1-Amino-1-deoxy-d-fructose ("fructosamine") and its derivatives: An update.

1-Amino-1-deoxy-d-fructose (fructosamine, FN) derivatives are omnipresent in all living organisms, as a result of non-enzymatic condensation and Amadori rearrangement reactions between free glucose and biogenic amines such as amino acids, polypeptides, or aminophospholipids. Over decades, steady interest in fructosamine was largely sustained by its role as a key intermediate structure in the Maillard reaction that is responsible for the organoleptic and nutritional value of thermally processed foods, and for pathophysiological effects of hyperglycemia in diabetes. New trends in fructosamine research include the discovery and engineering of FN-processing enzymes, development of advanced tools for hyperglycemia monitoring, and evaluation of the therapeutic potential of both fructosamines and FN-recognizing proteins. This article covers developments in the field of fructosamine and its derivatives since 2010 and attempts to ascertain challenges in future research.

1-Amino-1-deoxy-d-fructose ("fructosamine") and its derivatives.

Fructosamine has long been considered as a key intermediate of the Maillard reaction, which to a large extent is responsible for specific aroma, taste, and color formation in thermally processed or dehydrated foods. Since the 1980s, however, as a product of the Amadori rearrangement reaction between glucose and biologically significant amines such as proteins, fructosamine has experienced a boom in biomedical research, mainly due to its relevance to pathologies in diabetes and aging. In this chapter, we assess the scope of the knowledge on and applications of fructosamine-related molecules in chemistry, food, and health sciences, as reflected mostly in publications within the past decade. Methods of fructosamine synthesis and analysis, its chemical, and biological properties, and degradation reactions, together with fructosamine-modifying and -recognizing proteins are surveyed.

Comprehensive analysis of the anti-glycation effect of peanut skin extract.

Advanced glycation end-products (AGEs) are produced during protein glycation and associated with diabetic complications. Peanut skin is rich in procyanidins, which may be used as an inhibitor of glycation. This study evaluated the potential anti-glycation effect of peanut skin extract (PSE) and dissected the underlying mechanism. PSE could effectively inhibit the formation of AGEs in BSA-Glc and BSA-MGO/GO models, with 44%, 37% and 82% lower IC50 values than the positive control (AG), respectively. The inhibitory effect of PSE on BSA glycation might be ascribed to its binding interaction with BSA, attenuated formation of early glycation products and trapping of reactive dicarbonyl compounds. Notably, PSE showed a remarkably stronger inhibitory effect on Amadori products than AG. Furthermore, three new types of PSE-MGO adducts were formed as identified by UPLC-Q-TOF-MS. These findings suggest that PSE may serve as an inhibitor of glycation and provide new insights into its application.

In vitro formation of Maillard reaction products during simulated digestion of meal-resembling systems.

The aim of the present research was to study the formation of Maillard reaction products (MRPs) during digestive process of meal-resembling systems. An average meal (protein, starch and oil) and sugar-containing meals (protein and glucose or fructose or high fructose corn syrup (HCFS)) were tested. Intestinal simple amino acid systems were also analyzed to gain insight into their contribution to the Maillard reaction (MR). Decrease of lysine (11.7-34%), arginine (24-35%) and other amino acids occurred after digestion of the meals. Fructosamine (42.6±4.7 and 332.9±10.4µg/ml) and fluorescent adducts (22,270±119.6 and 9283±188.3 RFU) were detected in digests of those meals containing HCFS and starch, respectively. Carboxymethyllysine (CML) (5.03±1.09µg/ml) and MGO-derivative AGEs (12.2±1.5µg/ml) were found in the meals composed of fructose and only MGO-derivative AGEs (12.2±1.6µg/ml) in presence of glucose. Physiological intestinal concentrations (43mM) of sugars in simplified systems composed by single amino acids caused formation of MRPs under digestive conditions. Arginine and fructose (314mM) showed formation of fructosamine and different AGEs. Fructose (43mM) gave rise to CML by interaction with lysine, which was observed within 1h of incubation at intestinal conditions. These conditions are possible in the intestine during fructose malabsorption. The results suggest the importance of using meal systems for better understanding of complex chemical events taking place during digestion such as MR. This is the first study proposing the formation of non-fluorescent AGEs associated to the pathogenesis of diabetes during digestion of sugar containing and average meals. This formation may be possible in conditions where sugar absorption is delayed such as fructose malabsorption or intake of a fatty meal. The occurrence of the MR during the digestion process may reduce the bioavailability of essential amino acids and increase the production of MRPs causing health disorders.

Effects of glycation on human γd-crystallin proteins by different glycation-inducing agents.

Human γd-crystallin (Hγd-crystallin), a major protein component of the human eye lens, is associated with the development of juvenile- and mature-onset cataracts. Evidence suggests that nonenzymatic protein glycation plays an important role in the aetiology of cataract and diabetic sequelae. This research compared the effects of various glycation modifiers on Hγd-crystallin aggregation, by treating samples of Hγd-crystallin with ribose, galactose, or methylglyoxal using several biophysical techniques. To measure advanced glycation end products, an Nε-(carboxyethyl)lysine enzyme-linked immunosorbent assay was performed on the glycating agent-treated Hγd-crystallin samples. Fructosamine production detection was performed for both ribose-treated and galactose-treated samples. Methylglyoxal-treated samples had the highest level of aggregation and the greatest extent of unfolding, and upon incubation for a minimum of 12 days, exhibited a marked enhancement in the amount of Nε-(carboxyethyl)lysine. The molecular profiles and morphological features of the glycated samples were highly correlated to the type of glycation agent used. These findings highlight a close connection between the type of glycation modifier and the various aggregation species that form. Thus, these results may facilitate deciphering of the molecular mechanism of diabetic cataractogenesis.

Reactions between methylglyoxal and its scavengers in-vivo appear to be catalyzed enzymatically.

Methylglyoxal (MGO) is thought to be an important contributor to the development of diabetic complications. In this paper I propose that MGO, not detoxified by the glyoxalase system, is removed from circulation by MGO-scavengers. Furthermore, since intrinsic rates of reactions between MGO and its scavengers are low, I propose that, in-vivo, these reactions are catalyzed enzymatically.

Evolution of protein bound Maillard reaction end-products and free Amadori compounds in low lactose milk in presence of fructosamine oxidase I.

Thermal treatments and storage influence milk quality, particularly in low lactose milk as the higher concentration of reducing sugars can lead to the increased formation of the Maillard reaction products (MRPs). The control of the Amadori products (APs) formation is the key step to mitigate the Maillard reaction (MR) in milk. The use of fructosamine oxidases, (Faox) provided promising results. In this paper, the effects of Faox I were evaluated by monitoring the concentration of free and bound MRPs in low lactose milk during shelf life. Results showed that the enzyme reduced the formation of protein-bound MRPs down to 79% after six days at 37°C. Faox I lowered the glycation of almost all the free amino acids resulting effective on basic and polar amino acids. Data here reported corroborate previous findings on the potentiality of Faox enzymes in controlling the early stage of the MR in foods.

Electrochemical sensing system employing fructosamine 6-kinase enables glycated albumin measurement requiring no proteolytic digestion.

Currently available enzymatic methods for the measurement of glycated proteins utilize fructosyl amino acid/peptide oxidases (FAOXs/FPOXs) as sensing elements. FAOXs/FPOXs oxidize glycated amino acids or glycated dipeptides but they are not able to accept longer glycated peptides or intact glycated proteins as substrates. Therefore, pretreatment via proteolytic digestion is unavoidable with the current enzymatic methods, and there remains a need for simpler measurement methods for glycated proteins. In this study, in order to develop a novel sensing system for glycated albumin (GA), a marker for diabetes, with no requirement for proteolytic digestion, we created an electrochemical sensor based on fructosamine 6-kinase (FN6K) from Escherichia coli. Uniquely, FN6K can react directly with intact GA unlike FAOXs/FPOXs. The concentration of GA in samples was measured using a carbon-printed disposable electrode upon which FN6K as well as two additional enzymes, pyruvate kinase and pyruvate dehydrogenase were overlaid. A clear correlation between the response current and the concentration of GA was observed in the range of 20-100 µM GA, which is suitable for measurement of GA in diluted blood samples from both healthy individuals and patients with diabetes. The sensing system reported here could be applied to point-of-care-testing devices for measurement of glycated proteins.

Kinetics of glycoxidation of bovine serum albumin by glucose, fructose and ribose and its prevention by food components.

The aim of this study was to compare the kinetics of the glycoxidation of bovine serum albumin (BSA) as a model protein by three sugars: glucose, fructose and ribose, using fluorometric measurements of the content of advanced glycation end products (AGEs), protein-bound fructosamine, dityrosine, N'-formylkynurenine, kynurenine, tryptophan, the content of advanced oxidation protein products (AOPP), protein carbonyl groups, as well as thiol groups. Moreover, the levels of glycoalbumin and AGEs were determined by using an enzyme-linked immunosorbent assay. Based on the kinetic results, the optimal incubation time for studies of the modification of the glycoxidation rate by additives was chosen, and the effects of 25 compounds of natural origin on the glycoxidation of BSA induced by various sugars were examined. The same compounds were found to have different effects on glycoxidation induced by various sugars, which suggests caution in extrapolation from experiments based on one sugar to other sugars. From among the compounds tested, the most effective inhibitors of glycoxidation were: polyphenols, pyridoxine and 1-cyano-4-hydroxycinnamic acid.

Potential of birds to serve as pathology-free models of type 2 diabetes, part 2: do high levels of carbonyl-scavenging amino acids (e.g., taurine) and low concentrations of methylglyoxal limit the production of advanced glycation end-products?

In our previous publication, we reported on the advantages of using birds as a pathology-free model of type 2 diabetes mellitus (T2DM). Using this new perspective, we observed that birds are missing the RAGE gene, considered an important factor in the development of diabetic complications. In this article, we identify two additional Maillard reaction-related characteristics of birds that have the potential to account, in part, for avian ability to cope successfully with chronic hyperglycemia. First, compared to mammals, blood plasma of birds has significantly higher concentrations of taurine and other free amino acids that act as scavengers of reactive carbonyls. Second, there are also indications that avian blood plasma contains lower concentrations of methylglyoxal (MG) due, in part, to its decreased production by avian erythrocytes. Our deductions are based on relatively meager experimental data and are therefore speculative. One certain outcome of our study, however, is the idea that birds can be a useful model for the study of Maillard reactions and etiology of diabetic complications. We anticipate and hope that results of future studies will support the hypothesis identifying MG as a key intermediate in the etiology of diabetic complications. If this is indeed the case, then prevention and control of diabetic complications may become transformed into a more circumscribed, defined, and tractable problem whose goals will be to minimize the production of MG and to maximize its elimination by detoxification or scavenging.

Faox enzymes inhibited Maillard reaction development during storage both in protein glucose model system and low lactose UHT milk.

Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (ß-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the ß-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.

Evaluation of biological variation of glycated albumin (GA) and fructosamine in healthy subjects.

BACKGROUND: Glycated albumin (GA) and fructosamine are nonenzymatically glycated proteins still frequently utilized for monitoring glycemic control in diabetics. To investigate the analytical variation and the degree of individuality of these glycemic markers, we have performed an experimental study under a well designed and standardized protocol. METHODS: We collected five specimens from each of 18 apparently healthy subjects (9 men and 9 women, ages 26-52 years), on the same day, every two weeks for two months. Samples were stored at -80°C until analysis and assayed in duplicate in a single analytical run. GA and fructosamine were measured using enzymatic (Lucica®GA-L, Asahi Kasei Pharma, AKP, Tokyo, Japan) and colorimetric assays, respectively, on a Modular P Roche system (Roche Diagnostics GmbH, Mannheim, Germany). Data were analyzed by ANOVA. RESULTS: Analytical coefficient of variation (CVA) was 1.7%, 2.3% and 2.8% for GA, albumin and fructosamine, respectively. Within-subject (CVW) and between-subject (CVG) coefficients of variation were 2.1% and 10.6% for GA, 2.3% and 2.9% for albumin, and 2.3% and 6.3% for fructosamine. The estimated critical difference (CD) was 7.5% for GA, 9% for albumin and 10% for fructosamine. CONCLUSIONS: The good quality achieved by the analytical method for GA assessment and the reduced within-subject biological variation would allow to recommend this test in clinical practice for evaluation of glycemic control along with measurement of glycated hemoglobin.

Oxygen reactivity in flavoenzymes: context matters.

Many flavoenzymes--oxidases and monooxygenases--react faster with oxygen than free flavins do. There are many ideas on how enzymes cause this. Recent work has focused on the importance of a positive charge near N5 of the reduced flavin. Fructosamine oxidase has a lysine near N5 of its flavin. We measured a rate constant of 1.6 × 10(5) M(-1) s(-1) for its reaction with oxygen. The Lys276Met mutant reacted with a rate constant of 291 M(-1) s(-1), suggesting an important role for this lysine in oxygen activation. The dihydroorotate dehydrogenases from E. coli and L. lactis also have a lysine near N5 of the flavin. They react with O(2) with rate constants of 6.2 × 10(4) and 3.0 × 10(3) M(-1) s(-1), respectively. The Lys66Met and Lys43Met mutant enzymes react with rate constants that are nearly the same as those for the wild-type enzymes, demonstrating that simply placing a positive charge near N5 of the flavin does not guarantee increased oxygen reactivity. Our results show that the lysine near N5 does not exert an effect without an appropriate context; evolution did not find only one mechanism for activating the reaction of flavins with O(2).

The cation-π interaction between Lys53 and the flavin of fructosamine oxidase (FAOX-II) is critical for activity.

Fructosamine oxidases (FAOXs) are flavin-containing enzymes that catalyze the oxidative deglycation of low molecular weight fructosamines or Amadori products. The fructosamine substrate is oxidized by the flavin in the reductive half-reaction, and the reduced flavin is then oxidized by molecular oxygen in the oxidative half-reaction. The crystal structure of FAOX-II from Aspergillus fumigatus reveals a unique interaction between Lys53 and the isoalloxazine. The ammonium nitrogen of the lysine is in contact with and nearly centered over the aromatic ring of the flavin on the si-face. Here, we investigate the importance of this unique interaction on the reactions catalyzed by FAOX by studying both half-reactions of the wild-type and Lys53 mutant enzymes. The positive charge of Lys53 is critical for flavin reduction but plays very little role in the reaction with molecular oxygen. The conservative mutation of Lys53 to arginine had minor effects on catalysis. However, removing the charge by replacing Lys53 with methionine caused more than a million-fold decrease in flavin reduction, while only slowing the oxygen reaction by ∼30-fold.

Enzymatic deglycation of Amadori products in bacteria: mechanisms, occurrence and physiological functions.

Amadori products (fructosamines)-ubiquitously occurring in nature-are precursors of the toxic and cell damaging 'advanced glycation endproducts'; thus, it is not surprising that numerous organisms have developed systems to degrade such compounds. The deglycating enzymes differ with respect to their mechanisms as well as to their substrate specificities. Furthermore, different physiological functions are proposed for the different enzymes. The fructosamine 3-kinases of mammals and homologous proteins (fructosamine 3-kinase related proteins), which are common to all taxa, are thought to focus on intracellular repair functions. In contrast, in Bacillus subtilis and Escherichia coli, the cooperative action of a kinase and a deglycase facilitates Amadori degradation. As genes encoding these enzymes are co-transcribed with ABC transporter genes, it is thought that these genes facilitate the utilisation of extracellular Amadori products. Indeed, it has been shown that fructosamines can serve as the sole carbon and nitrogen sources. Here, we provide an overview of known deglycating systems with the emphasis on Amadori product degradation in bacteria.

1-Amino-1-deoxy-D-fructose ("fructosamine") and its derivatives.

Fructosamine has long been considered as a key intermediate of the Maillard reaction, which to a large extent is responsible for specific aroma, taste, and color formation in thermally processed or dehydrated foods. Since the 1980s, however, as a product of the Amadori rearrangement reaction between glucose and biologically significant amines such as proteins, fructosamine has experienced a boom in biomedical research, mainly due to its relevance to pathologies in diabetes and aging. In this chapter, we assess the scope of the knowledge on and applications of fructosamine-related molecules in chemistry, food, and health sciences, as reflected mostly in publications within the past decade. Methods of fructosamine synthesis and analysis, its chemical, and biological properties, and degradation reactions, together with fructosamine-modifying and -recognizing proteins are surveyed.

Crystal structure of the deglycating enzyme fructosamine oxidase (amadoriase II).

Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6A resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12A deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.

Intrinsic toxicity of glucose, due to non-enzymatic glycation, is controlled in-vivo by deglycation systems including: FN3K-mediated deglycation of fructosamines and transglycation of aldosamines.

Along with oxygen, glucose is an essential macronutrient for most cells, a source of carbons for biosynthesis and energy. However, alongside this indispensable role for cell survival and growth, glucose is intrinsically toxic by reacting with primary amines such as lysine in proteins in a non-enzymatic glycation process (a.k.a. Maillard reaction) especially important in long-lived, homeothermic organisms where temperatures of 37-44 degrees C accelerate its rate. Products of Maillard reactions are known to have adverse effects on protein function and have been implicated in the development of diabetic complications and possibly in neurodegenerative diseases. Because of the unavoidable nature of non-enzymatic glycation and its deleterious effects, we propose that glucose-utilizing organisms, especially the homeothermic ones, possess mechanisms to control this process at its earliest stages. In the intracellular milieu two such mechanisms are apparent at present; a fructosamine-3-kinase(FN3K)-dependent process which is ubiquitous in all warm-blooded animals and a FN3K-independent deglycation pathway present in all animals, including ones which do not have FN3K, such as insects. We propose that of the two pathways, the FN3K-independent mechanism is more important due to the fact that it breaks down the very first intermediate of the Maillard reaction, the Schiff base (a.k.a aldosamine). We postulate that this, FN3K-independent, deglycation occurs by transglycation, in which carbohydrate moieties of glycated amines, such as glucoselysines on proteins, are removed by intracellular nucleophiles including free amino acids and peptides such as glutathione, carnosine and anserine. Furthermore, we hypothesize that one or more of these nucleophile-aldose adducts, formed as by-products of transglycation, are actively removed from cells by one or more of the multi-drug-resistance [MDR] proteins or similar pumps. In the extracellular space, non-enzymatic glycation and deglycation occur as well. We also postulate that, in that setting, transglycation products are removed from the system by the kidneys or similar excretory organs. Our hypothesis leads to several testable predictions including: The deglycation hypothesis offers new paradigm for thinking about non-enzymatic glycation and diabetic complications and offers possible strategies for intervention in this and possibly other degenerative conditions.

Identification of enzymes acting on alpha-glycated amino acids in Bacillus subtilis.

We have characterized the Bacillus subtilis homologs of fructoselysine 6-kinase and fructoselysine-6-phosphate deglycase, two enzymes that specifically metabolize the Amadori compound fructose-epsilon-lysine in Escherichia coli. The B. subtilis enzymes also catalyzed the phosphorylation of fructosamines to fructosamine 6-phosphates (YurL) and the conversion of the latter to glucose 6-phosphate and a free amino acid (YurP). However, their specificity was totally different from that of the E. coli enzymes, since they acted on fructoseglycine, fructosevaline (YurL) or their 6-phosphoderivatives (YurP) with more than 30-fold higher catalytic efficiencies than on fructose-alpha-lysine (6-phosphate). These enzymes are therefore involved in the metabolism of alpha-glycated amino acids.