Impact of wheat aleurone on biomarkers of cardiovascular disease, gut microbiota and metabolites in adults with high body mass index: a double-blind, placebo-controlled, randomized clinical trial.

PURPOSE: Aleurone is a cereal bran fraction containing a variety of beneficial nutrients including polyphenols, fibers, minerals and vitamins. Animal and human studies support the beneficial role of aleurone consumption in reducing cardiovascular disease (CVD) risk. Gut microbiota fiber fermentation, polyphenol metabolism and betaine/choline metabolism may in part contribute to the physiological effects of aleurone. As primary objective, this study evaluated whether wheat aleurone supplemented foods could modify plasma homocysteine. Secondary objectives included changes in CVD biomarkers, fecal microbiota composition and plasma/urine metabolite profiles. METHODS: A parallel double-blind, placebo-controlled and randomized trial was carried out in two groups of obese/overweight subjects, matched for age, BMI and gender, consuming foods supplemented with either aleurone (27 g/day) (AL, n = 34) or cellulose (placebo treatment, PL, n = 33) for 4 weeks. RESULTS: No significant changes in plasma homocysteine or other clinical markers were observed with either treatment. Dietary fiber intake increased after AL and PL, animal protein intake increased after PL treatment. We observed a significant increase in fecal Bifidobacterium spp with AL and Lactobacillus spp with both AL and PL, but overall fecal microbiota community structure changed little according to 16S rRNA metataxonomics. Metabolomics implicated microbial metabolism of aleurone polyphenols and revealed distinctive biomarkers of AL treatment, including alkylresorcinol, cinnamic, benzoic and ferulic acids, folic acid, fatty acids, benzoxazinoid and roasted aroma related metabolites. Correlation analysis highlighted bacterial genera potentially linked to urinary compounds derived from aleurone metabolism and clinical parameters. CONCLUSIONS: Aleurone has potential to modulate the gut microbial metabolic output and increase fecal bifidobacterial abundance. However, in this study, aleurone did not impact on plasma homocysteine or other CVD biomarkers. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov (NCT02067026) on the 17th February 2014.

New Advanced Glycation End Products Observed in Rat Urine by Untargeted Metabolomics after Feeding with Heat-Treated Skimmed Milk Powder.

SCOPE: Milk powder is commonly consumed throughout the world. However, advanced glycation end products (AGEs) will form in milk powder during thermal processing and long-term storage. This study aimed to identify such compounds with potential as new urinary biomarkers of intake of heat-treated skimmed milk powder (HSMP). METHODS AND RESULTS: A parallel study is performed with different dosages of HSMP as well as hydrolyzed HSMP and untreated skimmed milk powder (SMP) in 36 rats. The 24-h urine samples on day 7 or 8 are collected and profiled by untargeted UPLC-Qtof-MS metabolomics. Statistical analysis revealed 25 metabolites differentiating SMP and HSMP; nineteen of these structures are proposed as lysine- and arginine-derived AGEs, and heterocyclic compounds. CONCLUSION: These metabolites may potentially serve as biomarkers of food intake pending further validation to assess intakes of heat-processed dairy foods and thus help to elucidate the effects of HSMP consumption or dietary AGEs on human health.

Urine Metabolome Profiling Reveals Imprints of Food Heating Processes after Dietary Intervention with Differently Cooked Potatoes.

Heat treatment is a widely used method for food processing, and the compounds formed by heat processes may serve as biomarkers of heated food intake in nutrition studies. Therefore, we aimed to characterize the differential metabolic signatures resulting from intake of different potato products and identify potential intake biomarkers. In a randomized, controlled, crossover meal study, healthy volunteers consumed boiled rice, boiled potatoes, and two deep-fried potato products, potato chips and French fries. The urine metabolome was acquired by LC-MS-based untargeted metabolomics. Twenty-two selected metabolites were found for deep-fried potatoes, two for potato intake in general, and one for boiled rice. Fourteen of the 22 selected metabolites were tentatively identified as furan-, pyrrole- and pyrazine-derived compounds indicative of Maillard reactions. With further validation, these candidate biomarkers will be important tools to investigate the influence of heated foods on human health.

Two apples a day modulate human:microbiome co-metabolic processing of polyphenols, tyrosine and tryptophan.

PURPOSE: Validated biomarkers of food intake (BFIs) have recently been suggested as a useful tool to assess adherence to dietary guidelines or compliance in human dietary interventions. Although many new candidate biomarkers have emerged in the last decades for different foods from metabolic profiling studies, the number of comprehensively validated biomarkers of food intake is limited. Apples are among the most frequently consumed fruits and a rich source of polyphenols and fibers, an important mediator for their health-protective properties. METHODS: Using an untargeted metabolomics approach, we aimed to identify biomarkers of long-term apple intake and explore how apples impact on the human plasma and urine metabolite profiles. Forty mildly hypercholesterolemic volunteers consumed two whole apples or a sugar and energy-matched control beverage, daily for 8 weeks in a randomized, controlled, crossover intervention study. The metabolome in plasma and urine samples was analyzed via untargeted metabolomics. RESULTS: We found 61 urine and 9 plasma metabolites being statistically significant after the whole apple intake compared to the control beverage, including several polyphenol metabolites that could be used as BFIs. Furthermore, we identified several endogenous indole and phenylacetyl-glutamine microbial metabolites significantly increasing in urine after apple consumption. The multiomic dataset allowed exploration of the correlations between metabolites modulated significantly by the dietary intervention and fecal microbiota species at genus level, showing interesting interactions between Granulicatella genus and phenyl-acetic acid metabolites. Phloretin glucuronide and phloretin glucuronide sulfate appeared promising biomarkers of apple intake; however, robustness, reliability and stability data are needed for full BFI validation. CONCLUSION: The identified apple BFIs can be used in future studies to assess compliance and to explore their health effects after apple intake. Moreover, the identification of polyphenol microbial metabolites suggests that apple consumption mediates significant gut microbial metabolic activity which should be further explored.

Two apples a day lower serum cholesterol and improve cardiometabolic biomarkers in mildly hypercholesterolemic adults: a randomized, controlled, crossover trial.

BACKGROUND: Apples are rich in bioactive polyphenols and fiber. Evidence suggests that consumption of apples or their bioactive components is associated with beneficial effects on lipid metabolism and other markers of cardiovascular disease (CVD). However, adequately powered randomized controlled trials are necessary to confirm these data and explore the mechanisms. OBJECTIVE: We aimed to determine the effects of apple consumption on circulating lipids, vascular function, and other CVD risk markers. METHODS: The trial was a randomized, controlled, crossover, intervention study. Healthy mildly hypercholesterolemic volunteers (23 women, 17 men), with a mean ± SD BMI 25.3 ± 3.7 kg/m2 and age 51 ± 11 y, consumed 2 apples/d [Renetta Canada, rich in proanthocyanidins (PAs)] or a sugar- and energy-matched apple control beverage (CB) for 8 wk each, separated by a 4-wk washout period. Fasted blood was collected before and after each treatment. Serum lipids, glucose, insulin, bile acids, and endothelial and inflammation biomarkers were measured, in addition to microvascular reactivity, using laser Doppler imaging with iontophoresis, and arterial stiffness, using pulse wave analysis. RESULTS: Whole apple (WA) consumption decreased serum total (WA: 5.89 mmol/L; CB: 6.11 mmol/L; P = 0.006) and LDL cholesterol (WA: 3.72 mmol/L; CB: 3.86 mmol/L; P = 0.031), triacylglycerol (WA: 1.17 mmol/L; CB: 1.30 mmol/L; P = 0.021), and intercellular cell adhesion molecule-1 (WA: 153.9 ng/mL; CB: 159.4 ng/mL; P = 0.028), and increased serum uric acid (WA: 341.4 µmol/L; CB: 330 µmol/L; P = 0.020) compared with the CB. The response to endothelium-dependent microvascular vasodilation was greater after the apples [WA: 853 perfusion units (PU), CB: 760 PU; P = 0.037] than after the CB. Apples had no effect on blood pressure or other CVD markers. CONCLUSIONS: These data support beneficial hypocholesterolemic and vascular effects of the daily consumption of PA-rich apples by mildly hypercholesterolemic individuals.This trial was registered at clinicaltrials.gov as NCT01988389.

Discovery and Validation of Banana Intake Biomarkers Using Untargeted Metabolomics in Human Intervention and Cross-sectional Studies.

BACKGROUND: Banana is one of the most widely consumed fruits in the world. However, information regarding its health effects is scarce. Biomarkers of banana intake would allow a more accurate assessment of its consumption in nutrition studies. OBJECTIVES: Using an untargeted metabolomics approach, we aimed to identify the banana-derived metabolites present in urine after consumption, including new candidate biomarkers of banana intake. METHODS: A randomized controlled study with a crossover design was performed on 12 healthy subjects (6 men, 6 women, mean ± SD age: 30.0 ± 4.9 y; mean ± SD BMI: 22.5 ± 2.3 kg/m2). Subjects underwent 2 dietary interventions: 1) 250 mL control drink (Fresubin 2 kcal fiber, neutral flavor; Fresenius Kabi), and 2) 240 g banana + 150 mL control drink. Twenty-four-hour urine samples were collected and analyzed with ultra-performance liquid chromatography coupled to a quadrupole time-of-flight MS and 2-dimensional GC-MS. The discovered biomarkers were confirmed in a cross-sectional study [KarMeN (Karlsruhe Metabolomics and Nutrition study)] in which 78 subjects (mean BMI: 22.8; mean age: 47 y) were selected reflecting high intake (126-378 g/d), low intake (47.3-94.5 g/d), and nonconsumption of banana. The confirmed biomarkers were examined singly or in combinations, for established criteria of validation for biomarkers of food intake. RESULTS: We identified 33 potentially bioactive banana metabolites, of which 5 metabolites, methoxyeugenol glucuronide (MEUG-GLUC), dopamine sulfate (DOP-S), salsolinol sulfate, xanthurenic acid, and 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-ß-carboline sulfate, were confirmed as candidate intake biomarkers. We demonstrated that the combination of MEUG-GLUC and DOP-S performed best in predicting banana intake in high (AUCtest = 0.92) and low (AUCtest = 0.87) consumers. The new biomarkers met key criteria establishing their current applicability in nutrition and health research for assessing the occurrence of banana intake. CONCLUSIONS: Our metabolomics study in healthy men and women revealed new putative bioactive metabolites of banana and a combined biomarker of intake. These findings will help to better decipher the health effects of banana in future focused studies. This study was registered at clinicaltrials.gov as NCT03581955 and with the Ethical Committee for the Protection of Human Subjects Sud-Est 6 as CPP AU 1251, IDRCB 2016-A0013-48; the KarMeN study was registered with the German Clinical Trials Register (DRKS00004890). Details about the study can be obtained from https://www.drks.de.

Isotopic dilution method for bile acid profiling reveals new sulfate glycine-conjugated dihydroxy bile acids and glucuronide bile acids in serum.

An ultrahigh performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed for the determination of 41 target and 8 additional bile acids isomers (BAs) in biological fluids. BAs were analysed by solid-phase extraction on 50 µL biofluid-aliquots, followed by a properly optimised 27 min-chromatographic run. The method provided high sensitivity (limits of detection 0.0002-0.03 µM, limits of quantitation 0.0007-0.11 µM), linearity (R2>0.99) and precision (relative standard deviations ≤16%). A strategy of scheduled/ unscheduled injections of real samples together with neutral loss (80 Da and 176 Da) scans allowed us to find additional bile acid isomers not a priori included in the method, while high resolution full scan and MS/MS fragmentation analysis confirmed their structural adherence to the bile acid family. Moreover this is the first study quantifying four sulfate glycine conjugated-dihydroxy bile acid isomers, independently of the diet and postprandial time. Application to a dietary intervention kinetic study confirmed the existence of possible metabotypes amongst the study population (n = 20). A trend differentiating males from females was observed suggesting that serum samples from women contained smaller amounts of certain bile acids.

Host: Microbiome co-metabolic processing of dietary polyphenols - An acute, single blinded, cross-over study with different doses of apple polyphenols in healthy subjects.

Apples are one of the most commonly consumed fruits and their high polyphenol content is considered one of the most important determinants of their health-promoting activities. Here we studied the nutrikinetics of apple polyphenols by UHPLC-HRMS metabolite fingerprinting, comparing bioavailability when consumed in a natural or a polyphenol-enriched cloudy apple juice. Twelve men and women participated in an acute single blind controlled crossover study in which they consumed 250 mL of cloudy apple juice (CAJ), Crispy Pink apple variety, or 250 mL of the same juice enriched with 750 mg of an apple polyphenol extract (PAJ). Plasma and whole blood were collected at time 0, 1, 2, 3 and 5 h. Urine was collected at time 0 and 0-2, 2-5, 5-8, and 8-24 h after juice consumption. Faecal samples were collected from each individual during the study for 16S rRNA gene profiling. As many as 110 metabolites were significantly elevated following intake of polyphenol enriched cloudy apple juice, with large inter-individual variations. The comparison of the average area under the curve of circulating metabolites in plasma and in urine of volunteers consuming either the CAJ or the PAJ demonstrated a stable metabotype, suggesting that an increase in polyphenol concentration in fruit does not limit their bioavailability upon ingestion. Faecal bacteria were correlated with specific microbial catabolites derived from apple polyphenols. Human metabolism of apple polyphenols is a co-metabolic process between human encoded activities and those of our resident microbiota. Here we have identified specific blood and urine metabolic biomarkers of apple polyphenol intake and identified putative associations with specific genera of faecal bacteria, associations which now need confirmation in specifically designed mechanistic studies.

Assessment of diet-related GHG emissions using the environmental hourglass approach for the Mediterranean and new Nordic diets.

Food production and preparation affect the environment in many ways, with effects on greenhouse gases, use of land, biodiversity, etc. The impact is influenced by consumer demand and eating habits. Two different recommended dietary models were considered, the Mediterranean Diet and the New Nordic Diet, with quantitative analysis of GHG emissions through LCA. An environmental hourglass (EH) approach based on LCA was introduced to help translate health-promoting dietary recommendations that consider regional circumstances and cultural diversity into practical eating habits, to promote sustainable and environmentally friendly consumption. Using the environmental hourglass approach, we examined whether dietary choices based on nutritional recommendations can minimise certain negative effects on the food production environment. Using two examples of health-enhancing, regionally-oriented and culturally appropriate dietary patterns - the Mediterranean Diet and the New Nordic Diet - we showed that consumption of high protein foods has a similar and comparable environmental impact to fruit and vegetable consumption. The results of this work may provide a starting point for integrated policy addressing issues related to the healthy diet of the population, aware food choices and sustainable agriculture.

Fate and transformation products of amine-terminated PAMAM dendrimers under ozonation and irradiation.

This article deals with the degradation of a third-generation (G3) poly(amidoamine) (PAMAM) dendrimer under ozonation and irradiation. The identification and quantification of G3 PAMAM dendrimer and its transformation products has been performed by liquid chromatography-electrospray ionization-hybrid quadrupole time-of-flight-mass spectrometry. The dendrimer was completely depleted by ozone in less than 1 min. The effect of ultraviolet irradiation was attributed to hydroxyl-mediated oxidation. The transformation products were attributed to the oxidation of amines, which resulted in highly oxidized structures with abundance of carboxylic acids, which started from the formation of amine oxide and the scission of the CN bond of the amide group. We studied the toxicity of treated mixtures for six different organisms: the acute toxicity for the bacterium Vibrio fischeri and the microcrustacean Daphnia magna, the multigenerational growth inhibition of the alga Pseudokirchneriella subcapitata, and the seed germination phytotoxicity of Licopersicon esculentum, Lactuca sativa and Lolium perenne. Ozonation and irradiation originated transformation products are more toxic than the parent dendrimer. The toxicity of the dendrimer for the green alga was linked to a strong increase of intracellular reactive oxygen species with intense lipid peroxidation.

Simultaneous measurement in mass and mass/mass mode for accurate qualitative and quantitative screening analysis of pharmaceuticals in river water.

A new approach for the analysis of pharmaceuticals (target and non-target) in water by LC-QTOF-MS is described in this work. The study has been designed to assess the performance of the simultaneous quantitative screening of target compounds, and the qualitative analysis of non-target analytes, in just one run. The features of accurate mass full scan mass spectrometry together with high MS/MS spectral acquisition rates - by means of information dependent acquisition (IDA) - have demonstrated their potential application in this work. Applying this analytical strategy, an identification procedure is presented based on library searching for compounds which were not included a priori in the analytical method as target compounds, thus allowing their characterization by data processing of accurate mass measurements in MS and MS/MS mode. The non-target compounds identified in river water samples were ketorolac, trazodone, fluconazole, metformin and venlafaxine. Simultaneously, this strategy allowed for the identification of other compounds which were not included in the library by screening the highest intensity peaks detected in the samples and by analysis of the full scan TOF-MS, isotope pattern and MS/MS spectra - the example of loratadine (histaminergic) is described. The group of drugs of abuse selected as target compounds for evaluation included analgesics, opioids and psychostimulants. Satisfactory results regarding sensitivity and linearity of the developed method were obtained. Limits of detection for the selected target compounds were from 0.003 to 0.01 µg/L and 0.01 to 0.5 µg/L, in MS and MS/MS mode, respectively - by direct sample injection of 100 µL.

Interpreting PCB levels in breast milk using a physiologically based pharmacokinetic model to reconstruct the dynamic exposure of Italian women.

Polychlorinated biphenyls (PCBs) are persistent contaminants suspected to cause adverse health effects in humans. As PCBs levels in food have not been monitored frequently in the past, modeling approaches based on environmental data have been proposed to predict the human dietary intake. In this work, we propose to improve these approaches by taking into account internal levels of PCBs in humans. This methodology is based on the analysis of biomonitoring data using exposure and physiologically based pharmacokinetic (PBPK) modeling to determine the most probable scenario of exposure. Breast milk concentrations were measured in Italian women for PCB-138, PCB-153 and PCB-180. For each congener, three exposure scenarios were derived and a PBPK model was used to relate the lifetime exposure to the breast milk levels. For the three PCBs, we determined the most probable scenario of exposure. Our results support the adequacy of the exposure and the PBPK models for PCB-180 and PCB-153, whereas we observed discrepancies between the models and the biomonitoring data for PCB-138. Our intake estimates are in good agreement with previous exposure assessments based solely on food contamination demonstrating the relevance of our approach to reconstruct accurately the exposure and to fill in data gaps on exposure.

Exposure of the main italian river basin to pharmaceuticals.

This study give a preliminary survey of pharmaceutical contamination and accumulation in surface waters and sediments along the river Po basin (74,000 km(2), the largest in Italy), a strategic region for the Italian economy: it collects sewage from a vast industrialized area of Italy (Autorità di Baciono del fiume Po, 2006, 2009). 10 pharmaceuticals (atenolol, propanolol, metoprolol, nimesulide, furosemide, carbamazepine, ranitidine, metronidazole, paracetamol, and atorvastatin) from several therapeutic classes were searched in 54 sampling points along the river Po from the source to the delta, and at the mouth of its major effluents. In water samples were found pharmaceuticals in the range of 0.38-0.001 µg/L, except for furosemide (max conc. 0.605 µg/L), paracetamol (max conc. 3.59 µg/L), metoprolol (never detected) and for atenolol (not analysed). In sediment samples, only paracetamol was not detected, while the others were generally found in the range of 0.4-0.02 µg/kg ww with high concentrations for atenolol (max conc. 284 µg/kg ww) and furosemide (max conc. 98.4 µg/kg ww). The findings confirm also STPs as point sources of contamination. Despite of the much evidence for the adverse effects of pharmaceuticals in the aquatic environment, the observed low levels cannot be considered to pose a serious risk to human health; further studies are necessary for a comprehensive risk assessment.

The effect of waste combustion on the occurrence of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) in breast milk in Italy.

Levels of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were measured in the breast milk of mothers living in Giugliano (Campania, Italy), an area at increased risk of PCDD and PCDF exposure caused by recent and serious open-air waste-combustion accidents. Polychlorinated biphenyls (PCBs) in milk samples were also studied. Breast milk was also monitored in two cities in northern Italy, Milan and Piacenza, which were selected as controls. Other variables, such as diet and age of the mothers, were also studied. PCDD/F and PCB levels and congener profiles in breast-milk samples were similar in all these cities, though total PCDD/F and DL-PCB TEQs in samples from Giugliano were significantly lower than those in Milan and Piacenza (8.65 pg WHO-TEQ in Giugliano vs. 11.0 and 9.94 pg WHO-TEQ in Milan and Piacenza respectively). Thus no direct correlations were found between PCDD/F and PCB levels in breast milk and the suspected increased exposure through open-air waste combustion in Giugliano. Diet did not seem to affect PCDD/F and PCB levels, whereas a significant correlation was observed between the mother's age and an increased concentration of PCDDs/Fs and PCBs in milk. Comparison of these findings with those of previous surveys in Italy in 2000-2001 showed a marked reduction of PCDD/F (about 60%) and DL-PCB (about 20%) levels in breast milk occurring over the last 10 years. This might well be a result of well functioning legislation, for example European Directives on of the reduction of the emission limits of PCDDs/Fs and PCBs from waste incineration set by EU Directive 2000/76/EC implemented in 2000, or Commission Regulations such as (EC) No. 466/2001 and 1881/2006 setting maximum levels for certain contaminants in foodstuffs.