Diana Bunzel

Stability of Individual Maillard Reaction Products in the Presence of the Human Colonic Microbiota

Maillard reaction products (MRPs) are taken up in substantial amounts with the daily diet, but the majority are not transported across the intestinal epithelium. The aim of this study was to obtain first insights into the stability of dietary MRPs in the presence of the intestinal microbiota. Four individual MRPs, namely, N-ε-fructosyllysine (FL), N-ε-carboxymethyllysine (CML), pyrraline (PYR), and maltosine (MAL), were anaerobically incubated with fecal suspensions from eight human volunteers at 37 °C for up to 72 h. The stability of the MRPs was measured by HPLC with UV and MS/MS detections. The Amadori product FL could no longer be detected after 4 h of incubation. Marked interindividual differences were observed for CML metabolism: Depending on the individual, at least 40.7 ± 1.5% of CML was degraded after 24 h of incubation, and the subjects could thus be tentatively grouped into fast and slow metabolizers of this compound. PYR was degraded by 20.3 ± 4.4% during 24 h by all subjects. The concentration of MAL was not significantly lowered in the presence of fecal suspensions. In no case could metabolites be identified and quantified by different mass spectrometric techniques. This is the first study showing that the human colonic microbiota is able to degrade selected glycated amino acids and possibly use them as a source of energy, carbon, and/or nitrogen.

Tocopherol and tocotrienol analysis in raw and cooked vegetables: A validated method with emphasis on sample preparation

Vegetables can be important dietary sources of vitamin E. However, data on vitamin E in raw and cooked vegetables are in part conflicting, indicating analytical pitfalls. The purpose of the study was to develop and validate an HPLC-FLD method for tocochromanol (tocopherols and tocotrienols) analysis equally suitable for raw and cooked vegetables. Significant instability of tocochromanols was observed in raw broccoli and carrot homogenates. Tocochromanols could be stabilized by freeze-drying or ascorbic acid addition prior to homogenization. The optimized protocol for tocochromanol analysis included knife and ball milling of freeze-dried vegetable pieces. Direct acetone extraction of vegetable powders allowed for satisfactory recoveries and precisions. A significant decrease of tocochromanols in baked compared to raw vegetables was shown, the extent of which varied largely between vegetables. For some raw vegetables, such as spinach or broccoli, underestimation of vitamin E in nutrient databases cannot be ruled out and should be examined.

Metabolite patterns predicting sex and age in participants of the Karlsruhe Metabolomics and Nutrition (KarMeN) study

Physiological and functional parameters, such as body composition, or physical fitness are known to differ between men and women and to change with age. The goal of this study was to investigate how sex and age-related physiological conditions are reflected in the metabolome of healthy humans and whether sex and age can be predicted based on the plasma and urine metabolite profiles. In the cross-sectional KarMeN (Karlsruhe Metabolomics and Nutrition) study 301 healthy men and women aged 18–80 years were recruited. Participants were characterized in detail applying standard operating procedures for all measurements including anthropometric, clinical, and functional parameters. Fasting blood and 24 h urine samples were analyzed by targeted and untargeted metabolomics approaches, namely by mass spectrometry coupled to one- or comprehensive two-dimensional gas chromatography or liquid chromatography, and by nuclear magnetic resonance spectroscopy. This yielded in total more than 400 analytes in plasma and over 500 analytes in urine. Predictive modelling was applied on the metabolomics data set using different machine learning algorithms. Based on metabolite profiles from urine and plasma, it was possible to identify metabolite patterns which classify participants according to sex with > 90% accuracy. Plasma metabolites important for the correct classification included creatinine, branched-chain amino acids, and sarcosine. Prediction of age was also possible based on metabolite profiles for men and women, separately. Several metabolites important for this prediction could be identified including choline in plasma and sedoheptulose in urine. For women, classification according to their menopausal status was possible from metabolome data with > 80% accuracy. The metabolite profile of human urine and plasma allows the prediction of sex and age with high accuracy, which means that sex and age are associated with a discriminatory metabolite signature in healthy humans and therefore should always be considered in metabolomics studies.

Structural Transformation of 8–5-Coupled Dehydrodiferulates by Human Intestinal Microbiota

Ingested dehydrodiferulates (DFAs) are partially released from cereal dietary fiber by human colonic microbiota, but little research has explored the further microbial metabolism of 8-5-coupled DFAs. This study investigated the in vitro microbial metabolism and elucidated major metabolites of free 8-5-DFAs (benzofuran and open forms) and an esterified analogue, 8-5-DFA diethyl ester (benzofuran). Synthesized standard compounds were incubated with fresh human fecal suspensions. Metabolites were isolated and structurally elucidated using high-resolution-LC-time-of-flight-(ToF)-MS, GC-MS, and NMR. Nine metabolite structures were unambiguously characterized with NMR, and four additional metabolites were tentatively identified to reveal structural conversion motifs: propenyl side chain hydrogenation (all substrates), O-demethylation and reductive ring-opening (8-5-DFA diethyl ester and free 8-5-DFA [benzofuran]), and de-esterification (8-5-DFA diethyl ester). A pathway of microbial 8-5-DFA metabolism was proposed based on metabolite formation kinetics. Importantly, de-esterification of the 8-5-DFA diethyl ester occurred primarily after and/or concurrently with other metabolism steps. Cleavage to monomers was not observed.

Formation of phosphoglycosides in Caenorhabditis elegans: a novel biotransformation pathway.

Background Caenorhabditis elegans (C. elegans) has become a widely used model to explore the effect of food constituents on health as well as on life-span extension. The results imply that besides essential nutrients several flavonoids are able to impact the aging process. What is less investigated is the bioavailability and biotransformation of these compounds in C. elegans. In the present study, we focused on the soy isoflavone genistein and its metabolism in the nematode as a basis for assessing whether this model system mimics the mammalian condition. Principal Findings C. elegans was exposed to 100 µM genistein for 48 hours. The worm homogenate was extracted and analyzed by liquid chromatography (LC). 11 metabolites of genistein were detected and characterized using LC electrospray ionization mass spectrometry. All genistein metabolites formed by C. elegans were found to be sugar conjugates, primarily genistein-O-glucosides. The dominant metabolite was identified as genistein-7-O-phosphoglucoside. Further interesting metabolites include two genistein-di-O-glycosides, a genistein-O-disaccharide as well as a genistein-O-phosphodisaccharide. Conclusions/Significance Our study provides evidence for a novel biotransformation pathway in C. elegans leading to conjugative metabolites which are not known for mammals. The metabolism of genistein in mammals and in C. elegans differs widely which may greatly impact the bioactivity. These differences need to be appropriately taken into consideration when C. elegans is used as a model to assess possible health or aging effects.

Identification of 8-O-4/8-5(Cyclic)- and 8-8(Cyclic)/5-5-Coupled Dehydrotriferulic Acids, Naturally Occurring in Cell Walls of Mono- and Dicotyledonous Plants.

Besides ferulate dimers, higher oligomers of ferulic acid such as trimers and tetramers were previously demonstrated to occur in plant cell walls. This paper reports the identification of two new triferulic acids. 8-O-4/8-5(cyclic)-triferulic acid was synthesized from ethyl ferulate under oxidative conditions using copper(II)-tetramethylethylenediamine [CuCl(OH)-TMEDA] as a catalyst, whereas 8-8(cyclic)/5-5-triferulic acid was isolated (preparative size exclusion chromatography, reversed-phase HPLC) from saponified insoluble maize fiber. Structures of both trimers were unambiguously elucidated by high-resolution LC-ToF-MS/MS and one- ((1)H) and two-dimensional (HSQC, HMBC, COSY, NOESY) NMR spectroscopy. The newly described trimers were identified by LC-MS/MS in alkaline hydrolysates of insoluble fibers from maize, wheat, and sugar beet, indicating that ferulic acid cross-links between cell wall polymers are more diverse than previously recognized. Saponification experiments also suggest that the newly identified 8-O-4/8-5(cyclic)-triferulic acid is the naturally occurring precursor of the previously identified 8-O-4/8-5(noncyclic)-triferulic acid in plant cell walls.

Alternaria toxins of the alternariol type are not metabolised by human faecal microbiota

The metabolism of the Alternaria toxins alternariol (AOH), alternariol-9-O-methyl ether (AME) and altenuene (ALT) by the microbiota present in faeces from three human volunteers was studied. Faecal cultures were prepared as a 5% faeces suspension in brain-heart infusion broth and incubated with 50 μM of the toxins under anaerobic conditions for 72 h at 37 °C. The metabolism of AOH was also studied in pure bacterial cultures with either Escherichia coli DH5α or Lactobacillus plantarum BFE 5092 for 72 h at 37 °C. The three parent toxins were stable in uninoculated, heat-treated medium over a 72 h incubation period with a recovery of more than 90%. As a control for the activity of the faecal microbiota, the isoflavone daidzein was incubated with the faecal cultures and was transformed to its expected metabolites. In contrast, no metabolites of AOH, AME and ALT could be detected in the faecal cultures from the same volunteers, indicating that the gut microbiota was not capable of metabolising these substances. The Alternaria toxins could be shown to be at least partially bound to bacterial cells in a non-covalent manner, which may serve as a mechanism for their removal from the gut.

Metabolization of Maillard reaction products by the human colonic microbiota

Objectives: Heat-treatment of foods leads to glycation of individual protein-bound amino acids such as lysine and arginine. Substantial amounts of up to 1000 mg of Amadori compounds (mainly fructoselysine) and up to 75 mg of advanced glycation end products (AGEs, mainly pyrraline and carboxymethyllysine) are ingested with the daily diet [4]. The majority of these products can not pass the intestinal barrier and enter into circulation and could therefore serve as a substrate for bacterial fermentation [2]. Degradation of fructoselysine has already been shown in rat feces cultures [1]. Methodology: Four MRPs (fructoselysine, carboxymethyllysine, pyrraline, maltosine) were synthesized and anaerobically incubated in the presence of fecal suspensions from 8 human volunteers at 37 °C for up to 72 h. The stability of the compounds was analyzed by HPLC-MS/MS (fructoselysine, CML) or HPLC-UV (pyrraline, maltosine) [3]. Results: Fructoselysine was degraded nearly completely in the presence of fecal suspensions of all subjects during 4 h, whereas maltosine proved to be completely stable. Pyrraline was degraded by 25-40% in 72 h by the gut microbiota of all subjects. Strong inter-individual differences in the kinetic and extent of degradation appeared when CML was incubated. However, no metabolites (e.g., biogenic amines) could as yet be identified. Conclusions: The human large intestinal microbiota is able to degrade individual MRPs. Especially Amadori products such as fructoselysine, which are ingested in comparatively high amounts, can thus possibly have an influence on the composition and viability of the gut microbiota. Further studies will focus on identification of the degrading species and in-depth metabolite analysis.