Lack of acute or chronic effects of epicatechin-rich and procyanidin-rich apple extracts on blood pressure and cardiometabolic biomarkers in adults with moderately elevated blood pressure: a randomized, placebo-controlled crossover trial.

Background: The reported effects of flavanol-rich foods such as cocoa, dark chocolate, and apples on blood pressure and endothelial function may be due to the monomeric flavanols [mainly (-)-epicatechin (EC)], the oligomeric flavanols [procyanidins (PCs)], or other components. Reports of well-controlled intervention studies that test the effects of isolated oligomeric flavanols on biomarkers of cardiovascular health are lacking. Objective: We studied the acute and chronic effects of an EC-rich apple flavanol extract and isolated apple PCs on systolic blood pressure (BP) and other cardiometabolic biomarkers. Design: Forty-two healthy men and women with moderately elevated BP completed this randomized, double-blind, placebo-controlled, 4-arm crossover trial. Participants ingested a single dose of an apple flavanol extract (70 mg monomeric flavanols, 65 mg PCs), a double dose of this extract (140 mg monomeric flavanols, 130 mg PCs), an apple PC extract (130 mg PCs, 6.5 mg monomeric flavanols), or placebo capsules once daily for 4 wk, in random order. Biomarkers of cardiovascular disease risk and vascular function were measured before and 2 h after ingestion of the first dose and after the 4-wk intervention. Results: Compared with placebo, none of the isolated flavanol treatments significantly (P < 0.05) changed systolic or diastolic BP (peripheral and aortic), plasma nitric oxide (NO) reaction products, or measures of arterial stiffness (carotid femoral pulse-wave velocity, brachial-ankle pulse-wave velocity, or Augmentation Index) after 2 h or 4 wk of the intervention. There were no changes in plasma endogenous metabolite profiles or circulating NO; endothelin 1; total, HDL, or LDL cholesterol; triglycerides; fasting glucose; fructosamine; or insulin after 4 wk of the intervention. Conclusions: Our data suggest that, in isolation, neither monomeric flavanols nor PCs affect BP, blood lipid profiles, endothelial function, or glucose control in individuals with moderately elevated BP. The reported benefits of consuming flavanol-rich cocoa, chocolate, and apple products appear to be dependent on other components, which may work in combination with monomeric flavanols and PCs. This trial was registered at www.clinicaltrials.gov as NCT02013856.

Bioavailability of Glucoraphanin and Sulforaphane from High-Glucoraphanin Broccoli.

SCOPE: Broccoli accumulates 4-methylsulphinylbutyl glucosinolate (glucoraphanin) which is hydrolyzed to the isothiocyanate sulforaphane. Through the introgression of novel alleles of the Myb28 transcription factor from Brassica villosa, broccoli genotypes have been developed that have enhanced levels of glucoraphanin. This study seeks to quantify the exposure of human tissues to glucoraphanin and sulforaphane following consumption of broccoli with contrasting Myb28 genotypes. METHODS AND RESULTS: Ten participants are recruited into a three-phase, double-blinded, randomized crossover trial (NCT02300324), with each phase comprising consumption of 300 g of a soup made from broccoli of one of three Myb28 genotypes (Myb28B/B , Myb28B/V , Myb28V/V ). Plant myrosinases are intentionally denatured during soup manufacture. Threefold and fivefold higher levels of sulforaphane occur in the circulation following consumption of Myb28V/B and Myb28V/V broccoli soups, respectively. The percentage of sulforaphane excreted in 24 h relative to the amount of glucoraphanin consumed varies among volunteers from 2 to 15%, but does not depend on the broccoli genotype. CONCLUSION: This is the first study to report the bioavailability of glucoraphanin and sulforaphane from soups made with novel broccoli varieties. The presence of one or two Myb28V alleles results in enhanced delivery of sulforaphane to the systemic circulation.

Acute Consumption of Flavan-3-ol-Enriched Dark Chocolate Affects Human Endogenous Metabolism.

Flavan-3-ols and methylxanthines have potential beneficial effects on human health including reducing cardiovascular risk. We performed a randomized controlled crossover intervention trial to assess the acute effects of consumption of flavan-3-ol-enriched dark chocolate, compared with standard dark chocolate and white chocolate, on the human metabolome. We assessed the metabolome in urine and blood plasma samples collected before and at 2 and 6 h after consumption of chocolates in 42 healthy volunteers using a nontargeted metabolomics approach. Plasma samples were assessed and showed differentiation between time points with no further separation among the three chocolate treatments. Multivariate statistics applied to urine samples could readily separate the postprandial time points and distinguish between the treatments. Most of the markers responsible for the multivariate discrimination between the chocolates were of dietary origin. Interestingly, small but significant level changes were also observed for a subset of endogenous metabolites. 1H NMR revealed that flavan-3-ol-enriched dark chocolate and standard dark chocolate reduced urinary levels of creatinine, lactate, some amino acids, and related degradation products and increased the levels of pyruvate and 4-hydroxyphenylacetate, a phenolic compound of bacterial origin. This study demonstrates that an acute chocolate intervention can significantly affect human metabolism.

The contribution of alliaceous and cruciferous vegetables to dietary sulphur intake.

Despite its importance in many areas of human metabolism, there are no recommended daily intake guide lines for sulphur. It is generally assumed that most dietary sulphur originates from intake of methionine and cysteine. We estimated sulphur intake from food diaries, and validated the results with the use of a duplicate diet analyses. Sulphur intake estimations were highly correlated with that obtain through an elemental analysis of duplicate diets, with a mean±sd daily intakes of 956±327.9mg estimated from diet diary analyses and 935±329.9mg estimated by a duplicate diet analyses. Sulphur intake from alliaceous and cruciferous vegetables contributed up to 42% of total sulphur intake. Daily intake estimation comparisons through diet diary analyses and duplicate diet for other elements showed good agreement, except for sodium and zinc, in which analyses of 24h diet dairies overestimated intake by 35% and 52%, respectively.

Consumption of a diet rich in Brassica vegetables is associated with a reduced abundance of sulphate-reducing bacteria: A randomised crossover study.

SCOPE: We examined whether a Brassica-rich diet was associated with an increase in the relative abundance of intestinal lactobacilli and sulphate-reducing bacteria (SRB), or alteration to the composition of the gut microbiota, in healthy adults. METHODS AND RESULTS: A randomised crossover study was performed with ten healthy adults who were fed a high- and a low-Brassica diet for 2-wk periods, with a 2-wk washout phase separating the diets. The high-Brassica diet consisted of six 84 g portions of broccoli, six 84 g portions of cauliflower and six 300 g portions of a broccoli and sweet potato soup. The low-Brassica diet consisted of one 84 g portion of broccoli and one 84 g portion of cauliflower. Faecal microbiota composition was measured in samples collected following 2-wk Brassica-free periods (consumption of all Brassica prohibited), and after each diet, whereby the only Brassica consumed was that supplied by the study team. No significant changes to the relative abundance of lactobacilli were observed (p = 0.8019). The increased consumption of Brassica was associated with a reduction in the relative abundance of SRB (p = 0.0215), and members of the Rikenellaceae, Ruminococcaceae, Mogibacteriaceae, Clostridium and unclassified Clostridiales (p < 0.01). CONCLUSION: The increased consumption of Brassica vegetables was linked to a reduced relative abundance of SRB, and therefore may be potentially beneficial to gastrointestinal health.

Nutritional factors and gender influence age-related DNA methylation in the human rectal mucosa.

Aberrant methylation of CpG islands (CGI) occurs in many genes expressed in colonic epithelial cells, and may contribute to the dysregulation of signalling pathways associated with carcinogenesis. This cross-sectional study assessed the relative importance of age, nutritional exposures and other environmental factors in the development of CGI methylation. Rectal biopsies were obtained from 185 individuals (84 male, 101 female) shown to be free of colorectal disease, and for whom measurements of age, body size, nutritional status and blood cell counts were available. We used quantitative DNA methylation analysis combined with multivariate modelling to investigate the relationships between nutritional, anthropometric and metabolic factors and the CGI methylation of 11 genes, together with LINE-1 as an index of global DNA methylation. Age was a consistent predictor of CGI methylation for 9/11 genes but significant positive associations with folate status and negative associations with vitamin D and selenium status were also identified for several genes. There was evidence for positive associations with blood monocyte levels and anthropometric factors for some genes. In general, CGI methylation was higher in males than in females and differential effects of age and other factors on methylation in males and females were identified. In conclusion, levels of age-related CGI methylation in the healthy human rectal mucosa are influenced by gender, the availability of folate, vitamin D and selenium, and perhaps by factors related to systemic inflammation.

Impact of steam explosion on biogas production from rape straw in relation to changes in chemical composition.

An 81day trial compared the cumulative production of methane from rape straw pre-treated by steam explosion at 15 levels of severity. The final methane yields were similar. The temporal variation in production rate exhibited two peaks: maximum production occurred in the first peak at around 21days with heights that increased with severity; the height of the second peak reduced with severity and peaked between 32 and 36days. Changes in the straw composition were investigated using mid-infrared spectroscopy. These were also strongly related to the degree of severity, allowing good predictive models to be built of severity and subsequently the rate of methane production. The main spectral changes showed the degradation of cellulose and xylose-containing hemicelluloses and production of furfural-like components commonly associated with biomass pre-treatments. Only small changes to lignin were associated with increased methane generation suggesting a structural rather than chemical role in this process.

Evaluation of multiple variate selection methods from a biological perspective: a nutrigenomics case study.

Genomics-based technologies produce large amounts of data. To interpret the results and identify the most important variates related to phenotypes of interest, various multivariate regression and variate selection methods are used. Although inspected for statistical performance, the relevance of multivariate models in interpreting biological data sets often remains elusive. We compare various multivariate regression and variate selection methods applied to a nutrigenomics data set in terms of performance, utility and biological interpretability. The studied data set comprised hepatic transcriptome (10,072 predictor variates) and plasma protein concentrations [2 dependent variates: Leptin (LEP) and Tissue inhibitor of metalloproteinase 1 (TIMP-1)] collected during a high-fat diet study in ApoE3Leiden mice. The multivariate regression methods used were: partial least squares "PLS"; a genetic algorithm-based multiple linear regression, "GA-MLR"; two least-angle shrinkage methods, "LASSO" and "ELASTIC NET"; and a variant of PLS that uses covariance-based variate selection, "CovProc." Two methods of ranking the genes for Gene Set Enrichment Analysis (GSEA) were also investigated: either by their correlation with the protein data or by the stability of the PLS regression coefficients. The regression methods performed similarly, with CovProc and GA performing the best and worst, respectively (R-squared values based on "double cross-validation" predictions of 0.762 and 0.451 for LEP; and 0.701 and 0.482 for TIMP-1). CovProc, LASSO and ELASTIC NET all produced parsimonious regression models and consistently identified small subsets of variates, with high commonality between the methods. Comparison of the gene ranking approaches found a high degree of agreement, with PLS-based ranking finding fewer significant gene sets. We recommend the use of CovProc for variate selection, in tandem with univariate methods, and the use of correlation-based ranking for GSEA-like pathway analysis methods.

Does prolonged chewing reduce food intake? Fletcherism revisited.

Horace Fletcher (1849-1919) spread his doctrine to chew each mouthful thoroughly in order to prevent gaining weight. We sought to test this idea by manipulating chewing instructions whilst using electromyography to monitor chewing behaviour. Comparing 35 with 10 chews per mouthful, we showed that higher chewing counts reduced food intake despite increasing chewing speed, and despite doubling meal duration for achieving a subjective reference point for feeling 'comfortably full'. Although limited by a low sample size, our preliminary findings confirm Mr. Fletcher's doctrine, and provide a basis for further research in this area. Outcomes and implications are discussed.

Patterns of DNA methylation in individual colonic crypts reveal aging and cancer-related field defects in the morphologically normal mucosa.

Methylation of CpG islands (CGIs) in the promoter regions of tumour suppressor genes is common in colorectal cancer and occurs also in an age-dependent manner in the morphologically normal colorectal mucosa. In this study, we quantified the level of methylation of six genes associated with the Wnt signalling pathway (adenomatous polyposis coli, DKK1, WIF1, SFRP1, SFRP2 and SFRP5) together with long-interspersed nuclear element-1 as a surrogate for global methylation. DNA methylation was analysed in 260 individual colorectal crypts obtained from eight female patients with no evidence of colorectal disease and five with colorectal cancer. Significant variation in methylation levels for each of the six genes existed between crypts from the same biopsy. The variation in both global and gene-specific CGI methylation between crypts from the same individual was significantly less than that between individuals. Bisulphite sequencing provided insight into the mechanism of aberrant methylation showing that CGI methylation occurs in an 'all-or-none' manner by the directional spreading of methylation from further upstream. Univariate statistical analyses revealed that there were significant differences in crypt-specific methylation associated with both aging and disease status. A multivariate statistical modelling approach was able to distinguish both subject age and health status based on crypt-specific methylation profiles. Our results indicate that the differential methylation of genes associated with the Wnt signalling pathway affecting individual morphologically normal crypts may contribute to the age-dependent generation of the colonic field defect and, in combination with mutations, to the stepwise development of colorectal neoplasia.

Effect of environmental stress during grain filling on the soluble proteome of wheat (Triticum aestivum) dough liquor.

The influence of genotype and environment on a soluble wheat dough liquor proteome was studied for four cultivars grown under field conditions and under hot/dry and cool/wet regimes by two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole time-of-flight mass spectrometry. Although the four cultivars had similar patterns, differences in the relative abundances of some components were observed. Similarly, some differences were observed between the control samples and the samples grown under cool/wet and hot/dry conditions. These included differences in the abundances of storage proteins belonging to the 7S globulin (vicilin-like) and alpha-globulin families and of protective proteins including members of the serpin, described as allergens, and chitinase families. A number of novel annotations were made as compared to previous work on the dough liquor of cv. Hereward, including two 19 kDa alpha-globulins, precursors of endochitinases A and C, and several polypeptides belonging to the 7S globulin (vicilin-like) family.

Multivariate techniques and their application in nutrition: a metabolomics case study.

The post-genomic technologies are generating vast quantities of data but many nutritional scientists are not trained or equipped to analyse it. In high-resolution NMR spectra of urine, for example, the number and complexity of spectral features mean that computational techniques are required to interrogate and display the data in a manner intelligible to the researcher. In addition, there are often multiple underlying biological factors influencing the data and it is difficult to pinpoint which are having the most significant effect. This is especially true in nutritional studies, where small variations in diet can trigger multiple changes in gene expression and metabolite concentration. One class of computational tools that are useful for analysing this highly multivariate data include the well-known 'whole spectrum' methods of principal component analysis and partial least squares. In this work, we present a nutritional case study in which NMR data generated from a human dietary Cu intervention study is analysed using multivariate methods and the advantages and disadvantages of each technique are discussed. It is concluded that an alternative approach, called feature subset selection, will be important in this type of work; here we have used a genetic algorithm to identify the small peaks (arising from metabolites of low concentration) that have been altered significantly following a dietary intervention.

FTIR spectroscopy and multivariate analysis can distinguish the geographic origin of extra virgin olive oils.

This work investigates whether Fourier transform infrared spectroscopy (FTIR), in combination with multivariate analysis, can distinguish extra virgin olive oils from different producing countries. Duplicate spectra were collected from 60 oils from four European countries. Two approaches to data analysis were used as follows: first, the "whole spectrum" method of partial least squares (PLS) followed by distance-based linear discriminant analysis (LDA) applied to the PLS scores, and second, a genetic algorithm (GA) for variate selection from the raw data, followed by LDA applied to the selected subset. The PLS-LDA approach produced a cross-validation success rate of 96%, whereas the GA-LDA approach achieved a 100% cross-validation success rate, from subsets comprising only eight variates. Neither the selected variate nor the whole spectrum approach was able to offer insight into the origin of the discrimination in biochemical terms. However, FTIR analysis is rapid, and this work shows that it has the required discriminatory power to potentially offer a "black box" method of screening oils to verify their country of origin.