Cite asBronowicka-Szydełko A, Krzystek-Korpacka M, Kuzan A, et al. Non-standard AGE4 epitopes that predict polyneuropathy independently of obesity can be detected by slot dot-blot immunoassay.
AbstractBackground. Advanced glycation end-products (AGEs) are formed during cascade reactions between reducing sugars or reactive aldehydes and proteins, lipids or DNA molecules. They constitute a group of various stable compounds. Advanced glycation end-products are considered potential biomarkers of metabolic disorders. However, so far only a few methods to determine the level of individual AGEs have been developed.Objectives. The aim of the study was to compare the efficiency of the slot-dot blot method and direct enzyme-linked immunosorbent assay (ELISA) in detecting non-standard epitopes of methylglyoxal (MGO)modified proteins (AGE4) found in diabetes serum in trace amounts, and to assess AGE4 in diabetes and associated metabolic abnormalities.Material and methods. The presence of AGE4 was detected using 2 methods: direct ELISA and the slotdot blot method -a newly developed immunoassay based on monoclonal, commercially available antibody detection of non-standard AGE epitopes. AGE4 quantification, expressed as median AGE4 in arbitrary units (AU) and AGE4 positivity (the percent of samples with detectable AGE4) was related to diabetes-associated metabolic abnormalities, complications and treatment.Results. Slot-dot blot was significantly more efficient than ELISA in detecting non-standard AGE4 epitopes. AGE4 positivity was less frequent in patients with microangiopathy and in those with polyneuropathy. In patients with abnormal glucose metabolism, metformin treatment was associated with higher AGE4. AGE4 positivity was significantly lower in gliptin-treated patients. Multivariate analysis showed that polyneuropathy and obesity were independently associated with AGE4 positivity, with odds ratios (ORs) of 0.21 and 3.02, respectively. Moreover, logistic regression showed that AGE4 positivity and HbA1c are independent predictors of polyneuropathy. Considering both indicators allows correct classification of 70.4% of cases with a general accuracy of 76%.
Conclusions.The slot dot-blot method detects compounds found in serum in trace amounts. Accumulation of AGE4 was associated with glucose metabolism abnormalities. A tendency toward AGE4 positivity was less frequent in patients with microangiopathy and in non-treated and gliptin-treated diabetes patients.
Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (Ks), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991 × 10(3)/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced Ks (-38%, p < 0.0001) and prolonged CLT (+34%, p < 0.0001) were observed in ET. The differences remained significant after adjustment for fibrinogen and platelet count. ET was associated with a slightly shorter lag phase (-5%, p = 0.01) and higher maximum absorbency of the turbidimetric curve (+6%, p < 0.001). The ET patients had higher plasma P-selectin by 193% (p < 0.00001) and platelet factor 4 (PF4) by 173% (p < 0.00001), with higher P-selectin observed in 19 (44%) patients with JAK-2 gene V617F mutation. Higher t-PA (+20%, p < 0.001), 23% higher plasminogen activator inhibitor-1, PAI-1 (+23%, p < 0.01) and unaltered thrombin-activatable fibrinolysis inhibitor, plasminogen and α2-antiplasmin activity were found in the ET group. Ks inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.
Advanced glycation end-products (AGEs) contribute to vascular complications and organ damage in diabetes. The unique AGE epitope (AGE10) has recently been identified in human serum using synthetic melibiose-derived AGE (MAGE). We aimed at developing ELISA for AGE10 quantification, determining whether AGE10 is present in diabetic patients (n = 82), and evaluating its association with diabetic complications. In a competitive ELISA developed, the reaction of synthetic MAGE with anti-MAGE was inhibited by physiological AGE10 present in serum. In this assay, new murine IgE anti-MAGE monoclonal antibodies, which do not recognize conventional AGEs, a synthetic MAGE used to coat the plate, and LMW-MAGE (low molecular mass MAGE) necessary to plot a standard curve were used. AGE10 was significantly higher in patients with microangiopathy, in whom it depended on treatment, being lower in patients treated with aspirin. AGE10 levels were positively correlated with estimated glomerular filtration rate (eGFR) and negatively with creatinine. As a marker of stage ≥3 chronic kidney disease or microangiopathy, AGE10 displayed moderate overall accuracy (respectively, 69% and 71%) and good sensitivity (82.6% and 83.3%) but poor specificity (58.1% and 57.8%). In conclusion, newly developed immunoassay allows for AGE10 quantification. AGE10 elevation is associated with microangiopathy while its decrease accompanies stage ≥3 chronic kidney disease.
Objective. The status of metabolites of the nitric oxide (NO) pathway in patients with chronic wounds in the course of cardiometabolic diseases is largely unknown. Yet arginine supplementation and citrulline supplementation as novel therapeutic modalities aimed at increasing NO are tested. Material and Methods. Targeted metabolomics approach (LC-MS/MS) was applied to determine the concentrations of L-arginine, L-citrulline, asymmetric and symmetric dimethylarginines (ADMA and SDMA), and arginine/ADMA and arginine/SDMA ratios as surrogate markers of NO and arginine availability in ulnar and femoral veins, representing systemic and local levels of metabolites, in patients with chronic wounds in the course of cardiometabolic diseases (n=59) as compared to patients without chronic wounds but with similar cardiometabolic burden (n=55) and healthy individuals (n=88). Results. Patients with chronic wounds had significantly lower systemic L-citrulline and higher ADMA and SDMA concentrations and lower L-arginine/ADMA and L-arginine/SDMA as compared to healthy controls. The presence of chronic wounds in patients with cardiometabolic diseases was associated with decreased L-arginine but with increased L-citrulline, ADMA, and SDMA concentrations and decreased L-arginine/ADMA and L-arginine/SDMA. Serum obtained from the ulnar and femoral veins of patients with chronic wounds differed by L-arginine concentrations and L-arginine/SDMA ratio, both lower in the femoral vein. Wound etiology affected L-citrulline and SDMA concentrations, lower and higher, respectively, in patients with venous stasis, and the L-arginine/SDMA ratio—lower in venous stasis. The wound type affected L-arginine/ADMA and citrulline—lower in patients with ulcerations or gangrene. IL-6 was an independent predictor of L-arginine/ADMA, VEGF-A of ADMA, G-CSF of L-arginine/SDMA, and GM-CSF of L-citrulline and SDMA. Conclusion. Chronic wounds in the course of cardiometabolic diseases are associated with reduced NO and arginine availability due to ADMA and SDMA accumulation rather than arginine deficiency, not supporting its supplementation. Wound character seems to affect NO bioavailability and wound etiology—arginine bioavailability. Arginine concentration and its availability are more markedly reduced at the local level than the systemic level.
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