Biomedical subjects

Fabien Robert

Publications and source records attributed to Fabien Robert.

At least 19 recordsLinked to original sources

PubMed2006

Sugar fragmentation in the maillard reaction cascade: isotope labeling studies on the formation of acetic acid by a hydrolytic beta-dicarbonyl cleavage mechanism.

The formation of acetic acid was elucidated based on volatile reaction products and related nonvolatile key intermediates. The origin and yield of acetic acid were determined under well-controlled conditions (90-120 degrees C, pH 6-8). Experiments with various 13C-labeled glucose isotopomers in the presence of glycine revealed all six carbon atoms being incorporated into acetic acid: C-1/C-2 ( approximately 70%), C-3/C-4 ( approximately 10%), and C-5/C-6 (approximately 20%). Acetic acid is a good marker of the 2,3-enolization pathway since it is almost exclusively formed from 1-deoxy-2,3-diulose intermediates. Depending on the pH, the acetic acid conversion yield reached 85 mol % when using 1-deoxy-2,3-hexodiulose (1) as a precursor. Hydrolytic beta-dicarbonyl cleavage of 1-deoxy-2,4-hexodiuloses was shown to be the major pathway leading to acetic acid from glucose without the intermediacy of any oxidizing agents. The presence of key intermediates was corroborated for the first time, i.e., tetroses and 2-hydroxy-3-oxobutanal, a tautomer of 1-hydroxy-2,3-butanedione, also referred to as 1-deoxy-2,3-tetrodiulose. The hydrolytic beta-dicarbonyl cleavage represents a general pathway to organic acids, which corresponds to an acyloin cleavage or a retro-Claisen type reaction. Although alternative mechanisms must exist, the frequently reported hydrolytic alpha-dicarbonyl cleavage of 1 can be ruled out as a pathway forming carboxylic acids.

Acetic Acid↗

PubMed2006

Sugar fragmentation in the maillard reaction cascade: formation of short-chain carboxylic acids by a new oxidative alpha-dicarbonyl cleavage pathway.

The formation of short-chain carboxylic acids was studied in Maillard model systems (90 degrees C, pH 6-10) with emphasis on the role of oxygen and water. The total amount of acetic acid formed did not depend on the reaction atmosphere. In the presence of labeled dioxygen or water (18O2, H2 17O), labeled oxygen was partially incorporated into acetic acid. Thermal treatment of 1-deoxy-d-erythro-2,3-hexodiulose (1) and 3-deoxy-d-erythro-hexos-2-ulose in the presence of 17O-enriched water under alkaline conditions led to acetic and formic acid, respectively, as indicated by 17O NMR spectroscopy. The suggested mechanism involves an oxidative alpha-dicarbonyl cleavage leading to an intermediary mixed acid anhydride that releases the acids, e.g., acetic and erythronic acid, from 1. Similarly, glyceric and lactic acids were formed from 1-deoxy-3,4-hexodiuloses, corroborated by complementary analytical techniques. This paper provides for the first time evidence for the direct formation of acids from C6-alpha-dicarbonyls by an oxidative mechanism and incorporation of a 17OH group into the carboxylic moiety. The experimental data obtained support the coexistence of at least two newly described reaction mechanisms leading to carboxylic acids, i.e., (i) a hydrolytic beta-dicarbonyl cleavage as a major pathway and (ii) an alternative minor pathway via oxidative alpha-dicarbonyl cleavage induced by oxidizing species.

Acetic Acid↗

PubMed2006

Combining protein identification and quantification: C-terminal isotope-coded tagging using sulfanilic acid.

Two methods of differential isotopic coding of carboxylic groups have been developed to date. The first approach uses d0- or d3-methanol to convert carboxyl groups into the corresponding methyl esters. The second relies on the incorporation of two 18O atoms into the C-terminal carboxylic group during tryptic digestion of proteins in H(2)18O. However, both methods have limitations such as chromatographic separation of 1H and 2H derivatives or overlap of isotopic distributions of light and heavy forms due to small mass shifts. Here we present a new tagging approach based on the specific incorporation of sulfanilic acid into carboxylic groups. The reagent was synthesized in a heavy form (13C phenyl ring), showing no chromatographic shift and an optimal isotopic separation with a 6 Da mass shift. Moreover, sulfanilic acid allows for simplified fragmentation in matrix-assisted laser desorption/ionization (MALDI) due the charge fixation of the sulfonate group at the C-terminus of the peptide. The derivatization is simple, specific and minimizes the number of sample treatment steps that can strongly alter the sample composition. The quantification is reproducible within an order of magnitude and can be analyzed either by electrospray ionization (ESI) or MALDI. Finally, the method is able to specifically identify the C-terminal peptide of a protein by using GluC as the proteolytic enzyme.

Caseins↗

PubMed2005

Chemical synthesis and NMR characterization of structured polyunsaturated triacylglycerols.

The chemical synthesis of pure triacylglycerol (TAG) regioisomers, that contain long chain polyunsaturated fatty acids, such as arachidonic acid (AA) or docosahexaenoic acid (DHA), and saturated fatty acids, such as lauric acid (La) or palmitic acid (P), at defined positions, is described. A single step methodology using (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), an activator of carboxyl group commonly used in peptide synthesis and occasionally used in carboxylic acid esterification, has been developed for structured TAG synthesis. Identification of the fatty acyl chains for each TAG species was confirmed by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) and fatty acid positional distribution was determined by (1)H and (13)C NMR spectra. The generic described procedures can be applied to a large variety of substrates and was used for the production of specific triacylglycerols of defined molecular structures, with high regioisomeric purity. Combination of MS and NMR was shown to be an efficient tool for structural analysis of TAG. In particular, some NMR signals were demonstrated to be regioisomer specific, allowing rapid positional analysis of LC-PUFA containing TAG.

Carbon Isotopes↗

PubMed2005

Acrylamide formation from asparagine under low moisture Maillard reaction conditions. 2. Crystalline vs amorphous model systems.

The formation of acrylamide was investigated in model systems based on asparagine and glucose under low moisture Maillard reaction conditions as a function of reaction temperature, time, physical state, water activity, and glass transition temperature. Equimolar amorphous glucose/asparagine systems with different water activities were prepared by freeze drying and were shown to quickly move to the rubbery state already at room temperature and a water activity of above 0.15. The acrylamide amounts were correlated with physical changes occurring during the reaction. Pyrolysis and kinetics of acrylamide release in amorphous and crystalline glucose/asparagine models indicated the importance of the physical state in acrylamide formation. In amorphous systems, acrylamide was generated in higher concentrations and at lower temperatures as compared to the crystalline samples. Time and temperature are covariant parameters in both systems affecting the acrylamide formation by thermal processes. On the other side, the water activity and glass transition temperature do not seem to be critical parameters for acrylamide formation in the systems studied.

Acrylamide↗

PubMed2005

Analysis of Amadori compounds by high-performance cation exchange chromatography coupled to tandem mass spectrometry.

High-performance cation exchange chromatography coupled to tandem mass spectrometry or electrochemical detection was found to be an efficient tool for analyzing Amadori compounds derived from hexose and pentose sugars. The method allows rapid separation and identification of Amadori compounds, while benefiting from the well-known advantages of mass spectrometry, such as specificity and sensitivity. Glucose- and xylose-derived Amadori compounds of several amino acids, such as glycine, alanine, valine, leucine/isoleucine, methionine, proline, phenylalanine, and glutamic acid, were separated or discriminated using this new method. The method is suitable for the analysis of both model reaction mixtures and food products. Fructosylglutamate was found to be the major Amadori compound in dried tomatoes (approximately 1.5 g/100 g) and fructosylproline in dried apricots (approximately 0.2 g/100 g). Reaction of xylose and glycine at 90 degrees C (pH 6) for 2 h showed rapid formation of xylulosylglycine (approximately 12 mol %, 15 min) followed by slow decrease over time. Analysis of pentose-derived Amadori compounds is shown for the first time, which represents a major breakthrough in studying occurrence, formation, and decomposition of these labile Maillard intermediates.

Chromatography, Ion Exchange↗

PubMed2005

Elucidation of chemical pathways in the maillard reaction by 17O-NMR spectroscopy.

17O-NMR spectroscopy was employed as an innovative method to help understand mechanistic pathways in sugar fragmentation. Elucidation of reaction mechanisms to final Maillard end products was achieved by starting from specific intermediates obtained by synthesis, such as 1-deoxy-D-erythro-hexo-2,3-diulose. This alpha-dicarbonyl was thermally treated in the presence of 17O-enriched water under alkaline conditions. The reaction products were monitored by 17O-NMR spectroscopy and their structures corroborated by complementary techniques. For the first time, evidence is shown for the direct formation of acetic acid from 1-deoxy-D-erythro-hexo-2,3-diulose by an oxidative alpha-dicarbonyl cleavage and incorporation of a 17OH group into the acetic acid released as sugar fragment.

Carbohydrates↗

PubMed2005

The effect of reaction conditions on the origin and yields of acetic acid generated by the maillard reaction.

The effect of the reaction conditions on the origin and yields of acetic acid from glucose was studied in the system containing equimolar concentrations of 13C-labeled glucose and glycine. Acetic acid was quantified by GC-MS using isotope dilution assay. The beta-dicarbonyl cleavage of 1-deoxyhexo-2,4-diulose is proposed to be a major pathway leading to the formation of acetic acid in the glucose-based Maillard reaction systems under food processing conditions. Acetic acid was built up from all six carbon atoms of glucose. The relative distribution of acetic acid was independent of the reaction time. Temperature and pH had only small effects.

Acetic Acid↗

PubMed2004

Acrylamide formation from asparagine under low-moisture Maillard reaction conditions. 1. Physical and chemical aspects in crystalline model systems.

The formation of acrylamide in crystalline model systems based on asparagine and reducing sugars was investigated under low-moisture reaction conditions. The acrylamide amounts were correlated with physical changes occurring during the reaction. Molecular mobility of the precursors turned out to be a critical parameter in solid systems, which is linked to the melting behavior and the release of crystallization water of the reaction sample. Heating binary mixtures of asparagine monohydrate and anhydrous reducing sugars led to higher acrylamide amounts in the presence of fructose compared to glucose. Differential scanning calorimetry measurements performed in open systems indicated melting of fructose at 126 degrees C, whereas glucose and galactose fused at 157 and 172 degrees C, respectively. However, glucose was the most reactive and fructose the least efficient sugar in anhydrous liquid systems, indicating that at given molecular mobility the chemical reactivity of the sugar was the major driver in acrylamide formation. Furthermore, reaction time and temperature were found to be covariant parameters: acrylamide was preferably formed by reacting glucose and asparagine at 120 degrees C for 60 min, whereas 160 degrees C was required at shorter reaction time (5 min). These results suggest that, in addition to the chemical reactivity of ingredients, their physical state as well as reaction temperature and time would influence the formation of acrylamide during food processing.

Acrylamide↗

PubMed2004

In-depth mechanistic study on the formation of acrylamide and other vinylogous compounds by the maillard reaction.

The formation of acrylamide was studied in low-moisture Maillard model systems (180 degrees C, 5 min) based on asparagine, reducing sugars, Maillard intermediates, and sugar degradation products. We show evidence that certain glycoconjugates play a major role in acrylamide formation. The N-glycosyl of asparagine generated about 2.4 mmol/mol acrylamide, compared to 0.1-0.2 mmol/mol obtained with alpha-dicarbonyls and the Amadori compound of asparagine. 3-Hydroxypropanamide, the Strecker alcohol of asparagine, generated only low amounts of acrylamide ( approximately 0.23 mmol/mol), while hydroxyacetone increased the acrylamide yields to more than 4 mmol/mol, indicating that alpha-hydroxy carbonyls are much more efficient than alpha-dicarbonyls in converting asparagine into acrylamide. The experimental results are consistent with the reaction mechanism based on (i) a Strecker type degradation of the Schiff base leading to azomethine ylides, followed by (ii) a beta-elimination reaction of the decarboxylated Amadori compound to afford acrylamide. The beta-position on both sides of the nitrogen atom is crucial. Rearrangement of the azomethine ylide to the decarboxylated Amadori compound is the key step, which is favored if the carbonyl moiety contains a hydroxyl group in beta-position to the nitrogen atom. The beta-elimination step in the amino acid moiety was demonstrated by reacting under low moisture conditions decarboxylated model Amadori compounds obtained by synthesis. The corresponding vinylogous compounds were only generated if a beta-proton was available, for example, styrene from the decarboxylated Amadori compound of phenylalanine. Therefore, it is suggested that this thermal pathway may be common to other amino acids, resulting under certain conditions in their respective vinylogous reaction products.

Acetone↗

PubMed2004

Formation and hydrolysis of triacylglycerol and sterols epoxides: role of unsaturated triacylglycerol peroxyl radicals.

Epoxidation of unsaturated pure triacylglycerols (TAGs), cholesterol, and phytosterols was investigated using air and 18O2 oxidation experiments. Oxidized lipids were analyzed using both triple quadrupole mass spectrometry (MS), ion-trap MS in the direct infusion mode, and triple quadrupole MS in tandem with a liquid chromatograph (LC-MS/MS). Pure 1,2-distearoyl-3-oleoyl-glycerol (SSO) samples were heated in sealed vials under air or 18O2 atmosphere at 160 degrees C for 1 h. LC-MS/MS analysis of 18O-labeled oxidized TAGs revealed that hydroperoxides and epoxide TAGs are formed mainly during this first step. Then, oxidized TAGs were incubated under an inert atmosphere, separately with 1,2-dipalmitoyl-3-oleoyl-glycerol (PPO) at 160 degrees C for 90 min, and with cholesterol and stigmasterol at 100 degrees C for 10 min. Subsequent LC-MS/MS analysis revealed the occurrence of epoxidation products of PPO, cholesterol, and sitosterol. Therefore, we showed the epoxidation of unsaturated lipids proceeds readily in contact with hydroperoxide TAGs, in the absence of molecular oxygen. Dual oxidation experiments using both air and 18O2 allowed investigation of oxygen atom transfer during epoxidation of lipids. Moreover, the experimental oxidation design presented can be used to study fragmentation pathways, as illustrated for 5,6-epoxycholesterol (CE) on both triple quadrupole and ion-trap MS. We report for the first time the occurrence of 5,6;22,23-diepoxystigmasterol (StDE) and 5,6;22,23-diepoxybrassicasterol (BDE) in autoxidized vegetable oils. Additionally, acid-catalyzed hydrolysis of epoxidized lipids, with emphasis on phytosterol polyol formation, was investigated using a model gastric medium. For confirmation, almost all identified products were synthesized and characterized by MS.

Cholesterol↗

PubMed2004

Synthesis and sensorial properties of 2-alkylalk-2-enals and 3-(acetylthio)-2-alkyl alkanals.

Parallel synthesis was applied to prepare a series of 3-(acetylthio)-2-alkyl alkanals by Michael addition of thioacetic acid under alkaline conditions to alpha,beta-unsaturated 2-alkyl-substituted aldehydes, which were obtained by aldol condensation of the corresponding primary aldehydes as starting materials. The target compounds were characterized in terms of GC, MS, and NMR data. The sensory properties of the odorants, such as odor quality and odor detection threshold value, were determined with a trained panel. Structure-activity relationships are discussed, suggesting that the 1,3-oxygen-sulfur functionality, required for the "olfactophore" of tropical/vegetable notes, can further be extended to the acetylthio derivatives.

Aldehydes↗

PubMed2004

Identification and synthesis of 2-heptanethiol, a new flavor compound found in bell peppers.

2-Heptanethiol was identified for the first time as a constituent of red and green bell pepper extracts. The chemical structure of this new aroma compound was proposed on the basis of mass spectra and retention indices and confirmed by chemical synthesis and nuclear magnetic resonance spectroscopy measurements. Its aroma properties were described as sulfury, onion-like, and vegetable-like, reminiscent of bell pepper at lower concentrations, with an orthonasal detection threshold of 10 microg/L of water. No differences in odor note and threshold value were observed for the enantiomeric forms, which were prepared from enantiopure 2-heptanol by tosylation, followed by thioacetylation and reduction, giving the target thiol enantiomers.

Capsicum↗

PubMed2003

Simultaneous quantitative analysis of maillard reaction precursors and products by high-performance anion exchange chromatography.

A new analytical setup allowing the simultaneous analysis of precursors and products of the Maillard reaction is described. It is based on high-performance anion exchange chromatography with electrochemical (ECD) and diode array detectors (DAD) coupled in series. Chromatography and detection were optimized to permit simultaneous monitoring of compounds relevant to the Maillard reaction, such as the sugar, the amino acid, and the corresponding Amadori compound as well as the cyclic intermediates 5-(hydroxymethyl)-2-furaldehyde, maltol, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one. Separation was achieved on a CarboPac PA-1 column using a gradient of sodium acetate in aqueous sodium hydroxide. The Amadori compound, glucose, and glycine were monitored by an ECD operating in the integrated amperometry mode. The number of analyzed compounds was further increased by coupling the ECD with a DAD for the analysis of ultraviolet-active constituents. This method was successfully applied to model Maillard reaction mixtures based on glucose and glycine.

Anions↗

PubMed2003

Synthesis and sensory characterization of novel umami-tasting glutamate glycoconjugates.

Two glycoconjugates of glutamic acid, namely, the N-glycoside dipotassium N-(d-glucos-1-yl)-l-glutamate (1) and the corresponding Amadori compound N-(1-deoxy-d-fructos-1-yl)-l-glutamic acid (2), have been synthesized in yields of 35 and 52%, respectively, using new Maillard-mimetic approaches, and their chemical structures have unequivocally been elucidated by 1D- and 2D-NMR and MS experiments. Systematic sensory studies revealed that both glycoconjugates exhibit pronounced umami-like taste with recognition taste thresholds of 1-2 mmol/L, close to that of monosodium glutamate (MSG). Contrary to an aqueous solution of MSG, 1 does not show the sweetish and slightly soapy by-note, but evokes an intense umami taste. Aqueous solutions of 2 were described by the descriptors umami, seasoning, and bouillon-like. Added to a bouillon base, which did not contain any taste enhancers, both glycoconjugates imparted a distinct umami character similar to the control sample containing the same amount of MSG on a molar basis. To the best of our knowledge, these types of glycoconjugates in general and, in particular, N-glucosyl glutamate and N-deoxyfructosyl glutamate have never been reported as taste active compounds having umami-like properties. Therefore, 1 and 2 represent a new class of umami-type taste compounds showing properties similar to the umami reference compound MSG. Systematic (13)C NMR measurements revealed that 1 was fairly stable in aqueous solutions under alkaline conditions (pH 8-10) as well as in dry form. However, it rapidly hydrolyzes in neutral and acidic solutions, giving rise to glucose and glutamate. In contrast, glycoconjugate 2 was observed to be rather stable in aqueous solution as well as in the presence of human saliva.

Flavoring Agents↗

PubMed2003

Racemic and enantiopure synthesis and physicochemical characterization of the novel taste enhancer N-(1-carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol inner salt.

Convenient syntheses were developed to obtain on a multigram scale the novel taste enhancer N-(1-carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol 1, called alapyridaine, as a racemic mixture and as pure (+)-(S) and (-)-(R) enantiomers, respectively. 5-(Hydroxymethyl)-2-furaldehyde was used as key intermediate and was reacted with l-alanine under alkaline conditions to obtain racemic 1. Alternatively, reductive amination of 5-(hydroxymethyl)-2-furaldehyde with Raney-Ni/hydrogen and l- or d-alanine followed by mild oxidation led to (+)-(S)-1 and (-)-(R)-1, respectively. Racemization was promoted under alkaline and boiling conditions via a carbanion, the formation of which was facilitated by the electron-withdrawing effect of the iminium cation and the resonance-stabilizing capacity of the pyridinium moiety. Under these conditions, 1 was obtained in a 1:1 mixture of the phenol (1) and phenolate (1-H) forms as shown by X-ray diffraction. Racemic 1 formed monoclinic crystals of high molecular organization in which the phenol-type (RS)-1, the phenolate-type (RS)-1-H, sodium cations, and ethanol molecules are present. The crystal structure of [Na(1)(1-H).(C(2)H(6)O)] shows one-dimensional mu(2)-bridging-oxygen polymers stabilized by a three-dimensional network of ionic, hydrogen bond, and pi-stacking interactions with channels occupied by solvent molecules.

Alanine↗

PubMed2003

Formation of odorants in Maillard model systems based on l-proline as affected by pH.

Formation of the odorants acetic acid, 4-hydroxy-2,5-dimethyl-3-(2H)-furanone (HDMF), 6-acetyl-1,2,3,4-tetrahydropyridine (ATHP), and 2-acetyl-1-pyrroline (AP) was monitored by isotope dilution assays at pH 6, 7, and 8 in Maillard model reactions containing glucose and proline (Glc/Pro) or the corresponding Amadori compound fructosyl-proline (Fru-Pro). In general, higher yields were obtained at pH 7 and 8. Acetic acid was the major odorant with up to 40 mg/mmol precursor followed by HDMF (up to 0.25 mg/mmol), the formation of which was favored in the Fru-Pro reaction systems. On the contrary, ATHP (up to 50 microg/mmol) and AP (up to 5 microg/mmol) were more abundant in Glc/Pro. However, the sensory relevance of the two N-heterocycles was more pronounced on the basis of odor activity values, confirming their contribution to the overall roasty note of the reaction samples. It was also found that formation and decomposition of Fru-Pro were faster at pH 7 as compared to pH 6, explaining in part the preferred formation of the four odorants studied under neutral and slightly alkaline conditions. After 4 h of reaction at pH 7 in the presence of proline, about one-fourth of the glucose was consumed leading to acetic acid with a transformation yield of almost 40 mol %.

Fructose↗

PubMed2003

Potential of gas chromatography-orthogonal acceleration time-of-flight mass spectrometry (GC-oaTOFMS) in flavor research.

Gas chromatography-orthogonal acceleration time-of-flight mass spectrometry (GC-oaTOFMS) is an emerging technique offering a straightforward access to a resolving power up to 7000. This paper deals with the use of GC-oaTOFMS to identify the flavor components of a complex seafood flavor extract and to quantify furanones formed in model Maillard reactions. A seafood extract was selected as a representative example for complex food flavors and was previously analyzed using GC-quadrupole MS, leaving several molecules unidentified. GC-oaTOFMS analysis was focused on these unknowns to evaluate its potential in flavor research, particularly for determining exact masses. N-Methyldithiodimethylamine, 6-methyl-5-hepten-2-one, and tetrahydro-2,4-dimethyl-4H-pyrrolo[2,1-d]-1,3,5-dithiazine were successfully identified on the basis of the precise mass determination of their molecular ions and their major fragments. A second set of experiments was performed to test the capabilities of the GC-oaTOFMS for quantification. Calibration curves were found to be linear over a dynamic range of 10(3) for the quantification of furanones. The quantitative data obtained using GC-oaTOFMS confirmed earlier results that the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone was favored in the xylose/glycine model reaction and 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone in the xylose/alanine model reaction. It was concluded that GC-oaTOFMS may become a powerful analytical tool for the flavor chemist for both identification and quantification purposes, the latter in particular when combined with stable isotope dilution assay.

Animals↗