Distillers dried grains with solubles (DDGS) is a major coproduct in corn-based bioethanol plants. As a staple feed ingredient for livestock and poultry, the nutrient profile of DDGS has been extensively documented. However, its fermentation metabolites and their association with pre- and postfermentation processing have not been well-defined. In the current study, paired whole stillage (WS) and DDGS samples from 10 biofuel plants in the eight Midwestern states of the United States were analyzed for their nutritional composition, and their fermentation metabolites were quantitatively determined by liquid chromatography-mass spectrometry analysis and then modeled by multivariate analysis. The protein, lipid, fiber, and ash concentrations of these industrial WS and DDGS samples were within the expected ranges of values. Glycerol and 2,3-butanediol were the most abundant alcohols besides ethanol, while lactic and acetic acids were the most abundant organic acids. Many alcohols, organic acids, aldehydes, ketones, and polyamines were present as minor components in highly variable concentrations. Interestingly, both WS and DDGS samples were clustered in their respective multivariate models based on two prefermentation processing platforms using different amounts of enzymes and heat, and this processing-related separation was mainly driven by the differences in multiple essential amino acids. In addition, the differences between WS and DDGS through correlation analysis showed the influences of postfermentation procedures on many organic coproducts. Overall, these results on the metabolic impacts of industrial fermentation and its pre- and postfermentation processing could be used as reference values for future development of processing and fermentation technology for bioethanol, DDGS, and valuable coproducts.

Deoxynivalenol (DON) is a highly reactive epoxy-sesquiterpenoid mycotoxin commonly present in cereal feed ingredients. Dietary DON contamination negatively affects feed intake, growth, and health status in all stages of swine production. Both in vivo biotransformation, including somatic xenobiotic metabolism and microbial metabolism, and ex vivo chemical mitigation reactions, such as bisulfite-based sulfonation, have been shown to reduce the reactivity and bioavailability of DON in pigs. However, the influences of age and maturation on the disposition of DON and its chemical mitigation in pigs have not been examined in detail. The objective of the current study was to evaluate growth performance and the metabolomic profiling of DON and its derivatives in feeds, feces, and urine through two feeding trials. In a 21-day nursery trial, 4 groups of pigs (n = 12/group) were fed low- and high-dose DON with and without 0.25% Notox D, a bisulfite agent. In a 49-day finishing trial, 5 groups of pigs (n = 12/group) were fed the DON-contaminated feed without a mitigant, or with 0.25% Notox D, and 0.25%, 0.50%, and 0.75% sodium metabisulfite. The performance results showed that sulfonation treatment restored the growth of nursery pigs under the high-dose DON exposure (3.7 ppm), but there was no difference in pig growth under the low-dose DON exposure (0.8 ppm for nursery pigs and 1.2 ppm for finishing pigs). Metabolomic profiling indicated that finishing pigs were more capable than nursery pigs in the microbial conversion of DON to deepoxy-deoxynivalenol (DOM), DOM absorption, and somatic production of their glucuronides. Bisulfite agents effectively and dose-dependently decreased DON in pig feeds by forming DON sulfonates (DON-S), which were further enriched and concentrated in feces. The decreases of free and total DON in urine after sulfonation treatment were correlated with the improved growth performance in nursery pigs. Overall, these results revealed extensive interactions among maturation, DON disposition, and sulfonation treatment in pigs and hence warrant further investigations to establish the reference values on the age of pigs, the level of DON contamination, and the dose of bisulfite agents to guide the mitigation practices in swine production.

This experiment was conducted to evaluate the effects of dietary supplementation level of a two-strain Bacillus subtilis probiotic on growth performance, blood parameters, fecal metabolites, and microbiome in nursery pigs. A total of 54 weaned piglets were allotted to three treatments in three replicate pens with six pigs/pen for a 28 d feeding trial. The treatments were as follows: control: no probiotic supplementation; Pro1x: B. subtilis supplementation at 1.875 × 105 CFU/g diet; and Pro10x: B. subtilis supplementation at 1.875 × 106 CFU/g diet. Body weight at d 14 postweaning (p = 0.06) and average daily gain for d 0 to 14 postweaning (p < 0.05) were greater in the Pro1x treatment than in the other treatments. Blood glucose levels were greater in both probiotic treatments than in the control treatment at d 14 postweaning (p < 0.05). In the fecal short-chain fatty acid (SCFA) concentrations, the butyrate concentrations were greater in the Pro1x treatment than in the other treatments (p < 0.05), and the acetate, propionate, and total SCFA concentrations were greater in the Pro1x treatment than in the Pro10x treatment (p < 0.05). The beta diversity of fecal microbiome composition at d 14 postweaning based on Unweighted Unifrac analysis was dissimilar between the Pro1x and Pro10x treatments (p < 0.05). In conclusion, dietary B. subtilis supplementation of two strains selected to reduce effects of pathogenic Escherichia coli to nursery diets at 1.875 × 105 CFU/g diet improved the growth rate in the early postweaning period, increased fecal SCFA concentrations and altered the fecal microbial community composition. A higher dose of B. subtilis did not improve the performance parameters over those of the control piglets.

Deoxynivalenol (DON) is a primary mycotoxin in cereal feed ingredients that negatively affects feed intake and immune function of pigs. Formation of DON sulfonate (DONS) with sulfites in feed has become an effective approach to mitigate the DON toxicities in practice, but the metabolic fate of DON under sulfonation-based decontamination was not well defined. In this study, 48 nursery pigs (35 d of age, 10.8 ± 1.3 kg) were stratified by weight and sex and grouped following a 2 x2 factorial design on the DON content in corn-soybean meal diets (1.2 vs. 4.1 ppm) and the inclusion of NoTox Ultimate D, a sulfite-based mitigant (0 vs. 0.25%). Four experimental diets, including low-DON feed with no mitigant treatment (LD-NT); low-DON feed with sulfonation treatment (LD-ST): high-DON feed with no mitigant treatment (HD-NT); high-DON feed with sulfonation treatment (HD-ST), were fed for 21 d for urine collection on day 0, 10, and 21 and fecal collection on day 21. Urine and fecal samples were analyzed by both targeted metabolite analysis and untargeted metabolomic modeling. DON itself was absent in all fecal and urine samples, indicating a complete biotransformation. As expected, sulfonation led to the formation of DONS in feces in correlation with the DON content in feed and the decrease of two DON glucuronides in urine (HD-ST < HD-NT). Interestingly, sulfonation did not decrease the concentrations of two deepoxy-deoxynivalenol (DOM) glucuronides in urine. Instead, it significantly increased DOM in feces (HD-ST > HD-NT). The results indicate that the formation of DONS by sulfite treatment alters the biotransformation and excretion of DON by decreasing the availability of free DON for absorption and simultaneously increasing its availability for microbial metabolism to form DOM, an inactivated metabolite, for fecal and urine excretion.

Rutin, a natural flavonol glycoside, elicits its diverse health-promoting effects from the bioactivities of quercetin, its aglycone. While widely distributed in the vegetables and fruits of human diet, rutin is either absent or inadequate in common animal feed ingredients. Rutin has been supplemented to dairy cows for performance enhancement, but its metabolic fate in vivo has not been determined. In this study, plasma, urine, and rumen fluid samples were collected before and after the intraruminal dosing of 100 mg/kg rutin to 4 Holsteins, and then characterized by both targeted and untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomic analysis. In plasma and urine, 4-methylcatechol sulfate was identified as the most abundant metabolite of rutin, instead of quercetin and its flavonol metabolites, and its concentration was inversely correlated with the concentration of p-cresol sulfate. In rumen fluid, the formation of 3,4-dihydroxyphenylacetic acid (DHPAA) and 4-methylcatechol after rapid degradation of rutin and quercetin concurred with the decrease of p-cresol and the increase of its precursor, 4-hydroxyphenylacetic acid. Overall, the formation of 4-methylcatechol, a bioactive microbial metabolite, as the dominant bioavailable metabolite of rutin and quercetin, could contribute to their beneficial bioactivities in dairy cows, while the decrease of p-cresol, a microbial metabolite with negative biological and sensory properties, from the competitive inhibition between microbial metabolism of rutin and tyrosine, has the potential to reduce environmental impact of dairy operations and improve the health of dairy cattle.

Abstract Camellia oil is widely recognized as a high‐quality culinary oil in East Asia for its organoleptic and health‐promoting properties, but its chemical composition and thermal stability have not been comprehensively defined by comparisons with other oils. In this study, the triacylglycerols (TAGs) in camellia, olive, and six other edible oils were profiled by the liquid chromatography‐mass spectrometry (LC‐MS)‐based chemometric analysis. Besides observing the similarity between camellia oil and olive oil, TAG profiling showed that OOO, POO, and OOG (O: oleic acid, P: palmitic acid, and G: gadoleic acid) can jointly serve as the identity markers of camellia oil. Thermal stability of virgin camellia oil (VCO) was further evaluated by extensive comparisons with virgin olive oil (VOO) in common lipid oxidation indicators, aldehyde production, and antioxidant and pro‐oxidant contents. The results showed that p‐anisidine value (AnV) was the sensitive lipid oxidation indicator, and C9‐C11 aldehydes, including nonanal, 2‐decenal, 2,4‐decadienal, and 2‐undecenal, were the most abundant aldehydes in heated VCO and VOO. Under the frying temperature, heated VCO had lower AnV and less aldehydes than heated VOO. Interestedly, the VCO had lower levels of pro‐oxidant components, including α‐linolenic acid, free fatty acids, and transition metals, as well as lower levels of antioxidants, including α‐tocopherol and phenolics, than the VOO. Overall, great similarities and subtle differences in TAG and aldehyde profiles were observed between camellia and olive oils, and the thermal stability of camellia oil might be more dependent on the balance among its unsaturation level, pro‐oxidant, and antioxidant components than a single factor.

Acetate is an intermediate metabolite originated from multiple important metabolic pathways. Even though blood acetate level has been associated with many health events, it is not commonly monitored in clinical practice, partially due to the needs for invasive blood collection and the challenges in acetate analysis. N-acetyltaurine (NAT) was earlier identified as a novel urinary metabolite of ethanol from the reaction between taurine and ethanol-derived acetate. As a direct metabolite of acetate, NAT has the potential to function as a urinary biomarker that reflects the acetate level inside the body. To test this hypothesis, this study examined the correlations between serum acetate level and urinary NAT level in three experimental animal models of hyperacetatemia. Glycerol-triacetate (GTA) dosing, ethanol dosing, and streptozotocin (STZ)-induced Type 1 diabetes were used to achieve hyperacetatemia in mice. In GTA model, serum samples were collected at 2 h and urine samples were collected for 24 h after dosing the mice with 5.8 g/kg GTA. In ethanol dosing, serum were collected at 2 h after intraperitoneal injection of 4 g/kg ethanol, while 24 h urine samples were collected before and after 14-day feeding of modified semi-solid diet containing 2.2%–6.7% (v/v) ethanol. In the Type I diabetes model, urine and serum samples were collected before and 5 days after the intraperitoneal injection of 180 mg/kg STZ. The concentrations of NAT and creatinine in urine, as well as acetate in serum, were measured using their respective liquid chromatography-mass spectrometry (LC-MS) methods. The occurrence of hyperacetatemia in three animal models was confirmed by the clear elevation of serum acetate concentrations. The concentrations of urinary NAT were also dramatically increased in three animal models, suggesting the correlations between serum acetate and urinary NAT. Urinary NAT is an effective metabolic marker of hyperacetatemia in three experimental models. The results warrant further investigation on its application in other pathophysiological conditions and in humans. This research was partially supported by the Agricultural Experiment Station project MIN-18–125 (C. C.) from the United States Department of Agriculture (USDA).

Choline and its metabolites have diverse and important functions in many physiological processes, especially for anabolic metabolism in growth and reproduction. Besides endogenous biosynthesis and direct choline supplement, choline esters in the diet are another source of choline in the body. Phenolic choline esters are a group of unique dietary choline esters rich in the seeds of Brassicaceae plants, among which sinapine is a choline ester of sinapic acid abundant in rapeseed. In this study, 40 nursery pigs were fed with rapeseed-derived feed ingredients (RSF) or soybean meal for 3 weeks (20 pigs/diet). The metabolic fate of sinapine-derived choline in RSF was examined by comparing the distribution of choline and its metabolites in digesta, liver, and serum samples by liquid chromatography-mass spectrometry analysis. The results showed that choline was released from extensive hydrolysis of sinapine in the small intestine. However, sinapine-derived choline did not increase the levels of choline and its major metabolites, including betaine, phosphocholine, and glycerophosphocholine, in the liver and serum. Instead, RSF feeding increased trimethylamine (TMA), the microbial metabolite of choline, in the large intestine and further increased trimethylamine N-oxide (TMAO), the oxidation metabolite of TMA, in the liver and serum. Overall, these results suggested that sinapine-derived choline from rapeseed feeding had limited influences on the post-absorption choline pool as a result of its low bioavailability but may serve as a major source of TMAO through microbial metabolism in nursery pigs. Improving the bioavailability of sinapine-derived choline might have the potential to modify the nutritional values and functionalities of rapeseed meal in swine feeding.

Abstract Mycoplasma hyopneumoniae, the primary pathogenic bacterium causing enzootic pneumonia, significantly affects worldwide swine production. The infection is usually persistent and bacterial identification and isolation of M. hyopneumoniae in clinical samples are challenging due to the fastidious requirements for its growth. Hence, new practical surveillance tools that improve or complement existing diagnostics on M. hyopneumoniae are desirable, especially in early infection. The objective of this study was to identify potential metabolite markers of early M. hyopneumoniae infection in pigs through metabolomics analysis. Samples obtained from pigs in a previous M. hyopneumoniae experimental infection were used in this study. Briefly, two pigs served as mock inoculated controls and ten pigs were intra-tracheally inoculated with M. hyopneumoniae. Sera, laryngeal swabs (LS), and tracheo-bronchial lavage fluid (TBLF) were collected from all pigs at 0, 2, 5, 9, 14, 21 and 28 days post-inoculation (dpi). Bronchial swabs (BS) were collected post-mortem at 28 dpi. Mycoplasma hyopneumoniae infection was confirmed by PCR in LS, TBLF and BS. Serum metabolites were profiled using high-resolution liquid chromatography–mass spectrometry (LC–MS) analysis. Metabolite markers were identified by structural analysis following multivariate analysis of LC–MS data. The results showed that M. hyopneumoniae infection time-dependently altered the serum levels of selective amino acids and fatty acids. α-Aminobutyric acid and long-chain fatty acids were markedly increased at 14 and 21 dpi in inoculated pigs (p < 0.05). These results indicated that M. hyopneumoniae infection caused systemic changes in host metabolism, warranting further studies to determine underlying biochemical and physiological mechanisms responsible for the observed changes.

Consumption of thermally oxidized oil is associated with metabolic disorders, but oxidized oil-elicited changes in the metabolome are not well defined. In this study, C57BL/6 mice were fed the diets containing either control soybean oil or heated soybean oil (HSO) for 4 weeks. HSO-responsive metabolic events were examined through untargeted metabolomics-guided biochemical analysis. HSO directly contributed to the presence of new HSO-derived metabolites in urine and the decrease of polyunsaturated fatty acid-containing phospholipids in serum and the liver. HSO disrupted redox balance by decreasing hepatic glutathione and ascorbic acid. HSO also activated peroxisome proliferator-activated receptors, leading to the decrease of serum triacylglycerols and the changes of cofactors and products in fatty acid oxidation pathways. Most importantly, multiple metabolic changes, including the decrease of tryptophan in serum; the increase of NAD+ in the liver; the increases of kynurenic acid, nicotinamide and nicotinamide N-oxide in urine; and the decreases of the metabolites from pyridine nucleotide degradation in the liver indicated that HSO activated tryptophan–NAD+ metabolic pathway, which was further confirmed by the upregulation of gene expression in this pathway. Because NAD+ and its metabolites are essential cofactors in many HSO-induced metabolic events, the activation of tryptophan–NAD+ pathway should be considered as a central metabolic response to the exposure of HSO.

Fecal microbiota transplantation (FMT) is a highly effective treatment of recurrent and recalcitrant Clostridium difficile infection (rCDI). In a recent study oral-delivery of encapsulated, freeze-dried donor material, resulted in comparable rates of cure to colonoscopic approaches. Here we characterize shifts in the fecal bacterial community structure of patients treated for rCDI using encapsulated donor material. Prior to FMT, patient fecal samples showed declines in diversity and abundance of Firmicutes and Bacteroidetes, with concurrent increases in members of the Proteobacteria, specifically Enterobacteriaceae. Moreover, patients who experienced recurrence of CDI within the 2-month clinical follow-up had greater abundances of Enterobacteriaceae and did not show resolution of dysbioses. Despite resolution of rCDI following oral-administration of encapsulated fecal microbiota, community composition was slow to return to a normal donor-like assemblage. Post-FMT taxa within the Firmicutes showed rapid increases in relative abundance and did not vary significantly over time. Conversely, Bacteroidetes taxa only showed significant increases in abundance after one month post-FMT, corresponding to significant increases in the community attributable to the donors. Changes in the associations among dominant OTUs were observed at days, weeks, and months post-FMT, suggesting shifts in community dynamics may be related to the timing of increases in abundance of specific taxa. Administration of encapsulated, freeze-dried, fecal microbiota to rCDI patients resulted in restoration of bacterial diversity and resolution of dysbiosis. However, shifts in the fecal microbiome were incremental rather than immediate, and may be driven by changes in community dynamics reflecting changes in the host environment.

Fecal microbiota transplantation (FMT) has become an effective therapy for recurrent Clostridium difficile infection (R‐CDI). Besides reestablishing a normal intestinal microbiome that offers better resistance to R‐CDI, the introduction of microflora from healthy donors through FMT is also expected to change the metabolomes in the intestine and the whole body due to diverse metabolic activities of transplanted microbiota. Therefore, monitoring the metabolic changes in biofluids and excretes provides the opportunities for identifying noninvasive biomarkers of microbial recolonization and understanding the metabolic influences of FMT on host and microflora. In this study, FMT‐induced metabolic changes were examined by the metabolomic comparison of pre‐FMT and post‐FMT urine samples from 18 R‐CDI patients who had symptoms rapidly resolved after receiving FMT from two donors. After the liquid chromatography‐mass spectrometry (LC‐MS) analysis of urine samples from the patients and the donors, the LC‐MS data were processed by partial‐least squares‐discriminant analysis (PLS‐DA), leading to the establishment of a multivariate model revealing the separation between pre‐ and post‐FMT urine samples as well as the similarity between post‐FMT and donor samples. Among a group of urinary metabolites affected by the FMT, the most prominent biomarkers are p‐cresol sulfate and secondary bile acid conjugates, which were absent in most pre‐FMT samples, but dramatically increased in the post‐FMT samples. The presence of p‐cresol sulfate and secondary bile acid conjugates in urine after FMT reflected the recovery of microfloral metabolism on tyrosine and primary bile acids in R‐CDI patients. Their levels in urine may function as effective and sensitive therapeutic indicators of the readiness of CDI patient for FMT as well as the recolonization of healthy microfloral colonies after FMT.Support or Funding InformationNIAID grant R21 AI114722 and NIFA project MIN‐18‐092

Background Avenanthramides (AVA) are a group of diphenolic acids found only in oats that have anti-inflammatory and antioxidant effects. Absorption of AVAs in humans after oral consumption of natural oat flour is unknown. Objective To examine the appearance of AVAs in plasma after oral ingestion of oat cookies and estimate key pharmacokinetic parameters. Methods Male and female nonobese participants (n = 16) consumed three cookies made with oat flour containing high (229.6 mg/kg, H-AVA) or low (32.7 mg/kg, L-AVA) amounts of AVAs, including AVA-A, AVA-B, and AVA-C. Blood samples were collected at 0, 0.5, 1, 2, 3, 5, and 10 h after ingestion. Plasma total (conjugated and free) AVA concentrations were quantified using UPLC-MS, and pharmacokinetic parameters for each AVA were estimated. Results AVAs reached peak concentrations in plasma between 2 and 3 h for the H-AVA group and between 1 and 2 h for the L-AVA group. Maximal plasma concentrations for AVAs were higher in the H-AVA than in the L-AVA group. AVA-B demonstrated a longer half-life and slower elimination rate than AVA-A and AVA-C. Conclusions AVAs found naturally in oats are absorbed in the plasma after oral administration in humans. AVA-B has the slowest elimination rate and the longest half-life compared to AVA-A and AVA-C, while AVA-C demonstrated the lowest plasma concentrations. This study is registered with ClinicalTrials.gov identifier NCT02415374.

Phytochemicals are under intensive investigation for their potential use as chemopreventive agents in blocking or suppressing carcinogenesis. Metabolic interactions between phytochemical and biological system play an important role in determining the efficacy and toxicity of chemopreventive phytochemicals. However, complexities of phytochemical biotransformation and intermediary metabolism pose challenges for studying phytochemical-elicited metabolic events. Metabolomics has become a highly effective technical platform to detect subtle changes in a complex metabolic system. Here, using green tea polyphenols as an example, we describe a workflow of LC-MS-based metabolomics study, covering the procedures and techniques in sample collection, preparation, LC-MS analysis, data analysis, and interpretation.

ObjectivesAvenanthramides (AVA) are a group of diphenolic acids that are found only in oats and have anti‐inflammatory and antioxidant effects. Although previous publications have reported AVA are bioavailable in humans, our study is the first to examine whether AVA are bioavailable in humans after acute consumption of oat cookies made with natural and non‐treated oat flour. We examined the metabolic fate of orally ingested oat AVA by measuring plasma AVA concentrations and their pharmacological characteristics.MethodsSixteen male and female non‐obese subjects were enrolled in a randomized, crossover trial. Subjects received three cookies made with oat flour containing high (229.6 mg/kg, H‐AVA) or low (32.7 mg/kg, L‐AVA) AVA on separate occasions. The H‐AVA cookie (7.2 mg/cookie; 21.6 mg total) contained 5.09 mg AVA‐A, 8.67 mg AVA‐B and 7.85 mg AVA‐C, whereas the L‐AVA (1.03 mg/cookie; 3.09 mg total) contained 0.57 mg AVA‐A, 0.95 mg AVA‐B and 1.55 mg AVA‐C. Subjects consumed the cookies in the morning after 10 h fast and blood samples were collected at 0 (baseline), 0.5, 1, 2, 3, 5 and 10h. Plasma total (conjugated and free) AVA were quantified with Ultra Performance Liquid Chromatography Quadrupole Time‐of‐Flight Mass Spectrometer (UPLC Q‐TOF‐MS), and pharmacokinetic (PK) parameters were calculated and presented as mean±SD. Half life time (T1/2) was calculated through logarithmic transformation and elimination constant (Kel) was calculated using equation Kel=0.693/T1/2. At least three time points in elimination phase were required to calculate T1/2. All plasma concentrations used for estimation of PK parameters were above assay limit of quantitation.ResultsAVA‐A, ‐B and ‐C were present at the peak concentration (Tmax) in plasma at 2.50±1.2 h, 2.04±0.9 h and 2.29±1.0 h in H‐AVA and at 1.80±1.3 h, 1.50±1.5 h and 1.32±1.0 h in L‐AVA, respectively. Maximal plasma concentrations (Cmax) for AVA‐A, ‐B and ‐C were 8.39±4.2 ng/ml, 8.44±2.3 ng/ml and 4.26±2.0 ng/ml in H‐AVA, and 1.98±1.3 ng/ml, 2.43±1.1 ng/ml and 1.33±0.7 ng/ml in L‐AVA, respectively (P<0.001). H‐AVA cookies led to significantly higher AVA‐A and ‐B than ‐C concentrations (P<0.05). Half‐life (T1/2), the time taken to fall to half initial value was 2.16±0.6 h, 4.23±0.8 h and 2.71±1.2 h in H‐AVA, and 2.44±0.8 h, 4.60±2.7 h and 2.87±1.4 h in L‐AVA, respectively (P<0.05, AVA‐B vs. AVA‐A or AVA‐C in H‐AVA).ConclusionsAVA found naturally in oats are bioavailable in humans. AVA‐B has the slowest elimination rate and highest half‐life compared to AVA‐A and AVA‐C, while AVA‐C has the lowest absorption rate.Support or Funding InformationThis study is sponsored by PepsiCo Nutrition.

SCOPE We previously showed that apiaceous but not cruciferous vegetables reduced DNA adducts formed by 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) in rats. Here, we report the effects of the putative chemopreventive phytochemicals from these vegetables on PhIP metabolism and DNA adduct formation. METHODS AND RESULTS Rats received three supplemented diets: P + I (phenethyl isothiocyanate and indole-3-carbinol), furanocoumarins (FC, 5-methoxypsoralen, 8-methoxypsoralen, and isopimpinellin), and combination (P + I and FC). Phytochemical supplementation matched the levels in vegetables fed in our previous study. After 6 days, rats were injected with PhIP (10 mg/kg body wt) and killed after 24-h urine collection. Compared to the control, P + I increased activity of hepatic cytochrome P450 (CYP) 1A1 (10.1-fold), CYP1A2 (3.62-fold), and sulfotransferase 1A1 (2.70-fold). The combination diet also increased CYP1A1 and CYP1A2 activity. Urinary metabolomics revealed that PhIP metabolite profiles generally agreed with biotransformation enzyme activities. P + I and combination diets reduced PhIP-DNA adducts by 43.5 and 24.1%, respectively, whereas FC had no effect on adducts, compared to the control diet. CONCLUSION Effects of phytochemicals on metabolic outcomes and markers of carcinogenesis might differ from fresh vegetables, thus limiting the inferences that one can draw from the effects of purified phytochemicals on the health benefits of the vegetables from which they derive.

BACKGROUND:Heterocyclic aromatic amines, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are carcinogenic compounds produced during heating of protein-containing foods. Apiaceous vegetables inhibit PhIP-activating enzymes, whereas cruciferous vegetables induce both PhIP-activating and -detoxifying enzymes.OBJECTIVE:We investigated the effects of these vegetables, either alone or combined, on PhIP metabolism and colonic DNA adduct formation in rats.METHODS:Male Wistar rats were fed cruciferous vegetables (21%, wt:wt), apiaceous vegetables (21%, wt:wt), or a combination of both vegetables (10.5% wt:wt of each). Negative and positive control groups were fed an AIN-93G diet. After 6 d, all groups received an intraperitoneal injection of PhIP (10 mg · kg body weight(-1)) except for the negative control group, which received only vehicle. Urine was collected for 24 h after the injection for LC-tandem mass spectrometry metabolomic analyses. On day 7, rats were killed and tissues processed.RESULTS:Compared with the positive control, cruciferous vegetables increased the activity of hepatic PhIP-activating enzymes [39.5% and 45.1% for cytochrome P450 (CYP) 1A1 (P = 0.0006) and CYP1A2 (P < 0.0001), respectively] and of uridine 5'-diphospho-glucuronosyltransferase 1A (PhIP-detoxifying) by 24.5% (P = 0.0267). Apiaceous vegetables did not inhibit PhIP-activating enzymes, yet reduced colonic PhIP-DNA adducts by 20.4% (P = 0.0496). Metabolomic analyses indicated that apiaceous vegetables increased the relative abundance of urinary methylated PhIP metabolites. The sum of these methylated metabolites inversely correlated with colonic PhIP-DNA adducts (r = -0.43, P = 0.01). We detected a novel methylated urinary PhIP metabolite and demonstrated that methylated metabolites are produced in the human liver S9 fraction.CONCLUSIONS:Apiaceous vegetables did not inhibit the activity of PhIP-activating enzymes in rats, suggesting that the reduction in PhIP-DNA adducts may involve other pathways. Further investigation of the importance of PhIP methylation in carcinogen metabolism is warranted, given the inverse correlation of methylated PhIP metabolites with a biomarker of carcinogenesis and the detection of a novel methylated PhIP metabolite.

Fecal microbiota transplantation (FMT) has emerged as a highly effective therapy for refractory, recurrent Clostridium difficile infection (CDI), which develops following antibiotic treatments. Intestinal microbiota play a critical role in the metabolism of bile acids in the colon, which in turn have major effects on the lifecycle of C. difficile bacteria. We hypothesized that fecal bile acid composition is altered in patients with recurrent CDI and that FMT results in its normalization. General metabolomics and targeted bile acid analyses were performed on fecal extracts from patients with recurrent CDI treated with FMT and their donors. In addition, 16S rRNA gene sequencing was used to determine the bacterial composition of pre- and post-FMT fecal samples. Taxonomic bacterial composition of fecal samples from FMT recipients showed rapid change and became similar to the donor after the procedure. Pre-FMT fecal samples contained high concentrations of primary bile acids and bile salts, while secondary bile acids were nearly undetectable. In contrast, post-FMT fecal samples contained mostly secondary bile acids, as did non-CDI donor samples. Therefore, our analysis showed that FMT resulted in normalization of fecal bacterial community structure and metabolic composition. Importantly, metabolism of bile salts and primary bile acids to secondary bile acids is disrupted in patients with recurrent CDI, and FMT corrects this abnormality. Since individual bile salts and bile acids have pro-germinant and inhibitory activities, the changes suggest that correction of bile acid metabolism is likely a major mechanism by which FMT results in a cure and prevents recurrence of CDI.

Fecal microbiota transplantation (FMT) has emerged as a highly effective therapy for refractory, recurrent Clostridium difficile infection (CDI), which develops following antibiotic treatments. Intestinal microbiota play a critical role in the metabolism of bile acids in the colon, which in turn have major effects on the lifecycle of C. difficile bacteria. We hypothesized that fecal bile acid composition is altered in patients with recurrent CDI and that FMT results in its normalization. General metabolomics and targeted bile acid analyses were performed on fecal extracts from patients with recurrent CDI treated with FMT and their donors. In addition, 16S rRNA gene sequencing was used to determine the bacterial composition of pre- and post-FMT fecal samples. Taxonomic bacterial composition of fecal samples from FMT recipients showed rapid change and became similar to the donor after the procedure. Pre-FMT fecal samples contained high concentrations of primary bile acids and bile salts, while secondary bile acids were nearly undetectable. In contrast, post-FMT fecal samples contained mostly secondary bile acids, as did non-CDI donor samples. Therefore, our analysis showed that FMT resulted in normalization of fecal bacterial community structure and metabolic composition. Importantly, metabolism of bile salts and primary bile acids to secondary bile acids is disrupted in patients with recurrent CDI, and FMT corrects this abnormality. Since individual bile salts and bile acids have pro-germinant and inhibitory activities, the changes suggest that correction of bile acid metabolism is likely a major mechanism by which FMT results in a cure and prevents recurrence of CDI.

Rodent animal models have been widely used for studying neurologic and toxicological events associated with cocaine abuse. It is known that the mouse is more susceptible to cocaine-induced hepatotoxicity (CIH) than the rat. However, the causes behind this species-dependent sensitivity to cocaine have not been elucidated. In this study, cocaine metabolism in the mouse and rat was characterized through LC-MS-based metabolomic analysis of urine samples and were further compared through calculating the relative abundance of individual cocaine metabolites. The results showed that the levels of benzoylecgonine, a major cocaine metabolite from ester hydrolysis, were comparable in the urine from the mice and rats treated with the same dose of cocaine. However, the levels of the cocaine metabolites from oxidative metabolism, such as N-hydroxybenzoylnorecgonine and hydroxybenzoylecgonine, differed dramatically between the two species, indicating species-dependent cocaine metabolism. Subsequent structural analysis through accurate mass analysis and LC-MS/MS fragmentation revealed that N-oxidation reactions, including N-demethylation and N-hydroxylation, are preferred metabolic routes in the mouse, while extensive aryl hydroxylation reactions occur in the rat. Through stable isotope tracing and in vitro enzyme reactions, a mouse-specific α-glucoside of N-hydroxybenzoylnorecgonine and a group of aryl hydroxy glucuronides high in the rat were identified and structurally elucidated. The differences in the in vivo oxidative metabolism of cocaine between the two rodent species were confirmed by the in vitro microsomal incubations. Chemical inhibition of P450 enzymes further revealed that different P450-mediated oxidative reactions in the ecgonine and benzoic acid moieties of cocaine contribute to the species-dependent biotransformation of cocaine.