A new statistical workflow (R-packages based) to investigate associations between one variable of interest and the metabolome.

INTRODUCTION: In metabolomics, the investigation of associations between the metabolome and one trait of interest is a key research question. However, statistical analyses of such associations are often challenging. Statistical tools enabling resilient verification and clear presentation are therefore highly desired. OBJECTIVES: Our aim is to provide a contribution for statistical analysis of metabolomics data, offering a widely applicable open-source statistical workflow, which considers the intrinsic complexity of metabolomics data. METHODS: We combined selected R packages tailored for all properties of heterogeneous metabolomics datasets, where metabolite parameters typically (i) are analyzed in different matrices, (ii) are measured on different analytical platforms with different precision, (iii) are analyzed by targeted as well as non-targeted methods, (iv) are scaled variously, (v) reveal heterogeneous variances, (vi) may be correlated, (vii) may have only few values or values below a detection limit, or (viii) may be incomplete. RESULTS: The code is shared entirely and freely available. The workflow output is a table of metabolites associated with a trait of interest and a compact plot for high-quality results visualization. The workflow output and its utility are presented by applying it to two previously published datasets: one dataset from our own lab and another dataset taken from the repository MetaboLights. CONCLUSION: Robustness and benefits of the statistical workflow were clearly demonstrated, and everyone can directly re-use it for analysis of own data.

Association between inflammatory markers and serum paraoxonase and arylesterase activities in the general population: a cross-sectional study.

BACKGROUND: Recent studies focused on modulating factors of paraoxonase-1 (PON1) activity. In some studies the association between pro-inflammatory markers and PON1 activity was examined, but so far no population-based investigations on this issue have been conducted. The present study investigated the relationships between the pro-inflammatory markers tumor necrosis factor (TNF)-α, leptin, interleukin (IL)-6, and high-sensitive C-reactive protein (hs-CRP) and paraoxonase and arylesterase, two hydrolytic activities of PON1, in the population-based Bavarian Food Consumption Survey II. METHODS: Based on 504 participants (217 men, 287 women), the relationship between the pro-inflammatory markers and the outcomes paraoxonase and arylesterase activities were investigated using multivariable linear models. RESULTS: Circulating plasma levels of leptin (P-value < 0.0001), hs-CRP (P-value = 0.031) and IL-6 (P-value = 0.045) were significantly non-linearly associated with arylesterase activity. Leptin levels were also significantly associated with paraoxonase activity (P-value = 0.024) independently from confounding factors, including high-density lipoprotein (HDL) cholesterol. With increasing levels of these inflammatory parameters, arylesterase and paraoxonase activities increased; however, at higher levels (> 75th percentile) the activities reached a plateau or even decreased somewhat. After Bonferroni-Holm correction, only leptin remained non-linearly but significantly associated with arylesterase activity (adjusted overall P-value < 0.0001). Neither age nor sex nor obesity modified the associations. No association was found between TNF-α and paraoxonase or arylesterase activity. CONCLUSIONS: The present findings suggest that in persons with very high levels of inflammation, PON1 activity may be impaired, a fact that might subsequently be accompanied by a higher risk for cardiometabolic diseases. Whether or not the measurement of PON1 activity in combination with a lipid profile and certain inflammatory markers could improve the prediction of cardiometabolic diseases in middle-aged individuals from the general population should be evaluated in clinical studies.

Targeted ultra-performance liquid chromatography/tandem mass spectrometric quantification of methylated amines and selected amino acids in biofluids.

RATIONALE: Methylated amino compounds and basic amino acids are important analyte classes with high relevance in nutrition, physical activity and physiology. Reliable and easy quantification methods covering a variety of metabolites in body fluids are a prerequisite for efficient investigations in the field of food and nutrition. METHODS: Targeted ultra-performance liquid chromatography/tandem mass spectrometric (UHPLC/MS) analysis was performed using HILIC separation and timed ESI-MRM detection, combined with a short sample preparation. Calibration in urine and blood plasma was achieved by matrix-matched standards, isotope-labelled internal standards and standard addition. The method was fully validated and the performance was evaluated using a subset from the Karlsruhe Metabolomics and Nutrition (KarMeN) study. RESULTS: Within this method, a total of 30 compounds could be quantified simultaneously in a short run of 9 min in both body fluids. This covers a variety of free amino compounds which are present in very different concentrations. The method is easy, precise and robust, and has a broad working range. As a proof of principle, literature-based associations of certain metabolites with dietary intake of respective foods were clearly confirmed in the KarMeN subset. CONCLUSIONS: Overall, the method turned out to be well suited for application in nutrition studies, as shown for the example of food intake biomarkers in KarMeN. Application to a variety of questions such as food-related effects or physical activity will support future studies in the context of nutrition and health.

Glyphosate and AMPA levels in human urine samples and their correlation with food consumption: results of the cross-sectional KarMeN study in Germany.

Glyphosate (N-[phosphonomethyl]-glycine) is the most widely used herbicide worldwide. Due to health concerns about glyphosate exposure, its continued use is controversially discussed. Biomonitoring is an important tool in safety evaluation and this study aimed to determine exposure to glyphosate and its metabolite AMPA, in association with food consumption data, in participants of the cross-sectional KarMeN study (Germany). Glyphosate and AMPA levels were measured in 24-h urine samples from study participants (n = 301). For safety evaluation, the intake of glyphosate and AMPA was calculated based on urinary concentrations and checked against the EU acceptable daily intake (ADI) value for glyphosate. Urinary excretion of glyphosate and/or AMPA was correlated with food consumption data. 8.3% of the participants (n = 25) exhibited quantifiable concentrations (> 0.2 µg/L) of glyphosate and/or AMPA in their urine. In 66.5% of the samples, neither glyphosate (< 0.05 µg/L) nor AMPA (< 0.09 µg/L) was detected. The remaining subjects (n = 76) showed traces of glyphosate and/or AMPA. The calculated glyphosate and/or AMPA intake was far below the ADI of glyphosate. Significant, positive associations between urinary glyphosate excretion and consumption of pulses, or urinary AMPA excretion and mushroom intake were observed. Despite the widespread use of glyphosate, the exposure of the KarMeN population to glyphosate and AMPA was found to be very low. Based on the current risk assessment of glyphosate by EFSA, such exposure levels are not expected to pose any risk to human health. The detected associations with consuming certain foods are in line with reports on glyphosate and AMPA residues in food.

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.

Association between alcohol consumption and serum paraoxonase and arylesterase activities: a cross-sectional study within the Bavarian population.

High alcohol consumption is an important risk factor for chronic disease and liver degeneration. Paraoxonase (PON1) and arylesterase (AE) are functions of the enzyme paraoxonase, which is synthesised by the liver. Paraoxonase circulates in plasma bound to HDL and hydrolyses lipid peroxides, protecting lipoproteins against oxidative modification. It has been shown that excessive alcohol consumption leads to a reduction of serum PON1 and AE activities; however, studies investigating the association with low and moderate alcohol consumption are scarce. We investigated the cross-sectional association between alcohol consumption and serum activities of PON1 and AE using data from the population-based Bavarian Food Consumption Survey II survey. PON1 and AE activities were quantified in serum samples of 566 male and female study participants (aged 18-80 years), and dietary intake including alcohol consumption was estimated from three 24-h dietary recalls. The association between alcohol consumption and PON1 and AE activities was analysed using linear regression, adjusted for age, sex and socio-economic status. There was no strong association between alcohol consumption and enzymatic activities of PON1 and AE in the Bavarian population. PON1 activity was seen to be lowest in non-drinkers (0 g/d) and highest in people who consumed 15·1-30 g of alcohol/d. AE activity increased across alcohol consumption categories, with a mean maximum difference of 14 U/ml (P for linear trend 0·04). These associations were attenuated after adjustment for blood concentrations of HDL. The results of this study do not support the hypothesis that alcohol consumption is related to important alterations in PON1 and AE activities.

Phase II metabolism of the soy isoflavones genistein and daidzein in humans, rats and mice: a cross-species and sex comparison.

Soy isoflavones (IF) are in the focus of biomedical research since more than two decades. To assess their bioactivity, IF are investigated in rats and mice as a model. As the biological activity of IF is affected by their biotransformation, our aim was to comprehensively compare the conjugative and microbial metabolism of daidzein and genistein in adult humans, rats and mice of both sexes. One identical soy extract and a validated LC-MS method were used for all studies. We detected considerable differences between the three species. In rats and mice, sex-specific differences were observed in addition. The major plasma phase II metabolites in humans were the 7-sulfo-4'-glucuronides (39-49 %) and, in case of genistein, also the diglucuronide (34 %), whereas in mice monosulfates (33-41 %) and monoglucuronides (30-40 %) predominated. In male rats the disulfates (23-62 %) and 7-sulfo-4'-glucuronides (19-54 %) were predominant, while in female rats the 7-glucuronides (81-93 %) exhibited highest concentrations. The portion of aglycones was low in humans (0.5-1.3 %) and rats (0.5-3.1 %) but comparatively high in mice (3.1-26.0 %), especially in the case of daidzein. Furthermore, substantial differences were observed between daidzein and genistein metabolism. In contrast to humans, all rats and mice were equol producer, independent of their sex. In conclusion, there are marked differences between humans, rats and mice in the profile of major metabolites following IF phase II metabolism. These differences may contribute to resolve inconsistencies in results concerning the bioactivity of IF and should be considered when applying findings of animal studies to humans, e.g., for risk assessment.

Influence of a probiotic Lactobacillus casei strain on the colonisation with potential pathogenic streptococci and Staphylococcus aureus in the nasopharyngeal space of healthy men with a low baseline NK cell activity.

The effect of a daily intake of the probiotic strain Lactobacillus casei Shirota (LcS) on the colonisation of pathogens, specifically streptococci and Staphylococcus aureus, in the nose and throat of healthy human volunteers with low natural killer cell activity, was investigated in a randomised and controlled intervention study. The study consisted of a 2-week run-in phase, followed by a 4-week intervention phase. The probiotic treatment group received a fermented milk drink with LcS, while the placebo group received an equally composed milk drink without the probiotic additive. To isolate potential pathogenic streptococci and Staph. aureus, samples from the pharynx, as well as of both middle nasal meati, were taken, once after the run-in phase and once at the end of the intervention phase. Isolated bacteria were identified as either Staph. aureus and α- or ß-haemolytic streptococci in a polyphasic taxonomical approach based on phenotypic tests, amplified ribosomal DNA restriction analysis genotyping, and 16S rRNA gene sequencing of representative strains. Salivary secretory immunoglobulin A (SIgA) was used as marker of protective mucosal immunity to evaluate whether LcS treatment influenced SIgA production. No statistically significant effect could be determined for intervention with LcS on the incidence of Staph. aureus in the nasal space, Staph. aureus in the pharyngeal space or for ß-haemolytic streptococci and Streptococcus pneumoniae in the pharyngeal space. Thus, the intervention did not influence the nasopharyngeal colonisation with Gram-positive potential pathogens. Production of salivary SIgA as a potential means of microbiota modulation was also not affected.

Prediction of fruit and vegetable intake from biomarkers using individual participant data of diet-controlled intervention studies.

Fruit and vegetable consumption produces changes in several biomarkers in blood. The present study aimed to examine the dose-response curve between fruit and vegetable consumption and carotenoid (α-carotene, ß-carotene, ß-cryptoxanthin, lycopene, lutein and zeaxanthin), folate and vitamin C concentrations. Furthermore, a prediction model of fruit and vegetable intake based on these biomarkers and subject characteristics (i.e. age, sex, BMI and smoking status) was established. Data from twelve diet-controlled intervention studies were obtained to develop a prediction model for fruit and vegetable intake (including and excluding fruit and vegetable juices). The study population in the present individual participant data meta-analysis consisted of 526 men and women. Carotenoid, folate and vitamin C concentrations showed a positive relationship with fruit and vegetable intake. Measures of performance for the prediction model were calculated using cross-validation. For the prediction model of fruit, vegetable and juice intake, the root mean squared error (RMSE) was 258.0 g, the correlation between observed and predicted intake was 0.78 and the mean difference between observed and predicted intake was - 1.7 g (limits of agreement: - 466.3, 462.8 g). For the prediction of fruit and vegetable intake (excluding juices), the RMSE was 201.1 g, the correlation was 0.65 and the mean bias was 2.4 g (limits of agreement: -368.2, 373.0 g). The prediction models which include the biomarkers and subject characteristics may be used to estimate average intake at the group level and to investigate the ranking of individuals with regard to their intake of fruit and vegetables when validating questionnaires that measure intake.

Dietary fat quality in regular fat diets has minor effects on biomarkers of inflammation in obese Zucker rats.

PURPOSE: Adipose tissue-associated chronic inflammation is involved in the pathogenesis of obesity-related diseases. Dietary fatty acids are known to influence inflammatory processes. The aim of this study was to investigate, whether diets with regular fat contents but variable fat qualities affect adipose tissue-associated inflammation through the fatty acid composition of mesenteric adipose tissue (MAT). METHODS: Obese Zucker rats were fed diets containing 7 % wt:wt rapeseed oil, corn oil, or lard for 10 weeks. Fatty acid composition and endocrine function regarding adipokines and cytokines of MAT, number of total CD3(+) T cells, and cytokine secretion of mesenteric lymph node (MLN)-derived lymphocytes were determined. Local effects in MAT and MLN were compared to systemic effects assessed in serum and peripheral blood mononuclear cells. RESULTS: Fatty acid composition of MAT reflected dietary fatty acid intake, without affecting endocrine function. Feeding the lard diet for 10 weeks increased the serum adiponectin and TNF-α secretion of blood lymphocytes, whereas CD3(+) T cells in blood were decreased. No effects were seen for the secretion of adipokines and cytokines from MAT, the amount of T cells in MLN, and cytokine secretion of MLN lymphocytes. CONCLUSIONS: In conclusion, feeding obese rats a diet with regular fat content but variable fat sources for 10 weeks, changed the fatty acid composition of MAT but not its secretory properties or MLN functions. Although the local immune system was not influenced, lard-feeding induced minor changes in systemic immune function.

Dietary walnut oil modulates liver steatosis in the obese Zucker rat.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. We aimed to clarify the impact of dietary walnut oil versus animal fat on hepatic steatosis, representing the initial step of multistage pathogenesis of NAFLD, in Zucker obese rats. METHODS: Zucker lean ad libitum (a.l.), Zucker obese a.l. or Zucker obese pair fed (p.f.) to the lean received isocaloric diets containing 8% walnut oil (W8), W14 or 14% lard (L14) (n = 10/group). Body weight, clinical serology, liver weight, lipid content and fatty acid composition and hepatic lipid metabolism-related transcripts were evaluated. RESULTS: Compared to lean, Zucker obese a.l. and p.f. showed hepatic triacylglyceride (TAG) accumulation. In Zucker obese p.f., W14 compared to W8 and L14 reduced liver lipids, TAG as well as hepatic omega-6 (n-6)/n-3 ratio and SCD activity index [(C18:0 + C18:1)/C18:0 ratio] paralleled by decreased lipoprotein lipase mRNA in obese p.f. and elevated microsomal triglyceride transfer protein mRNA in lean and obese. Further, W14 elevated the fasting blood TAG and reduced cholesterol levels in obese. CONCLUSIONS: In our model, consumption of W14 inhibited hepatic lipid accumulation along with modulated hepatic gene expression implicated in hepatic fatty acid influx or lipoprotein assembly. These results provide first indication that dietary lipids from walnut oil are modulators of hepatic steatosis as the initial step of progressive NAFLD pathogenesis.

Streptococcus rubneri sp. nov., isolated from the human throat.

The novel, Gram-stain-positive, ovoid, lactic acid bacterial isolates LMG 27205, LMG 27206, LMG 27207(T) and MRI-F 18 were obtained from throat samples of healthy humans. 16S rRNA gene sequence analyses indicated that these isolates belong to the genus Streptococcus, specifically the Streptococcus mitis group, with Streptococcus australis and Streptococcus mitis as the nearest neighbours (99.45 and 98.56 % 16S rRNA gene sequence similarity to the respective type strains). Genotypic fingerprinting by fluorescent amplified fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE), DNA-DNA hybridizations, comparative sequence analysis of pheS, rpoA and atpA and physiological and biochemical tests revealed that these bacteria formed a taxon well separated from its nearest neighbours and other species of the genus Streptococcus with validly published names and, therefore, represent a novel species, for which the name Streptococcus rubneri sp. nov. is proposed, with LMG 27207(T) ( = DSM 26920(T)) as the type strain.

Microbial Metabolism of the Soy Isoflavones Daidzein and Genistein in Postmenopausal Women: Human Intervention Study Reveals New Metabotypes.

BACKGROUND: Soy isoflavones belong to the group of phytoestrogens and are associated with beneficial health effects but are also discussed to have adverse effects. Isoflavones are intensively metabolized by the gut microbiota leading to metabolites with altered estrogenic potency. The population is classified into different isoflavone metabotypes based on individual metabolite profiles. So far, this classification was based on the capacity to metabolize daidzein and did not reflect genistein metabolism. We investigated the microbial metabolite profile of isoflavones considering daidzein and genistein. METHODS: Isoflavones and metabolites were quantified in the urine of postmenopausal women receiving a soy isoflavone extract for 12 weeks. Based on these data, women were clustered in different isoflavone metabotypes. Further, the estrogenic potency of these metabotypes was estimated. RESULTS: Based on the excreted urinary amounts of isoflavones and metabolites, the metabolite profiles could be calculated, resulting in 5 metabotypes applying a hierarchical cluster analysis. The metabotypes differed in part strongly regarding their metabolite profile and their estimated estrogenic potency.

Assessment of Energy and Nutrient Intake and the Intestinal Microbiome (ErNst Study): Protocol and Methods of a Cross-sectional Human Observational Study.

BACKGROUND: On the national level, nutritional monitoring requires the assessment of reliable representative dietary intake data. To achieve this, standardized tools need to be developed, validated, and kept up-to-date with recent developments in food products and the nutritional behavior of the population. Recently, the human intestinal microbiome has been identified as an essential mediator between nutrition and host health. Despite growing interest in this connection, only a few associations between the microbiome, nutrition, and health have been clearly established. Available studies paint an inconsistent picture, partly due to a lack of standardization. OBJECTIVE: First, we aim to verify if food consumption, as well as energy and nutrient intake of the German population, can be recorded validly by means of the dietary recall software GloboDiet, which will be applied in the German National Nutrition Monitoring. Second, we aim to obtain high-quality data using standard methods on the microbiome, combined with dietary intake data and additional fecal sample material, and to also assess the functional activity of the microbiome by measuring microbial metabolites. METHODS: Healthy female and male participants aged between 18 and 79 years were recruited. Anthropometric measurements included body height and weight, BMI, and bioelectrical impedance analysis. For validation of the GloboDiet software, current food consumption was assessed with a 24-hour recall. Nitrogen and potassium concentrations were measured from 24-hour urine collections to enable comparison with the intake of protein and potassium estimated by the GloboDiet software. Physical activity was measured over at least 24 hours using a wearable accelerometer to validate the estimated energy intake. Stool samples were collected in duplicate for a single time point and used for DNA isolation and subsequent amplification and sequencing of the 16S rRNA gene to determine microbiome composition. For the identification of associations between nutrition and the microbiome, the habitual diet was determined using a food frequency questionnaire covering 30 days. RESULTS: In total, 117 participants met the inclusion criteria. The study population was equally distributed between the sexes and 3 age groups (18-39, 40-59, and 60-79 years). Stool samples accompanying habitual diet data (30-day food frequency questionnaire) are available for 106 participants. Current diet data and 24-hour urine samples for the validation of GloboDiet are available for 109 participants, of which 82 cases also include physical activity data. CONCLUSIONS: We completed the recruitment and sample collection of the ErNst study with a high degree of standardization. Samples and data will be used to validate the GloboDiet software for the German National Nutrition Monitoring and to compare microbiome composition and nutritional patterns. TRIAL REGISTRATION: German Register of Clinical Studies DRKS00015216; https://drks.de/search/de/trial/DRKS00015216. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/42529.

Acute effects of moderate vs. vigorous endurance exercise on urinary metabolites in healthy, young, physically active men-A multi-platform metabolomics approach.

Introduction: Endurance exercise alters whole-body as well as skeletal muscle metabolism and physiology, leading to improvements in performance and health. However, biological mechanisms underlying the body's adaptations to different endurance exercise protocols are not entirely understood. Methods: We applied a multi-platform metabolomics approach to identify urinary metabolites and associated metabolic pathways that distinguish the acute metabolic response to two endurance exercise interventions at distinct intensities. In our randomized crossover study, 16 healthy, young, and physically active men performed 30 min of continuous moderate exercise (CME) and continuous vigorous exercise (CVE). Urine was collected during three post-exercise sampling phases (U01/U02/U03: until 45/105/195 min post-exercise), providing detailed temporal information on the response of the urinary metabolome to CME and CVE. Also, fasting spot urine samples were collected pre-exercise (U00) and on the following day (U04). While untargeted two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) led to the detection of 608 spectral features, 44 metabolites were identified and quantified by targeted nuclear magnetic resonance (NMR) spectroscopy or liquid chromatography-mass spectrometry (LC-MS). Results: 104 urinary metabolites showed at least one significant difference for selected comparisons of sampling time points within or between exercise trials as well as a relevant median fold change >1.5 or <0. 6 ¯ (NMR, LC-MS) or >2.0 or <0.5 (GC×GC-MS), being classified as either exercise-responsive or intensity-dependent. Our findings indicate that CVE induced more profound alterations in the urinary metabolome than CME, especially at U01, returning to baseline within 24 h after U00. Most differences between exercise trials are likely to reflect higher energy requirements during CVE, as demonstrated by greater shifts in metabolites related to glycolysis (e.g., lactate, pyruvate), tricarboxylic acid cycle (e.g., cis-aconitate, malate), purine nucleotide breakdown (e.g., hypoxanthine), and amino acid mobilization (e.g., alanine) or degradation (e.g., 4-hydroxyphenylacetate). Discussion: To conclude, this study provided first evidence of specific urinary metabolites as potential metabolic markers of endurance exercise intensity. Future studies are needed to validate our results and to examine whether acute metabolite changes in urine might also be partly reflective of mechanisms underlying the health- or performance-enhancing effects of endurance exercise, particularly if performed at high intensities.

Moderate effects of apple juice consumption on obesity-related markers in obese men: impact of diet-gene interaction on body fat content.

PURPOSE: The effect of polyphenol-rich cloudy apple juice (CloA) consumption on plasma parameters related to the obesity phenotype and potential effects of interactions between CloA and allelic variants in obesity candidate genes were assessed in obese men. METHODS: In this controlled, randomized, and parallel study, n = 68, non-smoking, non-diabetic men with a BMI ≥27 kg/m(2) received 750 mL/day CloA (802.5 mg polyphenols) or 750 mL/day control beverage (CB, isocaloric equivalent to CloA) for 4 weeks. Further, study participants were genotyped for single-nucleotide polymorphisms in PPARγ (rs1801282), UCP3 (rs1800849), IL-6 (rs1800795), FABP2 (rs1799883), INSIG2 (rs7566605), and PGC1 (rs8192678) genes. At the beginning and at the end of intervention plasma lipids, distinct adipokines and cytokines as well as anthropometric parameters were determined. RESULTS: CloA compared to CB had no significant effect on plasma lipids, plasma adipokine and cytokine levels, BMI, and waist circumference. However, CloA consumption significantly reduced percent body fat compared to CB (∆ % body fat: CloA: -1.0 ± 1.3 vs. CB: -0.2 ± 0.9, p < 0.05). The IL-6-174 G/C polymorphism showed an interaction with body fat reduction induced by CloA. Solely in C/C, but not in G/C or G/G variants, a significant reduction in body fat after 4 weeks of CloA intervention was detectable. CONCLUSION: The observed diet-gene interaction might be a first indication for the impact of individual genetic background on CloA-mediated bioactivity on obesity-associated comorbidities.

Critical review: vegetables and fruit in the prevention of chronic diseases.

BACKGROUND: Vegetables and fruit provide a significant part of human nutrition, as they are important sources of nutrients, dietary fibre, and phytochemicals. However, it is uncertain whether the risk of certain chronic diseases can be reduced by increased consumption of vegetables or fruit by the general public, and what strength of evidence has to be allocated to such an association. METHODS: Therefore, a comprehensive analysis of the studies available in the literature and the respective study results has been performed and evaluated regarding obesity, type 2 diabetes mellitus, hypertension, coronary heart disease (CHD), stroke, cancer, chronic inflammatory bowel disease (IBD), rheumatoid arthritis (RA), chronic obstructive pulmonary disease (COPD), asthma, osteoporosis, eye diseases, and dementia. For judgement, the strength of evidence for a risk association, the level of evidence, and the number of studies were considered, the quality of the studies and their estimated relevance based on study design and size. RESULTS: For hypertension, CHD, and stroke, there is convincing evidence that increasing the consumption of vegetables and fruit reduces the risk of disease. There is probable evidence that the risk of cancer in general is inversely associated with the consumption of vegetables and fruit. In addition, there is possible evidence that an increased consumption of vegetables and fruit may prevent body weight gain. As overweight is the most important risk factor for type 2 diabetes mellitus, an increased consumption of vegetables and fruit therefore might indirectly reduces the incidence of type 2 diabetes mellitus. Independent of overweight, there is probable evidence that there is no influence of increased consumption on the risk of type 2 diabetes mellitus. There is possible evidence that increasing the consumption of vegetables and fruit lowers the risk of certain eye diseases, dementia and the risk of osteoporosis. Likewise, current data on asthma, COPD, and RA indicate that an increase in vegetable and fruit consumption may contribute to the prevention of these diseases. For IBD, glaucoma, and diabetic retinopathy, there was insufficient evidence regarding an association with the consumption of vegetables and fruit. CONCLUSIONS: This critical review on the associations between the intake of vegetables and fruit and the risk of several chronic diseases shows that a high daily intake of these foods promotes health. Therefore, from a scientific point of view, national campaigns to increase vegetable and fruit consumption are justified. The promotion of vegetable and fruit consumption by nutrition and health policies is a preferable strategy to decrease the burden of several chronic diseases in Western societies.

Generation of Se-fortified broccoli as functional food: impact of Se fertilization on S metabolism.

Selenium (Se)-fortified broccoli (Brassica oleracea var. italica) has been proposed as a functional food for cancer prevention, based on its high glucosinolate (GSL) content and capacity for Se accumulation. However, as selenate and sulphate share the initial assimilation route, Se fertilization could interfere with sulphur metabolism and plant growth. Consequently, GSL accumulation could be compromised. To evaluate these potentially adverse effects of Se fertilization, we performed a comprehensive study on sand-grown young broccoli plants (weekly selenate applications of 0.8 µmol plant(-1) via the root) and field-grown adult broccoli plants during head formation (single foliar selenate application: 25.3 or 253 µmol plant(-1) ). The results show that under these conditions, Se application does not affect plant growth, contents of cysteine, glutathione, total GSL, glucoraphanin (major aliphatic GSL) or the expression of BoMYB28 (encoding a functionally confirmed master regulator for aliphatic GSL biosynthesis). Conversely, due to the changed expression of sulphate transporters (BoSULTR1;1, 1;2, 2;1, and 2;2), sulphate and total S contents increased in the shoot of young plants while decreasing in the root. We conclude that broccoli can be fertilized with Se without reduction in GSL content, even with Se accumulation exceeding the level recommended for human consumption.

Probiotic Lactobacillus casei Shirota supplementation does not modulate immunity in healthy men with reduced natural killer cell activity.

Oral intake of probiotic bacteria may beneficially modulate functions of NK cells. In healthy individuals, contradictory results exist as to whether NK cell functions can be modulated by probiotic bacteria. Therefore, the primary objective of our randomized, double-blind, placebo-controlled trial was to determine the effects of the probiotic strain Lactobacillus casei Shirota (LcS) on the activity of NK cells in healthy men who had been preselected for a reduced lytic function of their NK cells. Study participants (n = 68) were supplemented for 4 wk with a probiotic drink providing 1.95 × 10(10) CFU LcS/d or with a similar milk drink without probiotic additive. A run-in period of 2 wk preceded the probiotic supplementation followed by a 2-wk follow-up phase without the probiotic or control drink. Changes in the relative proportions of NK cells and other leukocytes as well as multiple functional measurements were determined longitudinally at baseline, after the 4-wk supplementation, and at the end of the follow-up. The probiotic supplementation had no significant effect on NK cell numbers and function or on phagocytosis, respiratory burst, or cytokine secretion of peripheral blood mononuclear cells. In conclusion, 4 wk of supplementation with LcS does not increase NK cell activity in healthy men with a reduced NK cell lytic activity. However, other doses of LcS, time of intervention, or differences, e.g. in the background diet, may result in a different outcome.

CLA does not impair endothelial function and decreases body weight as compared with safflower oil in overweight and obese male subjects.

OBJECTIVE: Conjugated linoleic acid (CLA) showed a wide range of beneficial biological effects with relevance for cardiovascular health in animal models and humans. Most human studies used olive oil as a reference. This study assessed the effect of CLA as compared with safflower oil on endothelial function and markers of cardiovascular risk in overweight and obese men. Heated safflower oil and olive oil were given for additional descriptive control. METHODS: Eighty-five overweight men (aged 45-68 years, body mass index 25-35 kg/m(2)) were randomized to receive 4.5 g/d of the CLA isomeric mixture, safflower oil, heated safflower oil, or olive oil in a 4-week double-blind study. Endothelial function was assessed by peripheral arterial tonometry (PAT) index determination in the fasting and postprandial state (i.e., 4 hours after consumption of a fat- and sucrose-rich meal). RESULTS: CLA as compared with safflower oil consumption did not impair fasting or postprandial PAT index but decreased body weight. CLA as compared with safflower oil did not change total, low-density lipoprotein (LDL), or high-density lipoprotein (HDL) cholesterol; triglycerides; insulin sensitivity indices; C-reactive protein; soluble adhesion molecules; oxidized LDL; lipoprotein a (Lp[a]); paraoxonase; or platelet-activating factor acetylhydrolase (PAF-AH) activity, but significantly reduced arylesterase activity and increased concentrations of the F(2)-isoprostane 8-iso-prostaglandin F (PGF)(2α). CONCLUSION: CLA did not impair endothelial function. Other parameters associated with metabolic syndrome and oxidative stress were not changed or were slightly improved. Results suggest that CLA does not increase cardiovascular risk. Increased F(2)-isoprostane concentrations in this context may not indicate increased oxidative stress.