DIRECT EVIDENCE FOR AN ACYLATED THIOL AS AN INTERMEDIATE IN PAPAIN- AND FICIN-CATALYSED HYDROLYSES.
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1. Methyl thionohippurate was prepared and shown to be a specific substrate for both papain and ficin. 2. The ultraviolet-absorption properties of the acyl-enzyme intermediate for both papain and ficin with methyl thionohippurate was that expected of an acyl-thiol. The possibility that other functional groups present in papain or ficin might be the site of acylation has been excluded. 3. The change in extinction at the absorption maximum with time was as expected for the acyl-enzyme on the basis of the known Michaelis-Menten parameters for methyl thionohippurate. 4. The variation of extinction with initial substrate concentration for both papain and ficin was that expected from the Michaelis-Menten parameters. 5. The extinction of the absorption maximum of the thionohippuryl-enzyme intermediate was suppressed by the addition of methyl hippurate to the extent predicted from the Michaelis-Menten parameters. 6. The decay of the extinction for the acyl-enzyme was arrested by adjusting the pH of the solution to 2.5. 7. These experiments provide compelling evidence that the acylation by substrate of both papain and ficin takes place through a thiol residue. (+info)
A STUDY OF SOME THIOL ESTER HYDROLYSES AS MODELS FOR THE DEACYLATION STEP OF PAPAIN-CATALYSED HYDROLYSES.
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1. The self-catalysed hydrolyses of the thiol esters, S-hippurylthioglycollic acid and S-ethyl monothiolsuccinate, have been shown to be slower than the deacylation step for the papain-catalysed hydrolysis of hippuric esters, by a factor approx. 10(5). This difference in rate constants largely reflects a difference in activation energy, which together with other evidence drawn from the literature make it unlikely that a carboxylate ion could be the nucleophile responsible for the deacylation of acyl-papain. 2. The imidazole-catalysed hydrolysis of S-hippurylthioglycollic acid and ethyl thiolacetate have activation energies similar to that for the deacylation step in papain-catalysed hydrolyses. This, together with other evidence drawn from the literature, suggests that the imidazole of a histidine residue is the nucleophile responsible for the deacylation of acyl-papain. (+info)
PAPAIN-CATALYSED HYDROLYSIS OF SOME HIPPURIC ESTERS. A NEW MECHANISM FOR PAPAIN-CATALYSED HYDROLYSIS.
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1. The Michaelis-Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k(0), is 2-3sec.(-1) and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k(0), less than 2-3sec.(-1). It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme-substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis-Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, sigma. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-alpha-amino acid derivatives. (+info)
A survey of urinary hippuric acid and subjective symptoms among occupational low toluene exposed workers.
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Biological monitoring of toluene exposure by urinary hippuric acid determination and a subjective symptom survey by self-administered questionnaire were performed in 20 workers at low toluene exposure in factories to evaluate the health hazard including dysfunction of nervous system. Environmental monitoring was carried out using toluene gas detection tubes. Urine samples were collected three times a day in order to measure hippuric acid: first before the commencement of work, then at the end of forenoon work, and lastly at the end of afternoon work. Toluene vapor concentrations of throughout the workday ranged from 15.3 to 31.4 ppm. The urinary hippuric acid concentrations correlated with the toluene concentrations of ambient air (r = 0.58, p = 0.01). The subjective symptoms increased in close association with the exposure to toluene; the prevalence rate of subjective symptoms "during work" in the exposed group was 15 times higher than the rate of the non-exposed group (p < 0.0001). The prevalence rate of subjective symptoms "off-work" in the exposed group was 2.4 times higher than the rate of the non-exposed group (p < 0.0001), and also the prevalence rate of "nineteen symptoms off-work which are apparently related to central nervous system (CNS) and autonomic nervous system (ANS)" in the exposed group was 1.8 times higher than the rate of the non-exposed group (p < 0.05). From these results, these subjective symptoms, which have been believed to be complained in high organic solvent exposure should be reassessed and reconsidered in evaluating the nervous system dysfunction and local irritation in relatively low toluene exposed workers. (+info)
Molecular and ultrastructural characterization of porcine hippurate-negative Brachyspira pilosicoli.
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Brachyspira pilosicoli, the causative agent of porcine intestinal spirochetosis, usually has hippurate-cleaving capacity. We have regularly isolated hippurate-negative B. pilosicoli from cases of porcine diarrhea. In this study, we show that these biochemically atypical B. pilosicoli isolates can be classified as B. pilosicoli. 16S ribosomal DNA was partially sequenced from eight hippurate-negative and two hippurate-positive B. pilosicoli-like isolates from seven herds. The differences in nucleotide sequence with B. pilosicoli P43/6/78 type strain were not associated with hippurate cleavage. In 877 bp, the hippurate-negative isolates had a similarity of 98.63 to 100% to the type strain, with the corresponding figures for the two hippurate-positive isolates being 98.86 and 100%. The nucleotide sequences of hippurate-positive isolates were identical to the respective sequences of hippurate-negative isolates from one herd. The DNA macrorestriction patterns of a total of 20 hippurate-negative and -positive B. pilosicoli isolates were diverse, and no clustering in conjunction with the hippurate reaction was found. In two herds, hippurate-positive and -negative B. pilosicoli isolates had a common macrorestriction pattern. The ultrastructure of hippurate-negative isolates was similar to the type strain. In conclusion, B. pilosicoli can be either hippurate positive or negative and, thus, the scheme for biochemical differentiation of porcine Brachyspira should be revised to include identification of hippurate-negative B. pilosicoli. (+info)
Effects of ethanol and phenobarbital treatments on the pharmacokinetics of toluene in rats.
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Rats were exposed to toluene at a wide range of concentrations from 50 to 4000 ppm for six hours, and the effects of ethanol and phenobarbital (PB) treatments on the pharmacokinetics of toluene metabolism were investigated. Ethanol treatment influenced toluene metabolism mainly at low exposure concentrations. Thus ethanol accelerated the clearance of toluene from blood only when the blood concentration of toluene was not high (less than 360 microM), and ethanol increased hippuric acid (HA) excretion in urine more significantly at low (less than 250 ppm) than at high atmospheric toluene concentrations. Ethanol also expressed a similar effect on p-cresol excretion as on HA, but had little effect on o-cresol. Phenobarbital treatment promoted the urinary excretion of all of the metabolites of toluene, especially after exposure to high toluene concentration. As well as HA, benzoylglucuronide (BG) and free benzoic acid were found in urine. These are the products of the side chain metabolism of toluene. Amounts of BG could be detected when the urinary excretion of free benzoic acid exceeded 5 mumol/kg/6 h, indicating that a great deal of benzoic acid is required for the formation of BG. The Michaelis constant (Km) and the maximum rate of metabolic excretion in urine during six hours exposure (Vmax) of isozymes involved in the excretion of toluene metabolites were calculated, and correlated with the subtypes of cytochrome P-450. The significance of the result was suggested in the biological monitoring of exposure to toluene. (+info)
Consumption of both black tea and green tea results in an increase in the excretion of hippuric acid into urine.
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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. (+info)
Effects of air pollutants on childhood asthma.
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Epidemiologic studies have suggested the association between environmental exposure to volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) and the increased risk of incurring asthma. Yet there is little data regarding the relationship between personal exposure to air pollution and the incidence of asthma in children. This study was designed to evaluate the effect of exposure to air pollution on children with asthma by using exposure biomarkers. We assessed the exposure level to VOCs by measuring urinary concentrations of hippuric acid and muconic acid, and PAHs by 1-OH pyrene and 2-naphthol in 30 children with asthma and 30 children without asthma (control). The mean level of hippuric acid was 0.158 +/- 0.169 micromol/mol creatinine in the asthma group and 0.148 +/- 0.249 micromol/mol creatinine in the control group, with no statistical significance noted (p=0.30). The mean concentration of muconic acid was higher in the asthma group than in the control group (7.630 +/- 8.915 micromol/mol creatinine vs. 3.390 +/- 4.526 micromol/mol creatinine p=0.01). The mean level of urinary 1-OHP was higher in the asthma group (0.430 +/- 0.343 micromol/mol creatinine) than the control group (0.239 +/- 0.175 micromol/mol creatinine), which was statistically significant (p=0.03). There was no difference in the mean concentration of 2-NAP between the two groups (9.864 +/- 10.037 micromol/mol in the asthma group vs. 9.157 +/- 9.640 micromol/mol in the control group, p=0.96). In conclusion, this study suggests that VOCs and PAHs have some role in asthma. (+info)