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 DIETARY AGEs
AND THEIR ROLE IN
HEALTH AND DISEASE
 DIETARY AGEs
AND THEIR ROLE IN
HEALTH AND DISEASE

EDITED BY
Jamie Uribarri
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742

© 2018 by Taylor & Francis Group, LLC

CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government works

Printed on acid-free paper

International Standard Book Number-13: 978-1-4987-2151-6 (Hardback)

This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been
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Library of Congress Cataloging‑in‑Publication Data

Names: Uribarri, Jaime, editor.

Title: Dietary AGEs and their role in health and disease / edited by Jaime
Uribarri.
Description: Boca Raton, Florida : CRC Press, [2018] | Includes
bibliographical references and index.
Identifiers: LCCN 2017022981| ISBN 9781498721516 (hardback : alk. paper) |
ISBN 9781315120041 (e-book) | ISBN 9781498721523 (e-book) | ISBN
9781351646352 (e-book) | ISBN 9781351636810 (e-book)
Subjects: | MESH: Glycosylation End Products, Advanced--adverse effects |
Glycosylation End Products, Advanced--metabolism | Glycosylation End
Products, Advanced--drug effects | Diet--adverse effects
Classification: LCC QP606.G6 | NLM QU 55.5 | DDC 572.792--dc23
LC record available at https://lccn.loc.gov/2017022981

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Contents

Preface....................................................................................................................................................... ix
Editor......................................................................................................................................................... xi
Contributors.............................................................................................................................................xiii

Section I What Are AGEs?

1. What Are AGEs, Their Chemical Structure, and How Can They Be Measured?..................... 3
Katarzyna Wrobel, Kazimierz Wrobel, Sarahi Jaramillo Ortiz, and Alma Rosa Corrales Escobosa

2. How AGEs Cause Disease: Cellular Mechanisms........................................................................ 19

Melinda M. Nugent and John C. He

3. AGE Clearance Mechanisms......................................................................................................... 37

Armando Rojas, Ileana Gonzalez, and Carolina Añazco

4. How Are AGEs Handled by the Kidney?.......................................................................................51

Alejandro Gugliucci

Section II The Modern Diet & AGEs: How Do Exogenous

AGEs Become Incorporated into Our Body?
5. Dietary Advanced Glycation End Products: Animal Studies..................................................... 63
Melpomeni Peppa

6. AGEs in Infant Formulas: Chemical and Physiological Aspects............................................... 75

Latifa Abdennebi-Najar and Ghada Elmhiri

7. Potentially Toxic Food Components Formed by Excessive Heat Processing............................ 87

Franco Pedreschi and Michael Murkovic

8. Is Part of the Fructose Effects on Health Related to Increased AGE Formation?................. 103
Halyna Semchyshyn

Section III Role of AGEs in the Pathogenesis of Chronic Diseases

9. Role of Advanced Glycation Products in Health and Disease in Children..............................115
Anshu Gupta and Tasnim Rahman

10. The Role of Advanced Glycation End Products in Cognitive Decline and Dementia............ 123
Roni Lotan, Aron M. Troen, and Michal Schnaider Beeri

v
vi Contents

11. Advanced Glycation End Products and Polycystic Ovarian Syndrome.................................. 137
Eleni A. Kandaraki and Evanthia Diamanti-Kandarakis

12. Dietary AGEs and Diabetic Complications.................................................................................147

Ma. Eugenia Garay-Sevilla, Armando Gómez-Ojeda, and Claudia Luevano-Contreras

13. Dietary AGEs and Aging.............................................................................................................. 159

Claudia Luevano-Contreras, Ma. Eugenia Garay-Sevilla, and Armando Gomez-Ojeda

14. AGE and Erectile Dysfunction: Any Role of Dietary AGEs?....................................................171

Delminda Neves

15. Biological Implications of Diet-Derived Advanced Glycation End Products

on Carcinogenesis...........................................................................................................................189
David P. Turner and Victoria J. Findlay

16. Advanced Glycation End Products and Their Receptors in Aspiration-Induced Acute
Respiratory Distress Syndrome................................................................................................... 203
Julie Ottosen, Peter Smit, and Weidun Alan Guo

17. Dietary AGEs in the Development and Progression of Chronic Kidney Disease....................213
Amelia K. Fotheringham, Linda A. Gallo, and Josephine M. Forbes

18. Dietary AGEs May Have Different Effects in People with Vegetarian versus
Omnivorous Eating Patterns........................................................................................................ 225
Katarina Šebeková and Katarína Brouder Šebeková

19. Effects of Dietary AGEs in the Gut Microbiota Composition.................................................. 239

Sergio Pérez-Burillo, Silvia Pastoriza, José Ángel Rufián-Henares, and
Cristina Delgado-Andrade

20. Associations of Circulating AGE Levels and Cardiovascular Disease—Incidence

and Outcome.................................................................................................................................. 247
Kristian F. Hanssen and Kari Anne Sveen

21. Pathological Role of AGEs in Osteoporosis................................................................................ 253

Sho-ichi Yamagishi

22. Is There a Relationship between Dietary AGEs and Food Allergies?..................................... 265
Masako Toda

23. Quantitation and Potential Health Effects of Advanced Glycation End Products in
Pet Foods........................................................................................................................................ 275
Guido Bosch and Wouter Hendriks

24. The Role of AGEs in the Pathogenesis of Macrovascular Complications in

Diabetes Mellitus........................................................................................................................... 283
Marisa Passarelli
Contents vii

Section IV Therapeutic Alternatives to Deal with Dietary AGEs

25. Plant-Derived Products with Antiglycation Activity................................................................. 295
Laura C. Cogoi and Rosana Filip

26. Dietary Intake of AGEs and ALEs and Inflammation: Nutritional Aspects.......................... 309
Stig Bengmark

27. Effects of a Low-AGE Diet on Insulin Sensitivity...................................................................... 329

Barbora de Courten and Estifanos Baye

28. Clinical Trials with an AGE-Restricted Diet............................................................................. 339

Jaime Uribarri

29. Blocking Gastrointestinal Absorption of AGEs......................................................................... 345

Rabi Yacoub

30. Antagonizing the Effects of Dietary Advanced Glycation End Products on

Endothelial Dysfunction................................................................................................................353
Ovidiu Alin Stirban

31. Methylglyoxal and Other AGEs: Good and Bad Dual Role in the Body................................. 365
Mayuri Gogoi, Kapudeep Karmakar, Kasturi Chandra, and Dipshikha Chakravortty

Index....................................................................................................................................................... 379
Preface

Modern “Western” society has brought with itself profound changes in lifestyle but at the same time a
much greater prevalence of chronic diseases such as cardiovascular disease, metabolic syndrome, insulin
resistance, obesity, and type-2 diabetes mellitus. All these diseases seem to have in common elevated
levels of markers of inflammation and oxidative stress. Of the many components of modern lifestyle,
which alone or in combination may play a role increasing inflammation and oxidative stress, diet is
a prominent one. Of the many dietary factors that may be associated with inflammation and oxida-
tive stress, we have been particularly interested in a specific group of food-derived pro-inflammatory
and pro-oxidant compounds, the so-called advanced glycation end products (AGEs). AGEs represent a
large heterogeneous group of compounds, which has made it difficult to standardize their measurement.
Although endogenous AGEs have been widely recognized as important factors in the pathogenesis of
diabetic complications, the importance of AGEs of dietary origin as a factor in human disease has been
largely unappreciated until recently. Over the past two decades, several clinical trials have tested the
effects of a low-AGE dietary intervention on a variety of conditions. These trials demonstrate that a
simple low-AGE dietary intervention decreases circulating levels of AGEs, markers of inflammation and
oxidative stress in healthy, chronic kidney disease, and diabetic patients, and improves insulin resistance
in type 2 diabetes patients. These data have generated a new paradigm of disease, widely unrecognized,
suggesting that excessive consumption of dietary AGEs secondary to a “Western lifestyle” represents an
independent risk factor for inappropriate chronic oxidative stress and inflammation during life, which
over time facilitates the emergence of the chronic diseases of the modern world, especially diabetes and
cardiovascular disease. Moreover, the data also show that reducing AGE content of common foods by
simple changes in culinary techniques is a feasible, safe, and easily applicable intervention in both health
and disease.
This book presents most of the data that have been accumulated in the past two decades on the role
of food-derived AGEs in causing chronic human disease. The book starts with a general definition of
the compounds passing through all the clinical diseases that have been associated with them and finally
offers different therapeutic options to deal with the problem. I have been extremely lucky to have the par-
ticipation of a selected group of national and international experts in the field to develop these concepts at
a highly academic level. I have also left room for presentation by scholars who may not fully agree with
the concept that dietary AGEs are toxic to the body. I am very indebted to all these collaborators. My
hope is that this book provides the basis to initiate a serious academic conversation about the real role of
dietary AGEs in human disease.

Jaime Uribarri, MD
Icahn School of Medicine at Mount Sinai
New York, NY

ix
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Editor

Dr. Jaime Uribarri is a physician and clinical investigator. He was born in Chile and received his
medical degree from the University of Chile School of Medicine. He did all his postgraduate training in
the United States. He has been in the Icahn School of Medicine at Mount Sinai, NY, since 1990, where
he is currently professor of medicine and director of the Renal Clinic and the Home Dialysis Program at
the Mount Sinai Hospital.
In parallel with his clinical activities, Dr. Uribarri has been very active in clinical investigation for
the past 30 years. His main areas of research have been on acid-base and fluid and electrolytes disorders
as well as nutrition in chronic kidney disease and diabetic patients. Dr. Uribarri, working together with
Dr. Helen Vlassara, were among the first to explore the role of food-derived advanced glycation end
products (AGEs) and their negative effects in healthy persons as well as in those with diabetes or with
kidney disease from different causes. This teamwork for more than 10 years was instrumental in estab-
lishing the first food AGE database and its application in the form of practical guidelines for everyone,
which is now widely used. He has published over 150 peer-reviewed papers and written many chapters in
books. He has lectured extensively on these research topics in New York City as well as in national and
international meetings. He serves as an ad hoc referee for numerous nutrition, medical, and other scien-
tific journals and he is an active member of several health organizations and professional associations,
including the American Society of Nephrology, the American Society of Nutrition, the International
Society of Nephrology, The New York Academy of Sciences, The Maillard Society, etc.

xi
Contributors

Latifa Abdennebi-Najar, PhD Dipshikha Chakravortty, PhD

Société Francophone pour la recherche et Department of Microbiology and Cell Biology
l’éducation sur les origines développementales, Indian Institute of Science
environnementales et épigénétiques de la santé Bangalore, India
et des maladies (SF-DOHaD) and
Chatillon, France Center for Biosystems Science and Engineering
Indian Institute of Science
Carolina Añazco Bangalore, India
Biomedical research Laboratories
Medicine Faculty, Catholic University of Maule Kasturi Chandra
Talca, Chile Department of Microbiology and Cell Biology
Indian Institute of Science
Estifanos Baye, BSc, MPH Bangalore, India
Monash Centre for Health Research and
Implementation Laura C. Cogoi
School of Public Health and Preventive Medicine Universidad de Buenos Aires
Monash University Facultad de Farmacia y Bioquímica
Clayton, VIC, Australia Departamento de Farmacología.
and
Michal Schnaider Beeri, PhD CONICET-Universidad de Buenos Aires
The Joseph Sagol Neuroscience Center Tel Instituto de Química y Metabolismo del Fármaco
Hashomer (IQUIMEFA)
Ramat Gan, Israel Buenos Aires, Argentina
and
Department of Medicine Alma Rosa Corrales Escobosa, PhD
Icahn School of Medicine at Mt Sinai Chemistry Department
New York, NY University of Guanajuato
and Guanajuato, Mexico
Interdisciplinary Center (IDC) Herzliya
School of Psychology Barbora de Courten, MD, PhD, MPH, FRACP
Herzliya, Israel Monash Centre for Health Research and
Implementation
Stig Bengmark, MD, PhD, FRACS (Hon), School of Public Health and Preventive Medicine
FRCPS (Hon) Monash University
Division of Surgery & Interventional Science Clayton, VIC, Australia
University College London and
London, United Kingdom Diabetes and Vascular Medicine Unit
Monash Health
Guido Bosch, PhD Clayton, VIC, Australia
Animal Nutrition Group
Wageningen University Cristina Delgado-Andrade, PhD
Wageningen, The Netherlands Department of Physiology and Biochemistry of
Animal Nutrition
Estación Experimental del Zaidín (EEZ-CSIC)
Granada, Spain

xiii
xiv Contributors

Evanthia Diamanti-Kandarakis, MD, PhD School of Biomedical Sciences

Department of Endocrinology and Metabolism University of Queensland
Hygeia Hospital St Lucia, Australia
Athens, Greece
Linda A. Gallo, PhD
Ghada Elmhiri
Glycation and Diabetes, Mater Research Institute
PRP-HOM/SRBE/LRTOX
University of Queensland, Translational
Laboratoire de Radiotoxicologie Expérimentale
Research Institute
Fontenay-aux-Roses, France
Woolloongabba, Australia
Rosana Filip, PhD
Universidad de Buenos Aires Ma. Eugenia Garay-Sevilla, MD, PhD
Facultad de Farmacia y Bioquímica Department of Medical Science
Departamento de Farmacología. Division of Health Science
and University of Guanajuato Campus León
CONICET-Universidad de Buenos Aires León, México
Instituto de Química y Metabolismo del Fármaco
(IQUIMEFA) Mayuri Gogoi
Buenos Aires, Argentina Department of Microbiology and Cell Biology
Indian Institute of Science
Victoria J. Findlay, PhD Bangalore, India
Department of Pathology & Laboratory Medicine
Hollings Cancer Center
Medical University of South Carolina Armando Gómez-Ojeda, PhD
Charleston, SC Department of Medical Science
Division of Health Science
Josephine M. Forbes, PhD University of Guanajuato Campus León
Glycation and Diabetes, Mater Research Institute León, México
University of Queensland, Translational
Research Institute Ileana Gonzalez
Woolloongabba, Australia Biomedical research Laboratories
and Medicine Faculty, Catholic University of Maule
School of Biomedical Sciences Talca, Chile
University of Queensland
St Lucia, Australia Alejandro Gugliucci, MD, PhD
and TGlycation Oxidation and Disease Laboratory
Mater Clinical School Department of Research
University of Queensland Touro University College of Osteopathic
St Lucia, Australia Medicine
and Vallejo, CA
Department of Medicine
The University of Melbourne
Weidun Alan Guo, MD, PhD
Austin Hospital
Department of Surgery
Heidelberg, Australia
University at Buffalo
and
Buffalo, NY
Baker IDI Heart and Diabetes Institute
Melbourne, Australia
Anshu Gupta, MBBS, MS
Amelia K. Fotheringham Department of Pediatrics
Glycation and Diabetes, Mater Research Institute Children’s Hospital of Richmond
University of Queensland, Translational Virginia Commonwealth University
Research Institute Richmond, VA
Woolloongabba, Australia
and
Contributors xv

Kristian F. Hanssen, MD, PhD Claudia Luevano-Contreras, PhD

Department of Endocrinology, Department of Medical Science
Morbid obesity and Preventive Medicine Division of Health Science
Oslo University Hospital and Institute of University of Guanajuato Campus León
Clinical Medicine León, México
University of Oslo
Oslo, Norway Michael Murkovic, PhD
Graz University of Technology
John C. He, MD, PhD Institute of Biochemistry
Division of Nephrology Graz, Austria
Department of Medicine
Icahn School of Medicine at Mount Sinai Delminda Neves, PhD
New York, NY Department of Biomedicine—Experimental
Biology Unit
Wouter Hendriks, PhD Faculty of Medicine of the University of Porto,
Animal Nutrition Group Al. Prof. Hernâni Monteiro
Wageningan University Porto, Portugal
Wageningan, The Netherlands and
and Instituto de Investigação e Inovação em Saúde
Faculty of Veterinary Medicine (I3S) Rua Alfredo Allen
Utrecht University Porto, Portugal
Utrecht, The Netherlands
Melinda M. Nugent, MD
Sarahi Jaramillo Ortiz, PhD Division of Nephrology
Chemistry Department Department of Medicine
University of Guanajuato Icahn School of Medicine at Mount Sinai
Guanajuato, Mexico New York, NY

Eleni A. Kandaraki, MD, PhD, MRCP(UK) Julie Ottosen, MD

Unit of Endocrinology and Metabolism Department of Surgery
Third Department of Internal Medicine University of Minnesota
Medical School of Athens University Minneapolis, MN
Athens, Greece
Marisa Passarelli, PhD
Kapudeep Karmakar Lipids Laboratory (LIM10)
Department of Microbiology and Cell Biology University of Sao Paulo Medical School
Indian Institute of Science Sao Paulo, SP, Brazil
Bangalore, India
Silvia Pastoriza, PhD
Roni Lotan, MSc Departamento de Nutrición y Bromatología
The Nutrition and Brain Health Laboratory Facultad de Farmacia
The Institute of Biochemistry, Food and Universidad de Granada
Nutrition Science Granada, Spain
Robert H. Smith Faculty of Agriculture, Food and
the Environment Franco Pedreschi, PhD
The Hebrew University of Jerusalem Departamento de Ingeniería Química y
Rehovot, Israel Bioprocesos
and Pontificia Universidad de Católica de Chile
The Joseph Sagol Neuroscience Center Tel Hashome Santiago de Chile, Chile
Ramat Gan, Israel
xvi Contributors

Melpomeni Peppa, MD Kari Anne Sveen, MD, PhD

Endocrine and Bone Metabolic Disorders Unit Department of Endocrinology
2nd Department of Internal Medicine Morbid Obesity and Preventive Medicine
Propaedeutic Oslo University Hospital and Institute of Clinical
Research Institute and Diabetes Center Medicine
National and Kapodistrian School of Athens University of Oslo
Attikon University Hospital Oslo, Norway
Athens, Greece
Katarina Brouder Šebeková, MD
Sergio Pérez-Burillo Intensive Care Unit
Departamento de Nutrición y Bromatología John Radcliffe Hospital
Facultad de Farmacia Oxford, United Kingdom
Universidad de Granada
Granada, Spain Masako Toda, PhD
Paul-Ehrlich-Institut
Langen, Germany
Tasnim Rahman, BS
Virginia Commonwealth University Aron M. Troen, DPhil
Richmond, VA The Nutrition and Brain Health Laboratory
The Institute of Biochemistry, Food and Nutrition
Armando Rojas, PhD Science
Biomedical research Laboratories The Robert H. Smith Faculty of Agriculture Food
Medicine Faculty and the Environment
Catholic University of Maule The Hebrew University of Jerusalem
Talca, Chile Rehovot, Israel

José Ángel Rufián-Henares, PhD David P. Turner, PhD

Departamento de Nutrición y Bromatología Department of Pathology & Laboratory Medicine
Facultad de Farmacia Hollings Cancer Center
Universidad de Granada Medical University of South Carolina
Granada, Spain Charleston, SC

Jaime Uribarri, MD
Katarina Šebeková, MD, DSc
Icahn School of Medicine at Mount Sinai
Institute of Molecular Biomedicine
New York, NY
Medical Faculty, Comenius University
Bratislava, Slovakia
Katarzina Wrobel, PhD
Chemistry Department
Halyna Semchyshyn, PhD University of Guanajuato
Department of Biochemistry and Biotechnology Guanajuato, Mexico
Vasyl Stefanyk Precarpathian National University
Ivano-Frankivsk, Ukraine Rabi Yacoub, MD
Department of Internal Medicine
Peter Smit, MD Jacobs School of Medicine and Biomedical
Department of Cardiothoracic Surgery Sciences
Wake Forest University University at Buffalo
Winston-Salem, NC Buffalo, NY

Sho-ichi Yamagishi, MD, PhD

Ovidiu Alin Stirban, MD
Department of Pathophysiology and Therapeutics
Department of Diabetes and Endocrinology
of Diabetic Vascular Complications
Sana Klinikum and MVZ Sana Artzpraxen
Kurume University School of Medicine
Remscheid, Germany
Kurume, Japan
 Section I

What Are AGEs?

1
What Are AGEs, Their Chemical Structure,
and How Can They Be Measured?

Katarzyna Wrobel, Kazimierz Wrobel, Sarahi Jaramillo Ortiz,

and Alma Rosa Corrales Escobosa
University of Guanajuato
Guanajuato, Mexico

CONTENTS
1.1 Formation and Structural Diversity of AGEs................................................................................... 3
1.2 Endogenous and Exogenous AGEs................................................................................................... 5
1.3 Determination of AGEs.................................................................................................................... 6
1.3.1 Simple Assays for Fluorescent AGEs................................................................................... 6
1.3.2 Immunochemical Methods................................................................................................... 7
1.3.3 Analytical Methods Based on Liquid Chromatography Separations.................................. 7
1.3.4 Analytical Methods Based on Gas Chromatography Separations..................................... 12
1.4 MS Tools in Explorative Studies of Glycation Processes............................................................... 12
1.5 Conclusions and Future Trends....................................................................................................... 13
Acknowledgment...................................................................................................................................... 13
References................................................................................................................................................. 13

KEY POINTS
• Nε -(carboxymethyl)-L-lysine (CML), Nε -(carboxyethyl)-L-lysine (CEL), and pentosidine—
three advanced glycation end products generally accepted as biomarkers of in vitro and
in vivo glycation processes.
• Analytical chemistry procedures for the determination of advanced glycation end products
(AGEs) provide high selectivity, sensitivity, and detection power demanded in clinical studies.
• Liquid chromatography–tandem mass spectrometry is a gold standard for the determination
of known AGEs in clinical samples and in food products.
• Proteomic-based mass spectrometry tools are required for full characterization of AGE
structures and their role in human aging and diseases.

1.1 Formation and Structural Diversity of AGEs

Advanced glycation end products (AGEs) are formed in a branched chain of nonenzymatic reactions that
start by condensation of free amino groups present in a given biomolecule with carbonyl group of reducing
sugars or other related chemical species. Initially, these processes had been referred to as Maillard reac-
tion and were studied within the context of food processing. Since mid-1950s, when glycated hemoglobin
(HbA1c) was first described, the research interest has partially moved to in vivo glycation processes.

3
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4 Dietary AGEs and Their Role in Health and Disease

Despite inevitable formation of AGEs in physiological conditions and their gradual accumulation during
normal aging, today there is no doubt that the excess of glycation products is harmful and is associated
with several health disorders including diabetes and its complications. In this regard, contribution of
endogenously formed and exogenous AGEs has to be considered.
During the last decades, extensive studies have been carried out in order to elucidate molecular
­mechanisms underlying glycation processes and to understand their role in human diseases and aging.
The detailed description of currently known glycation pathways can be found in several comprehensive
reviews [1–4]; a simplified scheme elaborated for the purposes of this chapter is presented in Figure 1.1.
The already mentioned amine-carbonyl reaction yields imine group in the condensed labile compound,
generically called Schiff base. Although at this early stage glycation is reversible, Shiff base is slowly
rearranged to a more stable open-chain ketoamine—still an intermediate glycation product (Amadori
product). In the classic pathway, Amadori products undergo dehydration, fragmentation, oxidation, and
cyclization reactions, which ultimately lead to the formation of irreversible AGEs.
Among reducing sugars, glucose itself is a relatively weak glycation agent, especially as compared
to the small alpha-dicarbonyl compounds produced from glucose auto-oxidation, from polyol path-
way, from degradation of Amadori products, from intermediates of glycolysis, and also from lipids
peroxidation. As shown in Figure 1.1, glyoxal (GO), methylglyoxal (MGO), and 3-­deoxyglucosone

Lipid peroxidation F-3-P Fructose Sorbitol

α-oxoaldehydes Glycolysis Sugar

Pyruvate
(GO, MGO, 3-DG)

Free AAs
R-NH2
Amines
Peptides
Proteins
Nucleic acids Lipids

Schiff base
Degradation
Amadori product

Advanced glycation end products*

O
NH2 HO
NH2
NH2 HO NH2
HO OH NH HO OH
NH O N NH
O O O O
N N
N -(carboxymethyl)-L-lysine
ε
Nε-(carboxyethyl)-L-lysine
Pentosidine

FIGURE 1.1 Simplified scheme of glycation pathways, highlighting the role of alpha-dicarbonyl compounds and the
formation of three often determined AGEs: CML, CEL, and pentosidine.
What Are AGEs, Their Chemical Structure, and How Can They Be Measured? 5

(3-DG) are the key metabolites that act as intermediates of glycation processes as well as the pre-
cursors and propagators of AGEs.
It should be stressed that actual physicochemical conditions play a critical role in the formation and
progression of AGEs; in particular, alkaline pH favors initial Schiff base formation, whereas rearrange-
ment to Amadori products occurs preferentially at physiological pH. Furthermore, transition metal ions,
complex redox equilibria, and, more generally, the increased oxidative stress exert catalytic effect of
AGEs generation.
The main feature of AGEs is their structural diversity and their ability for cross-linking the bio-
molecules. Consequently, relatively few individual compounds have been fully characterized and even
fewer have proved their utility as biomarkers of glycation processes or related diseases. Three impor-
tant compounds from the latter group are Nε -(carboxymethyl)-L-lysine (CML), Nε -(carboxyethyl)-​
L-lysine (CEL), and pentosidine; their structures are shown in Figure 1.1. On the contrary, glycation
processes are capable of modification of proteins, nucleic acids, and lipids (advanced lipoxidation end
products, ALEs). Unfortunately, majority of AGEs’ cross-links still remain unknown which at least in
part is due to their high stability and resistance to typical hydrolysis procedures. In relation to proteins,
explorative studies employing proteomics tools are in use for the full characterization of glycation
sites and their specific biological roles [5,6]. Finally, the intrinsic aspect of AGE biochemistry is their
role in prooxidant and proinflammatory signaling; in this regard, AGE receptors present on the cell
surface (RAGE) have been characterized and extensively studied [7]. The discovery of soluble circu-
lating receptors (sRAGE) and their potential in counterbalancing RAGE signaling has triggered new
directions in studies of AGEs [8].

1.2 Endogenous and Exogenous AGEs

Maillard reactions take place during food heating that is usually applied for processing, conserva-
tion, or may occur during storage of food [9]. A wide range of compounds (MRPs, Maillard reaction
­products) is generated in the course of heating, depending on the “cooking intensity,” specific raw prod-
uct, water content, pH conditions, etc. [10]. Due to the variety of sugars present in foods, their degrada-
tion products, and also different sources of amine groups, structural/functional complexity of MRP
is higher as compared to the glycation products generated in vivo, specifically when concerning low
molecular mass compounds. Food multiple reaction monitoring (MRM) may have positive or negative
health effects [11,12]. In particular, unsaturated heterocyclic nitrogenous compounds denominated as
melanoidins confer antioxidant and antimicrobial properties and are responsible for the brown color of
processed products [13,14]. Food aroma is determined by volatile MRP generated from fragmentation/
degradation of sugars (furans, pyrones, and carbonyls) and amino acids (aldehydes and sulfur com-
pounds); these products are further converted in pyrazines and alkylpyrazines [15,16]. On the contrary,
some of the MRPs are classified as prooxidant, toxic, and even carcinogenic agents; such is the case of
acrylamide [17]. Finally, Maillard reactions in food produce the same AGEs as are generated in vivo—
among them CML, CEL, and pentosidine [18].
Food AGEs deteriorate bioavailability of free amino acids (lysine, arginine, and histidine) and cause
protein cross-linking, thereby affecting the food texture and its digestibility. Furthermore, consumption
of AGE-rich diets has been associated with an increase in circulating AGEs and with progression of
AGE-related clinical conditions. Noteworthy, it has been demonstrated that restriction of dietary AGEs
is helpful in the prevention of diabetes, and vascular and kidney disorders [19].
Based on the above discussion, there is a clear need for the determination of AGEs in food, principally
in order to control/lower their daily intake. On the contrary, determination of AGEs that are common
for food products and for in vivo conditions is highly demanded for the elucidation of the putative role
that exogenous AGEs might play in aging, diabetes, and other chronic degenerative diseases [1,18,20].
The three AGEs most studied within this context have been CML, CEL, and pentosidine.
Nε -(carboxymethyl)-L-lysine is produced directly by binding the epsilon amine group of lysine to the
electrophilic carbonyl moiety of GO and by the subsequent reduction/oxidation. Owing to the variety
of possible sources of GO in vivo and during thermal treatment of food and also because CML can be
6 Dietary AGEs and Their Role in Health and Disease

derived from any stage of glycoxidation or from lipoxidation processes, this specific compound has
been accepted as a versatile biomarker of formation/accumulation of endogenous and exogenous AGEs.
In this regard, extensive database of CML content in a variety of food items had been obtained by means
of enzyme-linked immunosorbent assay (ELISA) [19]. It should be highlighted that CML in clinical and
food-related samples is present in free form and as bound to proteins, both of which are important and
need to be quantitatively evaluated in studies focused on the biological role of AGEs.
When glyoxal is substituted by methylglyoxal in the reaction with lysine, CEL is generated. There are
many sources of MGO in vivo; it is formed by enzymatic and nonenzymatic reactions from intermedi-
ates of glycolysis, during the metabolism of amino acids (glycine and threonine), during lipolysis, and
also as an intermediate of glycation reactions. In foods, MGO is formed from sugars, from the inter-
mediates of Maillard reactions, and from lipids. Microbial fermentation applied in food processing or
undesirably occurring during storage is an additional source of MGO. CEL is considered as endogenous
and exogenous AGE, and it has been determined both in foods and in clinical samples.
Pentosidine is the third AGE often reported in foods and in human tissues; this compound is generated
from linking arginine and lysine via pentose molecule, and it is indicative of protein cross-linking by
glycation processes.
In the following sections, an overview of the existing determination methodologies is presented with
an emphasis on CML, CEL, and pentosidine as the target compounds.

1.3 Determination of AGEs

Measurement of AGEs can be carried out by several approaches, depending on the number of target
compounds, their identity, required selectivity, expected concentration, and chemical composition of the
sample. The existing methods can be grouped as follows: (1) assays enabling estimation of AGEs based
on their native fluorescence, (2) immunochemical methods; (3) determination of individual compounds
by highly selective analytical procedures; and (4) explorative studies focused on the structural character-
ization of new compounds or AGEs cross-links. Procedural tools currently available for the determina-
tion of known AGEs are schematically presented in Figure 1.2 and briefly discussed below.

1.3.1 Simple Assays for Fluorescent AGEs

Structural rigidity of AGEs cross-links (mainly via lysine and arginine) is responsible for fluorescent
properties of several compounds such as pentosidine, GOLD (glyoxal-derived lysine dimer), MOLD
(methylglyoxal-derived dimer), and argypirimidine, pyrraline. Direct fluorimetric measurements pro-
vide useful information of cumulative tissue damage by AGEs, but they are unable to quantify individual
compounds. On the contrary, these assays are simple with no need for any sophisticated instrumentation
or trained analyst, and they can be adopted in routine laboratory control, especially when performed in

Immunochemical Fluorimetric
methods assays
in batch
ELISA
in flow

AGEs determination

Liquid Gas
chromatography chromatography
MS MS
FLD FID

FIGURE 1.2 Currently available tools for measurement of known AGEs.

What Are AGEs, Their Chemical Structure, and How Can They Be Measured? 7

a flow system or directly in exposed skin. Typically, excitation wavelengths in the range of 350–390 nm
are used with fluorescence emission measurement at 440–470 nm; it is recommended to remove lipids
from the analyzed sample and potential interferences from naturally fluorescent peptides/protein can
be eliminated by signal normalization using sample absorbance acquired at 280 nm [21]. Fluorimetric
assays have been preferentially used to assess endogenous AGEs in serum, saliva, or skin [22]. While
analyzing biological fluids, AGEs can be evaluated in low and high molecular mass fractions after pro-
tein precipitation with trichloroacetic or perchloric acid [21,23,24]. Commercially available AGE reader
from DiagnOptics B.V. (Groningen, The Netherlands) was designed for noninvasive measurement of skin
auto-fluorescence; relative skin reflectance is also measured by this device in order to compensate for
differences in skin pigmentation [25].
Despite obvious convenience of fluorescence assays, it remains uncertain what units should be used
to express AGE content, what AGEs are measured, what calibrator should be applied, and how to assure
analytical reliability of the obtained results.

1.3.2 Immunochemical Methods

The most common biochemical approach to AGE measurement is based on the ELISA. It had been gener-
ally accepted that CML would be the dominant epitope recognized by AGE antibodies (especially those
that are polyclonal) [26]; however, over the past two decades, several studies have reported production of
antibodies against other specific compounds (MGO-glycated albumin, CEL, pentosidine, among others)
that proved their feasibility in the analysis of clinical samples and food items [19,27–29]. Commercial
ELISA kits are also available from several international (e.g., OxiSelectTM from Cell Biolabs) or local
suppliers. These kits are preferentially used in clinical studies or more generally, when large sample
series have to be run and when accurate/precise quantification is not required. Although quite popular
and easy to use, ELISA kits present several drawbacks that have often been highlighted in scientific lit-
erature [26]. The main problems include uncertain specificity and possible cross-reactivity of the applied
antibodies, and a lack of universal and reliable quantification method, which in consequence will ham-
per the highly demanded direct comparisons of the results obtained in different studies. Furthermore,
the assessment of bound AGEs or cross-link structures requires protein digestion. Noteworthy, ELISA
results have often been validated by analytical procedures based on chromatographic separation and
mass spectrometry detection [18,26,30–32].

1.3.3 Analytical Methods Based on Liquid Chromatography Separations

The principal strength of analytical chemistry procedures relies on their high specificity toward indi-
vidual compounds and ability for precise and exact determination. The results obtained are expressed
directly as the mass of analyte per unit of mass or volume of the initial sample or per protein content;
such a quantitative approach is extremely important when concentrations of specific compounds are to
be compared among experimental groups, among different analytical procedures or clinical trials, in
dose–response studies, and also in the evaluation of dietary intake of exogenous AGEs.
Sample pretreatment is the critical step in AGE analysis. As aforementioned, data regarding free-,
bound-, and total AGE content are of interest, and for the second and the third contents, acid or enzy-
matic protein hydrolysis has to be performed [33]. Typical protocol involves extractive elimination of
lipids (hexane and chloroform), reduction in sodium borohydride necessary to prevent artifacts from
being oxidatively formed during hydrolysis, protein precipitation, recovery of the pellet, and finally acid
or enzymatic hydrolysis [34–38]. For further protection of the samples from oxidation, especially dur-
ing chemically harsh acid hydrolysis, the tubes are often flushed with nitrogen before being sealed [32].
Finally, the supernatant after protein precipitation can be used for the determination of free AGEs
[32,39–41].
For the determination of various compounds in a single analytical run and also for enhanced selectiv-
ity, chromatographic separation is usually coupled to a convenient detection system [42,43]. Because
AGEs are nonvolatile and individual compounds differ in polarity, high performance liquid chroma-
tography (HPLC) has been preferentially used with the reversed phase- or ion-pair reversed phase
8 Dietary AGEs and Their Role in Health and Disease

separation mode. Several representative examples of HPLC applications in the analysis of clinical and
food samples are briefly summarized below and in Table 1.1.
Fluorimetric detection (FLD) with the excitation/emission wavelengths tuned for specific compound(s)
offer high selectivity and sensitivity; this detection system is of special interest in the analysis of fluores-
cent AGEs, such as pentosidine or argypirimidine [44]. Several methods for the determination of these
two compounds in blood plasma, urine, human tissues, and food products can be found in the literature.
Ion-pair reversed phase separation with mobile phases containing trifluoroacetic acid or heptafluoro-
butyric acid has usually been reported, and the excitation/emission wavelengths were in the range of
320–330 and 378–385 nm, respectively [44–49]. On the contrary, fluorimetric detection has proved its
utility for nonfluorescent AGEs after their suitable pre-column derivatization. Noteworthy, derivatiza-
tion reaction not only confers a fluorescent tag but also alters physicochemical properties of the original
compounds facilitating their chromatographic separation; hence, the selection of derivatizing agent is
an important issue. In this regard, ortho-phthal-aldehyde (OPA) is typically used for primary amines
with excitation/emission detection wavelengths of 340/455 nm [43], respectively. Another derivatizing
agent is 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC); it binds to both primary and sec-
ondary amines and allows for fluorimetric detection at 230–245/395 nm [50,51]. HPLC-FLD systems
have been used for the determination of CML and CEL in clinical and food samples as described in
several research articles [43,51–55].
With the advent of instrumental and methodological development of mass spectrometry (MS), liquid
chromatography coupled with tandem MS is the gold standard in all areas referred for AGE determi-
nation [18,42,56]. Owing to the exceptional selectivity and detection capability of MS, baseline chro-
matographic separation of the target compounds and their pre-column derivatization are not necessary
in this technique. Typically, column effluent is directly introduced to the soft electrospray ionization
(ESI) source, and suitable parent ion formed for each compound is selected and fragmented enabling
for quantification in MRM or selective reaction monitoring (SRM) mode. Once specific ion transition
is established for each analyte, and quantification is based on the intensity of fragment (product) ion.
Although product intensity is lower as compared to that of parent ion, the great advantage of MRM
relies on the enhanced selectivity toward target species and efficient removal of spectral background
with obvious benefits for accuracy and precision. Of note, MRM is especially apposite for routine clini-
cal practice because it can be carried out using triple quadrupole (TQ) or ion trap spectrometers that
present low susceptibility to variation of chromatographic conditions, are easy to operate, and allow
for ion fragmentations.
Additional strength of MS detection is the use of isotopically enriched target compounds as internal
standards (IS). For a given compound, up to eight atoms of less abundant stable isotope of ­hydrogen (2H),
carbon (13C), or nitrogen (15N) can be introduced to the molecule and such prepared IS during the entire
procedure behaves identically as the analyte, although it is detected as a separate species. In such an
approach, any imprecision committed during sample manipulation is efficiently eliminated as also
any ionization suppression/enhancement problem. Typical MRM conditions for CML analysis involve
ion transition m/z 205→84 or 205→130 (207→84 for d2-CML and 209→88 for d4-CML); ion transi-
tions used for CEL are 219→130 or 219→84 (223→88 for d4-CML); for pentosidine, 379→135 transi-
tion is applied; in each case, mass precision would depend on the resolution capability of a specific
­instrument [32–41,57–62]. The IS technique not only assures enhanced accuracy and precision but also
enables for higher robustness and for the adoption of nonrigorous protocols, an important issue in routine
analysis of large series of biological/clinical samples.
Several applications of liquid chromatography—tandem mass spectrometry are briefly summarized in
Table 1.1. Pentosidine is a cationic species, CML and CEL are polar compounds, so retention/­separation
of these AGEs on the reversed phase columns has often been achieved in the presence of ion-pair
reagent, typically nonafluoropentanoic acid (NFPA) [32,35–38,40,57,59, 62]. Alternatively, hydrophilic
interaction chromatographic columns (HILIC) or silica columns modified with amino groups can also be
applied [41,58,61]. Finally, ultra performance liquid chromatography should be mentioned as the effec-
tive separation technique in terms of speed, sensitivity, and resolution, which makes it well suited for MS
detection [34,35,38,40].
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consistent when a predominant preference was found in
the original book; otherwise they were not changed.
Simple typographical errors were corrected.
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