Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Mining the human intestinal microbiota for biomarkers associated with metabolic disorders
Hermes, Gerben - \ 2016
Wageningen University. Promotor(en): Hauke Smidt; Erwin Zoetendal. - Wageningen : Wageningen University - ISBN 9789462579514 - 205
gastrointestinal microbiota - metabolic disorders - biomarkers - obesity - intestinal microorganisms - antibiotics - dna sequencing - rna - ribosomal rna - microbiota van het spijsverteringskanaal - stofwisselingsstoornissen - obesitas - darmmicro-organismen - antibiotica - dna-sequencing - ribosomaal rna

After birth, our gastrointestinal (GI) tract is colonized by a highly complex assemblage of microbes, collectively termed the GI microbiota, that develop intimate interactions with our body. Recent evidence indicates that the GI microbiota and its products may contribute to the development of obesity and related diseases. This, coupled with the current worldwide epidemic of obesity, has moved microbiome research into the spotlight of attention. Although the main cause of obesity and its associated metabolic complications is excess caloric intake compared with expenditure, differences in GI tract microbial ecology between individuals might be an important biomarker, mediator or even new therapeutic target. Nevertheless, it is currently still unclear which bacterial groups play a role in the development of the metabolic syndrome in humans. This might partly be explained by: 1. Biological factors such as the heterogeneity in genotype, lifestyle, diet; and the often complex aetiology of human disease of which the metabolic syndrome is no exception. 2. Technological factors, such as the use of miscellaneous incompatible methods to assess the gut microbiota, often enumerating specific groups rather than using broad 16S rRNA gene surveys or metagenomics. 3. Studies vary greatly in the populations considered, their designs, and the degree of control for potential confounding factors such as lifestyle and diet. Nevertheless, recent research on this matter has shown a conceptual shift by focusing on more homogenous subpopulations, based on stricter control over variables such age range or through the use of both anthropometric (weight, total body fat) as well as biochemical variables (insulin resistance, hyperlipidaemia) to define groups.

Perturbations in microbial diversity and community structure in adults with overweight and obesity may be partly due to long-term dietary habits or physiological changes in these subjects. As such, exploring the association between the gut microbiota and variation in BMI and weight in early life, prior to or close to the onset of overweight, might provide additional insights into these processes. Therefore, we studied the fecal microbiota of 295 six-seven year old children from the KOALA Birth Cohort, living in the south of the Netherlands. This age range is relatively uncharted microbiota territory. We found that its composition seems to conform to tot same ecosystem rules as that of adults. The bimodal distribution pattern of several bacterial groups as well as their co-correlating groups that were reported previously, including Uncultured Clostridiales II (UCII), Prevotella spp. and Dialister were confirmed. Furthermore, one of the previously described bimodal groups (Uncultured Clostridiales I) was shown before to exhibit very clear shifting state probabilities associated with ageing, where the high abundance state was mainly observed above 40 years of age. This was corroborated as no support for bimodality of this group was observed in the children included in the study described here. A large part of the variation in microbiota composition was explained by the abundance of aforementioned groups in contrast to the anthropometric outcomes, suggesting that in this group of healthy children within a relatively normal weight range, weight and associated parameters were not major drivers of overall genus-level microbial composition or vice versa. Hereafter, multiple linear and logistic regression models with rigorous adjustment for confounders were applied to investigate individual microbiota features association with weight related anthropometric outcomes. Previously reported parameters such as diversity, richness and Bacteroidetes to Firmicutes ratio, were not significantly associated with any of the outcomes. Nevertheless, the abundance of several specific bacterial taxa; Akkermansia, Sutterella wadsworthia et rel. and Bryantella formatexigens et rel. and the dichotomous abundance state of the bi-modally distributed UCII was consistently associated with weight-related outcomes.

Other biochemical features of the metabolic syndrome have been associated with the gut microbiome. Mainly rodent studies have indicated that antibiotic treatment may improve glucose homeostasis and metabolic impairments. Therefore, the effects of gut microbiota manipulation by antibiotics (7d administration of amoxicillin, vancomycin or a placebo) on tissue-specific insulin sensitivity, energy metabolism, gut permeability and inflammation in 57 obese, pre-diabetic men from the same geographical region, were investigated. Vancomycin decreased bacterial diversity and significantly reduced well known butyrate- producing Firmicutes from Clostridium clusters IV and XIVa and bacterial groups involved in bile acid metabolism. These changes occurred concomitantly with altered plasma and fecal concentrations of these metabolites. In adipose tissue, gene expression of oxidative pathways was upregulated by antibiotics, whereas immune-related pathways were downregulated by vancomycin. However, antibiotic treatment had no significant effects on tissue-specific insulin sensitivity, energy/substrate metabolism, postprandial hormones and metabolites, systemic inflammation, gut permeability and adipocyte size. Importantly, despite a still considerably altered microbial composition at eight weeks follow-up, energy harvesting, adipocyte size and whole-body insulin sensitivity (HOMA-IR) remained unaltered. Overall these data indicate that interference with adult microbiota by antibiotic treatment for 7 days had no clinically relevant impact on metabolic health in obese humans. These data are in contrast with several rodent studies as well as a human intervention. The present study, which was well-powered and placebo-controlled, indicates that the previously reported vancomycin-induced effects on human peripheral insulin sensitivity are probably of minor physiological significance.

The aforementioned group that was relatively homogeneous with regards to phenotype was combined with another cohort with similar phenotypical characteristics (obese, male and pre-diabetic) from another region of the Netherlands, to investigate whether tissue specific insulin sensitivity, as measured by the golden standard hyperinsulinemic-euglycemic clamp technique, is related to a specific microbial pattern. Remarkably, despite the fact that both cohorts were constructed based on comparable recruitment strategies, the average microbiota composition in both cohorts showed pronounced differences. Firstly, we found no consistent and significant association between liver, adipose tissue or skeletal muscle insulin sensitivity and the microbiota in both cohorts. Nevertheless, Random Forests classifiers using microbiota composition as predictors revealed taxa associated with fasting glucose concentrations and HbAc1 but only in one cohort. The top microbial features distinguishing classes were different Proteobacteria and groups involved in butyrogenesis, such as Faecalibacterium prausnitzii, Roseburia intestinalis, and Eubacterium rectale and related species, for fasting glucose levels. For HbAc1 these taxa were Oscillospira guillermondii, Sporobacter termitidis, Lactobacillus gasseri and Peptococcus niger and related species. The striking cohort-specific observations suggest that the relation between microbiota composition and type 2 diabetes mellitus as well as other characteristics of the metabolic syndrome is very dependent on the selected cohort of patients and their respective baseline microbiota composition. Similar observations have been made by other researchers as well. It could be that differences in microbiota composition are not associated with the insulin resistance phenotype when the overweight and/or obese state of the patient is already established, as is the case for our metabolic syndrome patients. In the latter case we cannot exclude that the composition of the fecal microbiota may play a role in the worsening of insulin sensitivity in an early stage in the development from a lean towards an overweight/obese phenotype. Furthermore, the observation of a subgroup- specific microbiota only observed in one of the cohorts might indicate an alternative state of microbiota composition driven by yet unknown forces. Nevertheless, this study clearly demonstrated that cohort-specific microbiota differences hamper finding a consensus biological interpretation between cross-sectional studies. This, combined with the complexity of individual disease pathogenesis, as well as the individual-specific differences in microbiota composition, may explain the inconsistency in observations between different studies concerning the identification of signature microbes for obesity, irritable bowel syndrome and other diseases.

Besides the biological drivers for cohort specific inconsistencies in identified microbial biomarkers, there are also technological factors. Although high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene has transformed the methodological landscape describing microbial diversity within and across complex biomes, evidence is increasing that methodology rather than the biological variation is responsible for observed sample composition and distribution. Large meta-analyses would aid in elucidating whether the basis for these observed inconsistencies is biological, technical or maybe a combination of both. To facilitate these meta-analyses of microbiota studies we developed NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different Mock Communities (MC). NG-Tax demonstrated high robustness against choice of region and other technical biases associated with 16S rRNA gene amplicon sequencing studies. The analysis of α- and β-diversity of these MC confirmed conclusions guided by biology rather than the methodological aspects. This pipeline was applied to biological samples to monitor the developing communities an in vitro gut model (TIM-2) fed either with a normal diet, or modified versions from which the carbohydrate (MPLC) or protein fraction was diluted (LPMC) for 72h. In combination with global metatranscriptomics and metabolomics this revealed that each diet produced distinct microbial communities and temporal patterns and ratios of metabolites. The microbiota in reactors fed diets containing normal carbohydrate levels were enriched in members of the genera Prevotella, Subdoligranulum, Blautia and Bifidobacterium, all associated with carbohydrate fermentation. In turn, the microbiota in the reactors fed the MPLC diet, containing ten-fold less carbohydrates, was enriched in the genus Bacteroides, which is associated with diets rich in protein and animal fat. This setup allows researchers to study the (trophic) interactions and task division within a community and how they are impacted by diet-related factors under controlled conditions, which may assist in defining causal links between specific diet-derived parameters microbial groups and their activities.

In conclusion, currently it seems that GI microbiota based biomarkers associated with metabolic impairments and anthropometric variables associated with the metabolic syndrome are cohort specific or possibly individual, which could partly be due to the use of incompatible analytical approaches. Nevertheless, there is growing evidence that human health is a collective property of the human body and its associated microbiome and thus requires to study the interface of two very complex systems, i.e. on one side the extraordinary coding capacity, high inter-individuality and complex dynamics of the microbiome and on the other side the multifactorial individual nature of human disease. In light of these observations the manifestation of individual dynamics of the microbiota with the host when homeostasis is lost seems plausible and likely.

Biomarkers and mechanisms of natural disease resistance in dairy cows
Altena, S.E.C. van - \ 2016
Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - Wageningen : Wageningen University - ISBN 9789462578005 - 158 p.
dairy cows - biomarkers - disease resistance - immunity - antibodies - proteomics - immune response - dendritic cells - immunology - melkkoeien - ziekteresistentie - immuniteit - antilichamen - eiwitexpressieanalyse - immuniteitsreactie - dendritische cellen - immunologie

The aim of this thesis was to define and test biomarkers for disease resistance in dairy cows and to determine the underlying mechanism in natural disease resistance. The health status of the cows is an important issue in dairy farming. Due to the mandatory reduction in the use of antibiotics, alternatives are required to prevent the development and expression of illness in dairy cows. The identification of biomarkers associated with such disease offers the opportunity to adapt the management of cows at risk, and in this way, prevent them from developing overt disease. Previously, natural antibodies (NAbs) in serum and milk were used as candidate biomarkers for natural disease resistance in cows. In this thesis, we continue on the occurrence and mode of action of NAbs and also focus on their source: the B-1 cells. We performed a literature study on the identification and function of B-1 cells in different species and defined the limitations in the current identification of these cells in pigs, sheep and cows (Chapter 2). B-1 cells were described in cows by using widely accepted cell surface markers CD5 and CD11b. However, in literature several findings suggest that these cell surface markers are not unique markers for B-1 identification. The similarities between mice and veterinary animals in foetal B-cell development and antibody production, implies that B-1 cells are present in cows. In chapter 3, we carefully studied new markers to selectively identify B-1 cells in cows. The combination of B-1 cell markers IgM++ and pSYK++ (indicator constitutive intracellular signalling) identifies a distinct cell population with essential B-1 characteristics such as high CD80 expression. In addition, the development of these B-1 cells in calves before colostrum intake and 3 weeks afterwards shows the same kinetics as the development of NAbs represented by IgM antibodies binding to the well-accepted NAb-antigen phosphatidylcholine (PtC). In calves up to half a year of age, it is shown that the production of such NAbs increases from birth and stabilises from 6 weeks onwards. This implies an endogenous NAb production, which follows the same age-related kinetics as can be expected from B-1 cell development. In contrast, the development of total IgM antibody levels in calves shows a bimodal distribution, which is caused by the uptake and breakdown of maternally-derived IgM and simultaneous endogenous production of specific and natural IgM. Chapter 4 describes the role of such NAbs in bovine immunity. NAbs were represented by the binding of IgM to the naïve antigen keyhole limpet hemocyanin (KLH). Cows with high serum NAb levels were shown to have more IgM and IgG antibodies binding to common microbial structures LPS, LTA and PGN, than cows with low serum NAb levels. In addition, they also have more IgM antibodies binding to intact, fixed E. coli and S. Typhimurium bacteria. However, the killing of live E. coli and S. Typhimurium bacteria via antibody-mediated complement killing does not differ between cows with high and low NAb levels. The antibody-mediated complement killing was determined in a newly developed serum bactericidal test. Cows that performed less in the bactericidal test were more likely to develop mastitis in the future. This association was observed for the killing of E. coli and S. Typhimurium and the development of mastitis within the next one year. For S. Typhimurium the association was still present for the cases of mastitis occurring within four years after testing. Alternative biomarkers for disease resistance in cows were defined in chapter 5 by using a contemporary proteomics approach. Milk samples from high and low disease resistant cows were selected from the “Resilient Cattle” (Weerbaar Vee) biobank. Comparing the spectrum of milk proteins of high and low disease-resistant cows showed potential candidate biomarkers that were elevated in the milk of low-resistant cows. Two candidate marker proteins were validated with ELISA in a new and larger group of high- and low-resistant cows. Lactoferrin (LF) levels were significantly increased in milk of low-resistant cows. In addition, LF levels in milk were associated with clinical manifestations of lameness and had a predictive value for subsequent culling.

In conclusion, we found that also in cows NAbs are produced by B-1 cells that can be identified based on the combined expression of cell surface IgM and internal pSYK. In addition, the frequency of these B-1 cells after birth follows a similar kinetics as described before in mice. These NAbs can be more precisely identified based on their PtC binding ability and their functional activity in a bactericidal test. However, the true predictive value of B-1 cells and NAbs for the health status and immunocompetence of dairy cattle remains to be established. Proteomics turned out to be a useful approach for identifying potential new biomarkers for health and disease in milk of cows. Application and further development of their predictive capacity is dependent on the availability of robust, sensitive and quantitative assays. This project was part of the “Resilient Cattle” project providing biological samples and essential data on the health status during respective lactation periods of individual dairy cows. The impact of this research now requires translation into management tools and principles for the individual farmer impacting on the overall health status and economic performance of his herd of dairy cattle.

Novel biomarkers for mitochondrial health
Boer, Vincent de - \ 2015
biomarkers - mitochondrial health
A technological and physiological integrated approach for appetite control : from identification of novel biomarkers to development of new functional ingredients
Mennella, I. - \ 2015
Wageningen University. Promotor(en): Vincenzo Fogliano, co-promotor(en): P. Vitaglione. - Wageningen : Wageningen University - ISBN 9789462575448 - 138
eetlustcontrole - perceptie - voedselvoorkeuren - speeksel - cannabinoïden - biomarkers - ingrediënten - ontwikkeling - gewichtscontrole - appetite control - perception - food preferences - saliva - cannabinoids - ingredients - development - weight control

A technological and physiological integrated approach for appetite control.

From identification of novel biomarkers to development of new functional ingredients.

Human dietary behaviour is driven by homeostatic, hedonic and environmental factors. Foods can

influence these factors throughout extrinsic (marketing suggestions, portion sizes, form) and

intrinsic characteristics (taste, flavour, smell, texture). In turn biochemical response and

psychological traits influenced food taste, flavour, smell and texture perception determining the

hedonic value of a meal. This interplay between the food and the subjective psychophysiological

response determine the control of energy intake, therefore must be considered in developing food

for appetite control.

In the present thesis four human studies are described. Of these two were conducted to investigate

the role of the saliva and the endocannabinoids system in the food preference and liking during the

cephalic phase of digestion. We found out that salivary enzymes activity are influenced by

nutritional status, food preference and food habits. Moreover, food palatability influenced some

plasma endocannabinoid and N-acylethanolamine concentrations during the cephalic phase

response and indicated that 2-arachidonoylglycerol and pancreatic polypeptide can be used as

biomarkers of food liking in humans. These findings can have interesting implications in designing

foods for appetite control:

­ salivary enzymatic activity must be considered because it influence taste and texture

perception and consequently food choice;

­ the measure of 2-arachidonoylglycerol can offer the possibility to merge the sensory and

biochemical approach to compare the satiating and rewarding capacity of foods.

The other two studies investigated the potential satiating effect on the short term energy intake of

specific food ingredients. As previous in animal studies shown, we demonstrated (chapter 4) that

also in humans the circulating oleoylethanolamide levels can be modulated by the fatty acid

composition of a meal and this can influence the short-term energy intake. Therefore, we

highlighted the anorexigenic effect of the oleoylethanolamide that can be a target of specific food

ingredients. In the study described in the chapter 5, we aimed in assessing the appetite control

capability of bitter compounds. The ingredient was microencapsulated with the double aim to avoid

the (not palatable) taste perception in the mouth and to deliver the compounds directly in the

gastrointestinal tract and target the enteroendocrine bitter taste receptors. We showed that

microencapsulated bitter compounds are effective to reduce daily energy intakes in humans. This

study demonstrated that sense the taste receptors directly in the gastrointestinal tract may be a valid

way to trigger satiety and control appetite.

The general conclusions of the present thesis are that a fine design of ingredients for appetite

control is necessary to develop novel foods for appetite control that has to take in account from one

side the hedonic value from the other side the functionality.

New applications of the interaction between diols and boronic acids
Duval, F.L. - \ 2015
Wageningen University. Promotor(en): Han Zuilhof, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789462574717 - 131
antilichamen - immobilisatie - boorzuur - biomarkers - vloeistofchromatografie - antibodies - immobilization - boric acid - liquid chromatography

Florine Duval - New applications of the interaction between diols and boronic acids – Summary

Chapter 1 introduces the theory and known applications of the interaction between boronic acids and diols, and explains the context of this thesis. Diagnosis of depression was the initial goal of this multidisciplinary project. The focus of the PhD project was the development of a strategy to immobilize antibodies on the surface of a chip in such a way that very low concentrations (~ 1 pM) of biomarkers for depression could be detected in urine. To achieve this, the immobilization of antibodies using boronic acids seemed promising.

However, preliminary experiments and further insights revealed the many challenges that this immobilization strategy faces, giving rise to Chapter 2. This chapter discusses several important points that need to be taken into account when one plans to immobilize antibodies via boronic acids: choice of the boronic acid structure and spacer to attach it to the surface, use of an antifouling polymer, choice of an antibody with suitable glycosylation, optimization of the conditions for antibody immobilization...

One big issue for antibody immobilization using boronic acids is the reversibility of the reaction between boronic acids and diols, hence the possible release of the antibody from the surface.

Chapter 3 describes the design and synthesis of boronic acid-containing linkers that would enable the oriented and irreversible immobilization of antibodies. Two linkers were designed with an amine for surface attachment, a boronic acid for capturing antibodies via the N-glycans in their Fc chain, and a diazirine for irreversible immobilization upon UV irradiation while maintaining antibody orientation. From a diazirine building-block that was obtained in three steps, the first linker was synthesized in four steps and the second linker was synthesized in three steps. Diol-functionalized silica was used for the chromatography of two boronic acid-containing intermediates, this method being novel (to the best of our knowledge) and likely based on boronic acid-diol interactions. High-resolution mass spectrometry, through matching exact masses, matching isotope patterns and observation of species corresponding to the esterification of boronic acids with MeOH, confirmed that both linkers were synthesized successfully.

During the synthesis of boronic acid-containing linkers, it was difficult to see which spots on TLC plates corresponded to boronic acids. To solve this problem, a new TLC staining method based on the reaction between boronic acids and alizarin was developed.

Chapter 4 presents this work in detail. After optimization experiments, 1 mM alizarin in acetone was shown to be the preferred staining solution. When the TLC plate was briefly dipped in this solution, allowed to dry in ambient air and observed under 365 nm light, bright yellow fluorescent spots were observed where boronic acids were present. Phenylboronic acid was detected at a concentration as low as 0.1 mM. A range of boronic acids and derivatives was successfully detected, and boron-free compounds resulted in no or very weak fluorescence. The staining method was further tested in the monitoring of three reactions involving boronic acids, and provided clear information about the consumption or formation of boronic acid-containing compounds.

Although TLC is useful to synthetic chemists, analysis of reaction mixtures by HPLC is sometimes necessary for obtaining more accurate information or for optimization of preparative HPLC conditions.

Chapter 5 presents the development and applicability of a method for the on-line HPLC detection of boronic acids using alizarin. After optimization experiments at an HPLC flow rate of 0.40 mL/min, the HPLC-separated analytes were mixed post-column with a solution of 75 μM alizarin and 0.1% triethylamine in ACN, which was delivered at a flow rate of 0.60 mL/min. The reaction between alizarin and boronic acids occurred in a reaction coil of dimensions of 3.5 m × 0.25 mm at a temperature of 50 °C, resulting in fluorescent complexes that were detected as positive peaks by a fluorescence detector (lexc 469 nm and lem 610 nm). The method enabled the selective detection of various boronic acids and derivatives, with a limit of detection of phenylboronic acid of 1.2 ng or 1 μM. It could successfully monitor the progress of two organic reactions involving boronic acid-containing compounds, and provided useful insights into the course of the reactions.

Chapter 6 provides a reflexion about the work presented in this thesis, suggestions for future research, and a general conclusion.

Improvement of risk assessment by integrating toxicological and epidemiological approaches: the case of isoflavones
Islam, M.A. - \ 2015
Wageningen University. Promotor(en): Ivonne Rietjens; Rolaf van Leeuwen; Tinka Murk. - Wageningen : Wageningen University - ISBN 9789462574649 - 174
isoflavones - glucosides - soyabeans - toxic substances - bioavailability - biomarkers - human nutrition research - risk-benefit analysis - gene expression - isoflavonen - glucosiden - sojabonen - toxische stoffen - biologische beschikbaarheid - voedingsonderzoek bij de mens - risico-baten analyse - genexpressie

Improvement of risk assessment by integrating toxicological and epidemiological approaches: the case of isoflavones

PhD-thesis Mohammed Ariful Islam

This thesis describes the results of a research project that aimed at the improvement of the risk/benefit assessment of soy isoflavones (SIF) by combining toxicological and epidemiological methods. The toxicological studies were carried out at the Department of Toxicology and part of the results were compared with the outcome of human intervention studies, that were carried out in parallel research project at the Division of Human Nutrition. In Chapter 1 it is explained why we considered such an integrated “tox-epi” approach to be useful for the prediction of possible effects of SIF in humans on the basis of animal data. SIF are constituents of soy based supplements, which became more and more popular in Western societies over the last decades, because of their putative beneficial health effects, that were related to the SIF present in these supplements. In spite of the long and safe history of soy consumption by the East and the South-East Asian population, the benefit and safety of soy have been challenged in recent years and concerns have been raised about possible adverse health effects. These concerns focussed primarily on the weak estrogenic and proliferative effects of SIF. Chapter 1 also provides some background information on the individual SIF, their structural similarity with the steroid hormone estradiol (E2) and their interaction with the estrogen receptors ERα and ERβ.

Chapter 2 describes the differences between rats and humans in the conversion of the three major soy isoflavone glucosides, daidzin, genistin and glycitin, and their aglycones in a series of in vitro models. Results of studies in a Caco-2 transwell model confirmed that deconjugation of the isoflavone glucosides is essential for their transport across the intestinal barrier. It was shown that both rat and human intestinal S9 fractions were able to deconjugate the glucosides, and that intestinal enzymes plaid an important role in this deconjugation reaction. It was demonstrated that in the rat lactase phlorizin hydrolase, glucocerebrosidase, and cytosolic broad-specificity β-glucosidase contribute significantly to this deconjugation, and that in humans deconjugation mainly appeared to occur through the activity of broad-specificity β-glucosidase. Species difference in glucuronidation and sulfation were smaller than for the deconjugation reaction, and it was shown that 7-O-glucuronides were the major metabolites for all the three isoflavone aglycones. The in vitro results also indicated that glucuronidation in rats might be more efficient than in humans, again pointing towards species differences in the metabolism of isoflavone glycosides between rats and humans. It was also shown that the reconjugation reaction has a larger catalytic efficiency than the deconjugation of the glucosides, which corroborates that the detection of aglycones in the systemic circulation is unlikely.

It has been reported in literature that following administration of SIF to humans or animals, these compounds are mainly (~98%) present in the systemic circulation in their conjugated form (i.e. as glucuronide and sulphate) of which the estrogenic potency is not yet clear. Chapter 3 provides evidence that in an intact cellular model the major SIF glucuronide metabolites in blood, genistein-7-O-glucuronide (GG) and daidzein-7-O-glucuronide (DG), only become estrogenic after deconjugation. The estrogenic potencies of genistein (Ge), daidzein (Da), GG and DG were determined using stably transfected U2OS-ERα, U2OS-ERβ reporter gene cells and proliferation was tested in T47D-ERβ and in T47D breast cancer cells. In all these assays the estrogenic potency of the aglycones was significantly higher than that of their corresponding glucuronides. UPLC analysis revealed that in the in vitro cell line assays, 0.2-1.6% of the glucuronides were deconjugated to their corresponding aglycones. It was also found that, under similar experimental conditions, rat breast tissue S9 fraction was about 30 times more potent in deconjugating these glucuronides than human breast tissue S9 fraction. The results presented in Chapter 3 confirm that SIF glucuronides are not estrogenic as such when tested in an intact cellular model system, and that the small fraction of aglycones account for the observed estrogenic effects. They also provide evidence for a significant species difference in the metabolism of SIF.

In Chapters 4 and 5 of this thesis, two rat studies are described, that were performed to further elucidate important modes of action underlying biological effects of SIF and to facilitate an interspecies comparison of the effects observed in rats with those observed in human intervention studies. In these studies inbred ovariectomized Fischer344 rats were used, as an animal model for (post)menopausal women. In the first study described in Chapter 4, two dose levels (i.e. 2 and 20 mg/kg bw) were used to characterise plasma bioavailability, urinary and faecal concentrations of SIF and to investigate changes in gene expression in peripheral blood mononuclear cells (PBMC). The low dose was in line with the type of dosing relevant for human supplement use. Animals were dosed at 0 and 48 hr and sacrificed 4 hr after the last dose. A clear dose dependent increase of SIF concentrations in plasma, urine and faeces was observed, together with a strong correlation in changes in gene expression between the two dose groups. In the transcriptomic analysis, all estrogen responsive genes (ERG) and related biological pathways (BPs) that were found to be affected by the SIF treatment were regulated in both dose groups in the same direction, and indicate possible beneficial effects of SIF. However, most of the common genes in PBMC of rats and of (post)menopausal women, exposed to a comparable dose of the same supplement, were regulated in opposite direction. Thus based on these results no correlation was found between the changes in gene expression in rats and humans, leading to the conclusion that rats might not be a suitable model for humans.

In Chapter 5 an animal experiment is described, in which rats received a dose of 2 mg SIF/kg body weight per day for a period of eight weeks. This dosing regimen was similar as that of the parallel human intervention study. Changes in gene expression in different target (i.e. breast (BT), uterus (UT) and sternum (ST)) and non-target (i.e. peripheral blood mononuclear cells (PBMC), adipose (AT) and liver (LT)) tissues were compared. Rank-rank scattered plots did not show any correlation in gene expression changes among different tissues. Out of 87 estrogen responsive genes (ERG), only 19 were found to be significantly regulated (p<0.05) in different tissues. The significantly regulated ERG were mostly found in LT, AT and UT. Surprisingly, no ERG were significantly regulated in BT and ST, although these are considered to be important estrogen sensitive target tissues. No correlation was observed with the changes in gene expression in the PBMC of two rat studies. Correlation was also not seen in the changes of gene expression in PBMC and adipose tissue between rat and humans.

In Chapter 6 the results of the research project described in this thesis are evaluated. It was the aim of thesestudies to contribute to theimprovement ofthe risk and/or benefit assessment of SIF for humans, by using in vitro and in vivo animal and human models, and gene expression data in various animal and human tissues, as early biomarkers of effects of exposure to SIF. Although important information has been gathered on the metabolism and the estrogenic activity of SIF and their aglycones, we were not able to predict possible effects in human target tissues based on the results of changes in gene expression in target tissues obtained in the 8 weeks rat study. Possibly aged rats might be a more appropriate model than young ovariectomized rats.

Allostasis and Resilience of the Human Individual Metabolic Phenotype
Ghini, V. ; Saccenti, E. ; Tenori, L. ; Assfalg, M. ; Luchinat, C. - \ 2015
Journal of Proteome Research 14 (2015)7. - ISSN 1535-3893 - p. 2951 - 2962.
nmr metabolomics - gut microbiota - health - stress - biomarkers - nutrition - disease - urine - load - discovery
The urine metabotype of 12 individuals was followed over a period of 8-10 years, which provided the longest longitudinal study of metabolic phenotypes to date. More than 2000 NMR metabolic profiles were analyzed. The majority of subjects have a stable metabotype. Subjects who were exposed to important pathophysiological stressful conditions had a significant metabotype drift. When the stress conditions ceased, the original metabotypes were regained, while an irreversible stressful condition resulted in a permanent metabotype change. These results suggest that each individual occupies a well-defined region in the broad metabolic space, within which a limited degree of allostasis is permitted. The insurgence of significant stressful conditions causes a shift of the metabotype to another distinct region. The spontaneous return to the original metabolic region when the stressful conditions are removed suggests that the original metabotype has some degree of resilience. In this picture, precision medicine should aim at reinforcing the patient's metabolic resilience, that is, his or her ability to revert to his or her specific metabotype rather than to a generic healthy one
Surface functionalization and analysis thereof by ambient mass spectrometry
Manova, R.K. - \ 2014
Wageningen University. Promotor(en): Han Zuilhof, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789462571570 - 214
biosensoren - detectie - biomarkers - allergenen - oppervlaktechemie - analytische methoden - synthese - unimoleculaire films - biosensors - detection - allergens - surface chemistry - analytical methods - synthesis - unimolecular films

A challenge in the global healthcare is the lack of suitable diagnostic tools for early disease detection. One possible solution is the use of biosensors in diagnostic tests. By definition, a biosensor is a bioanalytical device that detects the presence of a compound (analyte) in the sample. The detection relies on the specific interactions between the ligands that are attached onto the biosensor surface and the analytes in the sample.

This PhD dissertation is focused on developing an optimal protocol for attachment of ligands onto the biosensing surface. A step-wise approach was established for the versatile and reproducible modification and functionalization of a silicon nitride-based biosensor. This approach included the application of bioorthogonal copper-free reactions as a useful tool for oriented attachment of biomolecules. Additionally, a novel surface sensitive analytical method was developed for the identification of covalently bound molecules in monolayers. The method, which is fast and easy to apply, uses DART ionization coupled to a high-resolution mass spectrometer. The nm-thin layers were analysed, and interpretation rules for the obtained mass spectra were formulated. The method was applied in the identification of commercially available nm-thin coatings and biochips.

Plasma and dietary carotenoids and vitamines A,C and E and the risk of colon and rectal cancer in the European prospective investigation into cancer and nutrition
Leenders, M. ; Leufkens, A.M. ; Siersema, P.D. ; Duijnhoven, F.J.B. van; Vrieling, A. ; Hulshof, P.J.M. - \ 2014
International Journal of Cancer 135 (2014)12. - ISSN 0020-7136 - p. 2930 - 2939.
serum alpha-tocopherol - colorectal-cancer - oxidative stress - physical-activity - epic project - antioxidants - retinol - health - cohort - biomarkers
Carotenoids and vitamins A, C and E are possibly associated with a reduced colorectal cancer (CRC) risk through antioxidative properties. The association of prediagnostic plasma concentrations and dietary consumption of carotenoids and vitamins A, C and E with the risk of colon and rectal cancer was examined in this case-control study, nested within the European Prospective Investigation into Cancer and Nutrition study. Plasma concentrations of carotenoids (alpha- and beta-carotene, canthaxanthin, beta-cryptoxanthin, lutein, lycopene, zeaxanthin) and vitamins A (retinol), C and E (alpha-, beta- and gamma-and delta-tocopherol) and dietary consumption of beta-carotene and vitamins A, C and E were determined in 898 colon cancer cases, 501 rectal cancer cases and 1,399 matched controls. Multivariable conditional logistic regression models were performed to estimate incidence rate ratios (IRR) and corresponding 95% confidence intervals (CIs). An association was observed between higher prediagnostic plasma retinol concentration and a lower risk of colon cancer (IRR for highest quartile = 0.63, 95% CI: 0.46, 0.87, p for trend = 0.01), most notably proximal colon cancer (IRR for highest quartile = 0.46, 95% CI: 0.27, 0.77, p for trend = 0.01). Additionally, inverse associations for dietary beta-carotene and dietary vitamins C and E with (distal) colon cancer were observed. Although other associations were suggested, there seems little evidence for a role of these selected compounds in preventing CRC through their antioxidative properties.
Nutritional intervention in animals: benchmarking of strategies, monitoring biomarkers and immune competence
Krimpen, M.M. van; Hulst, M.M. ; Meulen, J. van der; Schokker, D. ; Savelkoul, H.F.J. ; Tijhaar, E.J. ; Rutten, V.P.M.G. - \ 2014
Wageningen : Wageningen UR Livestock Research (Livestock Research report 800) - 116
maatregel op voedingsgebied - biomarkers - adequate immuniteit - vee - diergezondheid - immuunsysteem - biologische processen - veehouderij - voeding en gezondheid - diervoedering - dierenwelzijn - nutritional intervention - immune competence - livestock - animal health - immune system - biological processes - livestock farming - nutrition and health - animal feeding - animal welfare
The current study covers a review of literature regarding a number of topics related to immune competence in farm animals, which are introduced in chapter 1. These topics are: 1) A demonstration of the relationship between functional feed components and the expression of genes/biological processes that are involved in gut health of farm animals; 2) A description of available models, that can be used to investigate the effects of nutritional interventions on immune related parameters in animals; 3) A review of the effects of nutritional interventions in the maternal, neonatal and post-neonatal phase on the development of the innate and acquired immune system; 4) A review of the relationship between the immune system in the gut and in the upper airways, whereas the question will be addressed how the immune system in the upper airways can be affected by nutritional interventions.
Effect of moderate alcohol consumption on fetuin-A levels in men and women: post-hoc analyses of three open-label randomized crossover trials
Joosten, M.M. ; Schrieks, I.C. ; Hendriks, H.F.J. - \ 2014
Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2014). - ISSN 1871-4021 - 5 p.
community-dwelling adults - insulin-resistance - cardiovascular-disease - rancho bernardo - older-adults - risk - association - metaanalysis - expression - biomarkers
Background Fetuin-A, a liver-derived glycoprotein that impairs insulin-signalling, has emerged as a biomarker for diabetes risk. Although moderate alcohol consumption has been inversely associated with fetuin-A, data from clinical trials are lacking. Thus, we evaluated whether moderate alcohol consumption decreases circulating levels of fetuin-A. Methods We analyzed data of three separate open-label, randomized, crossover trials: 1) 36 postmenopausal women consuming 250 ml white wine (25 g alcohol) or white grape juice daily for 6 weeks, 2) 24 premenopausal women consuming 660 ml beer (26 g alcohol) or alcohol-free beer daily for 3 weeks, and 3) 24 young men consuming 100 ml vodka (30 g alcohol) orange juice or only orange juice daily for 4 weeks. After each treatment period fasting blood samples were collected. Results Circulating fetuin-A concentrations decreased in men after vodka consumption (Mean¿±¿SEM: 441¿±¿11 to 426¿±¿11 µg/ml, p¿=¿0.02), but not in women after wine (448¿±¿17 to 437¿±¿17 µg/ml, p¿=¿0.16) or beer consumption (498¿±¿15 to 492¿±¿15 µg/ml, p¿=¿0.48) compared to levels after each corresponding alcohol-free treatment. Post-hoc power analyses indicated that the statistical power to detect a similar effect as observed in men was 30% among the postmenopausal women and 31% among the premenopausal women. Conclusions In these randomized crossover trials, moderate alcohol consumption decreased fetuin-A in men but not in women. This sex-specific effect may be explained by the relatively short intervention periods or the low statistical power in the trials among women.
Integrative cross-omics analysis in primary mouse hepatocytes unravels mechanisms of cyclosporin A-induced hepatotoxicity
Hof, W.F.P.M. ; Summeren, A. van; Lommen, A. ; Coonen, M.L.J. ; Brauers, K. ; Herwijnen, M. van; Wodzig, W.K.W.H. ; Kleinjans, J.C.S. - \ 2014
Toxicology 324 (2014). - ISSN 0300-483X - p. 18 - 26.
drug-induced hepatotoxicity - gene-expression - micrornas - repression - normalization - accumulation - metabonomics - translation - activation - biomarkers
The liver is responsible for drug metabolism and drug-induced hepatotoxicity is the most frequent reason for drug withdrawal, indicating that better pre-clinical toxicity tests are needed. In order to bypass animal models for toxicity screening, we exposed primary mouse hepatocytes for exploring the prototypical hepatotoxicant cyclosporin A. To elucidate the mechanisms underlying cyclosporin A-induced hepatotoxicity, we analyzed expression levels of proteins, mRNAs, microRNAs and metabolites.
The effect of a six-month resistance-type exercise training program on the course of high sensitive cardiac troponin T levels in (pre)frail elderly
Linden, N. van der; Tieland, C.A.B. ; Klinkenberg, L.J.J. ; Verdijk, L. ; Groot, C.P.G.M. de; Loon, L.J.C. van; Dieijen-Visser, M.P. ; Meex, S.J.R. - \ 2014
International Journal of Cardiology 175 (2014)2. - ISSN 0167-5273 - p. 374 - 375.
placebo-controlled trial - older-adults - double-blind - biomarkers - frailty - stress - people
Healthy diet indicator and mortality in Eastern European populations: prospective evidence from the HAPIEE cohort
Stefler, D. ; Pikhart, H. ; Jankovic, N. ; Kubinova, R. ; Pajak, A. ; Malyutina, S. ; Simonova, G. ; Feskens, E.J.M. ; Peasey, A. ; Bobak, M. - \ 2014
European Journal of Clinical Nutrition 68 (2014). - ISSN 0954-3007 - p. 1346 - 1352.
food-frequency questionnaire - coronary-heart-disease - physical-activity - risk-factors - elderly-men - carotenoids - biomarkers - nutrition - validity - quality
Background/Objectives: Unhealthy diet has been proposed as one of the main reasons for the high mortality in Central and Eastern Europe (CEE) and the former Soviet Union (FSU) but individual-level effects of dietary habits on health in the region are sparse. We examined the associations between the healthy diet indicator (HDI) and all-cause and cause-specific mortality in three CEE/FSU populations. Subjects/Methods: Dietary intakes of foods and nutrients, assessed by food frequency questionnaire in the Health, Alcohol and Psychosocial Factors in Eastern Europe (HAPIEE) cohort study, were used to construct the HDI, which follows the WHO 2003 dietary recommendations. Among 18¿559 eligible adult participants (age range: 45–69 years) without a history of major chronic diseases at baseline, 1209 deaths occurred over a mean follow-up of 7 years. The association between HDI and mortality was estimated by Cox regression. Results: After adjusting for covariates, HDI was inversely and statistically significantly associated with cardiovascular disease (CVD) and coronary heart disease (CHD) mortality, but not with other cause-specific and all-cause mortality in the pooled sample. Hazard ratios per one standard deviation (s.d.) increase in HDI score were 0.95 (95% confidence interval=0.89–1.00, P=0.068), 0.90 (0.81–0.99, P=0.030) and 0.85 (0.74–0.97, P=0.018) for all-cause, CVD and CHD mortality, respectively. Population attributable risk fractions for low HDI were 2.9% for all-cause, 14.2% for CVD and 10.7% for CHD mortality. Conclusions: These findings support the hypothesis that unhealthy diet has had a role in the high CVD mortality in Eastern Europe.
Diet quality and markers of endothelial function: The CARDIA study
Sijtsma, F.P.C. ; Meyer, K.A. ; Steffen, L.M. ; Horn, L. van; Shikany, J.M. ; Odegaard, A.O. ; Gross, M.D. ; Kromhout, D. ; Jacobs, D.R. - \ 2014
Nutrition, Metabolism & Cardiovascular Diseases 24 (2014)6. - ISSN 0939-4753 - p. 632 - 638.
cardiovascular-disease risk - plasma-concentrations - atherosclerosis mesa - young-adults - inflammation - patterns - dysfunction - biomarkers - men
Background and aim: Dietary patterns are associated cross-sectionally with cellular adhesion molecules (CAMs). We studied prospective associations of three dietary patterns with CAMs. Methods and results: In the Coronary Artery Risk Development in Young Adults (CARDIA) study, diet was assessed at years 0 (1985-86) and 7 (1992-93) examinations. Four circulating CAMs (E-selectin, P-selectin, soluble intercellular adhesion molecule 1 (sICAM-1), and vascular cellular adhesion molecule (VCAM)) were assayed at years 7 and 15 (2000-01). We created one index score "A Priori Diet Quality Score" and derived dietary patterns using principal components analysis (PCA). Multivariable linear regression models predicted year 15 CAMs from averaged (year 0/7) dietary patterns. The A Priori Diet Quality Score rated 46 food groups beneficial, neutral or adverse based on hypothesized health effects. We derived two PCA dietary patterns: "fruit and vegetables (FV)" (high intakes of fruit, vegetables, and whole grains) and "meat" (high intakes of red meat, refined grain, and butter). All dietary patterns were related to E-selectin and sICAM-1. P-selectin was not related to the FV dietary pattern. VCAM was only related to the A Priori Diet Quality Score. Strongest associations were for the meat dietary pattern with E-selectin (effect size 28% of an SD (+3.9/13.7 ng/mL)) and P-selectin (effect size 37% of an SD (+4.1/11.2 ng/mL)) and the A Priori Diet Quality Score with sICAM-1 (effect size 34% of an SD (-15.1/44.7 ng/mL)) and VCAM (effect size of 26% of an SD (-45.1/170.3 ng/mL)). Conclusion: This prospective analysis suggests that dietary patterns are associated with CAMs. (C) 2014 Elsevier B. V. All rights reserved.
Protein biomarker-based screening for detection of recombinant bovine somatotropin abuse in dairy cows
Ludwig, S.K.J. - \ 2014
Wageningen University. Promotor(en): Michel Nielen, co-promotor(en): Leen van Ginkel. - Wageningen : Wageningen University - ISBN 9789462570146 - 248
somatotropine - melkvee - besmetters - biomarkers - recombinant eiwitten - toxicologie - detectie - somatotropin - dairy cattle - contaminants - recombinant proteins - toxicology - detection

Recombinant bovine somatotropin (rbST) is a 22 kDa proteohormone, which can be used to increase milk production in dairy cows. It has been marketed since 1994 and while its use in food production is approved in several countries, such as the US, it is banned in the EU since 2000. To enforce the ban on rbST in the EU and to control for ‘rbST-free’ –labelling in the US, detection methods are required that identify whether rbST has been used. Existing rbST detection methods focus on the detection of rbST itself in bovine serum. The recombinant form of the hormone has one amino acid exchanged at the N-terminus of the protein. RbST can therefore be potentially discriminated from the endogenous bST by mass spectrometric methods. Other methods employ sandwich enzyme-linked immunosorbent assays (ELISAs) with antibodies having a higher affinity to rbST than bST. These methods, however mainly lack sensitivity, reproducibility or selectivity for rbST and are therefore not widely applied. Hence, no method has been implemented so far to monitor rbST abuse in dairy farming. Screening methods developed for veterinary drug residue control in the EU have to perform according to Commission Decision 2002/657/EC and have to identify at least 95 % of the treated animals.

An alternative approach for rbST abuse detection is the analysis of rbST-dependent biomarkers. A biomarker is defined as an indicator of normal physiological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Therefore, the levels of rbST-dependent protein biomarkers are either up- or downregulated after administration of rbST and rbST-specific biomarker profiles can be used to detect its abuse. RbST exerts similar physiological actions in the cow’s body as the endogenous bST. Therefore, proteins involved in the regulatory circuit of bST have been chosen as candidate biomarkers, such as insulin-like growth factor-1 (IGF-1), IGF binding protein 2 (IGFBP2) and osteocalcin. Additionally to that, the administration of rbST induces anti-rbST antibodies in the cow’s body, which can be detected as biomarkers. This approach is according to the growth hormone (GH) abuse detection in sports doping control, where solely protein biomarker profiles are used to identify the abuse.

Chapter 2 introduces protein biomarkers and how biomarkers can be used in sports doping and veterinary control to detect the abuse of illegal substances. The advantages of using biomarkers are that the biological effect of a substance usually lasts longer than the substance itself can be detected and therewith, the window of detection is expanded. Moreover, since different substances exert similar effects on physiological machineries for growth or production enhancement, biomarker-based-detection methods have the potential to detect a whole class of substances. Furthermore, low-dose mixtures of different banned substances, which might escape from direct detection of each individual substance used, could be still detected by the combined effect they exert. In this chapter, protein biomarker-based detection strategies are discussed against generic challenges in biomarker discovery and method development.

Part I of the thesis concerns biomarker analysis in serum and plasma samples from cattle, which are analysed using laboratory-based equipment.A triplex flow cytometric immunoassay (FCIA), which combines the detection of three rbST-dependent biomarkers, viz. IGF-1, IGFBP2 and anti-rbST antibodies is demonstrated in Chapter 3. Serum samples from treated and untreated dairy cows from a single animal study were analysed using this triplex FCIA. Characteristic treatment-dependent responses for all three individual biomarkers were shown. These results were combined using the statistical model k-nearest neighbours (kNN). This model discriminated rbST-treated from untreated cows with a truepositive rate of 89.1 % and a true-negative rate of 97.7 %.

This triplex FCIA was further extended with the biomarker osteocalcin and the resulting fourplex FCIA was used for biomarker profiling in serum samples from rbST-treated and untreated cows from two independent rbST treatment studies. In Chapter 4, different data analysis approaches were tested with the aim to detect the highest possible number of true-positive samples. The statistical model kNN was used on all 11 possible biomarker combinations and the combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95 % of the samples of treated cows starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12 % of the samples of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control.

It can be expected that rbST-dependent biomarkers also show a response upon other treatments. Therefore in Chapter 5, the fourplex FCIA for rbST abuse detection was applied to bovines treated with steroids, such as estradiol, dexamethasone and prednisolone. Each treatment resulted in a specific plasma biomarker profile for IGF-1, IGFBP2, osteocalcin and anti-rbST antibodies, which could be distinguished from the profile of untreated animals. Therefore, the fourplex biomarker FCIA is, apart from rbST, also capable of detecting treatment with other growth-promoting agents and clearly shows the potential of biomarker profiling as a screening method in veterinary control.

Part II of the thesis focusses on protein biomarker analysis in milk samples and the change from laboratory-based to on-site analysis. In Chapter 6, the detection of anti-rbST antibodies in raw milk samples was demonstrated, which discriminated rbST-treated from untreated cows with a 67 % true-positive and 94 % true-negative rate. The laboratory-based assay was also applied to simulated tank milk and pasteurized milk samples. Using milk as a sample matrix for detection has the advantages of non-invasive sampling, and for tank milk analysis at the farm only one milk sample is needed to screen the whole farm for rbST (ab)use.

As a next step in Chapter 7, this assay was translated to an on-site pre-screening platform including a cellphone. Using this on-site platform, samples can be tested at the point where they were taken. Only samples that are suspect are transported to a laboratory for further analysis. To this end, a cellphone-based fluorescence imaging platform for the detection of anti-rbST antibodies in milk extracts was developed, which is based on a microsphere fluorescence immunoassay. After performing the assay, the fluorescence is excited by UV LEDs embedded in a dedicated cellphone attachment and the emitted fluorescence light is imaged by the cellphone camera. The fluorescence micro-images were analysed using a custom-developed Android application running on the same cellphone and milk samples from rbST-treated and untreated cows were discriminated.

Also in milk samples, the simultaneous detection of several biomarkers is advantageous as they can increase the confidence of a positive finding. Therefore in Chapter 8, a protein microarray-based platform for multiple rbST biomarker detection on a cellphone is presented, which detects anti-rbST antibodies and IGF-1 in milk samples. The 48 microspots on the microarray were labelled with Quantum Dots depending on the biomarker levels in the sample. Quantum Dot fluorescence was detected by the cellphone camera and the same opto-mechanical attachment as in Chapter 7 and images were analysed by custom software. RbST-treated clearly showed a treatment-dependent biomarker profile in milk that could be discriminated from the profile of untreated cows.

Future research should focus on the simultaneous detection of different targets of interest in milk samples, such as hormones, allergens, antibiotics, contaminants and other substances, all at the same time using the microarray platform on the cellphone. Moreover, sample handling can be facilitated by the use of pre-fabricated microfluidic devices including all required assay reagents. With the work presented in this thesis, screening for rbST abuse in serum and milk becomes possible: in the laboratory and on-site. The future implementation of these testing platforms for rbST abuse detection is a major leap forward concerning the enforcement of the rbST ban in the EU and concerning the value of protein biomarker-based approaches in veterinary control.

Milk biomarker discovery for detection of recombinant bovine somatotropin abuse
Ludwig, S.K.J. ; Smits, N.G.E. ; Nielen, M.W.F. - \ 2013
somatotropine - melkeiwit - melkkoeien - biomarkers - identificatie - somatotropin - milk protein - dairy cows - identification
The objective of the research is the identification of proteins in milk, whose concentrations are significantly increased or decreased after treating dairy cows with rbST. These identified milk protein biomarkers will be the basis of a screening method to detect rbST doping in dairy cows.
N-6 and N-3 Fatty Acid Cholesteryl Esters in Relation to Fatal CHD in a Dutch Adult Population: A Nested Case-Control Study and Meta-Analysis
Goede, J. de; Verschuren, W.M.M. ; Boer, J.M.A. ; Verberne, L.D.M. ; Kromhout, D. ; Geleijnse, J.M. - \ 2013
PLoS ONE 8 (2013)5. - ISSN 1932-6203 - 9 p.
coronary-heart-disease - alpha-linolenic acid - cardiovascular-disease - dietary-intake - erythrocyte-membranes - adipose-tissue - humans - risk - biomarkers - cohort
Background: Dietary polyunsaturated fatty acids (PUFA) are inversely related to coronary heart disease (CHD) in epidemiological studies. We examined the associations of plasma n-6 and n-3 PUFA in cholesteryl esters with fatal CHD in a nested case-control study. Additionally, we performed a dose-response meta-analysis of similar prospective studies on cholesteryl ester PUFA. Methods: We used data from two population-based cohort studies in Dutch adults aged 20-65y. Blood and data collection took place from 1987-1997 and subjects were followed for 8-19y. We identified 279 incident cases of fatal CHD and randomly selected 279 controls, matched on age, gender, and enrollment date. Odds ratios (OR) were calculated per standard deviation (SD) increase of cholesteryl ester PUFA. Results: After adjustment for confounders, the OR (95% CI) for fatal CHD per SD increase in plasma linoleic acid was 0.89 (0.74-1.06). Additional adjustment for plasma total cholesterol and systolic blood pressure attenuated this association (OR:0.95; 95% CI: 0.78-1.15). Arachidonic acid was not associated with fatal CHD (OR per SD:1.11; 95% CI: 0.92-1.35). The ORs (95% CI) for fatal CHD for an SD increase in n-3 PUFA were 0.92 (0.74-1.15) for alpha-linolenic acid and 1.06 (0.88-1.27) for EPA-DHA. In the meta-analysis, a 5% higher linoleic acid level was associated with a 9% lower risk (relative risk: 0.91; 95% CI: 0.84-0.98) of CHD. The other fatty acids were not associated with CHD. Conclusion: In this Dutch population, n-6 and n-3 PUFA in cholesteryl esters were not significantly related to fatal CHD. Our data, together with findings from previous prospective studies, support that linoleic acid in plasma cholesteryl is inversely associated with CHD.
N-6 and n-3 fatty acid cholesteryl esters in relation to incident stroke in a Dutch adult population: A nested case-control study
Goede, J. de; Verschuren, W.M.M. ; Boer, J.M.A. ; Kromhout, D. ; Geleijnse, J.M. - \ 2013
Nutrition, Metabolism & Cardiovascular Diseases 23 (2013)8. - ISSN 0939-4753 - p. 737 - 743.
food frequency questionnaire - alpha-linolenic acid - cardiovascular-disease - dietary-intake - erythrocyte-membranes - adipose-tissue - fish intake - humans - risk - biomarkers
Background and aims - There are few prospective studies on fatty acid status in relation to incident stroke, with inconsistent results. We assessed the associations of plasma n-6 and n-3 PUFA in cholesteryl esters with the risk of total stroke and stroke subtypes in Dutch adults. Methods and results - We conducted a nested case–control study using data from a population-based cohort study in adults aged 20–65 years. Blood sampling and data collection took place during 1993–1997 and subjects were followed for 8–13 years. We identified 179 incident cases of stroke and 179 randomly selected controls, matched on age, gender, and enrollment date. Odds ratios (OR) with 95% confidence intervals (95%CI) were calculated per standard deviation (SD) increase of PUFA in cholesteryl esters using multivariable conditional logistic regression. Cases comprised 93 ischemic, 50 hemorrhagic, and 36 unspecified strokes. The n-6 PUFA linoleic acid and arachidonic acid contributed ~55% and ~6.5% respectively to total plasma fatty acids, whereas the n-3 PUFA alpha-linolenic acid contributed ~0.5% and eicosapentaenoic acid plus docosahexaenoic acid (EPA-DHA) ~1.3%. After adjustment for confounders, n-6 and n-3 PUFA were not associated with incident total stroke or stroke subtypes. The OR (95% CI) for total stroke was 0.95 (0.74–1.23) per SD increase in linoleic acid and 1.02 (0.80–1.30) per SD increase in arachidonic acid. ORs (95% CI) for total stroke were 0.94 (0.72–1.21) for alpha-linolenic acid and 1.16 (0.94–1.45) for EPA-DHA. Conclusion - In the present study, plasma n-6 or n-3 fatty acids were not related to incident stroke or stroke subtypes.
The challenge of evaluating health effects of organic food; operationalisation of a dynamic concept of health
Huber, M.A.S. ; Bakker, M.H. ; Dijk, W. ; Prins, H.A.B. ; Wiegant, F.A.C. - \ 2012
Journal of the Science of Food and Agriculture 92 (2012)14. - ISSN 0022-5142 - p. 2766 - 2773.
nutrition-related health - cell-mediated-immunity - biological robustness - nervous-system - free-radicals - stress - exercise - biomarkers - immunomodulation - resilience
The health benefits of consuming organically produced foods compared with conventional foods are unclear. Important obstacles to drawing clear conclusions in this field of research are (1) the lack of a clear operational definition of health and (2) the inability to distinguish between different levels of health using valid biomarkers. In this paper, some shortcomings of the current definition of health are outlined and the relevance of integrating a more dynamic and functional component is emphasised, which is reflected by the ability to adapt. The state of health could then be determined by challenging an individual with some form of stressor and by subsequent quantification and evaluation of the coherence in recovery of various physiological processes and parameters. A set of relevant parameters includes the activity of the immune system and the activity of the autonomous nervous system. A good recovery towards homeostasis is suggested to reflect a qualitatively good state of health. Furthermore, it would enable objective evaluation of health-optimising strategies, including the consumption of organically produced foods that aim to strengthen health. (C) 2012 Society of Chemical Industry
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