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Chemical modification of proteins by advanced glycation and lipoxidation end products is implicated in the pathogenesis of macrovascular disease in aging and diabetes. To identify biomarkers of the lipoxidative modification of protein, we studied the oxidation of phospholipids in the presence of the model protein RNase A and compared protein-bound products formed in these reactions with those formed during oxidation of plasma proteins. Metal-catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylcholine or 1-palmitoyl-2-linoleoyl-phosphatidylcholine in the presence of RNase led to the loss of amino groups in RNase and the incorporation of phosphate, hexanoate, pentanedioate, nonanedioate, and palmitate into protein. Protein-bound palmitate and phosphate correlated strongly with one another, and protein-bound pentanedioate and nonanedioate, derived from arachidonate and linoleate, respectively, accounted for approximately 20% of the cross-linking of lipid phosphorus to protein. Similar results were obtained on oxidation of total plasma or isolated LDL. We conclude that alkanedioic acids are quantitatively important linkers of oxidized phospholipids to proteins and that measurement of protein-bound phosphate and long-chain fatty acids may be useful for assessing long-term lipid peroxidative damage to proteins in vivo. Analyses of plasma proteins from control and diabetic patients indicated significant increases in lipoxidative modification of protein in diabetic compared with control subjects.

Aims/hypothesis. The accumulation of AGE is thought to play a role in the pathogenesis of chronic complications of diabetes mellitus and renal failure. All current measurements of AGE accumulation require invasive sampling. We exploited the fact that several AGE exhibit autofluorescence to develop a non-invasive tool for measuring skin AGE accumulation, the Autofluorescence Reader (AFR). We validated its use by comparing the values obtained using the AFR with the AGE content measured in extracts from skin biopsies of diabetic and control subjects.Methods. Using the AFR with an excitation light source of 300-420 nm, fluorescence of the skin was measured at the arm and lower leg in 46 patients with diabetes (Type 1 and 2) and in 46 age- and sex-matched control subjects, the majority of whom were Caucasian. Autofluorescence was defined as the average fluorescence per nm over the entire emission spectrum (420-600 nm) as ratio of the average fluorescence per nm over the 300-420-nm range. Skin biopsies were obtained from the same site of the arm, and analysed for collagen-linked fluorescence (CLF) and specific AGE: pentosidine, N-epsilon-(carboxymethyl)lysine (CML) and N-epsilon-(carboxyethyl)lysine (CEL).Results. Autofluorescence correlated with CLF, pentosidine, CML, and CEL (r=0.47-0.62, pless than or equal to0.002). In 32 of 46 diabetic patients (70%), autofluorescence values were above the 95% CI of the mean value in control subjects, and correlated with age, diabetes duration, mean HbA(1)c of the previous year and creatinine levels.Conclusions/interpretation. The AFR offers a simple alternative to invasive measurement of AGE accumulation and, to date, has been validated in non-pigmented skin. The AFR may prove to be a useful clinical tool for rapid risk assessment of AGE-related long-term complications in diabetes mellitus and in other conditions associated with AGE accumulation.

Levels of glycation (fructose-lysine, FL) and advanced glycoxidation and lipoxidation end-products (AGE/ALEs) were measured in total skeletal (gastrocnemius) muscle and myofibril protein and compared to levels of the same compounds in insoluble skin collagen of control and diabetic rats. Levels of FL in total muscle and myofibril protein were 3-5% the level of FL in skin collagen. The AGE/ALEs, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine, were also significantly lower in total muscle and myofibril protein, approximately 25% of levels in skin collagen. The newly described sulfhydryl AGE/ALE, S-(carboxymethyl)cysteine (CMC), was also measured in muscle; levels of CMC were comparable to those of CML and increased similarly in response to diabetes. Although FL and AGE/ALEs increased in muscle protein in diabetes, the relative increase was less than that seen in skin collagen. These data indicate that muscle protein is partially protected against the increase in both glycation and AGE/ALE formation in diabetes.

Hyperglycaemia is the major risk factor for the development of complications in both Type I and Type II diabetes; however, there is growing evidence from several clinical trials that dyslipidaemia, including hypertriglyceridaemia, is a significant and independent risk factor for diabetic complications. In this paper, we propose that chemical modification of proteins by lipids may be a underlying pathogenic mechanism linking dyslipidaemia to diabetic complications. Thus the major AGEs (advanced glycation end-products) in tissues, such as carboxymethyl-lysine, carboxyethyl-lysine and hydroimidazolones, may, in fact, be ALEs (advanced lipoxidation end-products), derived from lipids. Increased lipid peroxidation and accelerated ALE formation, possibly catalysed by hyperglycaemia and oxidative stress, may be the mechanistic link between dyslipidaemia and diabetic complications. If correct, this proposal would suggest that inhibition or reversal of glycation, which is a central theme of this symposium, may not be sufficient for protection against diabetic complications.

Pyridoxamine (PM), originally described as a post-Amadori inhibitor of formation of advanced glycation end-products (AGEs), also inhibits the formation of advanced lipoxidation end-products (ALEs) on protein during lipid peroxidation reactions. In addition to inhibition of AGE/ALE formation, PM has a strong lipid-lowering effect in streptozotocin (STZ)-induced diabetic and Zucker obese rats, and protects against the development of nephropathy in both animal models. PM also inhibits the development of retinopathy and neuropathy in the STZ-diabetic rat. Several products of reaction of PM with intermediates in lipid autoxidation have been identified in model reactions in vitro and in the urine of diabetic and obese rats, confirming the action of PM as an AGE/ ALE inhibitor. PM appears to act by a mechanism analogous to that of AGE-breakers, by reaction with dicarbonyl intermediates in AGE/ALE formation. This review summarizes current knowledge on the mechanism of formation of AGE/ALEs, proposes a mechanism of action of PM, and summarizes the results of animal model studies on the use of PM for inhibiting AGE/ALE formation and development of complications of diabetes and hyperlipidemia.

Maillard or browning reactions between reducing sugars and protein lead to formation of advanced glycation end products (AGEs) and are thought to contribute to the pathogenesis of diabetic complications. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) inhibit both the formation of AGEs and development of complications in animal models of diabetes. PM also inhibits the chemical modification of protein by advanced lipoxidation end products (ALEs) during lipid peroxidation reactions in vitro. We show here that several PM adducts, formed in incubations of PM with linoleate and arachidonate in vitro, are also excreted in the urine of PM-treated animals. The PM adducts N-nonanedioyl-PM (derived from linoleate), N-pentanedioyl-PM, N-pyrrolo-PM, and N-(2-formyl)-pyrrolo-PM (derived from arachidonate), and N-formyl-PM and N-hexanoyl-PM (derived from both fatty acids) were quantified by liquid chromatography-mass spectrometry analysis of rat urine. Levels of these adducts were increased 5-10-fold in the urine of PM-treated diabetic and hyperlipidemic rats, compared with control animals. We conclude that the PM functions, at least in part, by trapping intermediates in AGE/ALE formation and propose a mechanism for PM inhibition of AGE/ALE formation involving cleavage of alpha-dicarbonyl intermediates in glycoxidation and lipoxidation reactions. We also conclude that ALEs derived from polyunsaturated fatty acids are increased in diabetes and hyperlipidemia and may contribute to development of long term renal and vascular pathology in these diseases.

The AGE inhibitor pyridoxamine inhibits lipemia and development of vascular and renal disease in Zucker obese rats. Background In previous studies, pyridoxamine (PM) limited the formation of advanced glycation end products (AGEs) and development of nephropathy in streptozotocin-diabetic rats without affecting glycemic control. However, the lipid-lowering effects of PM and the correlation of plasma cholesterol and triglycerides with AGEs in skin collagen suggested that lipids might be an important source of AGEs in the diabetic rat. This study addresses the effects of hyperlipidemia on formation of advanced glycation and lipoxidation end products (AGE/ALEs) and the effects of PM on hyperlipidemia, hypertension, AGE/ALE formation, and development of nephropathy in the nondiabetic, Zucker obese rat. Methods Three groups of Zucker rats were studied: lean (Fa/ fa ), untreated fatty ( fa/fa ), and fa/fa treated with PM (2 g/L drinking water). Blood pressure, plasma lipids and creatinine, and urinary albumin were measured monthly. AGE/ALEs were measured in skin collagen by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Changes in wall thickness of the aorta and renal arterioles were evaluated by light microscopy. Results AGE/ALEs formation was increased two- to threefold in skin collagen of obese versus lean rats. PM inhibited the increases in AGE/ALEs in collagen, and significantly decreased the rise in plasma triglycerides, cholesterol, and creatinine, corrected hypertension and thickening of the vascular wall, and nearly normalized urinary protein and albumin excretion in Zucker fa/fa rats. Conclusion Lipids are an important source of chemical modification of tissue proteins, even in the absence of hyperglycemia. PM inhibited AGE/ALE formation and hyperlipidemia and protected against renal and vascular pathology in a nondiabetic model.

Advanced glycosylation end-products (AGEs) are believed to play a significant role in the development of vascular complications in diabetic patients. One such product, AGE-LDL, has been shown to be immunogenic. In this report, we describe the isolation and characterization of human AGE-LDL antibodies from the sera of seven patients with Type 1 diabetes by affinity chromatography using an immobilized AGE-LDL preparation that contained primarily the AGE N epsilon (carboxymethyl)lysine (CML, 14.6 mmol/mol lysine), and smaller amounts of N epsilon (carboxyethyl)lysine (CEL, 2.7 mmol/mol lysine). The isolated antibodies were predominantly IgG of subclasses 1 and 3, and considered proinflammatory because of their ability to promote Fc gamma R-mediated phagocytosis and to activate complement. We determined dissociation constants (Kd) for the purified antibodies. The average Kd values (4.76 +/- 2.52 x 10(-9) mol/l) indicated that AGE-LDL antibodies are of higher avidity than oxidized LDL antibodies measured previously (Kd = 1.53 +/- 07 x 10(-8) ml/l), but of lower avidity than rabbit polyclonal LDL antibodies (Kd = 9.34 x 10(-11)). Analysis of the apolipoprotein B-rich lipoproteins isolated with polyethylene glycol-precipitated antigen-antibody complexes from the same patients showed the presence of both CML and CEL, thus confirming that these two modifications are recognized by human autoantibodies. A comparative study of the reactivity of purified AGE-LDL antibodies with CML-LDL and CML-serum albumin showed no cross-reactivity.

The purpose of this study was to examine the effect of prolonged exercise on plasma lipid and lipoprotein concentrations and to identify caloric time-points where changes occurred. Eleven active male subjects ran on a treadmill at 70% of maximal fitness (VO2max) and expended 6278.7 kilojoules (Kj) energy (1500 kcal). Blood samples were obtained at the 4185.8 Kj (1000 kcal) timepoint during exercise and at each additional 418.6 Kj (100 kcal) expenditure until 6278.7 Kj was expended. After correcting for plasma volume changes, decreases in low-density lipoprotein cholesterol (LDL-C) were observed during exercise at time-points corresponding to 4604.4 and 5441.5 Kj (1100 and 1300 kcal) of energy expenditure, and immediately after exercise. Total cholesterol concentrations decreased significantly at exercise kilojoule expenditures of 4604.4, 5441.5 and 5860.1 (1100, 1300 and 1400 kcal). There were also exercise induced increases in high-density lipoprotein cholesterol (HDL-C) and HDL2-C concentrations immediately after exercise. Although acute lipid and lipoprotein changes are typically reported in the days following exercise, the current data indicate that some lipoprotein concentrations change during acute exercise. Our data suggest that a threshold of exercise may be necessary to change lipoproteins during exercise. Future work should identify potential mechanisms (lipoprotein lipase, cholesterol ester transport protein, LDL uptake) that alter lipoprotein concentrations during prolonged exercise.

We hypothesized that the beneficial effects of a variety of pharmacological agents on the progression of diabetic complications were mediated by a common pathway limiting the formation of advanced glycation and advanced lipoxidation end products (AGEs/ALEs) on protein. We studied the effects of the AGE/ALE inhibitor pyridoxamine (PM), the antioxidant vitamin E (VE) and the ACE inhibitor enalapril (EP) on the development of nephropathy and retinopathy in STZ-induced diabetic rats over 29 weeks. Blood glucose and glycohemoglobin were similar in all diabetic groups. Plasma lipids rose continuously in diabetic animals and only PM significantly attenuated this increase. Early nephropathy was indicated by increased plasma creatinine, and urinary albumin, protein and TGF-β excretion in untreated rats. While all interventions limited renal damage to some extent, PM was the most effective, although the increased expression of renal laminin β1 and fibronectin mRNA was normalized by all therapies. Measurement of retinal damage (acellular capillaries, vascular basement membrane-associated laminin) showed that only PM significantly limited signs of early retinopathy in diabetic rats. Only PM limited the increases in the AGE/ALEs in renal and retinal tissue, and in skin collagen, of diabetic rats. Our results suggest that limiting both dyslipidemia and AGE/ALE formation is required for maximum protection of renal and retinal function in the STZ-diabetic rat.

Purpose: Most studies that use either a single exercise session, exercise training, or a cross-sectional design have failed to find a relationship between exercise and plasma lipoprotein(a) [Lp(a)] concentrations. However, a few studies investigating the effects of longer and/or more strenuous exercise have shown elevated Lp(a) concentrations, possibly as an acute-phase reactant to muscle damage. Based on the assumption that greater muscle damage would occur with exercise of longer duration, the purpose of the present study was to determine whether exercise of longer duration would increase Lp(a) concentration and creatine kinase. (CK) activity more than exercise of shorter duration. Methods: Ten endurance-trained men (mean +/- SD: age, 27 +/- 6 yr; maximal oxygen consumption [(V)over dotO(2max)], 57 +/- 7 mL(.)kg(-1) min(-1)) completed two separate exercise sessions at 70% (V)over dotO(2max). One session required 900 kcal of energy expenditure (60 +/- 6 min), and the other required 1500 kcal (112 +/- 12 min). Fasted blood samples were taken immediately before (0-pre), immediately after (0-post), 1 d after (1-post), and 2 d after (2-post) each exercise session. Results: CK activity increased after both exercise sessions (mean +/- SE; 800 kcal: 0-pre 55 +/- 11, 1-post 168 +/- 64 U(.)L(-1.)min(-1); 1500 kcal: 0-pre 51 +/- 5, 1-post 187 +/- 30, 2-post 123 +/- 19 U(.)L(-1.)min(-1); P < 0.05). However, median Lp(a) concentrations were not altered by either exercise session (800 kcal: 0-pre 5.0 mg(.)dL(-1), 0-post 3.2 mg(.)dL(-1), 1-post 4.0 mg(.)dL(-1), 2-post 3.4 mg(.)dL(-1); 1500 kcal: 0-pre 5.8 mg(.)dL(-1), 0-post 4.3 mg(.)dL(-1), 1-post 3.2 mg(.)dL(-1), 2-post 5.3 mg(.)dL(-1)). In addition, no relationship existed between exercise-induced changes in CK activity and Lp(a) concentration (800 kcal: r = -0.26; 1500 kcal: r = -0.02). Conclusion: These results suggest that plasma Lp(a) concentration will not increase in response to minor exercise-induced muscle damage in endurance-trained runners.

Fatigue from voluntary muscular effort is a complex phenomenon involving the central nervous system (CNS) and muscle. An understanding of the mechanisms within muscle that cause fatigue has led to the development of nutritional strategies to enhance performance. Until recently, little was known about CNS mechanisms of fatigue, even though the inability or unwillingness to generate and maintain central activation of muscle is the most likely explanation of fatigue for most people during normal daily activities. A possible role of nutrition in central fatigue is receiving more attention with the development of theories that provide a clue to its biological mechanisms. The focus is on the neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] because of its role in depression, sensory perception, sleepiness, and mood. Nutritional strategies have been designed to alter the metabolism of brain 5-HT by affecting the availability of its amino acid precursor. Increases in brain 5-HT concentration and overall activity have been associated with increased physical and perhaps mental fatigue during endurance exercise. Carbohydrate (CHO) or branched-chain amino acid (BCAA) feedings may attenuate increases in 5-HT and improve performance. However, it is difficult to distinguish between the effects of CHO on the brain and those on the muscles themselves, and most studies involving BCAA show no performance benefits. It appears that important relations exist between brain 5-HT and central fatigue. Good theoretical rationale and data exist to support a beneficial role of CHO and BCAA on brain 5-HT and central fatigue, but the strength of evidence is presently weak.

Recent studies have concluded that a single exercise session has no immediate effect on the plasma concentration of leptin, a putative satiety factor. We tested the hypothesis that an increase in energy expenditure would decrease the leptin concentration but the effects would be manifest in a 48-hour period following exercise. Eleven active males completed two treadmill exercise sessions with different energy expenditure (800 or 1,500 kcal) at 70% maximal O2 consumption (Vo2max). Subjects maintained constant energy intake on the day before, the day of, and 2 days after exercise, as verified by dietary recall. Compared with preexercise in either exercise session, there were no differences in plasma leptin concentrations following exercise (0 and 24 hours postexercise) except at 48 hours postexercise, where an approximately 30% decrease (P.05) was observed. With either duration of exercise, plasma glucose increased about 10% (P.05), insulin decreased 35% to 46% (P.05), and cortisol increased 41% to 50% (P.05, 1,500 kcal only) immediately following exercise, but returned to preexercise values at 24 and 48 hours postexercise. A statistically significant correlation was observed between the changes in leptin and insulin (r = .49, P.0001). Single exercise sessions of varying energy expenditure decreased the plasma leptin concentration after 48 hours in association with a preceding decrease in insulin.

The purpose of this study was to determine the threshold of exercise energy expenditure necessary to change blood lipid and lipoprotein concentrations and lipoprotein lipase activity (LPLA) in healthy, trained men. On different days, 11 men (age, 26.7 ± 6.1 yr; body fat, 11.0 ± 1.5%) completed four separate, randomly assigned, submaximal treadmill sessions at 70% maximal O2consumption. During each session 800, 1,100, 1,300, or 1,500 kcal were expended. Compared with immediately before exercise, high-density lipoprotein cholesterol (HDL-C) concentration was significantly elevated 24 h after exercise ( P < 0.05) in the 1,100-, 1,300-, and 1,500-kcal sessions. HDL-C concentration was also elevated ( P < 0.05) immediately after and 48 h after exercise in the 1,500-kcal session. Compared with values 24 h before exercise, LPLA was significantly greater ( P < 0.05) 24 h after exercise in the 1,100-, 1,300-, and 1,500-kcal sessions and remained elevated 48 h after exercise in the 1,500-kcal session. These data indicate that, in healthy, trained men, 1,100 kcal of energy expenditure are necessary to elicit increased HDL-C concentrations. These HDL-C changes coincided with increased LPLA.

Lipoprotein(a) (Lp(a)) is bound to apolipoprotein B-100 by disulfide linkage and is associated in the upper density range of low density lipoprotein cholesterol. Persons with elevated concentrations of Lp(a) are regarded as having an increased risk for premature coronary artery disease. Although many studies exist evaluating the effects of a single session of exercise on lipids and lipoproteins, little information is available concerning the effects of exercise on Lp(a). Therefore, the purpose of this study was to determine the effects of a single exercise session on plasma Lp(a). Twelve physically active men completed two 30-min submaximal treadmill exercise sessions: low intensity (LI, 50% VO2max) and high intensity (HI, 80% VO2max). Blood samples were obtained immediately before and after exercise. Total cholesterol (LI: before 4.22 +/- 0.26, after 4.24 +/- 0.28; HI: before 4.24 +/- 0.31, after 4.11 +/- 0.28 mmol.l-1, mean +/- SE) and triglyceride (LI: before 1.14 +/- 0.16, after 1.06 +/- 0.16; HI: before 1.12 +/- 0.19, after 1.21 +/- 0.19 mmol.l-1) concentrations did not differ immediately after either exercise session, nor did Lp(a) concentrations differ immediately after either exercise session (LI: before 4.1 +/- 2.2, after 4.0 +/- 2.1: HI: before 3.9 +/- 2.2, after 3.7 +/- 2.0 mg.dl-1). These results suggest that neither a low nor a high intensity exercise session lasting 30 min in duration has an immediate effect on plasma Lp(a).

S-(2-Succinyl)cysteine (2SC) has been identified as a chemical modification in plasma proteins, in the non-mercaptalbumin fraction of human plasma albumin, in human skin collagen, and in rat skeletal muscle proteins and urine. 2SC increases in human skin collagen with age and is increased in muscle protein of diabetic vs. control rats. The concentration of 2SC in skin collagen and muscle protein correlated strongly with that of the advanced glycation/lipoxidation end-product (AGE/ALE), N(epsilon)-(carboxymethyl)lysine (CML). 2SC is formed by a Michael addition reaction of cysteine sulfhydryl groups with fumarate at physiological pH. Fumarate, but not succinate, inactivates the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase in vitro, in concert with formation of 2SC. 2SC is the first example of spontaneous chemical modification of protein by a metabolic intermediate in the Krebs cycle. These observations identify fumarate as an endogenous electrophile and suggest a role for fumarate in regulation of metabolism.

Endothelin-1 (ET-1) is a potent vasoconstricting peptide released mostly from vascular endothelial cells. Isolated exercise sessions of relatively long duration (=or30 min) have produced increases in plasma ET-1 concentration while shorter exercise sessions usually have not. The purpose of the present study was to verify an effect of exercise duration at a steady work rate on plasma ET-1 concentration.Eleven endurance-trained males (age 27+/-6 years; maximal oxygen consumption--VO2max--56+/-7 mLxkg-1xmin-1, body fat 11+/-5%; mean+/-SD) exercised on a treadmill at 70% VO2max on 2 occasions separated by at least 2 weeks. During a short-duration session, subjects expended approximately 3,360 kJ (60+/-2 min). During a long-duration session, subjects expended approximately 6,300 kJ (112+/-4 min). Six of the subjects performed the 3,360 kJ session before the 6,300 kJ session while the other 5 subjects performed the 6,300 kJ session first.The short-duration session did not cause plasma ET-1 concentration to change immediately after exercise (0.23+/-0.01 pmolxL-1 before exercise, 0.22+/-0.02 pmolxL-1 after exercise, mean+/-SE). However, 10 of 11 subjects had increased ET-1 after the long-duration session (0.28+/-0.02 pmolxL-1 before exercise, 0.32+/-0.02 pmolxL-1 after exercise, P=0.0004). A treatment-by-time effect was present (P=0.003).These results demonstrate an effect of exercise duration on plasma ET-1 concentration. Exercise duration is, therefore, an essential consideration when investigating exercise's effect on ET-1.

The effects of valsartan on the accumulation of circulating and renal advanced glycation end products in experimental diabetes. Background Blockade of the RAS with the ACE inhibitor ramipril prevents the accumulation of advanced glycation end products (AGEs) in experimental diabetes. Although AT 1 receptor antagonists may inhibit AGE formation in vitro, their effect in normotensive animals with type 1 diabetes has not been established. Methods Streptozotocin-induced diabetic and control animals were randomized ( N =10/group) to receive the AT 1 antagonist valsartan at a dose of 30 mg/kg/day by oral gavage for 24 weeks, or no intervention. Renal and plasma AGE accumulation was correlated with renal functional parameters. Results Valsartan reduced the albumin excretion rate consistent with its renoprotective effects. Renal and skin collagen accumulation of the non-fluorescent AGE carboxymethyllysine (CML) were increased in animals with diabetes, but normalized following treatment with valsartan. Renal fluorescence and skin collagen pentosidine levels were also increased by diabetes. However, valsartan only provided a modest attenuation of these parameters. In addition, diabetes was associated with increased plasma fluorescence, which was unaffected by AT 1 antagonism. Conclusion Renoprotective doses of valsartan are associated with a significant reduction in the accumulation of tissue and plasma CML. These effects were not the same for all AGEs, suggesting combination approaches will be required to optimize renoprotection in diabetes.

It has been suggested that low-density lipoprotein (LDL) modified by glycation may be more susceptible to oxidation and thus, enhance its atherogenicity. Using affinity chromatography, LDL glycated in vivo (G-LDL) and relatively nonglycated. (N-LDL) subfractions can be isolated from the same individual. The extent of and susceptibility to oxidation of N-LDL compared with G-LDL was determined in 15 type 1 diabetic patients. Total LDL was isolated and separated by boronate affinity chromatography into relatively glycated (G-) and nonglycated (N-) subfractions. The extent of glycation, glycoxidation, and lipoxidation, lipid soluble antioxidant content, susceptibility to in vitro oxidation, and nuclear magnetic resonance (NMR)-determined particle size and subclass distribution were determined for each subfraction. Glycation, (fructose-lysine) was higher in G-LDL versus N-LDL, (0.28 +/- 0.08 v 0.13 +/- 0.04 mmol/mol lysine, P < .0001). However, levels of glycoxidation/lipoxidation products and of antioxidants were similar or lower in G-LDL compared with N-LDL and were inversely correlated with fructose-lysine (FL) concentrations in G-LDL, but positively correlated in N-LDL. In vitro LDL (CuCl2) oxidation demonstrated a longer lag time for oxidation of G-LDL than N-LDL (50 +/- 0.16 v 37 +/- 0.15 min, P < .01), but there was no difference in the rate or extent of lipid oxidation, nor in any aspect of protein oxidation. Mean LDL particle size and subclass distribution did not differ between G-LDL and N-LDL. Thus, G-LDL from well-controlled type 1 diabetic patients is not more modified by oxidation, more susceptible to oxidation, or smaller than relatively N-LDL, suggesting alternative factors may contribute to the atherogenicity of LDL from type 1 diabetic patients.

This study was designed to determine whether inhibition of formation of AGE and advanced lipoxidation end-products (ALE) is a mechanism of action common to a diverse group of therapeutic agents that limit the progress of diabetic nephropathy. We compared the effects of the ACE inhibitor enalapril, the antioxidant vitamin E, the thiol compound lipoic acid, and the AGE/ALE inhibitor pyridoxamine on the formation of AGE/ALE and protection against nephropathy in streptozotocin diabetic rats.Renal function and AGE/ALE formation were evaluated in rats treated with the agents listed above. Plasma was monitored monthly for triglycerides, cholesterol, creatinine and TNF-alpha, and 24-h urine samples were collected for measurement of albumin and total protein excretion. After 29 weeks, renal expression of mRNA for extracellular matrix proteins was measured, and AGE/ALE were quantified in skin and glomerular and tubular collagen.Diabetic animals were both hyperglycaemic and dyslipidaemic, and showed evidence of early nephropathy (albuminuria, creatinaemia). All interventions limited the progression of nephropathy, without affecting glycaemia. The order of efficacy was: pyridoxamine (650 mg.kg(-1).day(-1))vitamin E (200 mg.kg(-1).day(-1))lipoic acid (93 mg.kg(-1).day(-1)) approximately enalapril (35 mg.kg(-1).day(-1)). Pyridoxamine also significantly inhibited AGE/ALE accumulation in tissues; effects of other agents were mixed, but the degree of renoprotection was consistent with their effects on AGE/ALE formation.All interventions inhibited the progression of nephropathy at the doses studied, but the maximal benefit was achieved with pyridoxamine, which also limited dyslipidaemia and AGE/ALE formation. These experiments indicate that the more effective the renoprotection, the greater the inhibition of AGE/ALE formation. For optimal protection of renal function, it would be beneficial to select drugs whose mechanism of action includes inhibition of AGE/ALE formation.

Humans and laboratory animals recognize human modified LDL as immunogenic. Immune complexes (ICs) isolated from human sera contain malondialdehyde-modified LDL (MDA-LDL) and N (epsilon)(carboxymethyl)lysine-modified LDL (CML-LDL) as well as antibodies reacting with MDA-LDL, copper-oxidized LDL (OxLDL), CML-LDL, and advanced glycosylation end product (AGE)-modified LDL. OxLDL and AGE-LDL antibodies isolated from human sera recognize the same LDL modifications and do not react with modified non-LDL proteins. Rabbit antibodies have different reactivity patterns: MDA-LDL antibodies react strongly with MDA-LDL and MDA-BSA but weakly with OxLDL; OxLDL antibodies react strongly with OxLDL and weakly with MDA-LDL; CML-LDL antibodies react with CML-LDL > CML-BSA > AGE-LDL > OxLDL; AGE-LDL antibodies react strongly with AGE-LDL, react weakly with OxLDL, and do not react with CML-LDL. Thus, human and rabbit antibodies seem to recognize different epitopes. Capture assays carried out with all rabbit antibodies showed binding of apolipoprotein B-rich lipoproteins isolated from ICs, suggesting that laboratory-generated epitopes are expressed by in vivo-modified LDL, although they are not necessarily recognized by the human immune system. Thus, the definition of immunogenic forms of modified LDL eliciting human autoimmune responses requires the isolation and characterization of autoantibodies and modified LDL from human samples, whereas rabbit antibodies can be used to detect in vivo-modified human LDL.

Mice heterozygous for the Sod2 gene ( Sod2+/− mice) have been used to study the phenotype of life-long reduced Mn-superoxide dismutase (MnSOD) activity. The Sod2+/− mice have reduced MnSOD activity (∼50%) in all tissues throughout life. The Sod2+/− mice have increased oxidative damage as demonstrated by significantly elevated levels of 8-oxo-2-deoxyguanosine (8oxodG) in nuclear DNA in all tissues of Sod2+/− mice studied. The levels of 8oxodG in nuclear DNA increased with age in all tissues of Sod2+/− and wild-type (WT) mice, and at 26 mo of age, the levels of 8oxodG in nuclear DNA were significantly higher (from 15% in heart to over 60% in liver) in the Sod2+/− mice compared with WT mice. The level of 8oxodG was also higher in mitochondrial DNA isolated from liver and brain in Sod2+/− mice compared with WT mice. The increased oxidative damage to DNA in the Sod2+/− mice is associated with a 100% increase in tumor incidence (the number of mice with tumors) in old Sod2+/− mice compared with the old WT mice. However, the life spans (mean and maximum survival) of the Sod2+/− and WT mice were identical. In addition, biomarkers of aging, such as cataract formation, immune response, and formation of glycoxidation products carboxymethyl lysine and pentosidine in skin collagen changed with age to the same extent in both WT and Sod2+/− mice. Thus life-long reduction of MnSOD activity leads to increased levels of oxidative damage to DNA and increased cancer incidence but does not appear to affect aging.

modification and cross-linking of tissue proteins with age. Accelerated formation of AGE/ALEs during hyperglycemia is such as fructoselysine. The Amadori product is a precurimplicated in the development of diabetic complications. In sor to AGEs, which are a more permanent, irreversible this study, we examined the effect of the AGE/ALE inhibitor modification of proteins. According to the AGE hypothpyridoxamine on chemical modification and cross-linking of esis [1, 2], the increased rate of chemical modification collagen and development of renal disease in the streptozotocin-diabetic rat. and cross-linking of tissue proteins during hyperglycemia Methods. Diabetic rats were treated with pyridoxamine; par- compromises the structure and function of proteins and allel experiments were conducted with aminoguanidine, the contributes to the development of chronic complications prototype AGE inhibitor. Progression of renal disease was in diabetes. The accumulation of AGEs in collagen, a evaluated by measurements of albuminuria and plasma creatilong-lived protein in the extracellular matrix, is thought nine concentration. Plasma triglycerides, cholesterol, lactate and pyruvate were measured by enzymatic assays, and AGE/ to effect changes in elasticity, ionic charge, thickness and ALEs in skin collagen by HPLC and GC-MS assays. turnover of basement membrane components, setting Results. Pyridoxamine significantly inhibited the increase in the stage for development of diabetic renal, retinal and albuminuria, plasma creatinine, hyperlipidemia and plasma lac- vascular disease. tate/pyruvate ratio in diabetic rats, without an effect on blood glucose or glycated hemoglobin. AGE/ALEs, fluorescence and Reactive carbonyl and dicarbonyl compounds derived cross-linking of skin collagen increased approximately twofold from sugars are intermediates in the formation of AGEs, in diabetic versus control rats after seven months of diabetes. and oxygen, redox active transition metal ions and reacPyridoxamine caused a significant (25 to 50%) decrease the tive oxygen species are recognized as catalysts of AGE AGE/ALEs, carboxymethyllysine and carboxyethyllysine, crossformation [3]. The precise chemical origin of specific linking and fluorescence in skin collagen of diabetic rats, but did not affect pentosidine. AGEs is not always clear. Some AGEs, such as pentosiConclusions. Pyridoxamine inhibits the progression of renal dine, are clearly derived from carbohydrates, but may disease, and decreases hyperlipidemia and apparent redox im- be formed from glucose, ascorbate, 3-deoxyglucosone balances in diabetic rats. Pyridoxamine and aminoguanidine or other sugars in vivo [4]. The major AGEs, including had similar effects on parameters measured, supporting a the lysine adducts N e

Dose-response relationships between exercise training volume and blood lipid changes suggest that exercise can favourably alter blood lipids at low training volumes, although the effects may not be observable until certain exercise thresholds are met. The thresholds established from cross-sectional literature occur at training volumes of 24 to 32 km (15 to 20 miles) per week of brisk walking or jogging and elicit between 1200 to 2200 kcal/wk. This range of weekly energy expenditure is associated with 2 to 3 mg/dl increases in high-density lipoprotein-cholesterol (HDL-C) and triglyceride (TG) reductions of 8 to 20 mg/dl. Evidence from cross-sectional studies indicates that greater changes in HDL-C levels can be expected with additional increases in exercise training volume. HDL-C and TG changes are often observed after training regimens requiring energy expenditures similar to those characterised from cross-sectional data. Training programmes that elicit 1200 to 2200 kcal/wk in exercise are often effective at elevating HDL-C levels from 2 to 8 mg/dl, and lowering TG levels by 5 to 38 mg/dl. Exercise training seldom alters total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C). However, this range of weekly exercise energy expenditure is also associated with TC and LDL-C reductions when they are reported. The frequency and extent to which most of these lipid changes are reported are similar in both genders, with the exception of TG. Thus, for most individuals, the positive effects of regular exercise are exerted on blood lipids at low training volumes and accrue so that noticeable differences frequently occur with weekly energy expenditures of 1200 to 2200 kcal/wk. It appears that weekly exercise caloric expenditures that meet or exceed the higher end of this range are more likely to produce the desired lipid changes. This amount of physical activity, performed at moderate intensities, is reasonable and attainable for most individuals and is within the American College of Sports Medicine's currently recommended range for healthy adults.