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

Marika Villa, Mariacristina Parravano, Arianna Micheli, Lucia Gaddini, Andrea Matteucci, Cinzia Mallozzi, Francesco Facchiano, Fiorella Malchiodi-Albedi, Flavia Pricci

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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

Original languageEnglish
Pages (from-to)64-69
Number of pages6
JournalMetabolism: Clinical and Experimental
Volume71
DOIs
Publication statusPublished - Jun 1 2017

Fingerprint

Advanced Glycosylation End Products
Glucose
Streptozocin
Diabetes Complications
In Vitro Techniques
Disease Progression
Retina
Theoretical Models
Biomarkers
Fluorescence
Western Blotting
Pharmaceutical Preparations

Keywords

  • Advanced glycation end-product
  • Diabetes
  • Plasma
  • Rat
  • Vitreous

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Endocrinology

Cite this

A quick, simple method for detecting circulating fluorescent advanced glycation end-products : Correlation with in vitro and in vivo non-enzymatic glycation. / Villa, Marika; Parravano, Mariacristina; Micheli, Arianna; Gaddini, Lucia; Matteucci, Andrea; Mallozzi, Cinzia; Facchiano, Francesco; Malchiodi-Albedi, Fiorella; Pricci, Flavia.

In: Metabolism: Clinical and Experimental, Vol. 71, 01.06.2017, p. 64-69.

Research output: Contribution to journalArticle

Villa, Marika ; Parravano, Mariacristina ; Micheli, Arianna ; Gaddini, Lucia ; Matteucci, Andrea ; Mallozzi, Cinzia ; Facchiano, Francesco ; Malchiodi-Albedi, Fiorella ; Pricci, Flavia. / A quick, simple method for detecting circulating fluorescent advanced glycation end-products : Correlation with in vitro and in vivo non-enzymatic glycation. In: Metabolism: Clinical and Experimental. 2017 ; Vol. 71. pp. 64-69.
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AU - Micheli, Arianna

AU - Gaddini, Lucia

AU - Matteucci, Andrea

AU - Mallozzi, Cinzia

AU - Facchiano, Francesco

AU - Malchiodi-Albedi, Fiorella

AU - Pricci, Flavia

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N2 - Objective Advanced glycation end-products (AGEs) constitute a highly heterogeneous family of compounds, relevant in the pathogenesis of diabetic complications, which could represent efficient biomarkers of disease progression and drug response. Unfortunately, due to their chemical heterogeneity, no method has been validated to faithfully monitor their levels in the course of the disease. In this study, we refine a procedure to quantitatively analyze fluorescent AGEs (fAGEs), a subset considered remarkably representative of the entire AGE family, and measure them in in vitro glycated BSA (gBSA) and in plasma and vitreous of diabetic rats, for testing its use to possibly quantify circulating AGEs in patients, as markers of metabolic control. Methods fAGE levels were evaluated by spectrofluorimetric analysis in in vitro and in vivo experimental models. BSA was glycated in vitro with increasing D-glucose concentrations for a fixed time or with a fixed D-glucose concentration for increasing time. In in vivo experiments, streptozotocin-induced diabetic rats were studied at 1, 3, 6 and 12 weeks to analyze plasma and vitreous. To confirm the presence of AGEs in our models, non-diabetic rat retinal explants were exposed to high glucose (HG), to reproduce short-term effects, or in vitro gBSA, to reproduce long-term effects of elevated glucose concentrations. Rat retinal explants and diabetic retinal tissues were evaluated for the receptor for advanced glycation end-product (RAGE) by Western blot analysis. Results In in vitro experiments, fluorescence emission showed glucose concentration- and time-dependent increase of fAGEs in gBSA (p ≤ 0.05). In streptozotocin-induced diabetic rats, fAGE in plasma and vitrei showed an increase at 6 (p ≤ 0.005) and 12 (p ≤ 0.05) weeks of diabetes, with respect to control. RAGE was time-dependently upregulated in retinas incubated with gBSA, but not with HG, and in diabetic retinal tissue, substantiating exposure to AGEs. Conclusions Applying the proposed technique, we could show that fAGEs levels increase with glucose concentration and time of exposure in vitro. Furthermore, in diabetic rats, it showed that circulating fAGEs are similarly upregulated as those in vitreous, suggesting a correlation between circulating and tissue AGEs. These results support the use of this method as a simple and reliable test to measure circulating fAGEs and monitor diabetes progression.

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