Produtos de glicação avançada (AGE) prejudicam o metabolismo de lipoproteínas e o transporte reverso de colesterol, o que contribui para a aterosclerose no diabete melito (DM). Em particular, a albumina modificada por AGE (albumina-AGE) reduz a remoção de colesterol por diminuir o conteúdo do receptor ABCA-1 em macrófagos. Isto se vincula ao insulto oxidativo e inflamatório, os quais são indutores do estresse do retículo endoplasmático (RE). O objetivo do presente estudo foi avaliar, em macrófagos, os efeitos do tratamento com albumina-AGE sobre o estresse do RE e suas vias adaptativas (UPR), relacionando-os com o prejuízo na expressão do ABCA-1 e efluxo de colesterol celular. Albumina-AGE foi produzida pela incubação de albumina isenta em ácidos graxos com glicolaldeído 10 mM e, albumina controle (albumina-C) com PBS apenas. Albumina foi isolada do soro de pacientes portadores de DM com controle glicêmico inadequado (albumina-DM) ou indivíduos controles (albumina não- DM) por cromatografia para separação rápida de proteínas seguida por purificação alcoólica. Macrófagos de peritônio de camundongos ou macrófagos da linhagem J774 foram tratados com os diferentes tipos de albumina na presença ou ausência de ácido fenil butírico (PBA; chaperona química que alivia o estresse do RE) ou MG-132 (inibidor do sistema proteasomal) por diferentes intervalos de tempo. A expressão de marcadores do estresse do RE, UPR, proteína dissulfeto isomerase (PDI), calreticulina e ubiquitina foi determinada por imunoblot e o conteúdo de ABCA-1, por citometria de fluxo e imunocitoquímica. O efluxo de 14Ccolesterol foi avaliado, utilizando-se apoA-I como aceptora de colesterol. A albumina-AGE induziu aumento tempo-dependente na expressão das chaperonas marcadoras do estresse do RE - Gr78 e Grp94 - e de proteínas da UPR (ATF6 e eIF2-P) em comparação à albumina-C. O conteúdo de ABCA-1 e o efluxo de colesterol foram reduzidos em, respectivamente, 33% e 47% e ambos foram restaurados pelo tratamento com PBA, o qual também reduziu o estresse do RE. A associação entre estresse de RE e redução de ABCA-1 foi confirmada pelo uso da tunicamicina (indutor clássico de estresse do RE), que diminuiu em 61% o conteúdo de ABCA-1, prejudicando em 82% o efluxo de colesterol. A albumina-AGE aumentou o conteúdo total de ubiquitina. A inibição do sistema proteasomal não foi capaz de restaurar o conteúdo de ABCA-1 em células tratadas com albumina-AGE. Em macrófagos expostos à albumina-DM evidenciou-se maior expressão da PDI e calreticulina, com tendência à maior expressão da Grp94. A albumina-AGE (produzida in vitro ou isolada de portadores de DM) induz estresse de RE, o qual se vincula à redução no conteúdo de ABCA-1 e efluxo de colesterol. Estes eventos podem contribuir para a aterosclerose no DM. Chaperonas químicas, que aliviam o estresse do RE, podem ser ferramentas úteis na prevenção e tratamento da aterosclerose / Advanced glycation end products (AGE) disturb lipoprotein metabolism and reverse cholesterol transport, contributing to atherosclerosis in diabetes mellitus (DM). Particularly, advanced glycated albumin (AGE-albumin) reduces cell cholesterol removal by impairing the expression of ABCA-1 in macrophages. This is ascribed to the oxidative and inflammatory stress, conditions that elicit endoplasmic reticulum (ER) stress. In this study it was investigated the effect of AGE-albumin on ER stress and adaptative pathways (UPR) development in macrophages, and its relationship to the reduction in ABCA-1 expression and cholesterol efflux. AGE-albumin was prepared by incubating fatty acid free albumin with 10 mM glycolaldehyde and control albumin (C-albumin) with PBS only. Albumin was isolated from poorly controlled DM patients (DM-albumin) and control individuals (nonDMalbumin) by fast liquid chromatography and purified by alchoolic extraction. Mouse peritoneal macrophages or J774 cells were treated along time with the different types of albumin in the absence or presence of phenyl butiric acic (PBA; a chaperone that aleviates ER stress) or MG132 (a proteasomal inhibitor). The expression of ER stress and UPR markers, protein disulfide isomerase (PDI), calreticulin and ubiquitin was determined by immunoblot and ABCA-1 protein level, by flow cytometry and imunocytochemistry. 14Ccholesterol efflux was evaluated utilizing apo A-I as cholesterol acceptor. AGE-albumin induced a time-dependent increase in the expression of ER stress chaperone markers - Gr78 and Grp94 - and UPR proteins (ATF6 and eIF2-P) in comparison to C-albumin. ABCA-1 content and cholesterol efflux were diminished by, respectively, 33% and 47% and both were recovered by the treatment with PBA. The association between ER stress and ABCA-1 reduction was confirmed by the reduction, induced by tunicanycin (a classical ER stress inductior) in ABCA-1 protein level (61%) and cholesterol efflux (82%). AGE-albumin increased the amount of cellular total ubiquitin. The inhibiton of proteasomal system was unable to restore ABCA-1 protein level in cells treated with AGE-albumin. In macrophages exposed to DM-albumin a higher expression of PDI and calreticulin was observed together with a trend of enhanced Grp94 expression. In conclusion, AGE-albumin (produced in vitro or isolated from DM patients) induces ER stress which is related to the reduction in ABCA-1 level and cholesterol efflux in macrophages. These events can contribute to atherosclerosis in DM. Chemical chaperones that alleviate ER stress may be useful in the prevention and treatment of atherosclerosis

Introdução: Diabetes Mellitus é caracterizada por hiperglicemia crônica, e este aumento excessivo de glicose circulante pode gerar danos vasculares e microvasculares pela deposição de produtos de gliclação avançada (AGE), principalmente em estruturas com alta vascularização, como é o caso da sinóvia. Por todas estas razões, o presente estudo estabeleceu, de maneira temporal, o processo de acomentimento sinovial, através do grau de remodelamento e as proteínas envolvidas neste processo, tido como o gatilho na lesão da articulação do joelho. Foram utilizados ratos wistar (n=60), divididos em três grupos, conforme tempo de indução ( 7, 30 e 60 dias), cada grupo era composto de 10 animais diabéticos, induzido por estreptozotocina (35mg/kg de peso) e 10 animais controle, recebendo infusão do mesmo volume de solução salina, após o tempo estipulado os animais foram sacrificados e a sinóvia coletada para as análises propostas. Análise morfológica através de colorações de hematoxilina-eosina para análise do perfil celular do tecido sinovial e Picrosírius para avaliação da histoarquitetura das fibras colágenas. A quantificação das fibras colágenas foi realizada pela coloração do Picrosírius em microscópio de luz polarizada e a caracterização e distribuição de seus tipos por imunofluorescência, para quantificação total da proteina de colágeno foi realizado a medição da 4-hidroxiprolina (HPO). Os produtos de glicação avançada foram analisados e quantificados por imufluorescência. A detecção e quantificação da imunoexpressão de marcadores bioquímicos como ET-1, TGF-B e IL17 foi realizado por método estereológico de contagem de pontos em reticulo, e como método de confirmação dos achados imunohistoquimicos foi realizado análise de expressão gênica dos Colágenos I,III, e V alfa- 1, alfa-2), por Reação de Transcrição Reversa com amplificação por PCR em Tempo Real (qRT-PCR). Resultados: Foi observado modificação da estrutura sinovial de forma temporal, acometendo inicialmente os vasos subsinoviais e tecidos adjacentes a ele, isso foi observado em tanto em análise morfológica como confirmado em quantificação por Picro em luz polarizada, as modificações se mostraram significantes nos grupos de 30 e 60 dias, quando comparado ao respectivo grupo controle, houve aumento do colágeno total, através do Picrosirius, como por dosagem de HOP. Os resultados foram confirmados por imunofluorescência com o aumento progressivo do COLI e diminuição de COLIII e COLV, o RAGE e AGE também tiveram sua expressão aumentada conforme a evolução no tempo de indução dos animais. Em análise da expressão de outras proteínas foi possível observar a detecção da ET-1 e da IL-17 nos animais diabéticos em comparação ao controle, houve também expressão significativa do TGF-B quando comparado ao respectivo controle. Na análise da expressão gênica foi possível observar aumento do COLV inicialmente, principalmente da cadeia alfa 2, do COLIII e COLI, confirmando achados histomorfométricos. Conclusão: O tecido sinovial demonstra remodelamento precoce ao redor dos vasos, essa mediação envolve o COL1 e os produtos de glicação avançada. Esta alteração no tecido sinovial pode ser responsável por desencadear o acometimento articular no diabetes mellitus / Introduction: Diabetes Mellitus is characterized by chronic hyperglycemia, and this excessive increase of circulating glucose can cause vascular and microvascular damage by the deposition of advanced glycation products (AGE), especially in structures with high vascularization, as is the case of synovium. For all these reasons, the present study established, in a temporal way, the process of synovial concomitance, through the degree of remodeling and the proteins involved in this process, considered as the trigger in the lesion of the knee joint. Wistar rats (n = 60), divided into three groups, according to induction time (7, 30 and 60 days), each group consisted of 10 diabetic animals, induced by streptozotocin (35 mg / kg body weight) and 10 animals control, receiving infusion of the same volume of saline solution, after the stipulated time the animals were sacrificed and the synovium collected for the proposed analyzes. Morphological analysis using hematoxylineosin staining for analysis of the cellular profile of the synovial tissue and Picrosírius for evaluation of the histoarchitecture of the collagen fibers. The quantification of the collagen fibers was performed by the Picrosírius staining in a polarized light microscope and the characterization and distribution of its types by immunofluorescence, the measurement of 4-hydroxyproline (HPO) was performed for the total quantification of the collagen protein. Advanced glycation products were analyzed and quantified by impuluorescence. The detection and quantification of the immunoexpression of biochemical markers such as ET- 1, TGF-B and IL17 was performed by stereological method of reticule dot counting, and as a method of confirming the immunohistochemical findings, the analysis of the collagen I, III , and V alpha-1, alpha-2), by Reverse Transcription Reaction with Real-Time PCR Amplification (qRT-PCR). Results: Modification of the synovial structure was observed temporally, initially affecting subsynovial vessels and tissues adjacent to it, this was observed in both morphological analysis and confirmed in quantification by Picro in polarized light, the modifications were significant in the groups of 30 and 60 days, when compared to the respective control group, there was increase of the total collagen, through Picrosirius, as per HOP dosage. The results were confirmed by immunofluorescence with progressive increase of COLI and decrease of COLIII and COLV, RAGE and AGE also had their expression increased as the evolution in the induction time of the animals. In the analysis of the expression of other proteins it was possible to observe the detection of ET-1 and IL-17 in diabetic animals in comparison to the control, there was also significant expression of TGF-B when compared to the respective control. In the analysis of the gene expression it was possible to observe an increase of the COLV initially, mainly of the alpha 2 chain, of the COLIII and COLI, confirming histomorphometric findings. Conclusion: Synovial tissue demonstrates early remodeling around vessels, this mediation involves COL1 and advanced glycation products. This change in synovial tissue may be responsible for triggering joint involvement in diabetes mellitus

A glicação não enzimática das proteínas é um fator comum para a fisiopatologia de uma série de transtornos relacionados ao envelhecimento e a doenças como o diabetes mellitus (DM). O geração dos produtos de glicação, os AGEs (do inglês: Advanced Glycation End Products) se dá através de reações de glicação da mariz extracelular (MEC) na derme e têm sido apontado como um dos fatores responsáveis pela perda de elasticidade e deficiência de cicatrização da pele. A permeação cutânea de compostos anti-AGE é uma limitação importante para eficiência terapêutica de compostos que devem atingir camadas mais profundas da pele. Modelos de pele reconstruída contendo equivalente dérmico glicado são estruturas tridimensionais geradas in vitro que mimetizam a pele humana e representam um eficiente modelo para o estudo de células e modificações provocadas na MEC no processo de envelhecimento e DM. O modelo 3D de pele reconstruída tem características metabólicas, de permeabilidade e atividade semelhantes à da pele original, potencializando seu papel nas investigações sobre permeabilidade de drogas, toxicidade, irritação, eficácia e segurança de compostos e diferenciação de queratinócitos. Uma série de compostos naturais ou sintéticos inibidores de AGEs têm sido descobertos e apresentados recentemente e podem representar inovação terapêutica no tratamento de modificações causadas pela a formação e acúmulo destes AGEs também na pele. Este estudo avaliou o desenvolvimento da pele reconstruída glicada e posteriormente, a avaliação da eficácia e toxicidade de compostos anti-glicação como aminoguanidina e carnosina em modelo de pele reconstruída glicada. Em perspectiva, este estudo contribuiu para o desenvolvimento de uma nova tecnologia in vitro, a pele reconstruída glicada, que auxiliará a compreensão da biologia da interação célula-MEC mimetizando processos fisiopatológicos importantes como o envelhecimento e o DM. / The Advanced Glycation End Products (AGEs) of proteins is a common factor to the pathophysiology of a number of disorders related to aging and diseases such as diabetes mellitus (DM). The generation of the AGEs products on skin occurs mainly through non-enzymatic glycation reactions of the dermal extracellular matrix and has been touted as one of the factors responsible for loss of elasticity and disability of skin healing. The skin permeation of compounds is an important limitation for therapeutic/cosmetic efficacy of anti-AGE compounds, which must reach the deepest layers of the skin. Reconstructed skin model containing dermal equivalent modified by in vitro glycation is able to mimic the elderly human skin and represent an efficient model for the study of cells interactions and changes in extracellular matrix induced by aging and diabetes. The 3D reconstructed skin model has metabolic characteristics, permeability and activity similar to the original skin, reinforcing its role in drug permeability of investigations toxicity, irritation, safety and efficacy evaluation of compounds and differentiation of keratinocytes. A number of natural or synthetic AGEs inhibitor compounds have been recently discovered and displayed and can represent therapeutic innovation for the treatment of changes caused by the aging of the skin. In this study we performed the development of reconstructed glycated skin model and evaluated the efficacy and toxicity of anti-glycation compounds such as aminoguanidine and carnosine. In perspective, this study has contributed to the development of a new technology in vitro, and for the understanding cell-extracellular matrix interaction during the aging of skin.

Les produits de réaction de Maillard apportent des caractéristiques organoleptiques souhaitables aux aliments (couleur, odeur et le goût), contribuant ainsi à leur acceptabilité par le consommateur. Toutefois, plusieurs effets délétères physiopathologiques (vieillissement, diabètes) ont été attribués aux produits de glycation avancés (AGE). Le but de ce travail a concerné la détermination simultanée par LC-MS/ESI (Q-ToF) des carboxyméthyllysine (CML), pentosidine (Pen) et pyrraline (Pyr) dans des échantillons de lait chauffés ou non, après purification et concentration en phase solide (SPE) par échange d'ions. Les échantillons de lait en suite ont été soumis à une hydrolyse acide avec HCl à 37%, une précipitation des protéines avec un mélange méthanol/acétone et une digestion enzymatique des protéines pendant 30 heures à 37°C. Après l'étape d'extraction, les échantillons ont été concentrés/purifiés par SPE avec cartouches échangeuses d'ions puis analysés par LC-MS/ESI (Q-ToF). Les cartouches d'échange d'ions ont permis d'obtenir une excellente récupération des AGEs (89%, 95% et 117% pour la CML, Pen et Pyr, respectivement), valeurs bien plus élevées que celles qui ont pu être obtenues avec des cartouches de type C18. La concentration/purification par SPE est une étape qui mérite une attention particulière pour la détermination et quantification des AGEs. Parmi les AGEs analysés, seule la Pyr a pu être retrouvée dans des échantillons de lait et celà à des valeurs comprises 0,021 ng/mg de protéines (lait écrémé) et 8,367 ng/mg (lait stérilisé). / Maillard reaction products provide desirable organoleptic characteristics to foods (color, odor and taste), thus contributing to their acceptability by the consumer. However, several deleterious pathophysiological effects (aging, diabetes) have been attributed to advanced glycation products (AGEs). The aim of this work was to determine the simultaneous determination by LC-MS/ESI (Q-ToF) of carboxymethyllysine (CML), pentosidine (Pen) and pyrraline (Pyr) in milk samples heated or non-heated after purification and solid phase concentration (SPE) by ion exchange. Subsequent the milk samples were subjected to acid hydrolysis with 37% HCl, protein precipitation with a methanol / acetone mixture and enzymatic protein digestion for 30 hours at 37°C. Après l'étape d'extraction, les échantillons ont été concentrés/purifiés par SPE avec cartouches échangeuses d'ions puis analysés par LC-MS/ESI (Q-ToF). Ion exchange cartridges have resulted in excellent recovery of AGEs (89%, 95% and 117% for the CML, Pen and Pyr, respectively), much higher than those obtained with C18 cartridges. The concentration/purification by SPE is a step that deserves special attention for the determination and quantification of AGEs. Between the AGEs analyzed, only Pyr could be found in milk samples and the values were 0.021 ng/mg protein (skimmed milk) and 8.367 ng/mg (sterilized milk). / Os produtos da reação de Maillard fornecem propriedades organolépticas desejáveis para alimentos (cor, cheiro e sabor), contribuindo assim para a sua aceitação pelos consumidores. No entanto, vários efeitos deletérios fisiopatológicos (envelhecimento, diabetes) são atribuídos aos produtos da glicação avançada (AGEs). O objetivo deste trabalho foi à determinação simultânea por LC-MS/ESI (Q-ToF) de carboximetillisine (CML), a pentosidina (PEN) e pirralina (Pyr) em amostras de leite aquecido ou não, após purificação e concentração fase sólida (SPE) por troca iônica. As amostras de leite foram subsequentemente submetidas a uma hidrólise ácida com HCl a 37%, a uma precipitação da proteína com uma mistura de metanol/acetona e uma digestão enzimática durante 30 horas a 37°C. Após a etapa de extração, as amostras foram concentradas/purificado por SPE, com cartuchos de troca iônica e analisadas por LC-MS/ESI (Q-ToF). Os cartuchos de troca iônica permitiram obter uma excelente recuperação dos AGEs (89%, 95% e 117% para CML, Pen e Pyr, respectivamente), sendo valores bem mais elevados do que os que podem ser obtidos com cartuchos de C18. A concentração/purificação por SPE é um etapa que merece uma atenção especial para a determinação e quantificação de AGEs. Entre os AGEs analisados, apenas Pyr foi encontrado nas amostras de leite, sendo observado valores para Pyr entre 0,021 ng/mg de proteína (leite desnatado) e 8367 ng/mg (leite esterilizado).

Peritoneal dialysis (PD) is a form of renal replacement therapy using the peritoneum as a dialysis membrane. PD solutions employed to remove nitrogen metabolites and excess plasma fluid, and to restore electrolyte and acid-base balance are being developed to minimize local and systemic inflammatory responses while maintaining peritoneal homeostasis and host defense. The effect of chronic action of PD solutions on the peritoneum results in its remodeling and, possibly, eventual loss of peritoneal ultrafiltration capacity. Factors most responsible for late complications and peritoneal remodeling include high glucose levels in PD solutions, and the presence and formation of glucose degradation products (GDP) and advanced glycation end - products (AGEs) in the peritoneal cavity. The aim of our study described in this dissertation was to test various PD solutions with different glucose content and GDP and, using AGEs receptor ligands, to define their systemic effects and identify PD solutions with highest biocompatibility. This part of the dissertation characterizes conventional glucose - based solutions, low - glucose and GDP load solutions as well as glucose polymer (icodextrin) - based PD solutions while determining the plasma and dialysate levels of soluble receptor for AGEs (s - RAGE) and its...

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Die Interaktion von S100-Proteinen mit dem Rezeptor für advanced glycation endproducts (RAGE) wird als hoch relevant bei der Entstehung, Manifestation und Progression verschiedener entzündlicher Erkrankungen sowie bei der Tumorigenese gewertet. Das tiefergehende Verständnis der Interaktion von S100-Proteinen mit RAGE in vivo stellt eine wissenschaftliche Herausforderung dar und ist ein Ansatz für therapeutische Interventionen. Darüber hinaus stellen Untersuchungen zum Metabolismus von extrazellulär zirkulierenden S100-Proteinen in vivo einen vielversprechenden Forschungsansatz zur Analyse von S100-Protein-assoziierten Erkrankungen dar. Die einzigartigen Eigenschaften der Positronen-Emissions-Tomographie (PET) als nicht-invasives bildgebendes Verfahren erlauben die Darstellung und quantitative Erfassung biochemischer Prozesse mit der Möglichkeit zelluläre und molekulare Reaktionswege aufzuzeigen sowie in vivo-Mechanismen von Krankheiten im Kontext eines physiologischen Umfeldes darzulegen. Ziel der vorliegenden Arbeit war es, Fluor-18-markierte S100-Proteine (18F-S100) herzustellen, diese biochemisch, radiochemisch und radiopharmakologisch zu charakterisieren und deren Metabolismus und Interaktion mit RAGE in vivo mittels Kleintier-PET am Tiermodell zu untersuchen. Es wurden die mit RAGE interagierenden S100-Proteine S100A1, S100A12 und S100B in biologisch funktioneller Form hergestellt. Dazu wurden die entsprechenden S100-Gene in den prokaryotischen Expressionsvektor pGEX-6P-1 kloniert. Mit diesen Konstrukten wurden E. coli-Zellen transformiert, aus denen nachfolgend die S100-Proteine isoliert und gereinigt werden konnten. Es konnte eine Reinigung unter nativen, milden Bedingungen etabliert werden, die es ermöglichte, S100A1, S100A12 und S100B in biologisch aktiver Form und in hohen Reinheitsgraden (> 95%) für die nachfolgenden Experimente bereitzustellen. Diese S100-Proteine wurden über den 18F-tragenden Aktivester N-Succinimidyl-4-[18F]fluorbenzoesäure ([18F]SFB) radioaktiv markiert und charakterisiert. Dabei konnte sichergestellt werden, dass die 18F-S100-Proteine in vitro und in vivo stabil sind. Weiterhin konnte nachgewiesen werden, dass die radioaktive Markierung keine Beeinträchtigung auf die biologische Funktionalität der S100-Proteine hat. Dies wurde anhand von sRAGE-Bindungsuntersuchungen sowie Zell-Interaktionsuntersuchungen an konfluenten Endothelzellen (HAEC) und an zu Makrophagen differenzierten THP-1-Zellen (THP-1-Makrophagen) verifiziert. Für die Untersuchung der RAGE-Bindung war die Produktion des löslichen sRAGE bzw. die Generation von flRAGE-berexprimierenden Zellen erforderlich. Beide Konstrukte wurden in geeigneten Zellsystemen exprimiert und das sRAGE-Protein wurde in biologisch aktiver Form synthetisiert und gereinigt (Reinheitsgrad > 97%). Die 18F-S100-Bindung an THP-1-Makrophagen und HAEC wurde in Gegenwart von glykierten LDL (glykLDL) sowie sRAGE signifikant inhibiert, was auf eine RAGE-Interaktion hinweist. Weiterhin konnten durch den Einsatz von Scavenger-Rezeptor-Liganden, wie z. B. Maleinanhydrid-modifiziertes BSA (malBSA) bzw. von Lektinen inhibierende Effekte erzielt werden. Dies ist ein Indiz für die 18F-S100-Interaktion mit Scavenger-Rezeptoren und Glykokonjugaten an der Zelloberfläche. Durch die Untersuchungen mittels konfokaler Laserscanning-Mikroskopie an THP-1-Makrophagen wurde eine Zellaufnahme des Fluoreszein-markierten S100A12 festgestellt. Weiterhin konnten Kolokalisationen mit Lektinen detektiert werden. Das metabolische Schicksal extrazellulär zirkulierender 18F-S100-Proteine in vivo wurde mit Hilfe dynamischer PET-Untersuchungen bzw. anhand von Bioverteilungs-Untersuchungen in männlichen Wistar-Ratten analysiert. Die Hauptakkumulation der Radioaktivität wurde in der Leber und in den Nieren detektiert. In diesen Organen findet der Metabolismus bzw. die glomeruläre Filtration der 18F-S100-Proteine statt. In den Untersuchungen zur Genexpression mittels Echtzeit-PCR sowie im immunchemischen Proteinnachweis am Western Blot wurde eine hohe Expression und Proteinbiosynthese des RAGE in der Lunge ermittelt. Die Lunge eignet sich daher als „Referenz“-Organ für eine funktionelle in vivo-Charakterisierung von RAGE mit 18FS100-Proteinen. Bei den durchgeführten PET-Untersuchungen konnte eine temporäre 18F-S100-Interaktion mit dem Lungengewebe festgestellt werden. Die Retention des 18FS100A12 in der Lunge wurde in Gegenwart von sRAGE inhibiert. Dies ist ein Hinweis dafür, dass 18F-S100-Proteine auch in vivo an RAGE binden können. Die Radioaktivitäts-Akkumulation in den Organen Leber und Milz, die eine Vielzahl von sessilen Makrophagen aufweisen, wurde durch die Applikation von malBSA inhibiert. Dies ist ein Indiz dafür, dass 18F-S100-Proteine in vivo mit Scavenger-Rezeptoren interagieren können. Die vorliegende Arbeit liefert deutliche Hinweise darauf, dass RAGE nicht der alleinige Rezeptor für 18F-S100-Proteine ist. Der Einsatz von 18F-S100-Proteinen als experimentelles Werkzeug in dynamischen PET-Untersuchungen birgt das Potential einer Charakterisierung von S100-Protein-assoziierten, pathophysiologischen Prozessen. / Members of the S100 family of EF-hand calcium binding proteins play important regulatory roles not only within cells but also exert effects in a cytokine-like manner on definite target cells once released into extracellular space or circulating blood. Accordingly, increased levels of S100 proteins in the circulating blood have been associated with a number of disease states, e.g., diabetes, cancer, and various inflammatory disorders. As the best known target protein of extracellular S100 proteins, the receptor for advanced glycation endproducts (RAGE) is of significant importance. However, the role of extracellular S100 proteins during etiology, progression, and manifestation of inflammatory disorders still is poorly understood. One reason for this is the shortage of sensitive methods for direct assessment of the metabolic fate of circulating S100 proteins and, on the other hand, measurement of functional expression of extracellular targets of S100 proteins, e.g., RAGE in vivo. In this line, small animal PET provides a valuable tool for noninvasive imaging of physiological processes and interactions like plasma or vascular retention, tissue-specific receptor binding, accumulation or elimination in vivo. To address this question, human S100 proteins were cloned in the bacterial expression vector pGEX-6P-1, expressed in E. coli BL21, and purified by affinity chromatography and anion exchange chromatography. Purified S100A1, S100B and S100A12 proteins were then radiolabeled with the positron emitter fluorine-18 (18F) by N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Radiolabeling of S100 proteins resulted in radiochemical yields of 3-10% (corrected for decay) and effective specific radioactivities of 1 GBq/µmol, respectively. For investigations about RAGE binding soluble RAGE (sRAGE) was expressed and purified using pSecTag2B. A radioligand binding assay confirmed specific binding of 18F-S100A12, 18F-S100A1, and 18F-S100B to immobilized sRAGE, also showing an order of affinity with S100A12 > S100A1 > S100B. These results indicate that radioactive labelling of S100 proteins did not affect their overall affinity to RAGE. Cellular association studies in human THP-1 macrophages and human aortic endothelial cells (HAEC) showed specific binding of all 18F-S100 proteins to the non-internalizing RAGE as confirmed by inhibitory effects exerted either by other RAGE ligands, e.g., glycated LDL, or by soluble RAGE. Of interest, 18F-S100 proteins were also shown to interact with other putative binding sites, e.g. scavenger receptors as well as proteoglycans. In this line, uptake of 18F-S100 proteins in THP-1 and HAEC could be inhibited by various scavenger receptor ligands, in particular by maleylated BSA as well as by lectines (e.g. ConA and SBA). Confocal laser scanning microscopy analysis showed a major part of the fluoresceinated S100A12 bound to the surface of THP-1 macrophages. Beyond this, uptake of S100A12 could be determined indicating an interaction of S100A12 with both non-internalizing, e.g., RAGE, and internalizing receptors, e.g. scavenger receptors. By evaluation of the relative contribution of 18F-S100A12 association to RAGE-overexpressed CHO cells (using pIres2-AcGFP1), 18F-S100A12 showed a significantly higher association to CHO-RAGE cells compared with CHO-mock cells. Based on these findings and due to their crucial role in inflammatory disorders the metabolic fate of S100 proteins was further investigated in dynamic small animal Positron emission tomography (PET) studies as well as in biodistribution studies in Wistar rats in vivo. For interpretation of in vivo investigations in rats, expression of RAGE was analyzed by quantitative real time RT-PCR as well as western blotting in various organs. Lung tissue expressed the highest level of RAGE protein compared to the other tissues. PET studies in rats revealed a comparatively long mean residence time of circulating 18F-S100 proteins. A major contributor to this phenomenon seems to be a sustained temporary interaction with tissues overexpressing RAGE, e.g., the lung. On the other hand, renal clearance of 18F-S100 via glomerular filtration is a major elimination pathway. However, scavenger receptor-mediated pathways in the liver, the spleen and, to a minor extent, in the kidneys, also seem to contribute to the overall clearance. The presence of sRAGE revealed a decreased retention of 18F-S100A12 in the lung, indicating in vivo binding to RAGE. In vivo blocking studies using maleylated BSA demonstrated a strong inhibition of putative binding sites in rat tissues enriched in cells expressing scavenger receptors like liver and spleen. In conclusion, 18F-labeling of S100 proteins and the use of small animal PET provide a valuable tool to discriminate the kinetics and the metabolic fate of S100 proteins in vivo. Furthermore, the results strongly suggest an involvement of other putative receptors beside RAGE in distribution, tissue association and elimination of circulating proinflammatory S100 proteins. Moreover, the approach provides novel probes for imaging of functional expression of RAGE and scavenger receptors in peripheral inflammatory compartments.

Die Interaktion von S100-Proteinen mit dem Rezeptor für advanced glycation endproducts (RAGE) wird als hoch relevant bei der Entstehung, Manifestation und Progression verschiedener entzündlicher Erkrankungen sowie bei der Tumorigenese gewertet. Das tiefergehende Verständnis der Interaktion von S100-Proteinen mit RAGE in vivo stellt eine wissenschaftliche Herausforderung dar und ist ein Ansatz für therapeutische Interventionen. Darüber hinaus stellen Untersuchungen zum Metabolismus von extrazellulär zirkulierenden S100-Proteinen in vivo einen vielversprechenden Forschungsansatz zur Analyse von S100-Protein-assoziierten Erkrankungen dar. Die einzigartigen Eigenschaften der Positronen-Emissions-Tomographie (PET) als nicht-invasives bildgebendes Verfahren erlauben die Darstellung und quantitative Erfassung biochemischer Prozesse mit der Möglichkeit zelluläre und molekulare Reaktionswege aufzuzeigen sowie in vivo-Mechanismen von Krankheiten im Kontext eines physiologischen Umfeldes darzulegen. Ziel der vorliegenden Arbeit war es, Fluor-18-markierte S100-Proteine (18F-S100) herzustellen, diese biochemisch, radiochemisch und radiopharmakologisch zu charakterisieren und deren Metabolismus und Interaktion mit RAGE in vivo mittels Kleintier-PET am Tiermodell zu untersuchen. Es wurden die mit RAGE interagierenden S100-Proteine S100A1, S100A12 und S100B in biologisch funktioneller Form hergestellt. Dazu wurden die entsprechenden S100-Gene in den prokaryotischen Expressionsvektor pGEX-6P-1 kloniert. Mit diesen Konstrukten wurden E. coli-Zellen transformiert, aus denen nachfolgend die S100-Proteine isoliert und gereinigt werden konnten. Es konnte eine Reinigung unter nativen, milden Bedingungen etabliert werden, die es ermöglichte, S100A1, S100A12 und S100B in biologisch aktiver Form und in hohen Reinheitsgraden (> 95%) für die nachfolgenden Experimente bereitzustellen. Diese S100-Proteine wurden über den 18F-tragenden Aktivester N-Succinimidyl-4-[18F]fluorbenzoesäure ([18F]SFB) radioaktiv markiert und charakterisiert. Dabei konnte sichergestellt werden, dass die 18F-S100-Proteine in vitro und in vivo stabil sind. Weiterhin konnte nachgewiesen werden, dass die radioaktive Markierung keine Beeinträchtigung auf die biologische Funktionalität der S100-Proteine hat. Dies wurde anhand von sRAGE-Bindungsuntersuchungen sowie Zell-Interaktionsuntersuchungen an konfluenten Endothelzellen (HAEC) und an zu Makrophagen differenzierten THP-1-Zellen (THP-1-Makrophagen) verifiziert. Für die Untersuchung der RAGE-Bindung war die Produktion des löslichen sRAGE bzw. die Generation von flRAGE-berexprimierenden Zellen erforderlich. Beide Konstrukte wurden in geeigneten Zellsystemen exprimiert und das sRAGE-Protein wurde in biologisch aktiver Form synthetisiert und gereinigt (Reinheitsgrad > 97%). Die 18F-S100-Bindung an THP-1-Makrophagen und HAEC wurde in Gegenwart von glykierten LDL (glykLDL) sowie sRAGE signifikant inhibiert, was auf eine RAGE-Interaktion hinweist. Weiterhin konnten durch den Einsatz von Scavenger-Rezeptor-Liganden, wie z. B. Maleinanhydrid-modifiziertes BSA (malBSA) bzw. von Lektinen inhibierende Effekte erzielt werden. Dies ist ein Indiz für die 18F-S100-Interaktion mit Scavenger-Rezeptoren und Glykokonjugaten an der Zelloberfläche. Durch die Untersuchungen mittels konfokaler Laserscanning-Mikroskopie an THP-1-Makrophagen wurde eine Zellaufnahme des Fluoreszein-markierten S100A12 festgestellt. Weiterhin konnten Kolokalisationen mit Lektinen detektiert werden. Das metabolische Schicksal extrazellulär zirkulierender 18F-S100-Proteine in vivo wurde mit Hilfe dynamischer PET-Untersuchungen bzw. anhand von Bioverteilungs-Untersuchungen in männlichen Wistar-Ratten analysiert. Die Hauptakkumulation der Radioaktivität wurde in der Leber und in den Nieren detektiert. In diesen Organen findet der Metabolismus bzw. die glomeruläre Filtration der 18F-S100-Proteine statt. In den Untersuchungen zur Genexpression mittels Echtzeit-PCR sowie im immunchemischen Proteinnachweis am Western Blot wurde eine hohe Expression und Proteinbiosynthese des RAGE in der Lunge ermittelt. Die Lunge eignet sich daher als „Referenz“-Organ für eine funktionelle in vivo-Charakterisierung von RAGE mit 18FS100-Proteinen. Bei den durchgeführten PET-Untersuchungen konnte eine temporäre 18F-S100-Interaktion mit dem Lungengewebe festgestellt werden. Die Retention des 18FS100A12 in der Lunge wurde in Gegenwart von sRAGE inhibiert. Dies ist ein Hinweis dafür, dass 18F-S100-Proteine auch in vivo an RAGE binden können. Die Radioaktivitäts-Akkumulation in den Organen Leber und Milz, die eine Vielzahl von sessilen Makrophagen aufweisen, wurde durch die Applikation von malBSA inhibiert. Dies ist ein Indiz dafür, dass 18F-S100-Proteine in vivo mit Scavenger-Rezeptoren interagieren können. Die vorliegende Arbeit liefert deutliche Hinweise darauf, dass RAGE nicht der alleinige Rezeptor für 18F-S100-Proteine ist. Der Einsatz von 18F-S100-Proteinen als experimentelles Werkzeug in dynamischen PET-Untersuchungen birgt das Potential einer Charakterisierung von S100-Protein-assoziierten, pathophysiologischen Prozessen. / Members of the S100 family of EF-hand calcium binding proteins play important regulatory roles not only within cells but also exert effects in a cytokine-like manner on definite target cells once released into extracellular space or circulating blood. Accordingly, increased levels of S100 proteins in the circulating blood have been associated with a number of disease states, e.g., diabetes, cancer, and various inflammatory disorders. As the best known target protein of extracellular S100 proteins, the receptor for advanced glycation endproducts (RAGE) is of significant importance. However, the role of extracellular S100 proteins during etiology, progression, and manifestation of inflammatory disorders still is poorly understood. One reason for this is the shortage of sensitive methods for direct assessment of the metabolic fate of circulating S100 proteins and, on the other hand, measurement of functional expression of extracellular targets of S100 proteins, e.g., RAGE in vivo. In this line, small animal PET provides a valuable tool for noninvasive imaging of physiological processes and interactions like plasma or vascular retention, tissue-specific receptor binding, accumulation or elimination in vivo. To address this question, human S100 proteins were cloned in the bacterial expression vector pGEX-6P-1, expressed in E. coli BL21, and purified by affinity chromatography and anion exchange chromatography. Purified S100A1, S100B and S100A12 proteins were then radiolabeled with the positron emitter fluorine-18 (18F) by N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Radiolabeling of S100 proteins resulted in radiochemical yields of 3-10% (corrected for decay) and effective specific radioactivities of 1 GBq/µmol, respectively. For investigations about RAGE binding soluble RAGE (sRAGE) was expressed and purified using pSecTag2B. A radioligand binding assay confirmed specific binding of 18F-S100A12, 18F-S100A1, and 18F-S100B to immobilized sRAGE, also showing an order of affinity with S100A12 > S100A1 > S100B. These results indicate that radioactive labelling of S100 proteins did not affect their overall affinity to RAGE. Cellular association studies in human THP-1 macrophages and human aortic endothelial cells (HAEC) showed specific binding of all 18F-S100 proteins to the non-internalizing RAGE as confirmed by inhibitory effects exerted either by other RAGE ligands, e.g., glycated LDL, or by soluble RAGE. Of interest, 18F-S100 proteins were also shown to interact with other putative binding sites, e.g. scavenger receptors as well as proteoglycans. In this line, uptake of 18F-S100 proteins in THP-1 and HAEC could be inhibited by various scavenger receptor ligands, in particular by maleylated BSA as well as by lectines (e.g. ConA and SBA). Confocal laser scanning microscopy analysis showed a major part of the fluoresceinated S100A12 bound to the surface of THP-1 macrophages. Beyond this, uptake of S100A12 could be determined indicating an interaction of S100A12 with both non-internalizing, e.g., RAGE, and internalizing receptors, e.g. scavenger receptors. By evaluation of the relative contribution of 18F-S100A12 association to RAGE-overexpressed CHO cells (using pIres2-AcGFP1), 18F-S100A12 showed a significantly higher association to CHO-RAGE cells compared with CHO-mock cells. Based on these findings and due to their crucial role in inflammatory disorders the metabolic fate of S100 proteins was further investigated in dynamic small animal Positron emission tomography (PET) studies as well as in biodistribution studies in Wistar rats in vivo. For interpretation of in vivo investigations in rats, expression of RAGE was analyzed by quantitative real time RT-PCR as well as western blotting in various organs. Lung tissue expressed the highest level of RAGE protein compared to the other tissues. PET studies in rats revealed a comparatively long mean residence time of circulating 18F-S100 proteins. A major contributor to this phenomenon seems to be a sustained temporary interaction with tissues overexpressing RAGE, e.g., the lung. On the other hand, renal clearance of 18F-S100 via glomerular filtration is a major elimination pathway. However, scavenger receptor-mediated pathways in the liver, the spleen and, to a minor extent, in the kidneys, also seem to contribute to the overall clearance. The presence of sRAGE revealed a decreased retention of 18F-S100A12 in the lung, indicating in vivo binding to RAGE. In vivo blocking studies using maleylated BSA demonstrated a strong inhibition of putative binding sites in rat tissues enriched in cells expressing scavenger receptors like liver and spleen. In conclusion, 18F-labeling of S100 proteins and the use of small animal PET provide a valuable tool to discriminate the kinetics and the metabolic fate of S100 proteins in vivo. Furthermore, the results strongly suggest an involvement of other putative receptors beside RAGE in distribution, tissue association and elimination of circulating proinflammatory S100 proteins. Moreover, the approach provides novel probes for imaging of functional expression of RAGE and scavenger receptors in peripheral inflammatory compartments.

Le syndrome de détresse respiratoire aiguë (SDRA) est caractérisé par des lésions alvéolaires diffuses menant à un œdème alvéolaire lésionnel et une insuffisance respiratoire aiguë hypoxémique. Malgré les progrès récents dans la prise en charge des patients de réanimation, le SDRA reste un syndrome fréquent et associé à une morbimortalité importante. Deux mécanismes principaux du SDRA semblent associés à une mortalité plus élevée et à des réponses thérapeutiques différentes : la déficience de la clairance liquidienne alvéolaire (AFC, pour alveolar fluid clearance), l’incapacité pour l’épithélium alvéolaire de résorber l’œdème alvéolaire, et la présence d’un phénotype « hyper-inflammatoire ». Les approches pharmacologiques du traitement du SDRA restent limitées et il est nécessaire de poursuivre l’étude des voies biologiques impliquées dans la pathogénie du SDRA et dans sa résolution afin de développer des approches innovantes des prises en charge diagnostique et thérapeutique du SDRA. RAGE, le récepteur des produits de glycation avancée, est un récepteur multi-ligands, exprimé abondamment par les cellules épithéliales alvéolaires du poumon (pneumocytes), qui module de nombreuses voies de signalisation intracellulaire. De nombreuses études récentes suggèrent que sRAGE, la forme soluble principale de RAGE, pourrait servir de marqueur lésionnel du pneumocyte de type I, et que RAGE pourrait jouer un rôle-pivot dans la pathophysiologie du SDRA, en initiant et en entretenant la réponse inflammatoire alvéolaire. Nos objectifs étaient de caractériser les rôles de RAGE au cours du SDRA, grâce à une approche translationnelle combinant études cliniques et précliniques. D’abord, des études cliniques observationnelles et interventionnelles ont été conduites afin de caractériser sRAGE comme un véritable biomarqueur dans le SDRA. Ensuite, des cultures in vitro de cellules épithéliales et de macrophages, ainsi qu’un modèle expérimental in vivo de SDRA murin par instillation trachéale d’acide chlorhydrique ont été utilisés pour décrire les effets de la voie RAGE sur les mécanismes d’AFC et l’inflammation macrophagique médiée par l’inflammasome « Nod-Like Receptor family, Pyrin domain containing 3 » (NLRP3). Enfin, l’effet d’une inhibition de RAGE, par sRAGE recombinant ou par anticorps monoclonal anti-RAGE, était testée en modèle murin. Nos résultats issus des études cliniques suggèrent que sRAGE présente toutes les caractéristiques d’un biomarqueur au cours du SDRA, avec un intérêt dans le diagnostic, le pronostic et la prédiction du risque de développer un SDRA dans une population à risque. Pris ensemble, notre travail suggère que la voie RAGE joue un rôle important dans la régulation de l’atteinte pulmonaire, de l’AFC et de l’activation macrophagique au cours du SDRA. Toutefois, les mécanismes précis de cette régulation restent incertains. La forme soluble de RAGE (sRAGE), lorsqu’elle est dosée dans le plasma, présente toutes les caractéristiques d’un biomarqueur pouvant être utile en pratique clinique, mais son intérêt dans la sélection de sous-groupes (ou « phénotypes ») de patients pouvant bénéficier de traitements ciblés reste à étudier. La voie RAGE pourrait enfin représenter une cible thérapeutique prometteuse. Bien que des études de validation restent nécessaires, ces résultats pourraient ouvrir de nouvelles perspectives dans la prise en charge des patients atteints de SDRA. / The acute respiratory distress syndrome (ARDS) is associated with diffuse alveolarinjury leading to increased permeability pulmonary edema and hypoxemic respiratory failure. Despite recent improvements in intensive care, ARDS is still frequent and associated with high mortality and morbidity. Two major features of ARDS may contribute to mortality and response to treatment: impaired alveolar fluid clearance (AFC), i.e. altered capacity of the alveolar epithelium to remove edema fluid from distal lung airspaces, and phenotypes of severe inflammation. Pharmacological approaches of ARDS treatment are limited and further mechanistic explorations are needed to develop innovative diagnostic and therapeutic approaches. The receptor for advanced glycation endproducts (RAGE) is a multiligand pattern recognition receptor that is abundantly expressed by lung alveolar epithelial cells andmodulates several cellular signaling pathways. There is growing evidence supporting sRAGE (the main soluble isoform of RAGE) as a marker of epithelial cell injury, and RAGE may be pivotal in ARDS pathophysiology through the initiation and perpetuation of inflammatory responses. Our objectives were to characterize the roles of RAGE in ARDS through a translational approach combining preclinical and clinical studies. First, observational and interventional clinical studies were conducted to test sRAGE as a biomarker during ARDS.Then, cultures of epithelial cells, macrophages and a mouse model of acidinduced lung injury were used to describe the effects of RAGE pathway on AFC and inflammation, with special emphasis on a macrophage activation through NodLikeReceptor family, Pyrindomain containing 3 (NLRP3) inflammasome. Acidinjured mice were treated with an antiRAGE monoclonal antibody or recombinant sRAGE to test the impact of RAGE inhibition on criteria of experimental ARDS. Results from clinical studies support a role of sRAGE as a biomarker of ARDS, withdiagnostic, prognostic and predictive values. In addition, plasma sRAGE is correlated with a lung imaging phenotype of nonfocal ARDS and could inform on therapeutic response. Herein, we also describe in vivo and in vitro effects of RAGE activation on transepithelial fluid transport and expression levels of epithelial channels (aquaporin 5, αNa,KATPaseandαENaC) and on macrophage activation through NLRP3 inflammasome. Finally, RAGE inhibition improves AFC and decreases lung injury in vivo. Taken together, our findings support a role of RAGE pathway in the regulation of lung injury, AFC and macrophage activation during ARDS, albeit precise regulatory mechanisms remain uncertain. sRAGE has most features of a validated biomarker that could be used in clinical medicine, but whether it may help to identify subgroups (or phenotypes) of patients that would benefit from tailored therapy remains underinvestigated. Modulation ofRAGE pathway may be a promising therapeutic target, and though validation studies are warranted, such findings may ultimately open novel diagnostic and therapeutic perspectivesin patients with ARDS.

Furosin entsteht bei der Salzsäurehydrolyse aus den Amadori-Produkten des Lysins und wird als Marker für den Fortschritt der frühen Maillard-Reaktion, zur Beurteilung von lebensmitteltechnologischen Prozessen sowie zur Berechnung des verfügbaren und des nicht verfügbaren Lysins in Lebensmitteln verwendet. Für die Nutzung von Furosin als Qualitätsparameter ist die reproduzierbare und konstante Bildung während der Salzsäurehydrolyse entscheidend. Dies wird in der Literatur jedoch kontrovers diskutiert. Im ersten Abschnitt dieser Arbeit galt es deshalb, die molaren Ausbeuten an Furosin und den weiteren Hydrolyseprodukten Lysin, Pyridosin und N[epsilon]-Carboxymethyl-lysin zu bestimmen und damit eine sichere Interpretation der Ergebnisse zu ermöglichen. Dazu wurden peptid-gebundene Amadori-Produkte des N[alpha]-Hippuryl-lysins in chromatographisch reiner Form dargestellt. Weiterhin wurden N[alpha]-Hippuryl-N[epsilon]-carboxymethyl-lysin und Pyridosin als Standard gewonnen. Bei den Hydrolyseexperimenten zeigten die Fructosyl-Amadori-Produkte ein ähnliches Verhalten. Nach Hydrolyse mit 6M Salzsäure wurden molare Ausbeuten an Furosin von 32% für Fructosyl-lysin und jeweils 34% für Lactulosyl- und Maltulosyl-lysin bestimmt. Signifikant höhere Ausbeuten an Furosin waren nach Hydrolyse mit 8M Salzsäure festzustellen, 46% für Fructosyl-lysin, 50% für Lactulosyl-lysin und 51% für Maltulosyl-lysin. Im Gegensatz zu den Fructosyl-Derivaten war die molare Ausbeute an Furosin bei Tagatosyl-lysin unabhängig von der verwendeten Salzsäurekonzentration (6 bis 8M) und wurde zu 42% bestimmt. Anhand der auf Basis der molaren Ausbeuten ermittelten Überführungsfaktoren kann nun erstmals die Lysin-Derivatisierung mittels der Analytik von Furosin sicher bestimmt werden. Das ermöglicht exakte Aussagen zum Fortschritt nichtenzymatischer Glykierungsreaktionen sowohl in Lebensmittel als auch in vivo. Aufgrund der Relevanz für biologische Systeme und für Lebensmittel wurden weiterhin Reaktionen von alpha-Dicarbonylverbindungen mit kurzkettigen Peptiden und dem Protein Insulin unter physiologischen Bedingungen (pH=7,4 und 37°C) untersucht. Bei der Reaktion von Glyoxal mit ausgewählten Tripeptiden wurde eine sehr schnelle Derivatisierung der Peptide und jeweils die gleichzeitige Bildung eines definierten Produktes festgestellt. Mittels nuklearmagnetischer Resonanzspektroskopie und massenspektroskopischer Analyse konnten die Produkte zweifelsfrei, jeweils als die am N-Terminus durch einen 2(1H)-Pyrazinon-Ring modifizierten Peptide, aufgeklärt werden. Das Hauptprodukt der Reaktion von Methylglyoxal mit dem Peptid Gly-Ala-Phe wurde ebenfalls als 2(1H)-Pyrazinon-Peptid aufgeklärt. Nach Inkubation von Insulin mit Glyoxal unter physiologischen Bedingungen in verdünnter Lösung konnte weiterhin gezeigt werden, dass die 2(1H)-Pyrazinon-Bildung ebenfalls an einem Protein erfolgt. Die identifizierten N-terminalen 2(1H)-Pyrazinone weisen charakteristische UV-Absorptions- sowie Fluoreszenz-Spektren auf. Um die Reaktivität des N-Terminus und damit die Bedeutung der 2(1H)-Pyrazinon-Bildung beurteilen zu können, wurden vergleichende Studien mit dem als Hauptreaktionspartner für alpha-Dicarbonylverbindungen angesehenen Arginin durchgeführt. Bei diesen Experimenten zeigte der N-Terminus und peptidgebundenes Arginin eine nahezu identische Reaktivität. Auf Grund dieser Ergebnisse ist fest davon auszugehen, dass es sich bei den identifizierten N-terminalen 2(1H)-Pyrazinonen um eine neue Klasse von sogenannten Advanced Glycation Endproducts (AGEs) mit Bedeutung in physiologischen Systemen und in Lebensmitteln handelt.