Plasma AGE and Oxidation Products, Renal Function, and Preeclampsia in Pregnant Women with Type 1 Diabetes: A Prospective Observational Study.

Aims: We aimed to determine whether plasma advanced glycation end products or oxidation products (AGE/oxidation-P) predict altered renal function and/or preeclampsia (PE) in pregnant women with type 1 diabetes. Methods: Prospectively, using a nested case-control design, we studied 47 pregnant women with type 1 diabetes, of whom 23 developed PE and 24 did not. Nineteen nondiabetic, normotensive pregnant women provided reference values. In plasma obtained at ~12, 22, and 32 weeks' gestation (visits 1, 2, and 3; V1-V3), we measured five AGE products (carboxymethyllysine (CML), carboxyethyl-lysine (CEL), methylglyoxal-hydroimidazolone (MGH1), 3-deoxyglucosone hydroimidazolone (3DGH), and glyoxal-hydroimidazolone (GH1)) and four oxidation products (methionine sulfoxide (MetSO), 2-aminoadipic acid (2-AAA), 3-nitrotyrosine (3NT), and dityrosine (DT)), by liquid chromatography/mass spectroscopy. Clinical outcomes were "estimated glomerular filtration rate" (eGFR) at each visit and onset of PE. Results: In diabetic women, associations between AGE/oxidation-P and eGFR were found only in those who developed PE. In this group, CEL, MGH1, and GH1 at V2 and CML, CEL, MGH1, and GH1 at V3 were inversely associated with contemporaneous eGFR, while CEL, MGH1, 3DGH, and GH1 at V2 were inversely associated with eGFR at V3 (all p < 0.05). There were no associations of plasma AGE or oxidation-P with pregnancy-related development of proteinuria or PE. Conclusions: Inverse associations of second and early third trimester plasma AGE with eGFR among type 1 diabetic women who developed PE suggest that these patients constitute a subset susceptible to AGE-mediated injury and thus to cardiorenal complications later in life. However, AGE/oxidation-P did not predict PE in type 1 diabetic women.

Advanced Glycation End Products and Inflammatory Cytokine Profiles in Maintenance Hemodialysis Patients After the Ingestion of a Protein-Dense Meal.

OBJECTIVE: The goal of this investigation was to evaluate circulating and skeletal muscle inflammatory biomarkers between maintenance hemodialysis (MHD) and demographic-matched control subjects (CON) before and after ingestion of a protein-rich meal. DESIGN AND METHODS: CON (n = 8; 50 ± 2 years; 31 ± 1 kg/m2) and MHD patients (n = 8; 56 ± 5 years; 32 ± 2 kg/m2) underwent a basal blood draw and muscle biopsy and serial blood draws after the ingestion of a mixed meal on a nondialysis day. Plasma advanced glycation end products (AGEs) and markers of oxidation were assessed via liquid chromatography-tandem mass spectrometry before and after the meal (+240 min). Circulating inflammatory cytokines and soluble receptors for AGE (sRAGE) isoforms (endogenous secretory RAGEs and cleaved RAGEs) were determined before and after the meal (+240 min). Basal muscle was probed for inflammatory cytokines and protein expression of related signaling components (RAGE, Toll-like receptor 4, oligosaccharyltransferase subunit 48, TIR-domain-containing adapter-inducing interferon-ß, total IκBα, and pIκBα). RESULTS: Basal circulating AGEs were 7- to 343-fold higher (P < .001) in MHD than those in CON, but only MG-H1 increased in CON after the meal (P < .001). There was a group effect (MHD > CON) for total sRAGEs (P = .02) and endogenous secretory RAGEs (P < .001) and a trend for cleaved RAGEs (P=.09), with no meal effect. In addition, there was a group effect (MHD < CON; P < .05) for circulating fractalkine, interleukin (IL)10, IL17A, and IL1ß and a trend (P < .10) for IL6 and macrophage inflammatory protein 1 alpha, whereas tumor necrosis factor alpha was higher in MHD (P < .001). In muscle, Toll-like receptor 4 (P = .03), TIR-domain-containing adapter-inducing interferon-ß (P = .002), and oligosaccharyltransferase subunit 48 (P = .02) expression was lower in MHD than that in CON, whereas IL6 was higher (P = .01) and IL8 (P = .08) tended to be higher in MHD. CONCLUSION: Overall, MHD exhibited an exaggerated, circulating, and skeletal muscle inflammatory biomarker environment, and the meal did not appreciably affect the inflammatory status.

Plasma Levels of Advanced Glycation Endproducts and Risk of Cardiovascular Events: Findings From 2 Prospective Cohorts.

Background Advanced glycation endproducts (AGEs) have been linked to cardiovascular disease (CVD) in cohorts with and without diabetes. Data are lacking on prospective associations of various α-dicarbonyl-derived AGEs and incident CVD in the general population. We tested the hypothesis that major plasma AGEs are associated with new-onset CVD in 2 population-based cohorts of differing age and comorbidities. Methods and Results Analyses involved a random subcohort (n=466) from the Cardiovascular Health Study and a case-cohort sample (n=1631) from the Multi-Ethnic Study of Atherosclerosis. Five AGEs and 2 oxidative products were measured by liquid chromatography tandem mass spectrometry. Associations with CVD (myocardial infarction and stroke) were evaluated with Cox regression. Participants in the Cardiovascular Health Study were older than the Multi-Ethnic Study of Atherosclerosis, and had more comorbidities, along with higher levels of all AGEs. During median follow-up of 11 years, 439 participants in the Multi-Ethnic Study of Atherosclerosis and 200 in the Cardiovascular Health Study developed CVD. After multivariable adjustment, carboxymethyl-lysine, 3-deoxyglucosone hydroimidazolones and a summary variable of all measured AGEs (principal component 1) were significantly associated with incident CVD in the Cardiovascular Health Study (HRs [95% CI]: 1.20 [1.01, 1.42], 1.45 [1.23, 1.72], and 1.29 [1.06, 1.56], respectively), but not the Multi-Ethnic Study of Atherosclerosis. Oxidative products were not associated with CVD in either cohort. Conclusions We found α-dicarbonyl-derived AGEs to be associated with CVD in an older cohort, but not in a healthier middle-aged/older cohort. Our results suggest that AGEs may exert detrimental cardiovascular effects only under conditions of marked dicarbonyl and oxidative stress. Further investigation of α-dicarbonyl derivatives could lead to potential new strategies for CVD prevention in high-risk older populations.

Advanced Glycation End Products Predict Loss of Renal Function and High-Risk Chronic Kidney Disease in Type 2 Diabetes.

OBJECTIVE: To evaluate the association of a multicomponent advanced glycation end product (AGE) panel with decline in kidney function and its utility in predicting renal function loss (RFL) when added to routine clinical measures in type 2 diabetes. RESEARCH DESIGN AND METHODS: Carboxymethyl and carboxyethyl lysine and methylglyoxal, 3-deoxyglucosone, and glyoxal hydroimidazolones were measured in baseline serum and plasma samples, respectively, from Action to Control Cardiovascular Risk in Diabetes (ACCORD) (n = 1,150) and Veterans Affairs Diabetes Trial (VADT) (n = 447) participants. A composite AGE score was calculated from individual AGE z scores. The primary outcome was a sustained 30% decline in estimated glomerular filtration rate (eGFR) (30% RFL in both cohorts). Secondary outcomes (in ACCORD) were 40% RFL, macroalbuminuria, and high-risk chronic kidney disease (hrCKD). RESULTS: After adjustment for baseline and follow-up HbA1c and other risk factors in ACCORD, the AGE score was associated with reduction in eGFR (ß-estimate -0.66 mL/min ⋅ 1.73 m2 per year; P = 0.001), 30% RFL (hazard ratio 1.42 [95% CI 1.13-1.78]; P = 0.003), 40% RFL (1.40 [1.13-1.74]; P = 0.003), macroalbuminuria (1.53 [1.13-2.06]; P = 0.006), and hrCKD (1.88 [1.37-2.57]; P < 0.0001). AGE score improved net reclassification (NRI) and relative integrated discrimination (IDI) for 30% RFL (NRI 23%; P = 0.02) (relative IDI 7%; P = 0.009). In VADT, the AGE score calculated by the ACCORD-derived coefficients was associated with 30% RFL (1.37 [1.03-1.82); P = 0.03) and improved NRI (24%; P = 0.03) but not IDI (P = 0.18). CONCLUSIONS: These data provide further support for a causal role of AGEs in diabetic nephropathy independently of glycemic control and suggest utility of the composite AGE panel in predicting long-term decline in renal function.

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

BACKGROUND: Despite 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. METHODS: ACTG 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. RESULTS: Overall, 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. CONCLUSIONS: AGE 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.

Advanced Glycation End Products Are Associated With Inflammation and Endothelial Dysfunction in HIV.

OBJECTIVE: To compare levels of advanced glycation end products (AGEs) between HIV-infected patients and uninfected controls and assess the relationship between AGEs, HIV, inflammation, and endothelial dysfunction. DESIGN: Cross-sectional study involving 90 individuals (68 HIV+ and 22 healthy controls matched by age and sex). METHODS: AGE levels were assessed using 3 different modalities: free AGEs were measured in the serum, skin autofluorescence (AF) was determined with a noninvasive reader, and dietary AGEs were estimated using 24-hour dietary recalls. Markers of inflammation, immune activation, and endothelial dysfunction were also measured. Wilcoxon rank-sum and χ tests were used to compare AGEs between groups. Spearman correlations were used to explore relationships between variables while adjusting for different covariates. RESULTS: Overall, 71% were men and 68% were African American, with a median age of 53 years. Among HIV-infected individuals, all participants were on antiretroviral therapy by design, and most participants (78%) had an undetectable HIV-1 RNA level (≤20 copies/mL). Skin AF and serum AGEs were significantly higher in HIV-infected participants compared with uninfected controls (P < 0.01), whereas no differences in dietary AGEs were found between groups (P = 0.2). In the HIV-infected group, but not in controls, skin AF and circulating AGEs were significantly associated with inflammatory and oxidative markers, and with markers of endothelial dysfunction. CONCLUSIONS: These results suggest intrinsic production of AGE in HIV-infected individuals. The relationship between serum/skin AGE and inflammatory, oxidative, and cardiovascular markers highlights the potential implications of AGEs in chronic inflammation and endothelial dysfunction in HIV, suggesting a new potential target for HIV-associated heightened inflammation and cardiovascular risk.

Experimental Hyperglycemia Alters Circulating Concentrations and Renal Clearance of Oxidative and Advanced Glycation End Products in Healthy Obese Humans.

The purpose of this investigation was to evaluate the effects of experimental hyperglycemia on oxidative damage (OX), advanced glycation end products (AGEs), and the receptor for AGEs (RAGE) through an in vivo approach. Obese subjects (n = 10; 31.2 ± 1.2 kg·m-2; 56 ± 3 years) underwent 24 h of hyperglycemic clamp (+5.4 mM above basal), where plasma at basal and after 2 h and 24 h of hyperglycemic challenge were assayed for OX (methionine sulfoxide, MetSO, and aminoadipic acid, AAA) and AGE-free adducts (Ne-carboxymethyllysine, CML; Ne-carboxyethyllysine, CEL; glyoxal hydroimidazolone-1, GH-1; methylglyoxal hydroimidazolone-1, MG-H1; and 3-deoxyglucosone hydroimidazolone, 3DG-H) via liquid chromatography⁻tandem mass spectrometry (LC⁻MS/MS). Urine was also analyzed at basal and after 24 h for OX and AGE-free adducts and plasma soluble RAGE (sRAGE) isoforms (endogenous secretory RAGE, esRAGE, and cleaved RAGE, cRAGE), and inflammatory markers were determined via enzyme-linked immunosorbent assay (ELISA). Skeletal muscle tissue collected via biopsy was probed at basal, 2 h, and 24 h for RAGE and OST48 protein expression. Plasma MetSO, AAA, CEL, MG-H1, and G-H1 decreased (-18% to -47%; p < 0.05), while CML increased (72% at 24 h; p < 0.05) and 3DG-H remained unchanged (p > 0.05) with the hyperglycemic challenge. Renal clearance of MetSO, AAA, and G-H1 increased (599% to 1077%; p < 0.05), CML decreased (-30%; p < 0.05), and 3DG-H, CEL, and MG-H1 remained unchanged (p > 0.05). Fractional excretion of MetSO, AAA, CEL, G-H1, and MG-H1 increased (5.8% to 532%; p < 0.05) and CML and 3DG-H remained unchanged (p > 0.05). Muscle RAGE and OST48 expression, plasma sRAGE, IL-1ß, IL-1Ra, and TNFα remained unchanged (p > 0.05), while IL-6 increased (159% vs. basal; p > 0.05). These findings suggest that individuals who are obese but otherwise healthy have the capacity to prevent accumulation of OX and AGEs during metabolic stress by increasing fractional excretion and renal clearance.

Divergent Changes in Plasma AGEs and sRAGE Isoforms Following an Overnight Fast in T1DM.

Advanced glycation end products (AGEs) promote the development of diabetic complications through activation of their receptor (RAGE). Isoforms of soluble RAGE (sRAGE) sequester AGEs and protect against RAGE-mediated diabetic complications. We investigated the effect of an overnight fast on circulating metabolic substrates, hormones, AGEs, and sRAGE isoforms in 26 individuals with type 1 diabetes (T1DM). Blood was collected from 26 young (18⁻30 years) T1DM patients on insulin pumps before and after an overnight fast. Circulating AGEs were measured via LC-MS/MS and sRAGE isoforms were analyzed via ELISA. Glucose, insulin, glucagon, and eGFRcystatin-c decreased while cortisol increased following the overnight fast (p < 0.05). AGEs (CML, CEL, 3DG-H, MG-H1, and G-H1) decreased (21⁻58%, p < 0.0001) while total sRAGE, cleaved RAGE (cRAGE), and endogenous secretory RAGE (esRAGE) increased (22⁻24%, p < 0.0001) following the overnight fast. The changes in sRAGE isoforms were inversely related to MG-H1 (rho = -0.493 to -0.589, p < 0.05) and the change in esRAGE was inversely related to the change in G-H1 (rho = -0.474, p < 0.05). Multiple regression analyses revealed a 1 pg/mL increase in total sRAGE, cRAGE, or esRAGE independently predicted a 0.42⁻0.52 nmol/L decrease in MG-H1. Short-term energy restriction via an overnight fast resulted in increased sRAGE isoforms and may be protective against AGE accumulation.

Advanced Glycation End Products, Oxidation Products, and Incident Cardiovascular Events in Patients With Type 2 Diabetes.

OBJECTIVE: The goal of this study was to determine whether plasma levels of advanced glycation end products (AGE) and oxidation products (OP) predict the incidence of cardiovascular disease (CVD) in type 2 diabetes. RESEARCH DESIGN AND METHODS: Five specific AGE (methylglyoxal hydroimidazolone, carboxymethyl lysine, carboxyethyl lysine, 3-deoxyglucosone hydroimidazolone, and glyoxal hydroimidazolone) and two OP (2-aminoadipic acid and methionine sulfoxide [MetSO]) were measured at baseline in two intensive glucose-lowering studies: 1) a subcohort of the Veterans Affairs Diabetes Trial (VADT) (n = 445) and 2) a nested case-control subgroup from the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study (n = 271). RESULTS: Increased levels of several AGE and OP were associated with older age, decreased kidney function, previous CVD, and longer diabetes duration, but not with hemoglobin A1c. In the VADT, increased risk of incident CVD events (n = 107) was associated with lower MetSO after adjusting for age, race/ethnicity, sex, prior CVD event, kidney function, treatment assignment, and diabetes duration (hazard ratio [HR] 0.53; 95% CI 0.28-0.99; P = 0.047). Individuals with both low MetSO and high 3-deoxyglucosone hydroimidazolone concentrations were at highest risk for CVD (HR 1.70; P = 0.01). In the ACCORD study, those with incident CVD events (n = 136) had lower MetSO (by 14%; P = 0.007) and higher glyoxal hydroimidazolone and carboxymethyl lysine (by 18% and 15%, respectively; P = 0.04 for both); however, only the difference in MetSO remained significant after adjustment for prior CVD event (P = 0.002). CONCLUSIONS: Lower levels of MetSO and higher levels of select AGE are associated with increased incident CVD and may help account for the limited benefit of intensive glucose lowering in type 2 diabetes.

Methylglyoxal-derived advanced glycation end products contribute to negative cardiac remodeling and dysfunction post-myocardial infarction.

Advanced glycation end-products (AGEs) have been associated with poorer outcomes after myocardial infarction (MI), and linked with heart failure. Methylglyoxal (MG) is considered the most important AGE precursor, but its role in MI is unknown. In this study, we investigated the involvement of MG-derived AGEs (MG-AGEs) in MI using transgenic mice that over-express the MG-metabolizing enzyme glyoxalase-1 (GLO1). MI was induced in GLO1 mice and wild-type (WT) littermates. At 6 h post-MI, mass spectrometry revealed that MG-H1 (a principal MG-AGE) was increased in the hearts of WT mice, and immunohistochemistry demonstrated that this persisted for 4 weeks. GLO1 over-expression reduced MG-AGE levels at 6 h and 4 weeks, and GLO1 mice exhibited superior cardiac function at 4 weeks post-MI compared to WT mice. Immunohistochemistry revealed greater vascular density and reduced cardiomyocyte apoptosis in GLO1 vs. WT mice. The recruitment of c-kit+ cells and their incorporation into the vasculature (c-kit+CD31+ cells) was higher in the infarcted myocardium of GLO1 mice. MG-AGEs appeared to accumulate in type I collagen surrounding arterioles, prompting investigation in vitro. In culture, the interaction of angiogenic bone marrow cells with MG-modified collagen resulted in reduced cell adhesion, increased susceptibility to apoptosis, fewer progenitor cells, and reduced angiogenic potential. This study reveals that MG-AGEs are produced post-MI and identifies a causative role for their accumulation in the cellular changes, adverse remodeling and functional loss of the heart after MI. MG may represent a novel target for preventing damage and improving function of the infarcted heart.

Advanced Glycation End Products, Oxidation Products, and the Extent of Atherosclerosis During the VA Diabetes Trial and Follow-up Study.

OBJECTIVE: To determine whether plasma levels of advanced glycation end products and oxidation products play a role in the development of atherosclerosis in patients with type 2 diabetes (T2D) over nearly 10 years of the VA Diabetes Trial and Follow-up Study. RESEARCH DESIGN AND METHODS: Baseline plasma levels of methylglyoxal hydroimidazolone, Nε-carboxymethyl lysine, Nε-carboxyethyl lysine (CEL), 3-deoxyglucosone hydroimidazolone and glyoxal hydroimidazolone (G-H1), 2-aminoadipic acid (2-AAA), and methionine sulfoxide were measured in a total of 411 participants, who underwent ultrasound assessment of carotid intima-media thickness (CIMT), and computed tomography scanning of coronary artery calcification (CAC) and abdominal aortic artery calcification (AAC) after an average of 10 years of follow-up. RESULTS: In risk factor-adjusted multivariable regression models, G-H1 was associated with the extent of CIMT and CAC. In addition, 2-AAA was strongly associated with the extent of CAC, and CEL was strongly associated with the extent of AAC. The combination of specific advanced glycation end products and oxidation products (G-H1 and 2-AAA) was strongly associated with all measures of subclinical atherosclerosis. CONCLUSIONS: Specific advanced glycation end products and metabolic oxidation products are associated with the severity of subclinical atherosclerosis over the long term and may play an important role in the "negative metabolic memory" of macrovascular complications in people with long-standing T2D.

Advanced Glycation End Products Predict Loss of Renal Function and Correlate With Lesions of Diabetic Kidney Disease in American Indians With Type 2 Diabetes.

We examined associations of advanced glycation end products (AGEs) with renal function loss (RFL) and its structural determinants in American Indians with type 2 diabetes. Data were from a 6-year clinical trial that assessed renoprotective efficacy of losartan. Participants remained under observation after the trial concluded. Glomerular filtration rate (GFR) was measured annually. Kidney biopsies were performed at the end of the trial. Five AGEs were measured in serum collected at enrollment and at kidney biopsy. RFL was defined as ≥40% decline of measured GFR from baseline. Of 168 participants (mean baseline age 41 years, HbA1c 9.2%, GFR 164 mL/min, and albumin-to-creatinine ratio 31 mg/g), 104 reached the RFL end point during median follow-up of 8.0 years. After multivariable adjustment, each doubling of carboxyethyl lysine (hazard ratio [HR] 1.60 [95% CI 1.08-2.37]) or methylglyoxal hydroimidazolone (HR 1.30 [95% CI 1.02-1.65]) concentration was associated with RFL. Carboxyethyl lysine, carboxymethyl lysine, and methylglyoxal hydroimidazolone correlated positively with cortical interstitial fractional volume (partial r = 0.23, P = 0.03; partial r = 0.25, P = 0.02; and partial r = 0.31, P = 0.003, respectively). Glyoxyl hydroimidazolone and methylglyoxal hydroimidazolone correlated negatively with total filtration surface per glomerulus (partial r = -0.26, P = 0.01; and partial r = -0.21, P = 0.046, respectively). AGEs improve prediction of RFL and its major structural correlates.

Methylglyoxal in diabetes: link to treatment, glycaemic control and biomarkers of complications.

Diabetic complications are major health problems worldwide, with the cost of caring for diabetes rising to US$245 billion in 2012 in the U.S.A. alone. It is widely recognized that non-enzymatic glycation in diabetes is a major cause of damage and dysfunction of key vascular cells. MG (methylglyoxal) is directly toxic to tissues, and is a major precursor of AGEs (advanced glycation end-products). Various propensities to diabetic complications are seen among individuals with diabetes, with accelerated rates occurring in some individuals with modest hyperglycaemia, while others never progress in spite of poor glycaemic control over many years. Since production and detoxification of MG is ultimately controlled by enzymatic mechanisms, both genetic and environmental factors could regulate tissue glycation and potentially account for these variable complication rates. Activation of pathways that determine MG levels occurs in susceptible patients, indicting an important role in pathogenesis. MG leads to formation of specific AGEs, which are likely to predict propensity to diabetic complications. We have shown recently that three specific plasma AGE biomarkers [MG-H1 (MG-derived hydroimidazolones), CEL (Nε-carboxyethyl-lysine) and CML (Nε-carboxymethyl-lysine)] predict biopsy-documented fast DN (diabetic nephropathy) progression. Since two of the predictive biomarkers are MG end-products, these outcomes support a role for MG in the development of DN. Our studies on MG and its end-products have also shown anti-complication effects of the drug metformin, which binds and inactivates MG, thus reducing MG-related AGEs. We have also shown that reducing post-meal glucose decreases MG levels, as well as levels of MG-related AGEs. Our clinical outcome studies have been based on the novel concept that the unique glycation products that we can measure reflect the activity of specific chemical pathways that are selectively activated by hyperglycaemia in patients that are inherently more susceptible to diabetic complications, and can be used to solve other diabetes-related medical questions.

Detection of diabetic nephropathy from advanced glycation endproducts (AGEs) differs in plasma and urine, and is dependent on the method of preparation.

Increased advanced glycation endproducts (AGEs) and oxidation products (OPs) have been proposed as pathogenic for diabetic nephropathy (DN). We investigated the relationship between AGEs and OPs measured in different plasma and urine preparations, and progression of DN in 103 young, normoalbuminuric, normotensive participants with type 1 diabetes in the Natural History of Diabetic Nephropathy Study. The primary endpoint was electron microscopy-measured change in glomerular basement membrane (GBM) width from baseline to 5 years; change in mesangial fractional volume was a secondary endpoint. Fast progressors (FP) were defined as the upper quartile (n = 24) of rate of GBM thickening; slow progressors (SP) were the remainder (n = 79). Four AGEs [3-deoxyglucosone and methylglyoxal hydroimidazolones (DG3H1, MGH1) and carboxymethyl and ethyl lysine (CML, CEL)], and two oxidation products methionine sulfoxide and aminoadipic acid were measured by liquid chromatography, triple quadrupole mass spectrometry. Measurements were done on 10 K plasma filtrates and plasma proteolytic digests (PPD) at year 5, and at four time points over 5 years for urinary 10 K filtrates. Urinary filtrate CEL levels were significantly higher in FP, but not after adjustment for HbA1c, sex, and duration of diabetes. MGHI, CEL, and CML plasma filtrate levels were significantly higher in FP relative to SP (p < 0.05). In PPD, only MGHI showed borderline significantly higher levels in FP relative to SP (p = 0.067), while no other product showed correlation. AGE and OP measurements were not correlated with mesangial expansion. In plasma filtrates, HbA1c at year 5 accounted for 4.7 % of the variation in GBM width. The proportion of variation in GBM width was increased to 11.6 % when MGHI, CEL, and CML were added to the model (6.9 % increase).

Early progression of diabetic nephropathy correlates with methylglyoxal-derived advanced glycation end products.

OBJECTIVE: Increased advanced glycation end products (AGEs) and oxidation products (OPs) are proposed to lead to progression of diabetic nephropathy (DN). We investigated the relationship between AGEs, OPs, and progression of DN in 103 subjects with type 1 diabetes participating in the Natural History of Diabetic Nephropathy Study. RESEARCH DESIGN AND METHODS: Mean age of subjects was 17.6±7.4 years, and mean duration of diabetes was 8.3±4.9 years. All patients were normoalbuminuric. Change in glomerular basement membrane (GBM) width from baseline to 5 years, measured using electron micrographs of renal biopsies, was our primary end point, and mesangial fractional volume was a secondary end point. Fast progressors (FPs) were defined as those in the upper quartile of GBM change, and the remaining patients were classified as slow progressors (SPs). AGEs (3-deoxyglucosone and methylglyoxal hydroimidazolones [MGHI]), carboxymethyl lysine (CML), carboxyethyl lysine (CEL), and OPs (methionine sulfoxide and 2-aminoadipic acid) were measured at year 5 by liquid chromatography/triple-quadruple mass spectroscopy on 10-K plasma filtrates. RESULTS: We found that MGHI, CEL, and CML levels were significantly higher in FPs relative to SPs. No product predicted mesangial expansion. A model containing only HbA1c accounted for 4.7% of GBM width variation, with the total variability explained by the model increasing to 11.6% when MGHI, CEL, and CML were added to the regression model (7.9% increase). MGHI was a significant independent predictor of FP. Using a logistic regression model to relate each biomarker to the probability of a subject's classification as an FP, CML, CEL, and MGHI, but not HbA1c, showed a significant relationship to the probability of FP. CONCLUSIONS: The results suggest that these three major AGEs may be early indicators of progression of important DN lesions.

Effects on post-prandial glucose and AGE precursors from two initial insulin strategies in patients with type 2 diabetes uncontrolled by oral agents.

OBJECTIVE: Progressive ß-cell dysfunction in Type 2 diabetes results in the need for insulin therapy in many patients. Yet the best regimen to prescribe to patients transitioning from oral anti-hyperglycemic drugs (OADs) is not clear. We sought to compare the effects of two standard initial insulin strategies (basal insulin alone versus premixed insulin) on post-prandial glucose metabolism and precursors of advanced glycation end-products in patients with type 2 diabetes suboptimally controlled on OADs. RESEARCH DESIGN AND METHODS: This was a 6-month, open-label, single-center study using a cross-over design. 14 subjects were randomized to one of two protocols: once daily insulin glargine or twice-daily 75%/25% neutral protamine lispro/lispro mix. At 12 weeks, the subjects were crossed-over to the opposite protocol. During each period, insulin doses were titrated to target fasting blood glucose of 90-110 mg/dL. At baseline and after the two 12-week treatment periods, subjects were studied in the Clinical Research Center; they consumed three liquid mixed isocaloric meals at 4-h intervals, and glucose, free fatty acids (FFA), lipids, and α-dicarbonyls (3-deoxyglucosone [3-DG] and methylglyoxal [MG]) were measured before and after each meal. Patient data were analyzed in the context of their assigned insulin strategy groups. RESULT: Both insulin regimens led to a significant improvement in glycemic profiles, including fasting glucose and HbA1c, compared to baseline. However, mean post-prandial glucose was lower with lispro mix than with glargine (153 ± 36 vs. 199 ± 49 mg/dL, respectively; P=0.001). Likewise, there was a reduction in both fasting (48 ± 13 vs. 57 ± 19, P=0.047) and post-prandial (53 ± 19 vs. 63 ± 23; P=0.007) 3DG levels with lispro mix as compared to glargine. No differences were noted in MG concentrations. CONCLUSION: In type 2 diabetes patients failing OAD therapy, an initial insulin regimen of twice daily premixed insulin results in significantly improved post-prandial glucose levels as well as a reduction in a precursor of AGEs. The effect of these two initial insulin regimens on long-term diabetic complications requires further study.

Glycation and biomarkers of vascular complications of diabetes.

Propensity to diabetic nephropathy (DN), retinopathy (DR), and cardiovascular disease (CVD) varies between individuals. Current biomarkers such as indicators of glycemia (HbA1c), retinal examinations, and albuminuria, cannot detect early tissue damage. HbAIc also doesn't reflect most glycative and oxidative chemical pathways that cause complications, and studies of new biomarkers to measure their end-products are needed. This review proposes the study of advanced glycation end products (AGEs) and oxidation end-products (OPs) in long-term diabetes outcome studies. AGEs integrate the activity of glycation pathways that form dicarbonyls, while OPs reflect superoxides, hydroxyl radicals, and peroxides. We discuss using these biomarkers to predict risk of development and progression of DN, DR, and CVD, and to determine if they confer risk independently of the level of HbA1c. We also discuss methods and guidelines to document sample quality in such studies. These studies have the potential to validate unique biomarkers during the early stages of diabetes in those who are at high risk of diabetic complications. Information on basic mechanisms responsible for complications could also stimulate development of therapeutic approaches to delay or arrest them. The ultimate goal is to predict those requiring aggressive therapies during the earliest stages, when prevention or reversal of complications is still possible.

Two fluorescent wavelengths, 440(ex)/520(em) nm and 370(ex)/440(em) nm, reflect advanced glycation and oxidation end products in human skin without diabetes.

BACKGROUND: Advanced glycation end products (AGEs) and oxidation products (OPs) play an important role in diabetes complications, aging, and damage from sun exposure. Measurement of skin autofluorescence (SAF) has been promoted as a noninvasive technique to measure skin AGEs, but the actual products quantified are uncertain. We have compared specific SAF measurements with analytically determined AGEs and oxidative biomarkers in skin collagen and determined if these measurements can be correlated with chronological aging and actinic exposure. METHODS: SAF at four excitation (ex)/emission (em) intensities was measured on the upper inner arm ("sun protected") and dorsal forearm ("sun exposed") in 40 subjects without diabetes 20-60 years old. Skin collagen from the same sites was analyzed by liquid chromatography-tandem mass spectrometry for three AGEs-pentosidine, carboxymethyllysine (CML), and carboxyethyllysine (CEL)-and the OP methionine sulfoxide (MetSO). RESULTS: There was poor correlation of AGE-associated fluorescence spectra with AGEs and OP in collagen, with only pentosidine correlating with fluorescence at 370(ex)/440(em) nm. A little-studied SAF (440(ex)/520(em) nm), possibly reflecting elastin cross-links, correlated with all AGEs and OPs. Levels of CML, pentosidine, and MetSO, but not SAF, were significantly higher in sun-exposed skin. These AGEs and OPs, as well as SAF at 370(ex)/440(em) nm and 440(ex)/520(em) nm, increased with chronological aging. CONCLUSIONS: SAF measurements at 370(ex)/440(em) nm and 335(ex)/385(em) nm, except for pentosidine, which correlated with fluorescence at 370(ex)/440(em), correlate poorly with glycated and oxidatively modified protein in human skin and do not reflect actinic modification. A new fluorescence measurement (440(ex)/520(em) nm) appears to reflect AGEs and OPs in skin.

Mealtime 50/50 basal + prandial insulin analogue mixture with a basal insulin analogue, both plus metformin, in the achievement of target HbA1c and pre- and postprandial blood glucose levels in patients with type 2 diabetes: a multinational, 24-week, randomized, open-label, parallel-group comparison.

BACKGROUND: In people without diabetes, approximately 50% of daily insulin secretion is basal and the remainder is postprandial. Hence, it would be expected that insulin replacement therapy in a 50/50 ratio with each meal would mimic physiologic insulin secretion better than treatment with once-daily basal insulin in patients with diabetes mellitus. Using lispro mix (LM) 50/50 before meals may be a logical approach to achieving glycemic targets (glycosylated hemoglobin [HbA(lc)] and pre- and postprandial blood glucose [BG] concentrations) in these patients. OBJECTIVE: The aim of this study was to test the hypothesis that treatment with a premixed insulin analogue containing 50/50 basal + prandial insulins administered before each meal would achieve lower overall and mealtime glycemic control than once-daily basal insulin analogue, both plus metformin (Met), in patients with type 2 diabetes mellitus. METHODS: This 24-week, randomized, open-label, parallel-group trial was conducted at 38 sites across Australia, Greece, India, The Netherlands, Poland, Puerto Rico, and the United States. Male and female patients aged 35 to 75 years with type 2 diabetes mellitus and an HbA(1c) level of 6.5% to 11.0%, who were receiving metformin and/or a sulfonylurea with a stable dose of 0 to 2 daily insulin injections over the previous 3 months were eligible. Patients were randomly assigned to receive LM50/50 (50% insulin lispro protamine suspension [ILPS] and 50% lispro) TID plus metformin (to a maximally tolerated daily dosage of 500-1000 mg BID) (LM50/50 + Met) or insulin glargine QD at bedtime plus metformin (500-1000 mg BID) (G + Met) for 24 weeks. With LM50/50 + Met, the insulin dose was titrated to target a fasting BG (FBG) level of <6.7 mmol/L (<120 mg/dL) and a 2-hour post-prandial BG (PPBG) level of <8.0 mmol/L (<144 mg/dL); those who did not reach the FBG target would be switched from presupper LM50/50 to LM75/25 (75% ILPS, 25% lispro). RESULTS: A total of 315 patients were randomized and received treatment (158 women, 157 men; mean age, 57.7 years; mean body mass index, 32.1 kg/m2; LM50/50 + Met, 157 patients; G + Met, 158 patients). At 24 weeks, the mean (SD)HbA(1c) level was significantly lower in the LM50/50 + Met group than in the G + Met group (7.1% [0.9%] vs 7.5% [1.0%]; P<0.001), and the proportion who reached an HbA(1c) target of < or = 7.0% was greater (88 [56.1%] vs 63 [39.9%]; P = 0.005). The G + Met group had a lower mean (SD)FBG value (6.5 [1.6] vs 8.1 [1.8] mmol/L; P<0.001). The LM50/50 + Met group had lower mean preprandial BG levels prelunch (7.4 [1.9] vs 7.9 [2.1] mmol/L; P=0.03) and presupper (8.3 [2.0] vs 8.9 [2.8] mmol/L; P=0.04). The LM50/50 + Met group also had lower mean 2-hour PPBG values postbreakfast (8.7 [2.2] vs 9.2 [2.5] mmol/L; P=0.03), postlunch (8.4 [1.9] vs 9.8 [2.6], mmol/L; p<0.001), and postsupper (8.7 [2.2] vs 10.7 [3.2], mmol/L; P<0.001). The mean (SD) total insulin doses at study end point were 0.7 (0.3) U/kg in the LM50/50 + Met group and 0.6 (0.3) U/kg in the G + Met group (P<0.001). The mean (SD)M-value (an expression of mean glycemia and the effect of glucose swings) was statistically similar between the 2 groups at baseline but significantly lower in the LM50/50 + Met group at end point (17.3 [13.8] vs 25.1 [24.8] mmol/L; P<0.001). During the entire treatment period, mean (SD) overall and nocturnal hypoglycemia rates (episodes per patient for 30 days) were statistically similar between the 2 groups (overall, 0.8 [1.4] vs 0.5 [1.0]; nocturnal, 0.2 [0.7] vs 0.3 [0.6]). At end point, the mean (SD) nocturnal hypoglycemia rates were similar between the 2 groups (0.2 [0.9] vs 0.2 [0.6]), but the overall and non-nocturnal hypoglycemia rates were higher with LM50/50 + Met (overall, 0.7 [1.7] vs 0.3 [0.8]; P=0.02; non-nocturnal, 0.5 [1.2] vs 0.1 [0.4]; P=0.002). CONCLUSION: In these patients with type 2 diabetes, mealtime LM50/50 + Met was associated with lower overall (HbA(1c)) and preprandial BG and PPBG levels (except for FBG), with similar nocturnal hypoglycemia and less glycemic variability, compared with G + Met.