Biotransformation and Kinetics of Excretion of tert-Amyl-methyl Ether in Humans and Rats after Inhalation Exposure

Alexander Amberg, Elisabeth Rosner and Wolfgang Dekant1

Institut für Toxikologie, Universität Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany

Received November 16, 1999; accepted February 4, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
tert-Amyl methyl ether (TAME) may be widely used as an additive to gasoline in the future. The presence of this ether in gasoline reduces the tail pipe emission of pollutants. Therefore, widespread human exposure to TAME may occur. To contribute to the characterization of potential adverse effects of TAME, its biotransformation was compared in humans and rats after inhalation exposure. Human volunteers (three males and three females) and rats (five males and five females) were exposed to 4 (3.8 ± 0.2) and 40 (38.4 ± 1.7) ppm TAME for 4 h in a dynamic exposure system. Urine samples were collected for 72 h in 6-h intervals and blood samples were taken at regular intervals for 48 h in humans. In urine, the TAME metabolites tert-amyl alcohol (t-amyl alcohol), 2-methyl-2,3-butane diol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid were quantified. TAME and t-amyl alcohol were determined in blood samples. After the end of the exposure period, blood concentrations of TAME were 4.4 ± 1.7 µM in humans and 9.6 ± 1.4 µM in rats after 40 ppm TAME, and 0.6 ± 0.1 µM in humans and 1.4 ± 0.8 µM in rats after 4 ppm. TAME was rapidly cleared from blood in both rats and humans. The blood concentrations of t-amyl alcohol were 9.2 ± 1.8 µM in humans and 8.1 ± 1.5 µM in rats after 40 ppm TAME, and 1.0 ± 0.3 µM in humans and 1.8 ± 0.2 µM in rats after 4 ppm TAME. t-Amyl alcohol was also rapidly cleared from blood. In urine of humans, 2-methyl-2,3-butane diol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid were recovered as major excretory products in urine. In rats, 2-methyl-2,3-butane diol and its glucuronide were major TAME metabolites. t-Amyl alcohol and its glucuronide were minor TAME metabolites in both species. All metabolites of TAME excreted with urine in rats were rapidly eliminated, with elimination half-lives of less than 6 h. Metabolite excretion in humans was slower and elimination half-lives of the different metabolites were between 6 and 40 h in humans. The obtained data indicate differences in TAME biotransformation and excretion between rats and humans. In rats, TAME metabolites are rapidly excreted. In humans, metabolic pathways are different and metabolite excretion is slower. Recovery of TAME metabolites in urine was higher in humans as compared to rats, suggesting more intensive biotransformation of TAME in humans.

Key Words: biotransformation; F344 NH rats; humans; inhalation exposure; tert-Amyl methyl ether (TAME).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased oxygen content in gasoline is required by the 1990 Amendments to the Clean Air Act in certain areas of the United States that fail to meet the National Ambient Air Quality standard for carbon monoxide or ozone. The chemicals blended with gasoline hydrocarbons to meet the required oxygen content are referred to as oxygenates (Costantini, 1993Go). At present, the oxygenates most often used are methyl tert-butyl ether and methanol. However, other ethers are also used or considered for use. tert-Amyl methyl ether (TAME) is a well-suited oxygenate due to its relatively low production costs, lower vapor pressure than other ethers, and its ability to act as a high-octane gasoline-blending compound (HEI, 1996; Vainiotalo et al., 1998Go).

Due to the potential widespread exposure of humans to oxygenates in fuel (HEI, 1996; Vainiotalo et al., 1999Go), studies on the toxicity of oxygenates including TAME are underway. The major toxic effect seen in rodents after inhalation of high concentrations of TAME is central nervous system depression. Subchronic inhalation studies with TAME for 4 weeks (6 h per day, 5 days per week) showed significantly reduced body weights and relative increases in adrenal, kidney, testes, brain, and lung weights in male rats exposed to 4000 ppm TAME, but no treatment-related histopathologic findings (White et al., 1995Go). The toxicity of the initial metabolite formed from TAME, tert-amyl alcohol (t-amyl alcohol), is also low. In rats exposed to concentrations of up to 1000 ppm TAME for 90 days by inhalation, increased liver weights in male rats were the only effect observed (Nolan et al., 1976Go, 1981Go).

The biotransformation of TAME has been studied in rats and humans, and metabolites have been identified (Fig. 1Go). Free and glucuronidated 2-methyl-2,3-butane diol and a glucuronide of t-amyl alcohol were major urinary metabolites excreted in rats; 2-hydroxy-2-methyl-butyric acid and 3-hydroxy-3-methyl-butyric acid were minor excretory products formed from TAME. In humans, 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid were major excretory products in addition to 2-methyl-2,3-butane diol (Amberg et al., 1999aGo). These results suggest further intensive biotransformation of t-amyl alcohol by metabolic oxidation reactions.



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FIG. 1. Biotransformation of TAME in mammals. Metabolites found in urine are underlined. 1, TAME; 2, t-amyl alcohol; 3, 2-methyl-2,4-butane diol; 4, 3-hydroxy-3-methyl-butyric acid; 5, 2-methyl-2,3-butane diol; 6, glucuronide of 2-methyl-2,3-butane diol; 7, 2-methyl-1,2-butane diol; 8, 2-hydroxy-2-methyl-butyric acid; 9, glucuronide of t-amyl alcohol.

 
In this work, we studied the uptake of TAME and quantified excretion of metabolites in humans and rats after exposure to TAME by inhalation to obtain data on blood levels, extent of metabolism in both species, and kinetics of metabolite excretion.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
TAME (97+% purity), t-amyl alcohol (99+% purity), 2-hydroxy-2-methylbutyric acid, (98+% purity), tert-butanol (99.5+% purity), 1,2-propane diol (99.5+ purity), 2-hydroxyvaleric acid (98+% purity), D-glucuronic acid (98+ purity), boron trifluoride methanol-solution (14%), and P2O5 (97% purity) were obtained from Sigma-Aldrich Chemical Company (Deisenhofen, Germany). 2-Methyl-2,3-butane diol was prepared as described (Amberg et al., 1999aGo). 3-Hydroxy-3-methylbutyric acid was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan), hexamethyldisilazane (98% purity), trimethylchlorosilane solution (1M in tetrahydrofuran), and pyridine were obtained from Fluka Chemie AG (Deisenhofen, Germany). All other reagents and solvents were reagent grade or better and obtained from several commercial suppliers. All GC columns were obtained from J&W Scientific (Folsom, CA).

Exposure of volunteers to TAME.
Three healthy female and three healthy male volunteers (Table 1Go) were exposed to targeted concentrations of 4 and 40 ppm TAME for 4 h in a dynamic exposure chamber (Ertle et al., 1972Go). TAME concentrations in chamber air were determined at 15-min intervals by GC/MS. Actual TAME concentrations were 3.8 ± 0.2 and 38.4 ± 1.7 ppm. The volunteers had to refrain from alcoholic beverages and drugs 2 days before and throughout each experiment. Subjects did not abuse alcohol and were nonsmokers or occasional smokers. Subjects were healthy as judged by medical examination and clinical blood chemistry, stated no previous occupational exposure to TAME, and did not refuel their cars during the 2 days prior to exposure and during sample collection period. Exposures started at 8 am. The study was carried out according to the Declaration of Helsinki, after approval by the Regional Ethical Committee of the University of Wuerzburg, Germany, and after written informed consent by the volunteers. A time interval of 4 weeks was kept between the two exposures. No significant differences in temperature in the chamber, number of air exchanges, and relative humidity were observed between the exposures.


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TABLE 1 Characteristics of Human Volunteers Participating in the Study
 
The design of the chamber and the generation of the chemical/air mixtures has been described previously (Ertle et al., 1972Go; Müller et al., 1972Go, 1974Go, 1975Go). The chamber had a total volume of 8 m3, airflow rate was 28 m3/h at a temperature of 22°C, and a relative humidity of 50–60 %. After the exposure, the urine of the volunteers was collected at fixed intervals for 72 h, urine volumes were determined by the volunteers, and two aliquots (60 ml each) were rapidly frozen after collection and stored at –20°C until sample preparation. Metabolite concentrations in each urine sample collected were determined in duplicate.

Exposure of rats to TAME.
Five male (210–230 g, 12 weeks of age) and five female (190–210 g, 12 weeks of age) F344 NH rats from Harlan-Winkelmann (Borchen, Germany) were exposed to targeted concentrations of 4 and 40 ppm TAME in the exposure chamber as described above for human volunteers. During the exposure, rats were kept separately in Macrolon® cages with free access to food and water. After the end of the exposure, the cages were checked for urine and feces, the animals were transferred to metabolic cages, and urine was collected on ice for 72 h in 6-h intervals. Blood samples from the tail vein (100 µl) were taken from each rat after the end of the exposure period to quantify TAME and t-amyl alcohol blood concentrations.

Quantification of TAME concentration in the exposure chamber.
Samples (50 µl) of the chamber air were taken every 15 min with a gas-tight syringe. TAME in the atmosphere of the exposure chamber was quantified by capillary gas chromatography using a Fisons 8000 gas chromatograph coupled to a Fisons MD 800 mass spectrometer. Separation was performed with a DB-1 fused silica column (30 m, 0.25 mm I.D., film thickness 1 µm) at an oven temperature of 35°C. Injector temperature was 150°C and detector temperature 200°C; split injection with a split ratio of 5:1. During the separation (run time of 5 min), the intensity of the major fragment ion in the electron impact mass spectrum of TAME (m/z = 73) was monitored, with a dwell time of 80 msec. Quantitation was based on calibration curves obtained with metered TAME concentrations.

Quantitation of TAME and t-amyl alcohol in blood.
Blood samples (10 ml) from the volunteers were taken with heparinized syringes. Volumes for blood samples from rats were 100 µl. Blood samples from humans (0.5 ml) and rats (0.025 ml) were transferred into GC autosampler vials (2-ml volume for human samples and 0.2-ml volume for rat blood samples) immediately after blood sampling to avoid loss of the volatile analytes during manipulations. The vials were capped and stored at –20°C for a maximum of 4 weeks.

For TAME and t-amyl alcohol quantitation, 5 µl of an aqueous solution of the internal standard tert-butanol (1000 nmol/ml) was added through the septum with a microliter syringe, and the vials were then heated to 70°C for 1 h. TAME and t-amyl alcohol-concentrations were quantified by headspace GC/MS by injecting 200 µl of the headspace from the vials using split injection (split ratio of 10:1). Samples were separated using a DB-1 coated fused silica column (30 m x 0.25 mm ID, 1.0-µm film) at a temperature of 40°C. In addition to monitoring m/z 73 (for TAME), m/z 59 (most intensive fragment ion in the electron impact mass spectrum of t-amyl alcohol and the internal standard t-butanol) were monitored during the separation, with dwell times of 80 msec. Quantitation was performed relative to the content of t-butanol and referenced to calibration curves with fortified aliquots of blood samples from controls containing 0–20 nmol TAME and 0–20 nmol t. amyl alcohol/ml blood. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 10–20 samples) The method permitted the quantitation of 0.1 nmol TAME and 0.2 nmol t-amyl alcohol/ml of blood with a signal-to-noise ratio of 5:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 10% (n = 8). TAME and t-amyl alcohol concentrations reported in blood samples are based on duplicate analysis of samples from every individual.

Quantitation of TAME and TAME metabolites in urine.
TAME and free t-amyl alcohol in urine samples were quantified by headspace GC/MS using 0.5 ml of human urine and 0.2 ml of rat urine. TAME and t-amyl alcohol in the urine samples was quantified as described above for blood samples. Two different methods were used to quantify t-amyl alcohol and 2-methyl-2,3-butane diol glucuronides. The first method involves direct analysis of the glucuronides by GC/MS-determination of trimethylsilyl derivatives. To quantify glucuronide excretion, 50 µl of a 1000-nmol/ml solution of the internal standard glucuronic acid were added to 100 µl of human or rat urine, and the mixtures were lyophilized. The obtained residues were treated for 30 min with 1 ml of a mixture of hexamethyldisilazane, trimethylchlorosilane solution, and pyridine (2:1:9, v:v:v) at 80°C in a closed reaction vial. From the obtained solution, 2 µl were injected into the GC/MS. Separation was performed using a DB-1 coated fused silica column (30 m x 0.25 mm ID, 1-µm film). Injector temperature was 310°C, and transfer line temperature was 310°C. Samples were injected using split injection (split ratio of 10:1); oven temperature was 100°C and increased to 310°C with a rate of 10°C/min. Samples were monitored using m/z 204 and 217. Quantitation was performed relative to the content of glucuronic acid and referenced to calibration curves with fortified aliquots of urine samples from controls containing 0–1000 nmol/ml of the glucuronides isolated from urine of TAME-treated rats by prep HPLC (Amberg et al., 2000Go). This method was not very sensitive and could be used only with samples containing high concentrations of the glucuronides. In addition, the injected mixture resulted in a rapid deterioration of the performance of the mass spectrometer. Therefore, all samples were analyzed by a simpler and more sensitive method using acid hydrolysis of the glucuronides. Enzymatic hydrolysis was not very effective with reference compounds. Glucuronidase did not completely cleave the glucuronides within 24 h. Under the conditions of the acid hydrolysis, the alcohols formed by the acid hydrolysis were further converted by an acid-catalyzed dehydration of t-amyl alcohol to give 2-methyl-2-butene and of 2-methyl-2,3-butane diol to form 3-methyl-2-butanone. The efficiency of the acid hydrolysis and the dehydration was checked by NMR using urine samples from rats treated with 13C-TAME (Amberg et al., 1999aGo) monitoring the disappearance of the glucuronide signals. To quantify content of free alcohols and glucuronides, t-butanol (25 µl from a 1000-nmol/ml solution in water; t-butanol is cleaved to 2-methylpropene under acidic conditions) as internal standard and 60 µl of 10 M sulfuric acid were added to 200 µl of urine in a closed vial. After 1 h at 90°C, 500 µl of the gas phase from the vial was analyzed by GC/MS. Separation was performed using a DB-1 coated fused silica column (30 m x 0.25 mm ID, 1-µm film). Samples were injected using splitless injection; oven temperature was 40° C. Samples were monitored using m/z 56, 70, and 86 by selected ion monitoring. Quantitation was performed relative to the formed 2-methylpropene and referenced to calibration curves with fortified aliquots of urine samples from controls containing 0–1000 nmol/ml of t-amyl alcohol and 2-methyl-2,3-butane diol. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 20–30 samples). The method permitted the quantitation of 0.1 nmol of t-amyl alcohol and 0.5 nmol 2methyl-2,3-butane diol glucuronide with a signal-to-noise ratio of 3:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 10% (n = 8). This method determined the content of free t-amyl alcohol and free 2-methyl-2,3-butane diol and their glucuronides in the samples. Concentrations of the two glucuronides were obtained by subtraction of the content of the free alcohols determined as described below.

To quantify 2-methyl 2,3-butane diol, 25 µl of a solution of the internal standard 1,2-propane diol (1000 nmol/ml in water) was added to 0.1 ml of human or rat urine. Urine samples were then diluted with 0.9 ml methanol, and 2-methyl-2,3-butane diol content was quantified by GC/MS by injecting 1 µl of the obtained mixtures. Separation was achieved using a fused silica column coated with DB-FFAP (30 m x 0.32 mm, film thickness 0.25 µm) with helium as carrier gas (2 ml/min). Samples were separated using a linear temperature program from 50°C to 230°C with a heating rate of 10°C/min. Injector and transfer line temperatures were 230°C. The concentrations of 2-methyl-2,3-butane diol were determined by monitoring m/z 59 and m/z 45 during the gas chromatographic separation, with dwell times of 80 msec. Split injection (split ratio of 10:1) was used. Quantitation was performed relative to the content of 1,2-propane diol and referenced to calibration curves with fortified aliquots of urine samples from controls containing 0–1000 nmol/ml 2-methyl 2,3-butane diol. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 20–30 samples) The method permitted the quantitation of 1 nmol 2-methyl 2,3-butane diol/ml of urine with a signal-to-noise ratio of 5:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 15% (n = 8).

Concentrations of 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid in urine were quantified by GC/MS after transformation to the corresponding methyl esters. Urine samples (0.1 ml for humans and rats) were mixed with 2-hydroxyvaleric acid (internal standard, 25 µl of a 1000-nmol/ml solution in water). Samples were then taken to dryness using anhydrous P2O5 in an evacuated desiccator. The obtained residues were treated with 500 µl of BF3/methanol (14%) at 60°C for 30 min. Samples were then diluted with 250 µl water and extracted with 1 ml chloroform. The chloroform layers were dried over sodium sulfate and 2 µl of the obtained solutions was analyzed by GC/MS (splitless injection). Samples were separated on a DB-WAX column (30 m x 0.25 mm, 0.25 µm film thickness) using a linear temperature program from 50°C to 230°C with a heating rate of 10°C/min. The intensities of m/z 55 and 59 were monitored during the separation with dwell times of 80 msec. Quantitation was performed relative to the content of 2-hydroxyvaleric acid and referenced to calibration curves with fortified aliquots of urine samples from controls containing 0–1000 nmol/ml 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 20–30 samples) The method permitted the quantitation of 3 nmol 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric /ml of urine with a signal-to-noise ratio of 3:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 10% (n = 8).

GC/MS analysis.
GC/MS analyses were performed on a Fisons MD 800 mass spectrometer coupled to a Fisons 8000 GC and equipped with an AS 800 autosampler and an electron impact source (Fisons Instruments, Mainz, Germany). Samples were also analyzed with electron impact ionization using a Hewlett-Packard 5970 mass spectrometer coupled to a 5890 GC or a Hewlett-Packard 5973 mass spectrometer coupled to a 6890 GC. Both instruments were equipped with a CTC Combi-PAL autoinjector with capability for headspace injection.

Statistical analysis.
Statistical analyses of the data were performed using Student's t-test in Microsoft Excel spreadsheets. p-Values less than 0.05 were considered significant. To determine possible sex-differences, all data sets from the male and female animals and male and female human volunteers were compared using Student's t-test in Microsoft Excel spreadsheets. p-Values of less than 0.05 were considered significant. Half-lives were calculated using exponential regression in Microsoft ExcelR spreadsheets. The curve-fitting function of the program was used and curves were stripped based on correlation coefficients. r2-Values of > 0.95 were considered for separation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biotransformation of TAME in Humans
During all experiments, the deviations between the targeted concentrations and the actual concentrations of TAME in the chamber were less than 10% of the targeted values. Average concentrations of TAME in the chamber were 3.8 ± 0.2 ppm and 38.4 ± 1.7 ppm (mean ± SD of 16 determinations in 15-min intervals over 4 h). Experimental results on the excretion of TAME metabolites and half-lives in humans are given in Tables 2–4GoGoGo and in Figures 2 and 3GoGo. TAME and t-amyl alcohol were not detected in blood samples from the volunteers taken before the exposure. The maximal concentrations of TAME and t-amyl alcohol in blood were determined directly after the end of the exposure period. TAME concentrations rapidly decreased to reach the limit of detection 12 h (40 and 4 ppm) after the end of the exposure period. Elimination of TAME from blood was rapid and could be separated into two phases with half-lives of 1.2 h and 3.5 h. Blood samples taken from the volunteers after exposure to 4 and 40 ppm TAME showed detectable concentrations of t-amyl alcohol for the time period between the end of the exposure and the 36-h blood sampling after 40 ppm TAME and between the end of the exposure and the 6-h blood sampling point after 4 ppm TAME (data not shown). Clearance of t-amyl alcohol from blood followed first-order kinetics and was slower than that of TAME.


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TABLE 2 TAME and t-Amyl Alcohol Blood Concentrations in Humans Exposed to TAME for Four Hours
 

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TABLE 3 Cumulative Metabolite Excretion and Half-Lives of Urinary Excretion of TAME Metabolites in Humans (n = 6) after Exposure to 38.4 ± 1.7 and 3.8 ± 0.2 ppm TAME
 

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TABLE 4 Received Doses of TAME in Humans and Amount of Metabolites Recovered in Urine
 


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FIG. 2. Excretion with urine of 3-hydroxy-3-methylbutyric acid (square), 2-hydroxy-2-methylbutyric acid (octagon) (Panel A), and 2-methyl-2,3-butane diol (octagon), t-amyl alcohol glucuronide (square), t-amyl alcohol (triangle), and unchanged TAME (diamond) (Panel B) in six human volunteers exposed to 38.4 ± 1.7 ppm TAME for 4 h in a dynamic exposure chamber. Numbers (mean ± SD) given represent total amount of metabolite excreted in the urine samples collected in 6-h intervals. Each sample was analyzed in duplicate. Statistically significant differences as compared to background in controls (**p < 0.01; * p < 0.05).

 


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FIG. 3. Excretion with urine of 2-methyl-2,3-butane diol (octagon), t-amyl alcohol glucuronide (square), and t-amyl alcohol (triangle) in six human volunteers exposed to 3.8 ± 0.2 ppm TAME for 4 h in a dynamic exposure chamber. Numbers (mean ± SD) given represent total amount of metabolite excreted in the urine samples collected in 6-h intervals. Each sample was analyzed in duplicate. Statistically significant differences as compared to background in controls (** p < 0.01; * p < 0.05).

 
In urine samples of the volunteers collected before TAME exposure and in samples collected from control subjects, low concentrations of 2-methyl-2,3-butane diol were present. In addition, high and variable concentrations of 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid were observed. In the urine samples from TAME-exposed individuals, the concentrations of 2-methyl-2,3-butane diol were significantly increased in all urine samples collected until 72 h after the end of the exposure period after both 4 and 40 ppm TAME. Elimination of 2-methyl-2,3-butane diol was slow and not complete within the period of observation. Statistically significant increases in the concentrations of 2-hydroxy-2-methylbutyric acid were observed only in urine samples taken between 0 and 30 h after the end of the 40-ppm exposure. Significantly increased concentrations of 3-hydroxy-3-methylbutyric acid were seen only in urine samples taken between 0 and 12 h after the end of exposure. After exposure to 4 ppm TAME, none of the urine samples contained significantly increased concentrations of 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid due to the high and variable background.

Due to the absence of background, the concentrations of TAME and t-amyl alcohol could be quantified with higher precision. TAME was detectable in all urine samples from the volunteers collected between 0 and 12 h after the end of the exposure after 40 ppm TAME and between 0 and 6 h after the end of exposure after 4 ppm TAME. t-Amyl alcohol and its glucuronide were also detected in all urine samples collected between 0 and 36 h after the end of the 40-ppm exposure in low concentrations. Excretion of these compounds with urine was rapid and occurred with half-lives of less than 10 h.

Based on the recovered amounts of 2-methyl-2,3-butane diol and 2-hydroxy-2-methylbutyric acid, these compounds represent the major excretory metabolites formed from TAME (Tables 3 and 4GoGo) in humans. In addition, 3-hydroxy-3-methylbutyric acid was a TAME metabolite present in higher concentrations in urine, whereas free and conjugated t-amyl alcohol and unchanged TAME were only minor excretory products. Large variations in the extent of TAME biotransformation (Table 4Go) between the individuals and in the rates of excretion and the urinary concentrations of 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid were observed. However, no statistically significant differences in the amounts of these acids, of free and conjugated 2-methyl-2,3-butane diol or any of the other metabolites excreted, or in the rates of excretion of metabolites were seen between the male and female subjects in the study. The determined half-lives of elimination with urine were not significantly different after the 4 and 40 ppm TAME exposures (Tables 3 and 4GoGo).

Biotransformation of TAME in Rats
Rats were exposed to the same TAME concentrations as used in the human studies. The experimental results on metabolite concentrations and excretion are compiled in Tables 5–7GoGoGo and in Figures 4 and 5GoGo. The concentrations of TAME in blood of rats determined after the end of the 4-h exposure period were twice as high as those seen in humans after identical exposure concentrations. TAME was more rapidly cleared from rat blood as compared to human blood. The concentrations of t-amyl alcohol were not significantly different between rats and humans after 4 and 40 ppm TAME.


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TABLE 5 Blood Concentrations at the End of Exposure and Half-Lives of Elimination from Blood after Exposure of Rats (n = 10) to 38.4 ± 1.7 and 3.8 ± 0.2 ppm TAME for Four Hours
 

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TABLE 6 Cumulative Metabolite Excretion and Half-Lives of Urinary Excretion of TAME metabolites in Rats (n = 10) after Exposure to 38.4 ± 1.7 and 3.8 ± 0.2 ppm TAME
 

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TABLE 7 Received Doses of TAME in Rats and Amount of Metabolites Recovered in Urine
 


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FIG. 4. Excretion with urine of 2-methyl-2,3-butane diol (octagon), 2-methyl-2,3-butane diol glucuronide (diamond) (panel A), t-amyl alcohol glucuronide (square), 2-hydroxy-2-methylbutyric acid (octagon), and t-amyl alcohol (triangle) (panel B) in 10 rats exposed to 38.7 ± 3.2 ppm TAME for 4 h in a dynamic exposure chamber. Numbers (mean ± SD) given represent total amount of metabolite excreted in the urine samples collected in 6-h intervals. Each sample was analyzed in duplicate. Statistically significant differences as compared to background in controls (** p < 0.01; * p < 0.05).

 


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FIG. 5. Excretion with urine of 2-methyl-2,3-butane diol (octagon) and t-amyl alcohol glucuronide (square) in 10 rats exposed to 3.8 ± 0.2 ppm TAME for 4 h in a dynamic exposure chamber. Numbers given represent total amount of metabolite excreted in the urine samples collected in 6-h intervals. Statistically significant differences as compared to background in controls (** p < 0.01; * p < 0.05)

 
In urine samples of rats collected before TAME exposure, and in samples collected from control rats, low concentrations of 2-hydroxy-2-methylbutyric acid, 3-hydroxy-3-methylbutyric acid, 2-methyl-2,3-butane diol, and t-amyl alcohol were present. In the urine samples from exposed rats, the concentrations of 2-hydroxy-2-methylbutyric acid were significantly increased (as compared to controls) in only a few urine samples collected within 24 h after the end of 40-ppm TAME inhalation (Fig. 4Go). Significantly increased concentrations of 2-hydroxy-2-methylbutyric acid were not observed in any of the urine samples collected from the 4-ppm exposure of rats. Statistically significant increases above background excretion rates in the concentration of 3-hydroxy-3-methylbutyric acid were not observed in any of the urine samples collected from TAME-exposed rats. Due to much lower background levels, the concentrations of 2-methyl-2,3-butane diol were significantly higher than controls in all samples collected between 0 and 42 h after both 4 and 40 ppm TAME. Excretion of the glucuronide of 2-methyl-2,3-butane diol was detectable in all urine samples collected up to 48 h after the end of the exposure after 40 ppm, but not in urine samples after 4-ppm exposure. Excretion of the glucuronide of t-amyl alcohol was detectable in all urine samples collected up to 24 h after the end of exposure after 40 ppm and 4 ppm. The other metabolites quantified were rapidly excreted, and their concentration in urine samples was below the limit of detection after 24 h. The presence of these minor metabolites was not detected in urine samples collected from rats after exposure to 4 ppm TAME.

Based on the amounts of 2-methyl-2,3-butane diol and its glucuronide, these compounds and t-amyl alcohol glucuronide represent the major urinary metabolites of TAME (Table 6Go) in rat urine. Assuming identical retention of TAME after inhalation exposure, humans excreted a significantly higher proportion (p < 0.03) of the retained TAME as metabolites in urine as compared to rats (Tables 5 and 7GoGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this work, the biotransformation of TAME in rats and in humans was compared after inhalation exposure. After 4-h inhalation exposures, lower blood levels of TAME were obtained in humans as compared to rats immediately after the end of exposure to 4 and 40 ppm TAME. The higher blood levels observed in rats are likely due to higher alveolar ventilation rates, resulting in increased delivery and uptake of TAME in rats as compared to humans. In general, the time course of elimination of TAME and all metabolites quantified in this study shows that rats excrete TAME and its metabolites more rapidly than humans.

Half-lives of elimination of TAME from blood were also similar to those seen with other ethers intended for use as oxygenates determined in other studies. Elimination of methyl tert-butyl ether and ethyl tert-butyl ether also occurs with half-lives of less than 1 h in rats and (Miller et al., 1997Go) between 2 and 4 h in humans (Nihlen et al., 1998Go; Prah et al., 1994Go). As seen with these ethers, no sex differences in the apparent half-lives of elimination of these compounds were seen.

In urine of humans and rats, the known metabolites of TAME excreted with urine and unchanged TAME were quantified. A minor part of the TAME dose is excreted with urine in humans. Elimination of the parent ether with urine was also seen with MTBE and ETBE (Amberg et al., 1999bGo, 2000Go; Nihlen et al., 1998Go; Prah et al., 1994Go). With these compounds, urinary elimination of the ether also represents a minor pathway for the clearance of the ethers. In rat urine, the TAME concentrations in the urine samples were already below the limit of detection in the first available samples (6 h after the end of exposure).

The study confirms the structure of TAME metabolites formed in the rat and demonstrates that identical metabolites of TAME are formed in humans and excreted with urine. Moreover, the obtained results confirm and extend the semiquantitative findings from our previous study on differences in the metabolic pattern of TAME between humans and rats (Amberg et al., 1999aGo). In rats, TAME is mainly excreted as 2-methyl-2,3-butane diol and its glucuronide. Further oxidation of t-amyl alcohol to other products is of minor importance due to a more rapid elimination and glucuronide formation. In humans, 2-methyl-2,3-butane diol is eliminated more slowly as compared to rats. In addition, t-amyl alcohol seems to be more efficiently oxidized to 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid in humans. This reaction occurs only to a minor extent in rats. These differences are likely due to substrate specificities of the enzymes involved in the formation of these compounds from TAME and t-amyl alcohol (cytochrome P450 and glucuronyl transferase).

All TAME metabolites quantified in this study were eliminated in rats with apparent half-lives of elimination of less than 6 h; in humans, the elimination with urine of the metabolites formed from TAME was considerably slower.

The extent of TAME biotransformation in humans is significantly higher as compared to rats when the amounts of metabolites and the relative concentrations recovered in urine are compared to the doses received (Tables 5 and 7GoGo). Between 40% (rats) and almost 60% (humans) of the calculated doses of TAME received by inhalation were recovered as metabolites in urine. The rest of the TAME taken up by inhalation is likely exhaled. Exhalation of unchanged TAME was not determined in this study. However, due to the volatility of TAME and based on studies with the structurally similar compounds methyl tert-butyl ether and ethyl tert-butyl ether, it has to be assumed that the unaccounted part of the received TAME-dose is exhaled unchanged either by rats or by humans after the termination of the inhalation period. Exhalation of unchanged parent ether was a major pathway of elimination of both methyl tert-butyl ether and ethyl tert-butyl ether in humans and in rats. Extent of biotransformation of TAME in both rats and humans seems not to be saturated in the concentration range studied, as the percentage of dose recovered as metabolites was identical after both exposure concentrations. The interindividual differences in the extent of TAME biotransformation in humans are likely due to interindividual differences in the cytochrome P450 profile (Hong et al., 1999Go). More than 10-fold differences in the capacity of human liver microsomes to oxidize TAME to t-amyl alcohol were observed (Hong et al., 1999Go). Further studies identifying the cytochrome P450 enzymes involved in t-amyl alcohol biotransformation should address this problem.

The differences in biotransformation and kinetics of metabolite excretion suggest that humans may respond differently than rats to potential toxic effects of TAME based on biotransformation. However, as only limited data on toxic effects of TAME after long-term inhalation exposure in rats are available, conclusions must be taken with caution. The potential of TAME to induce acute and chronic toxicity in rats is low, and the role of biotransformation in the observed changes in relative organ weights is unknown. As the formation of reactive metabolites during TAME biotransformation is not suggested based on the structures of metabolites and their mechanisms of formation, covalent binding to macromolecules has to be regarded as unlikely; thresholds are likely for TAME toxicity. Moreover, the major TAME metabolites found in humans are also formed endogenously (Liebich and Forst, 1984Go). TAME exposures in low concentrations as expected from the environment are not likely to result in a significant increase in the human body burden of these compounds, making toxic effects of TAME in humans under realistic exposure conditions unlikely.

In conclusion, the biotransformation of TAME after inhalation is quantitatively different, but qualitatively similar in rats and humans. Sex differences in biotransformation were not seen in either species. 2-Methyl-2,3-butane diol concentrations in urine or blood may represent useful biomarkers of exposure to low levels of TAME expected from environmental exposures.


    ACKNOWLEDGMENTS
 
Research described in this article was conducted under contract from the Health Effects Institute (HEI, Research Agreement No. 96-3), an organization jointly funded by the United States Environmental Protection Agency (EPA) (Assistance Agreement X-816285) and certain motor vehicle and engine manufactures. The contents of this article do not necessarily reflect the views of HEI or its sponsors, nor do they necessarily reflect the views and policies of EPA or motor vehicle and engine manufacturers. Part of this work was also supported by the Biomed Program of the European Union, Contract No. BMH4-CT96-0184.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +49-0931-201-3865. E-mail: dekant{at}toxi.uni-wuerzburg.de. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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