Persistent Urinary Ethyl Sulfate in the Absence of Urinary Ethyl Glucuronide in a Patient with Alcohol Use Disorder Who Claimed Abstinence.

A 48-year-old nurse with an alcohol use disorder history was being monitored in a professional health program. She consistently produced low-to-moderate urinary ethyl sulfate (EtS) concentrations in the absence of detectable urinary ethyl glucuronide (EtG), blood phosphatidylethanol and breath alcohol. She denied intentional ethanol consumption. After prolonged monitoring in a drug treatment program, including a period in a controlled environment, we concluded that this individual's urinary EtS likely resulted from anatomical and microbial factors related to Roux-en-Y gastric bypass surgery, with possible contributions from hidden dietary sources of ethanol. We have no definitive explanation for the lack of urinary EtG.

Longitudinal metabolic and gut bacterial profiling of pregnant women with previous bariatric surgery.

OBJECTIVE: Due to the global increase in obesity rates and success of bariatric surgery in weight reduction, an increasing number of women now present pregnant with a previous bariatric procedure. This study investigates the extent of bariatric-associated metabolic and gut microbial alterations during pregnancy and their impact on fetal development. DESIGN: A parallel metabonomic (molecular phenotyping based on proton nuclear magnetic resonance spectroscopy) and gut bacterial (16S ribosomal RNA gene amplicon sequencing) profiling approach was used to determine maternal longitudinal phenotypes associated with malabsorptive/mixed (n=25) or restrictive (n=16) procedures, compared with women with similar early pregnancy body mass index but without bariatric surgery (n=70). Metabolic profiles of offspring at birth were also analysed. RESULTS: Previous malabsorptive, but not restrictive, procedures induced significant changes in maternal metabolic pathways involving branched-chain and aromatic amino acids with decreased circulation of leucine, isoleucine and isobutyrate, increased excretion of microbial-associated metabolites of protein putrefaction (phenylacetlyglutamine, p-cresol sulfate, indoxyl sulfate and p-hydroxyphenylacetate), and a shift in the gut microbiota. The urinary concentration of phenylacetylglutamine was significantly elevated in malabsorptive patients relative to controls (p=0.001) and was also elevated in urine of neonates born from these mothers (p=0.021). Furthermore, the maternal metabolic changes induced by malabsorptive surgery were associated with reduced maternal insulin resistance and fetal/birth weight. CONCLUSION: Metabolism is altered in pregnant women with a previous malabsorptive bariatric surgery. These alterations may be beneficial for maternal outcomes, but the effect of elevated levels of phenolic and indolic compounds on fetal and infant health should be investigated further.

Could resistant starch supplementation improve inflammatory and oxidative stress biomarkers and uremic toxins levels in hemodialysis patients? A pilot randomized controlled trial.

An imbalance of gut microbiota is considered a new cardiovascular risk factor for chronic kidney disease (CKD) patients, since it is directly associated with increased uremic toxin production, inflammation and oxidative stress. Strategies such as prebiotic supplementation have been suggested to mitigate these complications. We hypothesized that prebiotic-resistant starch could ameliorate uremic toxins levels, oxidative stress, and inflammatory states in hemodialysis (HD) patients. This pilot study evaluated 31 HD patients assigned to either resistant starch (16 g of resistant starch Hi-Maize® 260) or placebo (manioc flour) supplementation, which they received for 4 weeks on alternate days through cookies on dialysis days and powder in a sachet on non-dialysis days. Levels of interleukin (IL)-6, high-sensitive C-reactive protein, thiobarbituric acid reactive substances plasma (TBARS), protein carbonylation, indoxyl sulfate (IS) and p-cresyl sulfate were measured. Anthropometric and biochemical parameters, as well as, food intake were also evaluated. As expected, resistant starch group increased fiber intake (p > 0.01), in addition the prebiotic supplementation reduced IL-6 (p = 0.01), TBARS (p > 0.01), and IS (p > 0.01) plasma levels. No significant differences were evident in the placebo group. Prebiotic-resistant starch supplementation seems to be a promising nutritional strategy to improve inflammation, oxidative stress and to reduce IS plasma levels in CKD patients on HD.

Exploration of the Fecal Microbiota and Biomarker Discovery in Equine Grass Sickness.

Equine grass sickness (EGS) is a frequently fatal disease of horses, responsible for the death of 1 to 2% of the U.K. horse population annually. The etiology of this disease is currently uncharacterized, although there is evidence it is associated with Clostridium botulinum neurotoxin in the gut. Prevention is currently not possible, and ileal biopsy diagnosis is invasive. The aim of this study was to characterize the fecal microbiota and biofluid metabolic profiles of EGS horses, to further understand the mechanisms underlying this disease, and to identify metabolic biomarkers to aid in diagnosis. Urine, plasma, and feces were collected from horses with EGS, matched controls, and hospital controls. Sequencing the16S rRNA gene of the fecal bacterial population of the study horses found a severe dysbiosis in EGS horses, with an increase in Bacteroidetes and a decrease in Firmicutes bacteria. Metabolic profiling by 1H nuclear magnetic resonance spectroscopy found EGS to be associated with the lower urinary excretion of hippurate and 4-cresyl sulfate and higher excretion of O-acetyl carnitine and trimethylamine-N-oxide. The predictive ability of the complete urinary metabolic signature and using the four discriminatory urinary metabolites to classify horses by disease status was assessed using a second (test) set of horses. The urinary metabolome and a combination of the four candidate biomarkers showed promise in aiding the identification of horses with EGS. Characterization of the metabolic shifts associated with EGS offers the potential of a noninvasive test to aid premortem diagnosis.

Determination of bisphenol A, triclosan and their metabolites in human urine using isotope-dilution liquid chromatography-tandem mass spectrometry.

Bisphenol A (BPA) and triclosan (TCS) are ubiquitous environmental phenols exhibiting endocrine disrupting activities that may be involved in various health disorders in humans. There is a need to measure separately free forms and conjugated metabolites because only the former are biologically active. We have developed sensitive methods using isotope-dilution liquid chromatography-tandem mass spectrometry for individual measurements of free BPA and TCS as well as their metabolites, BPA glucuronide (BPAG), BPA monosulfate (BPAS), BPA disulfate (BPADS), TCS glucuronide (TCSG) and TCS sulfate (TCSS) in urine. Comparative analyses of urine samples from 46 volunteers living in the Quebec City area using the new methods and a GC-MS/MS method previously used in our laboratory revealed very strong correlations for total BPA (Spearman's rs=0.862, p<0.0001) and total TCS concentrations (rs=0.942, p<0.0001). Glucuronide metabolites were the most abundant BPA and TCS species in urine samples (>94% of total urinary concentrations). Unconjugated TCS concentrations represented a small proportion of total TCS species (median=1.6%) but its concentration was likely underestimated due to losses by adsorption to the surface of polypropylene tubes used for sample storage. To our knowledge, we are the first to report levels of free, sulfated and glucuronidated TCS levels in human urine.

Measurement of ethyl glucuronide, ethyl sulphate and their ratio in the urine and serum of healthy volunteers after two doses of alcohol.

AIMS: Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are minor metabolites of ethanol, and their presence in urine provides a strong indication of recent alcohol administration. In this study, we performed a drinking experiment to investigate the kinetics of EtG and EtS formation and elimination after the administration of two doses of alcohol. METHODS: Nineteen volunteers provided urine and serum (only 18) after administration of 4 and 8 units of alcohol (1 unit corresponds to 10 ml or ∼8 g of pure ethanol). The analysis was performed using a validated ultra-performance liquid chromatography-mass spectrometry (UPLC(®)-MS/MS) method. RESULTS: After 4 units, the median EtG maximum concentration (C(max)) was 0.4 µg/ml and the interquartile range (0.3 µg/ml) in serum and 3.5 mg/h (1.2 mg/h) in urine and were reached (T(max)) after 2.0 h (0.8 h) and 3.0 h (1.0 h), respectively. EtS C(max) was 0.2 µg/ml (0.1 µg/ml) in serum and 1.3 mg/h (0.6 mg/h) in urine, and the corresponding T(max) were 1.0 h (1.0 h) and 2.0 h (0.5 h). After 8 units, EtG C(max) was 1.3 µg/ml (0.4 µg/ml) in serum and 10 mg/h (3.4 mg/h) in urine and was reached after 4.0 h (1.8 h) and 4.0 h (2.0 h), respectively. EtS C(max) was 0.6 µg/ml (0.1 µg/ml) in serum and 3.5 mg/h (1.1 mg/h) in urine, the corresponding T(max) were 3.0 h (1.0 h) and 3.0 h (1.0 h). The EtG/EtS ratio increased as a function of the time after alcohol administration in both serum and urine samples but to a lesser extent after 8 units than 4. CONCLUSION: These results correlate with values obtained in previous studies. T(max) of EtG and EtS increased between 4 and 8 units. The EtG:EtS ratio increased in the serum and urine samples of all volunteers as a function of time at least up to 4 h after alcohol administration.

Identification of O-methyl-(-)-epicatechin-O-sulphate metabolites by mass-spectrometry after O-methylation with trimethylsilyldiazomethane.

(-)-Epicatechin, an abundant dietary polyphenol found mainly in cocoa and tea, is known to extensively undergo metabolism after ingestion giving rise to a complex series of conjugated metabolites including numerous isomers. In the present study, the combination of fractionation, chemical derivatization and various mass spectrometric approaches is described to determine the exact position of sulphate group in methylated epicatechin metabolites. Four O-methyl-(-)-epicatechin-O-sulphate metabolites isolated from human urine samples were derivatized under mild condition using trimethylsilyldiazomethane (TMSD) in the presence of methanol. The resulting methylated reaction products were then analyzed by high resolution and multistage mass spectrometry for the subsequent identification of the sulphate positional isomers. Results show that O-methylation affects the charge delocalization in negatively charged ions and hereby the fragmentation pattern of the sulphate isomers allowing the identification of diagnostic ions. In addition, this study demonstrates that methoxy derivatives of polyphenol metabolites can be prepared using TMSD. Subsequently, the localization of the sulphate group in the polyphenol metabolites can be achieved by analyzing the methoxy derivatives by multistage mass spectrometry. Using an enzymatic reaction for identification of the O-methyl position, and a chemical O-methylation with TMSD follow by high resolution and multistage tandem MS for the identification of the sulphate group position, we were able to identify the previously unknown O-methyl-(-)-epicatechin-O-sulphate. Accordingly, we identified 3'-O-methyl-(-)-epicatechin-5-O-sulphate and 3'-O-methyl-(-)-epicatechin-7-O-sulphate as the main O-methyl-(-)-epicatechin-sulfates(-)-epicatechin metabolites in humans.

First synthesis, characterization, and evidence for the presence of hydroxycinnamic acid sulfate and glucuronide conjugates in human biological fluids as a result of coffee consumption.

A systematic investigation of the human metabolism of hydroxycinnamic acid conjugates was carried out. A set of 24 potential human metabolites of coffee polyphenols has been chemically prepared, and used as analytical standards for unequivocal identifications. These included glucuronide conjugates and sulfate esters of caffeic, ferulic, isoferulic, m-coumaric and p-coumaric acids as well as their dihydro derivatives. A particular focus has been made on caffeic and 3,4-dihydroxyphenylpropionic acid derivatives, especially the sulfate conjugates, for which regioselective preparation was particularly challenging, and have so far never been identified as human metabolites. Ten out of the 24 synthesized conjugates have been identified in human plasma and/or urine after coffee consumption. A number of these conjugates were synthesized, characterized and detected as hydroxycinnamic acid metabolites for the first time. This was the case of dihydroisoferulic acid 3'-O-glucuronide, caffeic acid 3'-sulfate, as well as the sulfate and glucuronide derivatives of 3,4-dihydroxyphenylpropionic acid.

Characterization of inflammatory bowel disease with urinary metabolic profiling.

OBJECTIVES: Distinguishing between the inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC) is important for both management and prognostic reasons. Discrimination using noninvasive techniques could be an adjunct to conventional diagnostics. Differences have been shown between the intestinal microbiota of CD and UC patients and controls; the gut bacteria influence specific urinary metabolites that are quantifiable using proton high-resolution nuclear magnetic resonance (NMR) spectroscopy. This study tested the hypothesis that such metabolites differ between IBD and control cohorts, and that using multivariate pattern-recognition analysis, the cohorts could be distinguished by urine NMR spectroscopy. METHODS: NMR spectra were acquired from urine samples of 206 Caucasian subjects (86 CD patients, 60 UC patients, and 60 healthy controls). Longitudinal samples were collected from 75 individuals. NMR resonances specific for metabolites influenced by the gut microbes were studied, including hippurate, formate, and 4-cresol sulfate. Multivariate analysis of all urinary metabolites involved principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA). RESULTS: Hippurate levels were lowest in CD patients and differed significantly between the three cohorts (P<0.0001). Formate levels were higher and 4-cresol sulfate levels lower in CD patients than in UC patients or controls (P=0.0005 and P=0.0002, respectively). PCA revealed clustering of the groups; PLS-DA modeling was able to distinguish the cohorts. These results were independent of medication and diet and were reproducible in the longitudinal cohort. CONCLUSIONS: Specific urinary metabolites related to gut microbial metabolism differ between CD patients, UC patients, and controls. The emerging technique of urinary metabolic profiling with multivariate analysis was able to distinguish these cohorts.

Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice.

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for gamma-radiation biodosimetry in a mouse model. Mice were gamma-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and beta-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose-response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to gamma radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

The effects of milk as a food matrix for polyphenols on the excretion profile of cocoa (-)-epicatechin metabolites in healthy human subjects.

The effect of different food matrices on the metabolism and excretion of polyphenols is uncertain. The objective of the study was to evaluate the possible effect of milk on the excretion of (2)-epicatechin metabolites from cocoa powder after its ingestion with and without milk. Twenty-one volunteers received the following three test meals each in a randomised cross-over design with a 1-week interval between meals: (1) 250 ml whole milk as a control; (2) 40 g cocoa powder dissolved in 250 ml whole milk (CC-M); (3) 40 g cocoa powder dissolved in 250 ml water (CC-W). Urine was collected before consumption and during the 0-6, 6-12 and 12-24 h periods after consumption. (2)-Epicatechin metabolite excretion was measured using liquid chromatography-MS. One (2)-epicatechin glucuronide and three (2)-epicatechin sulfates were detected in urine excreted after the intake of the two cocoa beverages (CC-M and CC-W). The results show that milk does not significantly affect the total amount of metabolites excreted in urine. However, differences in metabolite excretion profiles were observed; there were changes in the glucuronide and sulfate excretion rates, and the sulfation position between the period of excretion and the matrix. The matrix in which polyphenols are consumed can affect their metabolism and excretion, and this may affect their biological activity. Thus, more studies are needed to evaluate the effect of these different metabolite profiles on the body.

Evaluation of the role of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4) in the urinary excretion of sulfate and glucuronide metabolites of edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one).

Edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the treatment of acute cerebral infarction. Edaravone is mainly excreted into the urine after conjugation to glucuronide or sulfate. Previous studies have demonstrated that edaravone sulfate is a good substrate of human organic anion transporter (OAT) 1 (SLC22A6) and human OAT3 (SLC22A8). In this study, we examined the involvement of breast cancer resistance protein [BCRP (ABCG2)] and [multidrug resistance-associated protein 4 MRP4 (ABCC4)] in the luminal efflux in the kidney. Increased ATP-dependent uptake of edaravone sulfate but not edaravone glucuronide was observed in BCRP-expressing membrane vesicles compared with control vesicles (Km = 16.5 microM). In contrast, edaravone glucuronide, but not edaravone sulfate, exhibited greater ATP-dependent uptake in MRP4-expressing membrane vesicles than that in control vesicles (Km = 9.85 microM). Unlike taurocholate uptake, S-methylglutathione had no effect on the ATP-dependent uptake of edaravone glucuronide by MRP4. The functional importance of BCRP and MRP4 in the urinary excretion of edaravone sulfate and edaravone glucuronide, respectively, was investigated using Bcrp and Mrp4 knockout mice. The renal clearance with respect to the kidney concentration of edaravone sulfate was reduced significantly but not abolished in Bcrp knockout mice compared with wild-type mice (3.62 versus 4.85 ml/min/kg b.wt.). The renal clearance of edaravone glucuronide was lower in Mrp4 knockout mice than wild-type mice (2.01 versus 5.06 ml/min/kg BW). Our results suggest that Bcrp and Mrp4 are partly involved in the luminal efflux of edaravone sulfate and edaravone glucuronide, respectively.

Human urine: epicatechin metabolites and antioxidant activity after cocoa beverage intake.

Associations between cocoa consumption in humans, excreted metabolites and total antioxidant capacity (TAC) have been scarcely investigated. The aims of the study were to investigate the epicatechin (( - )-Ec) metabolites excreted in urine samples after an intake of 40 g of cocoa powder along with the TAC of these urine samples and the relation between both the analyses. Each of the 21 volunteers received two interventions, one with a polyphenol-rich food (PRF) and one with a polyphenol-free food (PFF) in a randomized cross-over study. Urine samples were taken before and during 24 h at 0-6, 6-12 and 12-24 h periods after test intake. The excreted ( - )-Ec metabolites and the TAC were determined in urine samples by LC-MS/MS and TEAC assay, respectively. The maximum excretion of ( - )-Ec metabolites and the maximum TAC value were observed in urine samples excreted between 6 and 12 h after PRF consumption. Significance of TAC increase was found in urine samples excreted during 0-6 and 6-12 h (66.6 and 72.67%, respectively, with respect to the 0 h).

The requirement of ammonium or other cations linked with p-cresol sulfate for cross-reactivity with a peptide of myelin basic protein.

Urinary myelin basic protein-like material (MBPLM), so designated because of its immunoreactivity with a polyclonal antibody directed against a cryptic epitope located in residues 83-89 of myelin basic protein (MBP), exists in humans normally but increases in concentration in patients with multiple sclerosis who have progressive disease. Given its possible role in reflecting events of neural tissue destruction occurring in multiple sclerosis, urinary MBPLM is a candidate surrogate marker for this phase of the disease. Previously, it has been demonstrated that p-cresol sulfate (PCS) is the dominant component of MBPLM; however, another component(s) was essential in enabling p-cresol sulfate to have molecular mimicry with MBP peptide 83-89 detected by immunoreactivity. In the present investigation, this remaining component(s) was characterized by a combination of high performance size exclusion chromatography followed by nuclear magnetic resonance spectroscopy and shown to be ammonium. The monovalent cation ammonium could be substituted in vitro by several different monovalent and divalent cations, most notably zinc, in restoring to deprotonated p-cresol sulfate its immunoreactivity as MBPLM. These findings indicate the basis for the unexpected molecular mimicry between an epitope of an encephalitogenic protein and a complex containing a small organic molecule, p-cresol sulfate. Furthermore, the reaction of either ammonium or other cations with p-cresol sulfate may represent an in vivo process directly related to damage of axonal membranes.

Lignan and isoflavonoid conjugates in human urine.

Lignans and isoflavonoids are two groups of diphenolic phytoestrogens of plant origin which have gained increasing interest because of their possible cancer protective properties. High excretion of these compounds occur in populations at low risk of breast, prostate and colon cancer consuming either high amounts of whole-grain (lignans and some isoflavonoids) or soy products (isoflavonoids and some lignans). We determined the pattern of conjugation of the phytoestrogens in four urine samples from vegetarian or semivegetarian women and in two samples from men. Seven compounds were investigated: enterodiol, enterolactone, matairesinol, diadzein, equol, genistein and O-desmethylangolensin. The fractions quantified are the free fraction, mono- and disulfate, as well as the mono-, di- and sulfoglucuronide fractions. For the fractionation and purification we used ion-exchange chromatography and the determination of the concentrations of each compound in all fractions was done by isotope dilution gas chromatography-mass spectrometry (GLC-MS) using deuterated internal standards of all diphenols. More than 60% of all compounds determined, occurred in the monoglucuronide fraction. Daidzein, enterodiol and equol are excreted to a relatively high extent as sulfoglucuronides and genistein as diglucuronide. We conclude that the general pattern of lignan and isoflavonoid conjugates in urine is similar to that of endogenous estrogens.

Gas chromatographic determination of sulfuric acid and application to urinary sulfate.

A new gas chromatographic method for the determination of sulfate was developed. In this method, sulfate was quantitatively converted to a volatile derivative, dimethyl sulfate, by a two-step procedure. First, sulfate was converted to silver sulfate by reaction with silver oxide, and then to dimethyl sulfate by reaction with methyl iodide. The derivative was analyzed by gas chromatography. Methyl methanesulfonate was used as an internal standard. The method was applied to the determination of total urinary sulfate. Phosphate and chloride ions, which interfered with the present method, were eliminated with the use of basic magnesium carbonate and an excess of silver oxide, respectively. Recovery was over 96% when 5 to 40 mumol/ml of sulfate was added to human urine samples.

A comparative study of biliary and urinary 2-hydroxylated metabolites of [6,7-3H]17 alpha-ethynylestradiol in women.

The biliary and urinary metabolites of [6,7-3H]17 alpha-ethynylestradiol (EE2) in women were studied with reference to the possibility of estimating EE2 2-hydroxylation by analysing urinary metabolites alone. Five subjects received 50 micrograms of 3H-EE2 orally. Bile was obtained by either endoscopy or T-tube drainage. The metabolites excreted in bile and urine were largely glucuronides and arylsulphates, but in variable proportions. The glutathione adduct of 2-hydroxyethynylestradiol was not observed in bile. EE2 was the predominant component of the glucuronide fractions of bile and urine. Additionally, the proportion of glucuronylated EE2 in a subject's urine quantitatively paralleled that in bile. HPLC analyses indicated that the proportions of EE2 and 2-methoxy-EE2 in urine are predictive of EE2 2-hydroxylation in most women. With some subjects, however, urinary analysis alone considerably underestimates the extent of 2-hydroxylation.

Urinary excretion of inorganic sulfate, ester sulfate, total sulfur and taurine in cancer patients.

The urinary excretion of inorganic sulfate, ester sulfate, taurine and total sulfur has been estimated in 18 patients with malignancy, and compared with those of an age-matched reference group. The absolute amount of inorganic sulfate was significantly lower in the cancer group, whereas an increased ester sulfate excretion was observed. Reduced excretion of total sulfate (inorganic plus ester sulfate) and increased excretion of "neutral sulfur" (total sulfur minus total sulfate) in the cancer group was calculated from these data. If these values were recalculated with respect to creatinine, no significant differences were found for inorganic and total sulfate between the two patient groups, whereas ester sulfate and the "neutral sulfur" fractions were still increased. The enlarged "neutral sulfur" could to a large extent be explained by an increased excretion of taurine. The mechanism behind these findings is discussed.

Radioactive conversion products of intramuscularly injected [4-14C]formononetin including sulfates in the urine of hens.

The phytoestrogen formononetin was injected intramuscularly as [4-14C]formononetin into two adult hens. Radioactive materials in the urine for the succeeding 14 days (hen 1) or 16 days (hen 2) were fractionated on DEAE-Sephadex-25 columns by elution with a gradient of NaCl; the four major fractions thus separated were examined by solvent partition, thin-layer chromatography, and enzymic cleavage. The following seven radioactive components were identified in the urine, the average proportions of each being given in terms of percentage of total 14C recovered from the urine: [14C]formononetin (4.3%); [14C]diaidzein (11.4%); [14C]equol (6.8%); [14C]daidzein monosulfate (30.4%); [14C]equol monosulfate (5.8%); [14C]diadzein disulfate (19.8%); and [14C]equol disulfate (6.5%). Small proportions of sulfates of unidentified radioactive phenols were present. Tests for presence of glucosiduronates of 14C-labelled material gave negative results. Radioactive formononetin sulfate was not detected in the urine of either hen.