Unravelling mechanisms of protein and lipid oxidation in mayonnaise at multiple length scales

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Adapting cryogenic correlative light and electron microscopy (cryo-CLEM) for food oxidation studies

Lipid oxidation is a major cause of product deterioration in protein stabilised oil-in-water food emulsions. The impact of protein emulsifiers on lipid oxidation and the stability depends on the specific type of protein emulsifiers used and the redox conditions in the emulsion. However, the exact impact of these protein emulsifiers at the oil-water interface on lipid oxidation and the mechanism of lipid-protein co-oxidation are currently unknown. Here, we developed a cryo-correlative light and electron microscopy (cryo-CLEM) platform for co-localising the oxidation of lipids and proteins. For this first implementation of cryo-CLEM for food oxidation studies we optimised specifically the part of cryo-fluorescence microscopy (cryo-FM) by adding parts that prevent fogging on the sample and enable homogeneous laser illumination. We showed that lipid oxidation in food emulsions can be observed at cryogenic temperature using fluorescence imaging of the fluorophore BODIPY 665/676 that we employed earlier as a lipid oxidation sensor at room temperature. Using cryo-transmission electron microscopy (cryo-TEM), we observed that more protein aggregates are found at the droplet interfaces in oxidized emulsions compared to fresh emulsions. Our cryo-CLEM platform paves the way for future cryo-correlative oxidation studies of food emulsions.</p

Mitosene-DNA adducts. Characterization of two major DNA monoadducts formed by 1,10-bis(acetoxy)-7-methoxymitosene upon reductive activation

Reductive activation of racemic 1,10-bis(acetoxy)-7-methoxymitosene WV15 in the presence of DNA, followed by enzymatic digestion and HPLC analysis, revealed the formation of various DNA adducts. Reduction is a necessary event for adduct formation to occur. This reductive activation was performed under hypoxic conditions in various ways:  (1) chemically, using a 2-fold excess of sodium dithionite (Na2S2O4), (2) enzymatically using NADH-cytochrome c reductase, (3) electrochemically on a mercury pool working electrode, and (4) catalytically, using a H2/PtO2 system. Five different mitosene−DNA adducts were detected. These adducts were also present when poly(dG-dC) was used instead of DNA, but were absent with poly(dA-dT). All were shown to be adducts of guanine. Reduction of 1,10-dihydroxymitosene WV14 in the presence of DNA did not result in detectable adduct formation, demonstrating the importance of good leaving groups for efficient adduct formation by these mitosenes. Finally, two of the adducts were isolated and their structures elucidated, using mass spectrometry, 1H NMR and circular dichroism (CD). The structures were assigned as the diastereoisomers N2-(1‘ ‘-n-hydroxymitosen-10‘ ‘-yl), 2‘-deoxyguanosine (n = α or β). These type of adducts, in which the mitosene C-10 is covalently bonded to the N-2 of a guanosylic group, are different from the well-known mitomycin C 2‘-deoxyguanosine monoadducts, that is linked via the mitomycin C C-1 position, demonstrating that the order of reactivity of the C-1 and C-10 in these mitosenes is reversed, as compared to mitomycin C. The 7-methoxy substituent of WV15 is a likely factor causing this switch. Evidence is presented that the 7-substituent of mitosenes also influences their DNA alkylation site. Adducts 4 and 5 represent the first isolated and structurally characterized covalent adducts of DNA and a synthetic mitosene

Assessment of dietary exposure and effect in humans : The role of NMR

In human nutritional science progress has always depended strongly on analytical measurements for establishing relationships between diet and health. This field has undergone significant changes as a result of the development of NMR and mass spectrometry methods for large scale detection, identification and quantification of metabolites in body fluids. This has allowed systematic studies of the metabolic fingerprints that biological processes leave behind, and has become the research field of metabolomics. As a metabolic profiling technique, NMR is at its best when its unbiased nature, linearity and reproducibility are exploited in well-controlled nutritional intervention and cross-sectional population screening studies. Although its sensitivity is less good than that of mass spectrometry, NMR has maintained a strong position in metabolomics through implementation of standardisation protocols, hyphenation with mass spectrometry and chromatographic techniques, accurate quantification and spectral deconvolution approaches, and high-throughput automation. Thus, NMR-based metabolomics has contributed uniquely to new insights into dietary exposure, in particular by unravelling the metabolic fates of phytochemicals and the discovery of dietary intake markers. NMR profiling has also contributed to the understanding of the subtle effects of diet on central metabolism and lipoprotein metabolism. In order to hold its ground in nutritional metabolomics, NMR will need to step up its performance in sensitivity and resolution; the most promising routes forward are the analytical use of dynamic nuclear polarisation and developments in microcoil construction and automated fractionation

Solid-state structure, dynamical properties in solution and computational studies of a new sodium hemispherand complex

The solid-state structure of 1·NaClO4 has been determined by X-ray diffraction and shows the Na+ complexed in an approximate hexagonal bipyrimidal fashion. The six ether oxygens form the ground plane, the inner carbonyl group and one of the two outer carbonyl groups occupy the apical positions. The solution structure in methanol, as determined by NMR spectroscopy, has a time-averaged plane of symmetry through the inner phenyl ring. This plane of symmetry is the result of a fast interconversion of conformations in which either one of the outer carbonyl groups is coordinated to the Na+. The enthalpy of activation in methanol determined by T1 measurements is 5 ± 1 kcal mol–1. This process of fast exchange was supported by TRAVEL/CHARMm simulations which revealed a transition-state structure with the two outer carbonyl groups coordinated to the Na+ with a plane of symmetry through the inner phenyl ring. The calculated activation energy is 6.1 kcal mol–1, in very good agreement with the experimental value. A significant influence of the solvent on the structure of 1·Na+ could be ruled out by an MD simulation in methanol. The structure is very similar to the solid-state structure. \u

Solubilization of NaX salts in chloroform by bifunctional receptors

First the cation, then the anion. This describes the complexation of alkali metal halides with bifunctional receptors like that on the right (R = n‐octyl) based on calix[4]arenes. The uncomplexed ligand has a strong intramolecular hydrogen bond between the urea moieties, which is disrupted by the conformational change induced by complexation of a Na+ ion. Only then is an anion like Cl− bound in the upper portion of the calixarene

Rapid Quantitative Profiling of Lipid Oxidation Products in a Food Emulsion by 1H NMR

Lipid oxidation is one of the most important reasons for the compromised shelf life of food emulsions. A major bottleneck in unravelling the underlying mechanisms is the lack of methods that provide a rapid, quantitative, and comprehensive molecular view on lipid oxidation in these heterogeneous systems. In this study, the unbiased and quantitative nature of 1H NMR was exploited to assess lipid oxidation products in mayonnaise, a particularly oxidation-prone food emulsion. An efficient and robust procedure was implemented to produce samples where the 1H NMR signals of oxidation products could be observed in a well resolved and reproducible manner. 1H NMR signals of hydroperoxides were assigned in a fatty acid and isomer specific way. Band-selective 1H NMR pulse excitation allowed immediate and precise (RSDR = 5.9%) quantification of both hydroperoxides and aldehydes with high throughput and large dynamic range at levels of 0.03 mmol/kg. Explorative multivariate data modeling of the quantitative 1H NMR profiles revealed that shelf life temperature has a significant impact on lipid oxidation mechanisms