The method of choice for the determination of 2,5-hexanedione as an indicator of occupational exposure to n-hexane

Summary To identify the method of choice for analysis of urine for 2,5-hexanedione (2,5-HD) as an indicator of occupational exposure to n-hexane, the end-of-shift urine samples of 36 n-hexane exposed male workers and 30 non-exposed male workers were analyzed for 2,5-HD under three conditions of hydrolysis, i.e. enzymic hydrolysis at pH 4.8, acid hydrolysis at pH 0.5, and without hydrolysis. The 2,5-HD concentrations thus determined were examined for correlation with 8-h, time-weighted average exposure concentrations of n-hexane measured by diffusive sampling. The regression analysis showed that the 2,5-HD concentrations without any hydrolysis correlated best with the intensity of exposure to n-hexane. No 2,5-HD was detected in the urine of the non-exposed subjects under the analytical conditions with no hydrolysis. Thus, the analysis without hydrolysis was considered to be the method of choice from the viewpoint of simplicity in analytical procedures, sensitive separation of the exposed from the non-exposed, and quantitative increase in the amount of 2,5-HD after n-hexane exposure.A part of this work was presented at the 63rd Annual Meeting of Japan Association of Industrial Health, held in Kumamoto, Japan, on 3rd–6th April, 1990     

Behaviour of urinary 2,5-hexanedione in occupational co-exposure to n-hexane and acetone

We analysed the relationship between free 2,5-hexanedione (2,5-HD) and total 2,5-HD in the urine of 87 workers exposed to n-hexane and other solvents (hexane isomers, acetone and toluene), in relation to different working conditions. The concentration of free 2,5-HD in urine of workers exposed to n-hexane was about 12% of total urinary 2,5-HD. The most significant correlation (r = 0.936) was that of total 2,5-HD in urine with environmental n-hexane and exhaled air. With equal exposure to n-hexane, the concentrations in urine of free and total 2,5-HD increased when cutaneous absorption was involved (gloves not used), during the working week and with co-exposure to acetone. An analysis of the relationship between combined exposure to acetone and urinary concentrations of the various forms of 2,5-HD suggests that acetone might influence the toxicokinetics of n-hexane, increasing the proportion of free 2,5-HD.     

2-Acetylfuran,a confounder in urinalysis for 2,5-hexanedione as an n-hexane exposure indicator

Summary The apparent amount of 2,5-hexanedione, a biomarker of n-hexane exposure in occupational health, in the urine of both exposed and non-exposed subjects varied not only as a function of the pH at which the urine sample was hydrolyzed but also depending on the capillary column used for gas chromatographic (GC) analysis of the urinary hydrolyzates after extraction with dichloromethane. The formation of a compound, identified by gas chromatography-mass spectrometry (GC-MS) as 2-acetylfuran, following acid hydrolysis was a major cause of confounding effects. This compound was hardly separated from 2,5-hexanedione on a capillary column such as DB-WAX, whereas separation could be achieved on a DB-1 capillary column. 2-Acetylfuran was formed when a urine sample was heated at a pH of < 2 for hydrolysis, and the amount detected in urine did not differ between exposed and non-exposed subjects, indicating that the formation of 2-acetylfuran is independent of n-hexane exposure. When urinary hydrolysis is used, hydrolysis at a pH of < 0.5, extraction with dichloromethane, and GC analysis on a non-polar capillary column are proposed to be the best analytical conditions for 2,5-hexanedione analysis in biological monitoring of exposure to n-hexane.     

Biological monitoring of occupational exposure to n-hexane by exhaled air analysis and urinalysis

Summary To compare two methods of biological monitoring for the evaluation of risk of occupational exposure to n-hexane, we analyzed the relationship between environmental exposure to this solvent and urinary excretion of 2,5-hexanedione and n-hexane in exhaled air in 69 workers employed in the shoe industry. Environmental exposure to the solvent was monitored with personal diffusive samplers, which were desorbed with carbon sulfide and analyzed by gas chromatography. To measure 2,5-hexanedione, urine was subjected to acid hydrolysis, separation in octadecyl silane columns, elution with 5% aqueous acetonitrile solution and extraction with dichloromethane, followed by gas chromatography. In exhaled air, n-hexane was measured with a sampling system that permitted concentration of aliquots of end-exhaled air (alveolar air) from one or more exhalations in a tube packed with activated charcoal, which was then desorbed with carbon sulfide and analyzed by gas chromatography. Concentrations of n-hexane in breathing zone air were significantly correlated with urinary concentrations of 2,5-hexanedione (r = 0.88) and with exhaled air n-hexane (r = 0.86); in addition, the two biological indicators correlated significantly (r = 0.70). Analyses in both exhaled air and urine were thus considered useful for biological monitoring of the risk of exposure to n-hexane.     

Biochemical and physiological aspects of 2,5-hexanedione: endogenous or exogenous product?

Summary This article reports results regarding two different physiological aspects of 2,5-hexanedione (2,5-HD). The first is the relationship between free 2,5-HD (the fraction of real 2,5-HD) and total 2,5-HD (2,5-HD obtained from acid hydrolysis) in urine and blood of workers exposed ton-hexane. The second part of the study is an attempt to clarify physiological excretion of 2,5-HD in subjects not occupationally exposed ton-hexane. The concentration of free 2,5-HD in urine of workers exposed ton-hexane is about 8% of total urinary 2,5-HD. In blood, free 2,5-HD is about 50% of the total. The serum concentration range of total and free 2,5-HD in workers from whom blood was taken was 33–418 g/l and 14–283 g/l respectively. In subjects not exposed ton-hexane, urinary concentration of 2,5-HD ranged between 0.17 and 0.98 mg/1, the urinary excretion rate between 0.23 and 0.57g/min, and renal clearance between 14 and 66 ml/min. The blood concentration of 2,5-HD in nonexposed subjects was 6–30g/1. Fluctuations typical of a circadian rhythm were not observed for 2,5-HD in blood or urine. We think that 2,5-HD is mainly a product of intermediate metabolism in the human body. Only a minimal part could derive fromn-hexane as a ubiquitous micropollutant.     

Toxicokinetic study of pyrrole adducts and its potential application for biological monitoring of 2,5-hexanedione subacute exposure

Purpose

The formation of pyrrole adducts might be responsible for peripheral nerve injury caused by n-hexane, but there is not an effective biomarker for monitoring occupational exposure of n-hexane. The current study was designed to investigate the changes of pyrrole adducts in serum and urine of rats exposed to 2,5-hexanedione (2,5-HD) and analyze the correlation between pyrrole adducts and 2,5-HD.

Methods

Two groups of male Wistar rats (n = 8) were administered a single dose of 200 and 400 mg/kg 2,5-HD (i.p.), and another two groups (n = 8) were given daily dose of 200 and 400 mg/kg 2,5-HD (i.p.) for 5 days. Pyrrole adducts and 2,5-HD in serum and urine were determined, at different time points after dosing, using Ehrlich’s reagent and gas chromatography, respectively.

Results

The levels of pyrrole adducts in serum accumulated in a time-dependant manner after repeated exposure to 2,5-HD, while pyrrole adducts in urine, and 2,5-HD in serum and urine were kept stable. The half-life times (t _1/2) of 2,5-HD and pyrrole adducts in serum were 2.27 ± 0.28 and 25.3 ± 3.34 h, respectively. Furthermore, the levels of pyrrole adducts in urine were significantly correlated with the levels of 2,5-HD in serum (r = 0.736, P < 0.001) and urine (r = 0.730, P < 0.001), and the levels of pyrrole adducts in serum were correlated with the cumulative dosage of 2,5-HD (r = 0.965, P < 0.001).

Conclusion

The results suggested that pyrrole adducts in serum and urine might be markers of chronic exposure to n-hexane or 2,5-HD.     

Biological monitoring of occupational exposure ton-hexane by measurement of urinary 2,5-hexanedione

Summary Occupational exposure ton-hexane in shoe factory workers was monitored by measuring urinary 2,5-hexanedione, the major metabolite of this solvent and the probable cause of peripheral neuropathy in exposed workers. Solvent pollution was monitored in the work environments of 189 employees, of whom 123 (65%) worked in Alicante, Spain, and 66 (35%) in Veneto, Italy. 2,5-Hexanedione was measured in spot urine samples collected from workers at the end of the shift. Information on working conditions was obtained from a previous study. A significant linear correlation was found between mean environmental concentration ofn-hexane and urinary concentration of 2,5-hexanedione. The variability in the correlation may have been due to the variable use of protective clothing (gloves), and to variations in exposure during the working week. In numerous workers, percutaneous absorption ofn-hexane represented as much as 50% of the total absorbed dose. Urinary concentrations of 2,5-hexanedione tended to increase during the working week. Simultaneous exposure ton-hexane and toluene tended to reduce urinary excretion of 2,5-hexanedione, whereas exposure ton-hexane and methyl ethyl ketone tended to increase excretion of the metabolite.     

Urinary 2,5-dichlorophenol as biological index for p-dichlorobenzene exposure in the general population

The relationship between exposure to p-dichlorobenzene (p-DCB) and urinary excretion of 2,5-dichlorophenol (2,5-DCP), the major metabolite of p-DCB, was examined to evaluate the usefulness of the metabolite as a biological index for low-level exposure of p-DCB in the general population. Personal exposure concentrations of p-DCB and concentrations of 2,5-DCP excreted in the urine of 119 adults living in Osaka were determined. Airborne p-DCB was collected for 24 h by passive gas sampling tubes packed with charcoal. The tubes were exchanged every 12 h. The sampling was started immediately after the subject woke up in the morning (7 A.M.). The collected p-DCB was desorbed with toluene and measured using a gas chromatograph with an electron capture detector (GC-ECD). On the other hand, the first morning urine samples were collected at the endpoint of airborne p-DCB sampling (7 A.M. the next morning). The urine samples were hydrolyzed with concentrated sulfuric acid. 2,5-DCP in the hydrolysates was extracted with n-hexane and measured by GC-ECD.Both p-DCB and 2,5-DCP were detected in more than 99% of the air and urine samples, respectively, from the participants. The median of p-DCB exposure concentrations for 24 h was 2.5 ppb, with a maximum concentration of 33.3 ppb. The median of urinary 2,5-DCP concentrations was 0.39 mg/g creatinine, with the maximum concentration of 3.32 mg/g creatinine. The regression line between the urinary 2,5-DCP concentration (y) and the p-DCB exposure concentration (x) was y = 0.080 x + 0.181, with the Pearson correlation coefficient of 0.81 (p < 0.001), demonstrating a strong association between these measurements. Consequently, urinary 2,5-DCP should be suitable as an index for monitoring low-level exposure of p-DCB in the general population.     

Urinary 2,5-Dichlorophenol as Biological Index for p-Dichlorobenzene Exposure in the General Population

The relationship between exposure to p-dichlorobenzene (p-DCB) and urinary excretion of 2,5-dichlorophenol (2,5-DCP), the major metabolite of p-DCB, was examined to evaluate the usefulness of the metabolite as a biological index for low-level exposure of p-DCB in the general population. Personal exposure concentrations of p-DCB and concentrations of 2,5-DCP excreted in the urine of 119 adults living in Osaka were determined. Airborne p-DCB was collected for 24 h by passive gas sampling tubes packed with charcoal. The tubes were exchanged every 12 h. The sampling was started immediately after the subject woke up in the morning (7 A.M.). The collected p-DCB was desorbed with toluene and measured using a gas chromatograph with an electron capture detector (GC-ECD). On the other hand, the first morning urine samples were collected at the endpoint of airborne p-DCB sampling (7 A.M. the next morning). The urine samples were hydrolyzed with concentrated sulfuric acid. 2,5-DCP in the hydrolysates was extracted with n-hexane and measured by GC-ECD. Both p-DCB and 2,5-DCP were detected in more than 99% of the air and urine samples, respectively, from the participants. The median of p-DCB exposure concentrations for 24 h was 2.5 ppb, with a maximum concentration of 33.3 ppb. The median of urinary 2,5-DCP concentrations was 0.39 mg/g creatinine, with the maximum concentration of 3.32 mg/g creatinine. The regression line between the urinary 2,5-DCP concentration (y) and the p-DCB exposure concentration (x) was y = 0.080 x + 0.181, with the Pearson correlation coefficient of 0.81 (p < 0.001), demonstrating a strong association between these measurements. Consequently, urinary 2,5-DCP should be suitable as an index for monitoring low-level exposure of p-DCB in the general population. Received: 25 October 2001/Accepted: 4 March 2002     

Comparison of unchanged n-hexane in alveolar air and 2,5-hexanedione in urine for the biological monitoring of n-hexane exposure in human volunteers

Introduction and aim Biological monitoring of n-hexane (HEX) is based on the measurement of urinary 2,5-hexanedione (2,5-HD). In 2001, the American Conference of Governmental Industrial Hygienists modified the biological exposure index (BEI) for HEX and suggested measuring free urinary 2,5-HD (without hydrolysis) (3.5 mol/l) instead of total 2,5-HD (acid hydrolysis). This BEI value was derived from four field studies that involved worker exposures to variable concentrations of HEX and other solvents. This study was undertaken to characterize, for 5 consecutive days, the relationship between HEX exposure (25 ppm and 50 ppm) and (1) 2,5-HD urinary excretion and (2) HEX in alveolar air.Methods Five volunteers (three women, two men) were exposed to HEX in an exposure chamber for 2 non-consecutive weeks (7 h/day). They were exposed to 50 ppm HEX, during the first week and to 25 ppm during the second week. Alveolar air and urine samples were collected at different intervals before, during and after the exposures. The concentration of unchanged HEX in alveolar air and the concentration of urinary 2,5-HD under three analytical conditions (with acid, or enzymatic hydrolysis and without hydrolysis) were measured.Results The results show that the mean concentrations of HEX in alveolar air were 18 ppm (25 ppm) and 37 ppm (50 ppm), which indicates that approximately 73% of inspired HEX was expired unchanged in alveolar air by the volunteers. The mean (± SD) concentrations of urinary 2,5-HD for the last 4 h of exposure at the end of the week (day 5) following exposure to 50 ppm HEX were 30.4 mol/l (±7.8 mol/l) (acid hydrolysis); 5.8 mol/l (±1.0 mol/l) (enzymatic hydrolysis); 6.2 mol/l (±0.9 µmol/l) (without hydrolysis). Following the volunteers exposure to 25 ppm HEX, the urinary excretion concentrations were 15.2 mol/l ± 1.9 mol/l, 3.1 mol/l ± 0.7 mol/l and 3.7 mol/l ± 0.5 mol/l, respectively.Conclusion Both free urinary 2,5-HD and HEX in alveolar air measurements could be used for the biological monitoring of HEX. Between these two indicators, HEX in alveolar air is less variable than 2,5-HD in urine, but the sampling time is more critical. Therefore, biological monitoring of HEX based on the measurement of free urinary 2,5-HD is preferable to HEX in alveolar air. Additionally, we believe that the 2,5-HD values reported in this study better reflect the actual levels of exposure to HEX alone than what has been previously reported in studies that involved co-exposure to other solvents, and that the current BEI value for HEX is most likely more protective than what has been believed up until now.     

Urinary 2,5-hexanedione increases with potentiation of neurotoxicity in chronic coexposure to n-hexane and methyl ethyl ketone

Objective: MEK (methyl ethyl ketone) is widely and frequently used as an ingredient of mixed solvents together with n-hexane. MEK is known to decrease urinary levels of 2,5-hexanedione dose-dependently in an acute or chronic coexposure with a constant level of n-hexane. This change in urinary 2,5-hexanedione appears to contradict the potentiation effect of MEK on n-hexane-induced neurotoxicity because it is believed that the toxicity of n-hexane is activated through n-hexane metabolism. We aimed to clarify how the urinary level of 2,5-hexanedione changes when MEK modifies the degree of n-hexane-induced neurotoxicity. Method: A total of 32 male Wistar rats were divided into 4 groups of 8 each and were then exposed to fresh air only, 2000 ppm n-hexane only, 2000 ppm n-hexane plus 200 ppm MEK, and 2000 ppm n-hexane plus 2000 ppm MEK, respectively. Inhalation exposures were performed 12 h/day, 6 days/week, for 20 weeks. Motor-nerve conduction velocity (MCV), distal latency (DL), and urinary 2,5-hexanedione were measured every 4 weeks. Results: The MCV decreased, the DL increased, and urinary levels of 2,5-hexanedione increased in the 2000-ppm n-hexane plus 2000 ppm MEK group in comparison with the 2000-ppm n-hexane only group following 4 weeks' exposure. On the 1st day of exposure, however, coexposure to MEK decreased urinary levels of 2,5-hexanedione dose-dependently. Conclusions: The present study showed that urinary concentrations of 2,5-hexanedione increased with potentiation of n-hexane neurotoxicity. Urinary 2,5-hexanedione concentration does not necessarily reflect the exposure concentration of n-hexane in coexposure to n-hexane along with MEK or other solvents, but it may be useful as a marker in the assessment of neurotoxicity in coexposure to n-hexane and other solvents. Received: 21 March 1997 / Accepted: 10 July 1997     

Urinary 2,5-hexanedione assay involving its conversion to 2,5-dimethylpyrrole

Summary High-performance liquid chromatography (HPLC), gas chromatography (GC) and spectrophotometry were used to examine 2,5-dimethylpyrrole, derived from 2,5-hexanedione in the acid-hydrolyzed urine of subjects exposed to n-hexane. The urine of a subject exposed to n-hexane was hydrolyzed with hydrochloric acid and then neutralized. Thereafter, 2,5-hexanedione in the hydrolysate was heated with ammonium carbonate to yield 2,5-dimethylpyrrole. A methanol extract of the 2,5-dimethylpyrrole formed was subjected to HPLC and an ethylacetate extract of it was subjected to GC, with a flame thermoionic detector (FTD). For spectrophotometry, the ethylacetate extract was allowed to react with Ehrlich's reagent and then the absorbance of the colored compound formed was read at 525 nm. Pyrrole condensation with ammonia is specific to 1,4-diketones and these three assay methods can be used for determination of 2,5-hexanedione in the urine of workers exposed to n-hexane. The hydrolytic conditions for urine of subjects exposed to n-hexane for analysis of 2,5-hexanedione were discussed.     

Ambient monitoring and biomonitoring of workers exposed to N-methyl-2-pyrrolidone in an industrial facility

Objectives: The exposure of seven workers and three on-site study examiners to N-methyl-2-pyrrolidone (NMP) was studied in an adhesive bonding compound and glue production facility. Methods: Airborne NMP was analysed by personal and stationary sampling on activated charcoal tubes. NMP and its main metabolites, 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI), were analysed in pre-shift and post-shift spot urine samples by gas chromatography-mass spectrometry. The workers were examined with respect to irritation of the eyes, the mucous membranes and the skin, and health complaints before and after the work-shift were recorded. Results: The time-weighted average concentration of NMP in most work areas varied between 0.2 and 3.0 mg/m³. During the manual cleaning of stirring vessels, valves and tools, 8-h TWA exposures of up to 15.5 mg/m³ and single peak exposures of up to 85 mg/m³ were observed. NMP and its metabolites were detected in two pre-shift urine specimens. NMP and 5-HNMP concentrations in post-shift urine samples of five workers and three on-site study examiners were below 125 μg/g creatinine and 15 mg/g creatinine, respectively, while two vessel-cleaning workers showed significantly higher urinary NMP concentrations of 472 and 711 μg/g creatinine and 5-HNMP concentrations of 33.5 and 124 mg/g creatinine. 2-HMSI was detectable in four post-shift samples (range: 1.6–14.7 mg/g creatinine). The vessel cleaner with the highest NMP exposure reported irritation of the eyes, the upper respiratory tract and headaches. Conclusions: The results of this study indicate a relatively low overall exposure to NMP in the facility. An increased uptake of NMP occurred only during extensive manual vessel cleaning. Health complaints associated with NMP exposure were recorded in one case and might be related to an excessive dermal exposure due to infrequent and inadequate use of personal protective equipment.     

Comparative evaluation of blood and urine analysis as a tool for biological monitoring of n-hexane and toluene

Blood and urine samples were collected from 57 male Japanese solvent workers [exposed to n-hexane (Hex-A), ethyl acetate, and toluene (Tol-A) at 1.5, 2.3, and 2.3 ppm as GM-TWA, respectively] and also from 20 male nonexposed workers at the end of a 8-h shift, and analyzed for n-hexane (Hex-B) and toluene (Tol-B) in blood, and n-hexane (Hex-U), toluene (Tol-U), 2,5-hexanedione [both with (HD-U/cHYD) and without hydrolysis (HD-U/sHYD)] and hippuric acid (HA-U) in urine. Regression analysis showed that both Hex-B and Tol-B correlated significantly with corresponding exposure to the solvents. Solvents in urine (Hex-U and Tol-U) also correlated with solvents in air but with smaller correlation coefficients than the solvents in blood. Both HD-U/cHYD and HD-U/sHYD showed significant correlation with Hex-A, but HA-U failed to do so with Tol-A. Based on the correlation among biological exposure indicators and solvent concentration in air, sensitivity as an exposure indicator was compared between the solvent in blood and the metabolite in urine in terms of the lowest solvent concentration at which the exposed can be separated (with statistical significance) from the nonexposed (the lowest separation concentration; LSC). The LSC was 3.9 ppm for Hex-B, 1 to 2 ppm for HD-U/sHYD and 10 to 30 ppm for HD-U/cHYD, suggesting that HD-U/sHYD is superior even to Hex-B in detecting low n-hexane exposure; this high sensitivity of HD-U/sHYD is due to the absence of HD-U/sHYD in the urine from the nonexposed. In contrast, Tol-B (with LSC of 2.4 ppm) was more sensitive than HA-U; no LSC for HA-U could be obtained because of lack of correlation with Tol-A at low toluene exposure.     

Ubiquitous pollution by n-hexane and reference biological levels in the general population

Summary n-Hexane levels were determined by gas chromatography and mass spectrometry in environmental air and in the alveolar air, blood and urine of a group of subjects aged on average of 38 years who had not been occupationally exposed to this hydrocarbon. n-Hexane was found in all environmental air samples examined (n=49), with the mean concentration being 104 ng/l (limit values, 1–279 ng/l). It was also found in all 49 samples of alveolar air, with the mean concentration being 50 ng/l (variation limit, 1–304ng/l). In 64 samples of urine, n-hexane was found in only 50 samples, with the mean concentration being 1,417 ng/l (limit values, 34–8,820 ng/l). In 77 of the 90 blood samples taken, a mean concentration of 608 ng/l was detected (variation limit, 15–7,684ng/l). Particularly the haematic and urinary concentration showed significant differences among the nine groups of individuals classified according to their work activity. The lowest levels were found in the blood and urine of farmers: 270 and 298 ng/l, respectively. The highest values were found for chemical workers (1,377 and 411 ng/1), respectively printers (585 and 2,691 ng/l respectively), and traffic wardens (740 and 8,820 ng/l, respectively). In all, 95% of the determinations of n-hexane yielded values of < 255 ng/l in environmental samples, < 105 ng/l in alveolar air, < 1,475 ng/l in blood and < 5,875 ng/l in urine. A comparison of these data revealed a significant correlation between environmental levels and alveolar (r/s = 0.769; P<0.00001), haematic (r/s = 0.624; P<0.0002), and urinary (r/s = 0.597; P<0.0005) values for n-hexane.     

Neurotoxicity of petroleum benzine compared with n-hexane

Summary Petroleum benzine is one of the mixtures of organic solvents containing n-hexane. The occurrence of polyneuropathy in the workers using petroleum benzines is attributed mainly to n-hexane, though other hydrocarbons present are also suspected of having some neurotoxicity or some potential which could modify the neurotoxicity of n-hexane. The present experiment was performed in order to clarify the toxicity of petroleum benzine to the peripheral nerve and compare it with that of n-hexane.Forty rats were randomly divided into five groups. The groups were exposed to 200 ppm n-hexane, 500 ppm n-hexane, and petroleum benzine vapor containing 200 ppm n-hexane or 500 ppm n-hexane, together with aliphatic and aromatic hydrocarbons for 12 h a day for 24 weeks. The body weight, motor nerve conduction velocity, motor distal latency, and mixed nerve conduction velocities were measured before exposure and every 4 weeks of exposure. A rat from each exposed group was histopathologically examined after 24 weeks' exposure.The function of the peripheral nerve was conspicuously impaired by 500 ppm n-hexane, slightly impaired by 200 ppm n-hexane and petroleum benzine containing 500 ppm n-hexane, and even less impaired by petroleum benzine containing 200 ppm n-hexane. Degenerations of the myelin sheaths and axons were demonstrated in all exposed groups upon examination of the raveled tail nerves. Thus, the experiment revealed that petroleum benzine could impair the peripheral nerves, while some components of petroleum benzine were considered to inhibit the neurotoxicity of n-hexane.This investigation was partly supported by a grant for scientific research from the Chiyoda Mutual Life Foundation in 1980–1981     

Urinary excretion of phenols as an indicator of occupational exposure in the coke-plant industry

Objective: In the present study the relationship between the level of exposure to o-cresol and of 2,4- +2,5-, 3,4-, and 3,5-xylenols and the urinary excretion of their metabolites was examined. The mixed exposure to phenolic derivatives of exposed workers during their work shift was monitored by personal air sampling of the breathing-zone air and by measurements of phenol, o-cresol, and xylenol isomer concentrations in shift-end urine. Methods: The study subjects were 76 men working at a coke plant who were 22–58 years old and 34 nonexposed subjects. Concentrations of phenolic compounds were determined in the breathing-zone air during the work shift, whereas concentrations of phenol, cresol, and xylenol isomers were measured in urine collected after the work shift. Concentrations of phenols in air and urine were determined by gas chromatography with flame-ionization detection. Urine samples were extracted after acid hydrolysis of glucuronides and sulfates by solid-phase extraction. The gas chromatography-mass spectrometry method was applied to identify metabolites in urine samples. Results: The time-weighted average concentrations of phenol, cresol, and xylenol isomers detected in breathing-zone air showed that the exposure level of the workers was relatively low. The geometric mean values were as follows: 0.26 mg/m³ for phenol, 0.09 mg/m³ for o-cresol, 0.13 mg/m³ for p- and m-cresol, and 0.02–0.04 mg/m³ for xylenols at the tar-distillation process. Corresponding urinary concentrations were 10.39, 0.53, and 0.25–0.88 mg/g creatinine for phenol, o-cresol, and xylenol isomers, respectively. The correlation coefficients between the o-cresol and 2,4-, 2,5-, 3,4-, and 3,5-xylenol concentrations measured in urine and in the breathing-zone air were statistically significant, varying in the range of 0.54–0.74 for xylenol isomers and being 0.69 for o-cresol. Conclusion: We have found that the presence of o-cresol and xylenol isomers in urine can be used as a biomarker for phenol exposure. Analysis performed on workers at the tar-distillation process showed that they were exposed to relatively low concentrations of phenolic compounds. Received: 15 October 1996 / Accepted: 5 May 1997     

Interlaboratory quality control and status of n-hexane biological monitoring in Japan

This paper reports the problem of interlaboratory quality control and the actual condition of biological monitoring of n-hexane among the seven major laboratories specializing in clinical chemistry tests, where more than 80% of the total urine specimens in Japan have been analyzed after the beginning of biological monitoring of n-hexane in 1989. First, transportation conditions of urine specimens and transportation effects on measurement results were studied. The seven major laboratories carried urine specimens at distances of more than 450 km from the University of Occupational and Environmental Health (UOEH) to the respective laboratories and had at least one transit terminal where specimens from various areas were collected and transferred to the analytical laboratories. All laboratories used cooler boxes with dry ice or ice bars and stored the specimens in freezers or refrigerators. Specimens arrived at the final destinations within 24 hours. The transportation from institutes for health examination to the laboratories did not affect the measurement results of 2,5-hexanedione (HD), a determinant of n-hexane biological monitoring. Interlaboratory cross-checks were performed among the seven major laboratories and four institutes for occupational health examination that have their own analytical laboratories. All of the laboratories analyzed HD by acid hydrolysis (pH<1.0); interlaboratory differences were recognized at the first cross-check. After some laboratories intensified gas chromatography (GC) maintenance and changed to a new column, the interlaboratory variation lessened. However, the utilization of a common standard could not diminish the interlaboratory variation because the interlaboratory coefficient of variation (CV) was almost the same as that of daily reproducibility. Therefore, daily maintenance and careful measurement in each laboratory was important to improve quality control. The minor differences of analytical method, that is, standard, calibration, pre-treatment, extraction, internal standard and gas chromatography, did not cause systematic errors. The seven major laboratories have been measuring 26,000 to 32,000 specimens every six months since April 1990. Throughout the survey periods, over 98% of the examined urine had HD levels of 0–2.0 mg/L. Generally, the number of specimens with >5.0 mg/L HD decreased as the examination progressed in time. From the latest data, only 24 cases (0.08%) of the total 30,306 determinations exceeded 5.0 mg/L. The total tendency was the gradual improvement of urinary HD concentration with time.     

Metabolic interaction between n-hexane and toluene in vivo and in vitro

Summary Metabolic interference between n-hexane and toluene was studied both in vivo and in vitro. In in vivo experiments the urinary excretion of n-hexane and toluene metabolites was tested in rats treated with the two solvents separately or in combination. The same experimental program was repeated in rats pretreated with phenobarbital (PB). The urinary excretion of n-hexane metabolites in rats treated with the two solvents showed a significantly decreased excretion of all n-hexane metabolites in comparison with those treated with n-hexane alone. In rats pretreated with PB the excretion of n-hexane metabolites was significantly higher compared with that of unpretreated rats; the combined administration of the two solvents showed in this case, too, that n-hexane metabolite excretion was less than that found in rats treated with n-hexane alone. The biotransformation of toluene to o-cresol and hippuric acid studied in the urine of rats treated with or without n-hexane and pretreated or not with PB did not show any difference. The in vitro metabolic interference was studied by measuring the disappearance of solvents from rat's incubated liver microsomes. The maximum velocity (Vmax) of n-hexane was 2.8 nmol/g/min when incubated alone, 1.9 and 0.9 nmol/g/min when incubated with 5 and 20 M of toluene respectively. The Vmax of toluene was 14.9 nmol/g/min when incubated alone and 13.1 and 10.5 nmol/g/min when incubated with 10.4 and 20.9 M of n-hexane respectively. The inhibition constant (Ki) of toluene on n-hexane biotransformation was 7.5 M and that of n-hexane on toluene was 30 M. The data show that a mutual non-competitive interference exists in vitro betweeen n-hexane and toluene. The interference of toluene on n-hexane biotransformation was detectable also in vivo experiments, while n-hexane did not modify the biotransformation of toluene.     

Effects of methyl ethyl ketone, acetone, or toluene coadministration on 2,5-hexanedione concentration in the sciatic nerve, serum, and urine of rats

Objective: To clarify changes in the serum, nerve, and urinary levels of 2,5-hexanedione (2,5-HD) in rats on coadministration with methyl ethyl ketone (MEK), acetone (AC), and toluene (TO). Method: 2,5-HD alone or combined with MEK, AC, and TO was injected subcutaneously into a total of 306 male Wistar rats. The rats were divided as follows into 7 groups: (1) 2.6 mmol/kg 2,5-HD alone (HD) and (2) 2.6 mmol/kg 2,5-HD combined with 2.6 mmol/kg MEK (HD + MEK), (3) with 2.6 mmol/kg AC (HD + AC), (4) with 2.6 mmol/kg TO (HD + TO), (5) with 13.0 mmol/kg MEK (HD + 5MEK), (6) with 13.0 mmol/kg AC (HD + 5AC), and (7) with 13.0 mmol/kg TO (HD + 5TO). 2,5-HD concentrations in the serum, sciatic nerve, and urine of rats were determined within 16 h of the injections and pharmacokinetic parameters were estimated. Results: It was observed that (1) the 2,5-HD concentration and AUC value (area under concentration versus time curve) determined in the serum and nerve increased significantly in the cotreated groups as compared with the HD group; (2) the effect MEK had in elevating the 2,5-HD concentration and AUC in the serum and nerve was stronger than that of AC, and the effect AC had was stronger than that of TO; (3) a dose increase from 2.6 to 13.0 mmol/kg for MEK and AC induced further increases in the 2,5-HD concentration and AUC determined in the serum and nerve; (4) elimination constants recorded for 2,5-HD (K _e) from the serum and nerve decreased in all the cotreated groups, and the degree of the decrease correlated inversely with the elevation in 2,5-HD concentration and AUC in the serum and nerve; and (5) urinary 2,5-HD concentrations measured in the 13.0-mmol/kg cotreated groups increased in parallel with the elevation in serum 2,5-HD concentrations. Conclusion: Coadministration of 2,5-HD with MEK, AC, or TO can increase the concentration and AUC of 2,5-HD in serum and the sciatic nerve, and these increases can be further enhanced by an increase in the concomitant doses of MEK and AC. Received: 13 August 1997 / Accepted: 27 November 1997