Biotransformation of 12C- and 2-13C-labeled methyl tert-butyl ether, ethyl tert-butyl ether, and tert-butyl alcohol in rats: Identification of metabolites in urine by 13C nuclear magnetic resonance and gas chromatography/mass spectrometry

Article abstract of DOI:10.1021/tx970215v

The biotransformation of the fuel oxygenates methyl tert-butyl ether (MTBE) and ethyl tertbutyl ether (ETBE) was studied in rats after inhalation exposure; the biotransformation of the initial metabolite of these ethers, tert-butyl alcohol, was studied after oral gavage. To study ether metabolism, rats were exposed for 6 h to initial concentrations of 2000 ppm of MTBE or ETBE, respectively [2-¹³C]MTBE and [2-¹³C]ETBE. Urine was collected for 48 h after the end of the exposure, and urinary metabolites were identified by ¹³C NMR (¹³C-labeled ethers) and gas chromatography/mass spectrometry (GC/MS) (¹²C- and ¹³C-labeled ethers). To study tert-butyl alcohol metabolism, rats were dosed either with tert-butyl alcohol at natural carbon isotope ratio or with ¹³C-enriched tert-butyl alcohol (250 mg/kg of body weight), urine was collected, and metabolites were identified by NMR and GC/MS. tert-Butyl alcohol was identified as a minor product of the biotransformation of MTBE and ETBE. In addition, small amounts of a tert- butyl alcohol conjugate, likely a glucuronide, were present in the urine of the treated animals. Moreover, the mass spectra obtained indicate the presence of small amounts of [¹³C]acetone in the urine of [¹³C]MTBE and [¹³C]ETBE-treated rats. 2-Methyl-1,2-propanediol, 2-hydroxyisobutyrate, and another unidentified conjugate of tert-butyl alcohol, most probably a surf ate, were major urinary metabolites of MTBE and ETBE as judged by the intensities of the NMR signals. In [¹³C]-tert-butyl alcohol-dosed rats, [¹³C]acetone, tert-butyl alcohol, and its glucuronide represented minor metabolites; as with the ethers, 2-methyl-1,2-propanediol, 2- hydroxyisobutyrate, and the presumed tert-butyl alcohol sulfate were the major metabolites present. In one human individual given 5 mg/kg [¹³C]- tert-butyl alcohol orally, 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate were major metabolites in urine detected by ¹³C NMR analysis. Unconjugated tert-butyl alcohol and tert-butyl alcohol glucuronide were present as minor metabolites, and traces of the presumed tert-butyl alcohol sulfate were also present. Our results suggest that tert-butyl alcohol formed from MTBE and ETBE is intensively metabolized by further oxidation reactions. Studies to elucidate mechanisms of toxicity for these ethers to the kidney need to consider potential toxicities induced by these metabolites.

Full text of DOI:10.1021/tx970215v

Chem. Res. Toxicol. 1998, 11, 651-658
651
12
Biotr a n sfor m a tion of C- a n d 2-13C-La beled Meth yl
ter t-Bu tyl Eth er , Eth yl ter t-Bu tyl Eth er , a n d ter t-Bu tyl
Alcoh ol in Ra ts: Id en tifica tion of Meta bolites in Ur in e
by 13C Nu clea r Ma gn etic Reson a n ce a n d Ga s
Ch r om a togr a p h y/Ma ss Sp ectr om etr y
Ulrike Bernauer, Alexander Amberg, Dieter Scheutzow,† and Wolfgang Dekant*
Institut fu¨r Toxikologie, Universita¨t Wu¨rzburg, Versbacher Strasse 9, 97078 Wu¨rzburg, Germany,
and Institut fu¨r Organische Chemie, Universita¨t Wu¨rzburg, Am Hubland,
97070 Wu¨rzburg, Germany
Received December 5, 1997
The biotransformation of the fuel oxygenates methyl tert-butyl ether (MTBE) and ethyl tert-
butyl ether (ETBE) was studied in rats after inhalation exposure; the biotransformation of
the initial metabolite of these ethers, tert-butyl alcohol, was studied after oral gavage. To
study ether metabolism, rats were exposed for 6 h to initial concentrations of 2000 ppm of
MTBE or ETBE, respectively [2-13C]MTBE and [2-13C]ETBE. Urine was collected for 48 h
after the end of the exposure, and urinary metabolites were identified by 13C NMR (13C-labeled
ethers) and gas chromatography/mass spectrometry (GC/MS) (12C- and 13C-labeled ethers).
To study tert-butyl alcohol metabolism, rats were dosed either with tert-butyl alcohol at natural
carbon isotope ratio or with 13C-enriched tert-butyl alcohol (250 mg/kg of body weight), urine
was collected, and metabolites were identified by NMR and GC/MS. tert-Butyl alcohol was
identified as a minor product of the biotransformation of MTBE and ETBE. In addition, small
amounts of a tert-butyl alcohol conjugate, likely a glucuronide, were present in the urine of
the treated animals. Moreover, the mass spectra obtained indicate the presence of small
amounts of [13C]acetone in the urine of [13C]MTBE and [13C]ETBE-treated rats. 2-Methyl-
1,2-propanediol, 2-hydroxyisobutyrate, and another unidentified conjugate of tert-butyl alcohol,
most probably a sulfate, were major urinary metabolites of MTBE and ETBE as judged by the
intensities of the NMR signals. In [13C]-tert-butyl alcohol-dosed rats, [13C]acetone, tert-butyl
alcohol, and its glucuronide represented minor metabolites; as with the ethers, 2-methyl-1,2-
propanediol, 2-hydroxyisobutyrate, and the presumed tert-butyl alcohol sulfate were the major
metabolites present. In one human individual given 5 mg/kg [13C]-tert-butyl alcohol orally,
2-methyl-1,2-propanediol and 2-hydroxyisobutyrate were major metabolites in urine detected
by 13C NMR analysis. Unconjugated tert-butyl alcohol and tert-butyl alcohol glucuronide were
present as minor metabolites, and traces of the presumed tert-butyl alcohol sulfate were also
present. Our results suggest that tert-butyl alcohol formed from MTBE and ETBE is intensively
metabolized by further oxidation reactions. Studies to elucidate mechanisms of toxicity for
these ethers to the kidney need to consider potential toxicities induced by these metabolites.
to investigate the toxicology of these compounds are
underway (2).
In tr od u ction
The use of oxygen-containing compounds in fuels to
reduce harmful engine emissions from cars is required
in certain areas of the United States. Chemicals blended
with gasoline to meet the required oxygen content of 2.0%
(oxygenated gasoline), respectively 2.7% (reformulated
gasoline), are referred to as “oxygenates” (1). Methyl tert-
butyl ether (MTBE)1 and ethanol are the major oxygen-
ates presently in use; however, other oxygenates such as
ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether
(TAME) may be increasingly used in the future. Due to
possible widespread exposure of humans to these ethers
when refueling cars or during commute, several programs
The acute toxicity of MTBE and ETBE is low. Both
MTBE and tert-butyl alcohol have been studied in long-
term bioassays for tumorigenicity. MTBE (inhalation
exposure up to 8000 ppm, 6 h/day, 5 days/week for 24
months) and its presumed major metabolite, tert-butyl
alcohol (administration in doses up to 5 mg/L in drinking
water for 15 months), induce renal tumors in male rats
(3, 4). Renal tumor induction by these compounds may
be mediated by the accumulation of R2u-globulin (5, 6).
An impared degradation of this protein induced by bound
metabolites of tert-butyl alcohol and MTBE or by tert-
butyl alcohol or MTBE may cause renal toxicity, cell
proliferation, and, finally, renal tumors (7). MTBE
exposure also increased the incidence of liver tumors in
female mice and testicular tumors in male rats (8).
Testicular tumors in male rats were also observed
* Address correspondence to Dr. W. Dekant. Tel: +49(931)201 3449.
Fax: +49(931)201 3446. E-mail: dekant@toxi.uni-wuerzburg.de.
† Institut fu¨r Organische Chemie.
1
Abbreviations: MTBE, methyl tert-butyl ether; ETBE, ethyl tert-
butyl ether; TAME, tert-amyl methyl ether.
S0893-228x(97)00215-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/13/1998

652 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
Bernauer et al.
7 Hz, J HC ) 2 Hz, (CH3)3COCH2CH3]. 13C NMR (63 MHz,
D2O): δ ) 17.9 [s, (CH3)3COCH2CH3]; 29.3 [d, J CC ) 39 Hz,
(CH3)3COCH2CH3]; 60.2 [s, (CH3)3COCH2CH3]; 77.7 [s, d, with
0.5% of a satellite doublet, J CC ) 40 Hz, (CH3)3COCH2CH3]. MS
(70 eV): m/z ) 88 [94%, M+ - CH3]; 60 [100%]; 58 [88%]; 56
[12%]; 44 [23%]; 42 [45%]; 41 [16%].
Syn th esis of 2-Meth yl-1,2-p r op a n ed iol. 2-Methyl-1,2-
propanediol was prepared by reduction of the ethyl ester of
2-hydroxyisobutyrate; 4 mmol of NaBH4 (151 mg) was dissolved
in 12 mL of 2-propanol followed by addition of 10 mmol of
2-hydroxybutyric acid ethyl ester at room temperature. The
mixture was then stirred overnight. To dissolve the precipitated
solid 2 N HCl was added to the reaction mixture. After
extraction with ethyl ether the organic layers were combined
and dried over K2CO3. After removal of the solvent, 2-methyl-
1,2-propanediol was isolated by fractional distillation under
reduced pressure.
An alytical Ch ar acter ization of 2-Meth yl-1,2-pr opan ediol.
1H NMR (250 MHz, D2O): δ ) 1.19, 6 H [s, (CH3)2C(OH)CH2-
(OH)]; 3.42, 2 H [s, (CH3)2C(OH)CH2(OH)]. 13C NMR (63 MHz,
D2O): δ ) 27.4 [s, (CH3)2C(OH)CH2(OH)]; 72.6 [s, (CH3)2C(OH)-
CH2(OH)]; 74.2 [s, (CH3)2C(OH)CH2(OH)]. MS (70 eV): m/z )
75 [8%, M+ - CH3]; 59 [70%]; 57 [28%]; 55 [12%]; 43 [100%]; 42
[22%]; 41 [33%].
following oral administration of MTBE in a daily dose of
1000 mg/kg of body weight, 4 days/week for 104 weeks
(3). MTBE, ETBE, and their presumed major metabolite
tert-butyl alcohol are negative in standard genotoxicity
studies (9).
As part of the efforts to characterize the toxic effects
of MTBE and ETBE in this study, we investigated the
biotransformation of MTBE and ETBE. Intensive me-
tabolism of these ethers to tert-butyl alcohol in vivo and
further metabolism of tert-butyl alcohol are indicated by
a low recovery of tert-butyl alcohol in the urine of humans
exposed to MTBE and ETBE under controlled conditions
and a high percentage of retention of inhaled MTBE and
ETBE likely due to biotransformation to less volatile
metabolites (10, 11). The role of biotransformation in the
toxic effects of MTBE and ETBE is unknown; studies on
the metabolism of these ethers may delineate the role of
the parent compound and metabolites in the toxic effects
seen. Moreover, the identification of metabolites will
help to develop strategies for monitoring exposure of
individuals to MTBE and other oxygenates in fuel (12).
An im a ls a n d Tr ea tm en t. Male and female Fischer F344
rats (220-260 g) were obtained from Harlan Winkelmann
(Borchen, FRG). Animals were kept at constant humidity and
temperature (21 °C) in the animal facility of the department
with a 12-h light/dark cycle in steel cages (4 rats/cage). Food
(Altromin) and tap water were provided ad libitum. Before the
experiments, the animals were accustomed to the metabolic
cages for 3 days; control urine was collected during this time
for 12 h before the exposure.
Exp er im en ta l Design . Male and female rats were indi-
vidually exposed to [12C]- or [13C]MTBE (separate experiments
with two male and two female rats for each [12C]- and [13C]-
MTBE and [12C]- and [13C]ETBE) by inhalation for 6 h in a static
exposure chamber; initial concentrations of MTBE or ETBE
were 2000 ppm. After the end of the 6-h exposures, the animals
were individually housed in all glass metabolic cages with free
access to food (Altromin) and tap water in the animal facility of
the department. Urine samples were collected in 24-h intervals
for 48 h and analyzed by 13C NMR (experiments with 13C-labeled
ethers) and GC/MS. Male rats (n ) 3/experiment) were treated
with either [12C]- or [13C]-tert-butyl alcohol by gavage (250 mg/
kg in corn oil). All animals were individually kept in metabolic
cages for 72 h. After the termination of the experiments, all
animals were sacrificed by cervical dislocation.
Ma ter ia ls a n d Meth od s
Syn th esis of [2-13C]-ter t-Bu tyl Alcoh ol. An ethereal solu-
tion of 60 mmol of CH3J (Merck, Darmstadt, FRG) was slowly
added to equimolar amounts of Mg• turnings (1.5 g) covered by
5.0 mL of diethyl ether. The Grignard reaction was initiated
by the addition of traces of iodine (15, 16). The mixture was
then stirred for 30 min at flux, an ethereal solution of 50 mmol
of [2-13C]acetone (CIL Cambridge Isotope Laboratories, Andover,
MA, lot no. P-7787) was added within 30 min, and the mixture
was kept at flux for 2 h. After cooling, hydrolysis was performed
with 50 mL of an ice-cold, saturated NH4Cl solution. The layers
were separated, and the aqueous layer was extracted five times
with 10 mL of diethyl ether. The ethereal layers were combined
and dried over Na2CO3. After evaporation of the solvent the
residue was distilled to yield [2-13C]-tert-butyl alcohol (42% yield,
97% GC/FID purity). The structure of the reaction product was
confirmed by GC/MS and 1H and 13C NMR.
An a lytica l Ch a r a cter iza tion of [2-13C]-ter t-Bu tyl Alco-
h ol. 1H NMR (250 MHz, D2O): δ ) 1.25, 9 H [d, J HC ) 4 Hz,
(CH3)3COH]. 13C NMR (63 MHz, D2O): δ ) 32.4 [d, J CC ) 39.1
Hz, (CH3)3COH]; 72.6 [s, d, with 0.5% of a satellite doublet, J CC
) 39 Hz, (CH3)3COH]. MS (70 eV): m/z ) 60 [100%, M+ - CH3];
58 [40%]; 44 [31%]; 42 [40%].
Syn th esis of [13C]MTBE a n d [13C]ETBE fr om [2-13C]-
ter t-Bu tyl Alcoh ol. A mixture of 20 mmol of either methanol
(for the synthesis of MTBE) or ethanol (for the synthesis of
ETBE) and 3 mL of 10% H2SO4 in H2O was heated to 70 °C;
then 10 mmol of [2-13C]-tert-butyl alcohol was added with a
syringe. With increasing temperature an azeotropic mixture
of MTBE (ETBE), methanol (ethanol), and tert-butyl alcohol was
distilled off. Methanol (ethanol) was removed from the reaction
mixture by boiling over sodium for 1 h. Further purification
was performed by slow distillation with dry ice cooling on a
Bu¨chi-GKR-51 Kugelrohr apparatus to yield 26% [2-13C]MTBE
and 21% [2-13C]ETBE. The GC/FID purities of both reaction
products were >95%. The structures of the reaction products
were confirmed by GC/MS and 1H and 13C NMR (17).
An a lytica l Ch a r a cter iza tion of [2-13C]MTBE. 1H NMR
(250 MHz, D2O): δ ) 1.22, 9 H [d, J HC ) 4 Hz, (CH3)3COCH3];
3.24, 3 H [d, J HC ) 4 Hz, (CH3)3COCH3]. 13C NMR (63 MHz,
D2O): δ ) 28.8 [d, J CC ) 40 Hz, (CH3)3COCH3]; 51.6 [s, (CH3)3-
COCH3]; 77.9 [s, d, with 0.5% of a satellite doublet, J CC ) 40
Hz, (CH3)3COCH3]. MS (70 eV): m/z ) 74 [100%, M+ - CH3];
58 [59%]; 56 [21%]; 44 [50%]; 42 [38%].
Exp osu r e of On e Hu m a n Volu n teer to [13C]-ter t-Bu tyl
Alcoh ol. [13C]-tert-Butyl alcohol was orally administered in a
gel capsule at a dose of 5 mg/kg to one human volunteer (male,
age 44 years, body weight 80 kg). Urine was collected in 12-h
intervals for 48 h and analyzed by 13C NMR as described below.
The human experiment was approved by the institutional
review board of the University of Wu¨rzburg.
Ch a m ber Design a n d Tr ea tm en t. The design of the
exposure chamber has been described in detail (13). Volume of
the system built from stainless steel was 1.89 L. The chamber
atmosphere was circulated (2 L/min) by a stainless steel-bellows
pump. Carbon dioxide was removed with barium hydroxide
contained in a scrubber on the output side of the chamber
downstream from a moisture condenser and a flow meter.
Chamber oxygen concentrations were maintained within the
range of 20-22% by an oxygen monitor which was connected
to a solenoid valve that metered oxygen into the chamber as
required. The animals were introduced into the chamber at 10
a.m., and the calculated amounts of the 12C- or 13C-labeled ethers
were introduced with a microliter syringe. The concentrations
of the ethers in the gas phase of the chamber were monitored
every 10 min by an automatic gas sampling valve; 100 µL of
the gas phase was introduced into a capillary gas chromatograph
(HP 5970) and separated with a 40-m DB1-coated fused silica
An a lytica l Ch a r a cter iza tion of [2-13C]ETBE. 1H NMR
(250 MHz, D2O): δ ) 1.15, 3 H [t, J ) 7 Hz, (CH3)3COCH2CH3];
1.23, 9 H [d, J HC ) 4 Hz, (CH3)3COCH2CH3]; 3.54, 2 H [q, J )

Biotransformation of Fuel Oxygenates
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 653
Sch em e 1. Syn th etic Rou tes to [13C]MTBE a n d [13C]ETBE fr om [13C]Aceton e
column (DB1, J &W Scientific; 40 m, i.d. ) 0.18 mm, film
thickness ) 0.4 µm) at an oven temperature of 30 °C using
hydrogen as carrier gas. Ether concentrations in the air were
quantified by flame ionization detection (FID).
does not interfere with the intended identification of
metabolites by NMR spectrometry since the alcohol is
also formed as a metabolite from the ethers in vivo.
Moreover, the synthetic procedures applied also yielded
[2-13C]-tert-butyl alcohol which could be also used for
metabolism studies for a better definition of the fate of
this first intermediary metabolite of MTBE and ETBE
in vivo in rats and humans.
Monitoring of the exposure chamber air concentrations
in MTBE and ETBE exposures of rats indicated a
continuous decrease of the air concentrations of the
compounds due to uptake of the ethers by the rats and
metabolism to less volatile metabolites. At the end of
the exposure after 6 h the ether concentrations in the
chamber were below 100 ppm indicating intensive me-
tabolism of the ethers by the rats. Analysis of the gas
phase of the chamber did not indicate the formation of
volatile or exhaled ether metabolites detectable by flame
ionization detection. Urine samples were collected in
24-h intervals for 48 h and analyzed by GC/MS and 13C
NMR. A typical NMR spectrum of a urine sample is
shown in Figure 1.
Biotr a n sfor m a tion of MTBE. The NMR spectra
obtained from the urine of [13C]MTBE-exposed rats
showed several resonances which were also present in
the urine from control animals (not shown in Figure 1).
The structures of those endogenous products were as-
signed by comparison with literature data and reference
spectra (18, 19). In addition, the range of chemical shifts
between 70 and 90 ppm, where only one resonance was
observed in the 13C NMR spectra of control urine (δ )
78.3), showed several signals indicative of MTBE me-
tabolites (δ ) 73.7, 74.3, 76.6, 80.5). The structures of
these metabolites were elucidated by a combination of
13C NMR and GC/MS.
In str u m en ta l An a lysis a n d Sa m p le P r ocessin g. Before
the recording of 13C NMR spectra, the urines obtained from the
animals exposed to 13C-labeled ethers and control urine (720
µL) were introduced into a NMR tube (5-mm i.d.) and 80 µL of
D2O was added. These samples were directly analyzed by NMR.
Some of the urine samples were treated with â-glucuronidase
(Sigma, G7846, lot no. 72H6836) for 30 min at 37 °C or sulfatase
(Sigma, S9751, lot no. 621-07881) for 48 h at 37 °C (14); aliquots
of the treated urine samples were then also analyzed by NMR.
For nonenzymatic cleavage urine samples were acidified to pH
2 with concentrated hydrochloric acid and incubated for 1 h at
37 °C.
For gas chromatography/mass spectrometry, 0.5 mL of the
urine samples was introduced into 1.5-mL sample vials, acidified
with 2 N HCl to pH 4, and kept at 80 °C for 30 min; 100 µL of
the headspace from the incubations was then injected into the
GC. To analyze less volatile metabolites, some samples (0.5 mL)
were extracted with 0.5 mL of ethyl acetate and 1 µL of the
ethyl acetate layer was analyzed via GC/MS. Separation was
performed on a 30-m × 0.25-mm fused silica column coated with
DB-WAX, film thickness 0.25 µm. For analysis, a temperature
gradient from 35 to 230 °C with a heating rate of 10 °C/min
was applied. Electron impact mass spectra (70 eV) were
recorded and metabolite peaks identified by comparison of the
chromatograms of the urine from treated rats with those of
untreated controls.
1H NMR and composite pulse-decoupled 13C NMR spectra
were recorded with a Bruker AC 250 spectrometer or a Bruker
Avance DMX 600 NMR spectrometer under standard conditions.
Usually, 2000 scans were aquired for Fourier transformation.
Gas chromatography/mass spectrometry was performed with a
Fisons MD 800 mass spectrometer coupled to a Carlo Erba GC
8000 series gas chromatograph. Split injection (split ratio 1:5)
was used, and spectra were recorded from m/z 30 to 300 in 1-s
intervals.
The NMR signal at δ ) 73.7 most likely represents a
glucuronide of tert-butyl alcohol since this signal disap-
peared after treatment of urine with glucuronidase with
concomitant appearance of the signal for tert-butyl
alcohol at δ ) 72.6. Moreover, the signal also disap-
peared and the tert-butyl alcohol signal appeared when
the urines were treated with acid to cleave acid-labile
conjugates such as glucuronides.
Calculations to predict the structure based on the
chemical shift of the signal at δ ) 74.2 suggest that this
metabolite may represent 2-methyl-1,2-propanediol. The
13C NMR spectrum of synthetic 2-methyl-1,2-propanediol
also showed a resonance at δ ) 74.2 attributed to the C2
atom. The presence of 2-methyl-1,2-propanediol is also
suggested by the results of mass spectrometric analysis
of urine from MTBE-treated rats (Figure 2). The mass
Resu lts
Syn th esis of 13C-La beled Eth er s. To identify me-
tabolites of the ethers without the expensive synthesis
of 14C-labeled material and to be able to detect nonvolatile
metabolites, 13C-labeled ethers (>99% 13C in one carbon
atom) were prepared (Scheme 1). The use of stable
isotope-labeled ethers permits metabolite analysis in
urine by 13C NMR in sufficient sensitivity, also permits
the identification of metabolites not anticipated, and
confirms the structure of metabolites. The 13C-labeled
ethers were synthesized according to standard proce-
dures; the purity of all compounds used for animal
exposures was >95%. The major impurity present was
[2-13C]-tert-butyl alcohol. The presence of this compound

654 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
Bernauer et al.
F igu r e 3. Mass spectra of [13C]acetone present in the urine of
a male Fischer 344 rat exposed to [13C]MTBE (spectrum B). The
major fragments are shifted by 1 mass unit indicating the
presence of 13C in these fragments.
was greatly increased when authentic hydroxyisobu-
tyrate was added to the urine sample. This observation
confirms hydroxyisobutyrate as a metabolite of [13C]-
MTBE. The structure of 2-hydroxyisobutyrate was also
confirmed by mass spectrometry (data not shown).
The NMR signal at δ ) 80.6 which also represents a
major metabolite of MTBE in rat urine was not conclu-
sively identified. The chemical shift of the signal indi-
cates the presence of an electron-withdrawing group next
to the C atom carrying the 13C label but no major
structural change in the molecule. The NMR signal was
decreased in intensity by 80% (relative to the urea signal
at δ ) 165.5) when urine samples were treated for a
prolonged time (48 h at 37 °C) with sulfatase with a
parallel increase in the intensity of the signal represent-
ing tert-butyl alcohol. Moreover, the signal disappeared
and a tert-butyl alcohol resonance appeared when urine
samples were treated with acid to cleave acid-labile
conjugates such as sulfates. In summary, these data
suggest that the metabolite represents a conjugate of tert-
butyl alcohol, likely a sulfate.
F igu r e 1. (A) 63-MHz 13C NMR spectrum of urine from an
untreated female Fischer 344 rat serving as control; (B) 63-MHz
13C NMR spectrum of a 24-h urine sample from a female rat
exposed to 2000 ppm [13C]MTBE for 6 h; 2000 scans were
recorded for Fourier transformation. Only the shift range
between 70 and 90 ppm is shown. The following structural
assignments were made: δ ) 80.5, tert-butyl alcohol sulfate; δ
) 76.6, 2-hydroxyisobutyrate; δ ) 74.3, 2-methyl-1,2-pro-
panediol; δ ) 73.7, tert-butyl alcohol glucuronide.
Comparison of the NMR spectra recorded from the
urine of the treated male and female rats did not indicate
relevant differences between the individual animals and
also did not indicate sex differences in the structures of
metabolites formed or in relative signal intensities when
the spectra were recorded under identical acquisition
conditions. Identical metabolites were also present,
albeit at lower concentrations, in the urines of both male
and female rats treated with [13C]MTBE collected be-
tween 24 and 48 h after exposure. In addition to
structure elucidation of metabolites by 13C NMR, all urine
samples obtained in these studies were also analyzed by
headspace GC/MS. In addition to the metabolites sug-
gested by NMR analysis in the urines of rats treated with
[13C]MTBE, the peak representing tert-butyl alcohol was
shifted by 1 mass unit and the peak representing acetone
showed fragments representing both [12C]acetone and
[13C]acetone (Figure 3) indicating the formation of [13C]-
acetone as a metabolite of [13C]MTBE. The concentra-
tions of [13C]acetone present were, however, too low to
result in a signal in the 13C NMR spectra.
F igu r e 2. Mass spectrum of 2-methyl-1,2-propanediol present
in the urine (collected for 24 h after the end of exposure) of a
male Fischer 344 rat exposed to [12C]MTBE (2000 ppm) for 6 h.
A compound with a similar mass spectrum with several frag-
ments shifted by 1 mass unit was observed in the urine of rats
exposed to [13C]MTBE.
spectrum of the [12C]MTBE metabolite in urine and that
of the synthetic reference were identical confirming
2-methyl-1,2-propanediol as a urinary metabolite of
MTBE. Moreover, in the mass spectrum of the peak
representing 2-methyl-1,2-propanediol after separation
of the urine of rats treated with [13C]MTBE, several
signals were shifted by 1 mass unit, further supporting
the assigned structure (m/z ) 76, 60, 58, 56, 44, 42, 40).
The NMR signal at δ ) 76.6 exhibited an identical
chemical shift as the C2 atom of hydroxyisobutyrate and
Biotr a n sfor m a tion of ETBE. A typical NMR spec-
trum of a urine sample from [13C]ETBE-exposed rats is
shown in Figure 4. The chemical shifts of the observed
signals were identical to the signals observed in the
urines of animals exposed to [13C]MTBE. Therefore, the
metabolites formed are identical with those resulting
from MTBE metabolism (δ ) 73.7, tert-butyl alcohol

Biotransformation of Fuel Oxygenates
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 655
F igu r e 5. 63-MHz 13C NMR spectrum of a 24-h urine sample
from a male Fischer 344 rat administered [13C]-tert-butyl alcohol
(250 mg/kg) by gavage; 2000 scans were recorded for Fourier
transformation. The following structural assignments were
made: δ ) 80.4, tert-butyl alcohol sulfate; δ ) 76.6, 2-hydroxy-
isobutyrate; δ ) 74.3, 2-methyl-1,2-propanediol; δ ) 73.7, tert-
butyl alcohol glucuronide; δ ) 72.6, tert-butyl alcohol.
F igu r e 4. 63-MHz 13C NMR spectrum of a 24-h urine sample
from a female Fischer 344 rat exposed to 2000 ppm [13C]ETBE
for 6 h; 2000 scans were recorded for Fourier transformation.
Only the shift range between 70 and 90 ppm is shown. The
following structural assignments were made: δ ) 80.5, tert-
butyl alcohol sulfate; δ ) 76.6, 2-hydroxyisobutyrate; δ ) 74.3,
2-methyl-1,2-propanediol; 73.7, tert-butyl alcohol glucuronide.
glucuronide; δ ) 74.3, 2-methyl-1,2-propanediol; δ ) 76.6,
2-hydroxyisobutyrate; δ ) 80.5, tert-butyl alcohol sulfate).
This indicates that the major ETBE metabolites, as well
as the major MTBE metabolites, result from tert-butyl
alcohol formed in the first metabolic step of the ether
biotransformation. Again, no significant differences in
signal intensities were observed in the urine of male and
female rats exposed to [13C]ETBE. In the urine collected
between 24 and 48 h after exposure, only the signal at δ
) 76.6 ppm was present, indicating that 2-hydroxyisobu-
tyrate is excreted slowly and over a relatively long period.
Neither high-resolution NMR nor GC/MS analysis of
the urine samples from ETBE-exposed rats indicated the
presence of ETBE metabolites formed by oxidation of the
â-carbon of the ethyl group in ETBE (e.g., tert-butyl glycol
or tert-butoxyacetic acid). Small amounts of [13C]acetone
were also present in the urines of rats exposed to [13C]-
ETBE as indicated by GC/MS of the headspace from
urine samples.
F igu r e 6. 63-MHz 13C NMR of a 24-h urine sample from a
male volunteer administered 5 mg/kg [13C]-tert-butyl alcohol.
The following structural assignments were made: δ ) 76.6,
2-hydroxyisobutyrate; δ ) 74.3, 2-methyl-1,2-propanediol; δ )
73.7, tert-butyl alcohol glucuronide; δ ) 72.6, tert-butyl alcohol;
δ ) 72.2 unknown.
Biotr a n sfor m a tion of ter t-Bu tyl Alcoh ol. Studies
on the metabolism of [13C]-tert-butyl alcohol were in-
cluded to confirm the structures of metabolites “down-
stream” from the formation of tert-butyl alcohol and to
identify if metabolic reactions of MTBE at sites other
than the methyl ether moiety may occur. Moreover, [13C]-
tert-butyl alcohol was available in sufficient amounts
from the synthesis of the 13C-labeled ethers. A typical
NMR spectrum of a urine sample from tert-butyl alcohol-
treated rats is shown in Figure 5.
Again, at chemical shifts between 70 and 90 ppm,
where only one resonance (δ ) 78.5) of an endogenous
compound was present, several signals indicative of tert-
butyl alcohol metabolites (δ ) 72.6, 73.7, 74.3, 76.6, 80.4)
were observed. In addition, several minor signals rep-
resenting either minor metabolites (not observed in other
experiments due to lower concentrations of 13C in urine)
or coupling of the 13C atom with 12C atoms were observed.
The signal at δ ) 72.6 represents tert-butyl alcohol.
This compound was identified by comparison of the
δ-value with that of a synthetic reference. Moreover,
addition of [13C]-tert-butyl alcohol greatly increased the
intensity of this signal. Analysis of the headspace of
urine samples from [12C]- and [13C]MTBE-treated rats by
GC/MS also confirmed the presence of [12C]- and [2-13C]-
tert-butyl alcohol. The chemical shifts of the other signals
were identical to those seen in urine after [13C]MTBE
inhalation exposure. On the basis of these observations
and MS data identical to those obtained with MTBE, tert-
butyl alcohol, tert-butyl alcohol glucuronide, and tert-
butyl alcohol sulfate, 2-hydroxyisobutyrate and 2-methyl-
1,2-propanediol are identified as urinary metabolites of
tert-butyl alcohol.
Identical metabolites were also present, albeit at lower
concentrations, in the urines collected between 24 and
48 h after exposure. In contrast to the MTBE inhalation
exposure, where 2-hydroxyisobutyrate was the most
prominent metabolite based on signal intensities after
acquiring spectral data under identical conditions, after
tert-butyl alcohol exposure, the presumed tert-butyl
alcohol sulfate was the main metabolite excreted. Head-
space analysis by GC/MS also indicated the presence of
small amounts of [13C]acetone in the urines of [13C]-tert-
butyl alcohol-treated animals.
In the urine of the human individual administered
[13C]-tert-butyl alcohol orally, the 13C NMR spectra
recorded (Figure 6) showed the presence of tert-butyl
alcohol, tert-butyl alcohol glucuronide, 2-hydroxyisobu-
tyrate and 2-methyl-1,2 propanediol. Moreover, an ad-
ditional NMR resonance (δ ) 72.2) not observed in control
urine taken before or 1 week after tert-butyl alcohol

656 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
Bernauer et al.
Sch em e 2. Biotr a n sfor m a tion of MTBE/ETBE a n d ter t-Bu tyl Alcoh ol (TBA) in Ra tsa
a
Excreted metabolites in urine are underlined.
administration was present in the urine samples. The
structures of this metabolite could not be elucidated. The
metabolites were present in all urine samples analyzed;
in contrast to rat urine samples where the presumed tert-
butyl alcohol sulfate was observed as a major product
excreted after tert-butyl alcohol administration, the
sulfate was only present in traces in the human urine
samples analyzed. In the human urine, 2-hydroxyisobu-
tyrate was the major metabolite excreted based on
relative intensities of the 13C NMR signals.
urine of rats within 48 h after exposure to 400 ppm [14C]-
MTBE for 6 h indicating intensive metabolism (21).
On the basis of the structures of the metabolites
elucidated, MTBE biotransformation in the rat proceeds
by oxidation of the methyl group in MTBE to give an
intermediate hemiacetal which decomposes to tert-butyl
alcohol with release of formaldehyde (Scheme 2).
tert-Butyl alcohol is also formed in vitro from MTBE
by a cytochrome P450-mediated oxidation reaction (22,
23). The involvement of cytochromes P450 2E1 and P450
2A6 in the transformation of MTBE to tert-butyl alcohol
in human liver microsomes has been established.
Discu ssion
In [13C]ETBE-exposed rats, identical metabolites were
identified as observed in MTBE-treated rats. On the
basis of relative signal intensities after identical acquisi-
tion conditions, the concentrations of metabolites present
in the urines of [13C]ETBE-treated animals within 48 h
after the end of the exposures were similar to those
observed in the urine of [13C]MTBE animals exposed
under identical conditions. This observation indicates
that there is a similar extent of metabolism of both ethers
to metabolites excreted in urine of rats after inhalation
exposure. These metabolite structures also suggest that
tert-butyl alcohol is also a major intermediate metabolite
of ETBE formed by a cytochrome P450-mediated oxida-
tion reaction. With ETBE, oxidation by cytochrome P450
seems to occur exclusively at the R-carbon atom of the
ethyl ether moiety since metabolites whose formation
could be explained by oxidation of the â-carbon (such as
tert-butyl glycol) were not detected. The finding of a
preferred oxidation on the R-carbon is in line with
observations on the biotransformation of ethyl ether
where ether cleavage by R-carbon oxidation is the major
The biotransformation of MTBE, ETBE, and their
presumed major metabolite tert-butyl alcohol was studied
in rats. In addition, the biotransformation of tert-butyl
alcohol was studied in one human individual. The major
aim of the study was to identify metabolites formed and
excreted in urine. The use of 13C-labeled ethers permit-
ted detection and identification of metabolites by 13C
NMR. Moreover, comparison of the mass spectra of urine
constituents from animals treated with 12C- or 13C-labeled
compounds also permitted identification of peaks repre-
senting metabolites due to the difference of 1 mass unit
in selected fragments of 13C-containing metabolites and
identification of metabolites of the [13C]ethers which were
also formed endogenously (e.g., acetone).
The MTBE studies confirmed tert-butyl alcohol, 2-meth-
yl-1,2-propanediol, and 2-hydroxyisobutyrate as MTBE
metabolites excreted in urine (20). Metabolite excretion
was not quantified in this study; however, other studies
have demonstrated that biotransformation significantly
contributes to the disposition of MTBE in rats. For
example, 65% of the radioactivity was recovered in the

Biotransformation of Fuel Oxygenates
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 657
pathway of biotransformation (24). Moreover, aliphatic
hydrocarbons such as n-hexane are also preferentially
oxidized at the w - 1 atom (25).
In ETBE biotransformation, the intermediate hemi-
acetal also decomposes to give tert-butyl alcohol and
acetaldehyde. The further fate of the aldehydes formed
in MTBE and ETBE biotransformation has not been
investigated; they are, however, expected to be rapidly
metabolized to formate and acetic acid.
The low amounts of tert-butyl alcohol recovered as the
free alcohol in urine of MTBE- and ETBE-exposed rats
and the presence of tert-butyl alcohol conjugates and
further metabolites downstream from tert-butyl alcohol
indicate intensive further metabolism of tert-butyl alcohol
in rats. Intensive metabolism of tert-butyl alcohol likely
formed as an intermediate in human MTBE biotrans-
formation is also indicated by the low recovery of tert-
butyl alcohol in the urines of human volunteers exposed
to MTBE (10).
Intensive biotransformation of tert-butyl alcohol in
rodents both by conjugation and by oxidation is confirmed
by results of our experiments with administration of
[13C]-tert-butyl alcohol. The NMR spectra obtained from
the urines of [13C]-tert-butyl alcohol-treated rats and the
human individual exposed to [13C]-tert-butyl alcohol
showed identical metabolites (tert-butyl alcohol conju-
gates, 2-methyl-1,2-propanediol, and 2-hydroxyisobu-
tyrate) as observed in the urines of MTBE- and ETBE-
treated rats.
Conjugation of tert-butyl alcohol with activated glucu-
ronic acid results in the excretion of the glucuronide
conjugate. A glucuronide conjugate of tert-butyl alcohol
has been described in rabbits (26), and these authors
recovered approximately 25% of the dose of tert-butyl
alcohol as glucuronide. Our study also provides indirect
evidence for the formation of a sulfate conjugate of tert-
butyl alcohol which, based on the intensities of the 13C
signal in the NMR spectra of the urine from animals
treated with 13C-labeled ethers and [13C]-tert-butyl alco-
hol, represents a major pathway of tert-butyl alcohol
biotransformation in the rat. The low recovery of the
presumed tert-butyl alcohol sulfate in the urine of the
human individual exposed to [13C]-tert-butyl alcohol is
likely based on a low affinity of human sulfotransferase-
(s) for tert-butyl alcohol. Species differences in the
capacities and substrate specificities of sulfotransferases
have been described (27). The two other metabolites
present in the urine of tert-butyl alcohol-treated animals
and also in urine of the rats exposed to MTBE and ETBE
(2-methyl-1,2-propanediol and 2-hydroxyisobutyrate) sug-
gest further oxidative metabolism of tert-butyl alcohol.
The likely pathway for the formation of these metabolites
involves oxidation of tert-butyl alcohol by cytochromes
P450 to give 2-methyl-1,2-propanediol. tert-Butyl alcohol
is not a substrate for alcohol dehydrogenase but is
oxidized by rat liver microsomes to formaldehyde and
acetone under conditions consistent with an involvement
of cytochromes P450 (28, 29). Cytochrome P450-medi-
ated oxidation of a C-H bond in one of the methyl groups
of tert-butyl alcohol results in the excretion of the diol
metabolite. Further oxidation of 2-methyl-1,2-propanediol
results in 2-hydroxyisobutyrate which is also excreted as
a major metabolite of tert-butyl alcohol, MTBE, and
ETBE.
Butyl alcohol as the origin of the excreted [13C]acetone
is confirmed by the obtained mass spectra confirming the
presence of 13C. Acetone is likely formed by further
oxidation of 2-hydroxyisobutyrate. Acetone was recov-
ered only as a very minor metabolite in the urine; other
studies on the biotransformation of tert-butyl alcohol in
rodents revealed inconsistent results in regard to acetone
formation: some authors reported significant increases
in blood acetone concentrations after tert-butyl alcohol
administration, while others failed to detect acetone as
a product formed in vivo from tert-butyl alcohol (28, 30).
Based on the known capacity of the human liver to
oxidize MTBE and ETBE to tert-butyl alcohol and
formaldehyde (23), and the results on the metabolism of
tert-butyl alcohol in one human, it may be anticipated
that there is a qualitatively identical biotransformation
of MTBE and ETBE in rats and humans. Presently, the
contribution of biotransformation reactions and of me-
tabolites formed to the toxic effects observed after
repeated administration of MTBE, ETBE, and tert-butyl
alcohol is unclear. Both MTBE and tert-butyl alcohol
induce nephropathy in rats and were found to increase
the incidence of renal tumors in male rats (3, 4, 31). The
R2u-globulin nephropathy syndrome has been suggested
to be involved in formation of renal tumors in male rats
after MTBE and tert-butyl alcohol. R2u-Globulin nephr-
opathy involves an impaired degradation of the male rat-
specific protein R2u-globulin in the kidney induced by
xenobiotics bound to this protein. The impaired degrada-
tion in the lysosomes of the kidney results in accumula-
tion of the modified R2u-globulin in the kidney tubular
epithelial cells, lysosomal rupture, cell death, and cell
proliferation. The induced cell proliferation is suggested
to be a major contributor to tumor formation. Tumors
are only observed in male rats due to the sex and species-
specific biosynthesis of R2u-globulin (7). The chemical
responsible for a possible binding to R2u-globulin in MTBE
and tert-butyl alcohol effects is not defined (4, 32). On
the basis of in vitro studies, unchanged MTBE may be
responsible for binding (33); however, a role for tert-butyl
alcohol formed as a metabolite may not be ruled out.
However, MTBE is only a weak inducer of R2u-globulin
accumulation, and other mechanisms may contribute to
toxic effects of this ether to the kidney. Our results
demonstrate that other metabolites than tert-butyl alco-
hol are also formed in vivo from MTBE and ETBE and
effects induced by these metabolites may also contribute
to the toxicities observed. The MTBE metabolites ob-
served in this study in rodents after inhalation of high
concentrations of MTBE (necessary for obtaining suf-
ficient amounts of metabolites for conclusive identifica-
tion) are also excreted in humans exposed to low and
occupationally relevant concentrations of MTBE (40 ppm)
(Amberg et al., unpublished).
Ack n ow led gm en t. Research desribed in this article
was conducted under contract from the Health Effects
Institute (HEI, Research Agreement No. 96-3), an orga-
nization jointly funded by the United States Environ-
mental Protection Agency (EPA) (Assistance Agreement
X-816285) and certain motor vehicle and engine manu-
facturers. 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. Parts of this work
were also supported by the Biomed Program of the
In this study, tert-butyl alcohol metabolism to small
amounts of acetone in vivo was also observed. [13C]-tert-

658 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
Bernauer et al.
of tris(2,3-dibromopropyl)phosphate on rat urinary metabolites:
assessment from 13C NMR spectra of urines and biochemical and
histopathological examinations. J . Appl. Toxicol. 7, 23-34.
European Union, Contract No. BMH4-CT96-0184.
Refer en ces
(19) Nicholson, J . K., and Wilson, I. D. (1987) High-resolution nuclear
magnetic resonance spectroscopy of biological samples as an aid
to drug development. Prog. Drug Res. 31, 427-479.
(20) Miller, M. J ., Ferdinandi, E. S., Klan, M., Andrews, L. S., Douglas,
J . F., and Kneiss, J . J . (1997) Pharmacokinetics and disposition
of methyl-tert-butyl ether in Fischer 344 rats. J . Appl. Toxicol.
17, S3-S12.
(21) HEI. (1996) The potential health effects of oxygenates added to
gasoline. Health Effects Institute, Cambridge, MA.
(22) Brady, J . F., Xiao, F., Ning, S. M., and Yang, C. S. (1990)
Metabolism of methyl tertiary-butyl ether by rat hepatic mi-
crosomes. Arch. Toxicol. 64, 157-160.
(23) Hong, J .-Y., Yang, C. S., Lee, M., Wang, Y.-Y., Huang, W.-Q., Tan,
Y., Patten, C. J ., and Bondoc, F. Y. (1997) Role of cytochromes
P450 in the metabolism of methyl tert-butyl ether in human livers.
Arch. Toxicol. 71, 266-269.
(24) Brady, J . F., Lee, M. J ., Li, M., Ishizaki, H., and Yang, C. S. (1988)
Diethyl ether as substrate for acetone/ethanol-inducible cyto-
chrome P-450 and as an inducer for cytochrome(s) P-450. Mol.
Pharmacol. 33, 148-154.
(25) Kra¨mer, A., Staudinger, H., and Ullrich, V. (1974) Effect of
n-hexane inhalation on the monooxygenase system in mice liver
microsomes. Chem.-Biol. Interact. 8, 11-18.
(26) Kamil, I. A., Smith, J . N., and Williams, R. T. (1953) The
metabolism of aliphatic alcohols. The glucuronic acid conjugation
of acyclic aliphatic alcohols. Biochem. J . 53, 129-136.
(1) Costantini, M. G. (1993) Health effects of oxygenated fuels.
Environ. Health Perspect. 6, 151-160.
(2) White, R. D., Daughtrey, W. C., and Wells, M. S. (1995) Health
effects of inhaled tertiary amyl methyl ether and ethyl tertiary
butyl ether. Toxicol. Lett. 82/83, 719-724.
(3) Burleigh-Flayer, H. D., Chun, J . S., and Kintigh, W. J . (1992)
Methyl tertiary butyl ether: vapor inhalation oncogenicity study
in CD-1 mice. BRRC Project Number 91N0013A, Bushy Bun
Research Center, Export, PA.
(4) Takahashi, K., Lindamood, C., and Maronport, R. R. (1993)
Retrospective study of possible R2u-globulin nephropathy and
associated cell proliferation in male Fischer 344 rats dosed with
tert-butyl alcohol. Environ. Health Perspect. 101, 281-286.
(5) Borghoff, S. J ., Prescott-Mathews, J . S., and Poet, T. S. (1996)
The mechanism of male rat kidney tumors induced by methyl tert-
butyl ether and its relevance in assessing human risk. CIIT
Activities 16, 1-8.
(6) Scientific Advisory Board on Toxic Air Pollutants. (1995) Sum-
mary of the carcinogenicity assessment of MTBE. Environ. Health
Perspect. 103, 420-422.
(7) Swenberg, J . A., Short, B., Borghoff, S., Strasser, J ., and Char-
bonneau, M. (1989) The comparative pathobiology of R2u-globulin
nephropathy. Toxicol. Appl. Pharmacol. 97, 35-46.
(8) Belpoggi, F., Soffritti, M., and Maltoni, C. (1995) Methyl-tertiary-
butyl ether (MTBE) - a gasoline additive - causes testicular and
lymphohaematopoietic cancers in rats. Toxicol. Ind. Health 11,
119-149.
(9) Duffy, J . S., Del Pup, J . A., and Kneiss, J . J . (1992) Toxicological
evaluation of methyl tertiary butyl ether (MTBE): testing
performed under TSCA consent agreement. J . Soil Contam. 1,
29-37.
(27) Falany, C. N., and Wilborn, T. W. (1994) Biochemistry of cytosolic
sulfotransferases involved in bioactivation. In Conjugation-de-
pendent carcinogenicity and toxicity of foreign compounds (Anders,
M. W., and Dekant, W., Eds.) pp 330-329, Academic Press, San
Diego.
(10) J ohanson, G., Nihle´n, A., and Lo¨f, A. (1995) Disposition and acute
effects of inhaled MTBE and ETBE in male volunteers. The
International Toxicologist, International Congress of Toxicology-
VII, Seattle, WA, J uly 2-6, 68-W-62.
(11) J ohanson, G., Nihle´n, A., and Lo¨f, A. (1995) Toxicokinetics and
acute effects of MTBE and ETBE in male volunteers. Toxicol. Lett.
83, 713-718.
(12) Gordian, M. E., Huelsman, M. D., Brecht, M. L., and Fisher, D.
G. (1995) Health effects of methyl tertiary butyl ether (MTBE)
in gasoline in Alaska. Alaska Med. 37, 101-103.
(13) Loizou, G. D., Urban, G., Dekant, W., and Anders, M. W. (1994)
Gas-uptake pharmacokinetics of 2,2-dichloro-1,1,1-trifluoroethane
(HCFC-123). Drug Metab. Dispos. 22, 511-517.
(14) Henderson, R. F., Sabourin, P. J ., Bechtold, W. E., Steinberg, B.,
and Chang, I. Y. (1993) Disposition of inhaled isobutene in F344/N
rats. Toxicol. Appl. Pharmacol. 123, 50-61.
(15) Kropf, H. (1975) Herstellung von Alkoholen durch Aufbaureak-
tionen. In Houben-Weyl. Handbuch der organischen Chemie, pp
1021-1022.
(28) Cederbaum, A. I., and Cohen, G. (1980) Oxidative demethylation
of tert-butyl alcohol by rat liver microsomes. Biochem. Biophys.
Res. Commun. 97, 730-736.
(29) Cederbaum, A. I., Qureshi, A., and Cohen, G. (1983) Production
of formaldehyde and acetone by hydroxyl-radical generating
systems during the metabolism of tertiary butyl alcohol. Biochem.
Pharmacol. 32, 3517-3524.
(30) Poet, T. S., Valentine, J . L., and Borghoff, S. J . (1997) Pharma-
cokinetics of tertiary butyl alcohol in male and female Fischer
344 rats. Toxicol. Lett. 92, 179-186.
(31) Lindamood, C. D., Farnell, D. R., Giles, H. D., Prejean, J . D.,
Collins, J . J ., Takahashi, K., and Maronpot, R. R. (1992) Sub-
chronic toxicity studies of tert-butyl alcohol in rats and mice.
Fundam. Appl. Toxicol. 19, 91-100.
(32) Prescott-Mathews, J . S., Wolf, D. C., Wong, B. A., and Borghoff,
S. J . (1997) Methyl tert-butyl ether causes R2u-globulin nephr-
opathy and enhanced renal cell proliferation in male Fischer-344
rats. Toxicol. Appl. Pharmacol. 143, 301-314.
(33) Poet, T. S., and Borghoff, S. J . (1997) In vitro uptake of methyl
tert-butyl ether in male rat kidney: use of a two-compartment
model to describe protein interactions. Toxicol. Appl. Pharmacol.
145, 340-348.
(16) Hu¨nig, S., Ma¨rkl, G., and Sauer, J . (1979) Integriertes organisches
Praktikum, Verlag Chemie, Weinheim.
(17) Meerwein, H. (1975) A¨ ther. In Houben-Weyl. Handbuch der
organischen Chemie, p 14.
(18) Fukuoka, M., Takahashi, T., Tanaka, A., Yamaha, T., Naito, K.,
Nakaji, Y., Kobayashi, K., and Tobe, M. (1987) Nephrotoxic effect
TX970215V