Urinary pyrraline as a biochemical marker of non-oxidative Maillard reactions in vivo

The presence of pyrraline, a non-oxidative glucose-derived Maillard reaction product in plasma proteins has been established previously. In this study we have investigated the presence of pyrraline in human urine to determine whether pyrraline-containing proteins are metabolized or selectively retained. Pyrraline was detected by means of HPLC, and its presence was confirmed by UV and electrospray-mass spectrometry. The quantification of pyrraline in urine from healthy individuals showed 1.21 +/- 0.4 micrograms/mg creatinine. In urine from diabetic patients, pyrraline levels varied considerably, although the mean level was higher than in healthy subjects (1.37 +/- 0.6 micrograms/mg creatinine). These data further support the presence of a catabolic pathway for advanced non-oxidative Maillard reaction products in vivo and suggest their role in the pathogenesis of diabetes.     

Effect of curcumin on the advanced glycation and cross-linking of collagen in diabetic rats

A close association between increased oxidative stress and hyperglycemia has been postulated to contribute significantly to the accelerated accumulation of advanced glycation end products (AGEs) and the cross-linking of collagen in diabetes mellitus. In the present work, we report the influence of curcumin, an efficient antioxidant, on the level of AGEs and the cross-linking of collagen in diabetic rats. Diabetic rats were given curcumin (200 mg/kg body wt) orally for a duration of 8 weeks. The antioxidant status in serum and the level of AGEs, cross-linking and browning of collagen in tail tendons and skin were investigated. The oxidative stress observed in diabetic rats was reduced significantly by curcumin administration. Nonenzymic antioxidants such as vitamin C, vitamin E, and glutathione were maintained at near normal values in curcumin-treated diabetic animals. Similarly, the accumulation of lipid peroxidation products in diabetic serum was reduced significantly by curcumin. Accelerated accumulation of AGE-collagen in diabetic animals, as detected by ELISA, was prevented by curcumin. Extensive cross-linking of collagen in the tail tendon and skin of diabetic animals was also prevented to a greater extent by curcumin treatment. A correlation between the level of AGEs and collagen cross-linking was noted, suggesting the involvement of advanced glycation in cross-linking. It was also noted that the preventive effect of curcumin on the advanced glycation and cross-linking of collagen was more pronounced than its therapeutic effect. However, the Maillard reaction fluorescence in both tail and skin collagen remained unaltered by curcumin. This study confirms the significance of free radicals in the accumulation of AGEs and cross-linking of collagen in diabetes. It supports curcumin administration for the prevention of AGE-induced complications of diabetes mellitus.     

The platysma myocutaneous flap. Indications and caveats

Arterial hypertension has been identified as a major secondary risk factor for diabetic retinopathy. However, the mechanisms by which hypertension worsens retinopathy are unknown. Inhibition of advanced glycation product formation prevents the development of experimental diabetic retinopathy in normotensive diabetic rats. In this study the effect of hypertension on the rate of diabetic retinopathy development and the formation of arteriolar thrombosis was evaluated. We also evaluated the effect of aminoguanidine, an inhibitor of advanced glycation and product formation on retinal pathology of diabetic hypertensive rats. After 26 weeks of diabetes, hypertension accelerated the development of retinopathy despite a lower mean blood glucose level than in the non-hypertensive group (diabetic spontaneous hypertensive rats (SHR) 16.00 +/- 6.83 mmol/l; diabetic normotensive Wistar Kyoto rats (WKY) 34.9 +/- 3.64 mmol/l; p < 0.0001). Diabetic SHR had nearly twice as many acellular capillaries as diabetic WKY (SHR diabetic: 91.9 +/- 7.5 acellular capillaries per mm2 of retinal area vs WKY diabetic: 53.7 +/- 8.5 acellular capillaries per mm2 of retinal area), and a 3.8-fold increase in the number of arteriolar microthromboses (SHR diabetic 23,504 +/- 5523 microns2 vs SHR non-diabetic 6228 +/- 2707 microns2). Aminoguanidine treatment of SHR diabetic rats reduced the number of acellular capillaries by 50%, and completely prevented both arteriolar deposition of PAS-positive material and abnormal microthrombus formation. These data suggest that hypertension-induced deposition of glycated proteins in the retinal vasculature plays a central role in the acceleration of diabetic retinopathy by hypertension.     

Accumulation of pyrraline-modified albumin in phagocytes due to reduced degradation by lysosomal enzymes

Previous studies suggested that the interaction between proteins modified by advanced glycation end products (AGEs) and cells, such as macrophages, may be involved in diabetic angiopathy. Pyrraline is one of the AGEs and known to be elevated in plasma of diabetic rats and humans, and is present in vascular lesions of diabetic and elderly subjects. We examined whether modification of albumin by pyrraline influences its degradation by macrophage-like cell line, P388D1 cells. Degradation of pyrraline-modified albumin by these cells was diminished, causing accumulation of the albumin in these cells. The susceptibility of pyrraline-modified albumin to lysosomal proteolytic enzymes was reduced by approximately 40% in vitro, while lysosomal activity in the cells per se was not affected. This phenomenon was also observed when human monocytes were used instead of P388D1 cells. Our results suggest that accumulation of pyrraline-modified albumin in P388D1 cells is due to the reduced susceptibility of the protein to lysosomal enzymatic degradation. Such alterations in the interaction between AGEs-modified protein and phagocytes may contribute to angiopathy in elderly subjects and patients with diabetes.     

Potential benefit of inhibitors of advanced glycation end products in the progression of type II diabetes: a study with aminoguanidine in C57/BLKsJ diabetic mice

Prolonged hyperglycemia in type II diabetic patients is linked both with diabetic complications and with further impairment of glucose homeostasis, possibly due to glucose toxicity of the beta cell. While the connection between the accumulation of extracellular advanced glycation end products (AGEs) and the development of complications is well established, it has only recently been suggested that intracellular glycation may be equally adverse and could be involved in the pathogenesis of glucose toxicity in vitro. Aminoguanidine is a recognized inhibitor of the formation of both extracellular and intracellular AGEs. In this study, we show that the development of diabetes, measured by increased water intake and concomitant midday blood glucose levels in type II genetically diabetic mice, is reduced by treatment with aminoguanidine at a dosage of 500 mg/kg/d for 12 weeks in the diet. In addition, at the end of the study, aminoguanidine reduced the decline in serum and pancreatic insulin levels and the degree of pancreatic islet morphological degeneration, all of which are associated with pancreatic insufficiency following prolonged hyperglycemia in this animal model. These results suggest that AGEs may be involved in the aggravation of type II diabetes in vivo and aminoguanidine may be beneficial in its treatment.     

Pyrone hydroperoxide formation during the Maillard reaction and its implication in biological systems

Hydroperoxide formation during Maillard reaction (amino-carbonyl reaction) was investigated using luminol-chemiluminescence-high performance liquid chromatography (CL-HPLC). From the equimolar reaction mixture of 1 M beta-alanine/D-glucose in phosphate buffer (pH 8.0) at 95 degrees C, two hydroperoxides and H2O2 were detected as chemiluminescent products in CL-HPLC, and the yields were proportional to the browning development. One of these hydroperoxides was isolated and identified as 3-hydroxy-5-hydroperoxy-2-methyl-5,6-dihydropyran-4-one (HMDP, pyrone hydroperoxide) by fast atom bombardment mass spectrometry. The HMDP formation was also confirmed in L-lysine/D-glucose and in bovine serum albumin/D-glucose with the physiological incubation at 37 degrees C for 4 days and 3 wk, respectively. Incubation at 37 degrees C of human plasma containing 5.5-25.0 mM of D-glucose for 60 h showed the glucose concentration-dependent formation of HMDP (10-35 microM of H2O2 equivalence). The HMDP was negative to thiobarbituric acid reaction and was degraded by peroxidases such as horseradish peroxidase, Athromyces ramosus peroxidase, heated cytochrome c, and microperoxidase. The results strongly suggested the formation of such hydroperoxide even in biological Maillard reaction termed as glycation, and implied its contribution in pathogenesis and oxidative lesions associated with hyperglycemia.     

Advanced glycation endproducts and cigarette smoking

The incidence of certain ageing sequelae such as lung and cardiovascular disease and cataract are higher in smokers than in non-smokers. We recently proposed that certain components of mainstream cigarette smoke can react with plasma and extracellular matrix proteins to form covalent adducts with many of the properties of advanced glycation endproducts (AGE). AGEs have been implicated previously in the pathogenesis of the end-organ complications of diabetes and ageing, including cataract, atherosclerosis and renal insufficiency. In these circumstances, AGEs arise in vivo from the non-enzymatic reaction of reducing sugars with amino groups. Over time the initial Schiff base and Amadori products that form gradually undergo dehydration and rearrangement to produce reactive, carbonyl containing compounds with characteristic fluorescence and covalent crosslinking properties. Recent studies indicate that in smokers, tobacco-derived AGEs accumulate on plasma low density lipoprotein (LDL), structural proteins present within the vascular wall, and the lens proteins of the eye. These data point to a new and significant source of Maillard products in the human environment, significantly broaden the role of Maillard chemistry in pathological processes, and provide new insight into the pathogenesis of atherosclerosis and other diseases associated with tobacco usage.     

Age-related increase in an advanced glycation end product in penile tissue

Nonenzymatic glycosylation (glycation) of proteins, often referred to as the Maillard reaction, has been proposed to play a role in age and diabetes-related processes by forming protein and DNA adducts and cross-links. These cross-links may contribute to erectile dysfunction by scavenging nitric oxide, which is needed for erection. As the basis for a possible role of the advanced Maillard reaction in age-related erectile dysfunction, we investigated the presence of the specific advanced glycation endproduct (AGE) pentosidine in penile corpus cavernosum tissue and penile tunica albuginea tissue as a function of age. A total of 23 penile tissue specimens were obtained at autopsy, from which 19 samples of tunica albuginea and 21 samples of corpus cavernosum were derived. In addition, 13 penile corporal and tunical specimens were procured at the time of insertion of a penile prosthesis, from which 12 tunica albugineal specimens and 10 samples of corpus cavernosum were derived. Collagen was extracted with acetic acid and pepsin digestion, and the final insoluble collagen product was acid-hydrolyzed with 6 N HCL for 24 h at 110 degrees C. Pentosidine was quantified by high-performance liquid chromatography using a reverse-phase column. The level of pentosidine (expressed in picomoles per milligram of insoluble collagen) was found to increase with age in cadaver as well as living penile corporal and tunical albugineal tissues. Best-fit analysis revealed an exponential increase in both types of cadaver penile tissue, with regression equations of y = 15.29 x 10(9.9e-3x), R2 = 0.79, being obtained in the tunica and y = 13.2 x 10(7.63e-3x), R2 = 0.56, in the corpora. These correspond to 6- and 4-fold increases in pentosidine levels from puberty to the age of 100 years (P < 0.05), respectively. Mean pentosidine levels were higher in the tunica than in the corpora. Comparison of pentosidine levels in the tunica versus the corpora revealed a weakly linear correlation (y = 24.88 + 1.08x, R2 = 0.32). Levels in the tunical and corporal specimens from the living human specimens fell with the predicted confidence intervals of the cadaveric tissue. Tunical specimens from patients who underwent repair or revision of a previously inserted penile prosthesis had very low levels of pentosidine. The exponential age-related increase in pentosidine observed in both types of penile tissue suggests an impairment of collagen turnover, which could be related to the advanced glycation reaction in aging. It is not known whether pentosidine itself is directly associated with erectile dysfunction, but its formation is usually accompanied by extensive tissue modification. Formation of advanced Maillard reaction products, which is greatly accelerated in aging, diabetes, and uremia, could contribute to erectile dysfunction in these syndromes.     

Protein modification by a Maillard reaction intermediate methylglyoxal. Immunochemical detection of fluorescent 5-methylimidazolone derivatives in vivo

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes, is a common intermediate in nonenzymatic glycation (Maillard reaction) in vivo. Here we describe the immunochemical approach to the detection of MG adducts in proteins in vitro and in atherosclerotic lesions of human aorta in vivo. The reaction of protein (bovine serum albumin) with MG led to selective loss of arginine and lysine residues, accompanied by the formation of 5-methylimidazolone (N delta-(5-methylimidazolon-2-yl)ornithine) and imidazolysine (1,3-di-lysino-4-methylimidazole) derivatives, respectively. The anti-5-methylimidazolone antibody was prepared by immunizing rabbits with a MG-keyhole limpet hemocyanin conjugate and purifying the serum on an affinity gel prepared by covalent attachment of the 5-methylimidazolone derivative. The antibody cross-reacted with the proteins treated with not only MG but trioses, such as hydroxyacetone, dihydroxyacetone, and glyceraldehyde. The immunohistochemical analysis revealed that atherosclerotic lesions of human aorta contained 5-methylimidazolone derivatives whose distributions were identical to those of advanced glycation end products (AGEs) detected by the anti-AGE antibody.     

High galactose levels in vitro and in vivo impair ascorbate regeneration and increase ascorbate-mediated glycation in cultured rat lens

In contrast to conventional view that glucose is the sole glycating agent, ascorbate has now emerged as a potential precursor of advanced glycation products in lenses during cataractogenesis, owing to the high concentration present in human lens. The effects of high hexose environment in vitro and in vivo on the disruption of redox equilibrium of ascorbate (ASA) to dehydroascorbate (DHA), which is required for ascorbate-mediated crystallin modification by the Maillard reaction during cataractogenesis were examined. Organ culture experiments were performed with rat lenses that were first exposed to high galactose levels in vitro and in vivo and then incubated with 1-14C-labeled ASA, DHA or DKG (2,3-diketogulonic acid). Formation of ASA degradation products as a function of time was assessed by radiometric TLC method. Upon incubation with ASA or DHA, an elevated level of the degradation product, DKG, was detected in lenses exposed to galactose in vivo and in vitro. ASA uptake was significantly enhanced in the galactosemic lenses as compared to controls (P = 0.01). Regeneration of ASA from DHA in both galactose treated and galactosemic lenses was impaired when compared to control lens which completely converted DHA from the medium into ASA. Surprisingly, the galactose exposed lenses showed enhanced permeability to DKG which was picked up readily from the medium in contrast to normal healthy lenses which remained impermeable to DKG. Galactose exposed lenses both in vitro and in vivo showed a 5-9-fold increase in crystallin bound Schiff base-linked radioactivity when incubated with 1-14C-labeled ASA or DHA. As a preamble to the question of whether lens pigmentation predisposes towards ascorbate oxidation, lens homogenate from normal young and old pigmented cataractous lenses were incubated with [1-14C]ASA. After 2 days, ASA levels were found to have decreased by 74% and DKG levels increased by 48% in brunescent lens as compared to the young lens. These data demonstrated that profound abnormalities in ASA metabolism exist in lenses exposed to a high sugar environment suggestive of a breakdown of the redox equilibrium of ASA to DHA and a loss of membrane permeability barrier for DKG. The latter would further contribute toward a ASA-catalysed Maillard reaction in the redox impaired lens.     

A novel biomarker for hyperglycemia, MRX isolated from hydrolysate of glycated proteins

Long-lived proteins can undergo non-enzymatic glycation to form highly crosslinked structures with characteristic fluorescence during aging and diabetes processes. In this paper, a typical fluorophore, named Maillard reaction product X (MRX), was isolated from the hydrolysate of glycated proteins. MRX could be formed by incubation of bovine serum albumin with glucose, followed by acid hydrolysis. The structure of MRX was determined to be 8-hydroxy-5-methyldihydrothiazolo[3,2-alpha] pyridinium-3-carboxylate. MRX was also found to be formed by the incubation of cysteine and arginine with glucose, followed by hydrolysis. We found the formation of MRX in the recently developed genetically diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and compared them with that in the control Long-Evans Tokushima Otsuka (LETO) rats. Significantly higher levels of MRX were observed from the serum (p < 0.005) and urinary protein (p < 0.001) of OLETF rats in comparison with those of LETO rats. MRX must be a potential candidate as a biomarker for hyperglycemia.     

Importance of advanced glycation end products--AGE products

Recent experimental findings suggest that free oxygen radicals and AGEs may be significantly involved in the onset and development of chronic diabetic complications and Alzheimer's disease. The presented review summarizes knowledge on structure and rise of these products in vitro and in vivo and the chemical and biological properties of advanced glycation endproducts are discussed. Strategy of influencing development and prevention of diabetic complications in the near future involves a potentially promising antiglycation therapy and supplementation by antioxidants.     

Determination of urinary and serum pentosidine and its application to elder patients

Pentosidine, a fluorescent cross-linking compound, accumulates in extracellular matrix, especially in the collagen, and is formed by the nonenzymatic process of advanced Maillard reaction. We developed a method of determination of pentosidine and tried to examine its level in urine and serum of elder patients. The method, which involves the hydrolysis of samples, pretreatment using a CF-11 cellulose column and HPLC quantification, resulted in a high recovery (94.3%) of pentosidine with low coefficient of variation (8-10%) of total analysis. Serum and urinary levels of pentosidine in control subjects gradually increased with age. Elder patients with cerebral infarction showed higher levels of serum and urinary pentosidine as compared with those with senile dementia and other geriatric disorders. These results suggest that serious damage to systemic vascular tissues has already occurred in these patients due to glycation.     

Running biomechanics

Nonenzymatic glycation of proteins and oxidative stress are considered independent factors important in the development of the complications of diabetes but may be interrelated by the process of autoxidative glycation. This pathway involves monosaccharide autoxidation to a reactive ketoaldehyde analogue and subsequent reaction with protein to form a ketoimine adduct. This study demonstrates that delta-gluconolactone (delta-GL), an oxidised analogue of glucose, is a potent glycating agent in vitro of haemoglobin present in blood samples from insulin-dependent diabetic and non-diabetic human subjects and from spontaneously diabetic, insulin-dependent BB/Edinburgh (BB/E) rats. The percentage glycated haemoglobin after incubation (37 degrees C, 5 h) with delta-GL (25 mmol/l) was significantly (P < 0.002) higher than that observed using an equimolar concentration of glucose. Intravenous administration of delta-GL (1 g/kg) to non-diabetic BB/E rats also significantly increased glycation of haemoglobin (6.0 +/- 0.1% vs 4.9 +/- 0.1%, P < 0.01) whereas intravenous injection of an identical dose of glucose had no significant effect (5.1 +/- 0.1% vs 5.0 +/- 0.2%). These results support the hypothesis that nonenzymatic glycation of proteins involves attachment by both native and oxidised monosaccharides. Further investigation of the interactions between diabetes-associated increases in oxidative stress and glycation on the development and progression of the vascular complications of diabetes is necessary.     

Leigh syndrome associated with mitochondrial DNA 8993 T-->G mutation and ragged-red fibers

Increased protein glycation has been mechanistically linked to accelerated vascular pathobiology in diabetes. Because glycated albumin induces biosynthetic abnormalities in cultured aortic endothelial cells that resemble those associated with macrovascular disease, we sought evidence that increased glycated albumin is operative in the genesis of diabetic vasculopathy in vivo. Plasma concentrations of fibronectin, a sensitive marker of endothelial cell damage, were increased two-fold in diabetic db/db mice compared with their nondiabetic db/m littermates. Treatment with monoclonal antibodies specifically reactive with albumin modified by Amadori glucose adducts normalized fibronectin in diabetic animals despite persistent hyperglycemia. These findings suggest that increased glycated albumin causally contributes to diabetic vasculopathy, and that blocking this influence ameliorates vascular damage.     

Effect of diabetes, insulin, and glucose load on lipid peroxidation in the rat

Lipid peroxidative activity in rats made diabetic with streptozocin and rats made acutely hyperglycemic by intraperitoneal dextrose administration was determined by measurement of exhaled ethane during exposure in vivo to ethane-free air (EFA). Diabetic rats demonstrated increased ethane in the expired breath while breathing EFA (5.82 +/- 0.56 pmol/min/100 g) compared with control rats (4.02 +/- 0.23 pmol/min/100 g). Insulin treatment of diabetic rats attenuated the ethane produced (4.88 +/- 0.23 pmol/min/100 g). Acute hyperglycemia increased exhaled ethane to levels higher than those seen in diabetic rats (9.87 +/- 0.98 pmol/min/100 g). Saline injected intraperitoneally to control rats produced ethane levels similar to those of untreated nondiabetic controls (4.11 +/- 0.52 pmol/min/100 g). Chronic uncontrolled hyperglycemia and acute hyperglycemia are associated with increased in vivo ethane production.     

The effects of streptozocin-induced diabetes on renal plasma flow

To evaluate the significance of collagen levels, nonenzymatic glycation, and effective renal plasma flow in diabetes, we studied 52 Streptozocin induced-diabetic rats. After 10 weeks of diabetes, rats were injected i.v. 0.5 microCi/g 99mTechnetium Mercaptoacetyl triglycine and effective renal plasma flow was calculated from the renograms obtained. The collagen content and hydroxymethyl furfural levels of kidney and tail tissue as well as renal plasma flow increased significantly in diabetic rats (p < 0.05). The increase in renal plasma flow indicates that hyperperfusion may play a role in diabetic nephropathy mechanism. The correlation between renal plasma flow and glycation was not as high as the correlations between blood glucose concentrations and hydroxymethyl furfural and collagen levels, suggesting that factors other than glycation are important in increasing the renal plasma flow.     

Benfluorex normalizes hyperglycemia and reverses hepatic insulin resistance in STZ-induced diabetic rats

We have examined the effect of chronic (20 days) oral administration of benfluorex (35 mg/kg) in a rat model of NIDDM, induced by injection of STZ 5 days after birth and characterized by frank hyperglycemia, hypoinsulinemia, and hepatic and peripheral insulin resistance. We assessed the following: 1) basal blood glucose and insulin levels, 2) glucose tolerance and glucose-induced insulin release in vivo and in vitro, and 3) basal and insulin-stimulated in vivo glucose production and glucose utilization, using the insulin-clamp technique in conjunction with isotopic measurement of glucose turnover. The in vivo insulin response of several individual tissues also was evaluated under the steady-state conditions of the clamp, using the uptake of the glucose analogue 2-deoxy-D-glucose as a relative index of glucose metabolism. In the benfluorex-treated diabetic rats, postabsorptive basal plasma glucose levels were decreased (8.1 +/- 0.2 mM compared with 10.5 +/- 0.5 mM in the pair-fed untreated diabetic rats and 6.1 +/- 0.2 mM in the benfluorex-treated nondiabetic rats), whereas the basal and glucose-stimulated intravenous glucose tolerance test plasma insulin levels were not improved. Such a lack of improvement in the glucose-induced insulin release after benfluorex treatment was confirmed under in vitro conditions (perfused pancreas). In the pair-fed untreated diabetic rats, the basal glucose production and overall glucose utilization were significantly increased, and during hyperinsulinemia both liver and peripheral tissues revealed insulin resistance. In the benfluorex-treated diabetic rats, the basal glucose production and basal overall glucose utilization were normalized. After hyperinsulinemia, glucose production was normally suppressed, whereas overall glucose utilization was not significantly improved.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renin inhibitory effect of 2-[4-(4'-chlorophenoxy)phenoxyacetylamino]-ethylphosphorylethanolamine (PE-104) in vitro and in vivo

The renin inhibitory activity of 2-[4-(4'-chlorophenoxy)phenoxyacetylamino]ethylphosphorylethanolamine (PE-104) was examined in vitro and in vivo. PE-104 inhibited the reaction between dog renal renin and homologous plasma angiotensinogen. The Ki value was 2 mM and the inhibitory mode was competitive and reversible. Data concerning the relationship between renin inhibitory activity and the chemical structure indicated that the whole structure was required for inhibitory activity of PE-104. PE-104 did not inhibit the caseinolytic activities of pepsin, papain and trypsin at 10 mM, the dose of which inhibited renin activity by more than 80%. In normotensive rats, infusion of PE-104 (20 mg/kg/min) abolished increases in blood pressure, plasma renin activity and plasma angiotensin I concentration after injection of renin. In two kidney model renal hypertensive rats, infusion of PE-104 resulted in decreases in blood pressure, plasma renin activity and plasma angiotensin I concentration.     

Glycemia and regulation of endothelial adhesion molecules

Hyperglycemia is one of the most prominent factors associated with diabetic vascular disease, the development of which is initated by functional and morphological alterations of the endothelium. The review summarizes several issues of glycemia and advanced glycation end products induced changes concerning soluble as well as cell bound endothelial adhesion molecules, and further addresses the aspect of apoptosis in the light of diabetic vascular complications.