Low protective PON1 lactonase activity in an Arab population with high rates of coronary heart disease and diabetes.

BACKGROUND: Recent studies showing that high density lipoproteins (HDL) can effect plaque regression indicate that recent trial failures do not exclude an atheroprotective role of HDL. Instead, they highlight differences between HDL function and measured HDL-cholesterol (HDL-C). PON1 is one key functional activity of HDL. Urban Palestinians have lower HDL-C and a higher incidence and mortality of coronary heart disease than those of Israelis. We hypothesized that the cardioprotective PON1 lactonase and arylesterase activities and PON1 functional genotype may differ between Palestinians and Israelis. METHODS: We measured PON1 activities in a cross-sectional population-based study of Palestinian (n=960) and Israeli (n=694) residents in Jerusalem, 1654 participants in all. RESULTS: Palestinians had high prevalences of obesity and diabetes and low mean concentrations of HDL-cholesterol (0.97 mmol/l in men and 1.19 mmol/l in women). Lactonase and arylesterase activities were lower by 10.8% (p=1.2∗10(-14)) and 2.7% (p<0.0005), respectively, in Palestinians as compared to Israelis. The functional genotype distribution, demonstrated by plotting paraoxonase vs lactonase activities, showed a modest between-group difference (p=0.024), with 12.1% RR in Palestinian Arabs vs 8.4% in Israeli Jews, but no overall difference in allele frequencies. Lactonase correlated inversely with age (Spearman's rho=-.156), weakly with BMI (-.059), positively with HDL-C (.173) and non-HDL-C (.103), but was not associated with triglycerides or fasting glucose. Palestinians showed consistently lower lactonase activity in logistic regression models adjusted for multiple covariates and for functional genotype (odds ratios of 1.81 and 1.98, respectively, for the lower fifth vs the upper 4 fifths of lactonase activity p<0.0001). CONCLUSION: We showed a lower physiologically-significant lactonase PON1 activity in an Arab population, a finding consistent with the high cardiovascular and diabetes risk of Palestinians.

Circulating soluble receptor for advanced glycation end products is inversely correlated to oxidized low-density lipoproteins in asymptomatic subjects.

OBJECTIVE: There is growing evidence that circulating soluble receptor for advanced glycation end products (sRAGE) exerts antiatherogenic effects as a decoy receptor that abolishes RAGE signalling. A previous study reported that oxidized low-density lipoprotein (oxLDL) can be one of the RAGE ligands. The present cross-sectional study investigated the clinical association between sRAGE and oxLDL in humans. METHODS: Serum levels of the conventional atherosclerotic risk factors, sRAGE and malondialdehyde-modified low-density lipoprotein (MDA-LDL) were analysed in asymptomatic subjects; MDA-LDL was measured as a biomarker of oxLDL. RESULTS: Mean serum levels of sRAGE and MDA-LDL were 1101 ng/l and 57.6 IU/l, respectively, in 33 subjects of mean age 65 years. Simple linear regression analysis showed a significant inverse correlation between sRAGE and MDA-LDL. Stepwise multiple linear regression analysis confirmed MDA-LDL to be independently, significantly and inversely correlated with sRAGE. CONCLUSIONS: An independent, significant and inverse correlation was shown to exist between circulating levels of sRAGE and oxLDL (MDA-LDL), which suggests that part of the antiatherosclerotic effects of sRAGE may be related to oxLDL quenching.

Correlation between ischaemia-modified albumin and intermediate-density lipoprotein in haemodialysis patients with end-stage renal disease.

This study investigated the association between ischaemia-modified albumin (IMA), a biomarker of cardiac ischaemia, and increases in the levels of intermediate-density lipoprotein (IDL), an atherogenic particle that can cause oxidative stress, in haemodialysis patients with end-stage renal disease (ESRD). Fasting levels of serum IMA and lipids/lipoproteins were analysed in 15 patients and 15 healthy control subjects. There was a close positive correlation between IMA and IDL levels in ESRD patients but no significant correlation between IMA and lipids/lipoproteins in control subjects. This suggests a possible link between the characteristic dyslipoproteinaemia found in ESRD and levels of IMA and, if confirmed in studies with larger sample sizes, may lead to further studies on the potential of the relationship between IMA and IDL as a biomarker in haemodialysis patients with ESRD.

Recent advances on Ilex paraguariensis research: minireview.

Ilex paraguariensis dried and minced leaves are made into a brewed tea, prepared in a sui generis manner by large populations in South America, having evolved from a tea drunk by the Guarani ethnic group to a beverage that has a social and almost ritualistic role in some South American modern societies. It is used both as a source of caffeine, in lieu or in parallel with tea and coffee, but also as a therapeutic agent for its alleged pharmacological properties. Although with some exceptions, research on biomedical properties of this herb has had a late start and strongly lags behind the impressive amount of literature on green tea and coffee. However, in the past 15 years, there was a several-fold increase in the literature studying Ilex paraguariensis properties showing effects such as antioxidant properties in chemical models and ex vivo lipoprotein studies, vaso-dilating and lipid reduction properties, antimutagenic effects, controversial association with oropharyngeal cancer, anti-glycation effects and weight reduction properties. Lately, promising results from human intervention studies have surfaced and the literature offers several developments on this area. The aim of this review is to provide a concise summary of the research published in the past three years, with an emphasis on translational studies, inflammation and lipid metabolism. Ilex paraguariensis reduces LDL-cholesterol levels in humans with Ilex paraguariensis dyslipoproteinemia and the effect is synergic with that of statins. Plasma antioxidant capacity as well as expression of antioxidant enzymes is positively modulated by intervention with Ilex paraguariensis in human cohorts. A review on the evidence implicating Ilex paraguariensis heavy consumption with some neoplasias show data that are inconclusive but indicate that contamination with alkylating agents during the drying process of the leaves should be avoided. On the other hand, several new studies confirm the antimutagenic effects of Ilex paraguariensis in different models, from DNA double breaks in cell culture models to mice studies. Novel interesting work has emerged showing significant effect on weight reduction both in mice and in rat models. Some mechanisms involved are inhibition of pancreatic lipase, activation of AMPK and uncoupling of electron transport. Intervention studies in animals have provided strong evidence of anti-inflammatory effects of Ilex paraguariensis, notably protecting cigarette-induced lung inflammation acting on macrophage migration and inactivating matrix-metalloproteinase. Research on the effects of Ilex paraguariensis in health and disease has confirmed its antioxidant, anti-inflammatory, antimutagenic and lipid-lowering activities. Although we are still waiting for the double-blind, randomized prospective clinical trial, the evidence seems to provide support for beneficial effects of mate drinking on chronic diseases with inflammatory component and lipid metabolism disorders.

"Blinding" of AMP-dependent kinase by methylglyoxal: a mechanism that allows perpetuation of hepatic insulin resistance?

AMP-dependent kinase (AMPK) is a regulatory carrefour and a key target for therapeutics. The role of AMPK in regulating cellular energy status (by sensing low energy using [AMP] as its signal) and activating catabolic pathways while inhibiting anabolic routes, places this enzyme at a central control point in maintaining energy homeostasis. The exquisite allosteric sensing of AMP is done by a domain with three arginine residues, which make it very vulnerable to glycation, especially by the alpha-dicarbonyl methylglyoxal (MG). MG accumulates in hyperglycemia, insulin resistance, diabetes and when there is excess flux of reactive oxygen species coming from the mitochondria. We hypothesize that excess MG in the above-mentioned conditions blocks the sensing of AMP by AMPK, thereby favoring gluconeogenesis (thus hepatic glucose output and hyperglycemia) and lipogenesis (hepatic steatosis and high VLDL), hallmarks of insulin resistance and diabetes. Our hypothesis may explain, for instance, the perpetuation of hepatic insulin resistance, as well as part of the action of metformin, which is a potent anti-glycation agent. Future targets for type 2 diabetes treatments will likely be those that can effect beneficial changes in the activity of AMPK, and our theory predicts that anti-glycation agents may become part of that armamentarium.

Caffeic and chlorogenic acids in Ilex paraguariensis extracts are the main inhibitors of AGE generation by methylglyoxal in model proteins.

The present study concentrates on the evaluation of the anti-glycation effect of some bioactive substances present in yerba maté (Ilex paraguariensis): 5-caffeoylquinic acid, caffeic acid and a sapogenin (oleanolic acid). Bovine serum albumin and histones were incubated in the presence of methylglyoxal with or without the addition of 5-caffeoylquinic acid, caffeic acid and oleanolic acid. After the incubation period, advanced glycation end product (AGE) fluorescence spectra were performed and protein structural changes were evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. Chlorogenic acid, caffeic acid are the main substances responsible for the anti-glycation effect of maté tea.

Alternative antiglycation mechanisms: are spermine and fructosamine-3-kinase part of a carbonyl damage control pathway?

Spermine is an ubiquitous molecule that bears unique structural features of regularly spaced positive charges interrupted by hydrophobic methylene bridges. In previous studies we have shown significant antiglycation effects of physiological concentrations of spermine and spermidine. The effect is apparent in four different protein models, two targeting structural changes on histones and ubiquitin, and two targeting impairment of catalytic activities of AT III and plasminogen. We hypothesize that polyamines inhibit glycation and that might be one of their elusive molecular functions. A mammalian fructosamine-3-kinase (FN-3-K), which phosphorylates fructoselysine (FL) residues on glycated proteins, to FL-3-phosphate has been isolated and cloned by two independent groups. This enzyme may function as a deglycating enzyme. Being its Km for FL two orders of magnitude lower than for its protein substrate, we propose the enzyme has a dual role and also functions as a recycler of spermine-carbonyl adducts. Spermine and FN-3-K may be part of a carbonyl damage control pathway. Thirdly, due to critically functional lysine residues, we underscore the vulnerability to glycation of ornithine decarboxylase, the main enzyme in spermine biosynthesis. If glycation is modulated by polyamines and glycation itself impairs polyamine synthesis, a dangerous loop of excessive spermine consumption and slower spermine biosynthesis might ensue in chronic hyperglycemic conditions. In this perspective, small changes in flow rates in the spermine (where ODC and antizyme are key players) and/or FN-3-K pathway could contribute to enhance the effects of hyperglycemia and explain why there are diabetic subjects with higher glycation phenotypes and incidence of complications. They could have altered steady state levels of polyamines and/or decreased FN-3-K expression or activity.

Polyamines as clinical laboratory tools.

Since their discovery by Antoni van Leeuwenhoek in 1678 until the recent development of transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner, polyamines have been the object of intense research efforts which have shed light on several biological and pathological processes. From the discovery of a particular form of proteasome regulation of the catabolism of the key regulatory enzyme in their synthetic pathway, to the experimental cancer treatment or prevention with polyamine antagonists or inhibitors of the latter enzyme, a whole spectrum of interests can be revealed. Still, many aspects of their functions remain elusive and difficulties inherent in their analysis, which relies on sophisticated high-performance liquid chromatographic (HPLC) methods, and the lack of standardization; have hampered the transit from the research realm to the standard clinical laboratory domain. Their assay in biological fluids has been used for cancer diagnosis and for monitoring anticancer treatment. In this article, we attempt to provide an overview of polyamine structure, nutritional value, metabolism, and physiological roles. Next, we will summarize the main analytical methods on which we count, and finally we will address their role in diagnosis of cancer as well their proposed role as antioxidant and antiglycation agents.

The polyamines spermine and spermidine protect proteins from structural and functional damage by AGE precursors: a new role for old molecules?

Due to the importance of glycation in the genesis of diabetic complications, an intense search for synthetic new antiglycation agents is ongoing. However, a somewhat neglected avenue is the search for endogenous compounds that may inhibit the process and be a source of protodrugs. Based on their ubiquity, their polycationic nature, their essential role in growth, their relatively high concentrations in tissues, and their high concentrations in sperm, we hypothesized that polyamines inhibit glycation and that might be one of their so far elusive functions. In this study we demonstrate a potent antiglycation effect of physiological concentrations of the polyamines spermine and spermidine. We employed two approaches: in the first, we monitored structural changes on histones and ubiquitin in which polyamines inhibit glycation-induced dimer and polymer formation. In the second we monitored functional impairment of catalytic activity of antithrombin III and plasminogen. Protection is afforded against glycation by hexoses, trioses and dicarbonyls AGE precursors and is comparable to those of aminoguanidine and carnosine.

The botanical extracts of Achyrocline satureoides and Ilex paraguariensis prevent methylglyoxal-induced inhibition of plasminogen and antithrombin III.

Endogenously produced dicarbonyls, such as methylglyoxal (MG), are involved in advanced glycation end-product formation and thus linked to the pathophysiology of diabetic chronic complications. While the search for synthetic new antiglycation agents continues, little attention has been paid to putative antiglycation agents in natural compounds. Given the link between glycation and oxidation, in this work, we study the effects of methylglyoxal on two model systems; plasminogen and antithrombin III (AT III), then we set out to unravel a possible antiglycation effect for extracts of the flavonoid-rich common herbal species Achyrocline satureoides (AS) and Ilex paraguariensis (IP). Using SAR-PRO-ARG-pNA as a specific thrombin substrate, we show that incubation of plasma with MG decreases heparin activation of AT III by up to a 70%, in a dose-dependent manner. A parallel dose-dependent decrease in plasminogen activity reaching more than 50% was shown using D-BUT-CHT-lys-pNA as a plasmin-specific substrate. Extracts of AS and IP display a dose dependent inhibition of the action of the dicarbonyl, already significant at a 1/100 dilution of the herbal infusions. The inhibition was comparable to that obtained by using millimolar concentrations of known AGE inhibitors such as aminoguanidine and carnosine as well as micromolar concentrations of the antioxidant ascorbic acid. We believe our system of whole plasma glycation over 16 h with micromolar concentrations of MG, coupled with the measurement of activities of plasminogen and AT III by specific substrates provides a straightforward, practical method for monitoring the action of putative antiglycation agents. If predictably milder glycated forms of AT III and plasminogen were to be secreted in vivo, the loss of activities shown here could act synergistically to generate hyperthrombicity.

Is diabetic hypercoagulability an acquired annexinopathy? Glycation of annexin II as a putative mechanism for impaired fibrinolysis in diabetic patients.

Diabetics die mainly from thrombotic complications and there is clear evidence that diabetes is a hypercoagulable state. Epidemiological and prospective intervention data link hyperglycemia to vascular complications and glycation of proteins is one favored molecular basis to explain this fact. Cell surface receptors may support fibrinolytic surveillance in both intravascular and extravascular locations by stimulating plasmin generation and by protecting plasmin from its inhibitors. The existing experimental evidence suggests that annexin II in its tetrameric form is the main physiological receptor for plasminogen on the extracellular surface of endothelial cells. We have recently shown that annexin II is an extremely vulnerable target for glycation, quickly responding to restoration of normoglycemia. We hypothesize that glycation of endothelial membrane annexin II impairs the appropriate formation of the plasminogen/tissue plasminogen activator/annexin II complex, disrupting a key regulatory mechanism in fibrinolytic vigilance. This would in turn produce decreased fibrinolytic activity and indirectly promote a thrombophilic state in diabetic patients. We base our hypothesis on our observation and on evidence for the mechanism of action of two major independent risk factors for CV events: lipoprotein (a) and hyperhomocysteinemia. Binding of plasminogen to annexin II is inhibited by Lp (a) and binding of tissue plasminogen activator to annexin II is blocked by homocysteine. If our hypothesis is correct, one of the components of the increased thrombogenicity seen in diabetic patients might then be an acquired annexinopathy.

Three different pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides.

In this study we investigated the antioxidant properties of one herbal preparation widely used in complementary and alternative medicine in large areas of the world: Achyrocline satureoides (AS), popularly known as "marcela". Although rich in flavonoids, the ethnopharmacological uses of this plant do not include atherosclerosis prevention. Furthermore, no study had been conducted so far exploring the antioxidant activity of Achyrocline satureoides vis-à-vis human LDL oxidation, which is the compelling issue in pinpointing potential cardioprotective new uses for a traditional remedy. We explored the effects of AS extracts on human LDL oxidation, employing 3 different systems which are thought to play a role in oxidation of LDL in the arterial wall: copper, peroxynitrite, and lipoxygenase. Oxidation was monitored by conjugate dienes, TBARS formation and aggregation of apoB using SDS-PAGE. In copper-initiated oxidation a dose dependent inhibition of the initiation and propagation of lipid oxidation is shown by an increase in the lag phase for conjugate diene production which was 60 +/- 15 min in the absence and 120 +/- 20 min in the presence of 4 microg/ml AS extracts (p < 0.001). TBARS production was reduced by 95% after 3 h incubation at 5 microg/ml. Aggregation of apoB was abolished at the same concentrations. SIN-1 (3-morpholinosydnonimine) produces peroxynitrite via generation of NO and O2-. When LDL was incubated in its presence, a milder oxidation was observed as compared with Cu2+, and AS produced over 70% inhibition. Finally, we show a striking dose-dependent inhibitory effect of lipoxygenase conjugate diene production, which is over 95% at AS concentrations of 5 microg/ml. When compared with other antioxidants, AS effect is greater but in the same order of magnitude than that of ascorbic acid and similar to the popular herbal tea Ilex paraguariensis. In all three systems employed an effect is already substantiated at a concentration of the AS extract of 4 microg/ml, which corresponds to a 1/100 dilution of the preparations usually drunk.

Actin and annexins I and II are among the main endothelial plasmalemma-associated proteins forming early glucose adducts in experimental diabetes.

An immunochemical and biochemical study was performed to reveal which of the endothelial plasma membrane proteins become glycated during the early phases of diabetes. The blood front of the lung microvascular endothelial plasmalemma was purified by the cationic colloidal silica method from normal and diabetic (streptozotocin-induced) rats and comparatively analyzed by two-dimensional electrophoresis. No major qualitative differences in the general spectrum of endothelial plasmalemmal proteins were recorded between normoglycemic and hyperglycemic animals. By probing with anti-glucitollysine antibodies, we found that at 1 month after the onset of diabetes, several endothelial membrane polypeptides contained glucose covalently linked to their lysyl residues. Ten days of insulin treatment restored euglycemia in the diabetic animals and completely abolished the membrane nonenzymatic glycosylation. All the glycated polypeptides of the endothelial plasma membrane belong to the peripheral type and are associated with its cytoplasmic face (cell cortex). They were solubilized by buffers of high pH and were not detected in the lung cytosolic fraction (100,000 g). By microsequencing, the major proteins labeled by the anti-glucitollysine have been identified as being actin, annexin I, annexin II, the p34 subunit of the Arp2/3 complex, and the Ras suppressor protein-1. Conversely, the intrinsic endothelial membrane proteins do not seem to be affected by hyperglycemia. This defines the internal face of the endothelial plasma membrane, particularly the cortical cytoskeleton, as a preferential target for nonenzymatic glycosylation in diabetes, with possible consequences on the fluidity of the endothelial plasmalemma and impairment of the endothelial mechanotransducing ability.

Glycation as the glucose link to diabetic complications.

Hyperglycemia is considered a key causal factor in the development of diabetic vascular complications and can mediate its adverse effects through multiple pathways. This was confirmed for microangiopathy in the Diabetes Control and Complications Trial study for type 1 diabetes and corroborated for type 2 diabetes by the United Kingdom Prospective Diabetes Study published in 1998. Prevention of diabetic complications requires at least control of glycemia. This article briefly summarizes the evidence that strongly supports the role of hyperglycemia in vascular complications. After outlining the role of the polyol pathway, protein kinase C, and oxidative stress, the author focuses on one of the key biochemical mechanisms for this pathologic process: the direct deleterious action of glucose and other sugars on proteins, known as glycation or nonenzymatic glycosylation. Results of animal studies and phase III clinical trials reveal that the inhibition of this process attenuates the development of a range of these complications.

Expression, partial purification and functional properties of themuscle-specific calpain isoform p94.

The muscle-specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His-tagged p94 bound calcium with high affinity, and their autolytic activity required Ca2+. The sensitivity of the catalytic activity of the recombinant protease to Ca2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m- and micro-calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle-specific calpain isoform.

Circulating advanced glycation peptides in streptozotocin-induced diabetic rats: evidence for preferential modification of IgG light chains.

As the glycation/glycoxidation hypothesis for the genesis of diabetic complications is achieving widespread acceptance, much attention is being paid to the role of low molecular weight advanced glycation (AGE) adducts, as second generation glycating agents. We set out a study with the objective of attesting the presence of increased amounts of AGE-peptides in the circulation of streptozotocin-induced diabetic rats and to determine the nature of the plasma proteins which are main targets for advanced glycation. AGE (Ex 370/Em 440 nm) and pentosidine fluorescence (Ex 335/Em 385 nm) were significantly higher in plasma from diabetic rats after only one month of hyperglycemia as compared to controls (35 +/- 7 vs 25 +/- 2 AU, p< 0.05 and 54 +/- 14 vs 27 +/- 3 AU, p< 0.01 respectively). AGE-peptides (<10 kDa) were more than two-fold higher in diabetic animals. Immunoblots after SDS-PAGE of plasma proteins showed that AGE-IgG displayed a selective predominant increment in the same animals. When native rat IgG was incubated in the presence of AGE-peptides isolated from diabetic animals, AGE modification was already apparent after only 24 h of incubation, and was particularly important for light chains. AGE-immunoreactive light chains displayed an apparent increase in molecular weight. Aminoguanidine prevented, while copper enhanced AGE binding to IgG light chains. Our data validate the streptozotocin-induced diabetic rat as a model reproducing the presence of circulating AGE-peptides, give evidence that IgG are preferential targets for advanced glycation in plasma and suggest that this modification, mediated by AGE-peptides, can be prevented by aminoguanidine.

Glycation of hepatocyte cytosolic proteins in streptozotocin-induced diabetic rats.

A role for glycation in diabetic pathology appears beyond doubt and one of the present trends is to focus the poorly explored field of intracellular glycation. In this work we studied the pattern of early glycation in hepatocyte cytosolic proteins from streptozotocin-induced diabetic rats (n=14) compared to control animals (n=8). Glycated proteins were present in the cytosol of control rats and increased three-fold after one month of diabetes, while glycated Hb and glycated plasma proteins rose two- and three-fold, respectively. A good correlation (r=0.82, p<0.001) was found between glycated cytosolic proteins and glycated plasma proteins, suggesting the latter could provide an indirect indication of intracellular glycation. Using PBA affinity chromatography followed by SDS-PAGE we detected 7 major glycated bands in cytosols from control animals which increased dramatically in diabetic rats. Moreover, other glycated proteins, which were undetectable in control animals, became prominent, and more than 15 major bands can thus be resolved. No major differences in the patterns can be seen after 1, 5, or 12 months of diabetes, suggesting that early glycation in cytosolic proteins reaches an equilibrium in a short period of one to two weeks (further supported by the tight correlation with glycated plasma proteins). Through comparison of the patterns obtained with an antiglucytollysine antibody on Western blots with those of silver stained gels from the PBA eluates we present evidence that intracellular glycation is mediated by glucose but mainly by other sugars.

Antioxidant effects of Ilex paraguariensis: induction of decreased oxidability of human LDL in vivo.

We have recently demonstrated that Ilex paraguariensis extracts inhibit LDL oxidation in vitro exhibiting a potency comparable to that of ascorbic acid (Gugliucci, A. and Stahl, A.J.C. 1995; Biochem Mol Biol Int 35, 47-56). In the present work we extend our observations to the in vivo situation. We first examined the oxidability of LDL in whole plasma from healthy fasted human subjects before and after intake of Ilex paraguariensis. Intake of water extracts of Ilex paraguariensis inhibit copper-induced autoxidation of LDL in whole plasma as shown by the end-term production of TBARS, and as a consequence are able to impair the appearance of Schiff base induced fluorescence, higher electrophoretic mobility and fragmentation of apoB. When LDL was isolated from plasma prior to oxidation no significant differences in lag-time, slope or maximum rate of oxidation could be detected. We then conclude that antioxidants in Ilex paraguariensis are absorbed and reach sufficient high levels in plasma to inhibit copper-induced LDL autoxidation by increasing aqueous-phase antioxidant capacity.

Renal fate of circulating advanced glycated end products (AGE): evidence for reabsorption and catabolism of AGE-peptides by renal proximal tubular cells.

The presence of excessive amounts of advanced glycation end products (AGE) in tissues or in the circulation may critically affect the progression of diabetic nephropathy. Circulating AGE levels, mainly in the form of small peptides, increase in diabetic patients or in patients with end-stage renal disease. This rise correlates with the severity of the nephropathy. However, so far little is known about the fate of AGE-proteins and AGE-peptides in renal tissue, and in order to elucidate this issue we undertook the present study. AGE-bovine serum albumin (AGE-BSA) and AGE-peptides were prepared, characterized by spectrophotometry, spectrofluorometry, chromatography and SDS-PAGE. AGE-peptides reacted in vitro with LDL producing biochemical and ultrastructural modifications. Using colloidal gold post-embedding immunoelectron microscopy with an anti-AGE antibody generated in our laboratory, we followed, in a short-term kinetic study, the cellular and sub-cellular localisation of circulating AGE-products throughout the nephron. AGE-peptides or AGE-BSA were injected into otherwise normal rats and detected by protein A-gold immuno-cytochemistry after 15, 30 or 45 min of circulation. Most of the AGE-BSA was found in the lumen of capillary vessels and distributed along the endothelial side of the glomerular basement membrane. Presence on mesangial matrix was also apparent. AGE-peptides were easily filtered and actively reabsorbed by the proximal convoluted tubule. At 15 min, little labelling was found in the glomerular wall. Instead, the labelling was present in the urinary space and microvilli of epithelial cells. Early endosomes displayed intense labelling as well. At 45 min, late endosomes and lysosomes added to the pattern of labelling. The distal tubule epithelial cells were devoid of labelling for any of the intervals studied. AGE-peptides but not AGE-BSA could be detected in the urine of injected rats. These observations point to participation of the endo-lysosomal apparatus of the proximal convoluted tubule to the disposal of AGE-peptides, while giving an ultrastructural support for a key role of the kidney in AGE catabolism.

Histones from diabetic rats contain increased levels of advanced glycation end products.

In a recent report we have demonstrated the in vitro formation of advanced glycation end products (AGEs) on histones in a time and sugar concentration dependent fashion. In the present work we examined histone advanced glycation in vivo. Diabetes was induced in rats by streptozotocin injection and the hyperglycemic state was maintained and surveyed for up to 24 weeks. Diabetic rats showed accumulation of early glycation products in plasma proteins and in hemoglobin. Histones from the liver of diabetic rats showed AGEs levels three-fold higher than those of their age-matched controls. Histone AGEs increased with the duration of diabetes and tended to increase with the age as well. Similar tendencies were apparent in skin collagen. Our data demonstrate that diabetes induces an increase in the accumulation of AGE products on histones. This reinforces the concept that advanced glycation occurs in intracellular proteins and suggests a possible role for intracellular glycation in the increased theratogeny associated with diabetes mellitus.