Pre-diagnostic plasma enterolactone concentrations are associated with lower mortality among individuals with type 2 diabetes: a case-cohort study in the Danish Diet, Cancer and Health cohort.

AIMS/HYPOTHESIS: The phytoestrogen enterolactone is a gut microbiota-derived metabolite of plant lignans with suggested beneficial properties for health. In the current study, we investigated the association between pre-diagnostic plasma enterolactone concentrations and mortality among individuals diagnosed with type 2 diabetes. METHODS: In a population of people diagnosed with diabetes, nested within the Danish Diet, Cancer and Health cohort, we conducted a case-cohort study including a random sample of n = 450 cases (deceased) and a randomly selected subcohort of n = 850 (in total n = 617 deaths). Information on diagnosis, vital status and cause of death was obtained from Danish registers. Cox proportional hazard models with special weighting were applied to assess all-cause and cause-specific mortality. RESULTS: The median enterolactone concentration of the current population was low, 10.9 nmol/l (5th percentile to 95th percentile: 1.3-59.6), compared with previously reported concentrations from the Diet, Cancer and Health cohort. Pre-diagnostic enterolactone concentrations were associated with lower all-cause mortality when assessed linearly per doubling in concentration (log2) (HR 0.91 [95% CI 0.85, 0.96]) and according to quartiles (HR 0.63 [95% CI 0.48, 0.84]) for the highest quartile of enterolactone compared with the lowest quartile. For cause-specific mortality, only death from diabetes (registered as underlying cause of death) reached statistical significance. CONCLUSIONS/INTERPRETATION: Based on this large cohort of people with diabetes with detailed and complete baseline and follow-up information, pre-diagnostic enterolactone concentrations were inversely associated with mortality. To our knowledge, this is the first study on enterolactone and type 2 diabetes mortality. Our findings call for further exploration of enterolactone in type 2 diabetes management.

Prediagnosis plasma concentrations of enterolactone and survival after colorectal cancer: the Danish Diet, Cancer and Health cohort.

The association between lifestyle and survival after colorectal cancer has received limited attention. The female sex hormone, oestrogen, has been associated with lower colorectal cancer risk and mortality after colorectal cancer. Phyto-oestrogens are plant compounds with structure similar to oestrogen, and the main sources in Western populations are plant lignans. We investigated the association between the main lignan metabolite, enterolactone and survival after colorectal cancer among participants in the Danish Diet, Cancer and Health cohort. Prediagnosis plasma samples and lifestyle data, and clinical data from time of diagnosis from 416 women and 537 men diagnosed with colorectal cancer were used. Enterolactone was measured in plasma using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Participants were followed from date of diagnosis until death or end of follow-up. During this time, 210 women and 325 men died (170 women and 215 men died due to colorectal cancer). The Cox proportional hazards model was used to estimate hazard ratios (HR) and 95 % CI. Enterolactone concentrations were associated with lower colorectal cancer-specific mortality among women (HRper doubling: 0·88, 95 % CI 0·80, 0·97, P=0·0123). For men, on the contrary, enterolactone concentrations were associated with higher colorectal cancer-specific mortality (HRper doubling: 1·10, 95 % CI 1·01, 1·21, P=0·0379). The use of antibiotics affects enterolactone production, and the associations between higher enterolactone and lower colorectal cancer-specific mortality were more pronounced among women who did not use antibiotics (analysis on a subset). Our results suggest that enterolactone is associated with lower risk of mortality among women, but the opposite association was found among men.

Effect of Antibiotics and Diet on Enterolactone Concentration and Metabolome Studied by Targeted and Nontargeted LC-MS Metabolomics.

High plant lignan intake is associated with a number of health benefits, possibly induced by the lignan metabolite enterolactone (ENL). The gut microbiota plays a crucial role in converting dietary lignans into ENL, and epidemiological studies have shown that use of antibiotics is associated with lower levels of ENL. Here we investigate the link between antibiotic use and lignan metabolism in pigs using LC-MS/MS. The effect of lignan intake and antibiotic use on the gut microbial community and the pig metabolome is studied by 16S rRNA sequencing and nontargeted LC-MS. Treatment with antibiotics resulted in substantially lower concentrations of ENL compared with concentrations detected in untreated animals, whereas the plasma concentrations of plant lignans were unchanged. Both diet and antibiotic treatment affected the clustering of urinary metabolites and significantly altered the proportions of taxa in the gut microbiota. Diet, but not antibiotic treatment, affected the plasma lipid profile, and a lower concentration of LDL cholesterol was observed in the pigs fed a high lignan diet. This study provides solid support for the associations between ENL concentrations and use of antibiotics found in humans and indicates that the lower ENL concentration may be a consequence of the ecological changes in the microbiota.

Rye Bran Modified with Cell Wall-Degrading Enzymes Influences the Kinetics of Plant Lignans but Not of Enterolignans in Multicatheterized Pigs.

Background: Whole-grain intake is associated with a lower risk of chronic Western-style diseases, possibly brought about by the high concentration of phytochemicals, among them plant lignans (PLs), in the grains.Objective: We studied whether treatment of rye bran with cell wall-degrading enzymes changed the solubility and kinetics of PLs in multicatheterized pigs.Methods: Ten female Duroc × Danish Landrace × Yorkshire pigs (60.3 ± 2.3 kg at surgery) fitted with permanent catheters were included in an incomplete crossover study. The pigs were fed 2 experimental diets for 1-7 d. The diets were rich in PLs and based on nontreated lignan-rich [LR; lignan concentration: 20.2 mg dry matter (DM)/kg] or enzymatically treated lignan-rich (ENZLR; lignan concentration: 27.8 mg DM/kg) rye bran. Plasma concentrations of PLs and enterolignans were quantified with the use of targeted LC-tandem mass spectrometry. Data were log transformed and analyzed with mixed-effects, 1-compartment, and asymptotic regression models.Results: The availability of PLs was 38% greater in ENZLR than in LR, and the soluble fraction of PLs was 49% in ENZLR compared with 35% in LR diets. PLs appeared in the circulation 30 min after intake of both the ENZLR and LR diets. Postprandially, consumption of ENZLR resulted in a 4-times-greater (P < 0.0001) plasma PL concentration compared with LR. The area under the curve (AUC) measured 0-360 min after ENZLR intake was ∼2 times higher than after LR intake. A 1-compartment model could describe the postprandial increase in plasma concentration after ENZLR intake, whereas an asymptotic regression model described the plasma concentrations after LR intake. Despite increased available and soluble PLs, ENZLR did not increase plasma enterolignans.Conclusion: The modification of rye bran with cell wall-degrading enzymes resulted in significantly greater plasma concentrations of PLs and the 4-h AUC, particularly syringaresinol, in multicatheterized pigs.

High-Throughput LC-MS/MS Method for Direct Quantification of Glucuronidated, Sulfated, and Free Enterolactone in Human Plasma.

Sulfation and glucuronidation constitute a major pathway in humans and may play an important role in biological activity of metabolites including the enterolignan, enterolactone. Because the aromatic structure of enterolactone has similarities to steroid metabolites, it was hypothesized that enterolactone may protect against hormone-dependent cancers. This led to numerous epidemiological studies. In this context, there has been a demand for rapid, sensitive, high-throughput methods to measure enterolactone in biofluids. Different methods have been developed using GC-MS, HPLC, LC-MS/MS and a fluoroimmunoassay; however, most of these methods measure the total concentration of enterolactone, without any specification of its conjugation pattern. Here for the first time we present a high-throughput LC-MS/MS method to quantify enterolactone in its intact form as glucuronide, sulfate, and free enterolactone. The method has shown good accuracy and precision at low concentration and very high sensitivity, with LLOQ for enterolactone sulfate at 16 pM, enterolactone glucuronide at 26 pM, and free enterolactone at 86 pM. The short run time of 2.6 min combined with simple sample clean up and high sensitivity make this method attractive for the high-throughput of samples needed for epidemiological studies. Finally, we have adapted the new method to quantify enterolactone and its conjugates in 3956 plasma samples from an epidemiological study. We found enterolactone glucuronide to be the major conjugation form and that conjugation pattern was similar between men and women.

Quantitative distribution of flavan-3-ols, procyanidins, flavonols, flavanone and salicylic acid in five varieties of organic winter dormant Salix spp. by LC-MS/MS.

Willow trees (Salix spp.) exhibit remarkable genetic and phenotypic diversity, yielding a broad spectrum of bioactive compounds, notably valuable phenolic compounds such as condensed tannins (phenolic polymers), flavonoids, salicylic glucosides, and phenolic compounds. These enhance the economic value of willow crops and make them suitable for circular bioeconomy. Phenolic compounds known for their diverse applications as antioxidants, antimicrobial agents, pharmaceuticals, nutraceuticals and antiseptics and more, find a natural source in willow. This study aimed to elucidate the composition of 12 flavonoids and salicylic acid in different segments of five organic winter dormant willow species (S. daphnoides, S. fragilis, S. dasyclados, S. viminalis, and S. dasyclados x viminalis) using quantitative analysis and providing valuable insights into their high-value phenolic compounds. Separation into buds, wood and bark segments allowed for a precise characterization of the location of certain phenolic compounds and quantification using LC-MS/MS techniques. LC-MS/MS is an analytical technique known for its increased sensitivity and chromatographic precision. Among the findings, catechin emerged as the predominant flavan-3-ol in all Salix species, with the highest concentration in the buds of Salix viminalis (7.26 mg/g DM). Naringenin exhibited species-specific variations, with S. dasyclados and S. viminalis recording the highest levels. Salicylic acid concentrations peaked in S. dasyclados (5.38 mg/g DM) and S. daphnoides (4.43 mg/g DM), particularly within the bark. When evaluating other individual flavonoids and total polyphenol content (TPC), disparities between buds, bark, and wood became evident, with wood consistently displaying the lowest content. Notably, the higher concentration of polyphenolic compounds in willow bark can be attributed to its susceptibility to external threats and its role as a robust defense mechanism against pathogens and herbivores. This study underscores the significance of diverse willow species as a source of high-value phenolic compounds, distributed differentially across plant parts and species. This knowledge holds promise for their potential applications in the circular bioeconomy.

Multicompartmental nontargeted LC-MS metabolomics: explorative study on the metabolic responses of rye fiber versus refined wheat fiber intake in plasma and urine of hypercholesterolemic pigs.

A multicompartmental nontargeted LC-MS metabolomics approach was used to study the metabolic responses on plasma and urine of hypercholesterolemic pigs after consumption of diets with contrasting dietary fiber composition (whole grain rye with added rye bran versus refined wheat). To study the metabolic responses, we performed a supervised multivariate data analyses used for pattern recognition, which revealed marked effects of the diets on both plasma and urine metabolic profiles. Diverse pools of metabolites were responsible for the discrimination between the diets. Elevated levels of phenolic compounds and dicarboxylic acids were detected in urine of pigs after rye consumption compared to refined wheat. Furthermore, consumption of rye was characterized by lower levels of linoleic acid derived oxylipins and cholesterol in the plasma metabolic profiles. These results indicate that higher consumption of nonrefined dietary fiber is reflected in higher excretion of phenolic compounds and dicarboxylic acids in urine and lower levels of linoleic acid derived oxylipins and cholesterol in plasma, which can be linked to beneficial health effects of rye components. On the other hand, pro-inflammatory lipid mediators were detected in higher concentration after rye consumption compared to refined wheat, which is opposite to what would be expected. These may indicate that even though a positive lowering effect with respect to cholesterol and fatty acids was achieved, this effect of rye dietary fiber was not sufficient to prevent inflammation in pigs. Moreover, we performed an alignment of the metabolic profiles between the breads consumed by pigs, plasma, and urine with the purpose to follow the metabolic fate of the compounds and to identify their pathways. One metabolite was identified in all three compartments, 16 metabolites were similar between bread and plasma, 3 were similar between plasma and urine, and 2 were similar between bread and urine. The use of multicompartmental metabolomics offered higher order information, including intercompartment relationships, and provided novel targets for future research.

Comprehensive quantification of flavonoids and salicylic acid representative of Salix spp. using microLiquid Chromatography-Triple Quadrupole Mass Spectrometry: the importance of drying procedures and extraction solvent when performing classical solid-liquid extraction.

Willow (Salix spp.) is gaining an increasing interest as a fast-growing tree with high biomass yield from low agricultural inputs, which contains potentially bioactive compounds. The present work aimed to develop a high-yield extraction procedure combined with robust, sensitive and fast microLiquid Chromatography-Triple Quadrupole Mass Spectrometry (LC-MS/MS) based method for comprehensively quantifying flavonoids and salicylic acid in the bark of Salix spp. We have investigated the effect of freeze- and oven-drying procedures and five extraction solvents on the yield of individual flavonoid and salicylic acid when performing classical solid-liquid extraction. The freeze-drying was the best drying procedure for preserving monomeric and polymeric flavan-3-ols, whereas other flavonoids were less affected. Salicylic acid was not affected by the drying procedures. The best extraction solvent in terms of the yield of individual flavonoid among the tested solvents in this study was the combination of methanol acidified with 1% hydrochloric acid. LC-MS/MS method has shown a high recovery percentage (≥80%), good precision and overall robustness.

Dose- and substrate-dependent reduction of enteric methane and ammonia by natural additives in vitro.

Ruminants contribute to global warming by emitting greenhouse gasses, particularly methane (CH4) which is a product of rumen fermentation. The use of feed additives able to modulate rumen fermentation is a promising strategy to reduce enteric CH4 and ammonia (NH3) emissions. Among the various strategies investigated, plant secondary metabolites (PSMs) have attracted attention due to their apparent potential to reduce enteric CH4 and NH3 emissions, and it would be possible to use such compounds as feed additives in organic production systems. In an in vitro system simulating rumen fermentation, we have tested the impact of different classes of naturally occurring PSMs; catechin and quercetin (flavonoids), salicylic acid (phenolic acid) and tannic acid (hydrolysable tannin). The PSMs were added to two different basal feeds (maize and grass silages) at three inclusion doses 1.5, 3 and 6% of the feed dry matter (DM). CH4 production was significantly lowered upon addition of quercetin to two basal feeds at doses of 3 and 6%, and this without changes in concentrations of total volatile fatty acid (VFA) produced during fermentation. Quercetin, as the only tested additive, reduced CH4 production, and when added to maize silage and grass silage, the reduction increased linearly with increasing dose, ie., by 51 and 43%, respectively, at a dose of 3% of feed DM and by 86 and 58%, respectively, at a dose of 6% of feed DM. Moreover, quercetin significantly reduced NH3 concentration by >12% at doses of 3 and 6% in feed DM irrespective of the basal feed used as compared to when the basal feeds were incubated alone. Although none of the other additives affected CH4 formation, several additives had significant impacts on concentrations of NH3 and VFAs in the incubated fluid after fermentation. This study demonstrated a dose-dependent ability of quercetin to reduce CH4 emission from rumen fermentation, however, the magnitude of the suppression of CH4 depended on the basal feed. Furthermore, quercetin reduced NH3 concentration irrespective of the basal feed type. These findings encourage to in vivo studies to verify whether quercetin can reduce CH4 emission also in cows.

Methane reduction by quercetin, tannic and salicylic acids: influence of molecular structures on methane formation and fermentation in vitro.

Plant secondary metabolites (PSMs) can potentially reduce ruminal methane formation. However, related to differences in their molecular structures, it is not yet clear what causes an anti-methanogenic effect. In an in vitro system simulating rumen fermentation, we investigated the impact of eight compounds with distinct chemical characteristics (gallic and salicylic acids, tannic acid, catechin, epicatechin, quercetin, rutin, and salicin) when added to a basal feed (maize silage) at a concentration of 12% of the feed dry matter. After 48 h of incubation in buffered rumen fluid, methane production was significantly lowered by quercetin (43%), tannic acid (39%) and salicylic acid (34%) compared to the control (maize silage alone) and without changes in total volatile fatty acid production during fermentation. No other PSM reduced methane formation as compared to control but induced significant differences on total volatile fatty acid production. The observed differences were related to lipophilicity, the presence of double bond and carbonyl group, sugar moieties, and polymerization of the compounds. Our results indicate the importance of distinct molecular structures of PSMs and chemical characteristics for methane lowering properties and volatile fatty acid formation. Further systematic screening studies to establish the structure-function relationship between PSMs and methane reduction are warranted.

Feeding level during the last week of gestation can influence performance of sows and their litters in the subsequent lactation.

This study investigated the impact of feeding level during the last week of gestation on performance of sows and their litters in the subsequent lactation. A total of 48 sows were assigned to one of six feeding levels (1.8, 2.4, 3.1, 3.7, 4.3, or 5.0 kg/d) from day 108 of gestation until farrowing. Post-farrowing, all sows were fed similarly during lactation with a gradual increase in feed allowance in accordance with Danish recommendation until it reached the maximum allowance of 9 kg/d on day 17 of lactation. Plasma samples were collected from the sows during farrowing and lactation, and sow's body weight and backfat thickness, and milk samples were taken during lactation. Litters were standardized to have 13 to 15 piglets each and weighed once weekly during lactation. Plasma concentrations of urea, acetate, and butyrate in sows linearly increased (P < 0.001), while non-esterified fatty acids linearly decreased (P < 0.001) during farrowing with increasing feeding level. Moreover, concentrations of triglycerides (P < 0.001), acetate (P = 0.007), and succinate (P < 0.001) were greater in plasma collected at the onset of farrowing compared to the end of farrowing. Conversely, concentrations of glucose, urea, and butyrate (P < 0.001) were lower in plasma collected at the onset of farrowing than at the end. Sows fed 2.4 and 3.1 kg/d exhibited greater triglyceride concentrations than those fed 3.7 (P = 0.03) and 5.0 (P = 0.02) kg/d. Sows fed 1.8 kg/d during the last week of gestation had lower milk yield in wk 1 (P < 0.001) and wk 2 (P = 0.001) of lactation compared to the other groups. Additionally, litter weight gain (P = 0.04) and litter weaning weight (P = 0.007) were lower in sows fed 1.8 kg/d compared to the other groups. The greatest milk yield, litter growth, and litter size were observed in sows fed 3.7 kg/d during the last week of gestation, whereas the estimate generated by the regression model revealed that sows should have been provided with 4.0 to 4.1 kg/d as an adequate feeding level to maximize these performances in the subsequent lactation. Interestingly, feeding level during the last week of gestation did not influence feed intake during lactation. In conclusion, this study highlights the significance of adequate feed supply (4.0 to 4.1 kg/d) during the last week of gestation in order to maximize performance of sows in the subsequent lactation.

Feeding sows at high or ad libitum level during an extended gestation period has been found to have a negative impact on the subsequent lactation performance. Conversely, in some European countries, it has been a common practice to reduce the feed allowance during the last 2 to 3 d before expected farrowing to mitigate issues like constipation and postpartum health problems such as mastitis, metritis, and agalactia. However, recent studies suggest that sows should be fed approximately 4 kg/d during transition period to improve farrowing performance. In the present study, we investigated the carry-over effects of feeding level (dose­response design) during the last week of gestation on lactation performance of sows and their litters in the subsequent lactation. The findings revealed that a lower feeding level during the last week of gestation reduced milk yield and litter size at weaning. Milk yield, litter growth, and litter size at weaning were greater in sows supplied with 3.7 kg/d during the last week of gestation. However, the estimate generated by the regression model indicates that sows should be provided with 4.0 to 4.1 kg/d during the final week of gestation to enhance their performance in the subsequent lactation. Therefore, the present results emphasize the benefits of maintaining an adequate feeding level during the last week of gestation to improve sows' lactation performance in the subsequent lactation.

Effects on Serum Hormone Concentrations after a Dietary Phytoestrogen Intervention in Patients with Prostate Cancer: A Randomized Controlled Trial.

Phytoestrogens have been suggested to have an anti-proliferative role in prostate cancer, potentially by acting through estrogen receptor beta (ERß) and modulating several hormones. We primarily aimed to investigate the effect of a phytoestrogen intervention on hormone concentrations in blood depending on the ERß genotype. Patients with low and intermediate-risk prostate cancer, scheduled for radical prostatectomy, were randomized to an intervention group provided with soybeans and flaxseeds (∼200 mg phytoestrogens/d) added to their diet until their surgery, or a control group that was not provided with any food items. Both groups received official dietary recommendations. Blood samples were collected at baseline and endpoint and blood concentrations of different hormones and phytoestrogens were analyzed. The phytoestrogen-rich diet did not affect serum concentrations of testosterone, insulin-like growth factor 1, or sex hormone-binding globulin (SHBG). However, we found a trend of decreased risk of increased serum concentration of estradiol in the intervention group compared to the control group but only in a specific genotype of ERß (p = 0.058). In conclusion, a high daily intake of phytoestrogen-rich foods has no major effect on hormone concentrations but may lower the concentration of estradiol in patients with prostate cancer with a specific genetic upset of ERß.

Untargeted Metabolomics Combined with Solid Phase Fractionation for Systematic Characterization of Bioactive Compounds in Hemp with Methane Mitigation Potential.

This study systematically evaluates the presence of methane mitigating metabolites in two hemp (Cannabis sativa L.) varieties, Futura 75 and Finola. Hemp metabolites were extracted with methanol and fractionated using Solid Phase Extraction (SPE). Extracts, fractions, and the remaining pulp were screened for their methane mitigating potential using an in vitro model of rumen fermentation. The bioactive metabolites were identified with Liquid Chromatography-Mass Spectrometry (LC-MS). When incubated with a standard feed (maize silage), the extract of Futura 75 significantly reduced methane production compared to that of control (without added extract) and without negative effects on feed degradability and volatile fatty acid patterns. The compounds responsible for the methane mitigating effect were assigned to flavonoid glycosides. However, none of the fractions of Futura 75 or the pulp exhibited similar effect on methane emission. Butyric acid concentration in the fermentation inoculum was significantly increased, which could indicate why methane production was higher, when incubated with the fractions and the pulp. The extract of Finola did not show a similar significant effect, however, there was a numerical tendency towards lower methane production. The difference in methane mitigating properties between Cannabis sativa L. Futura 75 and Finola, could be related to the content of bioactive flavonoids.

Determination of the Optimal Level of Dietary Zinc for Newly Weaned Pigs: A Dose-Response Study.

One hundred and eighty individually housed piglets with an initial body weight of 7.63 ± 0.98 kg (at 28 days of age) were fed a diet containing either 153, 493, 1022, 1601, 2052 or 2407 mg zinc/kg (added Zn as zinc oxide; ZnO) from day 0-21 post weaning to determine the optimal level of Zn for weaned piglets. Body weight, feed intake and faecal scores were recorded, and blood and faecal samples were collected. Dietary Zn content quadratically affected both feed intake and gain in the first two weeks, with an approximately 1400 mg Zn/kg diet and a Zn intake of 400 mg/day as the optimal levels. The relative risk of diarrhoea increased up to 60% at day 7 and 14 if serum Zn status dropped below the weaning level (767 µg/L), and maintain the weaning serum Zn status required approximately 1100 mg Zn/kg (166 mg Zn/day) during week 1. Blood markers of intestinal integrity (D-lactate and diamine oxidase) were unaffected by dietary Zn, and dietary Zn levels of 1022 and 1601 mg/kg did not affect the faecal numbers of total bacteria, Lactobacilli and E. Coli bacteria compared to 153 mg Zn/kg. These results indicate that the requirement for Zn in newly weaned piglets may be substantially higher than currently assumed.

Targeted and Untargeted Metabolic Profiling to Discover Bioactive Compounds in Seaweeds and Hemp Using Gas and Liquid Chromatography-Mass Spectrometry.

Greenhouse gas emissions are a global problem facing the dairy/beef industry. Novel feed additives consisting of seaweeds and hemp containing bioactive compounds are theorized to reduce enteric methane emissions. In this study we aimed to investigate the metabolic profiles of brown, red and green seaweeds and hemp using gas chromatography and liquid chromatography mass spectrometry. We used targeted and untargeted approaches, quantifying known halomethanes and phenolics, as well as identifying potentially novel bioactive compounds with anti-methanogenic properties. The main findings were: (a) Asparagopsis taxiformis contained halomethanes, with high concentrations of bromoform (4200 µg/g DW), six volatile halocarbons were tentatively identified; (b) no halomethanes were detected in the other studied seaweeds nor in hemp; (c) high concentrations of lignans were measured in hemp; (d) a high numbers of sulfated phenolic acids and unidentified sulfuric acid-containing compounds were detected in all seaweeds; (e) flavonoid glucosides and glucuronides were mainly identified in hemp; and (f) the condensed tannin gallocatechin was tentatively identified in Fucus sp. Using the combined metabolomics approach, an overview and in-depth information on secondary metabolites were provided. Halomethanes of Asparagopsis sp. have already been shown to be anti-methanogenic; however, metabolic profiles of seaweeds such as Dictyota and Sargassum have also been shown to contain compounds that may have anti-methanogenic potential.

Impact of feed glyphosate residues on broiler breeder egg production and egg hatchability.

Glyphosate is the active substance in glyphosate-based herbicides, e.g. Roundup. Its widespread application on feed crops leaves residues in the feed. Glyphosate has antimicrobial and mineral chelating properties and we investigated whether there is an association between feed residues of glyphosate on the one side and broiler breeder egg laying percent and egg hatchability on the other side. Twenty-six feed samples from five conventional flocks producing hatching eggs were analysed for glyphosate. Data on laying percent and egg hatchability from periods following each feed sampling were then associated with feed residues of glyphosate. The average glyphosate residue level was 0.09 mg/kg, maximum was 0.19 and minimum was 0.004 mg/kg. Average laying percent over observation days was 65% (SD = 5.4%) and average hatchability was 79% (SD = 5.8%). We found a negative association between feed glyphosate residue level and hatchability (P = 0.03) when adjusted for breeder age, storage time of eggs on farm before delivery and storage time at hatchery before incubation start. No association was found with laying percent (P = 0.59) adjusted for breeder age. The range of glyphosate concentrations in feed was narrow and should be kept in mind when interpreting both significant and non-significant associations with glyphosate residue concentrations. In nine of 24 analysed conventional eggs the concentration of glyphosate in yolk was above the detection limit however below the quantification limit indicating that traces of glyphosate are common in conventional eggs.

Obesity-Related Metabolome and Gut Microbiota Profiles of Juvenile Göttingen Minipigs-Long-Term Intake of Fructose and Resistant Starch.

The metabolome and gut microbiota were investigated in a juvenile Göttingen minipig model. This study aimed to explore the metabolic effects of two carbohydrate sources with different degrees of risk in obesity development when associated with a high fat intake. A high-risk (HR) high-fat diet containing 20% fructose was compared to a control lower-risk (LR) high-fat diet where a similar amount of carbohydrate was provided as a mix of digestible and resistant starch from high amylose maize. Both diets were fed ad libitum. Non-targeted metabolomics was used to explore plasma, urine, and feces samples over five months. Plasma and fecal short-chain fatty acids were targeted and quantified. Fecal microbiota was analyzed using genomic sequencing. Data analysis was performed using sparse multi-block partial least squares regression. The LR diet increased concentrations of fecal and plasma total short-chain fatty acids, primarily acetate, and there was a higher relative abundance of microbiota associated with acetate production such as Bacteroidetes and Ruminococcus. A higher proportion of Firmicutes was measured with the HR diet, together with a lower alpha diversity compared to the LR diet. Irrespective of diet, the ad libitum exposure to the high-energy diets was accompanied by well-known biomarkers associated with obesity and diabetes, particularly branched-chain amino acids, keto acids, and other catabolism metabolites.

Effects of whole-grain wheat, rye, and lignan supplementation on cardiometabolic risk factors in men with metabolic syndrome: a randomized crossover trial.

BACKGROUND: A whole-grain (WG)-rich diet has shown to have potential for both prevention and treatment of the metabolic syndrome (MetS), which is a cluster of risk factors that increase the risk of type 2 diabetes and cardiovascular disease. Different WGs may have different health effects. WG rye, in particular, may improve glucose homeostasis and blood lipids, possibly mediated through fermentable dietary fiber and lignans. Recent studies have also suggested a crucial role of the gut microbiota in response to WG. OBJECTIVES: The aim was to investigate WG rye, alone and with lignan supplements [secoisolariciresinol diglucoside (SDG)], and WG wheat diets on glucose tolerance [oral-glucose-tolerance test (OGTT)], other cardiometabolic outcomes, enterolignans, and microbiota composition. Moreover, we exploratively evaluated the role of gut microbiota enterotypes in response to intervention diets. METHODS: Forty men with MetS risk profile were randomly assigned to WG diets in an 8-wk crossover study. The rye diet was supplemented with 280 mg SDG at weeks 4-8. Effects of treatment were evaluated by mixed-effects modeling, and effects on microbiota composition and the role of gut microbiota as a predictor of response to treatment were analyzed by random forest plots. RESULTS: The WG rye diet (± SDG supplements) did not affect the OGTT compared with WG wheat. Total and LDL cholesterol were lowered (-0.06 and -0.09 mmol/L, respectively; P < 0.05) after WG rye compared with WG wheat after 4 wk but not after 8 wk. WG rye resulted in higher abundance of Bifidobacterium [fold-change (FC) = 2.58, P < 0.001] compared with baseline and lower abundance of Clostridium genus compared with WG wheat (FC = 0.54, P = 0.02). The explorative analyses suggest that baseline enterotype is associated with total and LDL-cholesterol response to diet. CONCLUSIONS: WG rye, alone or with SDG supplementation, compared with WG wheat did not affect glucose metabolism but caused transient LDL-cholesterol reduction. The effect of WG diets appeared to differ according to enterotype. This trial was registered at www.clinicaltrials.gov as NCT02987595.

Robust and highly sensitive micro liquid chromatography-tandem mass spectrometry method for analyses of polar pesticides (glyphosate, aminomethylphosfonic acid, N-acetyl glyphosate and N-acetyl aminomethylphosfonic acid) in multiple biological matrices.

Glyphosate is the most used herbicide in agriculture. To monitor glyphosate exposure, analytical methods have to fulfill requirements with regard to sensitivity, reproducibility, ease of handling/high-throughput and applicability to multiple biological matrices. Furthermore, the methods have to include the degradation product of glyphosate, aminomethylphosfonic acid (AMPA) and preferably metabolites of glyphosate and AMPA, N-acetyl AMPA and N-acetyl glyphosate. Majority of the published methods for glyphosate and AMPA require derivatization to be able to achieve high sensitivity. In this work, we present highly sensitive microLC-MS/MS method for simultaneous quantification of glyphosate, AMPA, N-acetyl AMPA and N-acetyl glyphosate in multiple biological matrices without derivatization. The combination of simple sample clean-up procedures for simultaneous handling of 96 sample and short chromatographic run of only 3.4 min, meets the requirements for high-throughput methods. Simple mobile phase of water containing formic and medronic acids and isocratic run provided robust chromatographic separation on hypercarb column. The use of micro-flow system decreased the background noise, increasing the sensitivity. Achieved Low Limits of Quantification (LOQs) for liquid samples (plasma/serum/urine) were 0.00005 mg L-1 and 0.0001 mg kg-1 for solid samples (grain and soybean based feed/stomach/gizzard/intestinal content), which is more than 100 time more sensitive compared to QuPPe-Method. The method was validated in representative matrices with minimum of five fortification levels, six measurements per spiked concentration and three batches. All the samples were spiked with corresponding internal standards for all four analytes before sample clean-up procedures, ensuring high accuracy and precision. Recoveries for plasma/serum ranged between 86-108%, urine 93-120%, feed 91-115% and stomach/gizzard/intestinal content 92-110% with precision below 20%. The method's applicability was tested on 2000 samples measured during one year period.

Concentrations of phytoestrogens in conventional, organic and free-range retail milk in England.

The effect of dairy management system (conventional, CNV; organic, ORG; free-range, FRG) and month on retail milk phytoestrogen composition was assessed for 12 consecutive months. ORG milk contained more secoisolariciresinol, matairesinol, lariciresinol, sum of plant lignans, daidzein, genistein, formononetin, naringenin, equol, sum of isoflavones and coumestrol, than CNV and FRG milk. This may be explained by the higher supply of pasture, and grazed or ensiled clover, in ORG dairy diets. Seasonal variation in milk phytoestrogen concentrations was higher for ORG than CNV and FRG systems. Phytoestrogen composition did not vary between FRG and CNV milk. Consuming organic milk can increase intake of potentially beneficial lignans and isoflavonoids, and in particular equol; but, any effects on human health from such milk compositional differences cannot be implied.