Personalized Nutrition Using Microbial Metabolite Phenotype to Stratify Participants and Non-Invasive Host Exfoliomics Reveal the Effects of Flaxseed Lignan Supplementation in a Placebo-Controlled Crossover Trial.

High-fiber plant foods contain lignans that are converted to bioactive enterolignans, enterolactone (ENL) and enterodiol (END) by gut bacteria. Previously, we conducted an intervention study to gain mechanistic insight into the potential chemoprotective effects of flaxseed lignan supplementation (secoisolariciresinol diglucoside; SDG) compared to a placebo in 42 men and women. Here, we expand on these analyses to further probe the impact of the microbial metabolite phenotype on host gene expression in response to lignan exposure. We defined metabolic phenotypes as high- or low-ENL excretion based on the microbial metabolism of SDG. RNA-seq was used to assess host gene expression in fecal exfoliated cells. Stratified by microbial ENL excretion, differentially expressed (DE) genes in high- and low-ENL excreter groups were compared. Linear discriminant analysis using the ENL phenotypes identified putative biomarker combinations of genes capable of discriminating the lignan treatment from the placebo. Following lignan intervention, a total of 165 DE genes in high-ENL excreters and 1450 DE genes in low-ENL excreters were detected. Functional analysis identified four common upstream regulators (master genes): CD3, IFNG, IGF1 and TNFRSF1A. Our findings suggest that the enhanced conversion of flaxseed lignan to ENL is associated with a suppressed inflammatory status.

Enterolignan Production in a Flaxseed Intervention Study in Postmenopausal US Women of African Ancestry and European Ancestry.

Lignans are phytochemicals studied extensively as dietary factors in chronic disease etiology. Our goal was to examine associations between the gut microbiota and lignan metabolism and whether these associations differ by ethnicity. We conducted a flaxseed (FS) dietary intervention in 252 healthy, postmenopausal women of African ancestry (AA) and European ancestry (EA). Participants consumed ~10 g/d ground flaxseed for 6 weeks and provided overnight urine collections and fecal samples before and after intervention. The gut microbiota was characterized using 16S rRNA gene sequencing and differences in microbial community composition compared by ethnicity and intervention status. We observed a significant difference in the composition of the microbiota measured as beta diversity (p < 0.05) between AA and EA at baseline that was attenuated with FS consumption. Genera that were significantly associated with ENL production (e.g., Klebsiella, Lactobacillus, Slackia, Senegalimassilia) were unique to each group. Bacteria (e.g., Fusobacteria, Pyramidobacter and Odoribacter) previously associated with colorectal cancer and cardiovascular disease, both diet-related chronic diseases, were unique to either AA or EA and were significantly reduced in the FS intervention. This study suggests that ethnic variation in ENL metabolism may be linked to gut microbiota composition, and its impact on disease risk deserves future investigation.

Limited effects of long-term daily cranberry consumption on the gut microbiome in a placebo-controlled study of women with recurrent urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) affect 15 million women each year in the United States, with > 20% experiencing frequent recurrent UTIs. A recent placebo-controlled clinical trial found a 39% reduction in UTI symptoms among recurrent UTI sufferers who consumed a daily cranberry beverage for 24 weeks. Using metagenomic sequencing of stool from a subset of these trial participants, we assessed the impact of cranberry consumption on the gut microbiota, a reservoir for UTI-causing pathogens such as Escherichia coli, which causes > 80% of UTIs. RESULTS: The overall taxonomic composition, community diversity, carriage of functional pathways and gene families, and relative abundances of the vast majority of observed bacterial taxa, including E. coli, were not changed significantly by cranberry consumption. However, one unnamed Flavonifractor species (OTU41), which represented ≤1% of the overall metagenome, was significantly less abundant in cranberry consumers compared to placebo at trial completion. Given Flavonifractor's association with negative human health effects, we sought to determine OTU41 characteristic genes that may explain its differential abundance and/or relationship to key host functions. Using comparative genomic and metagenomic techniques, we identified genes in OTU41 related to transport and metabolism of various compounds, including tryptophan and cobalamin, which have been shown to play roles in host-microbe interactions. CONCLUSION: While our results indicated that cranberry juice consumption had little impact on global measures of the microbiome, we found one unnamed Flavonifractor species differed significantly between study arms. This suggests further studies are needed to assess the role of cranberry consumption and Flavonifractor in health and wellbeing in the context of recurrent UTI. TRIAL REGISTRATION: Clinical trial registration number: ClinicalTrials.gov NCT01776021 .

Effect of a Flaxseed Lignan Intervention on Circulating Bile Acids in a Placebo-Controlled Randomized, Crossover Trial.

Plant lignans and their microbial metabolites, e.g., enterolactone (ENL), may affect bile acid (BA) metabolism through interaction with hepatic receptors. We evaluated the effects of a flaxseed lignan extract (50 mg/day secoisolariciresinol diglucoside) compared to a placebo for 60 days each on plasma BA concentrations in 46 healthy men and women (20-45 years) using samples from a completed randomized, crossover intervention. Twenty BA species were measured in fasting plasma using LC-MS. ENL was measured in 24-h urines by GC-MS. We tested for (a) effects of the intervention on BA concentrations overall and stratified by ENL excretion; and (b) cross-sectional associations between plasma BA and ENL. We also explored the overlap in bacterial metabolism at the genus level and conducted in vitro anaerobic incubations of stool with lignan substrate to identify genes that are enriched in response to lignan metabolism. There were no intervention effects, overall or stratified by ENL at FDR < 0.05. In the cross-sectional analysis, irrespective of treatment, five secondary BAs were associated with ENL excretion (FDR < 0.05). In vitro analyses showed positive associations between ENL production and bacterial gene expression of the bile acid-inducible gene cluster and hydroxysteroid dehydrogenases. These data suggest overlap in community bacterial metabolism of secondary BA and ENL.

Diet and Gut Microbes Act Coordinately to Enhance Programmed Cell Death and Reduce Colorectal Cancer Risk.

Diet is an important risk factor for colorectal cancer (CRC), and several dietary constituents implicated in CRC are modified by gut microbial metabolism. Microbial fermentation of dietary fiber produces short-chain fatty acids, e.g., acetate, propionate, and butyrate. Dietary fiber has been shown to reduce colon tumors in animal models, and, in vitro, butyrate influences cellular pathways important to cancer risk. Furthermore, work from our group suggests that the combined effects of butyrate and omega-3 polyunsaturated fatty acids (n-3 PUFA) may enhance the chemopreventive potential of these dietary constituents. We postulate that the relatively low intakes of n-3 PUFA and fiber in Western populations and the failure to address interactions between these dietary components may explain why chemoprotective effects of n-3 PUFA and fermentable fibers have not been detected consistently in prospective cohort studies. In this review, we summarize the evidence outlining the effects of n-3 long-chain PUFA and highly fermentable fiber with respect to alterations in critical pathways important to CRC prevention, particularly intrinsic mitochondrial-mediated programmed cell death resulting from the accumulation of lipid reactive oxygen species (ferroptosis), and epigenetic programming related to lipid catabolism and beta-oxidation-associated genes.

Modulation of Gut Microbiota by Glucosamine and Chondroitin in a Randomized, Double-Blind Pilot Trial in Humans.

Glucosamine and chondroitin (G&C), typically taken for joint pain, are among the most frequently used specialty supplements by US adults. More recently, G&C have been associated with lower incidence of colorectal cancer in human observational studies and reduced severity of experimentally-induced ulcerative colitis in rodents. However, little is known about their effects on colon-related physiology. G&C are poorly absorbed and therefore metabolized by gut microbiota. G&C have been associated with changes in microbial structure, which may alter host response. We conducted a randomized, double-blind, placebo-controlled crossover trial in ten healthy adults to evaluate the effects of a common dose of G&C compared to placebo for 14 days on gut microbial community structure, measured by 16S rRNA gene sequencing. Linear mixed models were used to evaluate the effect of G&C compared to placebo on fecal microbial alpha and beta diversity, seven phyla, and 137 genera. Nine genera were significantly different between interventions (False Discovery Rate < 0.05). Abundances of four Lachnospiraceae genera, two Prevotellaceae genera, and Desulfovibrio were increased after G&C compared to placebo, while Bifidobacterium and a member of the Christensenellaceae family were decreased. Our results suggest that G&C affect the composition of the gut microbiome which may have implications for therapeutic efficacy.

Colonic mucosal and exfoliome transcriptomic profiling and fecal microbiome response to a flaxseed lignan extract intervention in humans.

BACKGROUND: Microbial metabolism of lignans from high-fiber plant foods produces bioactive enterolignans, such as enterolactone (ENL) and enterodiol (END). Enterolignan exposure influences cellular pathways important to cancer risk and is associated with reduced colon tumorigenesis in animal models and lower colorectal cancer risk in humans. OBJECTIVES: The aim of this study was to test the effects of a flaxseed lignan supplement (50 mg secoisolariciresinol diglucoside/d) compared with placebo on host gene expression in colon biopsies and exfoliated colonocyte RNA in feces and fecal microbial community composition, and to compare responses in relation to ENL excretion. METHODS: We conducted a 2-period randomized, crossover intervention in 42 healthy men and women (20-45 y). We used RNA-seq to measure differentially expressed (DE) genes in colonic mucosa and fecal exfoliated cells through the use of edgeR and functional analysis with Ingenuity Pathway Analysis. We used 16S ribosomal RNA gene (V1-V3) analysis to characterize the fecal microbiome, and measured END and ENL in 24-h urine samples by gas chromatography-mass spectrometry. RESULTS: We detected 32 DE genes (false discovery rate <0.05) in the exfoliome, but none in the mucosal biopsies, in response to 60 d of lignan supplement compared with placebo. Statistically significant associations were detected between ENL excretion and fecal microbiome measured at baseline and at the end of the intervention periods. Further, we detected DE genes in colonic mucosa and exfoliome between low- and high-ENL excreters. Analysis of biopsy samples indicated that several anti-inflammatory upstream regulators, including transforming growth factor ß and interleukin 10 receptor, were suppressed in low-ENL excreters. Complementary analyses in exfoliated cells also suggested that low-ENL excreters may be predisposed to proinflammatory cellular events due to upregulation of nuclear transcription factor κB and NOS2, and an inhibition of the peroxisome proliferator-activated receptor γ network. CONCLUSIONS: These results suggest that ENL or other activities of the associated gut microbial consortia may modulate response to a dietary lignan intervention. This has important implications for dietary recommendations and chemoprevention strategies. This study was registered at clinicaltrials.gov as NCT01619020.

Plasma metabolite abundances are associated with urinary enterolactone excretion in healthy participants on controlled diets.

Enterolignans, products of gut bacterial metabolism of plant lignans, have been associated with reduced risk of chronic diseases, but their association with other plasma metabolites is unknown. We examined plasma metabolite profiles according to urinary enterolignan excretion in a cross-sectional analysis using data from a randomized crossover, controlled feeding study. Eighty healthy adult males and females completed two 28-day feeding periods differing by glycemic load, refined carbohydrate, and fiber content. Lignan intake was calculated from food records using a polyphenol database. Targeted metabolomics was performed by LC-MS on plasma from fasting blood samples collected at the end of each feeding period. Enterolactone (ENL) and enterodiol, were measured in 24 h urine samples collected on the penultimate day of each study period using GC-MS. Linear mixed models were used to test the association between enterolignan excretion and metabolite abundances. Pathway analyses were conducted using the Global Test. Benjamini-Hochberg false discovery rate (FDR) was used to control for multiple testing. Of the metabolites assayed, 121 were detected in all samples. ENL excretion was associated positively with plasma hippuric acid and melatonin, and inversely with epinephrine, creatine, glycochenodeoxycholate, and glyceraldehyde (P < 0.05). Hippuric acid only satisfied the FDR of q < 0.1. END excretion was associated with myristic acid and glycine (q < 0.5). Two of 57 pathways tested were associated significantly with ENL, ubiquinone and terpenoid-quinone biosynthesis, and inositol phosphate metabolism. These results suggest a potential role for ENL or ENL-metabolizing gut bacteria in regulating plasma metabolites.

Endogenous and exogenous equol are antiestrogenic in reproductive tissues of apolipoprotein e-null mice.

Equol is an isoflavone (IF) metabolite produced by intestinal microbiota in a subset of people consuming dietary soy. Equol producers may show different responses to soy foods and phenotypes related to cancer risk. Here, we assessed the effects of soy IF, endogenous microbial equol production, and dietary racemic equol in a 3 × 2 × 2 factorial experiment using gnotobiotic apoE-null mice (n = 9-11/group/sex). At age 3-6 wk, equol-producing microbiota were introduced to one-half of the colony (n = 122). At age 6 wk, mice were randomized to receive a diet that contained 1 of 3 protein sources: casein and lactalbumin, alcohol-washed soy protein (low IF), and intact soy protein (high IF), with total IF amounts of 0, 42, and 566 mg/kg diet, respectively. One-half of each diet group also received racemic equol (291 mg/kg diet). After 16 wk of dietary treatment, serum isoflavonoid profiles varied with sex, soy IF amount, and intestinal microbiota status. There were no treatment effects on tissues of male mice. In females, reproductive tissue phenotypes differed by equol-producing ability (i.e., microbiota status) but not dietary equol or IF content. Equol producers had lower uterine weight, vaginal epithelial thickness, total uterine area, endometrial area, and endometrial luminal epithelial height compared with nonproducers (P < 0.05 for all), with an association between microbiota status and estrous cycle (P > chi-square = 0.03). Exogenous equol reduced expression of progesterone receptor (PGR) and the proliferation marker Ki67 (P < 0.0001) in vaginal epithelium and endometrium; for endogenous equol, only PGR was reduced (P < 0.0005). Our findings indicate that equol diminishes estrogen-dependent tissue responses in apoE-null mice.

Assessing exposure to lignans and their metabolites in humans.

Phytoestrogens occur naturally in plants and are structurally similar to mammalian estrogens. The lignans are a class of phytoestrogen and can be metabolized to the biologically active enterolignans, enterodiol, and enterolactone by a consortium of intestinal bacteria. Secoisolariciresinol diglucoside (SDG), a plant lignan, is metabolized to enterodiol and, subsequently, enterolactone. Matairesinol, another plant lignan, is metabolized to enterolactone. Other dietary enterolignan precursors include lariciresinol, pinoresinol, syringaresinol, arctigenin, and sesamin. Enterolignan exposure is determined in part by intake of these precursors, gut bacterial activity, and host conjugating enzyme activity. A single SDG dose results in enterolignan appearance in plasma 8-10 h later--a timeframe associated with colonic bacterial metabolism and absorption. Conjugation of enterolignans with sulfate and glucuronic acid occurs in the intestinal wall and liver, with the predominant conjugates being glucuronides. Controlled feeding studies have demonstrated dose-dependent urinary lignan excretion in response to flaxseed consumption (a source of SDG); however, even in the context of controlled studies, there is substantial interindividual variation in plasma concentrations and urinary excretion of enterolignans. The complex interaction between colonic environment and external and internal factors that modulate it likely contribute to this variation. Knowledge of this field, to date, indicates that understanding the sources of variation and measuring the relevant panel of compounds are important in order to use these measures effectively in evaluating the impact of lignans on human health.