical identity of the modified amino acids in these protein populations were collected by using a newly developed two-dimensional LC-MS mapping technique supported by tandem mass analysis of the major species. The pooled water-insoluble proteins from aged normal human lenses, early stage brunescent cataract lenses and calf lens proteins reacted with or without 20 mM ascorbic acid in air for 4 weeks were digested with a battery of proteolytic enzymes under argon to release the modified amino acids. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column and four major A330nm-absorbing peaks were collected. Peaks 1, 2 and 3, which contained most of the modified amino acids were concentrated and subjected to RP-HPLC/ESI-MS, and the mass elution maps were determined. The samples were again analyzed and those peaks with a 10(4) - 10(6) response factor were subjected to MS/MS analysis to identify the daughter ions of each modification. Mass spectrometric maps of peaks 1, 2 and 3 from cataract lenses showed 58, 40 and 55 mass values, respectively, ranging from 150 to 600 Da. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values, respectively, of which 100 were identical to the peaks in the cataract lens proteins. A total of 40 of the major species from each digest were analyzed by LC-MS/MS and 36 were shown to be identical. Calf lens proteins incubated without ascorbic acid showed several similar mass values, but the response factors were 100 to 1000-fold less for every modification. Based upon these data, we conclude that the majority of the major modified amino acids present in early stage brunescent Indian cataract lens proteins appear to arise as a result of ascorbic acid modification, and are presumably advanced glycation end-products.     

Redox ribonucleosides. Isolation and characterization of 5-hydroxyuridine, 8-hydroxyguanosine, and 8-hydroxyadenosine from Torula yeast RNA.

Three hydroxyribonucleosides catalyzing the oxido-reduction of NADH and K3F3(CN)6 were purified from Torula yeast RNA by a series of steps including sodium dodecyl sulfate/phenol extraction, nuclease P1 digestion, alkaline phosphatase digestion, anion-exchange chromatography, and high performance liquid chromatography on an ODS column. Analysis by fast atom bombardment-mass spectrometry and 1H and 13C NMR spectroscopy led to identification of the redox ribonucleosides as 5-hydroxyuridine, 8-hydroxyguanosine, and 8-hydroxyadenosine. Their mass spectra, chromatographic behavior, UV spectra, NMR spectra, and IR spectra were identical to those from natural and synthetic sources. Oxidoreduction activities were specific for K3Fe(CN)6 as the oxidant and NADH as the reductant; and their magnitudes decreased in the order 5-hydroxycytidine, 5-hydroxyuridine, 8-hydroxyguanosine, and 8-hydroxyadenosine. The fact that these nucleosides have redox activities suggests new functional roles for RNAs as catalysts.