Fluorescent Advanced Glycation End-products Research Articles

Characterization of the acidic species of a monoclonal antibody using free flow electrophoresis fractionation and mass spectrometry

Antibody charge heterogeneity is one of the major product-related variants in recombinant biopharmaceuticals, which has been commonly monitored by imaged capillary isoelectric focusing (icIEF). Due to the challenges with sample recovery and fractionation, other charge-based analytical approaches have been explored as complementary methods allowing for further detailed charge variant characterization. This study describes the utilization of free flow electrophoresis (FFE) fractionation in combination with other analytical techniques, such as mass spectrometry for monoclonal antibody acidic variants characterization. The preparative FFE technique allowed for continuous sample separation and fluid phase fractionation of antibody charge isoforms. The monoclonal antibody starting material was fractionated by FFE, followed by purification and characterization. icIEF analysis demonstrated the purity of the fractions and comparability of the charge profiles between these two techniques. The intact molecular mass analysis revealed that glycation modification was highly enriched in the acidic fractions. SEC UV/Fluorescence method was developed to assess the levels of aggregation and fluorescent advanced glycation end-products (AGEs). Detailed peptide map was performed and revealed that acidic fractions were enriched in AGEs, methionine, tryptophan, histidine oxidation, asparagine deamidation, lysine glycation, carboxymethyl lysine, glycine to aspartic acid substitution compared to the main peak and starting material. The results indicate that acidic variants can account for a variety of low-level modifications present as very heterogeneous forms.

Serum Fluorescence of Advanced Glycation End Products: A Potential Screening Tool to Distinguish Between Diabetic Patients with and without Microvascular Complications

Introduction: Enhanced formation and accumulation of Fluorescent Advanced Glycation End products (F-AGEs) in diabetes mellitus have been linked to increased risk of developing the associated vascular complications. Aim: To evaluate the potential of serum fluorescence levels of F-AGEs as screening tools to distinguish between type 2 diabetic patients with and without microvascular complications such as retinopathy, neuropathy. Materials and Methods: This cross-sectional study was conducted between June 2016 and June 2017, included 95 type 2 diabetic patients with more than 1 year of diabetes duration. Fasting blood glucose, glycated haemoglobin and total protein levels were estimated by automated methods. Serum F-AGEs were estimated by using a simple spectrofluorometric method. Microvascular complications due to diabetes mellitus were studied in each patient from medical records data on fundus examination for retinopathy and touch, vibration sensation detection for neuropathy. Diabetic patients were categorised into two groups as those without microvascular complications and those with microvascular complications-retinopathy and neuropathy. Statistical tests used for comparisons between groups were chi-square test for gender distribution, independent t-test for other parameters and Pearson’s correlations. The p-value <0.05 indicated significant difference between variables. Results: Mean age of the population was 55.1±5.3 years. Diabetic patients with microvascular complications (n=26) in the form of retinopathy, neuropathy had significantly higher levels of serum F-AGEs with mean 7.4±1.8 AU/g protein compared with diabetic patients without complications with mean value 1.5±0.7 AU/g protein (p<0.01). Conclusion: Two categories of serum fluorescent AGE values, without overlap, could be distinguished between diabetic patients with and without complications. Measurement of serum F-AGEs products has the potential to emerge as a simple, valuable screening tool to distinguish between diabetic patients with and without microvascular complications.

Regional differences in turnover, composition, and mechanics of the porcine flexor tendon

ABSTRACT Purpose Recent data suggest that there is a lack of turnover in the core of human tendon, but it remains unknown whether there are regional differences between core and periphery of the cross section. The purpose of this project was to investigate regional differences in turnover as estimated by the accumulation of fluorescent Advanced Glycation End-products (AGEs) and regional differences in mechanical properties. Materials and methods Tendons were obtained from lean control (n = 4) and diabetic Göttingen minipigs (streptozotocin-induced, n = 6). The deep digital flexor tendon of one hind limb was separated into a proximal, central and distal part. Autofluorescence was measured in the core and periphery of the proximal and distal parts of the tendon, and mechanical properties were tested on fascicles taken from the core and periphery of the central tendon (only diabetic animals). Results Autofluorescence was greater in the proximal than the distal part. In the distal part of the lean control animals, autofluorescent AGE accumulation was also greater in the core than the periphery. Peak modulus in the core region (704 ± 79 MPa) was higher than the periphery (466 ± 53 MPa, p < 0.05) in diabetic tendons. Conclusion Taken together, autofluorescence varied both along the length and across the tendon cross section, indicating higher turnover in the distal and peripheral regions. In addition, mechanical properties differed across the tendon cross-section.

Curcumin combined with metformin decreases glycemia and dyslipidemia, and increases paraoxonase activity in diabetic rats

BackgroundCombination of current antidiabetic agents with natural antioxidants to manage diabetes mellitus and its complications has appeared as an emerging trend. Curcumin, a yellow pigment isolated from Curcuma longa rhizomes, has gained attention due to its beneficial effects in controlling the disturbances observed in diabetes mellitus. The purpose of this study was to investigate if yoghurt enriched with curcumin and metformin, individually or as mixtures, ameliorates physiometabolic parameters, glycoxidative stress biomarkers, and paraoxonase 1 (PON 1) activity in diabetic rats.MethodsStreptozotocin-diabetic rats (6-week-old Wistar rats) were treated for 30 days with curcumin and metformin, isolated or as mixtures in yoghurt (10 rats/group). After treatments, the plasma levels of glucose, triacylglycerol, cholesterol, thiobarbituric acid reactive substances (TBARS, a biomarker of lipid oxidation), fluorescent advanced glycation end products (AGEs), and the activity of PON 1, an antioxidant enzyme were assessed. Data were analyzed using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test.ResultsTreatment of diabetic rats with curcumin or metformin alone decreased the plasma levels of glucose, triacylglycerol, cholesterol, TBARS, and fluorescent AGEs, as well as increased the activity of PON 1. The combination of metformin with curcumin further decreased dyslipidemia and TBARS levels in diabetic rats, indicating synergy, and maintained the high levels of PON 1.ConclusionThese findings indicated that curcumin combined with metformin may act synergistically on dyslipidemia and oxidative stress, as well as increased PON 1 levels. Therefore, it might be a promising strategy for combating diabetic complications, mainly the cardiovascular events.

Dihydromyricetin as a Functional Additive to Enhance Antioxidant Capacity and Inhibit the Formation of Thermally Induced Food Toxicants in a Cookie Model.

Recently, there is a growing interest in fortifying food products with flavonoids to enhance health benefits. Naringenin, naringin, hesperetin, and dihydromyricetin are four typical flavonoids constituting a natural part of our diet. In the present work, they were fortified into a chia oil cookie model to evaluate their thermal stability during baking as well as their impact on antioxidant capacity and toxicant formation. Among them dihydromyricetin was the most unstable one (only 36.1% of which was left after baking at 180 °C for 20 min) and led to a loss of brightness in cookie. However, the antioxidant capacity of cookie fortified with dihydromyricetin was significantly enhanced compared with untreated cookie; on the other hand, dihydromyricetin showed the strongest effect to attenuate lipid and protein oxidation as well as decrease the level of fluorescent advanced glycation endproducts and carboxymethyl lysine in cookie model. Overall, among the four selected flavonoids, dihydromyricetin might be the most promising functional bakery additive enhancing the antioxidant capacity while decreasing the formation of toxicants.

Prognostic Values of Inflammatory and Redox Status Biomarkers on the Risk of Major Lower-Extremity Artery Disease in Individuals With Type 2 Diabetes.

Inflammation and oxidative stress play an important role in the pathogenesis of lower-extremity artery disease (LEAD). We assessed the prognostic values of inflammatory and redox status biomarkers on the risk of LEAD in individuals with type 2 diabetes. Plasma concentrations of tumor necrosis factor-α receptor 1 (TNFR1), angiopoietin-like 2, ischemia-modified albumin (IMA), fluorescent advanced glycation end products, protein carbonyls, and total reductive capacity of plasma were measured at baseline in the SURDIAGENE (Survie, Diabete de type 2 et Genetique) cohort. Major LEAD was defined as the occurrence during follow-up of peripheral revascularization or lower-limb amputation. Among 1,412 participants at baseline (men 58.2%, mean [SD] age 64.7 [10.6] years), 112 (7.9%) developed major LEAD during 5.6 years of follow-up. High plasma concentrations of TNFR1 (hazard ratio [95% CI] for second vs. first tertile 1.12 [0.62-2.03; P = 0.71] and third vs. first tertile 2.16 [1.19-3.92; P = 0.01]) and of IMA (2.42 [1.38-4.23; P = 0.002] and 2.04 [1.17-3.57; P = 0.01], respectively) were independently associated with an increased risk of major LEAD. Plasma concentrations of TNFR1 but not IMA yielded incremental information, over traditional risk factors, for the risk of major LEAD as follows: C-statistic change (0.036 [95% CI 0.013-0.059]; P = 0.002), integrated discrimination improvement (0.012 [0.005-0.022]; P < 0.001), continuous net reclassification improvement (NRI) (0.583 [0.294-0.847]; P < 0.001), and categorical NRI (0.171 [0.027-0.317]; P = 0.02). Independent associations exist between high plasma TNFR1 or IMA concentrations and increased 5.6-year risk of major LEAD in people with type 2 diabetes. TNFR1 allows incremental prognostic information, suggesting its use as a biomarker for LEAD.

Evaluation of Anti-glycation Activities of Phlorotannins in Human and Bovine Serum Albumin-glyceraldehyde Models

The anti-glycation activities of phlorotannins contained in the Japanese Lessoniaceae ( Ecklonia cava, Eck. kurome, Eck. stolonifera, Eisenia arborea, and Eis. bicyclis) were tested using serum albumin-glyceraldehyde (GA) models. In the human serum albumin (HSA)-GA model and the bovine serum albumin (BSA)-GA model, the concentrations of crude phlorotannins at 50% inhibition (IC50) of fluorescent advanced glycation end products (AGEs) formation was in the range of 0.48 to 0.70 mg/mL and 0.52 to 0.75 mg/mL, respectively. In tests using phloroglucinol and purified phlorotannins (eckol, fucofuroeckol A, phlorofucofuroeckol A, dieckol, and 8,8'-bieckol), dieckol had the highest inhibitory activity (IC50: 5.5 × 102 μM) against fluorescent AGEs formation in HSA-GA model and showed about 18 times inhibition compared with aminoguanidine hydrochloride of positive control. In the BSA albumin model, 8,8'-bieckol had about 27 times AGEs formation inhibitory activity (IC50: 6.2 × 102 μM) against aminoguanidine hydrochloride. In tests on GA scavenging activity, it was shown that compounds with phloroglucinol tetramer or higher had a scavenging rate of 70%, or more, with a reaction time of 120 minutes. These results suggest that among the phlorotannins, in particular the dimers of eckol (dieckol and 8,8'-bieckol), there are effective compounds for inhibiting the formation of AGEs derived from GA.

Effect of liquid-state fermentation on the antioxidant and functional properties of raw and roasted buckwheat flours

The influence of liquid-state fermentation (LSF) by selected lactic acid bacteria (LAB) and Rhizopus oligosporus fungi on the content of rutin and total phenolic compounds (TPC), antioxidant capacity measured by ABTS test, FRAP assay and photochemiluminescence technique, and the inhibitory activity against formation of fluorescent advanced glycation end-products (AGEs) in vitro of raw and roasted buckwheat flours was studied.LSF caused a slight, specific LAB-dependent increase in TPC and a decrease in rutin content. Fermented raw buckwheat flours contained higher amounts of rutin and TPC with one exception when the highest increase in TPC was noted in roasted flour fermented by fungi. A LAB-dependent difference in the antioxidant capacity of buckwheat flours was noted while the inhibitory activity of fermented flours against AGEs formation was generally reduced. It can be concluded that LSF with selected LAB and fungi may improve the antioxidant and functional properties of buckwheat flours.

Prognostic Values of Inflammation and Oxidative Stress Biomarkers on the Risk of Peripheral Arterial Disease in Type 2 Diabetes

Aim: We tested whether inflammation and oxidative stress biomarkers may predict the risk of peripheral arterial disease (PAD) in the SURDIAGENE (SURvie, DIAbete de type 2 et GENEtique) type 2 diabetes cohort. Methods: plasma concentrations of angiopoietin like 2 (ANGPTL2), TNF receptor 1 (TNFR1), fluorescent advanced glycation end products (F-AGE), advanced oxidation protein products (AOPP), protein carbonyl, ischemia-modified albumin (IMA), total reductive capacity of plasma (TRCP) and oxidative hemolysis inhibition assay (OxHLIA) were measured at baseline using ELISA, spectrofluorimetry, spectrophotometry and Folin-Ciocalteu methods. Cox model was fitted to assess the risk of severe PAD, requiring aortic or lower limb revascularization, by increasing tertiles of each biomarker at baseline adjusting for confounding variables. C-statistic was used to test PAD discrimination. Results: Among 1395 participants free for PAD at baseline (men 57%, mean±SD age 65±11 years, diabetes duration 14±10 years), aortic or lower limb revascularization was performed in 66 (4.7%) patients during 5.8±3.2 years of follow-up. The rate of revascularisation was higher in the upper tertiles (T2, T3) compared to the lower one (T1): TNFR1 (HR [95% CI] T2 vs. T1, 1.15 [1.01-1.32]; T3 vs. T1, 1.59 [1.34-1.88], trend p&amp;lt;0.0001), F-AGE (1.43 [1.25-1.64]; 2.[1.76-2.38], trend p&amp;lt;0.0001), and TRCP (1.00 [0.88-1.15]; 1.25 [1.09-1.44], trend p=0.002). Similar results were observed when the 3 markers were included together in the same model. TNFR1 (change in c-statistic 0.010 [0.004-0.015], p=0.001), F-AGE (0.026 [0.014-0.039], p&amp;lt;0.0001) and TRCP (0.017 [0.009-0.024], p&amp;lt;0.0001), considered individually or together (0.052 [0.038-0.065], &amp;lt;0.0001), improved PAD discrimination. Conclusion: Our results suggest that TNFR1, F-AGE, and TRCP can be used to screen severe PAD in type 2 diabetes, and support the role of inflammation and oxidative stress on the pathogenesis of PAD. Disclosure M. Nativel: None. F. Schneider: None. P. Saulnier: None. O. Meilhac: None. P. Rondeau: None. M. Cournot: None. L. Potier: Consultant; Self; Sanofi, Novo Nordisk A/S, Eli Lilly and Company. Board Member; Self; Merck Sharp &amp; Dohme Corp.. G. Velho: None. M. Marre: Board Member; Self; Abbott. Consultant; Self; AstraZeneca. Board Member; Self; Intarcia Therapeutics, Inc.. Consultant; Self; Eli Lilly and Company. Board Member; Self; Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S. Research Support; Self; Sanofi. Board Member; Self; Servier. R. Roussel: Advisory Panel; Self; AbbVie Inc., Abbott, Eli Lilly and Company, Sanofi, Novo Nordisk A/S, AstraZeneca. Speaker's Bureau; Self; Servier. Consultant; Self; Bayer AG. Advisory Panel; Self; Merck Sharp &amp; Dohme Corp.. Research Support; Self; Amgen Inc., Sanofi, Novo Nordisk A/S, Danone Research. Stock/Shareholder; Self; Iriade. Advisory Panel; Self; Physiogenex S.A.S.. V. Rigalleau: None. K. Mohammedi: Speaker's Bureau; Self; Novo Nordisk Inc.. Other Relationship; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Sanofi. Other Relationship; Self; Sanofi, Takeda Development Centre Europe Ltd., Boehringer Ingelheim Pharmaceuticals, Inc. S. Hadjadj: Consultant; Self; Abbott, Novo Nordisk A/S, Servier, AstraZeneca, Merck Sharp &amp; Dohme Corp.. Board Member; Self; Valbiotis. Consultant; Self; Sanofi, Eli Lilly and Company.

Thermal stability of collagen in naturally ageing and in vitro glycated rabbit tissues

The aim of the work was to compare glycation-related changes in thermal denaturation of collagen in naturally ageing and in vitro ribosylated tissues. Samples of cornea, meniscus and Achilles tendon from young adult and from ageing rabbits were compared. Moreover, in vitro glycated samples (ribose, 100 mg mL−1, 14 days) were prepared for both age groups. Collagen in tissues was characterized in terms of thermal denaturation parameters obtained from differential scanning calorimetry. The level of pentosidine and other fluorescent advanced glycation end products (AGEs) in the papain-digested tissue samples was evaluated using spectrofluorimetry (λex/em: 335/385 and 370/440 nm). In naturally ageing tissues, changes in properties of extracellular matrix collagen expressed in terms of parameters of thermal denaturation and fluorescent AGEs levels were tissue dependent and were related to differences in organization of matrix components. No signs of increased level of AGEs were found in the naturally ageing cornea, unlike in the tendon and meniscus. In vitro ribation proved by gains of fluorescent AGEs affected thermal stability of collagen in all tissues, both in young adult and in the ageing ones. The effects of ribation were considerably greater than the effects of natural ageing. The increase in denaturation temperature was similarly strong in all tissues and did not correlate with the increase in fluorescent AGEs. As a conclusion, parameters of collagen thermal denaturation are tissue and age dependent and an amount of fluorescent AGEs is not the only determinant of increasing thermal stability of glycated collagen.

Circulating Concentrations of Redox Biomarkers Do Not Improve the Prediction of Adverse Cardiovascular Events in Patients With Type 2 Diabetes Mellitus.

BackgroundDespite pathophysiological relevance and promising experimental data, the usefulness of biomarkers of oxidative stress for cardiac risk prediction is unclear. The aim of our study was to investigate the prognostic value of 6 biomarkers exploring different pathways of oxidative stress for predicting adverse cardiovascular outcomes in patients with type 2 diabetes mellitus beyond established risk factors.Methods and ResultsThe SURDIAGENE (Survie, Diabete de type 2 et Genetique) prospective cohort study consecutively recruited 1468 patients with type 2 diabetes mellitus. Assays were performed at baseline, and incident cases of major adverse cardiovascular events (MACE)—first occurrence of cardiovascular death, nonfatal myocardial infarction, or stroke—were recorded during a median of 64 months. Advanced oxidation protein products, oxidative hemolysis inhibition assay, ischemia‐modified albumin, and total reductive capacity of plasma were not associated with the risk of MACE in univariate analyses. Fluorescent advanced glycation end products and carbonyls were associated with MACE (hazard ratio=1.38 per SD, 95% confidence interval 1.24‐1.54, P<0.001 and hazard ratio=1.15 per SD, 95% confidence interval 1.04‐1.27, P=0.006, respectively) in univariate analysis, but when added to a multivariate predictive model including traditional risk factors for MACE, these markers did not significantly improve c‐statistics or integrated discrimination index of the model.ConclusionsThese plasma concentrations of 6 markers, which cover a broad spectrum of oxidative processes, were not significantly associated with MACE occurrence and were not able to improve MACE risk discrimination and classification beyond classical risk factors in type 2 diabetes mellitus patients.

Steamed bread enriched with quercetin as an antiglycative food product: its quality attributes and antioxidant properties.

Quercetin, a natural antiglycative agent, was incorporated into steamed bread to produce a functional food that has high potential to lower the risk of diabetes. With the incorporation of quercetin at 1.20, 2.40, and 3.60%, the volume of steamed bread significantly decreased and the hardness of the crumb correspondingly increased with incremental quercetin content, while incorporation levels below 1.20% had no impact. Within this range of enrichment (1.2-3.6%), quercetin negatively affected the yeast activity with significantly less CO2 produced in dough during proofing. The wheat protein structure was altered by quercetin in terms of a higher level of β-sheets and a lower level of β-turns. The antioxidant capacity of the steamed bread with quercetin (0.05-0.2%) was significantly enhanced dose-dependently. A high inhibitory activity of quercetin-enriched steamed bread (0.05-0.2%) against fluorescent advanced glycation endproducts (AGEs) via several different mechanisms was observed. The inhibition of total AGEs from 0.2% quercetin-enriched steamed bread was around 40% during in vitro protein glycation. Overall, the results support quercetin-enriched steamed bread to be a promising functional food with high antioxidant and antiglycation properties.

Impact of resveratrol, epicatechin and rosmarinic acid on fluorescent AGEs and cytotoxicity of cookies

In this study, cookies were incorporated with three polyphenols (rosmarinic acid, resveratrol, and epicatechin) at two concentrations (0.02% and 0.2%), and baked under three temperatures (150, 170, and 190 °C) to observe their interaction. They didn’t significantly change the cookie texture, but influence the color and pH. Temperature was a significant determinant to affect polyphenols’ stability and accordingly influenced cookies’ antioxidant capacity. 73.4%, 26.7% and 9.9% of rosmarinic acid, resveratrol, and epicatechin remained after baking at 190 °C for 10 min. Two addition levels of polyphenols decreased fluorescent advanced glycation endproducts (AGEs) by 18.58% to 40.29% and 28.60% to 62.05% respectively, while only resveratrol and epiatechin could reduce the level of glyoxal and methylglyoxal in cookies. In addition, we discovered that thermal processing could enhance the cytotoxicity of rosmarinic acid against cancer cells. Thus polyphenols were suggested as beneficial additives for enhancing antioxidant intake and lowering total fluorescent AGEs.

Evaluation of Anti-glycation Activities of Phlorotannins in Human and Bovine Serum Albumin-methylglyoxal Models

In this study, the anti-glycation activities of phlorotannins contained in the Japanese Lessoniaceae ( Ecklonia cava, Eck. kurome, Eck. stolonifera, Eisenia arborea, and Eis. bicyclis) were tested using serum albumin-methlglyoxal (MGO) models. In the human serum albumin (HSA)-MGO model and the bovine serum albumin (BSA)-MGO model, the concentrations of crude phlorotannins at 50% inhibition (IC50) of fluorescent advanced glycation end products (AGEs) formation was in the range of 0.45 to 0.53 mg/mL and 0.43 to 0.53 mg/mL, respectively. Among the purified phlorotannins, phlorofucofuroeckol A with the benzobisbenzofuran skeleton had the highest inhibitory activities (IC50: 4.8 × 102 μM in HSA-MGO model and 4.1 × 102 μM in BSA-MGO model) against fluorescent AGEs formation, and showed about 13 times the inhibition in the HSA-MGO model, and about 20 times the inhibition in the BSA-MGO model, with respect to aminoguanidine hydrochloride, which was used as a positive control. In the test on MGO-scavenging activity using HPLC analysis, it showed that eckol and its simple dimers (dieckol and 8,8′-bieckol) had higher scavenging rate than aminoguanidine hydrochloride. In addition, it was revealed by liquid chromatography-electrospray ionization-mass spectrometry that eckol reacted with MGO and formed two adducts with it. These results strongly suggest that phlorotannins are novel and effective anti-glycation substances derived from natural plants.

Characterization, antioxidant and antiglycation properties of polysaccharides extracted from the medicinal halophyte Carpobrotus edulis L

In this study, Box-Behnken design was used to optimize the ultrasonic extraction of Carpobrotus edulis polysaccharides (CEP), and the effect of time, extraction temperature and water to material ratio was evaluated. Optimum conditions were 1.77h, 78.0°C and 33.04mL/g to improved CEP yield (7.84%), which is in good agreement with the predicted yield 7.77%. Then, the physico-chemical, antioxidant and antiglycation properties of optimized CEP were studied, and the total sugar and galacturonic acid content were 89.7 and 63.2%, respectively. The composition of neutral monosaccharide was arabinose, xylose, rhamnose and mannose in the molar percentage of 71.84, 14.80, 8.57, and 4.79%, respectively. In addition, (1H, and 13C) NMR and FTIR analyses confirmed the presence of uronic acids in the free and methyl ester forms with a degree of esterification of 31.27%. Therefore, this finding showed that CEP is a low methoxyl pectic polysaccharide, with an average molecular weight about 65,000g/mol. Finally, the results indicated that CEP presents strong antioxidant activities in vitro (DPPH, chelating ability and reducing power), and significantly inhibits lipid peroxidation and the formation of fluorescent advanced glycation end products in glucose-BSA system model.

Syzygium cumini leaf extract inhibits LDL oxidation, but does not protect the liproprotein from glycation

Etnopharmacological relevanceSyzygium cumini (L.) Skeels is a plant widely used in folk medicine to treat diabetes mellitus (DM). The tea from its leaves is frequently used by diabetics for lowering hyperglycemia. There is a close relationship between DM and atherosclerosis, a chronic immuno-inflammatory disease, were the early stages encompass oxidative and glycative modifications in the structure of low density lipoprotein (LDL). Aim of this studyTo investigate the potential protective effects of aqueous-leaf extract from Syzygium cumini (S.cExt) against CuSO4-induced oxidation and methylglyoxal (MG)-induced glycation of human LDL in vitro. Materials and methodsLDL oxidative changes were evaluated by measuring conjugated dienes (CD) formation, thiobarbituric acid reactive substances (TBARS) levels, quenching of tryptophan (Trp) fluorescence and structural modifications in LDL particle. In LDL glycated by MG (glyLDL), we determined the levels of fluorescent advanced glycation end products (AGEs) and mobility by agarose gel electrophoresis. ResultsS.cExt blocked oxidative events induced by CuSO4 in human LDL, plasma and serum. Fourier transform infrared spectroscopy (FT-IR) revealed that specific regions of apoB100 were oxidized by CuSO4 in human LDL and that S.cExt reduced these oxidations. Unlike, the increased AGEs levels and eletrophoretic mobility observed in LDL MG-glycated were not modified by S.cExt. ConclusionThe findings herein indicate that S.cExt could be tested in atherogenesis models as potential protective agent against LDL oxidation.

Digestibility of Glyoxal-Glycated β-Casein and β-Lactoglobulin and Distribution of Peptide-Bound Advanced Glycation End Products in Gastrointestinal Digests.

This work reports the influence of glyoxal (GO)-derived glycation on the gastrointestinal enzymatic hydrolysis of β-lactoglobulin and β-casein. Reduced digestibility of glycated proteins was found in both gastric and intestinal stage. Distribution of Maillard reaction products in digests with different molecular weight ranges was investigated subsequently. The colorless and brown MRPs largely presented in the digests smaller than 20 kDa. However, the resistance of fluorescent advanced glycation end products (AGEs) to enzymatic hydrolysis gradually increased during glycation, rendering fluorescent AGEs largely present in the digests larger than 20 kDa. No free N (ε)-carboxymethyllysine (CML) was detected in digests. The relative amount of CML in digests larger than 1 kDa was higher than that of Lys, demonstrating the hindrance of CML to enzymatic hydrolysis. This study highlights the resistance of GO-derived AGEs to digestive proteases via blockage of tryptic cleavage sites or steric hindrance, which is a barrier to the absorption of dietary AGEs.

Formation of Pentosidine Cross-Linking in Myoglobin by Glyoxal: Detection of Fluorescent Advanced Glycation End Product.

Glyoxal, a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significance of protein modification by glyoxal-derived AGEs, we investigated the in vitro effect of glyoxal (200μM) on the monomeric heme protein myoglobin (Mb) (100μM) after incubation for one week at 25°C. Glyoxal-treated Mb exhibited increased absorbance around the Soret region, decreased α-helicity and thermal stability compared to control Mb. Intrinsic fluorescence spectrum of the treated Mb showed an additional signal in the 400-500nm region on excitation at 280nm that was absent in control Mb. When excited at 335nm, the glyoxal-treated sample gave a strong fluorescence indicating AGE formation. Mass spectrometric studies revealed formation of glyoxal-derived fluorescent AGE adduct pentosidine between Lys-145 and Arg-139 residues of Mb. Other than pentosidine, additional AGE adducts, namely, carboxymethyllysine at Lys-133, hydroimidazolone at Arg-31 and pyrrolidone-carboxymethyllysine at Lys-145 were also detected. Lys-145 was thus found to contain two different types of AGE adducts, indicating the heterogeneous nature of in vitro glycation reaction. AGE-induced protein modifications might be associated with complications in disease conditions.

Glycation of Lysozyme by Glycolaldehyde Provides New Mechanistic Insights in Diabetes-Related Protein Aggregation.

Glycation occurs in vivo as a result of the nonenzymatic reaction of carbohydrates (and/or their autoxidation products) with proteins, DNA, or lipids. Protein glycation causes loss-of-function and, consequently, the development of diabetic-related diseases. Glycation also boosts protein aggregation, which can be directly related with the higher prevalence of aggregating diseases in diabetic people. However, the molecular mechanism connecting glycation with aggregation still remains unclear. Previously we described mechanistically how glycation of hen egg-white lysozyme (HEWL) with ribose induced its aggregation. Here we address the question of whether the ribose-induced aggregation is a general process or it depends on the chemical nature of the glycating agent. Glycation of HEWL with glycolaldehyde occurs through two different scenarios depending on the HEWL concentration regime (both within the micromolar range). At low HEWL concentration, non-cross-linking fluorescent advanced glycation end-products (AGEs) are formed on Lys side chains, which do not change the protein structure but inhibit its enzymatic activity. These AGEs have little impact on HEWL surface hydrophobicity and, therefore, a negligible effect on its aggregation propensity. Upon increasing HEWL concentration, the glycation mechanism shifts toward the formation of intermolecular cross-links, which triggers a polymerization cascade involving the formation of insoluble spherical-like aggregates. These results notably differ with the aggregation-modulation mechanism of ribosylated HEWL directed by hydrophobic interactions. Additionally, their comparison constitutes the first experimental evidence showing that the mechanism underlying the aggregation of a glycated protein depends on the chemical nature of the glycating agent.

A quick, simple method for detecting circulating fluorescent advanced glycation end-products: Correlation with in vitro and in vivo non-enzymatic glycation

ObjectiveAdvanced glycation end-products (AGEs) constitute a highly heterogeneous family of compounds, relevant in the pathogenesis of diabetic complications, which could represent efficient biomarkers of disease progression and drug response. Unfortunately, due to their chemical heterogeneity, no method has been validated to faithfully monitor their levels in the course of the disease. In this study, we refine a procedure to quantitatively analyze fluorescent AGEs (fAGEs), a subset considered remarkably representative of the entire AGE family, and measure them in in vitro glycated BSA (gBSA) and in plasma and vitreous of diabetic rats, for testing its use to possibly quantify circulating AGEs in patients, as markers of metabolic control. MethodsfAGE levels were evaluated by spectrofluorimetric analysis in in vitro and in vivo experimental models. BSA was glycated in vitro with increasing D-glucose concentrations for a fixed time or with a fixed D-glucose concentration for increasing time. In in vivo experiments, streptozotocin-induced diabetic rats were studied at 1, 3, 6 and 12weeks to analyze plasma and vitreous. To confirm the presence of AGEs in our models, non-diabetic rat retinal explants were exposed to high glucose (HG), to reproduce short-term effects, or in vitro gBSA, to reproduce long-term effects of elevated glucose concentrations. Rat retinal explants and diabetic retinal tissues were evaluated for the receptor for advanced glycation end-product (RAGE) by Western blot analysis. ResultsIn in vitro experiments, fluorescence emission showed glucose concentration- and time-dependent increase of fAGEs in gBSA (p≤0.05). In streptozotocin-induced diabetic rats, fAGE in plasma and vitrei showed an increase at 6 (p≤0.005) and 12 (p≤0.05) weeks of diabetes, with respect to control. RAGE was time-dependently upregulated in retinas incubated with gBSA, but not with HG, and in diabetic retinal tissue, substantiating exposure to AGEs. ConclusionsApplying the proposed technique, we could show that fAGEs levels increase with glucose concentration and time of exposure in vitro. Furthermore, in diabetic rats, it showed that circulating fAGEs are similarly upregulated as those in vitreous, suggesting a correlation between circulating and tissue AGEs. These results support the use of this method as a simple and reliable test to measure circulating fAGEs and monitor diabetes progression.