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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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aspirin (acetylsalicylic acid) has been widely used as an over-the-counter drug to relieve pain throughout the world. In suicide attempts, aspirin is one of the most common drugs taken in large quantities. The concentration of salicylic acid shows a low-level distribution in the mouse brain, a site that may be critical regarding the expression of symptoms (e.g. hyperpnea, coma, convulsion and tremor) during acute aspirin toxicity. Therefore, it was suggested that sensitivity to salicylic acid concerning acute toxicity was higher in the brain than in other organs. Moreover, it is thought that it is common for aspirin and ethanol to be ingested at the same time. Therefore, the present study was designed to investigate the influence of ethanol on the distribution of salicylic acid, which is a primary metabolite of aspirin, and its related metabolite, salicyluric acid. The oral co-administration of aspirin (0.5g/kg) and ethanol (2.5g/kg; 10ml/kg of 25% (w/v)) enhanced the concentrations of salicylic acid in the plasma and organs, especially in the brain, compared with the aspirin alone-treated group. On the other hand, ethanol did not influence the concentrations of salicyluric acid in the plasma and kidney compared with the aspirin alone-treated group. These results suggest that ethanol enhances aspirin absorption from the gastrointestinal tract but has no influence on its metabolism. Thus, it is dangerous to ingest the alcohol and aspirin at the same time, as this may exacerbate the acute toxicity of aspirin.

A high-performance liquid chromatographic method for indirect determination of enalapril in human plasma, was developed and validated. An exogenous angiotensin converting enzyme after drug inhibition was determined by reacting with hippuryl-histidyl-leucine to produce hippuric acid (HA) which was inversely proportional to the amount of enalaprilat in plasma. The HPLC was carried out on a Lichrosphere 60RP-select B, C18, 5 microm (125 mm x 4.0 mm i.d.) column at flow rate of 1.0 ml/min. The analysis time per injection was within 6.5 min. The lowest concentration of enalaprilat to be quantitated was 3.0 ng/ml with the acceptable accuracy and precision. This successfully developed method was practically and accurately used for pharmacokinetics and bioequivalent study of enalapril.

A method is described for the determination of hippuric acid (HA) and o-, m-, and p-methylhippuric acids (o-, m-, p-MHAs) in urine using solid-phase extraction and gas chromatography-mass spectrometry (GC-MS). The extraction procedure uses an Empore disk, derivatized into the respective trimethyl silyl derivatives. All metabolites including the internal standard (I.S.) were clearly able to be analyzed by the DB-17 column. The calibration curves for the four acids show linearity in the range of 5-70 microg/ml. The detection limit of each acid was 1.0-2.5 microg/ml.

(R)- and (S)-Hippuryl-alpha-methylphenylalanine [(R)- and (S)-Hipp-alpha-MePhe] and (S)-hippuryl-alpha-methylphenyllactic acid [(S)-Hipp-alpha-MeOPhe] were synthesized and evaluated as substrates for carboxypeptidase A (CPA) in an effort to shed further light on the catalytic mechanism of the enzyme. The rate of CPA-catalyzed hydrolysis of (S)-Hipp-alpha-MePhe was reduced by 105-fold compared with that of (S)-Hipp-Phe, but the hydrolysis rate of (S)-Hipp-OPhe was lowered by only 6.8-fold by the introduction of a methyl group at the alpha-position. (R)-Hipp-alpha-MePhe failed to be hydrolyzed initially, then started to undergo hydrolysis in about 2 h at a much reduced rate. The results of present study may be envisioned on the basis of the proposition that while peptide substrate is hydrolyzed via a tetrahedral transition state formed by the attack of the zinc-bound water molecule at the peptide carbonyl carbon, ester hydrolysis takes the path that involves an anhydride intermediate generated by the attack of the carboxylate of Glu-270 at the ester carbonyl carbon.

A novel sensitive and specific method for the measurement of tissue angiotensin-converting enzyme (ACE) activity utilizing HPLC is described. ACE activity was determined in detergent-extracted canine hearts utilizing the synthetic ACE-specific substrate hippuryl histidyl leucine (HHL), both in the presence and the absence of the site-specific inhibitor captopril. Tissue ACE activity was quantitated from the moles of hippuric acid (HA) formed, in time-fixed assays, utilizing HPLC separation of HA from HHL and UV-spectrophotometry for quantitation of HA as in the standard Cushman and Cheung assay (Cushman DW and Cheung HS, Biochem Pharmacol 20: 1637-1648, 1971). Separation of HA from HHL was performed by reverse phase HPLC on a phenyl silica gel column with an eluent consisting of 20% acetonitrile in 0.1 M aqueous ammonium phosphate buffer, pH 6.8. After the standard liquid/liquid extraction procedure with ethyl acetate, HPLC analysis revealed the presence of unreacted substrate, HHL, in amounts comparable to the product of interest, HA, in the final assay; moreover, the amount of HA formed did not fall completely to zero in the presence of captopril. Regional studies of canine cardiac ACE activity utilizing the HPLC-based assay and the standard assay method showed a significantly higher ACE activity in the right ventricle compared with the left ventricle (2.37 +/- 0.7 vs 1.24 +/- 0.18 mU/g, P < 0.05 [N = 6], respectively) in the HPLC-based assay, but no difference in right and left ventricular ACE activities by the standard assay (0.25 +/- 0.08 vs 0.31 +/- 0.09 mU/g [N = 6], respectively). Kinetic studies utilizing the HPLC-based assay coupled with the use of captopril showed Km (1.34 +/- 0.08 mM) and Vmax (36.8 +/- 11.5 x 10(-10) M/min) values in agreement with those in the literature. Our results demonstrate that the application of HPLC to the standard Cushman and Cheung assay improves the sensitivity and specificity of the standard assay and enables the use of much smaller amounts (approximately 4 vs approximately 400 mg for the Cushman and Cheung assay) of tissue for ACE activity assay.

Objective: To evaluate glycine conjugation of para-aminobenzoic acid (PABA) to the hippurated metabolites, para-aminohippuric acid (PAHA), and para-acetamidohippuric acid (PAAHA) as a quantitative liver function test in patients with liver disease., Design and Methods: Serum concentrations of PABA and metabolites were measured by high pressure liquid chromatography in 24 controls and 50 patients with hepatobiliary disease., Results: Hippurate formation was significantly decreased in all patient groups with chronic liver disease versus controls. The hippurate ratio (% hippurated metabolites formed) correlated with severity of disease, serum albumin, and factor VII concentrations. PAHA concentration was a better prognostic indicator than factor VII concentrations in patients with acute liver disease; concentrations of zero correctly predicted a poor outcome in patients with fulminant liver failure., Conclusions: Glycine conjugation of PABA may be useful as a quantitative liver function test in patients with hepatobiliary disease and as a prognostic index in patients with fulminant liver failure.

The O2 dependence of the metabolism of benzylamine to benzaldehyde, benzoate and hippurate was studied in isolated rat hepatocytes. The initial oxidation to benzaldehyde, catalyzed by monoamine oxidase, had an apparent Kmo2 value of 34 microM in cells and 40 microM in isolated rat liver mitochondria. The conversion of benzaldehyde to benzoate was essentially independent of O2 concentration in spite of the dependence of the reaction upon cytosolic NAD+. The conversion of benzoate to hippurate was half-maximal at 2.4 microM O2 in hepatocytes and at about 0.5 microM O2 in rat liver mitochondria. These values are consistent with the O2 dependence of bioenergetic changes in these preparations and indicate that the O2 dependence of hippurate formation is due to ATP availability for synthesis of benzoyl-CoA. These studies show that the three metabolic processes involved in benzylamine metabolism have markedly different dependences upon O2 and that metabolism of benzylamine by monoamine oxidase is O2 dependent over a physiologically important range.

We have evaluated pH, chloride, calcium and several endogenous aromatic acids as possible causes of the impaired binding of drugs by plasma albumin in renal failure. Changes in pH, chloride and calcium over the range found in renal failure had minimal or no effects on the binding of [14C]salicylate, a model probe which binds to both of the major drug-binding loci of human albumin. Hippurate and indoxyl sulfate were weak inhibitors of binding by normal plasma. Ortho-hydroxy-hippurate was undetectable or minimally elevated, except among patients with elevated plasma salicylate concentration. Although plasma hippurate and indoxyl sulfate concentrations were elevated markedly in patients with renal failure, inhibition of salicylate binding was significantly correlated only with the concentration of indoxyl sulfate. Addition of hippurate and indoxyl sulfate separately and together to normal plasma showed that these ligands could account for only 15% of the impaired binding of salicylate by azotemic plasma. The retained solutes which account for most of this binding defect remain to be identified. This uremic disorder (and perhaps others) is due not to a single chemical but to the additive effect of a family of chemicals.