HPLC-DAD optimization of quantification of vescalagin, gallic and ellagic acid in chestnut tannins.

The quantification of hydrolysable polyphenols such as gallic, ellagic acid and vescalagin by HPLC-DAD is classically run after methanol extraction as a reference solvent. Water extraction is usually discarded because of a lower obtention of total polyphenol content compared to methanol extraction. In our study, methanol was compared to water extraction in both the total polyphenol content method and the HPLC-DAD analysis. Total polyphenol content in water extraction was lower than in methanol extraction, but water extraction gave better results on HPLC-DAD. In conclusion, total polyphenol content cannot be used as reference to choose the solvent of extraction to quantify some polyphenols by HPLC-DAD because of the specific properties of each polyphenol. Indeed, recovery results obtained on hydrolysable polyphenols with water extraction were better and with a lower variability than following methanol extraction.

Effect of leavening agent on Maillard reaction and the bifidogenic effect of traditional French bread.

The study aimed to evaluate the effect of using sourdough or yeast as a leavening agent for French bread making on the Maillard reaction and the bifidogenic effect. Sugars, proteins, and free asparagine were quantified in bread dough before and after fermentation. The levels of the Maillard reaction precursors were very different depending on the leavening agent used, which affected the Maillard reaction during baking. Strecker degradation was favored in the crust of sourdough bread (SB), generating about 7 times more aldehydes than in the crust of yeast bread (YB), thus improving the sensory quality of the bread. In the YB crust, the melanoidization pathway was predominant. The bifidogenic effect of crust and crumb from both breads was evaluated through the in vitro growth of Bifidobacterium adolescentis. SB showed a higher bifidogenic effect, probably due to its composition more favorable for bacteria growth.

Plasma Levels of Free NƐ-Carboxymethyllysine (CML) after Different Oral Doses of CML in Rats and after the Intake of Different Breakfasts in Humans: Postprandial Plasma Level of sRAGE in Humans.

N-carboxymethyl-lysine (CML) and other dietary advanced glycation end-products (AGEs) are chemically modified amino acids with potential toxicological effects putatively related to their affinity with the receptor for AGEs (RAGE). The goal of this study was to determine the postprandial kinetics of CML in both rodents and humans and, in the latter, to evaluate their relationship with the soluble RAGE isoforms (sRAGE). Four gavage solutions containing different forms of CML were given to rats, and blood was collected over 8 h. Three different breakfasts containing dietary CML (dCML) were administered to 20 healthy volunteers, and blood was collected over 2 h. Concentrations of CML, CEL, and lysine were quantified in plasma and human meals by LC-MS/MS, and sRAGE was determined in human plasma by ELISA. The results showed that dCML did not affect the concentrations of circulating protein-bound CML and that only free CML increased in plasma, with a postprandial peak at 90 to 120 min. In humans, the postprandial plasmatic sRAGE concentration decreased independently of the dAGE content of the breakfasts. This study confirms reports of the inverse postprandial relationship between plasmatic free CML and sRAGE, though this requires further investigation for causality to be established.

The Effect of Lactobacillus fermentum ME-3 Treatment on Glycation and Diabetes Complications.

SCOPE: Type 2 diabetes (T2D) induces organ damage associated with glycation, among other metabolic pathways. While therapeutic strategies have been tested to reduce the formation and impact of glycation products, results remain equivocal. Anti-diabetic therapies using probiotics have been proposed, but their effect upon glycation has not been reported. Here, the effects of the bacterial strain Lactobacillus fermentum ME-3 on glycation and T2D-related complications in a mouse model of T2D are investigated. METHODS & RESULTS: Wild-type LepRdb/+ and diabetic LepRdb/db littermates receive a daily gavage of either water or the probiotic ME-3 strain (1010 CFU). Glycation markers, fructoselysine-derived furosine (FL-furosine) and carboxymethyllysine (CML), are quantified in four major organs and plasma using stable-isotope dilution LC-MS/MS. After 12 weeks of ME-3 treatment, diabetic mice gain less weight and exhibit an apparently improved glucose tolerance. The ME-3 treatment reduces median renal levels of FL-furosine in both genotypes by 12-15%, and renal and pulmonary free-CML in diabetic mice by 30% and 18%, respectively. Attenuated hepatic steatosis and an improved plasma lipid profile are also observed with treatment in both genotypes, while the gut microbiota profile is unchanged. CONCLUSION: L. fermentum ME-3 has therapeutic potential for reducing the formation/accumulation of some glycation products in kidneys and attenuating some common diabetes-related complications.

Carbamylation and glycation compete for collagen molecular aging in vivo.

Tissue aging is a complex phenomenon involving molecular aging of matrix proteins, which mainly results from their progressive alteration by nonenzymatic post-translational modifications (NEPTMs) such as glycation and carbamylation. These two reactions, which correspond to the binding of reactive metabolites (i.e. reducing sugars and urea-derived cyanate, respectively) on amino groups of proteins, occur during aging and are amplified in various chronic diseases such as diabetes mellitus or chronic renal disease (CKD). Since these reactions target the same functional groups, they can reciprocally compete for protein modification. Determining which NEPTM is predominant in tissues is necessary to better understand their role in the development of long-term complications of chronic diseases. For that purpose, two different murine models were used for reproducing such a competitive context: a CKD-diabetic mice model and a cyanate-consuming mice model. The competition has been evaluated by quantifying glycation and carbamylation products by LC-MS/MS in skin and aorta total extracts as well as in skin type I collagen. The results showed that the simultaneous enhancement of glycation and carbamylation reactions resulted in a decrease of the formation of glycation products (especially Amadori products) whereas the concentrations of homocitrulline, a carbamylation product, remained similar. These results, which have been obtained in both tissues and in purified skin type I collagen, suggest that carbamylation takes precedence over glycation for the modification of tissue proteins, but only in pathological conditions favouring these two NEPTMs. While glycation has been considered for a long time the predominant NEPTM of matrix proteins, carbamylation seems to also play an important role in tissue aging. The existence of competition between these NEPTMs must be taken into account to better understand the consequences of molecular aging of matrix proteins in tissue aging.

The LepRdb/db mice model for studying glycation in the context of diabetes.

BACKGROUND: Early (furosine) and advanced (carboxymethyllysine, CML) products of glycation (AGEs) have been reported as increased in plasma, tissues, and organs of diabetic people, indicating a direct link between glycation and type 2 diabetes (T2D). While murine models present some of the characteristics observed in diabetic humans, their pertinence as models of glycation, particularly for T2D, remains poorly described. The aim of this study was to characterize and compare glycation in several organs of two commonly studied murine models of T2D using stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: Defining parameters of type 2 diabetes including body weight, fasting glycaemia, and glucose intolerance were measured in three different C57BL6 mouse models of T2D-the genetic LepRdb/db (db/db) model and two diet-induced obesity (DIO) models-and their respective controls. Furosine, free, and protein-bound CML were quantified in kidneys, lungs, heart, and liver by LC-MS/MS. RESULTS: The obesity, hyperglycaemia, and glucose intolerance in db/db mice was accompanied by an increase of furosine and protein-bound CML levels in all organs relative to controls. The DIO models took several months to become obese, exhibited less severe hyperglycaemia and glucose intolerance, while glycation products were not significantly different between these groups (with the exception of furosine in liver and CML in lungs). CONCLUSIONS: The db/db model better reflected the characteristics of human T2D compared with the DIO models and exhibited greater formation and accumulation of both furosine and protein-bound CML in all of the organs tested here.

Effects of Maillard Reaction Products on Sensory and Nutritional Qualities of the Traditional French Baguette.

The goals of this study were to evaluate the effect of baking time on the Maillard reaction products (MRPs) generated in the crust of the traditional French baguette and to estimate their impact on its sensory characteristics, its acrylamide content, and its bifidogenic effect. Melanoidins, volatile compounds, and acrylamide were evaluated in the crust of traditional French baguettes baked between 12 and 22 min at 225 °C. The increase in melanoidins was positively correlated to the baking time, while volatile compounds only increased until 18 min. The acrylamide content was estimated to be below 18 µg/kg, which confirms the findings of EFSA that bread is not a main contributor to dietary acrylamide. A descriptive sensory analysis showed that the baking time positively affected the sensory quality of the crust. The consumer test revealed the same trend and the panelists favorably judged the well-baked baguettes based on a better crust flavor and crispness. The bifidogenic effect of the crust and the crumb from the baguettes baked 22 min was evaluated on the in vitro growth of Bifidobacterium adolescentis. The results demonstrated that the crumb and the crust had exactly the same bifidogenic impact, therefore not caused by melanoidins. PRACTICAL APPLICATION: The consumption of bread in France has decreased since 2007, although bread is considered by French people as "healthy" and essential to maintain a balanced diet. The current study evaluated the global qualities of the French baguette in order to highlight its high sensory quality and its beneficial effect by inducing a possible growth of bifidobacteria, even in well-baked baguettes. These findings allow the French bakery industry to develop an argument to promote its technical know-how and to help consumers choose healthier and tastier bread. Moreover, this study provided some recommendations of baking processes to maintain a high sensory quality of the French baguette and limit the formation of undesirable compounds, such as acrylamide, in the crust.

Effect of added nitrogen fertilizer on pyrazines of roasted chicory.

BACKGROUND: Coffee substitutes made of roasted chicory are affected by the formation of acrylamide whose main precursor is asparagine. One strategy for limiting the formation of acrylamide is to reduce free asparagine in the chicory roots by lessening the supply of nitrogen in the field. However, decreasing nitrogen fertilizer could affect the formation of the volatile compounds and, consequently, the sensory characteristics of the roasted chicory. The present study aimed to investigate the impact of the nitrogen supply in five commercial varieties on their aroma profile. RESULTS: The addition of 120 kg ha-1 of nitrogen fertilizer in the field resulted in a greater amount of pyrazines in the roasted chicory. Triangle tests were performed to determine the effect of the nitrogen level on the sensory quality of the five varieties. The results revealed that the chicory aroma was modified in two out of five varieties. CONCLUSION: The results of the present study suggest that a strategy aiming to limit the amount of acrylamide could affect the sensory quality of some varieties of chicory. Further acceptance tests need to be conducted to assess the effect (whether favourable or otherwise) on the sensory quality of the coffee substitutes. © 2016 Society of Chemical Industry.

Quantitative assessment of organ distribution of dietary protein-bound 13 C-labeled Nɛ -carboxymethyllysine after a chronic oral exposure in mice.

SCOPE: Nɛ -Carboxymethyl-lysine (CML) is a prominent advanced glycation end-product which is not only found in vivo but also in food. It is known that a percentage of the dietary CML (dCML) is absorbed into the circulation and only partly excreted in the urine. Several studies have tried to measure how much dCML remains in tissues. However obstacles to interpreting the data have been found. METHODS AND RESULTS: A new protocol which discriminates dCML from native CML (nCML) has been developed. Three CML isotopes with different mass-to-charge ratios were used: nCML Nε -carboxymethyl-L-lysine, dCML Nε -[13 C]carboxy[13 C]methyl-L-lysine and internal standard Nε -carboxymethyl-L-[4,4,5,5-2 H4 ]lysine. Wild-type (n = 7) and RAGE-/- (n = 8) mice were fed for 30 days with either a control, or a BSA-bound dCML-enriched diet. Organs were analyzed for nCML and dCML using liquid chromatography-tandem mass spectrometry. Mice exposed to dCML showed an accumulation in all tissues tested except fat. The rate of deposition was high (81-320 µgdCML /g dry matter) in kidneys, intestine, and lungs and low (<5 µg/g) in heart, muscle, and liver. This accumulation was not RAGE dependent. CONCLUSION: The kidney is not the only organ affected by the accumulation of dCML. Its high accumulation in other tissues and organs may also, however, have important physiological consequences.

The impact of raw materials and baking conditions on Maillard reaction products, thiamine, folate, phytic acid and minerals in white bread.

The aim of this study was to develop a white bread with improved nutrient contents and reduced levels of potentially harmful Maillard reaction products such as N(ε)-carboxymethyllysine (CML) and 5-hydroxymethylfurfural (HMF). Assays were carried out through a full factorial experimental design allowing the simultaneous analysis of four factors at two levels: (1) wheat flour extraction rates (ash content: 0.60%-0.72%), (2) leavening agents (bakers' yeast - bakers' yeast and sourdough), (3) prebaking and (4) baking conditions (different sets of time and temperature). The baking conditions affected HMF and CML as well as certain mineral contents. A reduced baking temperature along with a prolonged heat treatment was found to be favourable for reducing both the CML (up to 20%) and HMF concentrations (up to 96%). The presence of sourdough decreased the formation of CML (up to 28%), and increased the apparent amounts of calcium (up to 8%) and manganese (up to 17.5%) probably through acidification of the dough. The extraction rate of flours as well as interactions between multiple factors also affected certain mineral content. However, compounds like folate, thiamine, copper, zinc, iron and phytic acid were not affected by any of the factors studied.

How Can Diet Affect the Accumulation of Advanced Glycation End-Products in the Human Body?

The accumulation of advanced glycation end products (AGEs) is associated with the complications of diabetes, kidney disease, metabolic disorders and degenerative diseases. It is recognized that the pool of glycation products found in the human body comes not only from an endogenous formation, but also from a dietary exposure to exogenous AGEs. In recent years, the development of pharmacologically-active ingredients aimed at inhibiting endogenous glycation has not been successful. Since the accumulation of AGEs in the human body appears to be progressive throughout life, an early preventive action against glycation could be effective through dietary adjustments or supplementation with purified micronutrients. The present article provides an overview of current dietary strategies tested either in vitro, in vivo or both to reduce the endogenous formation of AGEs and to limit exposure to food AGEs.

Maillard reaction products in bread: A novel semi-quantitative method for evaluating melanoidins in bread.

The aim of this study was to test the methods currently in use and to develop a new protocol for the evaluation of melanoidins in bread. Markers of the early and advanced stages of the Maillard reaction were also followed in the crumb and the crust of bread throughout baking, and in a crust model system. The crumb of the bread contained N(ε)-fructoselysine and N(ε)-carboxymethyllysine but at levels 7 and 5 times lower than the crust, respectively. 5-Hydroxymethylfurfural was detected only in the crust and its model system. The available methods for the semi-quantification of melanoidins were found to be unsuitable for their analysis in bread. Our new method based on size exclusion chromatography and fluorescence measures soluble fluorescent melanoidins in bread. These melanoidin macromolecules (1.7-5.6 kDa) were detected intact in both crust and model system. They appear to contribute to the dietary fibre in bread.

Impact of Variety and Agronomic Factors on Crude Protein and Total Lysine in Chicory; N(ε)-Carboxymethyl-lysine-Forming Potential during Drying and Roasting.

During the heat treatment of coffee and its substitutes some compounds potentially deleterious to health are synthesized by the Maillard reaction. Among these, N(ε)-carboxymethyl-lysine (CML) was detected at high levels in coffee substitutes. The objective of this study was to evaluate the impact of changes in agricultural practice on the lysine content present in chicory roots and try to limit CML formation during roasting. Of the 24 varieties analyzed, small variations in lysine content were observed, 213 ± 8 mg/100 g dry matter (DM). The formation of lysine tested in five commercial varieties was affected by the nitrogen treatment with mean levels of 176 ± 2 mg/100 g DM when no fertilizer was added and 217 ± 7 mg/100 g DM with a nitrogen supply of 120 kg/ha. The lysine content of fresh roots was significantly correlated to the concentration of CML formed in roasted roots (r = 0.51; p < 0.0001; n = 76).

Formula-derived advanced glycation end products are involved in the development of long-term inflammation and oxidative stress in kidney of IUGR piglets.

SCOPE: Formula-derived dietary advanced glycation end products (AGEs) may promote programming of inflammation and oxidative stress in the kidney of intrauterine growth retardation (IUGR) piglets. METHODS AND RESULTS: IUGR piglets received either a low temperature heated formula (n = 8) or a high temperature heated formula (HHF: n = 8) or suckled naturally for 3 wk postnatally. Then they were fed with normal ad libitum regular diet. N(ε)-carboxymethyllysine (CML) was measured in plasma, feces, and formula by HPLC/MS-MS. CML was detected by immunofluorescence in kidney cells. Target renin-angiotensin-apoptotic, pro-inflammatory genes-p62 NF-κB, and soluble receptor of AGE (sRAGE) levels were quantified. Compared with that in controls, free CML and plasma urea increased significantly in the HHF-fed group at PND36 (p < 0.05). CML was detected in the nuclei of renal tubular cells of formula-fed piglets but not in suckled ones. This presence of CML was associated with the activation of the soluble receptor of AGE. AT1, AT2, caspase 3, caspase 8, NF-κB, p62 NF-κB, and total protein oxidation in kidney were higher in HHF-fed group as compared to LHF-fed group (p < 0.05). CONCLUSION: Food processes aimed at reducing the concentration of AGEs in infant formula are urgently needed and may be therapeutically relevant for premature and/or IUGR babies.

Dietary CML-enriched protein induces functional arterial aging in a RAGE-dependent manner in mice.

SCOPE: Advanced glycation end-products (AGEs) are endogenously produced and are present in food. N(ε)-carboxymethyllysine (CML) is an endothelial activator via the receptor for AGEs (RAGEs) and is a major dietary AGE. This work investigated the effects of a CML-enriched diet and RAGE involvement in aortic aging in mice. METHODS AND RESULTS: After 9 months of a control diet or CML-enriched diets (50, 100, or 200 µg(CML)/g of food), endothelium-dependent relaxation, RAGE, vascular cell adhesion molecule-1, and sirtuin-1 expression, pulse wave velocity and elastin disruption were measured in aortas of wild-type or RAGE(-/-) male C57BL/6 mice. Compared to the control diet, endothelium-dependent relaxation was reduced in the wild-type mice fed the CML-enriched diet (200 µg(CML)/g) (66.8 ± 12.26 vs. 94.3 ± 2.6%, p < 0.01). RAGE and vascular cell adhesion molecule-1 (p < 0.05) expression were increased in the aortic wall. RAGE(-/-) mice were protected against CML-enriched diet-induced endothelial dysfunction. Compared to control diet, the CML-enriched diet (200 µg(CML)/g) increased the aortic pulse wave velocity (86.6 ± 41.1 vs. 251.4 ± 41.1 cm/s, p < 0.05) in wild-type animals. Elastin disruption was found to a greater extent in the CML-fed mice (p < 0.05). RAGE(-/-) mice fed the CML-enriched diet were protected from aortic stiffening. CONCLUSION: Chronic CML ingestion induced endothelial dysfunction, arterial stiffness and aging in a RAGE-dependent manner.

The rehabilitation of raw and brown butters by the measurement of two of the major Maillard products, N(ε)-carboxymethyl-lysine and 5-hydroxymethylfurfural, with validated chromatographic methods.

The fat food group, especially butter, has so far been thought to have a high N(ε)-carboxymethyl-lysine (CML) content. However recent data have challenged this opinion. The objective of this article was to determine not only CML content but also that of 5-hydroxymethylfurfural (HMF) in raw and cooked butters. The first aim of this study was to verify the accuracy of the LC-MS/MS and LC-UV methods used for the quantification of CML and HMF. The tests on fortified butter samples showed recovery values of 72% for CML and 78% for HMF. The amounts of CML in raw and cooked butters were 0.25 ± 0.03 and 2.22 ± 0.56 µg/g, respectively. The level of HMF in cooked butters was 61 ± 40 µg/g. No CML was detected in clarified butter, and no HMF was detected in raw and clarified butters. The results indicate that the contribution of butter alone to the exposure to CML and HMF is very low.

An advanced glycation end product (AGE)-rich diet promotes Nε-carboxymethyl-lysine accumulation in the cardiac tissue and tendons of rats.

The purpose of this study was to investigate the intake, excretion, and tissue accumulation of carboxymethyl-lysine (CML), after feeding rats a diet containing advanced glycation end products (AGEs) from a glucose-lysine (GL) model system. Rats were distributed into two groups and assigned to a control diet or a diet including 3% heated GL (GL diet) for three months. Feces and urine were collected over the last week. After sacrifice, serum was obtained and some organs were removed for CML analysis. The percentage of fecal CML was 2.5-fold higher in the animals fed the GL diet (33.2 vs 76.5% for control and GL group), whereby total recovery was 91.8% compared with a level of 54.6% in the animals fed the control chow, evidencing the importance of the chemical form and the net quantity of dietary CML on its elimination. We suggest that dietary dicarbonyl compounds from GL diet or dietary CML itself are responsible for CML accumulation in hearts and tendons. The most significant result of the present study is that the regular consumption of dietary AGEs in healthy individuals promotes CML accumulation in some organs.

Acrylamide, 5-hydroxymethylfurfural and N(ε)-carboxymethyl-lysine in coffee substitutes and instant coffees.

Sensitive analytical methods were developed and validated for the quantification of acrylamide, N(ε)-carboxymethyl-lysine (CML) and 5-hydroxymethylfurfural (HMF) in 24 commercial coffee substitutes (CSs) and 12 instant coffees (ICs). Acrylamide levels varied widely from 200 to 4940 µg kg(-1) with higher levels in CSs. Only two out of 24 CSs had a level of acrylamide above the indicative value set for this food category by the European Commission (4000 µg kg(-1)). None of the ICs tested in this study exceeded the indicative value set for this foodstuff (900 µg kg(-1)). CML ranged from 0.17 to 47 mg kg(-1) and it increased in proportion to the protein content of the samples. The highest concentrations were found in IC partly due to the relatively high protein content of this food group. HMF was the most abundant neoformed compound (NFC) found in the tested commercial samples. It was found between 0.59 and 13 g kg(-1). Among other food categories IC and CS could appear to be major contributors to the exposure to NFCs if consumed on a daily basis. Further investigations are needed to elucidate the acrylamide formation during processing and to determine the daily intake level of frequent consumers of these products.

Microorganisms and Maillard reaction products: a review of the literature and recent findings.

Research on the impact of Maillard reaction products (MRPs) on microorganisms has been reported in the literature for the last 60 years. In the current study, the impact of an MRP-rich medium on the growth of three strains of Escherichia coli was measured by comparing two classic methods for studying the growth of bacteria (plate counting and optical density at 600 nm) and by tracing MRP utilisation. Early stage and advanced MRPs in the culture media were assessed by quantifying furosine and N (ε) -carboxymethyllysine (CML) levels, respectively, using chromatographic methods. These measures were performed prior to and during bacterial growth to estimate the potential use of these MRPs by Escherichia coli CIP 54.8. Glucose and lysine, the two MRP precursors used in the MRP-rich medium, were also quantified by chromatographic means. Compared to control media, increased lag phases and decreased growth rates were observed in the MRP-rich medium for two out of the three Escherichia coli strains tested. In contrast, one strain isolated from the faeces of a piglet fed on a MRP-rich diet was not influenced by the presence of MRPs in the medium. Overall, CML as well as the products obtained by the thermal degradation of glucose and lysine, regardless of the Maillard reaction, did not affect the growth of the three strains tested. In addition, no degradation of fructoselysine or CML was found in the presence of Escherichia coli CIP 54.8.

Metabolic transit of N(ε)-carboxymethyl-lysine after consumption of AGEs from bread crust.

Our aim was to investigate carboxymethyl-lysine (CML) intake and excretion after feeding rats with diets containing advanced glycation end-products (AGEs) from bread crust (BC) or its soluble or insoluble fractions, and to identify the factors responsible for the effects observed. CML in serum and different tissues was measured to detect possible accumulations. For 88 days, weanling rats were fed with either a control diet or one containing BC, or its soluble low molecular weight (LMW), soluble high molecular weight (HMW) or insoluble fractions. In the last week of the assay, faeces and urine were collected daily and stored as a 1 week pool. After sacrifice, blood was drawn to obtain serum and some organs were removed. CML analysis was performed by HPLC/MS/MS in diets, faeces, urines, serum and tissues. Faecal excretion of CML was strongly influenced by dietary CML levels and represents the major route of excretion (i.e. 33.2%). However, the urinary elimination of CML was probably limited or saturated, especially when more complex compounds were present in the diet. BC consumption increased CML in the cardiac tissue (170 ± 18 vs. 97 ± 3 µmol per mol lysine for BC and control groups), which correlated with the CML intake. The levels of this AGE in bone were unaffected by the dietary treatment, but in tail tendons CML was greatly increased in the animals that consumed the BC diet (102 ± 13 vs. 51 ± 8 µmol per mol lysine for BC and control groups, P = 0.006), which was associated with the intake of soluble LMW compounds present in BC. Despite the CML accumulation detected in different tissues, serum levels of protein-bound CML were unchanged, indicating the importance of measuring the free CML in this fluid as a real index of dietary CML.