Physiologicomathematical model for studying human exposure to organic solvents: kinetics of blood/tissue n-hexane concentrations and of 2,5-hexanedione in urine.

The physiologicomathematical model with eight compartments described allows the simulation of the absorbtion, distribution, biotransformation, excretion of an organic solvent, and the kinetics of its metabolites. The usual compartments of the human organism (vessel rich group, muscle group, and fat group) are integrated with the lungs, the metabolising tissues, and three other compartments dealing with the metabolic kinetics (biotransformation, water, and urinary compartments). The findings obtained by mathematical simulation of exposure to n-hexane were compared with data previously reported. The concentrations of n-hexane in alveolar air and in venous blood described both in experimental and occupational exposures provided a substantial validation for the data obtained by mathematical simulation. The results of the urinary excretion of 2,5-hexanedione given by the model were in good agreement with data already reported. The simulation of an exposure to n-hexane repeated five days a week suggested that the solvent accumulates in the fat tissue. The half life of n-hexane in fat tissue equalled 64 hours. The kinetics of 2,5-hexanedione resulting from the model suggest that occupational exposure results in the presence of large amounts of 2,5-hexanedione in the body for the whole working week.     

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.     

Dose-dependent increase in 2,5-hexanedione in the urine of workers exposed to n-hexane

Summary The concentrations of 2,5-hexanedione (2,5-HD), an n-hexane metabolite, and 2-acetylfuran (2-AF) were measured in urine samples from 123 workers who had predominantly been exposed to n-hexane vapor and 53 workers who had experienced no exposure to solvents. The time-weighted average intensity of exposure to n-hexane vapor was determined by a diffusive sampling method. For biological monitoring of exposure, urine samples were collected late in the afternoon during the second half of a working week and were analyzed in the presence and absence of acid hydrolysis (at pH < 0.5) for 2,5-HD and 2-AF by gas chromatography on a non-polar capillary DB-1 column. The urinary 2,5-HD concentration increased as a linear function of the intensity of exposure to n-hexane, showing a correlation coefficient of 0.64–0.77 after acid hydrolysis and that of 0.730–0.83 in the absence of hydrolysis, depending on the correction for urinary density (P < 0.01 in all cases, with no improvement in the coefficient occurring after the corrections). In contrast, 2-AF levels were independent of n-hexane exposure. The geometric mean 2,5-HD concentration in urine samples from 53 nonexposed men was 0.26 mg/l as observed (i.e., with no correction), 0.19 mg/l after correction for a urinary specific gravity of 1.016, and 0.23 mg/g creatinine after correction for creatinine concentration, and the geometric standard deviation was approximately 2.     

Detection of 2,5-hexanedione in the urine of persons not exposed to n-hexane

Summary The neurotoxic n-hexane metabolite 2,5-hexanedione was detected by gas chromatography—mass spectrometry in urine samples from human subjects not exposed to n-hexane or related hydrocarbons. Quantitative determinations by multiple ion detection revealed an amount of 2,5-hexanedione ranging from 0.12 to 0.78 mg/l (0.45 ± 0.20 mg/l; The amounts of 2,5-hexanedione actually measured in urinary extracts depended on the pH employed for acid hydrolysis of the samples.     

Physiologically based modeling of n-hexane kinetics in humans following inhalation exposure at rest and under physical exertion: impact on free 2,5-hexanedione in urine and on n-hexane in alveolar air

We used a modified physiologically based pharmacokinetic (PBPK) to describe/predict n-hexane (HEX) alveolar air concentrations and free 2,5-HD urinary concentrations in humans exposed to n-HEX by inhalation during a typical workweek. The effect of an increase in workload intensity on these two exposure indicators was assessed and, using Monte Carlo simulation, the impact of biological variability was investigated. The model predicted HEX alveolar air concentrations at rest of 19.0 ppm (25 ppm exposure) and 38.7 ppm (50 ppm exposure) at the end of the last working day (day 5), while free 2,5-HD urinary concentrations of 3.4 micromol/L (25 ppm) and 6.3 micromol/L (50 ppm) were predicted for the same period (last 4.5 hours of Day 5). Monte Carlo simulations showed that the range of values expected to occur in a group of 1000 individuals exposed to 50 ppm of HEX (95% confidence interval) for free 2,5-HD (1.7-14.7 micromol/L) is much higher compared with alveolar air HEX (33.4-46 ppm). Simulations of exposure at 50 ppm with different workloads predicted that an increase in workload intensity would not greatly affect both indicators studied. However, the alveolar air HEX concentration is more sensitive to modifications of workload intensity and time of sampling, after the end of exposure, compared with 2,5-HD. The PBPK model successfully described the HEX alveolar air concentrations and free 2,5-HD urinary concentrations measured in human volunteers and is the first, to our knowledge, to describe the excretion kinetics of free 2,5-HD in humans over a 5-day period.     

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.     

"Dynamic" biological exposure indexes for n-hexane and 2,5-hexanedione, suggested by a physiologically based pharmacokinetic model

Biological exposure index (BEI) of n-hexane was studied for accuracy using a physiologically based pharmacokinetic (PB-PK) model. The kinetics of n-hexane in alveolar air, blood, urine, and other tissues were simulated for different values of alveolar ventilations and also for constant and variable exposures. The kinetics of 2,5-hexanedione, the toxic n-hexane metabolite, were also simulated. The ranges of n-hexane concentrations in biological media and the urinary concentrations of 2,5-hexanedione are discussed in connection with a mean n-hexane exposure of 180 mg/m3 (50 ppm) (threshold limit value [TLV] suggested by American Conference of Governmental Industrial Hygienists [ACGIH] for 1988-89). The experimental and field data as well as those predicted by simulation with the PB-PK model were comparable. The physiological-pharmacokinetic simulations are used to propose the "dynamic" BEIs of n-hexane and 2,5-hexanedione. The use of simulation with PB-PK models enables a better understanding of the limits, advantages, and issues associated with biological monitoring of exposures to industrial solvents.     

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.     

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     

大蒜油对正己烷在小鼠体内代谢成2,5.己二酮的影响

目的 探讨大蒜油对正己烷(n-hexane)在小鼠体内代谢成2,5.己二酮(2,5.hexanedione,2,5-HD)的影响.方法 取健康成年昆明种小鼠,随机分为正己烷染毒组和大蒜油干预组,分别灌胃给予正己烷和大蒜油,染毒结束后取血,分离血清经乙酸乙酯萃取后气相色谱法测定血清中2,5-HD含量.结果 (1)一次性给予4000 mg/kg正己烷后,小鼠血清中2,5-HD含量随时间延长而增加,10 h后达峰值,20 h后几乎不能检出;(2)对照组小鼠血清中未检出2,5-HD;随着正己烷染毒量的增加,小鼠血清中2,5-HD含量明显增加,2000、4000和6000 mg/kg组染毒8 h后小鼠血清2,5-HD含量分别为8.04、16.68和22.38μg/ml,呈明显的剂量一效应关系;(3)不同周龄小鼠给予正己烷8 h后血清中2,5-HD含量有明显差异,5周龄组(22.83;μg/ml)分别大于4周(19.59μg/ml)和6周龄组(16.42μg/ml),差异有统计学意义(P<0.05);(4)不同性别小鼠给予同剂量正己烷后,雌性小鼠血清中2,5-HD含量(13.22 t,g/na)高于雄鼠(10.34μg/ml),差异有统计学意义P<0.05;(5)正己烷染毒前后2 h分别给予80 mg/kg大蒜油,血清中2,5-HD含量与单纯染毒组相比均明显降低,差异有统计学意义(P<0.05),但染毒前给药组较染毒后给药组更低,但差异无统计学意义(P>0.05);染毒前2 h给予不同剂量大蒜油,10、20、40、80 mg/kg大蒜油+3 000 mg/kS正已烷组小鼠血清2,5-HD含量比染毒组分别降低16.2%、20.8%、22.8%和32.1%,差异有统计学意义(P<0.05,P<0.01),呈明显的剂量一效应关系.结论 在雌性小鼠血清中正己烷的代谢产物2,5-HD高于雄性;5周龄小鼠血清2,5-HD含量较4周和6周龄小鼠高;正己烷染毒前后给予大蒜油可明显减少2,5-HD的生成.     

Determination of urinary 2,5-hexanedione concentration by an improved analytical method as an index of exposure to n-hexane

2,5-Hexanedione is a main metabolite of n-hexane and is considered as the cause of n-hexane polyneuropathy. Therefore, it is useful to measure 2,5-hexanedione for biological monitoring of exposure to n-hexane. The analytical methods existing for n-hexane metabolites, however, were controversial and not established enough. Hence, a simple and precise method for determination of urinary 2,5-hexanedione has been developed. Five ml of urine was acidified to pH 0.5 with concentrated hydrochloric acid and heated for 30 minutes at 90-100 degrees C. After cooling in water, sodium chloride and dichloromethane containing internal standard were added. The sample was shaken and centrifuged. 2,5-Hexanedione concentration in an aliquot of dichloromethane extract was quantified by gas chromatography using a widebore column (DB-1701). Urinary concentration of 2,5-hexanedione showed a good correlation with exposure to n-hexane (n = 50, r = 0.973, p less than 0.001). This method is simple and precise for analysis of urinary 2,5-hexanedione as an index of exposure to n-hexane.     

Determination of urinary 2,5-hexanedione concentration by an improved analytical method as an index of exposure to n-hexane.

2,5-Hexanedione is a main metabolite of n-hexane and is considered as the cause of n-hexane polyneuropathy. Therefore, it is useful to measure 2,5-hexanedione for biological monitoring of exposure to n-hexane. The analytical methods existing for n-hexane metabolites, however, were controversial and not established enough. Hence, a simple and precise method for determination of urinary 2,5-hexanedione has been developed. Five ml of urine was acidified to pH 0.5 with concentrated hydrochloric acid and heated for 30 minutes at 90-100 degrees C. After cooling in water, sodium chloride and dichloromethane containing internal standard were added. The sample was shaken and centrifuged. 2,5-Hexanedione concentration in an aliquot of dichloromethane extract was quantified by gas chromatography using a widebore column (DB-1701). Urinary concentration of 2,5-hexanedione showed a good correlation with exposure to n-hexane (n = 50, r = 0.973, p less than 0.001). This method is simple and precise for analysis of urinary 2,5-hexanedione as an index of exposure to n-hexane.     

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     

Comparative evaluation of urinalysis and blood analysis as means of detecting exposure to organic solvents at low concentrations

Summary One hundred and forty-three workers exposed to one or more of toluene, xylene, ethylbenzene, styrene, n-hexane, and methanol at sub-occupational exposure limits were examined for the time-weighted average intensity of exposure by diffusive sampling, and for biological exposure indicators by means of analysis of shift-end blood for the solvent and analysis of shift-end urine for the corresponding metabolite(s). Urinalysis was also performed in 20 nonexposed control men to establish the background level. Both solvent concentrations in blood and metabolite concentrations in urine correlated significantly with solvent concentrations in air. Comparison of blood analysis and urinalysis as regards sensitivity in identifying low solvent exposure showed that blood analysis is generally superior to urinalysis. It was also noted that estimation of exposure intensity on an individual basis is scarcely possible even with blood analysis. Solvent concentration in whole blood was the same as that in serum in the case of the aromatics, except for styrene. It was higher in blood than in serum in the case of n-hexane, and lower in the cases of styrene and methanol.     

Gases and organic solvents in urine as biomarkers of occupational exposure: a review

A brief review of urine analysis in studies of occupational exposure to volatile organic compounds and gases is provided. Analysis of exhaled breath for volatile compounds does not have a long history in occupational medicine. A number of studies has been undertaken since the 1980s, and the methods are well enough accepted to be put forward as biological equivalents of threshold limit values (TLVs) for some volatile organic compounds (VOCs) such as acetone; methanol; methyl ethyl ketone (MEK); methyl isobutyl ketone (MIBK); tetrahydrofurane; dichloromethane. In the last 20 years many scientific articles have shown that the urinary concentrations of unchanged solvents are correlated with environmental exposure and could be used for biological monitoring. The use of urine analysis of unchanged solvents in occupational applications is not yet widespread. Nonetheless, in the short time since its application, a number of important discoveries has been made, and the future appears bright for this branch of analysis. In this paper, the basic concepts and methodology of urine analysis are briefly presented with a critical revision of the literature on this matter. The excretion mechanisms of organic solvents in urine are discussed, with regard to biological variability, and the future directions of research are described.     

Breath analysis by API/MS—human exposure to volatile organic solvents

Summary The expired breath of subjects, exposed for periods of ca. 90 min to atmospheres artificially contaminated with low levels of methanol, (ca. 100 ppm) toluene (ca. 50 ppm) or tetrachloroethylene, (ca. 50 ppm) was monitored during and after the exposure period using an atmospheric pressure ionization mass spectrometer, fitted with a direct breath analysis system. The retention of solvent by the subjects, estimated from steady state levels in the expired breath, averaged 82% of the inspired level for methanol, 83% for toluene and 87% for tetrachloroethylene. The elimination of unchanged solvent via respiration during the post exposure period followed first order kinetics with mean half life values of 24 min for methanol, 27 min for toluene and 79 min for tetrachloroethylene.     

Comparison of breath,blood and urine concentrations in the biomonitoring of environmental exposure to 1,3-butadiene, 2,5-dimethylfuran,and benzene

OBJECTIVES: To investigate and compare alveolar, blood, and urine concentrations of 1,3-butadiene, 2,5 dimethylfuran, and benzene, in non-occupational exposure to these products. METHODS: Benzene, 2,5-dimethylfuran and 1,3-butadiene were measured in the breath, blood, and urine samples of 61 subjects living in small mountain villages. All 61 were regularly employed as forestry workers. Sampling was done during the long winter-season non-working period. Samples were collected after overnight rest and analysed by headspace and GC-mass spectrometry methods. RESULTS: The median 1,3-butadiene level was 1.2 ng/l (range: <0.8-13.2 ng/l) in alveolar air, 2.2 ng/l (range: <0.5-50.2 ng/l) in blood, and 1.1 ng/l (range: <1-8.9 ng/l) in urine. The median benzene level was 5.7 ng/l (range: <1-24.9 ng/l) in alveolar air, 62.3 ng/l (range: 33.5-487.2 ng/l) in blood, and 63.4 ng/l (range: 25.8-1099.1 ng/l) in urine. The median 2,5-dimethylfuran level was 0.5 ng/l (range: <1-12.5 ng/l) in alveolar air, 2.5 ng/l (range: <5-372.9 ng/l) in blood, and 51.8 ng/l (range: <5-524.9 ng/l) in urine. In several cases, 2,5-dimethylfuran levels were below the detection limit in alveolar air and blood, especially in non-smokers. 1,3-Butadiene, 2,5-dimethylfuran and benzene levels were significantly higher in smokers than non-smokers in all biological media. CONCLUSIONS: 1,3-Butadiene and benzene, as ubiquitous pollutants, are detectable and quantifiable in human alveolar air, blood and urine. 2,5-Dimethylfuran, which is not a usual environmental pollutant, is almost always detectable in biological media, but only in smokers.     

2,5-己二酮对大鼠坐骨神经P0和NF表达影响

目的观察2,5-己二酮(2,5-HD)暴露对大鼠坐骨神经组织超微结构及P₀(myelin protein zero)和神经丝(neurofilament, NF)表达影响, 探讨它们之间的关联性。方法成年雄性SD大鼠40只, 随机分成对照组(生理盐水)和低、中、高剂量染毒组(100、200、400 mg/kg腹腔注射2,5-HD), 每组10只, 每周连续5 d, 1次/d。染毒5周后处死动物, 取坐骨神经, 电镜观察形态学改变, RT-PCR 和 Western blot 检测 P₀、NF-L、NF-M 和 NF-H 基因和蛋白表达水平。结果随染毒剂量升高, 大鼠坐骨神经髓鞘形状渐不规则, 并出现内折现象;轴索形状渐不规则, 部分发生萎缩;神经丝数量渐少, 排列稀疏。与对照组比较, 400 mg/kg 2,5-HD 组大鼠坐骨神经 P₀ mRNA 表达量(0.75±0.03)、NF-L、NF-M 和 NF-H mRNA 表达量[分别为(0.35±0.07)、(0.25±0.04)、(0.37±0.05)]明显下降(P<0.05);与对照组比较, 400 mg/kg 2,5-HD 组大鼠坐骨神经 P₀ 蛋白表达量(0.79±0.04)、NF-L、NF-M 和 NF-H 蛋白表达量[分别为(0.26±0.02)、(0.12±0.03)、(0.22±0.02)]明显降低(P<0.05)。结论亚慢性 2,5-HD 暴露导致的外周神经病变可能与 P₀ 和 NF 表达异常有关, 这些蛋白可能是 2,5-HD 攻击外周神经系统的靶点。