Hippuric Acids Research Articles

High performance liquid chromatographic procedure for quantitative determination of urinary phenylglyoxylic, mandelic, and hippuric acids as indices of styrene exposure.

A method is described for the quantitative determination of phenyl-glyoxylic, mandelic, and hippuric acids in urine are all metabolites of styrene monomer. After o-methylhippuric acid was added to urine as an internal standard, these three acids and o-methylhippuric acid were extracted with a mixture of ethyl ether and methanol (9/1, by vol.).

Possible metabolic interaction of styrene with organic solvents.

Excretion of metabolites of styrene (dose: 2.2 mmol/kg i.p.) in 24-h urine was suppressed when rats were coinjected intraperitoneally with either toluene or trichloroethylene (dose: 2.2--11.0 mmol/kg) in addition to styrene, while a coinjection of n-hexane, ethyl acetate, methylchloroform, or acetone at an equimolar dose was ineffective in all cases. Suppressive effects of toluene and trichloroethylene were further confirmed by 6-h vapor exposure to (a) 0 to 1,300 ppm of toluene in addition to 150--170 ppm of styrene and (b) 0 to 4,000 ppm of trichloroethylene together with 100 or 150 ppm of styrene. The relationship between the amounts of three metabolites (i.e., phenylglyoxylic, mandelic and hippuric acids) in urine and styrene concentration in air was also determined.

Studies on drug metabolism by use of isotopes. XXII. Identification of human urinary metabolites of 3-phenylpropyl carbamate by an ion cluster technique.

The human urinary metabolites of 3-phenylpropyl carbamate (I) were analysed by using an ion cluster technique. After oral administration of an equimolar mixture of non-labeled I and deuterium labeled I, neutral metabolites were extracted with ethyl acetate at pH 7.0, trimethylsilylated and subjected to gas chromatograph-mass spectro-meter. In the case of the analysis of acidic metabolites, the urine of human receiving deuterium labeled I alone was used. The obtained acidic metabolites were methylated with diazomethane and subjected to gas chromatograph-mass spectrometer. As neutral metabolites, 3-hydroxy-3-phenylpropyl carbamate, 2, 3-dihydroxy-3-phenylpropyl carbamate and 3, 4'-dihydroxy-3-phenylpropyl carbamate were identified. Unchanged I, however, was not detected. Benzoic and hippuric acids originated from I could be clearly distinguished from the corresponding compounds endogenously produced.

Measurement by gas chromatography of urinary hippuric acid and methylhippuric acid as indices of toluene and xylene exposure.

A gas chromatographic method was applied to the determination of the urinary glycine conjugates, hippuric, o-, m- and p-methylhippuric acids. These were extracted with ethyl acetate from urine after acidification with hydrochloric acid. The internal standard solution (heptadecanoic acid methanol solution) was added before extraction and a diazomethane-ether-ethanol solution was subsequently added to the dried extracts. The methylated residues were dissolved in methanol and injected into a gas chromatograph as described by Buchet and Lauwerys (1973). By the combined use of gas chromatography and mass spectrometry the methyl esters of hippuric acid and m-methylhippuric acid were identified in the urine of a volunteer who had been exposed to toluene and m-xylene vapours. When the urine specimen contained salicyluric acid (a urinary metabolite of salicylic acid) two sharp peaks were observed. The faster peak coincided with m- or p-methylhippuric acid. The upper limit of urinary hippuric acid concentration in healthy subjects with no occupational exposure was calculated by this method to be 1.026 microgram/ml (fiducial limit 5%) after correction to 1.024 for variation in urinary density.

Quantitative determination of hippuric and m-methylhippuric acids in urine by high-speed liquid chromatography.

High-speed liquid chromatography employing an ultraviolet photometric detector has been applied to the simultaneous determination of hippuric and m-methylhippuric acids in urine. Reversed-phase partition chromatography is carried out on a muBondapak C18 column with methanol-water as the eluent system. This method obviates the necessity for isolation or reaction of these acids before assay. The only pretreatment necessary is extraction of sample with ethyl acetate. A linear relationship is obtained between the peak heights and the hippuric or m-methylhippuric acid concentrations. Mean recovery of hippuric and m-methylhippuric acids in urine is 99.8% and 99.3% respectively. The determination of hippuric acid by this method gives lower concentrations in normal urine than does the colorimetric method of Umberger and Fiorese (1963).

個人サンプラーによる暴露濃度と尿中代謝産物量の測定 ＩＩ 造船所におけるトルエン蒸気の場合

Personal air samplers were applied to shipyard's painters putting on gas masks during the spraying work, and the levels of toluene vapour surrounding the workers were measured. On the other hand, levels of urinary hippuric acid (metabolites of toluene) of the workers were measured, and the levels of toluene vapour inhaled were calculated from the levels of urinary hippuric acid. Then the actual removing-efficiencies of toluene vapours by the use of gas masks were estimated from these two levels (i.e., toluene vapours exposed and inhaled). The values of removing-efficiencies were found to be 65.9-98. 1%.The concentrations of hippuric and methylhippuric acids in the urine of workers exposed to toluene and xylene for 3 hours, collected just after the exposure, are valuable indices of these organic solvent vapours inhaled.A minute amount of urinary methylhippuric acid can be determined by means of gas chromatography.

Simultaneous quantitation of 4-hydroxy-3-methoxymandelic (vanilmandelic) and 4-hydroxy-3-methoxyphenylacetic (homovanillic) acids in human urine.

We describe a simultaneous analysis for vanilmandelic and homovanillic acids in urine. An aliquot of a final extract, containing phenylpropriopionic acid as external standard, is dried, an internal standard (resorcinol) is added, and the mixture is silylated and chromatographed. The two compounds are identified from their characteristic retention times and from their elution behhavior in relation to four different marker compounds (the external and internal standards and two other naturally occurring compounds, p-hydroxyphenylacetic and hippuric acids) and are quantitated from their relative response factors. Within-run precision (CV) varied from 2 to 14% for vanilmandelic acid and 1 to 18% for homovanillic acid. The ratio of the amount of phenylpropionic acid extracted to the amount of added resorcinol found is a useful quality-control index. Values for vanilmandelic acid obtained by this method and by the technique of Pisano et al. [Clin. Chim. Acta 7, 285 (1962)] correlated well (r = 0.844).

Measurement of toluene and xylene metabolites by gas chromatography.

A new method for analyzing urinary hippuric and methyl hippuric acids by gas chromatography was developed. As well the direct analysis of hippuric acids as the determination of their alkaline hydrolytic products, benzoic and toluic acids, are described. Results of the two different methods are compared and discussed. The method was applied to workers occupationally exposed to paints containing mainly toluene and xylene. The urinary hippuric acid concentrations were related to the level of toluene and xylene in blood.

ChemInform Abstract: PH‐METRISCHE STUDIEN AN DEN BINAEREN KOMPLEXEN VON OXOVANADIN(IV) MIT HIPPUR‐ UND ANTHRANILSAEURE UND PYRIDIN‐2‐ALDOXIM

pH-metric studies on the interaction of oxovanadium(IV) with hippuric and anthranilic acids and pyridine-2-aldoxime indicate the formation of monohydroxo derivatives of 1:1 chelates. The equilibrium constants for the reaction, VO2++HA+H2O⇌VO(OH)A+2H+ have been calculated as 4.47±0.07 and 6.32±0.05 in the 1:1 VO2+-hippuric or anthranilic acid systems resp. and for the reaction, VO2++H2A++H2O⇋VO(OH)A+3H+ as 8.40±0.09 in the 1:1 VO2+-pyridine-2-aldoxime hydrochloride system at 30±0.5°C (μ=0.1-KNO3).

Thin-layer chromatographic evaluation of methylhippuric and hippuric acids in urine

Thin-layer chromatographic evaluation of methylhippuric and hippuric acids in urine

The Specificity of the Nucleophilic Site of α-Chymotrypsin and its Potential for the Resolution of Alcohols. Enzyme-catalyzed Hydrolyses of Some (+)-, (−)-, and (±)-2-Butyl, -2-Octyl, and -α-Phenethyl Esters

The α-chymotrypsin-catalyzed hydrolyses of a representative spectrum of (R)- and (S)-2-butyl, -2-octyl, and -α-phenethyl esters of N-acetyl-L-phenylalanine, N-acetylglycine, α-hydroxy-β-phenyl-propionic, dihydrocinnamic, and hippuric acids have been examined. The kinetic studies were carried out in order to provide data on the practicability of exploiting the stereospecificity of the nucleophilic region of the active site of the enzyme for the resolution of racemic alcohols via selective hydrolysis of their esters. The data obtained indicate that the structure and chirality of the alcohol moieties affect both the catalytic and binding constants of the substrates and that the greatest differences between enantiomeric alcohol moieties are observed for nonspecific substrates of the dihydrocinnamate and hippurate series. For racemic esters of the latter type, the stereospecificity of the alcohol site is such that significant resolution of racemic esters and alcohols can be achieved via α-chymotrypsin-catalyzed hydrolyses on a preparative scale.

Evaluation of hippuric, phenylglyoxylic and mandelic acids in urine as indices of styrene exposure.

In addition to those of phenylglyoxylic and mandelic acids, a distinct increase in hippuric acid level was observed in the urine of factory workers who were exposed to styrene at 50 to 200 ppm for 160 min. Comparison of the postexposure levels with respective non-exposure levels and supplements with rat exposure experiments revealed hippuric acid to be a poor indicator of styrene exposure at a “moderate degree”, while the other two acids are much more adequate indices. At a higher dose of styrene (e.g., 500 mg/kg i.p. or 100 ppm for 8 hrs for rats), however, the urinary levels of phenylglyoxylic and mandelic acids reach plateaus, while hippuric acid level remained proportional to the amount of styrene given.

Styrene作業者の尿中mandelic acid, phenylglyoxylic acidおよびhippuric acid排泄の経日変動

Styrene作業者の尿中mandelic acid, phenylglyoxylic acidおよびhippuric acid排泄の経日変動

Substituent effects in the acid solvolysis of para-substituted hippuric acids in acetic acid at low water concentrations. Neighbouring group participation by the carboxy-group

The rates of acid solvolysis of the para-substituted hippuric acids in acetic acid decrease with an increasing acid concentration for Cl, H, Me, and OMe indicating that the rate-controlling step is the ring closure reaction by the non-protonated amide to form an intermediate azlactone or its solvate. The acid–base equilibrium constants derived from the kinetic data vary in accordance with their substituent effects. Although the velocity constants show an irregular sequence, the products of the equilibrium and velocity constants vary as required by the substituent effects. This is in agreement with the reaction rate being dependent upon the zwitterion concentration through which the ring closure proceeds. The NO2 group behaves differently from the other groups and gives only increasing rates with increasing acid concentrations and this behaviour is consistent with that of other strongly electronegative groups such as the trifluoroacetyl and the positive terminal amino-group of a dipeptide. These groups favour ring closure so that the acid catalysed ring fission of the azlactone solvate becomes the slow reaction.

Urinary aromatic acid excretion by fed and fasted sheep in relation to protein metabolism in the rumen.

1. Two adult wether sheep were maintained on a diet of hay and two on a diet of dried grass for 3 weeks before starvation for a period of 10 d. Urinary excretion of the following acids was determined when the animals were fed and when they were fasted: total diethyl ethersoluble acids of hydrolysed and unhydrolysed urine, hippuric acid, benzoic acid and phenylacetic acid. By the 5th day of fasting, urinary output of all acids had attained stable levels that did not change during the remaining starvation period. The output of all urine fractions except phenylacetic acid declined rapidly during the first 4 d of fasting: phenylacetic acid output by all sheep increased to a maximum during the first 4 d of fast and then declined to stable values (0·42–0·73 g/24 h) which were greater than those observed when the sheep were fed. It is concluded that prolonged retention of food and microbial residues in the digestive tract is responsible for the large output of phenylacetic acid in the urine of fasted sheep.2. Solutions of casein which supplied between 6·3. and 26·5 g nitrogen/24 h were infused into the rumens (fifteen experiments) or abomasums (sixteen experiments) of eight adult wether sheep. Ruminal infusions of casein caused increments in the urinary excretion of diethyl ethersoluble acids and phenylacetic acid. Both these increments were described by linear regression equations (P< 0·001), the coefficients of which showed that 284 ± 44 and 220 ± 21 mg benzoic acid equivalent were excreted as diethyl ether-soluble acids and phenylacetic acid respectively per g casein N infused. The phenylacetic acid excreted was equivalent to 95% of the phenylalanine of the infused casein. No increments in urinary benzoic acid were observed. One sheep scoured when it was given an abomasal infusion of casein. This was the only animal to show any increment in urinary aromatic acids when casein was infused into the abomasum.3. When four sheep were given two rations containing an excess of carbohydrate as sugarbeet pulp or rolled barley, 11 and 16% respectively of their phenylalanine intakes were excreted in the urine as phenylacetic acid. When the same sheep were given two rations containing an excess of N as linseed meal or field beans, 51 and 59% respectively of their phenylalanine intakes appeared in the urine as phenylacetic acid.4. Methods for the determination of creatinine, and of benzoic, phenylacetic, 3-phenylpropionic, cinnamic, hippuric and phenaceturic acids are described.5. It is suggested that the amount of phenylacetic acid excreted in the urine is a measure of the equilibrium occurring in the rumen between catabolism of phenylalanine and reutilization of the products of catabolism for phenylalanine synthesis.

Measurement of urinary hippuric and m-methylhippuric acids by gas chromatography

Buchet, J. P., and Lauwerys, R. R. (1973).British Journal of Industrial Medicine,30, 125-128. Measurement of urinary hippuric and m-methylhippuric acids by gas chromatography. A gas chromatographic method for the determination of hippuric and m-methylhippuric acids in urine is described. A known amount of heptadecanoic acid (internal standard) is added to the urine before its extraction with ethyl acetate. After evaporation of the solvent, the acids are methylated with diazomethane. The residue is taken up in methanol and injected into the gas chromatograph. The ratio of the height of the hippuric or m-methylhippuric acid peak to the height of the heptadecanoic acid peak is calculated and by reference to a calibration curve prepared in the same conditions the urine concentration of the two acids is determined.

Action of drug conjugates on glucuronide biosynthesis

1. 1. Digitonin-activated UDP glucuronyltransferase ( p-nitrophenol) of rat liver microsomes was inhibited by sulphate esters of phenolphthalein and 4-methylumbelliferone. They competed with the glucuronyl donor, UDP-glucuronic acid ( K 1 0.8 m M and 4.0 m M respectively) but not with the aglycone, p-nitrophenol ( K 1) 3.7 m M and 9 m M respectively). 2. 2. The o-aminophenyl glucuronide synthesis in rat liver slices was inhibited irreversibly by inorganic sulphate (about 50 per cent at 10 m M concentration). Inorganic sulphate (10 m M) had no effect on liver UDP-glucose dehydrogenase, UDP glucuronyltransferase and intracellular UDP-glucose concentration, but it reduced by about 16 per cent the oxygen uptake of the slices. 3. 3. The glycine conjugates, salicyluric, hippuric and p-aminohippuric acids, only had a 5–20 per cent inhibitory effect on the enzyme at 10 m M concentration. Of acetyl conjugates, N-acetylsulphanilamide (5 m M) did not inhibit UDP glucuronyltransferase at all and the N-acetyl- o-toluidine (10 m M) caused about 20 per cent inhibition.

Investigation of exposure of ships' painters to organic solvents.

<b>Mikulski, P. I., Wiglusz, Renata, Bublewska, Anna, and Uselis, J. (1972).</b><i>Brit. J. industr. Med.,</i><b>29,</b> 450-453. <b>Investigation of exposure of ships9 painters to organic solvents.</b> An analysis was carried out of the hazard to ships9 painters working at different concentrations of toluene and xylene vapours in air. The authors point out that it is possible to use a joint determination of hippuric and methylhippuric acids for the assessment of mixed exposure. The problem of benzene contamination of solvents is considered. During the investigations exposure to toluene and xylene was found to impair the excretion of uric acid.

Studies on Components of Mammalian Urine

Hexahydrohippuric acid was detected from the urine of cattles together with hippuric and phenaceturic acids as one of the conjugated compounds with glycine. Furthermore, cyclohexanecarboxylic acid was also detected. These experimental results suggest the interesting synthetic processes in vivo or in rumen of the cattle, though both acids have not determined whether they are metabolites of cattle or synthesized by miccroorganisms in rumen.

Urinary excretion of hippuric acid and m- or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m- or p-xylene as a test of exposure.

<b>Ogata, M., Tomokuni, K., and Takatsuka, Y. (1970).</b><i>Brit. J. industr. Med.,</i><b>27,</b> 43-50. <b>Urinary excretion of hippuric acid and <i>m</i>- or <i>p</i>-methylhippuric acid in the urine of persons exposed to vapours of toluene and <i>m</i>- or <i>p</i>-xylene as a test of exposure.</b> Twenty-three male volunteers were exposed in groups of four or five to toluene and <i>m</i>- and <i>p</i>-xylene vapour for periods of 3 hours or of 7 hours with one break of an hour. Urine was collected at hourly intervals for several hours, and thereafter all urine was collected until 18 hours after the end of the exposure period, and was analysed for hippuric and methylhippuric acids. It was shown that hippuric acid was excreted equivalent to 68% of the toluene absorbed, and <i>m</i>-methylhippuric acid equivalent to 72% of the <i>m</i>-xylene absorbed. Up to hydrocarbon concentrations of 200 ppm the total quantity of hippuric acids excerted was proportional to the total exposure (ppm × hours). In descending order of precision the following were also related to exposure: rate of excretion during the exposure period; concentrations of hippuric acid in urine corrected to constant urine density; and concentrations in urine uncorrected for density. The last could not be used to calculate exposure, but the others could be to give screening tests to show whether workmen could have been exposed to concentrations greater than the maximum allowable. The effects of exposure on blood pressure, pulse rate, flicker value, and reaction time were measured. There were some variations which suggested that the MAC of toluene should be set higher than 200 ppm.