Methylglyoxal-derived Hydroimidazolone-1 Research Articles

Methylglyoxal-derived hydroimidazolone-1/RAGE axis induces renal oxidative stress and renal fibrosis in vitro and in vivo

Advanced glycation end products (AGEs) are important contributors to the progression of chronic kidney diseases (CKD), including renal fibrosis. Although the relationship between AGEs and renal fibrosis has been well studied, the mechanisms of individual AGE-induced renal injury remain poorly understood. This study investigated the adverse effect of methylglyoxal-derived hydroimidazolone-1 (MG-H1), a methylglyoxal (MG)-derived AGE generated by the glycation of MG and arginine residues, on kidney damage. We aimed to elucidate the molecular mechanisms of MG-H1-mediated renal injury and fibrosis, focusing on the receptor for AGEs (RAGE) signaling and its effects on the Wnt/β-catenin pathway, MAPK pathway, and inflammatory responses. Our results suggest that the MG-H1/RAGE axis plays a significant role in the pathogenesis of CKD and its downstream events involving MAPK kinase-related factors and inflammatory factors. MG-H1 treatment modulated the expression of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and MAPK proteins (ERK1/2, JNK, and p38).

Glycative stress inhibits hypertrophy and impairs cell membrane integrity in overloaded mouse skeletal muscle.

Glycative stress, characterized by the formation and accumulation of advanced glycation end products (AGEs) associated with protein glycation reactions, has been implicated in inducing a decline of muscle function. Although the inverse correlation between glycative stress and muscle mass and strength has been demonstrated, the underlying molecular mechanisms are not fully understood. This study aimed to elucidate how glycative stress affects the skeletal muscle, particularly the adaptive muscle response to hypertrophic stimuli and its molecular mechanism. Male C57BL/6NCr mice were randomly divided into the following two groups: the bovine serum albumin (BSA)-treated and AGE-treated groups. Mice in the AGE-treated group were intraperitoneally administered AGEs (0.5mg/g) once daily, whereas those in the BSA-treated group received an equal amount of BSA (0.5mg/g) as the vehicle control. After 7days of continuous administration, the right leg plantaris muscle of mice in each group underwent functional overload treatment by synergist ablation for 7days to induce muscle hypertrophy. In in vitro studies, cultured C2C12 myocytes were treated with AGEs (1mg/mL) to examine cell adhesion and cell membrane permeability. Continuous AGE administration increased the levels of fluorescent AGEs, Nε-(carboxymethyl) lysine, and methylglyoxal-derived hydroimidazolone-1 in both plasma and skeletal muscle. Plantaris muscle weight, muscle fibre cross-sectional area, protein synthesis rate, and the number of myonuclei increased with functional overload in both groups; however, the increase was significantly reduced by AGE treatment. Some muscles of AGE-treated mice were destroyed by functional overload. Proteomic analysis was performed to explore the mechanisms of muscle hypertrophy suppression and myofibre destruction by AGEs. When principal component analysis was performed on 4659 data obtained by proteomic analysis, AGE treatment was observed to affect protein expression only in functionally overloaded muscles. Enrichment analysis of the 436 proteins extracted using the K-means method further identified a group of proteins involved in cell adhesion. Consistent with this finding, dystrophin-glycoprotein complex proteins and cell adhesion-related proteins were confirmed to increase with functional overload; however, this was attenuated by AGE treatment. Additionally, the treatment of C2C12 muscle cells with AGEs inhibited their ability to adhere and increased cell membrane permeability. This study indicates that glycative stress may be a novel pathogenic factor in skeletal muscle dysfunctions by causing loss of membrane integrity and preventing muscle mass gain.

Effects of SGLT2 Inhibitors and DPP-4 Inhibitors on Advanced Glycation End Products.

Clinical trials have revealed that sodium glucose cotransporter 2 (SGLT2) inhibitors suppress the onset of heart failure and cardiovascular death in diabetic patients. On the other hand, few reports have been published concerning such effects of dipeptidyl peptidase-4 (DPP-4) inhibitors. We undertook the present study to evaluate the effects of SGLT2 inhibitors and DPP-4 inhibitors on the advanced glycation end products (AGEs), well known as a risk factor for the development of cardiovascular disorders.Type 2 diabetes mellitus were divided into two groups and treated with either SGLT2 inhibitors or DPP-4 inhibitors for 3 months. Before and after the 3-month treatment period with each drug, the AGEs and diabetes-related parameters were measured. Methylglyoxal-derived hydroimidazolone-1 (MG-H1) was measured as one of the AGEs.In the SGLT2 inhibitor group, both the blood HbA1c and MG-H1 levels decreased significantly after the 3-month treatment period. In the DPP-4 inhibitor group, only the blood HbA1c level decreased significantly, with no significant change of the blood MG-H1 level.SGLT2 inhibitor reduced both the blood levels of HbA1c and AGEs (MG-H1). Considering that the blood levels of AGEs are associated with the risk of heart failure and cardiovascular disorders, the results of the present study suggest that the effect of SGLT2 inhibitors in suppressing cardiovascular death might be mediated by the reduction in the blood levels of AGEs induced by this class of drugs. DPP-4 inhibitors showed no significant effects on the blood levels of AGEs.

Angiotensin II reduces glyoxalase 1 activity and expression in vascular smooth muscle cells: Implications for diabetic vascular complications.

Angiotensin II (Ang II), a key mediator of vascular diseases, is linked to methylglyoxal (MGO) formation, a by-product of glucose metabolism implicated in vascular complications. The glyoxalase system, consisting of glyoxalase 1 (Glo1) and reduced glutathione (GSH), is responsible for detoxifying MGO. This study investigated the effect of Ang II on Glo1 activity and expression in vascular smooth muscle cells (VSMCs). Primary VSMCs were isolated from rat aortas and exposed to Ang II under standard or high glucose conditions. We examined Glo1 activity, expression, intracellular GSH, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) levels. We also analyzed the expressions of nuclear factor-κB (NF-κB) p65 and nuclear factor erythroid 2-related factor 2 (Nrf2) as potential regulators of Glo1 expression. The results demonstrated that Ang II reduced Glo1 activity, expression, and GSH levels while increasing MG-H1 levels in VSMCs. Telmisartan and irbesartan, AT1R blockers, restored Glo1 activity, expression, and GSH levels and alleviated MG-H1 levels. Treatment with AT1R blockers or inhibitors targeting signaling pathways involved in Ang II-induced responses mitigated these effects. High glucose exacerbated the reduction in Glo1 activity and expression. In conclusion, this study provides evidence that Ang II reduces Glo1 activity and expression in VSMCs, which may contribute to developing vascular complications in diabetes. AT1R blockers and inhibitors targeting specific signaling pathways show potential in restoring Glo1 function and mitigating MGO-associated damage. These findings highlight the complex interactions between RAS, MGO, and vascular diseases, highlighting potential therapeutic targets for diabetic vascular complications.

Holothuria scabra Jaegar 1833 (Sandfish) extracts and collagens modulate protein‐bound Nε‐carboxymethyllysine, Nε‐carboxyethyllysine and methylglyoxal‐derived hydroimidazolone‐1 levels

SummaryIn this study, sea cucumber (Holothuria scabra; Jaegar 1833) extracts and collagens were evaluated for inhibitory properties of protein‐bound advanced glycation end products (AGEs). Processed dried sea cucumber with salt extract showed a significant lower IC50 value for fluorescent AGEs (9.19 ± 7.68 μg mL−1, P < 0.05) and fructosamine (503.47 ± 46.37 μg mL−1, P < 0.05), respectively. Processed dried with and without salt extracts significantly reduced the Nε‐carboxymethyllysine (CML) and methylglyoxal‐derived hydroimidazolone‐1 (MG‐H1) levels tested at 250 μg mL−1. Smoked dried and fresh‐dried extracts significantly reduced the Nε‐carboxyethyllysine (CEL) levels tested at 250 μg mL−1. Pepsin‐solubilised collagen and the crude collagen fibrils significantly reduced CML and MG‐H1 levels whereas CEL levels were unchanged. Pearson's correlation analysis showed that protein‐bound AGE and CML inhibition significantly correlated with the total phenolic and antioxidant activities, respectively. This study provides a strong rationale for further investigation aimed at identifying the active compounds responsible for the observed effects on biomarkers relevant to diabetes.

The Intake of Dicarbonyls and Advanced Glycation Endproducts as Part of the Habitual Diet Is Not Associated with Intestinal Inflammation in Inflammatory Bowel Disease and Irritable Bowel Syndrome Patients.

A Western diet comprises high levels of dicarbonyls and advanced glycation endproducts (AGEs), which may contribute to flares and symptoms in inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). We therefore investigated the intake of dietary dicarbonyls and AGEs in IBD and IBS patients as part of the habitual diet, and their association with intestinal inflammation. Food frequency questionnaires from 238 IBD, 261 IBS as well as 195 healthy control (HC) subjects were used to calculate the intake of dicarbonyls methylglyoxal, glyoxal, and 3-deoxyglucosone, and of the AGEs Nε-(carboxymethyl)lysine, Nε-(1-carboxyethyl)lysine and methylglyoxal-derived hydroimidazolone-1. Intestinal inflammation was assessed using faecal calprotectin. The absolute dietary intake of all dicarbonyls and AGEs was higher in IBD and HC as compared to IBS (all p < 0.05). However, after energy-adjustment, only glyoxal was lower in IBD versus IBS and HC (p < 0.05). Faecal calprotectin was not significantly associated with dietary dicarbonyls and AGEs in either of the subgroups. The absolute intake of methylglyoxal was significantly higher in patients with low (<15 μg/g) compared to moderate calprotectin levels (15−<50 μg/g, p = 0.031). The concentrations of dietary dicarbonyls and AGEs generally present in the diet of Dutch patients with IBD or IBS are not associated with intestinal inflammation, although potential harmful effects might be counteracted by anti-inflammatory components in the food matrix.

Formation of advanced glycation end‐products in glycation of silver carp myofibrillar protein with glucose: Relationship with its chemical indicators

SummaryThe formation of advanced glycation end products (AGEs), regarded as a potentially harmful substance, in the protein glycation course is still under investigation. This study investigated the glycation modification of silver carp myofibrillar protein (MF) with glucose via Maillard‐driven chemistry. The resultant conjugates and glycation extent were characterised by chemical indicators, such as Fourier transform infrared spectroscopy, sodium dodecyl sulphate–polyacrylamide gel electrophoresis, colour development, and AGEs level. As the primary responsibility for glycation, the loss of Lysine (Lys) and Arginine (Arg) in MF with longer glycation time (&gt;24 h) was more than 25%. While, the associated AGEs, including Nε‐carboxymethyl‐lysine (CML), Nε‐carboxyethyl‐lysine (CEL), and methylglyoxal‐derived hydroimidazolone 1 (MG‐H1), presented a gradually increasing tendency with glycation time. The kinetic studies indicated that the formation of CML and MG‐H1 presented a highly linear correlation with heating time, while the CEL development could be described as an exponential function of glycation time (r ≥ 0.95). Moreover, the correlation analysis among these chemical indicators indicated that colour development was positively correlated with AGEs (r ≥ 0.85), while negatively related to the loss of Lys and Arg (r ≤ −0.89). The findings may help to better control the glycation course of food protein based on the formation of AGEs.

High Molecular Weight Fucoidan Restores Intestinal Integrity by Regulating Inflammation and Tight Junction Loss Induced by Methylglyoxal-Derived Hydroimidazolone-1.

Fucoidan from brown seaweeds has several biological effects, including preserving intestinal integrity. To investigate the intestinal protective properties of high molecular weight fucoidan (HMWF) from Undaria pinnatifida on intestinal integrity dysfunction caused by methylglyoxal-derived hydroimidazolone-1 (MG-H1), one of the dietary advanced-glycation end products (dAGEs) in the human-colon carcinoma-cell line (Caco-2) cells and ICR mice. According to research, dAGEs may damage the intestinal barrier by increasing gut permeability. The findings of the study showed that HMWF + MG-H1 treatment reduced by 16.8% the amount of reactive oxygen species generated by MG-H1 treatment alone. Furthermore, HMWF + MGH-1 treatment reduced MG-H1-induced monolayer integrity disruption, as measured by alterations in transepithelial electrical resistance (135% vs. 75.5%) and fluorescein isothiocyanate incorporation (1.40 × 10−6 cm/s vs. 3.80 cm/s). HMWF treatment prevented the MG-H1-induced expression of tight junction markers, including zonula occludens-1, occludin, and claudin-1 in Caco-2 cells and mouse colon tissues at the mRNA and protein level. Also, in Caco-2 and MG-H1-treated mice, HMWF plays an important role in preventing receptor for AGEs (RAGE)-mediated intestinal damage. In addition, HMWF inhibited the nuclear factor kappa B activation and its target genes leading to intestinal inflammation. These findings suggest that HMWF with price competitiveness could play an important role in preventing AGEs-induced intestinal barrier dysfunction.

Studies about the Dietary Impact on "Free" Glycation Compounds in Human Saliva.

Glycation reactions play a key role in post-translational modifications of amino acids in food proteins. Questions have arisen about a possible pathophysiological role of dietary glycation compounds. Several studies assessed the metabolic fate of dietary glycation compounds into blood and urine, but studies about saliva are rare. We investigated here the dietary impact on salivary concentrations of the individual Maillard reaction products (MRPs) N-ε-fructosyllysine, N-ε-carboxymethyllysine (CML), N-ε-carboxyethyllysine (CEL), pyrraline (Pyr), and methylglyoxal-derived hydroimidazolone 1 (MG-H1). Quantitation was performed using stable isotope dilution analysis (LC-MS/MS). We describe here, that a low MRP diet causes a significant lowering of salivary levels of Pyr from 1.9 ± 0.4 ng/mL to below the LOD and MG-H1 from 2.5 ± 1.5 ng/mL to 0.7 ± 1.8 ng/mL. An impact on the salivary protein fraction was not observed. Furthermore, salivary Pyr and MG-H1 levels are modified in a time-dependent manner after a dietary intervention containing 1.2 mg Pyr and 4.7 mg MG-H1. An increase in mean salivary concentrations to 1.4 ng/mL Pyr and 4.2 ng/mL MG-H1 was observed within 30–210 min. In conclusion, saliva may be a useful tool for monitoring glycation compound levels by using Pyr and MG-H1 as biomarkers for intake of heated food.

879-P: Effect of Combined SGLT2 Inhibitor and DPP-4 Inhibitor Therapy on the Production of Advanced Glycation End Products

SGLT2 inhibitors (SGLT2is) prevent disease progression in diabetic patients with atherosclerotic disease. On the other hand, a similar beneficial effect on disease progression has not been reported for DPP-4 inhibitors (DPP-4is) , which are, however, widely used. To clarify the differences in effects between these two classes of drugs in diabetic patients, we focused on the production of advanced glycation end products (AGEs) and studied the effects of pretreatment with DPP-4i on the production of AGEs in patients who subsequently received SGLT2i. In the present study, Japanese patients with type 2 diabetes mellitus with unchanged glycemia control status for at least 6 months were administered SGLT2i (n = 11) for 3 months and then divided into a group that had already been receiving DPP-4i (n = 11) at the start of administration of the SGLT2i and the SGLT2i monotherapy group. We measured the blood levels of methylglyoxal-derived hydroimidazolone-1 (MG-H1) , which is an AGE, and also diabetes-related parameters before the start and after 3 months’ treatment with the SGLT2i. As a result, significant decreases of both HbA1c (from 8.6 ± 1.4 to 7.2 ± 1.4, P &amp;lt; 0.01) and circulating MG-H1 levels (from 0.48 ± 0.to 0.44 ± 0.07, P &amp;lt; 0.05) were observed in the SGLT2i monotherapy group. On the other hand, in the group that received SGLT2i in addition to a DPP-4i, the HbA1c value decreased significantly (from 7.2 ± 0.4 to 6.8 ± 0.5, P &amp;lt; 0.01) , whereas no significant change of the MG-H1 level was observed after the SGLT2i therapy. In conclusion, SGLT2i monotherapy decreased both the HbA1c and blood MG-H1 levels. However, no significant effect on the blood level of MG-H1 was observed in the combined DPP4i plus subsequent SGLT2i therapy group, suggesting that DPP4i possibly suppress the effect of the SGLT2i in reducing the production of MG-H1. Disclosure M.Kusunoki: Other Relationship; Taisho Pharmaceutical Holdings Co., Ltd. N.Wakazono: None. F.Hisano: None. T.Miyata: None.

Advanced Glycation End Products Associated With Cardiometabolic Biomarkers in Treated Human Immunodeficiency Virus Infection.

BackgroundDespite advances in antiretroviral therapy (ART), people living with human immunodeficiency virus (HIV) continue to be at increased risk of cardiometabolic complications compared to HIV-uninfected individuals. Advanced glycation end products (AGEs) are implicated in the development and progression of cardiometabolic complications in the general population. Their role in HIV remains unclear.MethodsACTG A5260s is a prospective open-label randomized trial in which ART-naive people living with HIV were randomized to tenofovir disoproxil fumarate /emtricitabine plus atazanavir/ritonavir, darunavir/ritonavir, or raltegravir over 96 weeks. Changes in circulating AGEs with ART initiation were assessed, and linear regression was used to examine the associations between serum AGEs with carotid intima-media thickness (cIMT), visceral and subcutaneous adipose tissue, total fat, lean mass, body mass index, insulin resistance, leptin, and adiponectin.ResultsOverall, 214 participants were included. Ninety percent were male, 48% were White, the median age was 36 years, median HIV-1 RNA was 4.58 log10 copies/mL, and median CD4 count was 338 cells/µL. Most AGEs remained relatively unchanged following 96 weeks of ART initiation, except for methylglyoxal-derived hydroimidazolone 1 (MG-H1), which increased following 96 weeks of ART (mean fold change, 1.15 [95% confidence interval, 1.02–1.30]). No differences were detected across ART regimens. Increases in AGE levels over time were associated with worsening body fat composition measures, insulin resistance, and cIMT, even after adjusting for clinically relevant factors.ConclusionsAGE levels did not decrease following ART initiation. Most AGE levels remained stable, except for MG-H1, which increased. In people with HIV on ART, the accumulation of circulating AGEs over time appears to be independently associated with worsening cardiometabolic biomarkers.Summary: Antiretroviral therapy (ART) does not appear to be effective in reducing advanced glycation end product (AGE) levels. On the contrary, AGE levels seem to increase following ART initiation. Accumulation of AGEs was found to be independently associated with cardiometabolic complications in treated people living with HIV.

Systemic inflammation down-regulates glyoxalase-1 expression: an experimental study in healthy males.

Background: Hypoxia and inflammation are hallmarks of critical illness, related to multiple organ failure. A possible mechanism leading to multiple organ failure is hypoxia- or inflammation-induced down-regulation of the detoxifying glyoxalase system that clears dicarbonyl stress. The dicarbonyl methylglyoxal (MGO) is a highly reactive agent produced by metabolic pathways such as anaerobic glycolysis and gluconeogenesis. MGO leads to protein damage and ultimately multi-organ failure. Whether detoxification of MGO into D-lactate by glyoxalase functions appropriately under conditions of hypoxia and inflammation is largely unknown. We investigated the effect of inflammation and hypoxia on the MGO pathway in humans in vivo.Methods: After prehydration with glucose 2.5% solution, ten healthy males were exposed to hypoxia (arterial saturation 80–85%) for 3.5 h using an air-tight respiratory helmet, ten males to experimental endotoxemia (LPS 2 ng/kg i.v.), ten males to LPS+hypoxia and ten males to none of these interventions (control group). Serial blood samples were drawn, and glyoxalase-1 mRNA expression, MGO, methylglyoxal-derived hydroimidazolone-1 (MG-H1), D-lactate and L-lactate levels, were measured serially.Results: Glyoxalase-1 mRNA expression decreased in the LPS (β (95%CI); -0.87 (-1.24; -0.50) and the LPS+hypoxia groups; -0.78 (-1.07; -0.48) (P<0.001). MGO was equal between groups, whereas MG-H1 increased over time in the control group only (P=0.003). D-Lactate was increased in all four groups. L-Lactate was increased in all groups, except in the control group.Conclusion: Systemic inflammation downregulates glyoxalase-1 mRNA expression in humans. This is a possible mechanism leading to cell damage and multi-organ failure in critical illness with potential for intervention.

789-P: Efficacy of SGLT2 Inhibitor on Circulating Advanced Glycation End Products in Japanese Patients with Type 2 Diabetes

Poor control of blood glucose could result in increase in advanced glycation end products (AGEs). AGEs cause various adverse vascular events such as atherosclerosis. Therefore, production of AGEs should be suppressed to prevent diabetic complications. In the present study, we evaluated effects of SGLT2 inhibitor (SGLT2i) and DPP-4 inhibitor (DPP-4i) on AGEs in patients with type 2 diabetes. The Japanese patients having poorly controlled blood glucose for 6 months were orally received SGLT2i (n=13; 2.5 mg/day luseogliflozin, 10 mg/day dapagliflozin, or 25 mg/day empagliflozin) or DPP-4i (n=11; 100 mg/day sitagliptin, 100 mg/day vildagliptin, or 25 mg/day alogliptin) for 3 months. Before and after the drug treatment, we measured HbA1c level and serum levels of lipids and hepatic function parameters. Besides, circulating methylglyoxal-derived hydroimidazolone-1 (MG-H1), one of AGEs, was determined optically. As shown in Table, SGLT2i significantly lowered HbA1c (P Disclosure M. Kusunoki: Other Relationship; Self; Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd. N. Wakazono: None. F. Hisano: None. T. Miyata: None.

Age-dependent accumulation of dicarbonyls and advanced glycation endproducts (AGEs) associates with mitochondrial stress

Aging is a strong risk factor for brain dementia and cognitive decline. Age-related accumulation of metabolites such as advanced glycation end products (AGEs) could serve as danger signals to initiate and accelerate disease process and neurodegeneration. The underlying causes and consequences of cerebral AGEs accumulation remain largely unknown. Here, we comprehensively investigate age-related accumulation of AGEs and dicarbonyls, including methylglyoxal (MG), glyoxal (GO), and 3-deoxyglucosone (3-DG), and the effects of mitochondrial reactive oxygen species (ROS) on cerebral AGEs accumulation, mitochondrial function, and oxidative stress in the aging human and mouse brain. We demonstrate that AGEs, including arginine and lysine derived N(6)-carboxymethyl lysine (CML), Nε-(1-Carboxyethyl)-l-lysine (CEL), and methylglyoxal-derived hydroimidazolone-1 (MG-H1), were significantly elevated in the cerebral cortex and hippocampus with advanced age in mice. Accordingly, aging mouse and human brains revealed decrease in activities of mitochondrial respiratory chain complexes I & IV and ATP levels, and increased ROS. Notably, administration of mitoTEMPO (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mTEMPO), a scavenger of mitochondrial ROS, not only suppressed ROS production but also reduced aged-induced accumulation of AGEs and dicarbonyls. mTEMPO treatment improved mitochondrial respiratory function and restored ATP levels. Our findings provide evidence linking age-related accumulation of toxic metabolites (AGEs) to mitochondrial oxidative stress. This highlights a novel mechanism by which AGEs-dependent signaling promotes carbonyl stress and sustained mitochondrial dysfunction. Eliminating formation and accumulation of AGEs may represent a new therapeutic avenue for combating cognitive decline and mitochondrial degeneration relevant to aging and neurodegenerative diseases including Alzheimer's disease.

Dietary sugars and related endogenous advanced glycation end-products increase chromosomal DNA damage in WIL2-NS cells, measured using cytokinesis-block micronucleus cytome assay

This study investigated the effect of glucose and fructose, and advanced glycation end-products (AGEs) on genome damage in WIL2-NS cells, measured using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The effect of AGEs was investigated using the bovine serum albumin (AGE-BSA) model system induced either with glucose (Glu-BSA) or with fructose (Fru-BSA). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed higher Nε-carboxymethyllysine (CML; 26.76 ± 1.09 nmol/mg BSA) levels in the Glu-BSA model. Nε-Carboxyethyllysine (CEL; 7.87 ± 0.19 nmol/mg BSA) and methylglyoxal-derived hydroimidazolone-1 (MG-H1; 69.77 ± 3.74 nmol/mg BSA) levels were higher in the Fru-BSA model. Genotoxic effects were measured using CBMN-Cyt assay biomarkers [binucleated(BN) cells with micronuclei (MNi), BN with nucleoplasmic bridges (NPBs) and BN with nuclear buds (NBuds)] following 9 days of treatment with either glucose, fructose, Glu-BSA or Fru-BSA. Fructose treatment exerted a significant genotoxic dose-response effect including increases of BN with MNi (R2 = 0.7704; P = 0.0031), BN with NPBs (R2 = 0.9311; P < 0.0001) and BN with NBuds (R2 = 0.7118; P = 0.0091) on cells, whereas the DNA damaging effects of glucose were less evident. High concentrations of AGEs (400-600 µg/ml) induced DNA damage; however, there was no effect on cytotoxicity indices (necrosis and apoptosis). In conclusion, this study demonstrates a potential link between physiologically high concentrations of reducing sugars or AGEs with increased chromosomal damage which is an important emerging aspect of the pathology that may be induced by diabetes. Ultimately, loss of genome integrity could accelerate the rate of ageing and increase the risk of age-related diseases over the long term. These findings indicate the need for further research on the effects of glycation on chromosomal instability and to establish whether this effect is replicated in humans in vivo.

Proteasomal degradation of glycated proteins depends on substrate unfolding: Preferred degradation of moderately modified myoglobin

The Maillard reaction generates protein modifications which can accumulate during hyperglycemia or aging and may have inflammatory consequences. The proteasome is one of the major intracellular systems involved in the proteolytic degradation of modified proteins but its role in the degradation of glycated proteins is scarcely studied. In this study, chemical and structural changes of glycated myoglobin were analyzed and its degradation by 20S proteasome was studied. Myoglobin was incubated with physiological (5–10 mM), moderate (50–100 mM) and severe levels (300 mM) of glucose or methylglyoxal (MGO, 50 mM). Glycation increased myoglobin's fluorescence and surface hydrophobicity. Severe glycation generated crosslinked proteins as shown by gel electrophoresis. The concentration of advanced glycation endproducts (AGEs) N-ε-carboxymethyl lysine (CML), N-ε-carboxyethyl lysine (CEL), methylglyoxal-derived hydroimidazolone-1 (MG-H1), pentosidine and pyrraline was analyzed after enzymatic hydrolysis followed by UPLC-MS/MS. Higher concentrations of glucose increased all analyzed AGEs and incubation with MGO led to a pronounced increase of CEL and MG-H1. The binding of the heme group to apo-myoglobin was decreased with increasing glycation indicating the loss of tertiary protein structure. Proteasomal degradation of modified myoglobin compared to native myoglobin depends on the degree of glycation: physiological conditions decreased proteasomal degradation whereas moderate glycation increased degradation. Severe glycation again decreased proteolytic cleavage which might be due to crosslinking of protein monomers. The activity of the proteasomal subunit β5 is influenced by the presence of glycated myoglobin. In conclusion, the role of the proteasome in the degradation of glycated proteins is highly dependent on the level of glycation and consequent protein unfolding.

Quantitation of free glycation compounds in saliva.

In the course of the Maillard reaction, which occurs during heating of food but also under physiological condition, a broad spectrum of reaction products is formed. Among them, the advanced glycation endproducts (AGEs) Nε-carboxymethyllysine (CML), pyrraline (Pyr), methylglyoxal-derived hydroimidazolone 1 (MG-H1) and Nε-carboxyethyllysine (CEL) are the quantitatively dominating compounds during later reaction stages. Those dietary glycation compounds are under discussion as to be associated with chronic inflammation and the pathophysiological consequences of diseases such as diabetes. In the present study, the concentration of individual glycation compounds in saliva was monitored for the first time and related to their dietary uptake. Fasting saliva of 33 metabolically healthy subjects was analyzed with HPLC-MS/MS. The observed levels of individual glycation compounds ranged from 0.5 to 55.2 ng/ml and differed both intra- and interindividually. Patterns did not correlate with subject-related features such as vegetarianism or sports activities, indicating that dietary intake may play an important role. Therefore, six volunteers were asked to eat a raw food diet free of glycation compounds for two days. Within two days, salivary Pyr was lowered from median 1.7 ng/ml to a minimum level below the limit of detection, and MG-H1 decreased from 3.6 to 1.7 ng/ml in in a time-dependent manner after two days. Salivary CML and CEL concentrations were not affected. Therefore, measuring Pyr and MG-H1 in saliva is a suitable diagnostic tool to monitor the dietary intake and metabolic transit of glycation compounds present in heated foods.

Analysis of Chemically Labile Glycation Adducts in Seed Proteins: Case Study of Methylglyoxal-Derived Hydroimidazolone 1 (MG-H1).

Seeds represent the major source of food protein, impacting on both human nutrition and animal feeding. Therefore, seed quality needs to be appropriately addressed in the context of viability and food safety. Indeed, long-term and inappropriate storage of seeds might result in enhancement of protein glycation, which might affect their quality and longevity. Glycation of seed proteins can be probed by exhaustive acid hydrolysis and quantification of the glycation adduct Nɛ-(carboxymethyl)lysine (CML) by liquid chromatography-mass spectrometry (LC-MS). This approach, however, does not allow analysis of thermally and chemically labile glycation adducts, like glyoxal-, methylglyoxal- and 3-deoxyglucosone-derived hydroimidazolones. Although enzymatic hydrolysis might be a good solution in this context, it requires aqueous conditions, which cannot ensure reconstitution of seed protein isolates. Because of this, the complete profiles of seed advanced glycation end products (AGEs) are not characterized so far. Therefore, here we propose the approach, giving access to quantitative solubilization of seed proteins in presence of sodium dodecyl sulfate (SDS) and their quantitative enzymatic hydrolysis prior to removal of SDS by reversed phase solid phase extraction (RP-SPE). Using methylglyoxal-derived hydroimidazolone 1 (MG-H1) as a case example, we demonstrate the applicability of this method for reliable and sensitive LC-MS-based quantification of chemically labile AGEs and its compatibility with bioassays.

Serum Levels of Protein-Bound Methylglyoxal-Derived Hydroimidazolone-1 are Independently Correlated with Asymmetric Dimethylarginine.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase, being involved in endothelial dysfunction. Furthermore, ADMA levels have been shown to predict future cardiovascular events in patients with coronary risk factors, such as diabetes and hypertension. We have previously found that glyceraldehyde-derived advanced glycation end products (glycer-AGEs) stimulate ADMA generation in vitro and the levels are associated with ADMA, endothelial dysfunction, and vascular inflammation in humans. However, it remains unclear what structurally distinct glycer-AGEs are independent correlates of ADMA. In this study, we addressed the issue. We measured serum levels of protein-bound and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) and argpyrimidine, two major structurally identified glycer-AGEs by liquid chromatography-tandem mass spectrometry in 128 outpatients, and examined the correlations of these AGEs, vascular stiffness, and inflammation with ADMA. Moreover, we examined whether the changes in serum MG-H1 and argpyrimidine levels after 4-month treatment with oral hypoglycemic agents (OHAs) were associated with those of ADMA in other 44 patients with impaired glucose tolerance or type 2 diabetes. Multiple stepwise regression analysis revealed that protein-bound MG-H1, high-density lipoprotein cholesterol (inversely), high-sensitivity C-reactive protein, and cardio-ankle vascular index were independently correlated with ADMA (R2 = 0.259). Treatment with OHAs significantly decreased ADMA levels in 44 glucose-intolerant or type 2 diabetic patients, and the changes in protein-bound MG-H1 levels were positively associated with those in ADMA values (p < 0.05). This study demonstrates that serum levels of protein-bound MG-H1 are independently correlated with ADMA and may be a therapeutic target for cardiovascular disease.

Individual Maillard reaction products as indicators of heat treatment of pasta — A survey of commercial products

During pasta production, drying processes promote glycation reactions (non-enzymatic browning, Maillard reaction). Certain “traditionally produced” pasta products are often labelled with claims suggesting high quality due to a gentle heat treatment. The impact of heat treatment of food can be assessed by the measurement of Maillard reaction products (MRPs). In the present study, the MRPs N-ε-fructosyllysine, N-ε-maltulosyllysine, furosine, N-ε-carboxymethyllysine (CML), N-ε-carboxyethyllysine (CEL), pyrraline, formyline, maltosine, methylglyoxal-derived hydroimidazolone 1 (MG-H1), 3-deoxyglucosone, 3-deoxygalactosone, glucosyl isomaltol, and HMF were quantified in 31 pasta products. N-ε-Maltulosyllysine was found to be the predominating MRP (up to 2.4 g/100 g of protein). Between 4 and 28% of the essential amino acid lysine was found to be modified due to glycation reactions. Taking ten MRPs into account, a low-MRP and a high-MRP cluster could be distinguished by cluster analysis. Closer examination of the clusters revealed that the MRPs N-ε-maltulosyllysine, pyrraline, maltosine, HMF, and glucosyl isomaltol differ most strongly and should further be considered as potential heating markers. Lastly, a study on 5 pasta products with different shapes provided evidence that glycated amino acids are not degraded during cooking. Maltosine is formed additionally during cooking. The consumption of pasta contributes substantially to the daily intake of Amadori rearrangement products and CML.