Higher habitual intake of dietary dicarbonyls is associated with higher corresponding plasma dicarbonyl concentrations and skin autofluorescence: the Maastricht Study.

BACKGROUND: Dicarbonyls are highly reactive compounds and major precursors of advanced glycation end products (AGEs). Both dicarbonyls and AGEs are associated with development of age-related diseases. Dicarbonyls are formed endogenously but also during food processing. To what extent dicarbonyls from the diet contribute to circulating dicarbonyls and AGEs in tissues is unknown. OBJECTIVES: To examine cross-sectional associations of dietary dicarbonyl intake with plasma dicarbonyl concentrations and skin AGEs. METHODS: In 2566 individuals of the population-based Maastricht Study (age: 60 ± 8 y, 50% males, 26% with type 2 diabetes), we estimated habitual intake of the dicarbonyls methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG) by combining FFQs with our dietary dicarbonyl database of MGO, GO, and 3-DG concentrations in > 200 commonly consumed food products. Fasting plasma concentrations of MGO, GO, and 3-DG were measured by ultra-performance liquid chromatography-tandem mass spectrometry. Skin AGEs were measured as skin autofluorescence (SAF), using the AGE Reader. Associations of dietary dicarbonyl intake with their respective plasma concentrations and SAF (all standardized) were examined using linear regression models, adjusted for age, sex, potential confounders related to cardiometabolic risk factors, and lifestyle. RESULTS: Median intake of MGO, GO, and 3-DG was 3.6, 3.5, and 17 mg/d, respectively. Coffee was the main dietary source of MGO, whereas this was bread for GO and 3-DG. In the fully adjusted models, dietary MGO was associated with plasma MGO (ß: 0.08; 95% CI: 0.02, 0.13) and SAF (ß: 0.12; 95% CI: 0.07, 0.17). Dietary GO was associated with plasma GO (ß: 0.10; 95% CI: 0.04, 0.16) but not with SAF. 3-DG was not significantly associated with either plasma 3-DG or SAF. CONCLUSIONS: Higher habitual intake of dietary MGO and GO, but not 3-DG, was associated with higher corresponding plasma concentrations. Higher intake of MGO was also associated with higher SAF. These results suggest dietary absorption of MGO and GO. Biological implications of dietary absorption of MGO and GO need to be determined. The study has been approved by the institutional medical ethical committee (NL31329.068.10) and the Minister of Health, Welfare and Sports of the Netherlands (Permit 131088-105234-PG).

Elevated serum levels of methylglyoxal are associated with impaired liver function in patients with liver cirrhosis.

Methylglyoxal (MGO) is a highly reactive dicarbonyl species that forms advanced glycation end products (AGEs). The binding of these AGEs to their receptor (RAGE) causes and sustains severe inflammation. Systemic inflammation is postulated to be a major driver in the progression of liver cirrhosis. However, the role of circulating MGO levels in liver cirrhosis remains unknown. In this study, we investigated the serum levels of two dicarbonyl species, MGO and glyoxal (GO) using tandem mass spectrometry (HPLC-MS/MS) and evaluated their association with disease severity. A total of 51 inpatients and outpatients with liver cirrhosis of mixed etiology and different disease stages were included. Elevated MGO levels were seen in an advanced stage of liver cirrhosis (p < 0.001). High MGO levels remained independently associated with impaired liver function, as assessed by the model for end-stage liver disease (MELD) (ß = 0.448, p = 0.002) and acute decompensation (AD) (ß = 0.345, p = 0.005) scores. Furthermore, MGO was positively correlated with markers of systemic inflammation (IL-6, p = 0.004) and the development of ascites (p = 0.013). In contrast, no changes were seen in GO serum levels. Circulating levels of MGO are elevated in advanced stages of liver cirrhosis and are associated with impaired liver function and liver-related parameters.

Metformin abrogates the voiding dysfunction induced by prolonged methylglyoxal intake.

Methylglyoxal (MGO) is a reactive carbonyl species found at high levels in blood of diabetic patients. The anti-hyperglycemic drug metformin can scavenger MGO and reduce the formation of advanced glycation end products (AGEs). Here, we aimed to investigate if MGO-induced bladder dysfunction can be reversed by metformin. Male C57/BL6 mice received 0.5% MGO in drinking water for 12 weeks, and metformin (300 mg/kg, daily gavage) was given in the last two weeks. The bladder functions were evaluated by performing voiding behavior assays, cystometry and in vitro bladder contractions. MGO intake markedly elevated the levels of MGO and fluorescent AGEs in serum and reduced the mRNA expression and activity of glyoxalase (Glo1) in bladder tissues. Glucose levels were unaffected among groups. MGO intake also increased the urothelium thickness and collagen content of the bladder. Void spot assays in conscious mice revealed an increased void volume in MGO group. The cystometric assays in anesthetized mice revealed increases of basal pressure, non-voiding contractions frequency, bladder capacity, inter-micturition pressure and residual volume, which were accompanied by reduced voiding efficiency in MGO group. In vitro bladder contractions to carbachol, α,ß-methylene ATP and electrical-field stimulation were significantly greater in MGO group. Metformin normalized the changes of MGO and AGEs levels, Glo1 expression and activity, urothelium thickness and collagen content. The MGO-induced voiding dysfunction were all restored by metformin treatment. Our findings strongly suggest that the amelioration of MGO-induced voiding dysfunction by metformin relies on its ability to scavenger MGO, preventing its accumulation in blood.

Reversal of Insulin Resistance in Overweight and Obese Subjects by trans-Resveratrol and Hesperetin Combination-Link to Dysglycemia, Blood Pressure, Dyslipidemia, and Low-Grade Inflammation.

The dietary supplement, trans-resveratrol and hesperetin combination (tRES-HESP), induces expression of glyoxalase 1, countering the accumulation of reactive dicarbonyl glycating agent, methylglyoxal (MG), in overweight and obese subjects. tRES-HESP produced reversal of insulin resistance, improving dysglycemia and low-grade inflammation in a randomized, double-blind, placebo-controlled crossover study. Herein, we report further analysis of study variables. MG metabolism-related variables correlated with BMI, dysglycemia, vascular inflammation, blood pressure, and dyslipidemia. With tRES-HESP treatment, plasma MG correlated negatively with endothelial independent arterial dilatation (r = -0.48, p < 0.05) and negatively with peripheral blood mononuclear cell (PBMC) quinone reductase activity (r = -0.68, p < 0.05)-a marker of the activation status of transcription factor Nrf2. For change from baseline of PBMC gene expression with tRES-HESP treatment, Glo1 expression correlated negatively with change in the oral glucose tolerance test area-under-the-curve plasma glucose (ΔAUGg) (r = -0.56, p < 0.05) and thioredoxin interacting protein (TXNIP) correlated positively with ΔAUGg (r = 0.59, p < 0.05). Tumor necrosis factor-α (TNFα) correlated positively with change in fasting plasma glucose (r = 0.70, p < 0.001) and negatively with change in insulin sensitivity (r = -0.68, p < 0.01). These correlations were not present with placebo. tRES-HESP decreased low-grade inflammation, characterized by decreased expression of CCL2, COX-2, IL-8, and RAGE. Changes in CCL2, IL-8, and RAGE were intercorrelated and all correlated positively with changes in MLXIP, MAFF, MAFG, NCF1, and FTH1, and negatively with changes in HMOX1 and TKT; changes in IL-8 also correlated positively with change in COX-2. Total urinary excretion of tRES and HESP metabolites were strongly correlated. These findings suggest tRES-HESP counters MG accumulation and protein glycation, decreasing activation of the unfolded protein response and expression of TXNIP and TNFα, producing reversal of insulin resistance. tRES-HESP is suitable for further evaluation for treatment of insulin resistance and related disorders.

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 (ß (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.

Recent Advances in Biomarkers and Regenerative Medicine for Diabetic Neuropathy.

Diabetic neuropathy is one of the most common complications of diabetes. This complication is peripheral neuropathy with predominant sensory impairment, and its symptoms begin with hyperesthesia and pain and gradually become hypoesthesia with the loss of nerve fibers. In some cases, lower limb amputation occurs when hypoalgesia makes it impossible to be aware of trauma or mechanical stimuli. On the other hand, up to 50% of these complications are asymptomatic and tend to delay early detection. Therefore, sensitive and reliable biomarkers for diabetic neuropathy are needed for an early diagnosis of this condition. This review focuses on systemic biomarkers that may be useful at this time. It also describes research on the relationship between target gene polymorphisms and pathological conditions. Finally, we also introduce current information on regenerative therapy, which is expected to be a therapeutic approach when the pathological condition has progressed and nerve degeneration has been completed.

Increase in the peripheral blood methylglyoxal levels in 10% of hospitalized chronic schizophrenia patients.

Increasing evidence indicates that enhanced peripheral carbonyl stress markers exist in subtype of schizophrenia, although it may not be the primary cause. This study aimed to investigate whether plasma concentrations of methylglyoxal, 3-deoxy-glucosone, and glyoxal, which are reactive intermediates of protein metabolism in carbonyl stress, are changed in patients with schizophrenia and can function as potential biomarkers for schizophrenia with enhanced carbonyl stress. Plasma concentrations of these di-carbonyls were simultaneously estimated in 40 patients with schizophrenia and 40 healthy controls. As a result, no statistically significant differences were observed in mean plasma concentrations of three di-carbonyls between patients and controls. However, a remarkable increase in methylglyoxal concentrations was observed in four patients but not in controls. This increase was not found with regard to 3-deoxyglucosone and glyoxal both of patients and controls. Our correlation analysis showed that both the plasma methylglyoxal and glyoxal concentrations were significantly correlated with 3-deoxyglucosone concentrations in 40 patients and 40 controls. However, the plasma methylglyoxal concentrations did not show any significant correlation with the glyoxal concentrations in the patients or the controls. In four patients with extremely high methylglyoxal levels, the plasma methylglyoxal and glyoxal concentrations were not correlated to the 3-deoxyglucosone concentrations. Methylglyoxal is a physiological substrate of the glyoxalase system, and the accelerated accumulation of this compound lowers the glyoxalase I activity. These results suggested that this increase in four patients with high methylglyoxal levels may indicate the presence of a subtype of chronic schizophrenia that is associated with enhanced carbonyl stress.

Evaluation of the nephrotoxicity and safety of low-dose aristolochic acid, extending to the use of Xixin (Asurum), by determination of methylglyoxal and d-lactate.

ETHNOPHARMACOLOGICAL RELEVANCE: Most Aristolochiaceae plants are prohibited due to aristolochic acid nephropathy (AAN), except Xixin (Asarum spp.). Xixin contains trace amounts of aristolochic acid (AA) and is widely used in Traditional Chinese Medicine. Methylglyoxal and d-lactate are regarded as biomarkers for nephrotoxicity. AIM OF THE STUDY: The use of Xixin (Asarum spp.) is essential and controversial. This study aimed to evaluate tubulointerstitial injury and interstitial renal fibrosis by determining urinary methylglyoxal and d-lactate after withdrawal of low-dose AA in a chronic mouse model. MATERIALS AND METHODS: C3H/He mice in the AA group (n = 24/group) were given ad libitum access to distilled water containing 3 µg/mL AA (0.5 mg/kg/day) for 56 days and drinking water from days 57 to 84. The severity of tubulointerstitial injury and fibrosis were evaluated using the tubulointerstitial histological score (TIHS) and Masson's trichrome staining. Urinary and serum methylglyoxal were determined by high-performance liquid chromatography (HPLC); urinary d-lactate were determined by column-switching HPLC. RESULTS: After AA withdrawal, serum methylglyoxal in the AA group increased from day 56 (429.4 ± 48.3 µg/L) to 84 (600.2 ± 99.9 µg/L), and peaked on day 70 (878.3 ± 171.8 µg/L; p < 0.05); TIHS and fibrosis exhibited similar patterns. Urinary methylglyoxal was high on day 56 (3.522 ± 1.061 µg), declined by day 70 (1.583 ± 0.437 µg) and increased by day 84 (2.390 ± 0.130 µg). Moreover, urinary d-lactate was elevated on day 56 (82.10 ± 18.80 µg) and higher from day 70 (201.10 ± 90.82 µg) to 84 (193.28 ± 61.32 µg). CONCLUSIONS: Methylglyoxal is induced after AA-induced tubulointerstitial injury, so methylglyoxal excretion and metabolism may be a detoxification and repair strategy. A low cumulative AA dose is the key factor that limits tubulointerstitial injury and helps to repair. Thus, AA-containing herbs, especially Xixin, should be used at low doses for short durations (less than one month).

Fasting and post-oral-glucose-load levels of methylglyoxal are associated with microvascular, but not macrovascular, disease in individuals with and without (pre)diabetes: The Maastricht Study.

AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MGO), rise during an oral glucose tolerance test (OGTT), particularly in (pre)diabetes. Fasting MGO levels are associated with chronic kidney disease (CKD) and cardiovascular disease (CVD) in patients with poorly controlled type 2 diabetes mellitus (T2DM). Yet, whether fasting or post-OGTT plasma MGO levels are associated with vascular disease in people with (pre)diabetes is unknown. METHODS: Subjects with normal glucose metabolism (n=1796; age: 57.9±8.2 years; 43.3% men), prediabetes (n=478; age: 61.6±7.6 years; 54.0% men) and T2DM (n=669; age: 63.0±7.5 years; 67.0% men) from the Maastricht Study underwent OGTTs. Plasma MGO levels were measured at baseline and 2h after OGTT by mass spectrometry. Prior CVD was established via questionnaire. CKD was reflected by estimated glomerular filtration rate (eGFR) and albuminuria; retinopathy was assessed using retinal photographs. Data were analyzed using logistic regression adjusted for gender, age, smoking, systolic blood pressure, total-to-HDL cholesterol ratio, triglycerides, HbA1c, BMI and medication use. Odd ratios (ORs) were expressed per standard deviation of LN-transformed MGO. RESULTS: Fasting and post-OGTT MGO levels were associated with higher ORs for albuminuria ≥30mg/24h [fasting: 1.12 (95% CI: 0.97-1.29); post-OGTT: 1.19 (1.01-1.41)], eGFR<60mL/min/1.73 m2 [fasting: 1.58 (95% CI: 1.38-1.82), post-OGTT: 1.57 (1.34-1.83)] and retinopathy [fasting: 1.59 (95% CI: 1.01-2.53), post-OGTT: 1.38 (0.77-2.48)]. No associations with prior CVD were found. CONCLUSION: Fasting and post-OGTT MGO levels were associated with microvascular disease, but not prior CVD. Thus, therapeutic strategies directed at lowering MGO levels may prevent microvascular disease.

The Effects of Taurine Supplementation on Metabolic Profiles, Pentosidine, Soluble Receptor of Advanced Glycation End Products and Methylglyoxal in Adults With Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVES: Advanced glycation end products, along with methylglyoxal (MGO) as their precursor, play a major role in increased complications of type 2 diabetes mellitus (T2DM). Taurine (2-aminoethanesulphonic acid), a conditionally essential amino acid, is found in most mammalian tissues. Taurine is known as an antiglycation compound. This study was designed to investigate the effects of taurine supplementation on metabolic profiles, pentosidine, MGO and soluble receptors for advanced glycation end products in patients with T2DM. METHODS: In this double-blind randomized controlled trial, 46 patients with T2DM were randomly allocated into taurine and placebo groups. Participants received either 3,000 mg/day taurine or placebo for 8 weeks. Metabolic profiles, pentosidine, MGO and soluble receptors for advanced glycation end products levels were assessed after 12 h of fasting at baseline and completion of the clinical trial. Independent t test, paired t test, Pearson correlation and analysis of covariance were used for analysis. RESULTS: The mean serum levels of fasting blood sugar (p=0.01), glycated hemoglobin (p=0.04), insulin (p=0.03), homeostasis model assessment-insulin resistance (p=0.004), total cholesterol (p=0.01) and low-density lipoprotein cholesterol (p=0.03) significantly were reduced in the taurine group at completion compared with the placebo group. In addition, after completion of the study, pentosidine (p=0.004) and MGO (p=0.006) were significantly reduced in the taurine group compared with the placebo group. CONCLUSIONS: The results of this trial show that taurine supplementation may decrease diabetes complications through improving glycemic control and advanced glycation end products.

Reduction in Circulating Advanced Glycation End Products by Mediterranean Diet Is Associated with Increased Likelihood of Type 2 Diabetes Remission in Patients with Coronary Heart Disease: From the Cordioprev Study.

SCOPE: It is hypothesized that decreased advanced glycation end products (AGEs) levels could affect type 2 diabetes mellitus (T2DM) remission in newly diagnosed patients through the consumption of two healthy diets. METHODS AND RESULTS: Patients from CORDIOPREV study, all with previous cardiovascular events, with T2DM at the beginning of the study are included. Patients are randomized to a Mediterranean or a low-fat diet for five years. No different diabetes remission rates are found among diets. Serum methylglioxal (MG) and carboximethyllysine (CML), levels dietary AGE, as well as gene expression of AGER1 and RAGE are measured. Serum MG decreases only after the consumption of the Mediterranean diet. Moreover, a COX regression analysis shows that each SD decrease in the MG, occurring after the Mediterranean diet, increases the probability of T2DM remission with HR:2.56(1.02-6.25) and p = 0.046 and each SD increase in disposition index at baseline increases the probability of remission with HR:1.94(1.32-2.87) and p = 0.001. CONCLUSIONS: It is demonstrated that the reduction of serum AGEs levels and the modulation of its metabolism, occurring after the consumption of a Mediterranean diet, might be involved in the molecular mechanism underlying the T2DM remission of newly diagnosed patients with coronary heart disease.

Irbesartan treatment does not influence plasma levels of the dicarbonyls methylglyoxal, glyoxal and 3-deoxyglucosone in participants with type 2 diabetes and microalbuminuria: An IRMA2 sub-study.

AIM: Angiotensin receptor blockers (ARBs) reduce vascular complications in diabetes independently of blood pressure. Experimental studies suggested that ARBs may restore the detoxifying enzyme glyoxalase 1, thereby lowering dicarbonyls such as methylglyoxal. Human data on the effects of ARBs on plasma dicarbonyl levels are lacking. We investigated, in individuals with type 2 diabetes, whether irbesartan lowered plasma levels of the dicarbonyls methylglyoxal, glyoxal, 3-deoxyglucosone and their derived advanced glycation end products (AGEs), and increased d-lactate, reflecting greater methylglyoxal flux. METHODS: We analysed a subset of the Irbesartan in Patients with T2D and Microalbuminuria (IRMA2) study. We measured plasma dicarbonyls methylglyoxal, glyoxal and 3-deoxyglucosone, free AGEs and d-lactate using ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) in the treatment arm receiving 300 mg irbesartan (n = 121) and a placebo group (n = 101) at baseline and after 1 and 2 years. Effect of treatment was analysed with repeated measurements ANOVA. RESULTS: There was a slight, but significant difference in baseline median methylglyoxal levels [placebo 1119 (907-1509) nmol/l vs. irbesartan 300 mg 1053 (820-1427) nmol/l], but no significant changes were observed in any of the plasma dicarbonyls over time in either group and there was no effect of irbesartan treatment on plasma free AGEs or d-lactate levels at either 1 or 2 years. CONCLUSION: Irbesartan treatment does not change plasma levels of the dicarbonyls methylglyoxal, glyoxal and 3-deoxyglucosone, free AGEs or d-lactate in type 2 diabetes. This indicates that increased dicarbonyls in type 2 diabetes are not targetable by ARBs, and other approaches to lower systemic dicarbonyls are needed in type 2 diabetes. (Clinical Trial Registry No: #NCT00317915).

Methylglyoxal, Glycated Albumin, PAF, and TNF-α: Possible Inflammatory and Metabolic Biomarkers for Management of Gestational Diabetes.

BACKGROUND: In gestational diabetes mellitus (GDM), pancreatic ß-cell breakdown can result from a proinflammatory imbalance created by a sustained level of cytokines. In this study, we investigated the role of specific cytokines, such as B-cell activating factor (BAFF), tumor necrosis factor α (TNF-α), and platelet-activating factor (PAF), together with methylglyoxal (MGO) and glycated albumin (GA) in pregnant women affected by GDM. METHODS: We enrolled 30 women whose inflammation and metabolic markers were measured at recruitment and after 12 weeks of strict dietetic therapy. We compared these data to the data obtained from 53 randomly selected healthy nonpregnant subjects without diabetes, hyperglycemia, or any condition that can affect glycemic metabolism. RESULTS: In pregnant women affected by GDM, PAF levels increased from 26.3 (17.4-47.5) ng/mL to 40.1 (30.5-80.5) ng/mL (p < 0.001). Their TNF-α levels increased from 3.0 (2.8-3.5) pg/mL to 3.4 (3.1-5.8) pg/mL (p < 0.001). The levels of methylglyoxal were significantly higher in the women with GDM (p < 0.001), both at diagnosis and after 12 weeks (0.64 (0.46-0.90) µg/mL; 0.71 (0.47-0.93) µg/mL, respectively) compared to general population (0.25 (0.19-0.28) µg/mL). Levels of glycated albumin were significantly higher in women with GDM (p < 0.001) only after 12 weeks from diagnosis (1.51 (0.88-2.03) nmol/mL) compared to general population (0.95 (0.63-1.4) nmol/mL). CONCLUSION: These findings support the involvement of new inflammatory and metabolic biomarkers in the mechanisms related to GDM complications and prompt deeper exploration into the vicious cycle connecting inflammation, oxidative stress, and metabolic results.

Endothelial Dysfunction and Advanced Glycation End Products in Patients with Newly Diagnosed Versus Established Diabetes: From the CORDIOPREV Study.

Endothelial dysfunction and intima-media thickness of common carotid arteries (IMT-CC) are considered subclinical markers of atherosclerotic cardiovascular disease (ASCVD). Advanced glycation end products (AGEs) are increased in type 2 diabetes mellitus (T2DM) patients, compared with non-diabetics, being implicated in micro- and macrovascular complications. Our aim was to compare serum AGEs levels and subclinical atherosclerotic markers between patients with established and newly diagnosed T2DM. Among 540 patients with T2DM and coronary heart disease from the CORDIOPREV study, 350 patients had established T2DM and 190 patients had newly diagnosed T2DM. Serum levels of AGEs (methylglyoxal (MG) and N-carboxymethyl lysine (CML)) and subclinical atherosclerotic markers (brachial flow-mediated vasodilation (FMD) and IMT-CC) were measured. AGEs levels (all p < 0.001) and IMT-CC (p = 0.025) were higher in patients with established vs. newly diagnosed T2DM, whereas FMD did not differ between the two groups. Patients with established T2DM and severe endothelial dysfunction (i.e., FMD < 2%) had higher serum MG levels, IMT-CC, HOMA-IR and fasting insulin levels than those with newly diagnosed T2DM and non-severe endothelial dysfunction (i.e., FMD ≥ 2%) (all p < 0.05). Serum CML levels were greater in patients with established vs. newly diagnosed T2DM, regardless of endothelial dysfunction severity. Serum AGEs levels and IMT-CC were significantly higher in patients with established vs. newly diagnosed T2DM, highlighting the progressively increased risk of ASCVD in the course of T2DM. Establishing therapeutic strategies to reduce AGEs production and delay the onset of cardiovascular complications in newly diagnosed T2DM patients or minimize ASCVD risk in established T2DM patients is needed.

Development of analytical methods GC-MS vs LC-UV for the serum monitoring of an inflammatory glycotoxin (methylglyoxal): A new biomarker of bovine hepatobiliary distomatosis.

Two analytical methods; high performance liquid chromatography and gas chromatography were used to determine the content of 2-methylquinoxaline, a methylglyoxal-derived agent in sera from cattle with fascioliasis. Methylglyoxal is a highly mutagenic and cytotoxic reactive dicarbonyl compound formed by non-enzymatic fragmentation of triose phosphate GAP and DHAP during glycolysis which regularly contributes to repositioning the energetic balance between physiological and pathological situations. The aim of this study was to propose the MGO as a new biomarker in the bovine fasciolosis. Strongly infected animals showed a correlation between the relatively high levels of Fasciola hepatica anti-f2 antibody and methylglyoxal compared to unharmed animals. Also, an acute hyperglycemia was recorded and closely related to hepatic parenchyma hyperplasia, inflammation, bile ducts obstruction and scléro-fibrous foci formation.Unlike HPLC, which has shown analytical flaws and irregularities, GC-MS remains an excellent diagnostic tool for detecting and quantifying methylglyoxal in biological fluids. The developed method has been validated under FDA guidelines. A full scan-range was set from m/z 39 to 144/999 and the molecular weight of the 2-methylquinoxaline was identified according to NIST Database and ES. Methylglyoxal was the only analyte successfully quantified in a relatively short run time. It was linear over a concentration range of 0.057-5.7  µg.ml-1with mean recoveries and RSD of 118% and 3.63% respectively. The intra and inter-day assays were satisfying and not exceed 3.00%. Results reflect the degree of precision of our method and indicate that MGO was an important contributor to understand the hepatic failure independently of other serum markers.

Improving Covalent Organic Frameworks Fluorescence by Triethylamine Pinpoint Surgery as Selective Biomarker Sensor for Diabetes Mellitus Diagnosis.

The nitrogen-containing imine or hydrazone linked covalent organic frameworks (COFs) are poorly luminescent due to the fluorescence quenching by nitrogen atoms in the linkages, even if highly luminescent units and linkers are employed. The fluorescence quenching pathway to prevent linkage-originated to mitigate the inherent limitations of the linkage is a promising method for luminescent COFs. The generation of N- by deprotonation of the N-H unit eliminates the electron transfer from N lone pair to COF (TpPa-1) and enhances the luminescence. In this work, TpPa-1 achieved turn-on luminescence response with good sensitivity and reproducibility toward triethylamine (TEA) vapor in the process of deprotonation. The fabricated detector offers a viable approach for sensing ppm-level TEA, which can remind people to take timely measures to reduce the environmental hazards caused by TEA. The fluorescent sensor TpPa-1@LE constructed by the products of TpPa-1 and TEA can quantitatively trace biomarker methylglyoxal (MGO) for diabetes mellitus diagnosis in serum system. Furthermore, using TEA and MGO as input signals and the two fluorescence emissions G476 and Y525 as output signals, an advanced analytical device based on two Boolean logic gates with INH and AND function is constructed. This work provides a new strategy for improving the weak luminescence of COF in aqueous solution and realizes selective response to biomarker (MGO) for diabetes mellitus diagnosis.

A dual-emitting Tb(iii)&Yb(iii)-functionalized coordination polymer: a "turn-on" sensor for N-methylformamide in urine and a "turn-off" sensor for methylglyoxal in serum.

Fluorescent materials with lanthanide cations encapsulated in MOFs are currently used in numerous applications, especially in biosensors. Therefore, herein, two novel composites were designed and developed based on a Tb(iii)&Yb(iii)-functionalized Cu(ii)-coordination polymer, possessing higher thermal and water stability and fascinating fluorescence properties. The first bimetallic composite Tb@Cu-Hcbpp demonstrated broad ligand-centered emission and weak typical Tb3+ ion emission; moreover, it was used as an excellent ratiometric fluorescent sensor for the metabolic product NMF of DMF in the human body (LOD = 0.02 µM). In addition, the Yb3+ ions were doped into Tb@Cu-Hcbpp to improve the fluorescence performance of the green Tb3+ ion emission. Among the series of Tb1-xYbx@Cu-Hcbpp samples (x = 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 and 0.40), Tb0.85Yb0.15@Cu-Hcbpp showed maximum enhanced fluorescence intensity (almost 9.6 times that of the pure terbium system), but exhibited high fluorescence quenching efficiency for methylglyoxal (MGO), which could be used for the sensitive detection of MGO (LOD = 0.25 µM). Furthermore, the developed biosensors were successfully applied for the detection of NMF and MGO in urine and serum samples, and satisfactory results were obtained, showing good potential of these biosensors in practical applications such as in disease diagnosis and biochemical research.

ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures.

Methylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases.

Dietary Genistein Inhibits Methylglyoxal-Induced Advanced Glycation End Product Formation in Mice Fed a High-Fat Diet.

BACKGROUND: Methylglyoxal (MGO), an important precursor of advanced glycation end products (AGEs), circulates at high concentrations in diabetic patients' blood and plays an important role in the pathogenesis of diabetes and other chronic diseases. OBJECTIVES: The aim of this study was to determine whether dietary genistein can prevent indicators of metabolic syndrome (MetS) induced by a very-high-fat (VHF) diet or a high-fat (HF) diet plus exogenous MGO, and the accumulation of MGO and AGEs in mice. METHODS: Male, 6-wk-old C57BL/6J mice (n = 15) were fed a low-fat (LF) diet (10% fat energy) or a VHF diet (60% fat energy) alone or including 0.25% genistein (VHF-G) for 16 wk in study 1. In study 2, 75 similar mice were fed the LF diet (LF) or the HF diet alone (HF) or in combination with up to 0.2% MGO in water (HFM) and 0.067% (HFM-GL) or 0.2% (HFM-GH) dietary genistein for 18 wk. Anthropometric and metabolic data were obtained in both studies to determine the effects of MGO and genistein on variables indicative of MetS. RESULTS: Body weight gain, fat deposits, dyslipidemia, hyperglycemia, and fatty liver were ameliorated by dietary genistein in both studies. The plasma MGO concentration in VHF-G mice was 52% lower than that in VHF mice. Moreover, the AGE concentrations in plasma, liver, and kidney of VHF-G mice were 73%, 52%, and 49%, respectively, lower than in the VHF group (study 1). Similarly, the concentrations of plasma MGO and AGE in plasma, liver, and kidney of HFM-GH mice were 33.5%, 49%, 69%, and 54% lower than in HFM mice (study 2). Genistein inhibited AGE formation by trapping MGO to form adducts and upregulating the expressions of glyoxalase I and II and aldose reductase in liver and kidney to detoxify MGO in both studies. CONCLUSIONS: Our data demonstrate for the first time that genistein significantly lowers MGO and AGE concentrations in 2 mouse MetS models via multiple pathways.

Ratiometric Fluorescent Probe for Monitoring Endogenous Methylglyoxal in Living Cells and Diabetic Blood Samples.

Optical imaging provides noninvasive powerful tools not only for better understanding the physiological and pathological roles of methylglyoxal (MGO) in living systems but also for potential clinical diagnosis of MGO-related diseases, such as diabetic complications. However, so far only very few "turn-on" MGO fluorescent sensors have been developed, and they are all based on the reaction between MGO and benzenediamines. Due to the possible reactions of benzenediamines with other cellular molecules, such as NO and FA, these sensors suffer from limited selectivity and potential deactivation in cells. Herein, we report a novel MGO recognition reaction using 2-aminoacetamide. The reaction between MGO and 2-aminoacetamide was found to be highly efficient and specific, with no interference from NO and FA in particular. This reaction was used to develop the first ratiometric fluorescent probe (CMFP) for MGO. We have proven that CMFP could detect MGO at physiological concentrations in both aqueous buffer and living cells with excellent selectivity and sensitivity. Furthermore, we successfully utilized CMFP to study intracellular MGO generation routes and evaluated MGO levels of clinic blood samples from healthy and diabetic patients. These results highlight the potential utility of this probe in both basic science research and clinical diagnosis.