Free Radical Biology & Medicine, Vol. 10, pp. 339-352, 1991

0891-5849/91 $3.00 + .00 Copyright©1991PergamonPress plc

Printedin the USA. All rightsreserved.

-~" Review Article PROTEIN GLYCATION AND OXIDATIVE STRESS IN DIABETES MELLITUS AND AGEING

SIMON P. WOL~* ZrmN Y. JIANG, and JAMES V. Huwr Toxicology Laboratory, Departmentof ClinicalPharmacology, UniversityCollege London, 5 UniversityStreet, London WC1E 6JJ, UK (Received 28 May 1990; Revised and Accepted 19 November 1990)

Abstract--Hyperglycemia is increasinglyregarded as the cause of the diabetic complications,in particularvia the ability of glucose to glycate proteins and generate Maillardbrowningproducts which cross-linkproteins and render them brown and fluorescent in vitro. Similar changes occur in vivo to long-livedproteins in diabetes mellitusas well as in ageing. The evidencesupportingthis route of glucose toxicity is discussed in the context of the ability of glucose to oxidize in vitro (catalyzed by trace amountsof transition metal) generatinghydrogenperoxide, highly reactive oxidants, and protein-reactiveketoaldehydecompounds. It is suggested that protein browningin vivo may not result from the reactionsof glucose with protein but from the transitionmetal-catalyzedreactions of other small autoxidisable substrates, such as ascorbate, with protein. Overall, studies of glycation and protein browning suggest a critical role for oxidative processes perhaps involvingdecompartmentalizedtransitionmetals and a variety of low molecular weight reducingagents in diabetes mellitusand ageing. Keywords--Diabetes, Free radicals, Glucose, Glycation,Glycosylation,Hyperglycaemia, Maillard, Protein modification

INTRODUCTION: THE DIABETIC COMPLICATIONS

result of gangrene, and a 2- to 6-fold increased risk of coronary heart disease and ischemic brain damage. 5 Almost half of those diagnosed as diabetic before age 31 die before they reach 50, largely as a result of cardiovascular or renal complications, often with many years of crippling and debilitating disease beforehand. 6

Before the introduction of insulin, the prognosis of insulin-dependent diabetes mellitus was extremely poor, with early death from ketoacidotic coma or infection. 1,2 It had been hoped that Banting and Best's discovery of insulin would allow diabetic individuals to lead normal lives. However, in the course of the following decades it became apparent that this was not so. The diabetic individual is prone to complications which are a major threat to both the quality and length of life. 3'4 These complications are a heterogeneous group of clinical disorders which affect the vascular system, the kidney, the retina, the peripheral nerves, the lens, and the skin. The individual with diabetes has a 25-fold increase in the risk of blindness, a 20-fold increase in the risk of renal failure, a 20-fold increase in the risk of amputation as a

HYPERGLYCAEMIA AND THE IDEA OF GLUCOSE TOXICITY The cause of the diabetic complications was initially an open question. Indeed, it was assumed that the complications were caused by the same underlying lesion which led to the initial loss of blood sugar control and abnormalities in lipid metabolism which characterize the syndrome. The complications subsequently became associated with hyperglycemia, since elevated plasma glucose levels became increasingly viewed as the single outstanding feature which distinguished the diabetic from the normal individual. Later, high blood sugar was regarded as causative in the diabetic complications. For example, Pirart, in an extensively cited study of 4,400 patients over a 26-year period, was firm in his proposition of a causal link between hyperglycaemia and retinopathy, neuropathy, and nephropathy since the presence of these complications appeared to be a function of the

Simon Wolff obtained his degrees (M.A., D. Phil.) from Oxford University. He graduated in 1984, went to Brunel University as a postdoctoral student, had a short period at ColumbiaUniversity,New York, in the Ophthalmology Department, before moving to his current post at UniversityCollege Londonin 1987. His research interests are diabetes, ageing, cataract, cell death, and leukemia. James Hunt took degrees at Chelsea College (BSc) and Brunel University(PhD) in Londongraduatingin 1988. Jiang is a graduate of the BeijingUnion Medical College. *Author to whom correspondenceshould be addressed. 339

340

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Fig. 1. Glycation and the Maillard Reactions. The addition of glucose to protein followed by rearrangements and dehydrations. The deoxyglucosones (ketoaldehydes)react with protein to form many of the AdvancedGlycationEndproducts (AGE).

duration of hyperglycemia and was inversely related to the previous year's glycemic control. 7 Wider acceptance of the view that excessive plasma and tissue glucose can exert pathological effects has been reinforced by the exploration and development of the nonenzymatic glycosylation (glycation) and aldose reductase (polyol pathway) theories of glucose toxicity. This review focuses on the experimental work concerning the role of glycation in diabetes mellitus as well as the possible importance of free radical oxidative processes in protein modification. Critical review of the aldose reductase hypothesis and the evidence that elevated levels of blood sugar contribute to the diabetic complications lie outside of the scope of this article but can be found elsewhere. 8

and can react with proteins to form cross-links, as well as chromo/fluorophoric adducts called Maillard products (or Advanced Glycation Endproducts (AGE)) 12 which result in the protein becoming "browned," fluorescent, and cross-linked in vitro 13 (Fig. 1). Evidence that at least the early stage of this reaction occurs in vivo was obtained through the study of minor hemoglobins which were elevated in diabetes. ~4 Later, borohydride reduction of hemoglobin permitted stabilization of the Amadori product and its identification and quantitation using amino acid analysis. 15,16 The extent of hemoglobin glycation is now used as a cumulative index of glycemia over the previous few weeks in the clinical management of diabetes. 17 DIABETES AND "ACCELERATED AGEING"

GLYCATION

Glucose can slowly condense nonenzymatically with protein amino groups forming, initially, a Schiff base which may rearrange to form the Amadori product (Fig. 1). This early stage of the reaction is called nonenzymatic glycosylation, or, more properly, "glycation". 9 The Amadori product subsequently degrades into alphaketoaldehyde compounds such as 1- and 3-deoxyglucosones (Fig. 1). These secondary compounds are more protein-reactive than the parent monosaccharide l°'H

In diabetes, arteries and joints are prematurely stiff 18'19 and elasticity as well as vital capacity of the lungs are prematurely decreased. 2° Collagen-associated fluorescence (370nm excitation; 440nm emission) increases with age and is increased in diabetic subjects, 2~ more so in those with complicationsY These changes have been postulated to result from the in vivo reaction of glucose with collagen since collagen, when incubated with glucose in vitro becomes browned, fluorescent, and cross-linked as well as altered with respect to tensile

Oxidation and glycation in diabetes

341

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Fig. 2. Monosaccharide Autoxidation. Note. From "Glucose Autoxidation and Protein Oxidation: The Role of Autoxidative Glycosylation in Diabetes Mellitus and Ageing" by S.P. Wolff and R.T. Dean, 1987, BiochemicalJournal, 245, pp. 243-250. Copyright 1987 by The Biochemical Society and Portland Press, Reprinted by permission.

properties. 23 Increases in plasma glucose concentration with age are also suggested to contribute to an increase in the glycation of long-lived proteins such as collagens and lens crystallins. 24 Many proteins are modified adversely when incubated with glucose in vitro. Albumin, for example, undergoes conformational alterations and shows diminished ligandbinding capacity.25 Similarly, superoxide dismutase (SOD)

loses activity when exposed to glucose in vitro; higher levels of glycated erythrocyte SOD are found in aged erythrocytes and in diabetes. 26'27 Lens crystallins are glycated in vivo and aggregate and undergo thiol oxidation when incubated with glucose in vitro. 28 This has been used as an explanation for the increased cataract risk in diabetes. 29 Similarly, when low density lipoprotein (LDL) is incubated with glucose in vitro it becomes

342

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Fig. 3. Glucose-MediatedTryptophanFluorescence Quenching and FluorophoreDevelopment.Bovine serum albumin was incubated for 3 weeks in the presenceof 25 mM glucoseat 37°C in 25mM potassiumphosphate buffer under sterile conditions and in the presenceof chelating agents (1 mm) or sodium cyanoborohydride(25 mM). A: Tryptophanfluorescence(280 nm, excitation), B: Novel fluorescence(350 nm, excitation). Note. From "Glucose Autoxidation and Protein Oxidation: The Role of Autoxidative Glycosylationin Diabetes Mellitus and Ageing" by S.P. Wolff and R.T. Dean, 1987, Biochemical Journal, 245, pp. 243-250. Copyright 1987 by The Biochemical Society and Portland Press. Reprinted by permission. poorly recognized by fibroblasts and preferentially accumulated by macrophages. 3°'31'32 This has been suggested to contribute to plasma LDL accumulation and atherosclerosis in diabetes. In essence, the conformational alterations and loss of recognition caused by protein exposure to glucose are related to cancellation of positive charges on the protein, to blocking of critical amino groups, loss of hydrogen bonding capacity, loss of cellular recognition and the formation of complex products capable of inter-/intra-molecular cross-linking. One of the postulated crosslinking and fluorescent AGE found in hydrolysates of in vitro glycated protein has been identified as 2-furoyl4[5]-[2-furanyl]-l-H-imidazole (FFI). 33 This compound has been suggested to be relevant to protein cross-linking and fluorescence in vivo since a specific macrophage receptor (distinct from the "scavenger" receptor) has been identified which recognizes proteins which have been exposed to glucose in vitro or to which this product is attached. 34 THE ROLE OF "AUTOXIDATIVEGLYCOSYLATION" Such evidence falls short, however, of establishing glycation as a causative factor in the complications of

diabetes mellitus. First, the reactions which occur when protein is exposed to glucose in vitro are considerably more complex than the simple addition of glucose to protein amino groups. Glucose, like other alphahydroxyaldehydes, can enolize and thereby reduce molecular oxygen under physiological conditions, catalyzed by transition metals, yielding alpha-ketoaldehydes and oxidizing intermediates 35'36 (Fig. 2). Evidence suggests that free radicals and hydrogen peroxide slowly produced by glucose "autoxidation" are a substantial cause of the structural damage which results when protein is exposed to glucose in vitro. For example, the conformational alterations which occur when bovine serum albumin (BSA) is exposed to glucose under physiological conditions in vitro are inhibited by the metal chelating agent diethylenetriaminepenta-acetic acid (DETAPAC) or ethylenediaminetetra-acetic acid (EDTA) (Fig. 3A). Chelating agents also inhibit protein browning induced by glucose (Fig. 3B). In contrast, if glycation is performed in the presence of the reducing agent sodium cyanoborohydride (NaCNBH3), which provides a reducing environment but also greatly increases the rate of glucose attachment by trapping the Schiff base, then virtually no fluorophore development is observed (Figs. 3B and 4). These obser-

343

Oxidation and glycation in diabetes

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Fig. 4. Effect of NaCNBH3, DETAPAC, and Glucose Irradiation on Glycation. Bovine serum albumin was incubated at a concentration of 10 mgs/mL with glucose (25 mM but containing 2.5 v.Ci/mL [y-lac] glucose) in 100 mM potassium phosphate buffer, pH 7.4 at 37°C under sterile conditions. At various time intervals, aliquots were withdrawn and precipitated with 5% trichloracetic acid. The pellet was extensively washed and dissolved in formic acid prior to scintillation counting. Concentration of NaCNBH3 was 25 mM; DETAPAC, 500 txM; Cu2+, 10 I~M; ketoaldehyde produced by glucose irradiation, 50 ixM. Inset: Mechanism of Trapping of the Schiff Base by NaCNBH3. Note. From "Glucose Autoxidation and Protein Oxidation: The Role of Autoxidative Glycosylation in Diabetes Mellitus and Ageing" by S.P. Wolff and R.T. Dean, 1987, Biochemical Journal, 245, pp. 243-250. Copyright 1987 by The Biochemical Society and Portland Press. Reprinted by permission.

vations confirm that oxidative reactions are critical for the production of glucose-induced protein alterations and distinguish protein alterations caused by the exposure of protein to glucose from the covalent attachment of monosaccharide to protein per se. DETAPAC also decreases the extent of attachment of radiolabeled monosaccharide to the protein, whereas prior irradiation of the

glucose (which generates ketoaldehydes) increases the rate of monosaccharide attachment (Fig. 4). This is consistent with the view that alpha-ketoaldehyde products of glucose autoxidation contribute to the covalent attachment of monosaccharide to protein, at least in vitro. 37 The rate of glucose autoxidation is slow, but the amounts of alpha-ketoaldehydes and oxidizing agents

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UNITS OF CATALASE Fig. 5. Role of H202 and Hydroxyl Radicals in Protein Fragmentation by Glucose. A: '4C-radiomethylated BSA (1 mg/mL) or benzoate (1 mM) was incubated with 25 mM glucose alone, or in the presence of 100 ixM Cu2+, 1 mM DETAPAC or 250 mM sorbitol together with 100 ixM Cu2÷ in 100 mM potassium phosphate buffer, pH 7.4, for 8 days at 37°C. SDS PAGE of albumin (1 mg/mL) exposed to glucose under analogous conditions. Track A, control-incubated albumin; B, Cu2÷ (100 I~M), glucose (25 mM), albumin; c, glucose, albumin; D, glucose, DETAPAC (1 mM), albumin. B: Radiomethylated albumin (1 mg/mL) was incubated with 25 mM glucose and 100 ixM in the presence of catalase over 3 days. Denatured catalase was prepared by incubation of the protein at 100°C for 15 min. Note. From "Hydroxyl Radical Production and Autoxidative Glycosylation: Glucose Autoxidation as the Cause of Protein Damage in the Experimental Glycation Model of Diabetes Mellitus and Ageing" by J.V. Hunt, R.T. Dean, and S.P. Wolff, 1988, Biochemical Journal, 256, pp. 205-212. Copyright 1988 by The Biochemical Society and Portland Press. Reprinted by permission.

formed over the typical time courses of in vitro glycation studies (days to weeks) are in the range consistent with protein damage and modification by this process. In a further study, 38 it was shown that glucose generates hydroxyl radicals (or some species with similar oxidizing ability) and that these are the proximal cause of the conformational alteration and fragmenta-

tion which occur when protein is exposed to glucose in vitro. The "hydroxyl radical scavenger," sorbitol, inhibits glucose-mediated fragmentation and benzoate hydroxylation (Fig. 5A). Similarly, if benzoate or deoxyribose are included in a reaction mixture containing glucose and protein then protein fragmentation is inhibited, the benzoate is hydroxylated, and the deoxyribose is oxi-

Oxidation and glycation in diabetes

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INCUBATION TIME (hours) Fig. 6. The Production of H202 by Autoxidizing Glucose. Glucose (5 raM, 10 mM, 25 mM, and 100 mM) with or without the presence of 1 mg/mL bovine serum albumin was incubated in potassium phosphate buffer (pH 7.4, 10 raM) at 37°C. After incubation, 100 ~L samples were added to 900 ~L of reagent (100 txM xylenol orange, 250 I~M Fe2÷, and 100 mM sorbitol in 25 mM H2504). Absorbance was read at 560 nm after 40 min incubation at room temperature following a 2-rain centrifugation at 12 000g to remove any flocculated protein. Values (peroxide concentration in the sample) are the mean _ standard deviation obtained from duplicate assays. Native (but not heat-denatured)catalase (Sigma Type C-40) added to the sample (at a concentration of 100 units/mL) prior to addition of reagent abolished the signal. Note. From "Hydrogen Peroxide Production During Experimental Protein Glycation" by Z-Y. Jiang, A.C.S. Woollard, and S.P. Wolff, 1990, FEBS Letters, 268, pp. 69-71. Copyright 1990 by Elsevier Science Publishers. Reprinted by permission.

dized to malondialdehyde. 38 H20 2 is presumably the precursor of the proximal protein oxidant since catalase inhibits glucose-stimulated fragmentation (Fig. 5B). The fragmentation pattern induced by glucose is rather specific (Fig. 5A) perhaps because autoxidation occurs at copper bound to histidine residues (rather than in free solution), which would localize the protein oxidative damage which results. 39 In this respect, it is interesting that the albumin residues most likely to be glycated in vivo are located in L y s - L y s , L y s - H i s , L y s - L y s - L y s , and L y s - H i s - L y s sequences, 4° which might bind transition metals and catalyze local formation of reactive ketoaldehydes.41 While there is little doubt that free radicals and tran-

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sition metals are involved in alterations to protein exposed to glucose in vitro, the production o f H202 by glucose in the presence of protein has only been inferred (by the inhibitory effect of catalase on protein fragmentation), since the steady-state concentrations of H202 are too low for measurement by most methods. The accumulation of H202 with respect to time and concentration can, however, be monitored using a sensitive assay 42 based upon the amplified oxidation of Fe 2+ to Fe 3+ in the presence of xylenol orange (Fig. 6). In the presence of serum albumin (1 mg/mL) the steady-state levels of H20 2 detected were approximately 6-fold lower than in the absence of the protein. Albumin chelates copper ion 43 and thus may inhibit glucose " a u t o x i d a t i o n " via chela-

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S.P. WOLFFet al.

346

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Fig. 8. The Contribution of Glucose Autoxidation and HydroxylRadical Production to Glucose-InducedProtein Damage. The contribution of ketoaldehyde-amine autoxidation to site-specificdamage is suggested. Note. From "Hydroxyl Radical Production and Autoxidative Glycosylation: Glucose Autoxidation as the Cause of Protein Damagein the ExperimentalGlycationModel of DiabetesMellitus and Ageing" by J.V. Hunt, R.T. Dean, and S.P. Wolff, 1988, Biochemical Journal, 256, pp. 205-212. Copyright 1988 by The BiochemicalSocietyand Portland Press. Reprinted by permission.

tion of trace amounts of copper. Reaction of the openchain form of glucose with protein amino groups will also retard the formation of H20 2. Although the steadystate concentrations of H20 2 are low in absolute terms they are in the range expected to contribute to protein damage. At 25 mM glucose a steady-state level of 200 nM H20 2 is achieved (compared with a protein concentration of 15 IxM) and this is in the concentration range previously observed for production of hydroxylating agents. 38

AMADORI PRODUCT AUTOXIDATION Work by Baynes and colleagues has shown that the Amadori adduct itself is able to oxidize, similarly catalyzed by transition metals, leading to the release of erythronic acid and the formation of carboxymethylated lysine residues 44 (Fig. 7), Using the model Amadori compound N-formylfructosyllysine (FFL) they found that its transition metal-catalyzed oxidation to carboxymethylysine (CML) is a pathway antagonistic to FFL rearrangement to brown chromophores. If FFL is

incubated under N 2, the solution browns rapidly; in contrast, incubation under 0 2 decreases FFL browning. CML has been observed in a cataractous lens hydrolysate (at levels in excess of the Amadori product) indicating that Amadori product oxidation occurs in vivo. 44 Copper accumulates in the lens with age, and more so in cataract. 45 The presence of CML would suggest that the metal is present in a form which can catalyze oxidations. Alpha-ketoaldehyde-protein adducts also autoxidize (from their ketoaminomethylol intermediates by a mechanism analogus to simple monosaccharide autoxidation46'47), and this process may also contribute to protein oxidation in vitro (Fig. 8). GLYCATION OR OXIDATION? The changes experienced by protein when exposed to glucose in vitro certainly result from chemistry other than the simple addition of glucose to amino groups. Indeed, glycation and oxidation appear to be inextricably linked. 48 Low density lipoprotein (LDL) and phosphatidylcholine liposomes, for example, have been shown to be massively peroxidized when exposed to

Oxidation and glycation in diabetes

347

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Fig. 9. Glucose-Induced LDL And Liposome Peroxidation. All incubations contained 100 mM potassium phosphate, pH 7.4 and glucose (200 raM), DETAPAC (50 IxM) and/or copper sulphate (20 p,M) where appropriate. Liposomes were incubated at a final concentration of 10 mgs/mL in a shaking water bath at 37°C. At defined time intervals, 100 IxL of the incubation suspension were removed and dissolved by the addition of 900 IxL of methanol. LDL was incubated at a concentration of 1 mg/mL in the same buffer at 37°C over an 8-day period. At the end of this time, the LDL suspension was treated in the same manner as the liposomes, except that the methanolic extract was subject to centrifugation at 12 000g for 2 rain in order to precipitate protein. Malondialdehyde, and similar stable aldehydic products of lipid peroxidation, formed in LDL or liposomes were determined using thiobarbituric acid (measuring "TBA-reactive material") in material soluble in 5% trichloroaeetic acid. 200 p,L aliquots of the trichloroacetic acid extracts were incubated with 800 p~L of 0.67% thiobarbituric acid at 100°C for 10 min. After cooling, the absorbance of the mixture was read at 532 nm. Lipid hydroperoxides present in the methanolic extracts were determined by their oxidation of Fe n to Fe in ill 25 m_M H2SO4, 90% methanol, 4mM butylated hydroxytoluene in the presence of xylenol orange using butyl peroxide as standard. Prior to the determination of lipid peroxides, hydrogen peroxide generated during autoxidative reactions was removed by the preincubation of 1 mL samples with 100 I.U. cataiase for 30 min. Values are the mean + / - SD. Note. From "Autoxidative Glycosylation and Its Possible Implications for Glycation Theory: Are Peroxides and Free Radicals Involved in LDL Modification by Glucose" by J.V. Hunt, C.C.T. Smith, and S.P. Wolff, 1990, Diabetes, 39, pp. 1420-1424. Copyright 1990 by The American Diabetes Association, Inc. Reprinted by permission.

348

S.P. WOLFFet

glucose in vitro, both in terms of the formation of thiobarbituric acid-reactive material as well as the formation of authentic peroxide: these changes are inhibited by the addition of DETAPAC but accelerated by Cu I! ions49 (Fig. 9). This phenomenon of LDL oxidation during exposure to glucose in vitro makes it difficult to ascribe concomitant behavioral changes to the addition of glucose groups to the protein per se, since LDL peroxidation induces similar changes and has also been implicated in atherogenesis. 5°'51 But we must acknowledge a substantial qualitative difference between glycation in vivo and in vitro. In vitro free radicals, hydrogen peroxide, and alphaketoaldehydes produced by glucose autoxidation appear to be primary mediators of protein modification and peroxidation of protein-associated lipid under physiological conditions of pH, temperature, and glucose concentration. In vivo, however, "autoxidative glycosylation" may play only a minor role and the classical route of glucose attachment, via the Amadori product, may be considerably more important. This current uncertainty, and lack of appreciation of the oxidative pathway makes it difficult to extrapolate from observations made on in vitro glycated proteins to the situation in vivo.

THE RELEVANCE

OF GLYCATIONIN VIVO?

Another question is still more pertinent: Is glycation important, by whatever route? The extent of glycation, although increased in diabetes, is small in absolute terms. In hemoglobin, for example, only about 1% of the total amino groups are glycated at the end of the 120-day lifespan of a normal red blood cell: this increases to about 2.5% in diabetes. 52 Similarly, levels of lysine glycation in plasma proteins are typically considerably less than 1%. 53 This may be substantially higher in longer-lived proteins such as collagen, but the concentration of Amadori products on collagen (in contrast to collagen-associated fluorescence) does not correlate with the presence of complications in individual diabetics. 54 Evidence suggests that such small levels of glycation may not be of pathophysiological significance in the absence of other factors. For example, although glycated hemoglobin shows altered oxygen binding, 55 there is no alteration of whole blood oxygen saturation curves in diabetes. 56 Similarly, while the exposure of albumin to glucose in vitro alters its conformation and drug-binding properties,57 there is no evidence to suggest that the level of albumin glycation affects drug pharmacokinetics in diabetes. 58 Glycation is also unlikely to influence the recognition of transferrin, fibrinogen, alpha-2-macroglobulin or immunoglobulins in vivo since the levels of glycation are lower than those seen to cause effects in vitro. 59"6° Even if we leave aside considerations of

al.

oxidative damage which occur to LDL when exposed to glucose in vitro, there are still problems relating glycation to atherosclerosis. Extensive glycation of LDL, particularly under the accelerated reducing conditions imposed by NaCNBH 3, certainly affects its catabolism and recognition. 61'62"63However, it appears unlikely that glycation of LDL to the extent observed in diabetes retards LDL catabolism 64 or stimulates macrophage cholesterol esterification,65 as has been proposed. 66 Similarly, levels of total erythrocyte SOD activity are the same in normal and diabetic subjects although SOD is inhibited by exposure to glucose in vitro and 32% of erythrocyte SOD appears to be glycated in diabetic subjects (compared with 20% in healthy controls). 26'27 The inhibitory effect of glucose on SOD in vitro could be related to the ability of H202 to inhibit this enzyme at low concentrations whereas in vivo, of course, H202 would be catabolized efficiently. There are also difficulties with the view that diabetic tissue damage is a form of accelerated ageing caused by hyperglycemia, and that glycation is the cause of ageing itself as is often proposed. First, average glycemia does not correlate with species longevity; average glycemia is very similar in mammals and can reach very high levels in birds which live as long as man. 67 Second, although age is a risk factor for many cancers, there is no evidence for any increased risk of cancer in diabetes, although t-cell function is frequently impaired. 68 Third, the Amadori adduct is reversible to the extent that, even taking some of the later reactions of the Amadori adduct into account, the reverse reaction is likely to have an important role in limiting the extent of glycation of structural proteins in vivo. Consistent with this it has been shown that the extent of glycation (Amadori product accumulation) of human lens protein remains essentially constant between the ages of 5 and 80. 69 Other data on rats show little change in crystallin glycation with age although lens browning is increased. 69'7° Neither is there evidence for an age-related increase in glycation of human plasma proteins 7z nor collagens. 72 The extent of glycation appears to reach a steady-state early in life. One could argue that the addition of glucose to proteins certainly occurs, but may well, in itself, be benign. RELEVANCE OF THE MAILLARD PATHWAY?

In response to these specific criticisms, it has been suggested that Maillard (AGE) products, not the Amadori adduct, are primarily responsible for tissue damage in diabetes and that formation and breakdown of AGE might be subject to control by unknown genetic or environmental factors. This would, in principle, account for individual variation in susceptibility to the complications. Current evidence suggests that Maillard products

Oxidation and glycationin diabetes result largely from the reaction of alpha-ketoaldehyde products with protein, formed as a result of Amadori product rearrangements, or transition metal-catalyzed glucose oxidation. 73'74 A great number of different products are formed when glucose is incubated with protein, but most are formed in only small yields and are subject to hydrolysis and other degradations during isolation 75 which can lead to methodological artefacts. FFI, the recently identified AGE, for example, is apparently not formed as a result of rearrangements of the Amadori adduct but is produced in vitro by the condensation between furosine (produced when glycated protein is hydrolyzed to amino acids) and ammonia when ammonium hydroxide is used to neutralize the hydrolysates. 76 This raises the interesting question of the products which the recently identified FFI receptor in the macrophage is able to recognize77 as well as the specificity of the antisera raised to FFI. 78 Presumably, there is extensive cross-reactivity of various AGE to this receptor and antibodies since there has been a recent report of an immunologically detectable pyrrole carboxyaldehyde in albumin from diabetic individuals. 79 The role of the Maillard products as a causative factor in the diabetic complications is certainly not clear. A study of collagen in streptozotocin-induced experimental diabetes showed that although collagen-associated fluorescence increased with age in diabetic and control animals, there was little difference at any age between the two groups; the fluorescence appeared not to be linked to collagen cross-linking.8° Maillard products may thus play only an indirect role in age/diabetes collagen alterations, at least in animal models of diabetes. Furthermore, although there is a correlation between the extent of skin collagen fluorescence and some complications in patients, this correlation is not strong (r < 0.5); some patients have high skin fluorescence but no diabetic complications, and vice versa. 28 Tissue fluorescence is a poor indicator of underlying tissue damage, and appears not to have a direct causative role in tissue degeneration. Thus, although diabetes is a risk factor for cataract, there is no difference in the levels of fluorescent compounds attached to lens crystallin extracted from the cataracts of diabetic and nondiabetic patients, although the level of Amadori products are expectedly higher in the former group. 8~ This observation suggests that it may be incorrect to assume that diabetic subjects with high fluorescence values have been exposed to a higher cumulative glycemia than those with lower values. It may also be invalid to assume that in vivo fluorescence is produced by glycation. ORIGINS OF TISSUEFLUORESCENCE Fructose, which is formed by the polyol pathway, has also been recently implicated as an in vivo collagen-

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browning agent s2 and many other small oxidizable molecules such as vitamin C and unsaturated fatty acids are efficient browning and cross-linking agents in vitro, when their oxidation is permitted. 83,84,85,86 Fluorescence and chromophore development in collagen, lens crystallin, and albumin is much more extensive with ascorbate and arachidonic acid than with glucose, and in these cases is similarly inhibited by DETAPAC. 87 In vitro, at least, browning reactions are dependent upon the presence of trace amounts of transition metals which catalyze the oxidative reactions required. OXIDATIVE STRESS AND TRANSITIONMETAL AVAILABILITY Whether glucose actually plays any substantial role in tissue fluorescence alterations or whether "decompartmentalized" transition metal is available in vivo for catalysis of the oxidative reactions which would appear to be required for fluorescence generation, from whatever source, are new and speculative questions. It is conceivable that iron-catalyzed oxidations could occur in some diabetic individuals, and may contribute to browning reactions, since diabetes is found commonly in transfusion siderosis, dietary iron overload, and idiopathic hemochromatosis. 8s'89 A possible link between transition metal overload and the complications is further suggested by the observation that desferrioxamine treatment decreases hyperglycemia and lowers hypercholesterolemia and hyperlipidemia in patients with high ferritin but lacking hemochromatosis. 9° Hyperglycemia, as well as increases in biochemical risk factors for atherosclerosis thus appear to be secondary to iron overload in this diabetic subgroup. Copper may also play a role since total Cun levels are higher in diabetic individuals than in normals, and are highest in diabetics with angiopathy and/or alterations in lipid metabolism. 91'92 It is by no means clear whether this copper increase is caused by an increase in ceruloplasmin (which might reflect abnormalities in iron metabolism) or represents, in part at least, an increase in the pool of copper associated with albumin or low molecular weight chelates. Patients with classical iron overload often possess low levels of serum and white blood cell vitamin C. 93'94 In diabetes, levels of plasma and white blood cell ascorbic acid are lower (despite similar levels of intake and excretion), and oxidation of this antioxidant to dehydroascorbate is higher than in normal individuals. 95'96 Oxidative stress, perhaps initiated by transition metal, may contribute to the pathogenesis of diabetes and its complications. 97'9s Oxidative stress has the potential for causing gross oxidative damage to biological structures, but may exert more subtle effects. 99 For example, enzymes involved in essential unsaturated fatty acid (EFA) metabolism, such as thromboxane and prostacyclin synthetases, are

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sensitive to the composition and level of ambient peroxides, the "peroxide tone". 1°°'~°1 Small alterations in peroxide tone may contribute to the abnormalities in EFA metabolism implicated in angiopathy, to2,1o3 The transition metal-catalyzed oxidation of low-molecular weight reductants (such as glucose, ascorbate, and polyunsaturated fatty acids) would contribute to oxidative stress via the production of a steady flux of hydrogen peroxide and/or lipid peroxides in vivo. Aldehydic products of such oxidations might accumulate on long-lived proteins as an index of the rate of such oxidation. CONCLUSION

Amadori product accumulation, AGE accumulation, and increases in tissue fluorescence do not, in themselves, adequately explain the pathogenesis of the diabetic complications, nor their individual variability. There is, however, underlying, if indirect, evidence for a systemic oxidative stress in diabetic pathogenesis. Transition metal overload, with a concomitant increased oxidation rate of reducing agents and/or lipid peroxidation, appears to be an attractive candidate for this stress. Acknowledgements -- We thank the Medical Research Council, Sir

Jules Thorn Trust, Research into Ageing, The Juvenile Diabetes Foundation and ONO Pharmaceuticals for providing financial support.

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