Increasing Doses of Blueberry Polyphenols Alters Colonic Metabolism and Calcium Absorption in Ovariectomized Rats.

SCOPE: Blueberries are rich sources of bioactive polyphenols that may provide health benefits when consumed regularly, leading to their increased marketing as dietary supplements. However, the metabolic changes associated with consuming concentrated doses of purified polyphenols, as may be present in dietary supplements, are unknown, especially when considering the colonic metabolites formed. This study aimed to evaluate the pharmacokinetics of high doses of purified blueberry polyphenols. METHODS AND RESULTS: 5-month old, ovariectomized Sprague-Dawley rats are acutely dosed with purified blueberry polyphenols (0, 75, 350, and 1000 mg total polyphenols per kg body weight (bw)) and 45 Ca to measure calcium absorption. Blood and urine are collected for 48 h after dosing and phenolic metabolites measured via ultra high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The most prominent metabolites are colonically generated cinnamic and hippuric acids. Smaller amounts of other phenolic acids, flavonols, and anthocyanins are also detected. Most metabolites follow a dose-response relationship, though several show saturated absorption. Maximal metabolite concentrations are reached within 12 h for a majority of compounds measured, while some (e.g., hippuric acid) peaked up to 24 h post-dosing. Calcium absorption is significantly increased in the highest dose group (p = 0.03). CONCLUSION: These results indicate that increased doses of blueberry polyphenols induce changes in intestinal phenolic metabolism and increase calcium absorption.

(1)H-NMR based metabolomics study for the detection of the human urine metabolic profile effects of Origanum dictamnus tea ingestion.

(1)H NMR spectroscopy was employed to investigate the repercussion of Origanum dictamnus tea ingestion in several volunteers' urine metabolic profiles, among them two with chronic inflammatory bowel diseases (IBD), mild IBD and Crohn's disease. Herein, we demonstrate that the concentrations of a lot of urinary metabolites such as hippurate, trimethylamine oxide (TMAO), citrate, and creatinine are altered, which prompts the intestinal microflora function/content perturbation as well as kidney function regulation by dictamnus tea. Interestingly, our preliminary results showed that a high dose of dictamnus tea intake appeared to be toxic for a person with Crohn's disease, since it caused high endogenous ethanol excretion in urine. All subjects' metabolic effects caused by the dictamnus tea appeared to be reversible, when all volunteers stopped its consumption. Finally, we highlight that individuals' metabolic phenotype is reflected in their urine biofluid before and after the dictamnus tea effect while all individuals have some common and different metabolic responses to this tea, implying that each phenotype has a quite different response to this tea consumption.

NMR-based metabolomics reveals urinary metabolome modifications in female Sprague-Dawley rats by cranberry procyanidins.

A (1)H NMR global metabolomics approach was used to investigate the urinary metabolome changes in female rats gavaged with partially purified cranberry procyanidins (PPCP) or partially purified apple procyanidins (PPAP). After collecting 24-h baseline urine, 24 female Sprague-Dawley rats were randomly separated into two groups and gavaged with PPCP or PPAP twice using a dose of 250 mg extracts per kilogram body weight. The 24-h urine samples were collected after the gavage. Urine samples were analyzed using (1)H NMR. Multivariate analyses showed that the urinary metabolome in rats was modified after administering PPCP or PPAP compared to baseline urine metabolic profiles. 2D (1)H-(13)C HSQC NMR was conducted to assist identification of discriminant metabolites. An increase of hippurate, lactate and succinate and a decrease of citrate and α-ketoglutarate were observed in rat urine after administering PPCP. Urinary levels of d-glucose, d-maltose, 3-(3'-hydroxyphenyl)-3-hydroxypropanoic acid, p-hydroxyphenylacetic acid, formate and phenol increased but citrate, α-ketoglutarate and creatinine decreased in rats after administering PPAP. Furthermore, the NMR analysis showed that the metabolome in the urine of rats administered with PPCP differed from those gavaged with PPAP. Compared to PPAP, PPCP caused an increase of urinary excretion of hippurate but a decrease of 3-(3'-hydroxyphenyl)-3-hydroxypropanoic acid, p-hydroxyphenylacetic acid and phenol. These metabolome changes caused by cranberry procyanidins may help to explain its reported health benefits and identify biomarkers of cranberry procyanidin intake.

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.

Effects of the Mediterranean diet supplemented with coenzyme q10 on metabolomic profiles in elderly men and women.

BACKGROUND: Characterization of the variations in the metabolomic profiles of elderly people is a necessary step to understand changes associated with aging. This study assessed whether diets with different fat quality and supplementation with coenzyme Q10 (CoQ) affect the metabolomic profile in urine analyzed by proton nuclear magnetic resonance spectroscopy from elderly people. METHODS: Ten participants received, in a cross-over design, four isocaloric diets for 4-week periods each: Mediterranean diet supplemented with CoQ (Med + CoQ diet); Mediterranean diet; Western diet rich in saturated fat diet; low-fat, high-carbohydrate diet enriched in n-3 polyunsaturated fat. RESULTS: Multivariate analysis showed differences between diets when comparing Med + CoQ diet and saturated fat diet, with greater hippurate urine levels after Med + CoQ diet and higher phenylacetylglycine levels after saturated fat diet in women. Following consumption of Med + CoQ, hippurate excretion was positively correlated with CoQ and ß-carotene plasma levels and inversely related to Nrf2, thioredoxin, superoxide dismutase 1, and gp91(phox) subunit of NADPH oxidase gene expression. After saturated fat diet consumption, phenylacetylglycine excretion was inversely related to CoQ plasma level and positively correlated with isoprostanes urinary level. CONCLUSIONS: The association between hippurate excretion and antioxidant biomarkers along with the relationship between phenylacetylglycine excretion and oxidant biomarkers suggests that the long-term consumption of a Med + CoQ diet could be beneficial for healthy aging and a promising challenge in the prevention of processes related to chronic oxidative stress, such as cardiovascular and neurodegenerative disease.

Hippuric acid in 24 h urine collections as a biomarker of fruits and vegetables intake in kidney stone formers.

This work aimed to underline the prospects of hippuric acid, a product of the metabolism of polyphenols, as a new biomarker of fruits and vegetables intake associated with lithogenic risk. Biochemical parameters of lithogenic risk and hippuric acid were measured in the 24 h urine collections of a cohort of 696 Italian kidney stone formers divided into two subgroups according to their different dietary habits. The link between lithogenic risk parameters and hippuric acid was assessed and this compound was revealed as a valuable biomarker of fruits and vegetables intake in kidney stone formers. A cut-off value of urinary excretion of hippuric acid, 300 mg/24 h, was set as the threshold of discrimination between low and high intake of fruits and vegetables for these patients. These results highlight the importance of monitoring of the excretion hippuric acid in urine to address proper dietary guidelines for the management of stone former patients.

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced by Semen Strychni.

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure, 12 typical biomarkers are selected and a simple LC-MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3-indoxyl sulfate, indole-3-acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C18 column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3-indoxyl sulfate, and indole-3-acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.

Phenolic acid concentrations in plasma and urine from men consuming green or black tea and potential chemopreventive properties for colon cancer.

SCOPE: Tea polyphenols are metabolized by the colonic microflora yielding phenolic metabolites, which may contribute to the health benefits of tea. We determined the serum and urine concentrations of phenolic acids, hippuric acid, and polyhydroxyphenyl-γ-valerolactones during green tea (GT) and black tea (BT) administration. The effects of (-)-epigallocatechin gallate (EGCG) and 3,4-dihydroxyphenylacetic acid (3,4-DHPAA) alone and in combination on bioavailability, intracellular metabolism, and antiproliferative activity were determined in HCT-116 colon cancer cells. METHODS AND RESULTS: The concentration of phenolic metabolites was quantified by HPLC with electrochemical detection and MS. Urine concentrations of 4-hydroxyphenylacetic acid (4-HPAA), 3-hydroxyphenylacetic acid (3-HPAA), and polyhydroxy-γ-valerolactones were increased significantly in men drinking GT compared to control. Urine concentration of 3-O-methylgallic acid (3OMGA) was significantly increased in men drinking BT compared to control. Serum 3,4-DHPAA was significantly increased after consumption of GT and BT and 4-HPAA after GT consumption. In vitro treatment of HCT-116 colon cancer cells with 3,4-DHPAA and EGCG exhibited an additive antiproliferative effect, while methylation of 3,4-DHPAA was significantly decreased. 3OMGA exhibited the strongest antiproliferative activity among the phenolic acids. CONCLUSION: The consumption of both, GT and BT, was associated with a significant increase in urinary and serum phenolic acids.

An investigation of the antidepressant action of xiaoyaosan in rats using ultra performance liquid chromatography-mass spectrometry combined with metabonomics.

A rapid, highly sensitive, and selective method was applied in a non-invasive way to investigate the antidepressant action of Xiaoyaosan (XYS) using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and chemometrics. Many significantly altered metabolites were used to explain the mechanism. Venlafaxine HCl and fluoxetine HCl were used as chemical positive control drugs with a relatively clear mechanism of action to evaluate the efficiency and to predict the mechanism of action of XYS. Urine obtained from rats subjected to chronic unpredictable mild stress (CUMS) was analyzed by UPLC-MS. Distinct changes in the pattern of metabolites in the rat urine after CUMS production and drug intervention were observed using partial least squares-discriminant analysis. The results of behavioral tests and multivariate analysis showed that CUMS was successfully reproduced, and a moderate-dose XYS produced significant therapeutic effects in the rodent model, equivalent to those of the positive control drugs, venlafaxine HCl and fluoxetine HCl. Metabolites with significant changes induced by CUMS were identified, and 17 biomarker candidates for stress and drug intervention were identified. The therapeutic effect of XYS on depression may involve regulation of the dysfunctions of energy metabolism, amino acid metabolism, and gut microflora changes. Metabonomic methods are valuable tools for measuring efficacy and mechanisms of action in the study of traditional Chinese medicines.

SPE-NMR metabolite sub-profiling of urine.

NMR-based metabolite profiling of urine is a fast and reproducible method for detection of numerous metabolites with diverse chemical properties. However, signal overlap in the (1)H NMR profiles of human urine may hamper quantification and identification of metabolites. Therefore, a new method has been developed using automated solid-phase extraction (SPE) combined with NMR metabolite profiling. SPE-NMR of urine resulted in three fractions with complementary and reproducible sub-profiles. The sub-profile from the wash fraction (100 % water) contained polar metabolites; that from the first eluted fraction (10 % methanol-90 % water) semi-polar metabolites; and that from the second eluted fraction (100 % methanol) aromatic metabolites. The method was validated by analysis of urine samples collected from a crossover human nutritional intervention trial in which healthy volunteers consumed capsules containing a polyphenol-rich mixture of red wine and grape juice extract (WGM), the same polyphenol mixture dissolved in a soy drink (WGM_Soy), or a placebo (PLA), over a period of five days. Consumption of WGM clearly increased urinary excretion of 4-hydroxyhippuric acid, hippuric acid, 3-hydroxyphenylacetic acid, homovanillic acid, and 3-(3-hydroxyphenyl)-3-hydroxypropionic acid. However, there was no difference between the excreted amounts of these metabolites after consumption of WGM or WGM_Soy, indicating that the soy drink is a suitable carrier for WGM polyphenols. Interestingly, WGM_Soy induced a significant increase in excretion of cis-aconitate compared with WGM and PLA, suggesting a higher demand on the tricarboxylic acid cycle. In conclusion, SPE-NMR metabolite sub-profiling is a reliable and improved method for quantification and identification of metabolites in urine to discover dietary effects and markers of phytochemical exposure.

Impact of short-term intake of red wine and grape polyphenol extract on the human metabolome.

Red wine and grape polyphenols are considered to promote cardiovascular health and are involved in multiple biological functions. Their overall impact on the human metabolome is not known. Therefore, exogenous and endogenous metabolic effects were determined in fasting plasma and 24 h urine from healthy male adults consuming a mix of red wine and grape juice extracts (WGM) for 4 days in a placebo-controlled, crossover study. Syringic acid, 3-hydroxyhippuric acid, pyrogallol, 3-hydroxyphenylacetic acid, and 3-hydroxyphenylpropionic acid were confirmed as the strongest urinary markers of WGM intake. Overall, WGM had a mild impact on the endogenous metabolism. Most noticeable were changes in several amino acids deriving from tyrosine and tryptophan. Reductions in the microbial metabolites p-cresol sulfate and 3-indoxylsulfuric acid and increases in indole-3-lactic acid and nicotinic acid were observed in urine. In plasma, tyrosine was reduced. The results suggest that short-term intake of WGM altered microbial protein fermentation and/or amino acid metabolism.

Consumption of Hibiscus sabdariffa L. aqueous extract and its impact on systemic antioxidant potential in healthy subjects.

BACKGROUND: To evaluate health benefits attributed to Hibiscus sabdariffa L. a randomized, open-label, two-way crossover study was undertaken to compare the impact of an aqueous H. sabdariffa L. extract (HSE) on the systemic antioxidant potential (AOP; assayed by ferric reducing antioxidant power (FRAP)) with a reference treatment (water) in eight healthy volunteers. The biokinetic variables were the areas under the curve (AUC) of plasma FRAP, ascorbic acid and urate that are above the pre-dose concentration, and the amounts excreted into urine within 24 h (Ae(0-24) ) of antioxidants as assayed by FRAP, ascorbic acid, uric acid, malondialdehyde (biomarker for oxidative stress), and hippuric acid (metabolite and potential biomarker for total polyphenol intake). RESULTS: HSE caused significantly higher plasma AUC of FRAP, an increase in Ae(0-24) of FRAP, ascorbic acid and hippuric acid, whereas malondialdehyde excretion was reduced. Furthermore, the main hibiscus anthocyanins as well as one glucuronide conjugate could be quantified in the volunteers' urine (0.02% of the administered dose). CONCLUSION: The aqueous HSE investigated in this study enhanced the systemic AOP and reduced the oxidative stress in humans. Furthermore, the increased urinary hippuric acid excretion after HSE consumption indicates a high biotransformation of the ingested HSE polyphenols, most likely caused by the colonic microbiota.

Influence of coffee intake on urinary hippuric acid concentration.

Intake of foods and drinks containing benzoic acid influences the urinary hippuric acid (HA) concentration, which is used to monitor toluene exposure in Japan. Therefore, it is necessary to control the intake of benzoic acid before urine collection. Recently, some reports have suggested that components of coffee, such as chlorogenic, caffeic, and quinic acids are metabolized to HA. In this study, we evaluated the influence of coffee intake on the urinary HA concentration in toluene-nonexposed workers who had controlled their benzoic acid intake, and investigated which components of coffee influenced the urinary HA concentration. We collected urine from 15 healthy men who did not handle toluene during working hours, after they had consumed coffee, and we measured their urinary HA concentrations; the benzoic acid intake was controlled in these participants during the study period. The levels of chlorogenic, caffeic, and quinic acids in coffee were analyzed by LC-MS/MS. Urinary HA concentration increased significantly with increasing coffee consumption. Spectrophotometric LC-MS/MS analysis of coffee indicated that it contained chlorogenic and quinic acids at relatively high concentrations but did not contain benzoic acid. Our findings suggest that toluene exposure in coffee-consuming workers may be overestimated.

Quantitative determination of hippuric and benzoic acids in urine by LC-MS/MS using surrogate standards.

An LC-MS/MS method for simultaneous determination of hippuric acid (HA) and benzoic acid (BA) in monkey urine after direct injection was developed. Since HA and BA are endogenous compounds in urine, surrogate standards ((13)C(6)-hippuric and (13)C(6)-benzoic acid) were employed to generate calibration curves. l-Phenylalanine-ring-D5 served as an internal standard. Multiple reaction monitoring in the negative ionization mode with an APCI source was used for detection of all components in the assay. The developed method is intended for determination of HA and BA in the range of 0.25-250 and 0.1-100microg/ml, respectively. Weighted (1/x) quadratic regression (r(2)>0.99) was used to generate calibration curves. Precision and accuracy of the method were assessed by analyzing 3 quality control samples (concentrations at low, medium, and high range of calibration curve) prepared in monkey urine. Stability for 48h at room temperature and after 3 freeze-thaw cycles was also evaluated. The proposed method was successfully utilized for analysis of urine samples from female monkeys following the administration of everninomicin alone and in combination with gentamicin. The concentrations of endogenous HA and BA were calculated based on the peak area ratio of the analyte to the internal standard using a regression equation for corresponding surrogate standard.

Influence of tea and coffee on biomonitoring of toluene exposure.

BACKGROUND: The intake of some benzoic acid-containing foods or drinks such as green tea and coffee is known to increase urinary hippuric acid (HA) concentrations, and, unless this can be accounted for, may result in false-positive findings during the biological monitoring of toluene exposure. AIMS: To investigate the influence of green tea and coffee ingestion on urinary HA concentrations. METHODS: Time-weighted average exposures to toluene were monitored by measuring urinary HA in 245 car painters in 2005 and again in 2006. Samples of both urine and blood were collected during work hours. All the participants were also asked about their alcohol intake, smoking and dietary and beverage habits, especially green tea and coffee. To clarify the relationship between green tea and coffee ingestion, workers were prohibited from consuming green tea and coffee for 1 day before the health check-up in 2006. RESULTS: The frequency of positive urinary HA concentrations (>1.0 g/l) in heavy consumers of green tea and/or coffee was significantly higher than that of the non-consumer or light consumers (OR 2.34, 95% CI 1.15-4.74). Prohibiting green tea and coffee ingestion was effective in decreasing the frequency of falsely elevated urinary HA concentrations (P < 0.01). CONCLUSIONS: The consumption of green tea and/or coffee can result in an overestimation of urinary HA concentrations and cause false-positive results during the biological monitoring of workers exposed to low doses of toluene.

Antioxidant metabolism induced by quinic acid. Increased urinary excretion of tryptophan and nicotinamide.

For over 50 years, hippuric/quinic acids were believed to have no biological efficacy. Here data are presented to support the hypothesis that quinic acid is not responsible for any efficacy, but rather that quinic acid nutritionally supports the synthesis of tryptophan and nicotinamide in the gastrointestinal (GI) tract, and that this in turn leads to DNA repair enhancement and NF-kB inhibition via increased nicotinamide and tryptophan production.Moreover, it is shown that quinic acid is a normal constituent of our diet, capable of conversion to tryptophan and nicotinamide via the GI tract microflora, thus providing an in situ physiological source of these essential metabolic ingredients to humans. The concentrations of quinic and hippuric acids in the diet were dependent on each other when analysed in urine, as was evidenced by a significant linear regression analysis that included unsupplemented control subjects (n = 45, p < 0.001). Thus, these ingredients were identified as major dietary components, and not simply originating from environmental pollution as previously had been thought.

Clinical applications of urinary organic acids. Part I: Detoxification markers.

Modern instrumentation allows the measurement of organic acids in urine in their physiological concentration ranges. Eight of the compounds that are reported can serve as markers for specific toxicant exposure or detoxification challenges. Xylene exposure causes elevation of 2-methylhippurate, and orotic acid elevation reveals ammonia challenge that exceeds the capacity of the urea cycle. General hepatic detoxification stimulation by natural compounds, drugs, or xenobiotic compounds causes elevated levels of glucaric acid. Abnormalities of alpha-hydroxybutyrate, pyroglutamate, and sulfate can indicate up-regulated glutathione biosynthesis, impaired reformation of glutathione in the gamma-glutamyl cycle, and depleted total body glutathione status, respectively. Patterns of these compounds measured in a simple overnight urine specimen help to identify focal areas of clinical concern and monitor patient responses to detoxification interventions.

Metabonomics approach to determine metabolic differences between green tea and black tea consumption.

The purpose of this study was to compare the effects of black and green tea consumption on human metabolism. Seventeen healthy male volunteers consumed black tea, green tea, or caffeine in a randomized crossover study. Twenty-four-hour urine and blood plasma samples were analyzed by NMR-based metabonomics, that is, high-resolution 1H NMR metabolic profiling combined with multivariate statistics. Green and black tea consumption resulted in similar increases in urinary excretion of hippuric acid and 1,3-dihydroxyphenyl-2-O-sulfate, both of which are end products of tea flavonoid degradation by colonic bacteria. Several unidentified aromatic metabolites were detected in urine specifically after green tea intake. Interestingly, green and black tea intake also had a different impact on endogenous metabolites in urine and plasma. Green tea intake caused a stronger increase in urinary excretion of several citric acid cycle intermediates, which suggests an effect of green tea flavanols on human oxidative energy metabolism and/or biosynthetic pathways.

[Integration of biological and workplace monitoring as an efficient control of chemical exposure]. / Integrazione dei piani di monitoraggio degli indicatori biologici di esposizione e dei piani di monitoraggio ambientale/espositivo.

Evaluation of professional exposure by means of biological monitoring is nowadays a consolidated method in the practice of Occupational Health. Generally biological monitoring is used simultaneously to ambient monitoring as a complementary method to obtain a mutual validation of exposure assessment. Experience gathered in the last years allowed us to verify that at low exposure levels, the values of biological indicators of dose are always markedly below their limits. Consequently, under standard conditions, it appears useful to alternate the two different exposure assessments (either biological or ambient monitoring), in order to obtain an efficient control of chemical exposure. Moreover, this methodological approach allows a better integration of all the professionals, who manage directly or indirectly the activities concerning Occupational Health and Industrial Hygiene, having as their first goal the health protection of employees and job environment.

Consumption of both black tea and green tea results in an increase in the excretion of hippuric acid into urine.

BACKGROUND: A major portion of the catechins in green tea is not absorbed in the small intestine. Bacteria in the colon convert nonabsorbed catechins into simpler phenolic compounds, which may also be absorbed. During the production of black tea, most catechins are polymerized to complex molecules called thearubigins. Little is known about the microbial degradation of these complex polyphenols, but hippuric acid has been identified as a major excretion product associated with black tea consumption. OBJECTIVE: To investigate whether green tea and black tea have the same metabolic fate in humans. DESIGN: Seventeen healthy male volunteers were studied with a randomized, full-crossover design. Each intervention period lasted 4 d, ie, a 2-d run-in period with a low-polyphenol diet followed by a 2-d treatment period. Volunteers consumed a daily dose of 6 g green tea solids, 6 g black tea solids, or 360 mg caffeine. Intervention periods were separated by a 10-d washout period. Twenty-four-hour urine samples were collected during the second day of each treatment period. Hippuric acid was analyzed with HPLC-tandem mass spectrometry. RESULTS: The mean excretion of urinary hippuric acid during black tea and green tea consumption was 3.75 +/- 0.28 mmol/24 h and 4.22 +/- 0.28 mmol/24 h, respectively (95% CI for the difference: -0.37 to +1.30 mmol/24 h). The hippuric acid excretion during the control treatment was much lower (1.89 +/- 0.28 mmol/24 h; P < 0.0001, compared with both black tea and green tea). CONCLUSION: The ingestion of either green tea or black tea results in a major increase in the excretion of hippuric acid into urine.