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R H Nagaraj

Publications and source records attributed to R H Nagaraj.

At least 37 records · Page 2Linked to original sources
PubMed1996

Protein cross-linking by the Maillard reaction. Isolation, characterization, and in vivo detection of a lysine-lysine cross-link derived from methylglyoxal.

The Maillard reaction, initiated by nonenzymatic glycosylation of amino groups on proteins by reducing sugars, has been studied for its potential role in aging and the complications of diabetes. One of the major consequences of the advanced Maillard reaction in proteins is the formation of covalently cross-linked aggregates. The chemical nature of the cross-linking structures is largely unknown. Recently, methylglyoxal has been shown to be a potential glycating agent in vivo and suggested to be a common intermediate in the Maillard reaction involving glucose. Methylglyoxal can form enzymatically or nonenzymatically from glycolytic intermediates and by retro-aldol cleavage of sugars. Its elevation in tissues in diabetes and its high potency to glycate and cross-link proteins led us to investigate the chemical nature of its advanced Maillard products. Using an approach in which a synthetic model peptide was reacted with methylglyoxal, we isolated and purified a cross-linked peptide dimer. Characterization of this dimer revealed that the peptides are linked through epsilon amino groups of lysine residues. The actual cross-link was shown to be a methylimidazolium, formed from the reaction of two lysines and two methylglyoxal molecules. We have named this cross-link imidazolysine. Imidazolysine was detected in proteins by high performance liquid chromatography using a postcolumn derivatization method. Proteins incubated with methylglyoxal showed a time-dependent formation of imidazolysine. Quantification of imidazolysine in human serum proteins revealed a significant increase (p < 0.05) in diabetic samples (mean +/- S.D., 313.8 +/- 52.7 pmol/mg protein) when compared with normal samples (261.3 +/- 50.4). These values correlated with glycohemoglobin (p < 0.05). These results provide chemical evidence for protein cross-linking by dicarbonyl compounds in vivo.

Blood Proteins↗
PubMed1996

Quantitative solubilization and analysis of insoluble paired helical filaments from Alzheimer disease.

In this study, we evaluate the ability of several solvents to solubilize insoluble paired helical filaments (PHF) of Alzheimer disease. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents of PHF. Using sequential categories of protein solvent to analyze insoluble PHF, only alkali or exhaustive proteolysis are effective in completely solubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosphorylation of PHF with hydrofluoric acid does not affect PHF solubility. These findings suggest that the 'hyperphosphorylation' of PHF proteins is not responsible for PHF insolubility. However the in vitro glycation of tau generates PHF that are insoluble in SDS and soluble in alkali. These findings suggest that protein crosslinks, including advanced glycation endproduct-derived crosslinks which were recently described in Alzheimer disease, play a major role in effecting PHF insolubility in vivo.

Adolescent↗
PubMed1996

The presence of a glucose-derived Maillard reaction product in the human lens.

Pyrraline is an advanced Maillard reaction product formed by the non-enzymatic reaction initiated by glucose with lysine residues on proteins. This reaction involves an intermediate, 3-deoxyglucosone, concentration of which is shown to be elevated in plasma and lenses during diabetes. Bovine lens alpha crystallins incubated with 3-deoxyglucosone showed that pyrraline formation was a major modification and its quantification by two different methods revealed time-dependent accumulation. Pyrraline was quantified in normal, senile cataractous and diabetic lenses. Although a wide variation was observed, the mean value in cataractous lenses (mean +/- S.E.: 48.4 +/- 12.67 pmol/mg protein) was higher than in age-matched normal lenses (30.9 +/- 10.26 pmol). Surprisingly, in diabetic lenses, the mean value was lower than normal lenses (28.4 +/- 15.3 pmol). These results suggest that glucose-specific advanced Maillard products occur in the human lens and such modifications may play a role in lens aging and cataract formation.

Animals↗
PubMed1996

Pyrraline ether crosslinks as a basis for protein crosslinking by the advanced Maillard reaction in aging and diabetes.

Pyrraline (epsilon 2-(formyl-5-hydroxymethyl-pyrrol-1-yl)-L- norleucine) is an advanced Maillard reaction product derived from the reaction of glucose with lysine amino group on proteins. Its presence in plasma and tissue proteins has been established by immunological and chromatographic methods. The purified preparation of pyrraline obtained from the reaction of glucose with lysine when stored at room temperature or at refrigeration turned pink in color, suggesting spontaneous formation of degradation products. These products were analyzed by high-performance liquid chromatography and one of the products was isolated to purity. The structure of the compound was established to be a dipyrraline formed by an ether bond between two pyrraline molecules. This finding led us to investigate the reactivity of pyrraline with thiol and hydroxy amino acids. The hydroxy amino acids were in general nonreactive, except hydroxy lysine and hydroxy proline which formed minor condensation products. While the reaction of cysteine resulted in the formation of two distinct thioethers, the reaction of glutathione resulted in the formation of two major unidentified compounds which gradually degraded upon incubation. These data suggest that pyrraline formed in vivo can further react with other amino acids on proteins to form crosslinks, which may explain in part increased protein crosslinking associated with aging and diabetes.

Aging↗
PubMed1996

Structure of advanced Maillard reaction products and their pathological role.

In this article we review recent progress and controversies relating to three areas of the field of advanced glycosylation end-products (AGE). A controversy exists as to whether pyrraline, an AGE detectable by immunohistochemistry in kidneys from patients with renal failure, exists in vivo. Recent data from the authors' laboratory revealed that pyrraline is present in alkaline or protease digests from human skin and plasma. However, the amounts are very low and pyrraline was found to undergo further reactions to form an ether with itself (dipyrraline) as well as a thioether with cysteine. This high reactivity of pyrraline may explain the difficulty of quantitating it accurately in biological material. In contrast, the glycoxidation products carboxymethyllysine (CML) and pentosidine are stable, very resistant to acid hydrolysis and easy to quantitate. They are present in elevated concentrations in the extracellular matrix in diabetes mellitus and ageing. In the diabetic human lens, CML is not elevated, in contrast to pentosidine, suggesting a different mechanism of formation. Recent data in diabetic dogs have shown that pentosidine is elevated only in lenses from poorly controlled dogs, in contrast to LM-1, a fluorophore thought to arise from ascorbate. Further studies are needed to clarify the intracellular mechanism of glycoxidation. The greatest concentrations of AGEs and glycoxidation products are found in patients with end-stage renal disease, and they are almost completely normalized by renal transplantation. Comparison of peritoneal dialysis (PD) with haemodialysis (HD) showed that PD is associated with lower plasma protein pentosidine, possibly due to selective transport of pentosidine-rich protein across the peritoneal wall. Fractionation of plasma proteins from ESRD patients by size showed that 90% of pentosidine is linked to HMW protein and 1-2% is in free form. The mechanism of accelerated glycoxidation in ESRD is still not understood.

Animals↗
PubMed1996

Evidence of a glycemic threshold for the formation of pentosidine in diabetic dog lens but not in collagen.

The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross-links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 +/- 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 +/- 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes.

Animals↗
PubMed1995

Protein modification by the degradation products of ascorbate: formation of a novel pyrrole from the Maillard reaction of L-threose with proteins.

Ascorbate (vitamin C) degradation products can undergo non-enzymatic glycation (Maillard reaction) with proteins to form highly crosslinked structures with brown pigmentation and characteristic fluorescence. Proteins in the body, especially the long-lived proteins develop similar changes during aging and diabetes. Several studies have shown excessive degradation of ascorbate in plasma in diabetes, and in ocular lens during aging and cataract formation. Recent studies have suggested that ascorbate degradation products-mediated glycation plays a role in lens pigmentation and cataract formation. However, the precise chemical nature of ascorbate-specific advanced glycation end-products are not known. Here, we report the purification and characterization of a glycation end-product derived from one of the major degradation products of ascorbate, L-threose. This compound was characterized to be 2-acetamido-6-(3-(1,2-dihydroxyethyl)-2-formyl-4-hydroxymethyl-1- pyrrolyl)hexanoic acid (formyl threosyl pyrrole or FTP) formed by the condensation of epsilon-amino group of lysine with two molecules of threose. Formation of FTP occurred rapidly in the incubation of threose and lysine and reached plateau level within a day. We have developed a sensitive assay for its quantification in proteins based on enzyme digestion followed by HPLC. Ribonuclease A and human lens crystallins incubated with L-threose showed time- and sugar concentration-dependent increases in FTP, reaching 8.2 and 2.48 nmol per mg protein, respectively after one week of incubation. Human plasma proteins showed a peak with identical retention time as that of purified FTP under two different HPLC conditions. FTP may be used as a sensitive marker to assess ascorbate-mediated protein glycation and modifications in aging and diabetes.

Aminocaproates↗
PubMed1995

Chromatographic evidence for pyrraline formation during protein glycation in vitro and in vivo.

Pyrraline (epsilon-2-(formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine) is an advanced Maillard reaction product formed from 3-deoxyglucosone in the non-enzymatic reaction between glucose and the epsilon-amino group of lysine residues on proteins. Although its presence in vivo as well as in in vitro incubations of proteins with sugars has been documented by immunochemical methods using polyclonal and monoclonal antibodies, its formation in proteins has recently been questioned by similar methodology. To clarify this issue, we investigated pyrraline formation in proteins following alkaline hydrolysis and quantitation by high-performance liquid chromatography on a C18 reverse-phase column. Time- and sugar concentration-dependent increase in pyrraline formation was noted in serum albumin incubated with either 100 mM glucose or 50 mM 3-deoxyglucosone. Formation of pyrraline from 3-deoxyglucosone was rapid at slightly acidic pH, confirming its synthetic pathway through this Maillard reaction intermediate. Low levels of pyrraline (< 10 pmol/mg protein) were also detected in a pool of human skin collagen by this method, but no age effect was apparent. Using a slightly different approach, pyrraline-like material was detected in human plasma proteins following enzyme digestion and analysis by high performance liquid chromatography. Plasma from diabetic patients showed a significant increase in pyrraline-like material compared to controls. The levels in diabetic and normal individuals were 21.6 +/- 9.56 and 12.8 +/- 5.6 pmol per mg protein, respectively (P = 0.005), reflecting thereby the elevated levels of the immediate precursor of pyrraline, 3-deoxyglucosone, in diabetic plasma.

Animals↗
PubMed1994

Suppression of pentosidine formation in galactosemic rat lens by an inhibitor of aldose reductase.

Recent work from our laboratory revealed a correlation between the degree of protein pigmentation in human cataractous lens and the advanced Maillard reaction as reflected by pentosidine formation. Although the data suggested a role for ascorbate in pentosidine formation in senile cataractous lenses, elevated pentosidine levels in diabetic cataracts suggested that glucosylation may be involved directly in pentosidine biosynthesis. To clarify this issue, we quantified pentosidine in lenses from rats with experimental galactosemia with and without aldose reductase inhibitor treatment. At 12 months, pentosidine-like fluorescence (335/385 nm) was three to six times higher (P < 0.0001) in water soluble and insoluble crystallins of galactosemic compared with nongalactosemic rats. Actual pentosidine levels increased shortly after onset of galactosemia. Contents in water-insoluble crystallins were 6.32 +/- 2.2 and 1.40 +/- 0.66 pmol/mg protein in galactosemic and control lenses, respectively (P < 0.001). Fluorescence and pentosidine were suppressed to almost control levels upon treatment with sorbinil. Incubation experiments showed that pentosidine could form slowly from galactose, but much more rapidly from ascorbate and its oxidation products. Its formation could be inhibited partly by both reduced and oxidized glutathione or epsilon-aminocaproic acid. The requirement of oxygen for pentosidine formation suggests that oxidative stress associated with glutathione depletion and ascorbate oxidation are plausible mechanisms for rapid pentosidine formation upon onset of galactosemia. In contrast, Maillard reaction by glycoxidation products may account for the sustained increase in pentosidine. Both these events may be linked to the newly recognized pseudohypoxic state of cells exposed to high sugar concentrations.

Aldehyde Reductase↗
PubMed1992

Isolation and characterization of a blue fluorophore from human eye lens crystallins: in vitro formation from Maillard reaction with ascorbate and ribose.

A blue fluorophore, named LM-1 was isolated from human eye lens crystallins. The fluorescence property of LM-1 (excitation/emission, 366/440 nm) is similar to the fluorescence originating during non-enzymatic glycation (Maillard reaction) of proteins with the reducing sugars. LM-1 accumulates linearly with age in highly cross-linked water insoluble crystallins and is present at higher levels in cataractous lenses. The fluorophore could be synthesized by incubation of bovine serum albumin (BSA) with ribose, but not with glucose or fructose. Incubation of bovine lens crystallins with ascorbic acid (ASA) and its oxidative products, dehydroascorbic acid (DHA) and 2,3-diketogulonic acid (DKG) in presence of oxygen resulted in LM-1 formation. When oxygen was removed from the system, only DHA and DKG could synthesize LM-1, but not ASA, suggesting that ASA oxidation is obligatory for LM-1 synthesis. Modification of lysine residues on BSA prior to incubation with ribose resulted in corresponding decrease in LM-1 formation. Since ASA concentration is unusually high in lens and has been found to be a powerful glycating agent of crystallins and since LM-1 does not form with hexoses, it is likely that ASA is the major precursor of LM-1.

Adult↗
PubMed1992

Maillard reaction-mediated molecular damage to extracellular matrix and other tissue proteins in diabetes, aging, and uremia.

Recent progress in structure elucidation of products of the advanced Maillard reaction now allows probing specifically for the role of this reaction in the pathogenesis of age- and diabetes-related complications. Pyrraline is a glucose-derived advanced glycation end product against which polyclonal and monoclonal antibodies have been raised. Immunohistochemical localization studies revealed that pyrraline is found predominantly in the sclerosed extracellular matrix of glomerular and arteriolar renal tissues from both diabetic and aged nondiabetic individuals. Pentosidine and carboxymethyllysine are Maillard end products derived from both glucose and ascorbate. In addition, pentosidine can be formed from several other sugars under oxidative conditions, and in vitro studies suggest that a common intermediate involving a pentose is a necessary precursor molecule. The highest levels of these advanced Maillard products are generally found in the extracellular matrix, but these products are also present in lens proteins and in proteins with a fast turnover such as plasma proteins. Diabetes, and especially uremia, greatly catalyzes pentosidine formation. Both conditions are characterized by accelerated cataractogenesis, atherosclerosis, and neuropathy, suggesting that molecular damage by advanced Maillard reaction products may be a common mechanism in their development.

Aging↗
PubMed1991

High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis.

Pentosidine is a recently discovered protein crosslink, involving lysine and arginine residues linked together in an imidazo [4,5,6] pyridinium ring formed by a 5-carbon sugar during nonenzymatic browning (Maillard reaction). The presence of high ascorbate levels in the human lens and its ability to undergo nonenzymatic browning led us to investigate pentosidine formation in the aging human lens. Incubation of lens crystallins with ascorbate and its oxidation products dehydroascorbate and 2,3-diketogulonate leads progressively to the formation of pentosidine crosslinks in the presence of oxygen. Under nitrogen, however, pentosidine forms only from 2,3-diketogulonate or xylosone, a degradation product of 2,3-diketogulonate. A high correlation between pentosidine crosslinks and the degree of lens pigmentation is noted in cataractous lenses. Pentosidine is found to be primarily associated with alpha-crystallin fractions of 300-5000 kDa. These results suggest that redox imbalance in cellular senescent systems such as the ocular lens may lead to irreversible ascorbate oxidation and protein crosslinking by xylosone. This mechanism may play an important role in the pathogenesis of "brunescent" cataracts.

Adult↗
PubMed1991

Pentosidine: a molecular marker for the cumulative damage to proteins in diabetes, aging, and uremia.

Collagen undergoes progressive browning with age and diabetes characterized by yellowing, fluorescence, and cross-linking. The present research was undertaken in order to investigate the nature of the collagen-linked fluorescence. Human collagen was exhaustively cleaved into peptides by enzymatic digestion. Upon purification, a highly fluorescent chromophore was identified and purified from old human collagen. Structure elucidation revealed the presence of an imidazo [4,5-b] pyridinium-type structure acting as a cross-link between arginine, lysine, and a pentose. This advanced glycosylation end-product and protein cross-link results from the reaction of pentoses with proteins and was named pentosidine. Further work indicated that long-term glycosylation of proteins with hexoses also leads to pentosidine formation through sugar fragmentation. The proposed mechanism of pentosidine formation involves the dehydration of the pentose-derived Amadori compound to form an intermediate which is attacked under base catalysis by the guanido group of arginine. The strict requirement for the Amadori rearrangement is uncertain. However, oxidation is definitely involved since pentosidine is not formed in the absence of oxygen. Five-carbon sugars contributing to pentosidine formation could be formed from larger sugars by oxidative fragmentation or from trioses, tetroses, and ketoses by condensation and/or reverse aldol reactions. Pentosidine increases exponentially in human skin at autopsy. Mean age-adjusted skin levels were significantly increased in subjects with uremia and especially in type 1 diabetics with uremia vs. controls. In skin biopsy, levels were significantly elevated in all diabetic (type 1) vs. control subjects. The highest degree of association was with the cumulative grade of diabetic complication (retinopathy, nephropathy, arterial stiffness, and joint stiffness). Pentosidine also forms in various proteins other than collagen, although to a much lesser extent. In blood, pentosidine is mainly associated with plasma proteins and is highly elevated during uremia. In the lens, it is associated with both water-soluble and -insoluble protein fractions and is especially elevated during brunescent cataract formation. The origin of pentosidine in vivo is uncertain. Evidence suggests that the pentoses are the most reactive sugars in pentosidine formation in vitro; however, the origin and importance of free pentoses in vivo, especially during the diabetic state, are not certain. Possible origins include hemolysis and/or a defect in the primary pentose metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1991

Mechanisms of protection against damage mediated by the Maillard reaction in aging.

In order to prevent or escape the ongoing damage to proteins and DNA resulting from amino-carbonyl reactions, the organism has to have powerful defense mechanisms. If the Maillard reaction played a role in determining longevity among mammalian species, one would expect protective mechanisms to be more developed in long- versus short-lived species. This article summarizes current knowledge in this field, and discusses the concept of anti-Maillard mechanisms as a basis for the genetic regulation of molecular damage by reducing sugars in aging and diabetes.

Aging↗
PubMed1990

Non-tryptophan fluorescence and high molecular weight protein formation in lens crystallins of rats with chronic galactosemia: prevention by the aldose reductase inhibitor sorbinil.

Crystallin-bound non-tryptophan fluorescence and protein cross-linking were studied in chronic galactosemia. Fluorescence (excitation/emission--370/440 nm) was significantly higher in galactosemic rats compared to controls (P less than 0.001). Accumulation of fluorescence was significantly reduced both in water soluble (P less than 0.001) and insoluble (P less than 0.005) lens fractions in galactosemic rats receiving the aldose reductase inhibitor sorbinil. High performance liquid chromatography of water soluble lens crystallins showed an increase in the content of high molecular weight proteins and the fluorescence associated with it. Sorbinil partly prevented the formation of such fluorescent high molecular weight proteins. Under reducing conditions, sodium dodecyl sulfate polyacrylamide gel electrophoresis of water soluble proteins revealed a distinct high molecular weight protein of 60 kDa in galactosemia. Sorbinil treatment completely abolished the formation of this protein. Western blotting using rabbit antiserum to bovine alpha-, beta- and gamma-crystallins revealed the presence of gamma, but not alpha- and beta-crystallins in the 60-kDa protein. Formation of this protein may result from trimerization of gamma-crystallins or from an association of gamma-crystallin with a non-crystallin cytosolic protein or mixed protein cross-linking of gamma-crystallins with membrane protein(s). The present study shows that polyol pathway in cataractogenesis also extends to protein cross-linking and formation of fluorescent compounds, the nature of which remains to be elucidated.

Aldehyde Reductase↗
PubMed1989

Aging of proteins: immunological detection of a glucose-derived pyrrole formed during maillard reaction in vivo.

Recent work from this laboratory revealed that glucose-derived pyrroles can form with model amines under physiological conditions (Niroge, F. G., Sayre, L. M., and Monnier, V. M. (1987) Carbohydr. Res. 167, 211-220). The major extractable product, 5-hydroxymethyl-1-alkylpyrrole-2-carbaldehyde (named by us pyrraline) was labile to acid hydrolysis. To allow its detection in proteins undergoing advanced glycosylation, an enzyme-linked immunosorbent assay was developed. An immunogen consisting of epsilon-caproyl pyrraline (hapten) was linked onto poly-L-lysine (114:1) and used to raise polyclonal antibodies in the rabbit. High antibody titers were obtained 16 weeks after immunization. The antibody cross-reacted with butyl pyrraline (88%), propyl pyrraline (8%), lysyl pyrraline (2%), and neopentyl pyrraline (1.3%). A time-related increase in pyrraline immunoreactivity was observed in bovine serum albumin incubated with glucose (1000 mM), glycated lysine (50 mM), and 3-deoxyglucosone (50 mM) which reached 25, 300, and 350 pmol/mg, respectively, after 30 days. Mean level of protein pyrraline immunoreactivity were 27.0 +/- 7.2 and 43.3 +/- 11.7 pmol/mg in serum albumin from control and diabetic subjects, respectively (p less than 0.001). The pathobiological relevance of pyrraline may relate to its reported antiproteolytic and mutagenic properties. In addition, glucose-derived pyrroles may play a role in diabetic neuropathy in analogy to pyrroles formed during hexane-induced neuropathy.

Aging↗