Karine Redeuil

Specific Dietary Preferences Are Linked to Differing Gut Microbial Metabolic Activity in Response to Dark Chocolate Intake

Systems biology approaches are providing novel insights into the role of nutrition for the management of health and disease. In the present study, we investigated if dietary preference for dark chocolate in healthy subjects may lead to different metabolic response to daily chocolate consumption. Using NMR- and MS-based metabolic profiling of blood plasma and urine, we monitored the metabolic response of 10 participants stratified as chocolate desiring and eating regularly dark chocolate (CD) and 10 participants stratified as chocolate indifferent and eating rarely dark chocolate (CI) to a daily consumption of 50 g of dark chocolate as part of a standardized diet over a one week period. We demonstrated that preference for chocolate leads to different metabolic response to chocolate consumption. Daily intake of dark chocolate significantly increased HDL cholesterol by 6% and decreased polyunsaturated acyl ether phospholipids. Dark chocolate intake could also induce an improvement in the metabolism of long chain fatty acid, as noted by a compositional change in plasma fatty acyl carnitines. Moreover, a relationship between regular long-term dietary exposure to a small amount of dark chocolate, gut microbiota, and phenolics was highlighted, providing novel insights into biological processes associated with cocoa bioactives.

First identification of dimethoxycinnamic acids in human plasma after coffee intake by liquid chromatography-mass spectrometry.

There is a substantial amount of published literature on the bioavailability of various coffee components including the most abundant metabolites, caffeic and ferulic acids. Surprisingly, to date, the appearance of dimethoxycinnamic acid derivatives in humans has not been reported despite the fact that methylated form of catechol-type polyphenols could help maintain, modify or even improve their biological activities. This study reports an LC-MS method for the detection of dimethoxycinnamic acid in human plasma after treatment with an esterase. Liquid chromatography, including the combination of methanol and acetonitrile as organic eluent, was optimized to resolve all interferences and enable reliable detection and identification of 3,4-dimethoxycinnamic and 3,4-dimethoxy-dihydrocinnamic acids. In addition to the good mass accuracy achieved (better than 5 ppm), tandem mass spectrometric and co-chromatography experiments further confirmed the identity of the compounds. The optimized method was applied to analyze samples obtained immediately, 1 and 10 h after coffee ingestion. The results show that in particular 3,4-dimethoxycinnamic acid appears in high abundance (∼380 nM at 60 min) in plasma upon coffee intake, indicating that it is important to consider these derivatives in future bioavailability and bioefficacy studies.

Quantification of Anthocyanins and Flavonols in Milk-Based Food Products by Ultra Performance Liquid Chromatography―Tandem Mass Spectrometry

The present article describes the development and validation of an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the comprehensive quantification of anthocyanin and flavonol constituents of milk-based food products. Protein precipitation by acidified methanol and ultrafiltration was utilized as sample preparation to preserve overall polyphenol composition but to eliminate milk proteins in order to comply with UPLC. Reversed-phase chromatography was optimized to achieve separation of 27 analytes in 10 min in order to reduce suppression effects, achieve a wide dynamic range, and most importantly, to resolve isomeric compounds. Positive-ion electrospray mass spectrometric detection and fragmentation of analytes was optimized, final transitions were selected for maximized selectivity, reliable quantification, and reduction of false positives. The quantitative performance of the method was validated, the main features include (1) range of lower limits of detection 0.3-30 ng/mL for glycosylated analytes, 10-300 ng/mL for aglycones, (2) lower limits of quantification 1-100 ng/mL for glycosylated analytes, 30-1,000 ng/mL for aglycones, (3) averaged intraday precision 9%, (4) calibrated range 2-180,000 ng/mL for glycosylated analytes, 60-600,000 ng/mL for aglycones, and (5) averaged accuracy 101%. Applications for yogurt and ice cream products are given. The presented data suggest that this method will help to better characterize the polyphenol composition of milk-based food products for quality control, for assessment of dietary intake, and for polyphenol bioavailability/bioefficacy studies.

Quantification of alkylresorcinols in human plasma by liquid chromatography/tandem mass spectrometry

Alkylresorcinols (AR) are of interest as biomarkers of wholegrain wheat and rye intake in epidemiological studies and are currently mainly measured by gas chromatography/mass spectrometry (GC/MS) after labour-intensive sample preparation including liquid-liquid extraction, solid-phase extraction (SPE) and chemical derivatization. This manuscript describes and validates an alternative approach based on normal-phase liquid chromatography/tandem mass spectrometry for the quantification of alkylresorcinols in human plasma. The method requires neither SPE nor chemical derivatization and has a shortened run time compared to GC/MS. Normal- and reversed-phase columns and various mobile phases were evaluated with and without previous SPE of the samples. Normal-phase chromatography allowed separation of AR from the interfering triacylglycerols, diacylglycerols and sterols and enabled detection of AR even without SPE of the samples. The described method has instrumental lower limits of detection in the 25-75 pg range, and lower limits of quantification in the 75-250 pg range. Pooled human plasma and (2)H(4)-nonadecylresorcinol (internal standard) was applied to calibrate the method in the 20-12 000 nM range. The overall method showed intra-batch precision of 8.6% and an averaged accuracy of 100.2%. Applications for diverse human plasma samples are presented and are compared with the results determined by GC/MS. Based on the presented data; this method requiring less sample preparation is suggested for further evaluation as an alternative to GC/MS for analysis of biomarkers of wholegrain wheat and rye intake in epidemiological studies.

Quantification of flavan-3-ols and phenolic acids in milk-based food products by reversed-phase liquid chromatography-tandem mass spectrometry

This article reports the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the comprehensive quantification of flavan-3-ol and phenolic acid constituents of milk-based food products. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with the stable isotope labelled internal standards and ultrafiltration to preserve overall polyphenol composition, but to eliminate milk proteins in order to comply with LC. Reversed-phase liquid chromatography was optimized to achieve separation of 22 analytes in 8 min in order to reduce suppression effects, achieve a wide dynamic range and, most importantly, to resolve isomeric compounds. Negative-ion electrospray mass spectrometric detection and fragmentation of analytes was optimized, final transitions were selected for maximized selectivity, reliable quantification and reduction of false positives. The quantitative performance of the method was validated, the main features include: (1) range of lower limits of detection 5-15 ng/mL for flavan-3-ols, 60-100 ng/mL for procyanidins, 1-60 ng/mL for other phenolic acids, (2) lower limits of quantification 15-45 ng/mL for flavan-3-ols, 200-300 ng/mL for procyanidins, 3-200 ng/mL for other phenolic acids, (3) averaged intra-day precision 9.5%, (4) calibrated range 60-300,000 ng/mL for flavan-3-ols, 900-900,000 ng/mL for procyanidins, 9-225,000 ng/mL for other phenolic acids, (5) averaged accuracy 99.5%. Applications for yoghurt and ice-cream products are given. The presented data suggest that this method will help to better characterize the polyphenol composition of milk-based food products for quality control, assessment of dietary intake and for polyphenol bioavailability/bioefficacy studies.

Double-balloon jejunal perfusion to compare absorption of vitamin E and vitamin E acetate in healthy volunteers under maldigestion conditions.

Background/Objectives:The vitamin E derivative, α-tocopheryl acetate, is often included in formulations used in enteral nutrition. In this respect, we compared α-tocopherol and α-tocopheryl acetate absorption under ‘maldigestion’ conditions, such as occurring during enteral tube feeding, using differentially labeled RRR-[5,7-methyl-(2H6)]-α-tocopherol and RRR-[5-methyl-2H3]-α-tocopheryl acetate allowing direct comparison between free and esterified forms.Subjects/Methods:The two derivatives were given together in a single dose to six volunteers directly into the jejunum using a double-balloon perfusion system. Perfusion lasted for 1 h, and the collected blood and effluent samples were analyzed by liquid chromatography–mass spectrometry.Results:In the isolated 20-cm length of exposed jejunum, on average ∼6% of the two vitamin E forms were absorbed >1 h based on subtraction of effluent from influent. There was substantial difference in the absolute absorbed quantity between individuals, but no significant differences were observed in the absorption between the two labeled forms as assessed in the plasma. 2H3-α-tocopherol was not present in the influent, but appeared in the effluent, indicating that the acetylated form of vitamin E is cleaved by brush border enzymes in the small intestine.Conclusions:This study shows that even in the absence of digestive enzymes and bile salts, the appropriately solubilized acetylated form of α-tocopherol exhibits the same bioavailability as free α-tocopherol. This suggests that both forms can be absorbed equally under maldigestion conditions such as present clinically during enteral tube feeding.

Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry

This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached <0.1-10nM and 0.2-25nM, respectively. Commercially available pooled human plasma was used to build matrix-matched calibration curves ranging 2-500, 5-1250, 20-5000 or 150-37500nM depending on the analyte. The overall performance of the method was considered adequate, with 2.8-20.9% and 5.2-20.0% intra and inter-day precision, respectively and averaged accuracy reaching 91-108%. Recovery experiments were also performed and reached in average 82%. This analytical approach was then applied for the quantification of circulating water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans.

Clinical and Vitamin Response to a Short-Term Multi-Micronutrient Intervention in Brazilian Children and Teens: From Population Data to Interindividual Responses

Scope Micronutrients are in small amounts in foods, act in concert, and require variable amounts of time to see changes in health and risk for disease. These first principles are incorporated into an intervention study designed to develop new experimental strategies for setting target recommendations for food bioactives for populations and individuals. Methods and results A 6‐week multivitamin/mineral intervention is conducted in 9–13 year olds. Participants (136) are (i) their own control (n‐of‐1); (ii) monitored for compliance; (iii) measured for 36 circulating vitamin forms, 30 clinical, anthropometric, and food intake parameters at baseline, post intervention, and following a 6‐week washout; and (iv) had their ancestry accounted for as modifier of vitamin baseline or response. The same intervention is repeated the following year (135 participants). Most vitamins respond positively and many clinical parameters change in directions consistent with improved metabolic health to the intervention. Baseline levels of any metabolite predict its own response to the intervention. Elastic net penalized regression models are identified, and significantly predict response to intervention on the basis of multiple vitamin/clinical baseline measures. Conclusions The study design, computational methods, and results are a step toward developing recommendations for optimizing vitamin levels and health parameters for individuals.

Concentrations of Carotenoids and Tocopherols in Breast Milk from Urban Chinese Mothers and their Associations with Maternal Characteristics：A Cross-Sectional Study

Milk composition remains the best estimate of infant requirements. The aims of this study were to quantify carotenoids and tocopherols in human milk from healthy Chinese mothers, and to explore their associations with lactation stage, region, socio-economic and obstetric characteristics, and dietary intake. Human milk was obtained from 509 healthy mothers, and concentrations of carotenoids and tocopherols were analyzed by Ultra High Performance Liquid Chromatography. The mothers’ socio-economic and obstetric characteristics and dietary intake through a single 24-h dietary recall were evaluated. The median concentrations (μg/100 mL) of each component of 0–4 days, 5–11 days, 12–30 days, 31–60 days, 61–120 days, and 121–240 days postpartum were respectively as follows: β-carotene 8.0, 2.8, 2.1, 1.7, 1.9, 1.8; β-cryptoxanthin 6.2, 3.4, 2.4, 1.7, 1.8, 2.1; lutein 5.7, 7.0, 2.2, 2.9, 2.8, 3.7; lycopene 6.3, 2.5, 1.8, 1.4, 1.4, 1.5; zeaxanthin 1.0, 1.4, 0.8, 0.8, 1.0, 1.1; α-tocopherol 645, 382, 239, 206, 212, 211; γ-tocopherol 68, 63, 70, 73, 68, 88. The levels of those components varied significantly among different lactation stages and presented regional differences. Associations of carotenoid contents with maternal education, delivery mode, and present body mass index were found in multivariate analyses. These results suggested that lactation stage, region, and socio-economic and obstetric factors were associated with human milk concentrations of carotenoids and tocopherols in healthy Chinese mothers.

A Novel Methodology for the Quantification of B-Vitamers in Breast Milk

With this report we present development, validation and application of an analytical methodology for the quantification of 18 water soluble vitamers and secreted or biological forms in breast milk. On a relatively low amount of breast milk (200 µL), we applied isotope dilution-based sample preparation based on a combination of enzymatic treatment and protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Compounds separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. To perform the quantification of 18 water soluble vitamers, procured pooled breast milk was used to build matrixmatched calibration curves, as labelled internal standards were not available for each vitamer. The analytical approach has been validated according to the EMA guidelines. The overall performance of the method was considered adequate, with 0.3- 28.3% and 0.9-32.6% intra and inter-day precision respectively and averaged accuracy reaching 92.2-107.5%. In addition, performed freeze/thaw stability studies showed the potential degradation of some vitamers. We therefore recommend particular attention in sample collection with rather having dedicated aliquots with small volumes. The feasibility of this analytical approach has been evaluated by quantifying various breast milk samples that were procured from an external supplier. The main forms found in breast milk were thiamine monophosphate for B1, flavin adenine nucleotide for B2, nicotinamide for B3, pyridoxal for B6 and 5-methyl tetrahydrofolic acid for B9. In addition, we newly reported nudifloramide as B3 form present in breast milk. With this analytical approach, it will give more confidence to provide a comprehensive assessment of the presence of water soluble vitamins in breast milk. This will enable the accurate evaluation of the nutritional requirements of infants.