Methylhippuric Acid In Urine Research Articles

Deep eutectic solvent-based dispersive liquid-liquid microextraction coupled with HPLC and central composite design for biological monitoring of hippuric acid and methyl hippuric acid in urine

Exposure assessment of toluene and xylene as two hazardous compounds in occupational settings are great of importance. Hippuric acid (HA) and methyl hippuric acid (MHA) are common urinary metabolites for biological monitoring of toluene and xylene exposure, respectively. In this study a dispersive liquid–liquid microextraction (DLLME) method based on a deep eutectic solvent (DES), was suggested to determine the urinary MHA and HA simultaneously. Optimizations of all parameters affecting the DLLME-DES were done using response surface methodology. In obtained optimized conditions, a linear calibration range of 0.024–20 µg mL−1 for HA and 0.033–20 µg mL−1 for MHA were achieved and R2 was above 0.99 for these two metabolites. The detection limit of HA and MHA was 0.008 and 0.010 µg mL−1 respectively. The quantification limit was reported as 0.024 and 0.033 µg mL−1 for HA and MHA respectively. The suggested analytical technique was used for quantitation of target biomarkers in urine samples of occupational workers with high accuracy (97–103.6% for HA and 92–104% MHA) and precision (RSD ≤ 4.5% for HA (RSD ≤ 6.2% for MHA). Applying the proposed method for urine sample analysis of exposed-workers and comparing with the standard method (National Institute for Occupational Safety & Health standard method) showed that HA and MHA can be pre-concentrated and quantified by a simple, fast and environmental friendly method.

Investigation of some effective factors on urinary metabolites in biological monitoring of benzene, toluene, and xylene compounds

ABSTRACT The present study was performed to identify possible factors affecting the biological monitoring of benzene, toluene, and xylene (BTX) metabolites. Fifty persons with occupational exposure to BTX and 28 administrative employees were evaluated. Urinary trans, trans-muconic acid (t,t-MA), Hippuric Acid (HA) and Methyl Hippuric Acid (MHA) were determined as the biomarkers of exposure. The mean levels of t,t-MA, HA, and MHA in the exposed group were 541.90 (µg‧g creatinine−1), 0.0199, and 0.00205 (g‧g creatinine−1), respectively, which were higher than the non-exposed group (significant difference, Pvalue <0.001). In the regression model obtained from the amount of urinary t,t-MA, level of education, work experience, residence, length of work per shift, use of personal protective equipment (PPE), and habit of consuming industrial food or fruit juice had a significant effect. HA levels were affected by variables including education level, residence, consumption of industrial food or fruit juice in the past three days, and use of masks, gloves, and PPE. Due to the small amount of MHA in urine, there was a weak correlation between it and independent variables. Based on results, it was concluded that the health effects of exposure to BTX were significantly different between the two groups.

Comparison of urinary biomarkers concentrations in exposed and non-exposed petrol station workers in the Eastern Economic Corridor (EEC), Thailand.

The Thai government has been developing its Eastern Economic Corridor (EEC), which spans three provinces, with the aim of improving connections with other Asian nations. Since this strategic development, the number of trucks, private car, and passenger car registrations have continued to grow, with a corresponding increase in related to benzene, toluene, ethylbenzene, and xylene (BTEX). This study aims to compare the levels of trans, trans-muconic acid (t, t MA); toluene (TU); mandelic acid (MA); and methyl hippuric acid (MHA) in the urine of gas station employees, considering demographic and occupational factors. These employees worked either near or away from the fuel dispenser, and there 100 people in each group. Data were collected using interviews and testing environmental air and urine samples for benzene, toluene, ethyl benzene, and xylene (BTEX). The results showed that BTEX concentrations were just detectable in all 200 cases (100%). The mean (±SD) urine level of t, t MA was 449.28 (±213.32) μg/g creatinine, while the median (min-max) was 428.23 (95.58-1202.56) μg/g creatinine. The mean TU was 0.011 (0.001) mg/L, while the median (min-max) was 0.011 (0.010-0.013) mg/L. MA levels were higher inside the pollution control zone than they were outside the zone (p=.009). Employees who practiced poor personal hygiene had relatively high urinary toluene and MHA levels (p=.009) and those who did not wear personal protective equipment (PPE) had relatively high MA levels (p=.040). The results of this study revealed statistically significant biomarkers influencing the levels of t, t MA; TU; MA; and MHA in urine. The recommendations of this study are that employers should provide their employees with suitable PPE, check regularly to ensure that it is worn, and strongly encourage employees to take care of their sanitation. Employees should take appropriate breaks and days off to minimize their exposure to BTEX.

Investigation of kidney function changes in sea salt workers during harvest season in Thailand.

Occupational factors have previously been mentioned as contributing to decreased kidney function and the development of chronic kidney disease of unknown cause. Sea salt workers are one of the occupations facing high outdoor temperatures and a highly, intensive workload. The purpose of the study was to examine whether the kidney function of sea salt workers at the beginning of the harvest season differs from kidney function at the end of the harvest season and to identify factors that can predict the change of kidney function. Data were collected from salt workers (n=50) who were between 18–60 years of age without hypertension, diabetes, and kidney disease in Samut Sakhon province, Thailand. Urine specific gravity (USG) was used for hydration status and the estimated glomerular filter rate (eGFR) was used to measure kidney function. The mixed model was used to find differences over the harvest season and prediction of factors. On average, the eGFR was estimated to decrease by 15.2 ml/min/1.73 m2 over the harvest season. The decline in eGFR of sea salt workers with moderate and heavy workloads were significantly faster than their light workload counterparts after controlling for other covariates. Similarly, dehydration (USG ≥ 1.030) significantly accelerated the rate of kidney function loss. Our study confirmed exposure to heat over the harvest season leads to decreased eGFR in sea salt workers. The rate of change of eGFR could be predicted by workload and hydration status. Workers with dehydration who performed medium to heavy workloads in farms showed faster kidney function decline than those who performed light workload.

An amino-functionalized mesoporous silica (KIT-6) as a sorbent for dispersive and ultrasonication-assisted micro solid phase extraction of hippuric acid and methylhippuric acid, two biomarkers for toluene and xylene exposure.

The authors described a new application of amino-functionalized KIT-6 for dispersive ultrasonication-assisted micro solid phase extraction of hippuric acid (HA) and methyl hippuric acid (MHA) from human urine and water samples. In the first step, an amino-functionalized mesoporous silica of type KIT-6 was synthesized. It was characterized by field emission scanning electron microscopy, Fourier transform infrared spectrometry, nitrogen adsorption-desorption analysis, thermogravimetry and X-ray diffraction. Following sorption and desorption with 1.0mL methanol/NH4OH (1%; v/v), HA and MHA were quantified by HPLC with UV detection. Various important parameters were optimized by Box-Behnken design. Under optimized conditions, The limit of detections (LOD) were calculated by a signal-to-noise ratio of 3, which were 0.5μgL-1 and 0.2μgL-1 for HA and MHA, respectively, and the calibration plot is linear in the 1-1000μgL-1 concentration range. Only small matrix effects were found. The method was successfully implemented for the sensitive determination of HA and MHA in (spiked) human urine samples. Graphical abstract Schematic of a sorbent for dispersive micro solid phase extraction coupled with ultrasonic power. It consists ofamino functionalized KIT-6 and was used for the simultaneous preconcentration and determination of low levels of hippuric and methyl hippuric acid in urine and water samples.

Urine Methyl Hippuric Acid Levels in Acute Pesticide Poisoning: Estimation of Ingested Xylene Volume and Association with Clinical Outcome Parameters.

To determine the relationship between the oral ingestion volume of xylene and methyl hippuric acid (MHA) in urine, we measured MHA in 11 patients whose ingested xylene volume was identified. The best-fit equation between urine MHA and ingested amount of xylene was as follows: y (ingested amount of xylene, mL/kg) = −0.052x2 + 0.756x (x = MHA in urine in g/g creatinine). From this equation, we estimated the ingested xylene volume in 194 patients who had ingested pesticide of which the formulation was not available. Our results demonstrated that oxadiazole, dinitroaniline, chloroacetamide, organophosphate, and pyrethroid were xylene-containing pesticide classes, while the paraquat, glyphosate, glufosinate, synthetic auxin, fungicide, neonicotinoid, and carbamate classes were xylene-free pesticides. Sub-group univariate analysis showed a significant association between MHA levels in urine and ventilator necessity in the pyrethroid group. However, this association was not observed in the organophosphate group. Our results suggest that MHA in urine is a surrogate marker for xylene ingestion, and high urine MHA levels may be a risk factor for poor clinical outcome with some pesticide poisoning.

Determination of hippuric acid and methylhippuric acid in urine by high performance liquid chromatography after extracted with acetonitrile

To establish pretreatment conditions of hippuric acid (HA)and methyl-hippuric acid (MHA)in urine and HPLC conditions. HA and MHA in urine were extracted with acetonitrile under acid condition and determinated by HPLC-DAD. The operating conditions by HPLC were C18 column (150 mm× 4.6 mm, 5 μm), methanol-0.2% acetic acid (contained 6.5 mmol/L potassium dihydrogen phosphate)(25∶75, V/V) as mobile phase,1 ml/min as flow rate and wavelength was at 254 nm. The standard curves for HA, 2-MHA and 3-MHA(4-MHA)showed good linearity between 9.91~2 974.20 μg/ml(r=0.999 98), 1.91~573.60 μg/ml (r=0.999 84)and 2.00~598.65 μg/ml (r=0.999 85), respectively. The mean recoveries were 96.38%~98.01%, 83.17%~94.05 %, 103.22%~104.45%, respectively. The within-run precision were 0.50%~1.20%, 0.51%~1.59%, 0.49%~0.95%, respectively, and the between-run precision were 1.70%~3.20%, 1.30%~2.67%, 0.86%~2.74%, respectively. The detection limit of HA, 2-MHA and 3-MHA(4-MHA)were 0.18 μg/ml, 0.46 μg/ml and 0.12 μg/ml, and the low determination concentrations of the method were 0.36 μg/ml, 0.92 μg/ml and 0.24 μg/ml(1 ml urine). The urine can be kept 15 days at 4 ℃ refrigerator without significantly loss. This method with simply pretreatment conforms to the relevant requirements of guide for establishing occupational health standards-part 5: determination methods of chemicals in biological materials. It can be used to detect HA and MHA in urine for occupational population exposure to toluene and xylene.

Neuropsychological Symptoms among Workers Exposed to Toluene and Xylene in Two Paint Manufacturing Factories in Eastern Thailand.

The study analyzed the exposure factors that may lead to neuropsychological symptoms among 92 workers who were exposed to xylene and toluene and 100 workers who were not exposed to the solvents. The airborne concentration of xylene and toluene was evaluated with personal passive badges. The levels of methyl hippuric acid and hippuric acid in urine were assessed, and interviews were performed to observe the neuropsychological symptoms that may result from exposure to the solvents. The result showed that the average concentration for the exposed group of xylene in the paint company working environment was 2.7 (SD = 2.4) ppm and the average concentration of toluene was 9.5 (SD = 10.4) ppm. The average level of methyl hippuric acid in urine was 78 (SD = 74.7) mg/g creatinine. Factors that affected the neuropsychological symptoms included the following. (1) The impact of age: the risk (adjusted odds ratio) for getting psychosomatic symptoms in persons over 40 and exposed to xylene was 9.5 and the aOR of those exposed to toluene was 8.3. (2) The impact of not providing personal protective equipment was found to be sleep disturbance; it was found that the aOR of those exposed to xylene was 3.9, and the aOR of those exposed to toluene was 4.4. In summary, periodic examination of workers by occupational physician is needed for detection of early neuropsychological effects, especially psychosomatic symptoms, and sleep disturbances.

Xylene-Induced Auditory Dysfunction in Humans

Animal data indicate that xylene induces cochlear dysfunction, characterized by the loss of outer hair cells. There is little evidence regarding xylene-induced ototoxicity in humans. The aim of the study was to investigate the possible adverse effects of xylene on the peripheral and central auditory system in humans. A total of 30 medical laboratory workers who had been exposed to a mixture of xylene isomers, together with 30 nonexposed control participants matched for gender, age, and educational level were selected. Participants of both groups were not exposed to noise levels above 85 dBA time-weighted average. All participants were evaluated with a comprehensive audiological test battery, which included measures of peripheral and central auditory function. Peripheral auditory measures included pure-tone audiometry and distortion product otoacoustic emissions. Behavioral measures of central auditory function included a pitch pattern sequence test, an adaptive test of temporal resolution, a dichotic digit test, and a masking level difference test. The auditory brainstem response was used to objectively evaluate the function of the auditory pathways at the brainstem level. Speech perception in quiet and in noise was evaluated using the Hearing In Noise Test (HINT). The xylene-exposed participants were extensively evaluated with regard to their exposure to both noise and xylene. Noise dosimetry was conducted over an 8-hr work shift to obtain noise-exposure levels for each xylene-exposed worker. Airborne xylene concentrations were obtained at 11 different workstations throughout the medical laboratories, and methyl hippuric acid levels per gram of creatinine in urine were obtained for each xylene-exposed subject. Finally, a detailed interview exploring current and past solvent and noise exposure was conducted. The xylene-exposed participants showed significantly worse pure-tone thresholds in comparison with the nonexposed participants. The xylene-exposed participants demonstrated significantly worse results than the control group participants for the pitch pattern sequence test, dichotic digit test, HINT, and the auditory brainstem response (absolute and interpeak latencies). No significant differences between the xylene-exposed and nonexposed participants were observed for distortion product otoacoustic emissions, adaptive test of temporal resolution, or the masking level difference test. A significant correlation between the concentrations of methyl hippuric acid in urine and pure-tone thresholds (2 to 8 kHz) was found in xylene-exposed workers. Also, participants with high cumulative dose of xylene exposure presented with poorer test results than participants with low cumulative dose of xylene exposure. The results of the present research suggest that xylene is associated with adverse central auditory effects and poorer sound detection abilities in humans. A major limitation of the study is that the results found among xylene-exposed participants cannot be proved to be permanent, and thus further research should be conducted to clarify this limitation. Workers exposed to this chemical should be routinely evaluated with a comprehensive audiological test battery, to detect early signs of auditory dysfunction.

Metabonomics study of atherosclerosis rats by ultra fast liquid chromatography coupled with ion trap-time of flight mass spectrometry

An ultra fast liquid chromatography coupled with IT-TOF mass spectrometry (UFLC/MS-IT-TOF) metabonomic approach was employed to study the plasma and urine metabolic profiling of atherosclerosis rats. Acquired data were subjected to principal component analysis (PCA) for differentiating the atherosclerosis and the control groups. Potential biomarkers were screened by using S-plot and were identified by the accurate mass and MSn fragments information obtained from UFLC/MS-IT-TOF analysis. 12 metabolites in rat plasma and 8 metabolites in urine were identified as potential biomarkers. Concentrations of leucine, phenylalanine, tryptophan, acetylcarnitine, butyrylcarnitine, propionylcarnitine and spermine in plasma and 3-O-methyl-dopa, ethyl N2-acetyl-l-argininate, leucylproline, glucuronate, t6A N(6)-(N-threonylcarbonyl)-adenosine and methyl-hippuric acid in urine decreased in atherosclerosis rats. Ursodeoxycholic acid, chenodeoxycholic acid, LPC (C16:0), LPC (C18:0) and LPC (C18:1) in plasma and hippuric acid in urine were in higher levels in atherosclerosis rats. The alterated metabolites demonstrated abnormal metabolism of phenylalanine, tryptophan, bile acids and amino acids. This research proved that metabonomics is a promising tool for disease research.

Exposición a Solventes Orgánicos y Efectos Genotóxicos en Trabajadores de Fábricas de Pinturas en Bogotá

Investigating the degree of exposure to organic solvents and related genotoxic consequences in paint-factory workers by using cytogenetic monitoring and determining micronuclei frequency in lymphocytes and DNA damage as assessed by comet assays. Excretion of phenol and hippuric and methylhippuric acids in urine was investigated as biomarkers for benzene, toluene and xylene exposure. Urine samples were taken in two paint-factories in Bogotá and in a non-exposed control group. Benzene, toluene and xylene concentrations in air were measured in the work setting. Micronuclei and simple DNA chain breakage frequency were evaluated in peripheral blood sample mononuclear cells to assess early genetic effects. Hippuric acid concentration was found to be within normal values; by contrast, phenol and methylhippuric acid sample concentrations were above normal values by 3,3 % and 50,8 %, respectively. Benzene concentration in air was above permissible limits in one of the factories. There were no statistical differences regarding genetic biomarkers between exposed and non-exposed workers. Organic solvent levels in the samples were apparently low, which is consistent with the absence of genotoxicity in cells. This study represents a step forward in research in occupational health surveillance.

Simultaneous detection of hippuric acid and methylhippuric acid in urine by empore™ disk and gas chromatography–mass spectrometry

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 μg/ml. The detection limit of each acid was 1.0–2.5 μg/ml.

Modified method for determination of hippuric acid and methylhippuric acid in urine by gas chromatography

A modified method for the simultaneous determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids ( o-, m- and p-MHAs) in urine is described. These metabolites were extracted, derivatized into their methyl ester derivatives and analyzed using a gas chromatograph equipped with flame ionization detector and a DB-1 capillary column. The derivatives of HA, o-, m- and p-MHAs were well separated within 11 min. The accuracy and precision in the present method were sufficient for quantitative analysis, and the results obtained by the GC method were highly correlated with those by the HPLC method (NIOSH 8301).

Occupational chronic exposure to organic solvents XVII. Ambient and biological monitoring of workers exposed to xylenes.

Ambient-air and biological monitoring of occupational xylene exposure were carried out on 2 groups of workers (13 and 10 men, respectively) exposed to a mixture of xylenes during the production of paints or during spraying. Personal ambient-air monitoring was performed for one complete work shift. Blood and urine samples were collected directly at the end of the shift. Biological monitoring was based on the determination of the concentration of xylenes in blood and on the quantification of the sum of the three methylhippuric acids in urine. Average xylene ambient-air concentrations were 29 ppm (production) and 8 ppm (spraying), ranging from 5 to 58 ppm and from 3 to 21 ppm, respectively. The concentrations of xylenes in blood ranged from 63 to 715 microg/l and from 49 to 308 microg/l, with average values being 380 and 130 microg/l, respectively. Accordingly, the workers engaged in paint production also excreted more methylhippuric acids in their urine (average 1221 mg/l, range 194 2333 mg/l) than did the sprayers (average 485 mg/l, range 65-1633 mg/l). Our results as well as a literature review indicate that occupational xylene exposure on average barely exceeds the threshold limit value of 100 ppm as proposed by both American and German institutions. Biological monitoring based on the determination of xylenes in blood and of methylhippuric acids in urine provides sufficient sensitivity and specificity for occupational health surveillance. The results also confirm the current limit values (BAT values) proposed by the Deutsche Forschungsgemeinschaft for xylenes in blood (1500 microg/l) and methylhippuric acids in urine (2000 mg/l).

Urinary methylhippuric acid isomer levels after occupational exposure to a xylene mixture

The quantitative relationship between exposure to xylene vapor and urinary excretion of methylhippuric acid (MHA) isomers were studied in the second half of a working week. The participants in the study were 121 male workers engaged in dip-coating of metal parts who were predominantly exposed to three xylene isomers. The intensity of exposure measured by diffusive sampling during an 8-h shift was such that the geometric mean vapor concentration was 3.8 ppm for xylenes (0.8 ppm for o-xylene, 2.1 ppm for m-xylene, and 0.9 ppm for p-xylene), 0.8 ppm for toluene, and 0.9 ppm for ethylbenzene. Urine samples were collected at the end of the shift and analyzed for metabolities by HPLC. The statistical analysis showed that there is a linear relationship between the intensity of exposure to xylenes and the concentration of MHA in urine, that the regression line passes very close to the origin, and that the increment in observed (i.e., noncorrected) MHA concentrations as a function of increasing xylene concentration was 17.8 mg x l-1 ppm-1. Further examination on the basis on individual xylene isomers showed that the slopes of the regression lines for o- and m-isomers were similar (i.e., 17.1 and 16.6 mg l-1 ppm-1, respectively), whereas that for p-xylene was larger (21.3 mg l-1 ppm-1).

Simultaneous high-performance liquid chromatographic analysis of hippuric acid and ortho-, meta-, and para-methylhippuric acids in urine.

A high-performance liquid chromatographic assay for the simultaneous analysis of hippuric and methylhippuric acids in urine is described. Compounds are first extracted from acidified urines after addition of o-methylbenzoylalanine (internal standard), using methyl-t-butyl ether. The organic phase is evaporated under nitrogen flow and the residue dissolved in the mobile phase which consisted of 91% potassium phosphate buffer (12.0mM, pH: 2.0), 4.5% methanol, and 4.5% tetrahydrofuran. The method described allows complete separation of meta- and para-methylhippuric acids in less than 20 min using a stainless steel column packed with octadecyl-dimethysilyl silica. The authors used this method to determine the urinary excretion of hippuric and methylhippuric acids in rabbits following exposure to a mixture of toluene and isomers of xylene. Data indicated that under the actual experimental conditions of exposure o-xylene is excreted to a lesser extent as a methylhippuric acid than the meta- and para-xylenes.

Isotachophoretic analysis of mandelic acid, phenylglyoxylic acid, hippuric acid and methylhippuric acid in urine after occupational exposure to styrene, toluene and/or xylene

A simple, rapid and sensitive analytical method has been developed for the determination of phenylglyoxylic acid, mandelic acid, hippuric acid and methylhippuric acid; o-, m- and p-methylhippuric acids are partly separated. These compounds are found as metabolites after occupational exposure to styrene, toluene and xylene. The method has been applied successfully to samples extracted from human urine by diethyl ether. The method can be used to accurately and simultaneously determine as little as 0.5 nmole of all of these acids in less than 20 min.

Quantitative determination of urinary hippuric acid and m- or p-methylhippuric acid as indices of toluene and m- or p-xylene exposure by high performance liquid chromatography.

A high performance liquid chromatographic method for the determination of hippuric and m-(p-)methylhippuric acids in urine, metabolites of toluene and m-(p-)xylene, is described. A stainless-steel column packed with octadecyl silanized silica gel was used and the mobile phase was a mixed solution of methanol/water/acetic acid (20/80/0.2). The method is simple and specific. Urine can be analyzed directly, without solvent extraction or pretreatment. The method has a lower detection limit of 0.2 μg on column. All the analysis and quantitative determination can be performed within about 30 min. for samples containing m-(p-)methylhippuric acid.

Quantitative determination in urine of hippuric acid and m- or p-methylhippuric acid, metabolites of toluene and m- or p-xylene.

<b>Ogata, M., Tomokuni, K., and Takatsuka, Y. (1969).</b><i>Brit. J. industr. Med.,</i><b>26,</b> 330-334. <b>Quantitative determination in urine of hippuric acid and <i>m</i>- or <i>p</i>-methylhippuric acid, metabolites of toluene and <i>m</i>- or <i>p</i>-xylene.</b> Improved and more specific methods for the quantitative determination of hippuric and <i>m</i>- and <i>p</i>-methylhippuric acids in urine are described. The acids were extracted from urine with ether/ethanol, which was dried with silica gel, or with ethyl acetate. After removing the solvent by evaporation coloured azlactones were formed by reaction with <i>p</i>-dimethylaminobenzaldehyde in acetic anhydride (DAB, only usable after ether/ethanol extraction) or benzenesulphonyl chloride in pyridine, and the absorbances were measured. The sensitivities were about 4 μg./ml. of urine using DAB, and 20 μg./ml. of urine using benzenesulphonyl chloride reagent. Separation of hippuric and methylhippuric acids was achieved by paper and thin-layer chromatography before estimation. A spot test is also described.

Relationship between Concentration of Toluene and Xylene in the Air and Amount of Hippuric Acid and Methyl Hippuric Acid in Urine of Volunteers Exposed to Toluene and Xylene in a Gas Chamber : Studies on Organic Solvents 12

Relationship between Concentration of Toluene and Xylene in the Air and Amount of Hippuric Acid and Methyl Hippuric Acid in Urine of Volunteers Exposed to Toluene and Xylene in a Gas Chamber : Studies on Organic Solvents 12