QComics: Recommendations and Guidelines for Robust, Easily Implementable and Reportable Quality Control of Metabolomics Data.

The implementation of quality control strategies is crucial to ensure the reproducibility, accuracy, and meaningfulness of metabolomics data. However, this pivotal step is often overlooked within the metabolomics workflow and frequently relies on the use of nonstandardized and poorly reported protocols. To address current limitations in this respect, we have developed QComics, a robust, easily implementable and reportable method for monitoring and controlling data quality. The protocol operates in various sequential steps aimed to (i) correct for background noise and carryover, (ii) detect signal drifts and "out-of-control" observations, (iii) deal with missing data, (iv) remove outliers, (v) monitor quality markers to identify samples affected by improper collection, preprocessing, or storage, and (vi) assess overall data quality in terms of precision and accuracy. Notably, this tool considers important issues often neglected along quality control, such as the need of separately handling missing values and truly absent data to avoid losing relevant biological information, as well as the large impact that preanalytical factors may elicit on metabolomics results. Altogether, the guidelines compiled in QComics might contribute to establishing gold standard recommendations and best practices for quality control within the metabolomics community.

Probing erythrocytes as sensitive and reliable sensors of metabolic disturbances in the crosstalk between childhood obesity and insulin resistance: findings from an observational study, in vivo challenge tests, and ex vivo incubation assays.

BACKGROUND: Although insulin resistance (IR) is among the most frequent and pathogenically relevant complications accompanying childhood obesity, its role in modulating and exacerbating obesity pathophysiology has not yet been completely clarified. METHODS: To get deeper insights into the interplay between childhood obesity and IR, we leveraged a comprehensive experimental design based on a combination of observational data, in vivo challenge tests (i.e., oral glucose tolerance test), and ex vivo assays (i.e., incubation of erythrocytes with insulin) using a population comprising children with obesity and IR, children with obesity without IR, and healthy controls, from whom plasma and erythrocyte samples were collected for subsequent metabolomics analysis. RESULTS: Children with concomitant IR showed exacerbated metabolic disturbances in the crosstalk between endogenous, microbial, and environmental determinants, including failures in energy homeostasis, amino acid metabolism, oxidative stress, synthesis of steroid hormones and bile acids, membrane lipid composition, as well as differences in exposome-related metabolites associated with diet, exposure to endocrine disruptors, and gut microbiota. Furthermore, challenge tests and ex vivo assays revealed a deleterious impact of IR on individuals' metabolic flexibility, as reflected in blunted capacity to regulate homeostasis in response to hyperinsulinemia, at both systemic and erythroid levels. CONCLUSIONS: Thus, we have demonstrated for the first time that metabolite alterations in erythrocytes represent reliable and sensitive biomarkers to disentangle the metabolic complexity of IR and childhood obesity. This study emphasizes the crucial need of addressing inter-individual variability factors, such as the presence of comorbidities, to obtain a more accurate understanding of obesity-related molecular mechanisms.

Identifying metabotypes of insulin resistance severity in children with metabolic syndrome.

BACKGROUND: Insulin resistance is a frequent precursor of typical obesity and metabolic syndrome complications. However, accurate diagnosis remains elusive because of its pathophysiological complexity and heterogeneity. Herein, we have explored the utility of insulin secretion dynamics in response to an oral glucose tolerance test as a surrogate marker to identify distinct metabotypes of disease severity. METHODS: The study population consisted of children with obesity and insulin resistance, stratified according to the post-challenge insulin peak timing (i.e., early, middle, and late peak), from whom fasting and postprandial plasma and erythrocytes were collected for metabolomics analysis. RESULTS: Children with late insulin peak manifested worse cardiometabolic health (i.e., higher blood pressure, glycemia, and HOMA-IR scores) than early responders. These subjects also showed more pronounced changes in metabolites mirroring failures in energy homeostasis, oxidative stress, metabolism of cholesterol and phospholipids, and adherence to unhealthy dietary habits. Furthermore, delayed insulin peak was associated with impaired metabolic flexibility, as reflected in compromised capacity to regulate mitochondrial energy pathways and the antioxidant defense in response to glucose overload. CONCLUSIONS: Altogether, these findings suggest that insulin resistance could encompass several phenotypic subtypes characterized by graded disturbances in distinctive metabolic derangements occurring in childhood obesity, which serve as severity predictive markers.

Exploration of differential responses to FODMAPs and gluten in people with irritable bowel syndrome- a double-blind randomized cross-over challenge study.

INTRODUCTION: There is large variation in response to diet in irritable bowel syndrome (IBS) and determinants for differential response are poorly understood. OBJECTIVES: Our aim was to investigate differential clinical and molecular responses to provocation with fermentable oligo-, di-, monosaccharides, and polyols (FODMAPs) and gluten in individuals with IBS. METHODS: Data were used from a crossover study with week-long interventions with either FODMAPs, gluten or placebo. The study also included a rapid provocation test. Molecular data consisted of fecal microbiota, short chain fatty acids, and untargeted plasma metabolomics. IBS symptoms were evaluated with the IBS severity scoring system. IBS symptoms were modelled against molecular and baseline questionnaire data, using Random Forest (RF; regression and clustering), Parallel Factor Analysis (PARAFAC), and univariate methods. RESULTS: Regression and classification RF models were in general of low predictive power (Q2 ≤ 0.22, classification rate < 0.73). Out of 864 clustering models, only 2 had significant associations to clusters (0.69 < CR < 0.73, p < 0.05), but with no associations to baseline clinical measures. Similarly, PARAFAC revealed no clear association between metabolome data and IBS symptoms. CONCLUSION: Differential IBS responses to FODMAPs or gluten exposures could not be explained from clinical and molecular data despite extensive exploration with different data analytical approaches. The trial is registered at www. CLINICALTRIALS: gov as NCT03653689 31/08/2018.

Low- versus High-Glycemic Index Mediterranean-Style Eating Patterns Improved Some Domains of Health-Related Quality of Life but Not Sleep in Adults at Risk for Type 2 Diabetes: The MEDGICarb Randomized Controlled Trial.

BACKGROUND: A healthy eating pattern such as the Mediterranean-style healthy eating pattern (MED-HEP) is associated with favorable effects on both cardiometabolic risk markers and self-reported health outcomes. Limited evidence exists regarding the influence of the glycemic index (GI) of carbohydrate foods consumed within a healthy eating pattern on self-reported health status and sleep. OBJECTIVES: To investigate the effects of a low- compared with high-GI MED-HEP on changes in health-related quality of life (HRQoL) and sleep. METHODS: The MEDGICarb-intervention trial is a 12-wk randomized, controlled, parallel multi-center trial in adults with ≥2 features of the metabolic syndrome. Participants consumed an eu-energetic diet profiled as a MED-HEP with either low GI (experimental) or high GI (control). HRQoL and sleep were measured with Medical Outcomes Study 36-item short-form health survey version 2, Pittsburgh sleep quality index, and Epworth Sleepiness Scale at baseline and postintervention. RESULTS: One hundred and sixty adults with ≥2 features of the metabolic syndrome completed the intervention [53% females, age 56 ± 10 y, body mass index (kg/m2) 31.0 ± 3.1]. Low- compared with high-GI MED-HEP resulted in differential changes between the groups in the HRQoL domains role physical [5.6 ± 2.2 arbitrary units (AU) compared with -2.5 ± 2.5 AU) and vitality (6.9 ± 1.7 AU compared with 0.0 ± 1.8 AU] (P < 0.05), which were driven mostly by improvements in the low-GI group. There were no significant differences between the MED-HEPs for changes in aggregated physical or mental components or for the other individual domains of HRQoL (physical functioning, bodily pain, general health, social functioning, role emotional, and mental health) or for sleep quality or daytime sleepiness. CONCLUSIONS: Low compared to high GI in the context of a MED-HEP resulted in modest improvements in some, but not all, health domains of HRQoL. No major differences were seen between the groups for measures of sleep. This trial was registered at clinicaltrials.gov as NCT03410719.

Effects of a Phytoestrogen Intervention and Estrogen Receptor ß Genotype on Prostate Cancer Proliferation and PSA Concentrations-A Randomized Controlled Trial.

A phytoestrogen-rich diet has been suggested to reduce tumor proliferation among men with prostate cancer, and the effect may differ between men with different polymorphisms of the estrogen receptor-beta gene (ERß). Patients with low- or intermediate-risk prostate cancer scheduled for radical prostatectomy were randomized to an intervention group (n = 71) provided with soybeans and flaxseeds (∼200 mg phytoestrogens/day) to eat until surgery (approximately 6 wk) or to a control group (n = 69). Tumor proliferation was assessed using Ki-67 indexes, prostate-specific antigen (PSA) concentrations were analyzed in blood, and ERß polymorphism was genotyped in all subjects. The intervention group had a 13% unit lower risk [95% confidence interval (CI): -28%, 1.8%] of a higher Ki-67 index compared to controls, but the effect was most pronounced among TT carriers of ERß [risk difference (RD) -19%, 95% CI: -45%, 6.8%]. Subjects with genotype TC/CC had a lower risk (RD -29%, 95% CI: -46%, -1.2%) and TT genotype a higher risk (RD 25%, 95% CI: 8.7%, 42%) of increased PSA concentration, comparing the intervention group to controls. In conclusion, a phytoestrogen-rich diet may cause lower tumor proliferation and concentration of PSA in men with prostate cancer with a specific genetic upset of ERß.

OMICs Signatures Linking Persistent Organic Pollutants to Cardiovascular Disease in the Swedish Mammography Cohort.

Cardiovascular disease (CVD) development may be linked to persistent organic pollutants (POPs), including organochlorine compounds (OCs) and perfluoroalkyl and polyfluoroalkyl substances (PFAS). To explore underlying mechanisms, we investigated metabolites, proteins, and genes linking POPs with CVD risk. We used data from a nested case-control study on myocardial infarction (MI) and stroke from the Swedish Mammography Cohort - Clinical (n = 657 subjects). OCs, PFAS, and multiomics (9511 liquid chromatography-mass spectrometry (LC-MS) metabolite features; 248 proteins; 8110 gene variants) were measured in baseline plasma. POP-related omics features were selected using random forest followed by Spearman correlation adjusted for confounders. From these, CVD-related omics features were selected using conditional logistic regression. Finally, 29 (for OCs) and 12 (for PFAS) unique features associated with POPs and CVD. One omics subpattern, driven by lipids and inflammatory proteins, associated with MI (OR = 2.03; 95% CI = 1.47; 2.79), OCs, age, and BMI, and correlated negatively with PFAS. Another subpattern, driven by carnitines, associated with stroke (OR = 1.55; 95% CI = 1.16; 2.09), OCs, and age, but not with PFAS. This may imply that OCs and PFAS associate with different omics patterns with opposite effects on CVD risk, but more research is needed to disentangle potential modifications by other factors.

Blood metabolite profiles linking dietary patterns with health-Toward precision nutrition.

Diet is one of the most important exposures that may affect health throughout life span. Investigations on dietary patterns rather than single food components are gaining in popularity because they take the complexity of the whole dietary context into account. Adherence to such dietary patterns can be measured by using metabolomics, which allows measurements of thousands of molecules simultaneously. Derived metabolite signatures of dietary patterns may reflect the consumption of specific groups of foods or their constituents originating from the dietary pattern per se, or the physiological response toward the food-derived metabolites, their interaction with endogenous metabolism, and exogenous factors such as gut microbiota. Here, we review and discuss blood metabolite fingerprints of healthy dietary patterns. The plasma concentration of several food-derived metabolites-such as betaines from whole grains and n - 3 polyunsaturated fatty acids and furan fatty acids from fish-seems to consistently reflect the intake of common foods of several healthy dietary patterns. The metabolites reflecting shared features of different healthy food indices form biomarker panels for which specific, targeted assays could be developed. The specificity of such biomarker panels would need to be validated, and proof-of-concept feeding trials are needed to evaluate to what extent the panels may mediate the effects of dietary patterns on disease risk indicators or if they are merely food intake biomarkers. Metabolites mediating health effects may represent novel targets for precision prevention strategies of clinical relevance to be verified in future studies.

IBS randomized study: FODMAPs alter bile acids, phenolic- and tryptophan metabolites, while gluten modifies lipids.

Diet is considered a culprit for symptoms in irritable bowel syndrome (IBS), although the mechanistic understanding of underlying causes is lacking. Metabolomics, i.e., the analysis of metabolites in biological samples may offer a diet-responsive fingerprint for IBS. Our aim was to explore alterations in the plasma metabolome after interventions with fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) or gluten versus control in IBS, and to relate such alterations to symptoms. People with IBS (n = 110) were included in a double-blind, randomized, crossover study with 1-wk provocations of FODMAPs, gluten, or placebo. Symptoms were evaluated with the IBS severity scoring system (IBS-SSS). Untargeted metabolomics was performed on plasma samples using LC-qTOF-MS. Discovery of metabolite alterations by treatment was performed using random forest followed by linear mixed modeling. Associations were studied using Spearman correlation. The metabolome was affected by FODMAP [classification rate (CR) 0.88, P < 0.0001], but less by gluten intake CR 0.72, P = 0.01). FODMAP lowered bile acids, whereas phenolic-derived metabolites and 3-indolepropionic acid (IPA) were higher compared with placebo. IPA and some unidentified metabolites correlated weakly to abdominal pain and quality of life. Gluten affected lipid metabolism weakly, but with no interpretable relationship to IBS. FODMAP affected gut microbial-derived metabolites relating to positive health outcomes. IPA and unknown metabolites correlated weakly to IBS severity. Minor symptom worsening by FODMAP intake must be weighed against general positive health aspects of FODMAP. The gluten intervention affected lipid metabolism weakly with no interpretable association to IBS severity. Registration: www.clinicaltrials.gov as NCT03653689.NEW & NOTEWORTHY In irritable bowel syndrome (IBS), fermentable oligo-, di-, monosaccharides, and polyols (FODMAPs) affected microbial-derived metabolites relating to positive health outcomes such as reduced risk of colon cancer, inflammation, and type 2 diabetes, as shown in previous studies. The minor IBS symptom induction by FODMAP intake must be weighed against the positive health aspects of FODMAP consumption. Gluten affected lipids weakly with no association to IBS severity.

Descriptive analysis of dietary (poly)phenol intake in the subcohort MAX from DCH-NG: "Diet, Cancer and Health-Next Generations cohort".

PURPOSE: (Poly)phenols are bioactive compounds widely distributed in plant-based foods. Currently, limited data exist on the intake distribution of (poly)phenols across meals. This study aimed to estimate dietary intakes of all individual (poly)phenols and total intake per class and subclass by meal event, and to identify their main food sources in the subcohort MAX from the Diet, Cancer and Health-Next Generations cohort (DCH-NG). METHODS: Dietary data were collected using three web-based 24-h dietary recalls over 1 year. In total, 676 participants completed at least one recall. The dietary data were linked to Phenol-Explorer database using standardized procedures and an in-house software. We categorized foods/drinks into five options of meal events selected by the participant: 'Breakfast', 'Lunch', 'Evening', 'Snack', and 'Drink'. RESULTS: Adjusted total (poly)phenols mean intake by meal was the highest in the drink event (563 mg/day in men and 423 mg/day in women) and the lowest in the evening event (146 mg/day in men and 137 mg/day in women). The main overall (poly)phenol class contributor was phenolic acids (55.7-79.0%), except for evening and snack events where it was flavonoids (45.5-60%). The most consumed (poly)phenol subclasses were hydroxycinnamic acids and proanthocyanidins. Nonalcoholic beverages (coffee accounted for 66.4%), cocoa products, and cereals were the main food sources of total (poly)phenols. CONCLUSION: This study provides data on the variability in the intake of classes and subclasses of (poly)phenols and their main food sources by meal event according to lifestyle data, age, and gender in a Danish population.

Dietary polyphenols, metabolic syndrome and cardiometabolic risk factors: An observational study based on the DCH-NG subcohort.

BACKGROUND AND AIMS: Polyphenol-rich foods have beneficial properties that may lower cardiometabolic risk. We aimed to prospectively investigate the relationship between intakes of dietary polyphenols, and metabolic syndrome (MetS) and its components, in 676 Danish residents from the MAX study, a subcohort of the Danish Diet, Cancer and Health-Next Generations (DCH-NG) cohort. METHODS AND RESULTS: Dietary data were collected using web-based 24-h dietary recalls over one year (at baseline, and at 6 and 12 months). The Phenol-Explorer database was used to estimate dietary polyphenol intake. Clinical variables were also collected at the same time point. Generalized linear mixed models were used to investigate relationships between polyphenol intake and MetS. Participants had a mean age of 43.9y, a mean total polyphenol intake of 1368 mg/day, and 75 (11.6%) had MetS at baseline. Compared to individuals with MetS in Q1 and after adjusting for age, sex, lifestyle and dietary confounders, those in Q4 - for total polyphenols, flavonoids and phenolic acids-had a 50% [OR (95% CI): 0.50 (0.27, 0.91)], 51% [0.49 (0.26, 0.91)] and 45% [0.55 (0.30, 1.00)] lower odds of MetS, respectively. Higher total polyphenols, flavonoids and phenolic acids intakes as continuous variable were associated with lower risk for elevated systolic blood pressure (SBP) and low high-density lipoprotein cholesterol (HDL-c) (p < 0.05). CONCLUSIONS: Total polyphenol, flavonoid and phenolic acid intakes were associated with lower odds of MetS. These intakes were also consistently and significantly associated with a lower risk for higher SBP and lower HDL-c concentrations.

Modest Conformity Between Self-Reporting of Bristol Stool Form and Fecal Consistency Measured by Stool Water Content in Irritable Bowel Syndrome and a FODMAP and Gluten Trial.

INTRODUCTION: Altered bowel habits constitute a criterion of irritable bowel syndrome (IBS), with the Bristol Stool Form Scale (BSFS) as the recommended tool for assessment of fecal consistency. However, BSFS is devoid of a comprehensive objective evaluation in subjects with IBS. Therefore, we aimed to evaluate the concordance between subjective reporting of BSFS and objective stool water content in subjects with IBS. Furthermore, we evaluated whether intake of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) or gluten would affect stool water content. METHODS: Data from a previous crossover trial in IBS with 1-week provocations of FODMAPs, gluten, or placebo were subanalyzed. After each intervention, fecal consistency was subjectively assessed using the BSFS and stool samples were collected. The stool water content was analyzed, where ≤68.5% water content was classified as hard stool, while ≥78% was classified as diarrhea. RESULTS: BSFS correlated to stool water content ( r = 0.36, P < 0.0001). The BSFS score increased in parallel with increasing water content, but with considerable overlap between BSFS scores. Stool water content differed between the BSFS categories 1-2, 3-5, and 6-7 (hard, normal, and loose, respectively) ( P < 0.0001). For BSFS categories 1-2, 77% had water content ≤68.5%, whereas for BSFS categories 6-7, 52% had water content ≥78%. There was no difference in stool water content after consumption of FODMAPs, gluten, or placebo ( P = 0.94). DISCUSSION: Subjective reporting of BSFS conforms only modestly with stool water content in IBS, warranting caution when subtyping IBS according to the BSFS. High intake of FODMAPs and gluten does not affect stool water content.

Pregnancy outcomes with differences in grain consumption: a randomized controlled trial.

OBJECTIVES: Contemporary obstetrics has begun to appreciate the importance of diet in pregnancy, but guidelines are not based on robust data. The hypothesis that a whole grains diet improves pregnancy outcomes is tested in this study. We compared maternal and neonatal outcomes for a pregnancy diet containing 75% of total carbohydrates as refined grains with outcomes for a diet with 75% of total carbohydrates as whole grains. METHODS: This was a randomized interventional study in a clinic population over the last 4-7 months of normal pregnancy with extensive compliance measures. Besides obstetrical and neonatal outcomes, anthropometric measurements were done. In addition to food frequency questionnaires (FFQs), total plasma alkyl resorcinols, a unique quantitative measure of whole grains, were used as a measure of whole grain consumption. RESULTS: The data show effective compliance and no difference in outcomes between the diets with regard to maternal weight gain, birth weights, subcutaneous fat and glucose tolerance. CONCLUSIONS: Ensuring compliance to a proper pregnancy diet resulted in satisfactory weight gain and normal outcomes even when the proportion of whole grains consumed is only 25% of total carbohydrates.

[Correlation between intake of fish or supplements containing omega-3 fatty acids and early pregnancy plasma concentrations.]

INTRODUCTION: Long-chain polyunsaturated omega-3 fatty acids are considered important for fetal development, but previous studies suggest suboptimal intake in part of pregnant women in Iceland. The study aim was to evaluate intake of food and supplements containing omega-3 fatty acids, among pregnant women in Iceland and correlations to fatty acid composition in plasma. MATERIALS AND METHODS: Subjects were 853 pregnant women attending their 11-14 weeks ultrasound appointment. Information on intake of food and supplements containing long-chain omega-3 fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) as well as background was obtained by a questionnaire. Blood samples were collected for analysis of plasma fatty acids. Correlation was evaluated using the Spearman correlation. RESULTS: Median intake of lean fish was 1.3 times per week, while fatty fish was consumed once monthly. About 50% of the women took omega-3 containing supplements daily. Higher intake of both fish and omega-3 containing supplements was reflected in higher omega-3 plasma levels (r=0.37 p<0.001). A positive correlation was seen between intake of cod liver oil/capsules (r=0.23, p=0.001), omega-3 oil/capsules (r=0.20, p=0.001) and plasma concentration of omega-3. However, no correlation was seen between intake of a maternal multivitamin containing omega-3 and corresponding plasma concentration (r=0.03, p=0.98). CONCLUSION: Intake of food and supplements containing omega-3 fatty acids was reflected in plasma concentration, except for an Icelandic maternal multivitamin. One third of the women followed the recommendation of eating fish at least twice weekly. About 50% had a daily intake of supplements containing omega-3 fatty acids.

Higher Alkylresorcinol Concentrations, a Consequence of Whole-Grain Intake, are Inversely Associated with Gestational Diabetes Mellitus in Iceland.

BACKGROUND: A diet rich in whole grains may provide benefits for pregnant women due to whole grains' high nutritional value and dietary fiber content. OBJECTIVES: To study the associations of whole-grain consumption, as well as the plasma alkylresorcinol concentration, a whole-grain consumption biomarker, in early pregnancy with gestational diabetes mellitus (GDM) diagnoses. METHODS: Subjects were women from the prospective study Pregnant Women in Iceland II (PREWICE II; n = 853) who attended their ultrasound appointment in gestational weeks 11-14 during the period from October 2017 to March 2018. During that visit, whole-grain consumption was estimated using a diet screening questionnaire, and blood samples were collected for analysis of plasma alkylresorcinols (ARs). Information on GDM diagnoses was later extracted from medical records. Multivariate log-binomial regression was used to evaluate the association of dietary whole-grain and AR concentrations with GDM. RESULTS: In total, 14.9% of the women adhered to the national food-based dietary guidelines (n = 127), which recommend 2 portions of whole grains daily. GDM was diagnosed in 127 women (14.9%). The frequency of whole-grain consumption was lower in women who were later diagnosed with GDM compared to the women without GDM (median, 5 times/week vs. 6 times/week, respectively; P = 0.02). This difference was reflected in the lower median concentration of total AR in women diagnosed with GDM (163 nmol/L vs. 209 nmol/L, respectively; P < 0.01). The quartile with the highest concentrations of AR had a RR of 0.50 (95% CI: 0.27-0.90) of being diagnosed with GDM, in comparison to the lowest quartile. There was a significant dose response in the GDM risk with higher AR levels. CONCLUSIONS: We found that a higher consumption of whole grains, reflected both by reported consumption according to the FFQ and AR biomarkers, was associated with a decreased risk of receiving a GDM diagnosis.

Evaluating an Intervention to Increase Cereal Fiber Intake in Children: A Randomized Controlled Feasibility Trial.

BACKGROUND: Observational studies have shown that higher cereal fiber intake is associated with reduced type 2 diabetes risk. However, it remains uncertain whether this association is causal. OBJECTIVE: This study evaluated the feasibility of an intervention to increase cereal fiber intake in children using breakfast cereals. METHODS: The study was a 2-arm parallel group randomized controlled trial in 9-10-y-old children, who received free supplies of high-fiber breakfast cereals (>3.5 g/portion) or low-fiber breakfast cereals (<1.0 g/portion) to eat daily for 1 mo with behavioral support to promote adherence. Children provided baseline and 1-mo fasting blood samples, physical measurements, and 24-h dietary recalls. The primary outcome was the group difference in change in plasma total alkylresorcinol (AR) concentration; secondary outcomes were group differences in nutrient intakes and adiposity indices. Analyses (complete case and multiple imputation) were conducted by regressing the final AR concentration on baseline AR in models adjusted for sex, ethnicity, age, and school (random effect). RESULTS: Two-hundred seventy-two children were randomly assigned (137 receiving a low-fiber and 135 a high-fiber diet) and 193 (71%) provided fasting blood samples at baseline and follow-up. Among randomized participants, median (IQR) of baseline AR was 43.1 (24.6-85.5) nmol/L and of cereal fiber intake was 4.5 (2.7-6.4) g; 87% of participants reported consuming the cereal on most or all days. Compared with changes in the low-fiber group, the high-fiber group had greater increases in AR (40.7 nmol/L; 95% CI: 21.7, 59.8 nmol/L, P < 0.0001) and in reported cereal fiber intake (2.9g/d; 95% CI: 2.0, 3.7 g; P < 0.0001). There were no appreciable differences in other secondary outcomes. CONCLUSIONS: We have developed a simple and acceptable nutritional intervention that increases markers of daily cereal fiber intake in children. This intervention could be used to test whether increases in cereal fiber intake in children might reduce insulin resistance. This trial was registered at www.isrctn.com as ISRCTN33260236.

The CRCbiome study: a large prospective cohort study examining the role of lifestyle and the gut microbiome in colorectal cancer screening participants.

BACKGROUND: Colorectal cancer (CRC) screening reduces CRC incidence and mortality. However, current screening methods are either hampered by invasiveness or suboptimal performance, limiting their effectiveness as primary screening methods. To aid in the development of a non-invasive screening test with improved sensitivity and specificity, we have initiated a prospective biomarker study (CRCbiome), nested within a large randomized CRC screening trial in Norway. We aim to develop a microbiome-based classification algorithm to identify advanced colorectal lesions in screening participants testing positive for an immunochemical fecal occult blood test (FIT). We will also examine interactions with host factors, diet, lifestyle and prescription drugs. The prospective nature of the study also enables the analysis of changes in the gut microbiome following the removal of precancerous lesions. METHODS: The CRCbiome study recruits participants enrolled in the Bowel Cancer Screening in Norway (BCSN) study, a randomized trial initiated in 2012 comparing once-only sigmoidoscopy to repeated biennial FIT, where women and men aged 50-74 years at study entry are invited to participate. Since 2017, participants randomized to FIT screening with a positive test result have been invited to join the CRCbiome study. Self-reported diet, lifestyle and demographic data are collected prior to colonoscopy after the positive FIT-test (baseline). Screening data, including colonoscopy findings are obtained from the BCSN database. Fecal samples for gut microbiome analyses are collected both before and 2 and 12 months after colonoscopy. Samples are analyzed using metagenome sequencing, with taxonomy profiles, and gene and pathway content as primary measures. CRCbiome data will also be linked to national registries to obtain information on prescription histories and cancer relevant outcomes occurring during the 10 year follow-up period. DISCUSSION: The CRCbiome study will increase our understanding of how the gut microbiome, in combination with lifestyle and environmental factors, influences the early stages of colorectal carcinogenesis. This knowledge will be crucial to develop microbiome-based screening tools for CRC. By evaluating biomarker performance in a screening setting, using samples from the target population, the generalizability of the findings to future screening cohorts is likely to be high. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01538550 .

Effects of substitution dietary guidelines targeted at prevention of IHD on dietary intake and risk factors in middle-aged Danish adults: the Diet and Prevention of Ischemic Heart Disease: a Translational Approach (DIPI) randomised controlled trial.

The objective was to investigate the effects of substitution (SUB) dietary guidelines (DG) targeted at the prevention of IHD on dietary intake and IHD risk factors in Danish adults with minimum one self-assessed IHD risk factor. A 6-month single-blinded parallel randomised controlled trial with a follow-up at month 12 included 219 subjects (median age 51 years, 59 % female, 73 % overweight or obese) randomised into an SUB DG, an official (OFF) DG or a control group following their habitual diet (HAB). Participants in the DG intervention groups received bi-weekly reminders of their DG and recipes for dishes and the HAB group received a greeting. Dietary intake and fasting blood, anthropometric and blood pressure measurements were obtained at baseline, month 6 and month 12. Linear regression analyses were applied. At month 6, when compared with the HAB, the SUB had a greater impact on the extent of dietary changes with increased intake of whole grains, dietary fibre and low fibre vegetables compared with the OFF DG, and both DG groups had similar decreased percentage of energy (E%) intake from SFA. The extent of dietary changes was similar at month 12. No overall significant changes from baseline were found in blood pressure, anthropometrics and IHD risk markers. In conclusion, both SUB and OFF DG resulted in cardioprotective dietary changes. However, neither the SUB nor the OFF DG resulted in any overall effects on the selected intermediate risk factors for IHD.

Variable selection and validation in multivariate modelling.

MOTIVATION: Validation of variable selection and predictive performance is crucial in construction of robust multivariate models that generalize well, minimize overfitting and facilitate interpretation of results. Inappropriate variable selection leads instead to selection bias, thereby increasing the risk of model overfitting and false positive discoveries. Although several algorithms exist to identify a minimal set of most informative variables (i.e. the minimal-optimal problem), few can select all variables related to the research question (i.e. the all-relevant problem). Robust algorithms combining identification of both minimal-optimal and all-relevant variables with proper cross-validation are urgently needed. RESULTS: We developed the MUVR algorithm to improve predictive performance and minimize overfitting and false positives in multivariate analysis. In the MUVR algorithm, minimal variable selection is achieved by performing recursive variable elimination in a repeated double cross-validation (rdCV) procedure. The algorithm supports partial least squares and random forest modelling, and simultaneously identifies minimal-optimal and all-relevant variable sets for regression, classification and multilevel analyses. Using three authentic omics datasets, MUVR yielded parsimonious models with minimal overfitting and improved model performance compared with state-of-the-art rdCV. Moreover, MUVR showed advantages over other variable selection algorithms, i.e. Boruta and VSURF, including simultaneous variable selection and validation scheme and wider applicability. AVAILABILITY AND IMPLEMENTATION: Algorithms, data, scripts and tutorial are open source and available as an R package ('MUVR') at https://gitlab.com/CarlBrunius/MUVR.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An LC-QToF MS based method for untargeted metabolomics of human fecal samples.

INTRODUCTION: Consensus in sample preparation for untargeted human fecal metabolomics is lacking. OBJECTIVES: To obtain sample preparation with broad metabolite coverage for high-throughput LC-MS. METHODS: Extraction solvent, solvent ratio and fresh frozen-vs-lyophilized samples were evaluated by metabolite feature quality. RESULTS: Methanol at 5 mL per g wet feces provided a wide metabolite coverage with optimal balance between signal intensity and saturation for both fresh frozen and lyophilized samples. Lyophilization did not affect SCFA and is recommended because of convenience in normalizing to dry matter. CONCLUSION: The suggested sample preparation is simple, efficient and suitable for large-scale human fecal metabolomics.