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

M. Villa, M. Parravano, A. Micheli, L. Gaddini, A. Matteucci, C. Mallozzi, F. Facchiano, F. Malchiodi-Albedi, F. Pricci

Research output: Contribution to journal › Article

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. © 2017 Elsevier Inc.
Original languageEnglish
Pages (from-to)64-69
Number of pages6
JournalMetabolism: Clinical and Experimental
Volume71
DOIs
Publication statusPublished - 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
  • advanced glycation end product
  • glucose
  • bovine serum albumin
  • fluorescent dye
  • animal experiment
  • animal model
  • animal tissue
  • Article
  • controlled study
  • glycation
  • in vitro study
  • in vivo study
  • nonhuman
  • priority journal
  • rat
  • retina tissue
  • spectrofluorometry
  • streptozotocin-induced diabetes mellitus
  • vitreous body
  • Western blotting
  • animal
  • blood
  • chemistry
  • diabetic retinopathy
  • experimental diabetes mellitus
  • male
  • metabolism
  • pathology
  • polyacrylamide gel electrophoresis
  • procedures
  • retina
  • Sprague Dawley rat
  • Animals
  • Diabetes Mellitus, Experimental
  • Diabetic Retinopathy
  • Electrophoresis, Polyacrylamide Gel
  • Fluorescent Dyes
  • Glycosylation End Products, Advanced
  • Male
  • Rats
  • Rats, Sprague-Dawley
  • Retina
  • Serum Albumin, Bovine
  • Spectrometry, Fluorescence

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, M.; Parravano, M.; Micheli, A.; Gaddini, L.; Matteucci, A.; Mallozzi, C.; Facchiano, F.; Malchiodi-Albedi, F.; Pricci, F.

In: Metabolism: Clinical and Experimental, Vol. 71, 2017, p. 64-69.

Research output: Contribution to journal › Article

Villa, M, Parravano, M, Micheli, A, Gaddini, L, Matteucci, A, Mallozzi, C, Facchiano, F, Malchiodi-Albedi, F & Pricci, F 2017, 'A quick, simple method for detecting circulating fluorescent advanced glycation end-products: Correlation with in vitro and in vivo non-enzymatic glycation', Metabolism: Clinical and Experimental, vol. 71, pp. 64-69. https://doi.org/10.1016/j.metabol.2017.03.004
Villa M, Parravano M, Micheli A, Gaddini L, Matteucci A, Mallozzi C et al. A quick, simple method for detecting circulating fluorescent advanced glycation end-products: Correlation with in vitro and in vivo non-enzymatic glycation. Metabolism: Clinical and Experimental. 2017;71:64-69. https://doi.org/10.1016/j.metabol.2017.03.004
Villa, M. ; Parravano, M. ; Micheli, A. ; Gaddini, L. ; Matteucci, A. ; Mallozzi, C. ; Facchiano, F. ; Malchiodi-Albedi, F. ; Pricci, F. / 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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title = "A quick, simple method for detecting circulating fluorescent advanced glycation end-products: Correlation with in vitro and in vivo non-enzymatic glycation",
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. {\circledC} 2017 Elsevier Inc.",
keywords = "Advanced glycation end-product, Diabetes, Plasma, Rat, Vitreous, advanced glycation end product, glucose, bovine serum albumin, fluorescent dye, animal experiment, animal model, animal tissue, Article, controlled study, glycation, in vitro study, in vivo study, nonhuman, priority journal, rat, retina tissue, spectrofluorometry, streptozotocin-induced diabetes mellitus, vitreous body, Western blotting, animal, blood, chemistry, diabetic retinopathy, experimental diabetes mellitus, male, metabolism, pathology, polyacrylamide gel electrophoresis, procedures, retina, Sprague Dawley rat, Animals, Diabetes Mellitus, Experimental, Diabetic Retinopathy, Electrophoresis, Polyacrylamide Gel, Fluorescent Dyes, Glycosylation End Products, Advanced, Male, Rats, Rats, Sprague-Dawley, Retina, Serum Albumin, Bovine, Spectrometry, Fluorescence",
author = "M. Villa and M. Parravano and A. Micheli and L. Gaddini and A. Matteucci and C. Mallozzi and F. Facchiano and F. Malchiodi-Albedi and F. Pricci",
note = "Export Date: 4 April 2018 CODEN: METAA Correspondence Address: Pricci, F.; Dept of Cardiovascular, Dysmetabolic and Aging-associated diseasesUnited States; email: flavia.pricci@iss.it Chemicals/CAS: glucose, 50-99-7, 84778-64-3; Fluorescent Dyes; Glycosylation End Products, Advanced; Serum Albumin, Bovine References: Giacco, F., Brownlee, M., Oxidative stress and diabetic complications (2010) Circ Res, 107 (9), pp. 1058-1070; Takeuchi, M., Kikuchi, S., Sasaki, N., Suzuki, T., Watai, T., Iwaki, M., Involvement of advanced glycation end-products (AGEs) in Alzheimer's disease (2004) Curr Alzheimer Res, 1, pp. 39-46; Baynes, J.W., The role of AGEs in aging: causation or correlation (2001) Exp Gerontol, 36, pp. 1527-1537; Brownlee, M., Biochemistry and molecular cell biology of diabetic complications (2001) Nature, 414 (6865), pp. 813-820; Singh, R., Barden, A., Mori, T., Beilin, L., Advanced glycation end-products: a review (2001) Diabetologia, 44, pp. 129-146; Di Mario, U., Pugliese, G., 15th Golgi lecture: from hyperglycaemia to the dysregulation of vascular remodelling in diabetes (2001) Diabetologia, 44, pp. 674-692; Ahmed, N., Advanced glycation endproducts-role in pathology of diabetic complications (2005) Diabetes Res Clin Pract, 67 (1), pp. 3-21; de Oliveira, S.C., Delbosc, S., Ara{\"i}s, C., Monnier, L., Cristol, J.P., Pares-Herbute, N., Modulation of CD36 protein expression by AGEs and insulin in aortic VSMCs from diabetic and non-diabetic rats (2008) Nutr Metab Cardiovasc Dis, 18 (1), pp. 23-30; Pricci, F., Leto, G., Amadio, L., Iacobini, C., Cordone, S., Catalano, S., Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C (2003) Free Radic Biol Med, 35 (6), pp. 683-694; Pinz{\'o}n-Duarte, G., Kohler, K., Arango-Gonz{\'a}lez, B., Guenther, E., Cell differentiation, synaptogenesis, and influence of the retinal pigment epithelium in a rat neonatal organotypic retina culture (2000) Vision Res, 40 (25), pp. 3455-3465; Sampathkumar, R., Balasubramanyam, M., Rema, M., Premanand, C., Mohan, V., A novel advanced glycation index and its association with diabetes and microangiopathy (2005) Metabolism, 54 (8), pp. 1002-1007; Zong, H., Ward, M., Stitt, A.W., AGEs, RAGE, and diabetic retinopathy (2011) Curr Diab Rep, 11 (4), pp. 244-252; Cohen, M.P., Hud, E., Shea, E., Shearman, C.W., Vitreous fluid of db/db mice exhibits alterations in angiogenic and metabolic factors consistent with early diabetic retinopathy (2008) Ophthalmic Res, 40 (1), pp. 5-9; G{\'e}hl, Z., Bakondi, E., Resch, M.D., Hegedűs, C., Kov{\'a}cs, K., Lakatos, P., Diabetes-induced oxidative stress in the vitreous humor (2016) Redox Biol, 9, pp. 100-103",
year = "2017",
doi = "10.1016/j.metabol.2017.03.004",
language = "English",
volume = "71",
pages = "64--69",
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TY - JOUR

T1 - A quick, simple method for detecting circulating fluorescent advanced glycation end-products: Correlation with in vitro and in vivo non-enzymatic glycation

AU - Villa, M.

AU - Parravano, M.

AU - Micheli, A.

AU - Gaddini, L.

AU - Matteucci, A.

AU - Mallozzi, C.

AU - Facchiano, F.

AU - Malchiodi-Albedi, F.

AU - Pricci, F.

N1 - Export Date: 4 April 2018 CODEN: METAA Correspondence Address: Pricci, F.; Dept of Cardiovascular, Dysmetabolic and Aging-associated diseasesUnited States; email: flavia.pricci@iss.it Chemicals/CAS: glucose, 50-99-7, 84778-64-3; Fluorescent Dyes; Glycosylation End Products, Advanced; Serum Albumin, Bovine References: Giacco, F., Brownlee, M., Oxidative stress and diabetic complications (2010) Circ Res, 107 (9), pp. 1058-1070; Takeuchi, M., Kikuchi, S., Sasaki, N., Suzuki, T., Watai, T., Iwaki, M., Involvement of advanced glycation end-products (AGEs) in Alzheimer's disease (2004) Curr Alzheimer Res, 1, pp. 39-46; Baynes, J.W., The role of AGEs in aging: causation or correlation (2001) Exp Gerontol, 36, pp. 1527-1537; Brownlee, M., Biochemistry and molecular cell biology of diabetic complications (2001) Nature, 414 (6865), pp. 813-820; Singh, R., Barden, A., Mori, T., Beilin, L., Advanced glycation end-products: a review (2001) Diabetologia, 44, pp. 129-146; Di Mario, U., Pugliese, G., 15th Golgi lecture: from hyperglycaemia to the dysregulation of vascular remodelling in diabetes (2001) Diabetologia, 44, pp. 674-692; Ahmed, N., Advanced glycation endproducts-role in pathology of diabetic complications (2005) Diabetes Res Clin Pract, 67 (1), pp. 3-21; de Oliveira, S.C., Delbosc, S., Araïs, C., Monnier, L., Cristol, J.P., Pares-Herbute, N., Modulation of CD36 protein expression by AGEs and insulin in aortic VSMCs from diabetic and non-diabetic rats (2008) Nutr Metab Cardiovasc Dis, 18 (1), pp. 23-30; Pricci, F., Leto, G., Amadio, L., Iacobini, C., Cordone, S., Catalano, S., Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C (2003) Free Radic Biol Med, 35 (6), pp. 683-694; Pinzón-Duarte, G., Kohler, K., Arango-González, B., Guenther, E., Cell differentiation, synaptogenesis, and influence of the retinal pigment epithelium in a rat neonatal organotypic retina culture (2000) Vision Res, 40 (25), pp. 3455-3465; Sampathkumar, R., Balasubramanyam, M., Rema, M., Premanand, C., Mohan, V., A novel advanced glycation index and its association with diabetes and microangiopathy (2005) Metabolism, 54 (8), pp. 1002-1007; Zong, H., Ward, M., Stitt, A.W., AGEs, RAGE, and diabetic retinopathy (2011) Curr Diab Rep, 11 (4), pp. 244-252; Cohen, M.P., Hud, E., Shea, E., Shearman, C.W., Vitreous fluid of db/db mice exhibits alterations in angiogenic and metabolic factors consistent with early diabetic retinopathy (2008) Ophthalmic Res, 40 (1), pp. 5-9; Géhl, Z., Bakondi, E., Resch, M.D., Hegedűs, C., Kovács, K., Lakatos, P., Diabetes-induced oxidative stress in the vitreous humor (2016) Redox Biol, 9, pp. 100-103

PY - 2017

Y1 - 2017

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. © 2017 Elsevier Inc.

AB - 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. © 2017 Elsevier Inc.

KW - Advanced glycation end-product

KW - Diabetes

KW - Plasma

KW - Rat

KW - Vitreous

KW - advanced glycation end product

KW - glucose

KW - bovine serum albumin

KW - fluorescent dye

KW - animal experiment

KW - animal model

KW - animal tissue

KW - Article

KW - controlled study

KW - glycation

KW - in vitro study

KW - in vivo study

KW - nonhuman

KW - priority journal

KW - rat

KW - retina tissue

KW - spectrofluorometry

KW - streptozotocin-induced diabetes mellitus

KW - vitreous body

KW - Western blotting

KW - animal

KW - blood

KW - chemistry

KW - diabetic retinopathy

KW - experimental diabetes mellitus

KW - male

KW - metabolism

KW - pathology

KW - polyacrylamide gel electrophoresis

KW - procedures

KW - retina

KW - Sprague Dawley rat

KW - Animals

KW - Diabetes Mellitus, Experimental

KW - Diabetic Retinopathy

KW - Electrophoresis, Polyacrylamide Gel

KW - Fluorescent Dyes

KW - Glycosylation End Products, Advanced

KW - Male

KW - Rats

KW - Rats, Sprague-Dawley

KW - Retina

KW - Serum Albumin, Bovine

KW - Spectrometry, Fluorescence

U2 - 10.1016/j.metabol.2017.03.004

DO - 10.1016/j.metabol.2017.03.004

M3 - Article

VL - 71

SP - 64

EP - 69

JO - Metabolism: Clinical and Experimental

JF - Metabolism: Clinical and Experimental

SN - 0026-0495

ER -

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