Urinary Concentrations of (+)-Catechin and (-)-Epicatechin as Biomarkers of Dietary Intake of Flavan-3-Ols in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study

This study examines the correlation of acute and habitual dietary intake of flavan-3-ol monomers, proanthocyanidins, theaflavins, and their main food sources with the urinary concentrations of (+)-catechin and (-)-epicatechin in the European Prospective Investigation into Cancer and Nutrition study (EPIC). Participants (N = 419, men and women) provided 24-h urine samples and completed a 24-h dietary recall (24-HDR) on the same day. Acute and habitual dietary data were collected using a standardized 24-HDR software and a validated dietary questionnaire, respectively. Intake of flavan-3-ols was estimated using the Phenol-Explorer database. Concentrations of (+)-catechin and (-)-epicatechin in 24-h urine were analyzed using tandem mass spectrometry after enzymatic deconjugation. Simple and partial Spearman's correlations showed that urinary concentrations of (+)-catechin, (-)-epicatechin and their sum were more strongly correlated with acute than with habitual intake of individual and total monomers (acute rpartial = 0.13-0.54, p < 0.05; and habitual rpartial = 0.14-0.28, p < 0.01), proanthocyanidins (acute rpartial = 0.24-0.49, p < 0.001; and habitual rpartial = 0.10-0.15, p < 0.05), theaflavins (acute rpartial = 0.22-0.31, p < 0.001; and habitual rpartial = 0.20-0.26, p < 0.01), and total flavan-3-ols (acute rpartial = 0.40-0.48, p < 0.001; and habitual rpartial = 0.23-0.33, p < 0.001). Similarly, urinary concentrations of flavan-3-ols were weakly correlated with both acute (rpartial = 0.12-0.30, p < 0.05) and habitual intake (rpartial = 0.10-0.27, p < 0.05) of apple and pear, stone fruits, berries, chocolate and chocolate products, cakes and pastries, tea, herbal tea, wine, red wine, and beer and cider. Moreover, all comparable correlations were stronger for urinary (-)-epicatechin than for (+)-catechin. In conclusion, our data support the use of urinary concentrations of (+)-catechin and (-)-epicatechin, especially as short-term nutritional biomarkers of dietary catechin, epicatechin and total flavan-3-ol monomers

Urinary excretions of 34 dietary polyphenols and their associations with lifestyle factors in the EPIC cohort study.

Urinary excretion of 34 dietary polyphenols and their variations according to diet and other lifestyle factors were measured by tandem mass spectrometry in 475 adult participants from the European Prospective Investigation into Cancer and Nutrition (EPIC) cross-sectional study. A single 24-hour urine sample was analysed for each subject from 4 European countries. The highest median levels were observed for phenolic acids such as 4-hydroxyphenylacetic acid (157 μmol/24 h), followed by 3-hydroxyphenylacetic, ferulic, vanillic and homovanillic acids (20-50 μmol/24 h). The lowest concentrations were observed for equol, apigenin and resveratrol ( 0.5) observed between urinary polyphenols and the intake of their main food sources (e.g., resveratrol and gallic acid ethyl ester with red wine intake; caffeic, protocatechuic and ferulic acids with coffee consumption; and hesperetin and naringenin with citrus fruit intake). The large variations in urinary polyphenols observed are largely determined by food preferences. These polyphenol biomarkers should allow more accurate evaluation of the relationships between polyphenol exposure and the risk of chronic diseases in large epidemiological studies

Identification Of Urinary Polyphenol Metabolite Patterns Associated With Polyphenol-rich Food Intake In Adults From Four European Countries

We identified urinary polyphenol metabolite patterns by a novel algorithm that combines dimension reduction and variable selection methods to explain polyphenol-rich food intake, and compared their respective performance with that of single biomarkers in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. The study included 475 adults from four European countries (Germany, France, Italy, and Greece). Dietary intakes were assessed with 24-h dietary recalls (24-HDR) and dietary questionnaires (DQ). Thirty-four polyphenols were measured by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS-MS) in 24-h urine. Reduced rank regression-based variable importance in projection (RRR-VIP) and least absolute shrinkage and selection operator (LASSO) methods were used to select polyphenol metabolites. Reduced rank regression (RRR) was then used to identify patterns in these metabolites, maximizing the explained variability in intake of pre-selected polyphenol-rich foods. The performance of RRR models was evaluated using internal cross-validation to control for over-optimistic findings from over-fitting. High performance was observed for explaining recent intake (24-HDR) of red wine (r = 0.65; AUC = 89.1%), coffee (r = 0.51; AUC = 89.1%), and olives (r = 0.35; AUC = 82.2%). These metabolite patterns performed better or equally well compared to single polyphenol biomarkers. Neither metabolite patterns nor single biomarkers performed well in explaining habitual intake (as reported in the DQ) of polyphenol-rich foods. This proposed strategy of biomarker pattern identification has the potential of expanding the currently still limited list of available dietary intake biomarkers

Patterns in metabolite profile are associated with risk of more aggressive prostate cancer: a prospective study of 3057 matched case‐control sets from EPIC

Metabolomics may reveal novel insights into the etiology of prostate cancer, for which few risk factors are established. We investigated the association between patterns in baseline plasma metabolite profile and subsequent prostate cancer risk, using data from 3,057 matched case-control sets from the European Prospective Investigation into Cancer and Nutrition (EPIC). We measured 119 metabolite concentrations in plasma samples, collected on average 9.4 years before diagnosis, by mass spectrometry (AbsoluteIDQ p180 Kit, Biocrates Life Sciences AG). Metabolite patterns were identified using treelet transform, a statistical method for identification of groups of correlated metabolites. Associations of metabolite patterns with prostate cancer risk (OR1SD ) were estimated by conditional logistic regression. Supplementary analyses were conducted for metabolite patterns derived using principal component analysis and for individual metabolites. Men with metabolite profiles characterized by higher concentrations of either phosphatidylcholines or hydroxysphingomyelins (OR1SD = 0.77, 95% confidence interval 0.66-0.89), acylcarnitines C18:1 and C18:2, glutamate, ornithine and taurine (OR1SD = 0.72, 0.57-0.90), or lysophosphatidylcholines (OR1SD = 0.81, 0.69-0.95) had lower risk of advanced stage prostate cancer at diagnosis, with no evidence of heterogeneity by follow-up time. Similar associations were observed for the two former patterns with aggressive disease risk (the more aggressive subset of advanced stage), while the latter pattern was inversely related to risk of prostate cancer death (OR1SD = 0.77, 0.61-0.96). No associations were observed for prostate cancer overall or less aggressive tumor subtypes. In conclusion, metabolite patterns may be related to lower risk of more aggressive prostate tumors and prostate cancer death, and might be relevant to etiology of advanced stage prostate cancer

Association between pre-diagnostic circulating lipid metabolites and colorectal cancer risk: a nested case–control study in the European Prospective Investigation into Cancer and Nutrition (EPIC)

Background: Altered lipid metabolism is a hallmark of cancer development. However, the role of specific lipid metabolites in colorectal cancer development is uncertain. Methods: In a case–control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC), we examined associations between pre-diagnostic circulating concentrations of 97 lipid metabolites (acylcarnitines, glycerophospholipids and sphingolipids) and colorectal cancer risk. Circulating lipids were measured using targeted mass spectrometry in 1591 incident colorectal cancer cases (55% women) and 1591 matched controls. Multivariable conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for associations between concentrations of individual lipid metabolites and metabolite patterns with colorectal cancer risk. Findings: Of the 97 assayed lipids, 24 were inversely associated (nominally p < 0.05) with colorectal cancer risk. Hydroxysphingomyelin (SM (OH)) C22:2 (ORper doubling 0.60, 95% CI 0.47–0.77) and acylakyl-phosphatidylcholine (PC ae) C34:3 (ORper doubling 0.71, 95% CI 0.59–0.87) remained associated after multiple comparisons correction. These associations were unaltered after excluding the first 5 years of follow-up after blood collection and were consistent according to sex, age at diagnosis, BMI, and colorectal subsite. Two lipid patterns, one including 26 phosphatidylcholines and all sphingolipids, and another 30 phosphatidylcholines, were weakly inversely associated with colorectal cancer. Interpretation: Elevated pre-diagnostic circulating levels of SM (OH) C22:2 and PC ae C34:3 and lipid patterns including phosphatidylcholines and sphingolipids were associated with lower colorectal cancer risk. This study may provide insight into potential links between specific lipids and colorectal cancer development. Additional prospective studies are needed to validate the observed associations

A Prospective Evaluation of Plasma Polyphenol Levels and Colon Cancer Risk

Polyphenols have been shown to exert biological activity in experimental models of colon cancer; however, human data linking specific polyphenols to colon cancer is limited. We assessed the relationship between pre-diagnostic plasma polyphenols and colon cancer risk in a case-control study nested within the European Prospective Investigation into Cancer and Nutrition study. Using high pressure liquid chromatography coupled to tandem mass spectrometry, we measured concentrations of 35 polyphenols in plasma from 809 incident colon cancer cases and 809 matched controls. We used multivariable adjusted conditional logistic regression models that included established colon cancer risk factors. The false discovery rate (qvalues ) was computed to control for multiple comparisons. All statistical tests were two-sided. After false discovery rate correction and in continuous log2 -transformed multivariable models, equol (odds ratio [OR] per log2 -value, 0.86, 95% confidence interval [95% CI] = 0.79-0.93; qvalue = 0.01) and homovanillic acid (OR per log2 -value, 1.46, 95% CI = 1.16-1.84; qvalue = 0.02) were associated with colon cancer risk. Comparing extreme fifths, equol concentrations were inversely associated with colon cancer risk (OR = 0.61, 95% CI = 0.41-0.91, ptrend = 0.003), while homovanillic acid concentrations were positively associated with colon cancer development (OR = 1.72, 95% CI = 1.17-2.53, ptrend < 0.0001). No heterogeneity for these associations was observed by sex and across other colon cancer risk factors. The remaining polyphenols were not associated with colon cancer risk. Higher equol concentrations were associated with lower risk, and higher homovanillic acid concentrations were associated with greater risk of colon cancer. These findings support a potential role for specific polyphenols in colon tumorigenesis

Circulating Metabolites Associated with Alcohol Intake in the European Prospective Investigation into Cancer and Nutrition Cohort.

Identifying the metabolites associated with alcohol consumption may provide insights into the metabolic pathways through which alcohol may affect human health. We studied associations of alcohol consumption with circulating concentrations of 123 metabolites among 2974 healthy participants from the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Alcohol consumption at recruitment was self-reported through dietary questionnaires. Metabolite concentrations were measured by tandem mass spectrometry (BIOCRATES AbsoluteIDQTM p180 kit). Data were randomly divided into discovery (2/3) and replication (1/3) sets. Multivariable linear regression models were used to evaluate confounder-adjusted associations of alcohol consumption with metabolite concentrations. Metabolites significantly related to alcohol intake in the discovery set (FDR q-value < 0.05) were further tested in the replication set (Bonferroni-corrected p-value < 0.05). Of the 72 metabolites significantly related to alcohol intake in the discovery set, 34 were also significant in the replication analysis, including three acylcarnitines, the amino acid citrulline, four lysophosphatidylcholines, 13 diacylphosphatidylcholines, seven acyl-alkylphosphatidylcholines, and six sphingomyelins. Our results confirmed earlier findings that alcohol consumption was associated with several lipid metabolites, and possibly also with specific acylcarnitines and amino acids. This provides further leads for future research studies aiming at elucidating the mechanisms underlying the effects of alcohol in relation to morbid conditions

Prospective analysis of circulating metabolites and breast cancer in EPIC

Background: Metabolomics is a promising molecular tool to identify novel etiologic pathways leading to cancer. Using a targeted approach, we prospectively investigated the associations between metabolite concentrations in plasma and breast cancer risk. Methods: A nested case-control study was established within the European Prospective Investigation into Cancer cohort, which included 1624 first primary incident invasive breast cancer cases (with known estrogen and progesterone receptor and HER2 status) and 1624 matched controls. Metabolites (n = 127, acylcarnitines, amino acids, biogenic amines, glycerophospholipids, hexose, sphingolipids) were measured by mass spectrometry in pre-diagnostic plasma samples and tested for associations with breast cancer incidence using multivariable conditional logistic regression. Results: Among women not using hormones at baseline (n = 2248), and after control for multiple tests, concentrations of arginine (odds ratio [OR] per SD = 0.79, 95% confidence interval [CI] = 0.70-0.90), asparagine (OR = 0.83 (0.74-0.92)), and phosphatidylcholines (PCs) ae C36:3 (OR = 0.83 (0.76-0.90)), aa C36:3 (OR = 0.84 (0.77-0.93)), ae C34:2 (OR = 0.85 (0.78-0.94)), ae C36:2 (OR = 0.85 (0.78-0.88)), and ae C38:2 (OR = 0.84 (0.76-0.93)) were inversely associated with breast cancer risk, while the acylcarnitine C2 (OR = 1.23 (1.11-1.35)) was positively associated with disease risk. In the overall population, C2 (OR = 1.15 (1.06-1.24)) and PC ae C36:3 (OR = 0.88 (0.82-0.95)) were associated with risk of breast cancer, and these relationships did not differ by breast cancer subtype, age at diagnosis, fasting status, menopausal status, or adiposity. Conclusions: These findings point to potentially novel pathways and biomarkers of breast cancer development. Results warrant replication in other epidemiological studies

Pre-diagnostic metabolite concentrations and prostate cancer risk in 1077 cases and 1077 matched controls in the European Prospective Investigation into Cancer and Nutrition.

BACKGROUND: Little is known about how pre-diagnostic metabolites in blood relate to risk of prostate cancer. We aimed to investigate the prospective association between plasma metabolite concentrations and risk of prostate cancer overall, and by time to diagnosis and tumour characteristics, and risk of death from prostate cancer. METHODS: In a case-control study nested in the European Prospective Investigation into Cancer and Nutrition, pre-diagnostic plasma concentrations of 122 metabolites (including acylcarnitines, amino acids, biogenic amines, glycerophospholipids, hexose and sphingolipids) were measured using targeted mass spectrometry (AbsoluteIDQ p180 Kit) and compared between 1077 prostate cancer cases and 1077 matched controls. Risk of prostate cancer associated with metabolite concentrations was estimated by multi-variable conditional logistic regression, and multiple testing was accounted for by using a false discovery rate controlling procedure. RESULTS: Seven metabolite concentrations, i.e. acylcarnitine C18:1, amino acids citrulline and trans-4-hydroxyproline, glycerophospholipids PC aa C28:1, PC ae C30:0 and PC ae C30:2, and sphingolipid SM (OH) C14:1, were associated with prostate cancer (p < 0.05), but none of the associations were statistically significant after controlling for multiple testing. Citrulline was associated with a decreased risk of prostate cancer (odds ratio (OR1SD) = 0.73; 95% confidence interval (CI) 0.62-0.86; p trend = 0.0002) in the first 5 years of follow-up after taking multiple testing into account, but not after longer follow-up; results for other metabolites did not vary by time to diagnosis. After controlling for multiple testing, 12 glycerophospholipids were inversely associated with advanced stage disease, with risk reduction up to 46% per standard deviation increase in concentration (OR1SD = 0.54; 95% CI 0.40-0.72; p trend = 0.00004 for PC aa C40:3). Death from prostate cancer was associated with higher concentrations of acylcarnitine C3, amino acids methionine and trans-4-hydroxyproline, biogenic amine ADMA, hexose and sphingolipid SM (OH) C14:1 and lower concentration of glycerophospholipid PC aa C42:4. CONCLUSIONS: Several metabolites, i.e. C18:1, citrulline, trans-4-hydroxyproline, three glycerophospholipids and SM (OH) C14:1, might be related to prostate cancer. Analyses by time to diagnosis indicated that citrulline may be a marker of subclinical prostate cancer, while other metabolites might be related to aetiology. Several glycerophospholipids were inversely related to advanced stage disease. More prospective data are needed to confirm these associations

Développement d’une nouvelle méthode analytique pour le dosage des polyphénols dans les fluides biologiques et application à l’épidémiologie du cancer dans la cohorte EPIC

Polyphenols are secondary plant metabolites found in diet and more than 500 different compounds are found in more than 450 foods. Along the last 30 years, many in vitro and animals studies have suggested a beneficial role of polyphenols in chronic diseases such as cancer. However, epidemiological studies based mainly on dietary questionnaires are inconsistent. New methodologies with broader polyphenol coverage are required to quantify a large diversity of compounds within large scale epidemiological studies.The goal of this thesis is to develop methods to quantify a large number of polyphenols representative of the main classes and sub-classes in blood and urine, evaluate urinary excretions of polyphenols and their association with food consumption in an European population, evaluate concentrations in blood in a nested case-control study on colorectal cancer within the European prospective investigation study on cancer and nutrition (EPIC).After a short presentation of polyphenols and colorectal cancer in chapter II, an original method based on differential isotopic dilution and analyse by tandem mass spectrometry is developed to quantify 38 polyphenols in urine and presented in chapter III. This method is based on differential 12C-/13C- isotope labelling of polyphenols through derivatisation with isotopic dansyl chloride reagents. Different conditions for enzymatic hydrolysis of conjugated polyphenols, extraction and dansylation of unconjugated aglycones have been tested, optimized and validated for the measure of 37 polyphenols.Urinary excretion levels of these 37 polyphenols in 475 subjects within EPIC cohort from 4 European countries are presented in chapter IV. Large urinary excretion variability in the 4 European countries has been shown and significant correlations with the consumption of specific food containing polyphenols have been observed suggesting the possible used of some polyphenols as biomarker for food consumption.A similar method on plasma for the 37 polyphenols, based on differential isotopic dilution, is developed, validated and applied to 1618 samples within the EPIC cohort (Chapter V). Main changes compared to the method developed on urine are essentially difficulties to hydrolyse conjugated polyphenols and huge matrix effects but reduced by the isotopic dilution method.Finally, the study on the association between polyphenols exposure measured owing to plasmatic marker with colorectal cancer risk has been driven in a nested case-control study within the EPIC cohort and is presented in chapter VI. An inverse association between plasmatic concentrations of equol, and a positive association between homovanillic acid and colon cancer risk have been found.Results obtained constitute a new approach for future applications in large scale epidemiology study on polyphenolsLes polyphénols sont un groupe de métabolites secondaires communément trouvés dans l’alimentation et plus de 500 composés différents sont retrouvés dans plus de 450 denrées alimentaires. Au cours des 30 dernières années, de nombreuses études in vitro et chez l’animal ont suggéré un rôle bénéfique des polyphénols dans des maladies chroniques comme le cancer 1-3. Les études épidémiologiques basées majoritairement sur des questionnaires alimentaires sont cependant plus contradictoires 4-6. De nouvelles méthodes sont requises pour quantifier un plus grand nombre de composés dans une même série afin de réaliser des études épidémiologiques à grande échelle. Le but de cette thèse est de développer des méthodes de quantification d’une large gamme de polyphénols représentatifs des principales classes et sous-classes de polyphénols dans le sang et urine, d’évaluer les excrétions urinaires de polyphénols et leurs associations avec la consommation d’aliments source dans une population Européenne, d’évaluer les concentrations dans le sang dans une étude cas-témoin sur cancer colorectal dans l’étude prospective européenne sur le cancer et la nutrition (EPIC). Après une brève présentation des polyphénols et du cancer colorectal dans le chapitre II, une méthode originale basée sur la dilution isotopique différentielle et l’analyse en spectrométrie de masse est développée pour quantifier 38 polyphénols dans l’urine et présentée dans le chapitre III. Cette méthode est basée sur un marquage différentiel en 12C et 13C des polyphénols grâce à une dérivatisation par les isotopes du chlorure de dansyl. Différentes conditions pour l’hydrolyse des polyphénols conjugués, l’extraction et la dérivatisation des aglycones ont été évaluées et la méthode a été validée pour la mesure de 37 polyphénols. Les niveaux d’excrétion urinaire de ces 37 polyphénols mesurés chez 475 individus de la cohorte EPIC issues de quatre pays européens sont présentés dans le chapitre IV. Une grande variabilité d’excrétion urinaire dans les quatre pays Européens a pu être montrée et des corrélations significatives avec la consommation de certains aliments source de ces composés ont été observées suggérant l’utilisation possible de plusieurs de ces polyphénols en tant que biomarqueurs de l’alimentation. Une méthode d’analyse pour les mêmes 37 polyphénols également basée sur la dilution isotopique différentielle est développée, validée et appliquée à 1618 échantillons dans la cohorte EPIC (chapitre V). Les changements majeurs en comparaison de la méthode sur la matrice urinaire sont essentiellement les difficultés d’hydrolyse des polyphénols conjugués et des effets de matrice importants mais réduits par la méthode de dilution isotopique. Enfin l’étude des associations entre l’exposition aux polyphénols mesurée à l’aide des marqueurs plasmatique avec le risque de cancer colorectal a été conduite dans une étude cas-témoin nichée dans la cohorte EPIC et est présentée dans le chapitre VI. Une association inverse entre les concentrations plasmatiques d’equol, et une association positive entre l’acide homovanillique et le risque de cancer du côlon ont été trouvées. Les résultats obtenus constituent une nouvelle base pour des applications futures dans le domaine de l’épidémiologie des polyphénol