Determination of total urinary 2,5-hexanedione in the Chinese general population.

OBJECTIVE: Determination of the urinary levels of 2.5-hexanedione (2,5-HD) was performed in subjects belonging to the Chinese general population to define the reference value for this metabolite. METHODS: Urine samples were collected from 8235 individuals (4216 men and 4019 women) from the healthy general population who had not been occupationally exposed to n-hexane or methyl-n-butyl ketone. The determination was performed by a gas chromatography mass spectrometry method using an ion-trap mass spectrometer. RESULTS: The result showed that the urinary 2,5-HD median level was 0.159mg/L for the total samples. Males had statistically significant higher excretion of 2,5-HD in urine than females (median 0.171mg/L compared to 0.147mg/L, Z=-8.21, P<0.001). There was a statistically significant difference in urinary 2,5-HD levels among age groups. The excretion of 2,5-HD in urine was related to increasing age (r=-0.160, P<0.05). There was statistically significant difference in urinary 2,5-HD levels among people from difference provinces. The results showed that there was also a statistically significant effect in urinary 2,5-HD levels between current smokers and non-smokers. CONCLUSION: Finding a measurable amount of 2,5-HD in urine does not mean that the level of 2,5-HD causes an adverse health effect. Biomonitoring studies on levels of urinary 2,5-HD can provide physicians and public health officials with reference values so that they can determine whether people have been exposed to higher levels of 2,5-HD than are found in the Chinese general population. These data can also provide a foundation for scientists to make a plan for further study.

[Readspace solid-phase microextraction-gas chromatography for determination of 2,5-hexanedione in urine].

OBJECTIVE: To establish a method for determination of 2,5-hexanedione in urine by headspace solid-phase microextraction-gas chromatography. METHODS: After extraction by solid-phase microextraction head, 2,5-hexanedione in urine was determined by gas chromatography and was quantified by external standard method. RESULTS: The concentration of 2,5-hexanedione in urine showed a linear relationship within the range of 0.1-20.0 µg/ml. The regression equation was y=261.36x-1.903 3, r=0.999 2. The minimum detectable concentration was 0.01 µg/ml. The recovery rate was 92.6%-97.1%, with a relative standard deviation (RSD) of 3.3%-5.8%. The intra-day and inter-day RSDs were 3.8%-6.2% and 4.7%-6.3% respectively. CONCLUSION: This determination method has no requirement for organic solvents, features simple and rapid operation, possesses higher detection sensitivity, and applies well to the determination of 2,5-hexanedione in urine.

Determination of 2,5-hexanedione in urine by headspace solid-phase microextraction and gas chromatography.

2,5-Hexanedione (2,5-HD) is the most important metabolite of n-hexane and methyl ethyl ketone in human urine. Urinary 2,5-HD is used as a biomarker for biological monitoring of workers exposed to n-hexane. A simple method using headspace solid-phase microextraction (HS-SPME) and gas chromatography (GC) equipped with a flame-ionization detector (FID) was developed. The parameters that affect the HS-SPME-GC-FID process were optimized (i.e., fiber coating, sample volume, adsorption and heating time, salt addition, and extraction temperature). The assay presented linearity in the range of 0.075 to 20.0 mg/L, precision (coefficient of variation < 7.0%), and detection limit of 0.025 mg/L for 2,5-HD in urine. The method was successfully applied to the analysis of 2,5-HD in urine samples from eight workers occupationally exposed to n-hexane in shoemaker's glue.

The effect of workload on biological monitoring of occupational exposure to toluene and n-Hexane: contribution of physiologically based toxicokinetic modeling.

A physiologically based toxicokinetic model was used to examine the impact of work load on the relationship between the airborne concentrations and exposure indicator levels of two industrial solvents, toluene and n-Hexane. The authors simulated occupational exposure (8 hr/day, 5 days/week) at different concentrations, notably 20 ppm and 50 ppm, which are the current threshold limit values recommended by ACGIH for toluene and n-hexane, respectively. Different levels of physical activity, namely, rest, 25 W, and 50 W (for 12 hr followed by 12 hr at rest) were simulated to assess the impact of work load on the recommended biological exposure indices: toluene in blood prior to the last shift of the workweek, urinary o-cresol (a metabolite of toluene) at the end of the shift, and free (nonhydrolyzed) 2,5-hexanedione (a metabolite of n-hexane) at the end of the shift at the end of the workweek. In addition, urinary excretion of unchanged toluene was simulated. The predicted biological concentrations were compared with the results of both experimental studies among human volunteers and field studies among workers. The highest predicted increase with physical exercise was noted for toluene in blood (39 microg/L at 50 W vs. 14 microg/L at rest for 20 ppm, i.e., a 2.8-fold increase). The end-of-shift urinary concentrations of o-cresol and toluene were two times higher at 50 W than at rest (for 20 ppm, 0.65 vs. 0.33 mg/L for o-cresol and 43 vs. 21 microg/L for toluene). Urinary 2,5-hexanedione predicted for 50 ppm was 1.07 mg/L at 50 W and 0.92 mg/L at rest (+16%). The simulations that best describe the concentrations among workers exposed to toluene are those corresponding to 25 W or less. In conclusion, toxicokinetic modeling confirms the significant impact of work load on toluene exposure indicators, whereas only a very slight effect is noted on n-hexane kinetics. These results highlight the necessity of taking work load into account in risk assessment relative to toluene exposure.

Inhalant abuse detection and evaluation in young Tunisians.

Occupational exposure biological monitoring techniques were applied for the diagnosis of inhalation abuse and for the evaluation of the levels of exposure to benzene, toluene, ethylbenzene, xylenes, and n-hexane, in 44 Tunisian adolescents and children suspected for volatile substance addiction. Urinary trans,trans-muconic acid, hippuric acid (HA), mandelic acid, and methylhippuric acids determinations were performed by high performance liquid chromatography with a photodiode array detector, and urinary o-cresol (o-Cr) and 2,5-hexanedione (HD) were extracted simultaneously and measured using gas chromatography with a flame ionization detector. Given the high linearity ranges, HD and o-Cr occupational exposure monitoring techniques could be applied without modification. However, urinary sample dilution was necessary before HA analysis. Concentrations were compared with the maxima of normal values (MNVs) in the general population and to the biological exposure indices (BEIs) used in occupational toxicology. Values as high as 6610-fold the MNV and 68 times the BEI were registered. The subjects showed high exposure to toluene and hexane. Measured metabolites HA and/or o-Cr and HD enabled the easy detection and evaluation of exposure levels. The problem of inhalant abuse should be given more attention and treated through an effective prevention strategy.

Microwave-assisted derivatization of 2,5-hexanedione in urine: evaluation using GC-MS and GC-ECD.

2,5-Hexanedione, the main metabolite of n-hexane, can be responsible for axonal degeneration symptoms via formation of pyrrol-adducts with several amino acids. In order to make it amenable to gas chromatographic analysis, a protocol including microwave assisted derivatization is presented and compared to state-of-the-art technique of urine analysis. The applied methodology includes derivatization with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine, extraction of the oximes and final analysis using either GC-MS or GC-muECD. Furthermore, the mass spectra of derivatized 2,5-hexanedione and 5-hydroxy-2-hexanone as well as preliminary excretion kinetics are provided. Orthogonal regression methodology demonstrated superior sensitivity for the microwave heating. Limits of detection were calculated to be approximately 20 ng mL(-1) with both MS and electron capture detection, the decompositon of excess derivatizing agent using sulfuric acid, following the reaction is beneficial. A matrix effect caused by urine was not observed, a calibration in aqueous matrix ensures accurate results therefore. Microwave heating yields excellent results regarding recovery, sensitivity and the time needed for sample preparation, furthermore, it is demonstrated that both mass selective as well as electron capture detection are of comparable suitability for this task.

Evaluation of 2,5-hexanedione in urine of workers exposed to n-hexane in Brazilian shoe factories.

Urinary 2,5-hexanedione (2,5-HD) is used as a biomarker for biological monitoring of workers exposed to n-hexane. The purpose of this study was to compare two types of treatment of urine samples during clean-up (with and without acidic hydrolysis) and to study the exposure situation of workers exposed to n-hexane during shoe manufacturing. There, various glues containing n-hexane are used. Quantification of 2,5-HD was carried out by gas chromatography and flame ionization detection (GC-FID). Fifty-two urine samples taken from workers of seven shoe factories were analyzed. Thirty-four persons from the administrative staff of the same factories served as controls. They were not known to be exposed to n-hexane. The samples treated with acidic hydrolysis showed levels (average 0.94 mg/l) approximately 10 times higher than samples without acidic hydrolysis (0.09 mg/l). The difference is predominantly caused by the conversion of other metabolites of n-hexane (e.g. 4,5-dihydroxy-2-hexanone) to 2,5-HD in the presence of acids. Our results also show, that exposure to n-hexane is different between various industries. Levels of 2,5-HD in urine are predominantly dependent on the type of operation (how the glue is applied on the leather during shoe manufacturing). Simple measures, e.g. using a glue handgun instead of a paintbrush significantly decreased exposure to n-hexane.

Free and total 2,5-hexanedione in biological monitoring of workers exposed to n-hexane in the shoe industry.

OBJECTIVES: To analyse the role of total 2,5-hexanedione (2,5-HD) compared with free 2,5-HD as a biological indicator of exposure to n-hexane at work. METHODS: One-hundred and thirty two workers in contact with this solvent during their occupation in the shoe industry in the province of Alicante (Spain) were studied. Environmental and biological tests were carried out analysing variations of the concentration of the metabolite in urine corresponding to different working conditions. Environmental exposure was evaluated in each work place using active personal monitors and measured by gas chromatography (GC). Dichloromethane extracts of the urine samples collected at the end of the working shifts were analysed, before (determining free 2,5-HD, the toxic metabolite) and after acid hydrolysis (pH 0.1) (yielding the total 2,5-HD) and also by GC. The concentration of conjugated metabolite 4,5-dihydroxy-2-hexanone was calculated from the difference between total and free 2,5-HD. RESULTS: Free 2,5-HD represented an average of 14.2% of the total 2,5-HD determined in urine, and this percentage increased significantly (P<0.01) with higher environmental levels of acetone. Other factors, such as absorption through the skin (depending on the use of gloves) and the day on which samples were taken also significantly affected the relation between the two indicators and their respective relationships with environmental concentrations of n-hexane. CONCLUSION: Although analyses of the relationship between the levels of atmospheric n-hexane and those of metabolites in urine show a greater correlation for total 2,5-HD than for free 2,5-HD, our results suggest that free 2,5-HD could be a better indicator in evaluating risk of exposure to n-hexane, since the concentration is directly related to the neurotoxic effect.

Determination of free and glucuronated hexane metabolites without prior hydrolysis by liquid- and gas-chromatography coupled with mass spectrometry.

Since n-hexane metabolites are excreted as glucuronide conjugates, most conventional analytical procedures require preliminary hydrolysis, yielding to the 'total' 2,5-hexanedione (2,5-HD), but also giving rise to a number of artifacts. The whole pattern of n-hexane metabolites, both conjugated and unconjugated, as well as different methods of sample pretreatment have been evaluated by hyphenated techniques (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS)). Aliquots of urine from rats exposed to n-hexane underwent enzymatic or acid hydrolysis or both; whereas one aliquot was applied to LC-MS, dichloromethane extracts were analyzed by GC-MS. In untreated urine, four glucuronides (-G) were identified and characterized by LC-MS: 2-hexanol-G, 5-hydroxy-2-hexanone-G, 4,5-dyhydroxy-2-hexanone-G, and 2,5-hexanediol-G. 'Free' 2,5-HD was detectable in non-hydrolyzed samples by both GC- and LC-MS. Whereas enzymatic hydrolysis did not increase the amount of 2,5-HD, acid hydrolysis led to increase 2,5-HD in variable amount and produced gamma-valerolactone as a result of a complete transformation of 4,5-dihydroxy-2-hexanone-G and the partial conversion from 5-hydroxy-2-hexanone-G. Further experiments showed that both 5-hydroxy-2-hexanone-G and 4,5-dihydroxy-2-hexanone-G, isolated by solid-phase extraction and hydrolyzed, yield comparable amount of 2,5-HD and gamma-valerolactone. In samples treated by acid hydrolysis, GC-MS only does not allow to understand the true source of 'total' 2,5-HD, which may be produced not only from 4,5-dihydroxy-2-hexanone-G but also from the more abundant 5-hydroxy-2-hexanone-G, which thus represents the main source of analytical artifacts. 'Free' 2,5-HD seems to be both suitable from an analytical point of view and meaningful for biological monitoring purposes, provided that conjugate metabolites are rapidly removed from the body leading to a negligible neurotoxic risk.

Urinalysis vs. blood analysis, as a tool for biological monitoring of solvent exposure.

Blood and urine samples were collected at the end of an 8-h workshift from 30 male workers exposed to a mixture of n-hexane, ethyl acetate and toluene (each being about 2 ppm as geometric means) and also from 20 nonexposed male workers. Blood samples were analyzed for n-hexane and toluene, and urine samples were analyzed for n-hexane, toluene, 2,5-hexanedione (both with and without hydrolysis) and hippuric acid. Based on the correlation between biological exposure indicators and solvent concentrations in air, sensitivity as an exposure indicator was compared between solvents in blood and solvents or metabolites in urine in terms of the lowest solvent concentration at which the exposed subjects can be statistically separated from the nonexposed. Both n-hexane and toluene in blood were sensitive enough to detect the exposure at 6.1 ppm and 1.4 ppm, respectively. n-Hexane exposure below 2 ppm was detectable also by urinalysis for 2,5-hexadione without hydrolysis. Urinary hippuric acid, however, failed to detect low toluene exposure under the conditions studied. Of additional interest is the fact that toluene in urine correlated significantly with toluene in air, which apparently deserves further study for confirmation.

Interaction of zinc on biomarker responses in rats exposed to 2,5-hexanedione by two routes of exposure.

The interaction of zinc(II) on the toxicokinetics of 2,5-hexanedione (2,5-HD), the ultimate toxic metabolite of n-hexane, was performed by quantifying the changes of two urinary biomarkers, free 2,5-HD and pyrrole derivatives, in rats exposed to 2,5-HD and to 2,5-HD plus zinc acetate. Eight groups of Wistar rats were exposed for 4 days (dietary and intraperitoneally) to 2,5-HD, zinc acetate and 2,5-HD plus zinc acetate and the 24 h urine was used to determine the excretion of these biomarkers. On comparing the results obtained by the two routes of exposure with different doses of 2,5-HD and zinc acetate, it was observed that there was a significant decrease (P<0.05) in the excretion of free 2,5-HD and pyrroles derivatives in rats exposed to the chemical mixture, when compared with the excretion of these biomarkers in rats exposed to 2,5-HD alone. To evaluate the mechanism of this interaction, further experiments were performed using one group of rat dietary pre-exposed to zinc acetate followed by 2,5-HD exposure. The results of our experiment suggest that zinc protect proteins of pyrrolization by coordination to amino groups, with the subsequent inhibition of protein cross-linking responsible by 2,5-HD neurotoxicity.

Large-scale biological monitoring in Japan.

Data from the large-scale biological monitoring program in Japan were assembled and analyzed and the following results were obtained. All workers handling lead and eight kinds of major organic solvents received physical examinations and biological monitoring at the same time. Therefore, the number of workers handling industrial chemicals and that received physical examinations and the number of workers been examined by biological monitorings were similar to each other. The total number of cases examined from 1989 to 1994 was about 661,000 for lead in the blood and about 4,173,000 for the urinary metabolites of eight organic solvents. The results were classified into three categories and category 3 consists of workers having exposure concentrations above the 1988-1989 biological exposure indices of the ACGIH with the exception of lead concentration in the blood where the limit in Japan was set at 40 micrograms/100 ml. The percentage of exposed workers in category 3 was 1.4% for blood lead and 0.2-2.4% for the urinary metabolites of the eight organic solvents. The percentage of exposed workers in category 3 for blood lead, delta-aminolevulinic acid, urinary mandelic acid, N-methylformamide and 2,5-hexanedione in the urine has decreased with time. In ambient monitoring, the percentage of workplaces in classification 3 for lead and styrene also has decreased with time.

The metabolism of n-nonane in male Fischer 344 rats.

The urinary metabolites of n-nonane in male Fischer 344 rats, after administering the hydrocarbon by gavage, included gamma-valerolactone, delta-hexanolactone, 2,5-hexanedione, delta-heptanolactone, 1-heptanol, 2-nonanol, 3-nonanol, 4-nonanol, 4-nonanone and 5-methyl-2-(3-oxobutyl)furan. Metabolism strongly favored the formation of monoalcohols and lactones, which are the products of appropriately substituted hydroxy carboxylic acids. The metabolites were identified using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). High pressure liquid chromatography (HPLC) permitted the detection of the dicarboxylic acids malonic acid and glutaric acid in the n-nonane dosed rat urines.

Gas chromatographic determination of 2,5-hexanedione in urine as an indicator of exposure to n-hexane.

A sensitive and specific method for determination of 2,5-hexanedione in urine is described. Treatment of the urine specimen directly with n-butylamine yields n-butyl 2,5-dimethyl pyrrole. The latter is extracted into diisopropylether and analyzed by gas chromatography (GC) with flame ionization detection (FID), thermionic specific detection (TSD) (N mode), or mass spectrometric detection (MS). The minimum detectable quantities are 1 mg/L urine when employing FID with a coefficient of variation of less than 6%. Recovery of 2,5-hexanedione added to the urine at the level of 10 mg/L was 78.9%.

Human polymorphonuclear leukocyte chemotaxis as a tool in detecting biological early effects in workers occupationally exposed to low levels of n-hexane.

Human polymorphonuclear leukocytes (PMN) were chosen to measure two cellular end points--chemotaxis and respiratory burst--and to verify whether they could function as biomarkers of early effect in detecting occupational exposure to n-hexane of apparently healthy shoe workers, without any electroneuromyographic (ENMG) abnormality. Chemotaxis, but not respiratory burst, was found to be impaired. A negative linear correlation between chemotaxis of PMN of those workers that had been exposed to n-hexane versus 2,5-hexanedione (2,5-HD) urinary concentrations were found. This negative trend is consistent with our previous in vitro experimental findings: it was observed that the progressive addition of 2,5-HD to PMN suspensions inhibited chemotaxis in a dose-dependent mode, while chemiluminescence was not modified. Now we have confirmed in vivo that chemotaxis is more sensitive than the respiratory burst response to 2,5-HD. Such results justify the interest in the behaviour of PMN harvested from workers exposed to n-hexane. Since significant inhibition of chemotactic activity was observed in some workers whose urinary 2,5-HD levels were lower than 5 mg l-1, which is the biological exposure index suggested by ACGIH, this study suggests that PMN chemotaxis may be proposed as a useful biomarker in detecting occupational exposure to low level of n-hexane.

Computerized posturography with sway frequency analysis: application in occupational and environmental health.

To examine the effects of occupational and environmental neurotoxicants on vestibular, cerebellar and spinocerebellar functions, the following three groups of subjects were examined, using a computerized posturography with sway frequency analysis: (1) 49 male chemical factory workers exposed to lead stearate, aged 27-63 (mean 43) years, with concurrent blood lead concentrations (BPbs) of 11-113 (mean 48) microg/100 g and past mean BPbs of 7-52 (mean 24) microg/100 g; (2) 29 male sandal, shoe and leather factory workers, aged 35-73 (mean 51) years, with urinary 2,5-hexanedione (HD) concentrations of 0.41-3.06 (mean 1.20) mg/g creatinine; and (3) 9 females, aged 19-58 (mean 29) years, who were exposed to sarin accidentally 6-8 months before the study (Tokyo Subway Sarin Poisoning, March 20,1995) and showed plasma cholinesterase (ChE) activities of 13-95 (mean 68) IU/l on the day of poisoning. The pattern of posturographic changes in lead workers suggested that the vestibulocerebellum (lower vermis), anterior cerebellar lobe and spinocerebellar afferent pathway were asymptomatically affected; the vestibulocerebellar change reflected concurrent lead absorption and the anterior cerebellar one reflected past absorption. Similarly, vestibulocerebellar and spinocerebellar functions were affected by n-hexane in solvent workers; the effect on the vestibulocerebellar function was probably inhibited by xylene. Also, the chronic (long-term) effect on the vestibulocerebellar function persisted in acute sarin poisoning. It is thus suggested that the vesitibulocerebellar function is most sensitive to all the three chemicals examined. It appears that the computerized posturography with frequency analysis is a useful technique for assessment of vestibular, cerebellar and spinocerebellar effects in occupational and environmental health.

FIOH external quality assurance scheme for organic solvent metabolites.

The scheme consists of analyses for phenol,2,5-hexanedione, and mandelic, trichloroacetic and methylhippuric acids in urine. The present participants are 31 laboratories from 14 countries. Samples are prepared by pooling urine obtained from occupationally exposed workers or by spiking with appropriate pure metabolites. Four sets of samples at two concentration levels for each analyte are distributed annually. The report includes information on the arithmetic means, standard deviations and CVs for overall results and separately for different methods. During the last three years, the CVs have varied rather non systematically, being 21-31% for mandelic acid, 24-26% for trichloroacetic acid, 24-35% for phenol, 55-110% for 2,5-hexanedione and 44-50% for methylhippuric acid.

[Quality control in a system for the biological surveillance of exposure to n-hexane]. / Verifica di qualità in un sistema di sorveglianza biologica dell'esposizione a n-esano.

The paper reports the results of a quality assurance programme for routine biological monitoring of n-hexane exposure via measurement of its urinary metabolite 2.5-hexanedione. The programme involved a number of local occupational health services and was coordinated by the industrial toxicology laboratory in Florence. The analytical results show good agreement (68.6%, i.e., 46/67) between high urinary levels of 2.5-hexanedione (equal to or higher than an action level of 3.2 mg/l) and a relatively poor environmental situation at the workplace as reported by the local occupational health services. The fall in the number of 2.5-hexanedione values equal to or higher than the action level in the course of the study period (1990-1991) could be a result of the prevention measures taken. To confirm this hypothesis, 69 enterprises with at least 3 determinations in both 1990 and 1991 were selected. A 58% decrease in the mean 2.5-hexanedione value between 1990 and 1992 was observed in the group of enterprises (24) with at least one sentinel health event in 1990 (a value equal to or higher than 3.2 mg/l). In the group of enterprises with no sentinel health event in 1990, the mean 2.5-hexanedione values were practically unchanged. The authors stress the need for further studies to test the hypotheses based on the data obtained in this study.