Chronic consumption of orange juice modifies urinary excretion of flavanone gut-derived metabolites through gut microbiota modulation.

The metabolism and absorption of citrus flavanones are intrinsically linked to the gut microbiota, creating a bidirectional relationship where these compounds influence the microbiome, and in turn, the microbiota affects their metabolism. This study evaluates the effect of acute and chronic consumption of orange juice (OJ) on the urinary excretion of gut-derived flavanone metabolites and the gut microbiota. Health volunteers ingested 500 mL of OJ for 60 days in a single-arm human intervention study. Blood and feces were collected at baseline and after 60 days, with an additional 24-hour urine collection after a single dose on day 1 and day 63. LC-MS/MS analyzed urinary flavanone metabolites, while 16S rRNA sequencing characterized gut microbiota. Total urinary hesperetin conjugates excretion significantly decreased over 60 days, while gut-derived total phenolic acids, particularly three hydroxybenzoic acids, increased. Moreover, the heterogeneity of the total amount of flavanone conjugates, initially categorizing individuals into high-, medium- and low- urinary excretor profiles, shifted towards medium-excretor, except for five individuals who remained as low-excretors. This alteration was accompanied by a decrease in intestinal ß-glucosidase activity and a shift in the relative abundance of specific genera, such as decreases in Blautia, Eubacterium hallii, Anaerostipes, and Fusicatenibacter, among which, Blautia was associated with higher urinary flavanone conjugates excretion. Conversely, an increase in Prevotella was observed. In summary, chronic OJ consumption induced transient changes in gut microbiota and altered the metabolism of citrus flavanones, leading to distinct urinary excretion profiles of flavanone metabolites.

The gut microbiome drives inter- and intra-individual differences in metabolism of bioactive small molecules.

The origin of inter-individual variability in the action of bioactive small molecules from the diet is poorly understood and poses a substantial obstacle to harnessing their potential for attenuating disease risk. Epidemiological studies show that coffee lowers the risk of developing type 2 diabetes, independently of caffeine, but since coffee is a complex matrix, consumption gives rise to different classes of metabolites in vivo which in turn can affect multiple related pathways in disease development. We quantified key urinary coffee phenolic acid metabolites repeated three times in 36 volunteers, and observed the highest inter- and intra-individual variation for metabolites produced by the colonic microbiome. Notably, a urinary phenolic metabolite not requiring the action of the microbiota was positively correlated with fasting plasma insulin. These data highlight the role of the gut microbiota as the main driver of both intra- and inter-individual variation in metabolism of dietary bioactive small molecules.

Identification of the Metabolites in Rat Urine after Oral Administration and Elucidation of the Metabolic Process of Naozhenning Granule Using LC-MS.

Naozhenning (NZN) granule, a Chinese herbal formula, is widely used to treat craniocerebral trauma and promote functional recovery. In our previous study, the chemical components, as well as the serum metabolites in the male Sprague-Dawley rats of the NZN granule after oral administration were characterized. In this study, the urine metabolites in the male Sprague-Dawley rats were further investigated by ultrahigh-performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry. In order to identify the urine metabolites comprehensively, three sample preparation methods were used, including solid-phase extraction, protein precipitation method and solvent partition. Based on the accurate molecular weight and the fragmentation information from the MS spectra, a total of 76 urine metabolites were identified, which including 17 prototypes and 59 metabolites. The results showed that the detected urine metabolites were different for the different pretreatment methods, as some metabolites could only be detected in the particular pretreatment method. In addition, the metabolic processes of the components from NZN granule to the serum and urine were also elucidated and discussed. The results will provide useful information for further studying the relationship between the chemical components and pharmacological activity of NZN granule.

Profiling Vaccinium macrocarpon components and metabolites in human urine and the urine ex-vivo effect on Candida albicans adhesion and biofilm-formation.

The aim of this work was to profile, by using an HPLC-MS/MS method, cranberry compounds and metabolites found in human urine after ingestion of a highly standardized cranberry extract (Anthocran®). Two different strategies were adopted for the data analysis: a targeted and an untargeted approach. These strategies allowed the identification of 42 analytes including cranberry components, known metabolites and metabolites hitherto unreported in the literature, including six valerolactones/valeric acid derivatives whose presence in urine after cranberry consumption has never been described before. Absolute concentrations of 26 over 42 metabolites were obtained by using pure available standards. Urine collected at different time points after the last dosage of Anthocran® were tested on the reference strain C. albicans SC5314, a biofilm-forming strain. Fractions collected after 12 h were found to significantly reduce the adhesion and biofilm formation compared to the control (p < 0.05). A similar effect was then obtained by using Anthocran™ Phytosome™, the lecithin formulation containing 1/3 of standardized cranberry extract and formulated to enhance the absorption of the cranberry components. The urinary profile of cranberry components and metabolites in the urine fractions collected at 1 h, 6 h and 12 h after the last capsule intake were then reproduced by using the pure standards at the concentration ranges found in the urine fraction, and tested on C. albicans. Only the mixture mimicking the urinary fraction collected at 12 h and containing as main components, quercetin and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone was found effective thus confirming the ex-vivo results.

Metabolisable energy of grass and red clover silages fed to sheep at maintenance level.

This study investigated the effect of forage type (grass or red clover) and harvesting time (primary growth or regrowth) of silage on energy and N utilisation by sheep fed at maintenance level. Specifically, the assumption of constant loss of energy of digestible organic matter from energy losses in urine and CH4 applied in evaluation of silage metabolisable energy (ME) was investigated. Urinary excretion of high-energy phenolic compounds related to solubilisation of lignin was assumed to affect urinary energy (UE) losses from sheep fed highly digestible grass silage (GS). A total of 25 primary growth and regrowth silages of timothy (Phleum pratense) and meadow fescue (Festuca pratensis) grass mixtures and red clover (Trifolium pratense) samples collected in digestibility trials with sheep, including faecal and urine samples, were used for energy and N determinations. Urinary concentration of monophenolic compounds and CH4 emissions in vitro were also analysed. Daily faecal N output, CH4 yield (MJ/kg DM intake), proportion of CH4 energy in digestible energy (DE) and proportion of UE in DE were greater (P ≤ 0.03) in sheep fed red clover silage (RCS) than GS. Furthermore, less (P = 0.01) energy was lost as UE of DE in sheep fed primary growth GS compared with the other treatments. The relationship between UE and silage N intake or urinary N output for both silage types (i.e. grass v. red clover) was strong, but the fit of the regressions was better for GS than RCS. The CH4/DE ratio decreased (P < 0.05) and the UE/DE ratio increased (P < 0.05) with increasing organic matter digestibility in RCS. These relationships were not significant (P < 0.05) for the GS diets. The regression coefficient was higher (P < 0.05) for GS than RCS when regressing ME concentration on digestible organic matter. The results of this study imply that ME/DE ratio is not constant across first-cut GS of different maturities. The ME production response may be smaller from highly digestible first-cut GS but could not be clearly related to urinary excretion of monophenols derived from solubilisation of lignin. Furthermore, energy lost in urine was not clearly defined for RCS and was much more predictable for GS from silage N concentration.

Phenolics of Green Pea (Pisum sativum L.) Hulls, Their Plasma and Urinary Metabolites, Bioavailability, and in Vivo Antioxidant Activities in a Rat Model.

Increased processing of pulses generates large volumes of hulls, which are known as an excellent source of phenolic antioxidants. However, the bioavailability and in vivo activity of these phenolics are rarely reported. This research was therefore carried out to study the absorption, metabolism, and in vivo antioxidant activities of green pea hull (GPH) phenolics using ultrahigh-pressure liquid chromatography with a linear ion trap-high-resolution Orbitrap mass spectrometry and an oxidative stress rat model. A total of 31 phenolics, including 4 phenolic acids, 24 flavonoids, and 3 other phenolics, were tentatively identified. Ten of these phenolics and 49 metabolites were found in the plasma and urine of rats, which helped to explain the favorable changes by GPH phenolics in key antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and glutathione) and indicators (total antioxidant capacity, malondialdehyde) in the plasma and different tissues of rats. This is the first comprehensive report on dry pea hull phenolics and their bioavailability, metabolic profiles, and mechanisms of in vivo antioxidant activities.

The urinary phenolic acid profile varies between younger and older adults after a polyphenol-rich meal despite limited differences in in vitro colonic catabolism.

PURPOSE: To investigate whether age influences colonic polyphenol metabolism. METHODS: Healthy participants, younger (n = 8; 23-43 years) and older (n = 13; 51-76 years), followed a 3-day low-polyphenol diet (LPD) and a 3-day high-polyphenol diet (HPD). Urinary phenolic acids (PA), short chain fatty acids (SCFA), pH and gas were monitored, alongside selected colonic bacteria. Human faecal in vitro fermentations of rutin with or without raftiline were used to evaluate the gut microbiota capacity in a subset of both groups. RESULTS: Total urinary PA were higher in the older group after HPD compared to the younger group (1.5-fold; p = 0.04), with no difference between groups in terms of a change between diets (Δ high-low diet). While 17 PA were detected in all younger participants after HPD, a narrower range (n = 8 to 16 PA) was detected in most (n = 9/13) older participants, with lower level of benzoic acid (19-fold; p = 0.03), vanillic acid (4.5-fold; p = 0.04) but higher hippuric acid (2.7-fold; p = 0.03). Faecal SCFA concentration did not change after HPD within group, with similar differential excretion (Δ high-low diet) between groups. There were no differences between groups for faecal pH, total, faecal bacteria including Flavonifractor plautii, bifidobacteria, and bacteroides. In human in vitro faecal fermentations, seven PAs were detected in both groups after 24 h of rutin fermentation, with no quantitative and modest qualitative differences between groups. Total SCFA in faecal fermentation did not differ between groups, except for butyric acid (twofold higher in the older group; p = 0.009) when rutin was fermented with raftiline over 24 h. CONCLUSIONS: Urinary phenolic acids were less diverse in older participants despite limited difference in functional capacity of in vitro faecal fermentations.

Resorcylic Acid Lactones in Urine Samples of Croatian Farm Animals.

Metabolic transformation of zearalenone (ZEA), a mycotoxin which can contaminate both food and feed, results in the formation of five metabolites, one of them being zeranol (α-ZAL), which can be abused in farm animals as a growth promoter. To the best of our knowledge, there is no analytical method that can distinguish whether α-ZAL is present in an animal urine sample as a result of ZEA biotransformation or as a result of anabolic abuse. This study aimed at monitoring resorcylic acid lactones (RALs) concentration in urine of farm animals over several years. Six hundred and three cattle and pig urine samples were collected on farms in different Croatian regions and analyzed for RAL presence. Based on the testing results, all RAL-positive samples were considered to be consequential to feed contamination. The difference in primary ZEA metabolites' ratio (α-zearalenol/ß-zearalenol) was observed between cattle (0.03-0.41) and pig (2.05-17.39) urine samples. If the animals are treated with α-ZAL and fed on ZEA-contaminated feed, α-ZAL and taleranol found in their organisms could come from two sources, so that the reliability of the statistical model might be questionable. Based on these findings, there exists the need for improving the approach to the distinction between α-ZAL abuse and ZEA feed contamination.

Quantification of phenolic acid metabolites in humans by LC-MS: a structural and targeted metabolomics approach.

AIM: Co-metabolism between a human host and the gastrointestinal microbiota generates many small phenolic molecules such as 3-hydroxy-3-(3-hydroxyphenyl)propanoic acid (3,3-HPHPA), which are reported to be elevated in schizophrenia and autism. Characterization of these chemicals, however, has been limited by analytic challenges. METHODOLOGY/RESULTS: We applied HPLC to separate and quantify over 50 analytes, including multiple structural isomers of 3,3-HPHPA in human cerebrospinal fluid, serum and urine. Confirmation of identity was provided by NMR, by MS and other detection methods. The highly selective methods support rapid quantification of multiple metabolites and exhibit superior chromatographic behavior. CONCLUSION: An improved ultra-HPLC-MS/MS and structural approaches can accurately quantify 3,3-HPHPA and related analytes in human biological matrices.

Cranberries attenuate animal-based diet-induced changes in microbiota composition and functionality: a randomized crossover controlled feeding trial.

Cranberries have multiple health effects but their impact on gut microbiota has not been examined in randomized controlled feeding trials. We evaluated the relationship between the microbiota and cranberries in the context of an animal-based diet. In a randomized, double-blind, cross-over, controlled design trial, 11 healthy adults consumed for 5 days each a control diet (animal-based diet plus 30 g/day placebo powder) and a cranberry diet (animal-based diet plus 30 g/day freeze-dried whole cranberry powder). The animal-based diet included meats, dairy products, and simple sugars. Stool, urine, and blood samples were obtained before and after each intervention phase. As compared to the pre-control diet, control diet modified 46 taxonomic clades, including an increase in the abundance of Firmicutes and decrease in Bacteroidetes. Moreover, it increased bacteria-derived deoxycholic acid and decreased acetate and butyrate in stool. As compared to the post-intervention phase of control diet, the cranberry diet modified 9 taxonomic clades, including a decrease in the abundance of Firmicutes and increase in Bacteroidetes. Further, the cranberry diet attenuated control diet-induced increase in secondary bile acids and decrease in short-chain fatty acids (SCFA), and increased urinary anthocyanins and bacterially derived phenolic acids. No changes were found in fecal trimethylamine and plasma cytokines. In conclusion, an animal-based diet altered the microbiota composition to a less favorable profile, increased carcinogenic bile acids, and decreased beneficial SCFA. Cranberries attenuated the impact of the animal-based diet on microbiota composition, bile acids, and SCFA, evidencing their capacity to modulate the gut microbiota.

Simultaneous electrochemical sensing of warfarin and maycophenolic acid in biological samples.

In this study, for the first time a rapid, selective and highly sensitive method was developed for simultaneous determination of warfarin and mycophenolic acid using carbon paste electrode modified by ß-cyclodextrin/multi-walled carbon nanotubes/cobalt oxide nanoparticles (ß-CD/MWCNTs/Co3O4NPs/CPE). The oxidation peaks of desired drugs was separated enough using the constructed electrode. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were utilized for study the electrochemical response of the fabricated electrode and its surface modification was investigated by electrochemical impedance spectroscopy (EIS). Under optimal conditions, the adsorptive stripping voltammetric responses were linear in the concentration ranges 0.05-150 µM and 0.5-200 µM for WAR and MPA, respectively. The correlation coefficients were greater than 0.99. The limits of detection for WAR and MPA were 0.02 and 0.03 µM. The fabricated electrode was applied for the simultaneous determination of WAR and MPA in urine and human serum samples with satisfactory results.

Association between different dietary polyphenol subclasses and the improvement in cardiometabolic risk factors: evidence from a randomized controlled clinical trial.

AIMS: Due to their different chemical structures and metabolism, polyphenol subclasses may have specific impact on cardiometabolic risk factors. Our aim was to evaluate whether the intake of different polyphenol subclasses is associated with clinical outcomes beneficially improved by polyphenols in a nutritional trial performed by our group (postprandial lipid response, glucose homeostasis, early insulin secretion and oxidative stress). METHODS: The present study is a secondary analysis of a nutritional intervention study with a diet naturally rich in polyphenols. The data are derived from 78 participants at high cardiovascular risk who completed the ETHERPATH trial. The associations between variations in polyphenol subclasses (phenolic acids, anthocyanidins, flavones, flavan-3-ols, flavonols and flavanones) and clinical outcomes beneficially influenced by polyphenols were firstly explored by Spearman's correlation. Thereafter, adjustment for gender, age and body mass index (BMI) was run. Linear regression analysis was used to assess the class of polyphenols that best predicted the outcome. RESULTS: Flavanone intake was inversely correlated with postprandial lipid response, whereas flavone intake was related to postchallenge glucose response. Anthocyanidins and flavan-3-ols associated positively with early insulin secretion. The decrease in urinary isoprostanes correlated with anthocyanidins, flavan-3-ols and flavonols. Correlations did not change after adjustment for gender, age, and BMI. Linear regression analysis showed an independent association between flavonols and urinary isoprostanes, whereas early insulin secretion was mainly associated with flavan-3-ols intake. CONCLUSIONS: The results of this study show that a polyphenol-rich diet may have a pleiotropic effect on cardiometabolic risk factors thanks to the specific action of different polyphenol subclasses.

Excretion of Avenanthramides, Phenolic Acids and their Major Metabolites Following Intake of Oat Bran.

SCOPE: Wholegrain has been associated with reduced chronic disease mortality, with oat intake particularly notable for lowering blood cholesterol and glycemia. To better understand the complex nutrient profile of oats, we studied urinary excretion of phenolic acids and avenanthramides after ingestion of oat bran in humans. METHODS AND RESULTS: After a 2-d (poly)phenol-low diet, seven healthy men provided urine 12 h before and 48 h after consuming 60 g oat bran (7.8 µmol avenanthramides, 139.2 µmol phenolic acids) or a phenolic-low (traces of phenolics) control in a crossover design. Analysis by ultra-high performance liquid chromatography (UPLC)-MS/MS showed that oat bran intake resulted in an elevation in urinary excretion of 30 phenolics relative to the control, suggesting that they are oat bran-derived. Mean excretion levels were elevated between 0-2 and 4-8 h, following oat bran intake, and amounted to a total of 33.7 ± 7.3 µmol total excretion (mean recovery: 22.9 ± 5.0%), relative to control. The predominant metabolites included: vanillic acid, 4- and 3-hydroxyhippuric acids, and sulfate-conjugates of benzoic and ferulic acids, which accounted collectively for two thirds of total excretion. CONCLUSION: Oat bran phenolics follow a relatively rapid urinary excretion, with 30 metabolites excreted within 8 h of intake. These levels of excretion suggest that bound phenolics are, in part, rapidly released by the microbiota.

Processing 'Ataulfo' Mango into Juice Preserves the Bioavailability and Antioxidant Capacity of Its Phenolic Compounds.

The health-promoting effects of phenolic compounds depend on their bioaccessibility from the food matrix and their consequent bioavailability. We carried out a randomized crossover pilot clinical trial to evaluate the matrix effect (raw flesh and juice) of 'Ataulfo' mango on the bioavailability of its phenolic compounds. Twelve healthy male subjects consumed a dose of mango flesh or juice. Blood was collected for six hours after consumption, and urine for 24 h. Plasma and urine phenolics were analyzed by electrochemical detection coupled to high performance liquid chromatography (HPLC-ECD). Five compounds were identified and quantified in plasma. Six phenolic compounds, plus a microbial metabolite (pyrogallol) were quantified in urine, suggesting colonic metabolism. The maximum plasma concentration (Cmax) occurred 2-4 h after consumption; excretion rates were maximum at 8-24 h. Mango flesh contributed to greater protocatechuic acid absorption (49%), mango juice contributed to higher chlorogenic acid absorption (62%). Our data suggests that the bioavailability and antioxidant capacity of mango phenolics is preserved, and may be increased when the flesh is processed into juice.

Novel urinary alkylresorcinol metabolites as biomarkers of whole grain intake in free-living Swedish adults.

SCOPE: Most studies on the role of whole grain for health rely on self-reported intake data, which are prone to measurement errors. There is a need for dietary biomarkers that can provide an objective measure of intake. Alkylresorcinols (AR) and their main metabolites 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA) have been proposed as biomarkers for whole grain (WG) wheat and rye intake. METHODS AND RESULTS: The medium-term reproducibility and relative validity of four putative urinary AR metabolites (3,5-dihydroxycinnamic acid (DHCA), 5-(3,5-dihydroxyphenyl) pentanoic acid (DHPPTA), 2-(3,5-dihydroxybenzamido)acetic acid (DHBA-glycine) and 3,5-dihydroxycinnamic acid amide (DHCA-amide)) as biomarkers for WG intake were investigated. Three-day weighed food records and 24-h urine samples from two occasions 2-3 months apart were obtained from 69 Swedish adults. WG intake was calculated and urinary AR metabolites were analyzed. The medium-term reproducibility determined for DHCA, DHPPTA, and DHBA-glycine varied from moderate-to-excellent (intra-class correlation coefficient = 0.63-0.85). Moreover, DHCA and DHPPTA excretion correlated well with self-reported total WG intake (r = 0.55, p < 0.001 and r = 0.42, p < 0.001, respectively). CONCLUSION: DHCA or DHPPTA excretion in 24-h urine might be a suitable medium- to long-term biomarker of WG wheat and rye intake. These findings need to be confirmed in populations with low and infrequent WG intake.

Graphene oxide for solid-phase extraction of bioactive phenolic acids.

A solid-phase extraction (SPE) method for the efficient analysis of trace phenolic acids (PAs, caffeic acid, ferulic acid, protocatechuic acid, cinnamic acid) in urine was established. In this work, a graphene oxide (GO) coating was grafted onto pure silica to be investigated as SPE material. The prepared GO surface had a layered and wrinkled structure that was rough and well organized, which could provide more open adsorption sites. Owing to its hydrophilicity and polarity, GO showed higher extraction efficiency toward PAs than reduced GO did, in agreement with the theoretical calculation results performed by Gaussian 09 software. The adsorption mechanism of PAs on GO@Sil was also investigated through static state and kinetic state adsorption experiments, which showed a monolayer surface adsorption. Extraction capacity of the as-prepared material was optimized using the response surface methodology. Under the optimized conditions, the as-established method provided wide linearity range (2-50 µg L-1 for protocatechuic acid and 1-50 µg L-1 for caffeic acid, ferulic acid, and cinnamic acid) and low limits of detection (0.25-1 µg L-1). Finally, the established method was applied for the analysis of urine from two healthy volunteers. The results indicate that the prepared material is a practical, cost-effective medium for the extraction and determination of phenolic acids in complex matrices. Graphical Abstract A graphene oxide coating was grafted onto pure silica as the SPE material for the extraction of phenolic acids in urines and the extraction mechanism was also mainly investigated.

Urinary Biomarkers of Whole Grain Wheat Intake Identified by Non-targeted and Targeted Metabolomics Approaches.

Mounting evidence suggests that whole grain (WG) intake plays an important role in chronic disease prevention. However, numerous human studies have failed to produce clear-cut conclusions on this topic. Here, a combination of non-targeted and targeted metabolomics approaches, together with kinetic studies, was used to investigate biomarkers of WG wheat intake and further explore the diet-disease associations. Via these integrated approaches, forty-one compounds were identified as the most discriminating endogenous metabolites after WG versus refined grain (RG) wheat bread consumption. The corresponding biological assessment of these endogenous changes suggests that, in contrast to RG consumption, WG wheat consumption may facilitate antioxidant defense systems and moderate the risk factors of cancer, cardiovascular diseases, and other chronic diseases. A panel of urinary markers consisting of seven alkylresorcinol metabolites and five benzoxazinoid derivatives as specific biomarkers, as well as five phenolic acid derivatives, was also established to cover multiple time points and longer time periods for correctly and objectively monitoring WG wheat intake. Through these findings, we have established a comprehensive biomarker pool to better assess WG wheat consumption, and to monitor the endogenous changes that are linked to health effects of WG wheat consumption.

Metabolic transformations of dietary polyphenols: comparison between in vitro colonic and hepatic models and in vivo urinary metabolites.

Studies on metabolism of polyphenols have revealed extensive transformations in the carbon backbone by colonic microbiota; however, the influence of microbial and hepatic transformations on human urinary metabolites has not been explored. Therefore, the aims of this study were (1) to compare the in vitro microbial phenolic metabolite profile of foods and beverages with that excreted in urine of subjects consuming the same foodstuff and (2) to explore the role of liver on postcolonic metabolism of polyphenols by using in vitro hepatic models. A 24-h urinary phenolic metabolite profile was evaluated in 72 subjects participating in an 8-week clinical trial during which they were randomly assigned to diets differing for polyphenol content. Polyphenol-rich foods and beverages used in the clinical trial were subjected to human fecal microbiota in the in vitro colon model. Metabolites from green tea, one of the main components of the polyphenol-rich diet, were incubated with primary hepatocytes to highlight hepatic conversion of polyphenols. The analyses were performed using targeted gas chromatography with mass spectrometer (GCxGC-TOFMS:colon model; GC-MS: urine and hepatocytes). A significant correlation was found between urinary and colonic metabolites with C1-C3 side chain (P=.040). However, considerably higher amounts of hippuric acid, 3-hydroxybenzoic acid and ferulic acid were detected in urine than in the colon model. The hepatic conversion showed additional amounts of these metabolites complementing the gap between in vitro colon model and the in vivo urinary excretion. Therefore, combining in vitro colon and hepatic models may better elucidate the metabolism of polyphenols from dietary exposure to urinary metabolites.

Comparison of plasma alkylresorcinols (AR) and urinary AR metabolites as biomarkers of compliance in a short-term, whole-grain intervention study.

PURPOSE: Alkylresorcinols (AR) are phenolic lipids present in the bran of wheat and rye. Plasma AR and their urinary metabolites may be suitable biomarkers of whole-grain (WG) wheat and rye consumption. The objective of this study was to examine plasma AR and urinary AR metabolites in response to WG wheat consumption. METHODS: In a randomized crossover study, 19 subjects (10 males, 9 females; BMI 22.0 kg/m(2); age 26 years) incorporated either 3 servings (48 g) or 6 servings (96 g) of WG wheat daily into their regular diet for 1 week. Subjects completed a 2-week washout period, abstaining from all WG consumption, before each intervention. Fasting blood and 24-h urine were collected before and after each intervention. Plasma AR homologues (C19:0, C21:0, C23:0) were quantified by GC-MS after diethyl ether and solid phase extraction and derivatization. Urinary AR metabolites [3,5-dihydroxybenzoic acid and 3-(3,5-dihydroxyphenyl)-propanoic acid] were determined using HPLC with electrochemical detection after enzymatic deconjugation and ethyl acetate extraction. RESULTS: Urinary total AR metabolites were significantly higher after 6 compared with 3 servings of WG wheat (56 vs. 32 µmol/day, P < 0.001). This dose-response relationship was independent of age, sex, energy intake, and baseline urinary AR metabolite concentration. Plasma total AR tended to be higher after 6 compared with 3 servings of WG wheat (103.0 vs. 86.9 nmol/L), but this difference was not significant (P = 0.42). CONCLUSION: The results suggest that urinary AR metabolites from 24-h urine collections may be useful as biomarkers of compliance in intervention studies of WG wheat.

Metabolism of Chuanxiong Rhizoma decoction: Identification of the metabolites in WZS-miniature pig urine.

Chuanxiong Rhizoma (CR), a well-known traditional Chinese medicine originated from the rhizome of Ligusticum chuanxiong Hort., was effective for treating various vascular diseases. To identify the metabolites of CR in vivo, the drug-containing urine samples of WZS-miniature pigs after orally administrated CR decoction were collected, after sequential column chromatography 17 metabolites (M1-M17) were isolated from the methanol extract of the urine samples. Their structures, including nine phthalides (M1-M9) and eight phenolic acids (M10-M17), were identified by spectroscopic means. Among them, 8 were new ones (M1-M6, M11-M12). On the basis of the structures of identified metabolites, seven original constituents, including 2 phthalides (senkyunolideI/H) and 5 phenolic acids (ferulic acid, isoferulic acid, caffeic acid, 3-hydroxycinnamoyl acid and 4-hydroxybenzonic acid) were deduced to be the major absorbed original constituents of CR in vivo. This is the first study on the metabolites of CR decoction in non-rodent animal (WZS-miniature pig), the results will give an insight into the metabolism profiles of phthalides and phenolic acids in CR decoction in vivo.