Inhibition of 1,4-butanediol metabolism in human liver in vitro

Article abstract of DOI:10.1007/s00210-011-0627-9

The conversion of 1,4-butanediol (1,4-BD) to gamma-hydroxybutyric acid (GHB), a drug of abuse, is most probably catalyzed by alcohol dehydrogenase, and potentially by aldehyde dehydrogenase. The purpose of this study was to investigate the degradation of 1,4-BD in cytosolic supernatant of human liver in vitro, and to verify involvement of the suggested enzymes by means of gas chromatography-mass spectrometry. The coingestion of 1,4-BD and ethanol (EtOH) might cause complex pharmacokinetic interactions in humans. Therefore, the effect of EtOH on 1,4-BD metabolism by human liver was examined in vitro. Additionally, the influence of acetaldehyde (AL), which might inhibit the second step of 1,4-BD degradation, was investigated. In case of a 1,4-BD intoxication, the alcohol dehydrogenase inhibitor fomepizole (4-methylpyrazole, FOM) has been discussed as an antidote preventing the formation of the central nervous system depressing GHB. Besides FOM, we tested pyrazole, disulfiram, and cimetidine as possible inhibitors of the formation of GHB from 1,4-BD catalyzed by human liver enzymes in vitro. The conversion of 1,4-BD to GHB was inhibited competitively by EtOH with an apparent K _i of 0.56 mM. Therefore, the coingestion of 1,4-BD and EtOH might increase the concentrations and the effects of 1,4-BD itself. By contrast AL accelerated the formation of GHB. All antidotes showed the ability to inhibit the formation of GHB. In comparison FOM showed the highest inhibitory effectiveness. Furthermore, the results confirm strong involvement of ADH in 1,4-BD metabolism by human liver.

Full text of DOI:10.1007/s00210-011-0627-9

Naunyn-Schmied Arch Pharmacol (2011) 383:647–654
DOI 10.1007/s00210-011-0627-9
ORIGINAL ARTICLE
Inhibition of 1,4-butanediol metabolism in human liver
in vitro
Daniel Lenz & Martin Jübner & Katja Bender &
Annette Wintermeyer & Justus Beike &
Markus A. Rothschild & Herbert Käferstein
Received: 29 August 2010 /Accepted: 23 March 2011 /Published online: 2 May 2011
#
Springer-Verlag 2011
Abstract The conversion of 1,4-butanediol (1,4-BD) to
gamma-hydroxybutyric acid (GHB), a drug of abuse, is
most probably catalyzed by alcohol dehydrogenase, and
potentially by aldehyde dehydrogenase. The purpose of this
study was to investigate the degradation of 1,4-BD in
cytosolic supernatant of human liver in vitro, and to verify
involvement of the suggested enzymes by means of gas
chromatography–mass spectrometry. The coingestion of
1,4-BD and ethanol (EtOH) might cause complex pharma-
cokinetic interactions in humans. Therefore, the effect of
EtOH on 1,4-BD metabolism by human liver was examined
in vitro. Additionally, the influence of acetaldehyde (AL),
which might inhibit the second step of 1,4-BD degradation,
was investigated. In case of a 1,4-BD intoxication, the
alcohol dehydrogenase inhibitor fomepizole (4-methylpyr-
azole, FOM) has been discussed as an antidote preventing
the formation of the central nervous system depressing
GHB. Besides FOM, we tested pyrazole, disulfiram, and
cimetidine as possible inhibitors of the formation of GHB
from 1,4-BD catalyzed by human liver enzymes in vitro.
The conversion of 1,4-BD to GHB was inhibited compet-
itively by EtOH with an apparent Ki of 0.56 mM.
Therefore, the coingestion of 1,4-BD and EtOH might
increase the concentrations and the effects of 1,4-BD itself.
By contrast AL accelerated the formation of GHB. All
antidotes showed the ability to inhibit the formation of
GHB. In comparison FOM showed the highest inhibitory
effectiveness. Furthermore, the results confirm strong
involvement of ADH in 1,4-BD metabolism by human
liver.
.
.
Keywords 1,4-Butanediol Gamma-hydroxybutyric acid
.
.
.
.
GHB Liquid ecstasy Metabolism Human liver enzymes
.
Alcohol dehydrogenase Aldehyde dehydrogenase
Introduction
Gamma-hydroxybutyric acid (GHB) is abused in doses of
20–30 mg/kg as a recreational drug (Couper and Marinetti
2002). Therefore, GHB is known as liquid ecstasy. If higher
amounts of this drug are ingested (≥50 mg/kg) it may cause
unconsciousness and coma, leading to its misuse as a date
rape drug at drug-facilitated sexual assaults. Therapeutical-
ly, the GHB sodium salt is used as anesthetic and for
treatment of narcolepsy and alcohol craving. Unlike GHB
its metabolic precursors gamma-butyrolactone (GBL) and
1,4-butanediol (1,4-BD) are not ranked as narcotic sub-
stances in many countries. Numerous lethal intoxications
caused either by intake of 1,4-BD, GBL, or GHB itself
have been reported (Kintz et al. 2005; Lenz et al. 2008;
Lora-Tamayo et al. 2003; Brent 2009).
After ingestion, 1,4-BD is metabolized to gamma-
hydroxybutyraldehyde and finally to GHB in a double-
stage, nicotinamide adenine dinucleotide (NAD)-dependent
reaction. It was shown that this reaction is most probably
catalyzed by alcohol dehydrogenase (ADH) and aldehyde
dehydrogenase (ALDH) using purified horse liver ADH
and cytosolic preparations from different rat organs con-
taining both ADH and ALDH (Fig. 1) (Bessman and
McCabe 1972; Poldrugo and Snead 1986).
:
:
:
:
D. Lenz (*) M. Jübner K. Bender A. Wintermeyer
:
:
J. Beike M. A. Rothschild H. Käferstein
Institute of Legal Medicine, University Hospital of Cologne,
Melatengürtel 60-62,
50823 Cologne, Germany
Ethanol (EtOH) is another substrate for ADH. After
administration of both EtOH and 1,4-BD to rats, increased
e-mail: daniel.lenz@uk-koeln.de

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Fig. 1 Putative metabolic pathway of 1,4-butanediol (1,4-BD) and
gamma-butyrolactone (GBL) in mammals: oxidation of 1,4-BD by
alcohol dehydrogenase (ADH) to gamma-hydroxybutyraldehyde,
which is converted to gamma-hydroxybutyric acid (GHB), potentially
by aldehyde dehydrogenase (ALDH). The hydrolysis of GBL is
catalyzed by serum lactonase. GHB is degraded by succinic semi-
aldehyde reductase. The resultant succinic semialdehyde undergoes
metabolism to succinic acid catalyzed by succinic semialdehyde
dehydrogenase. Both steps of GHB metabolism are nicotinamide
adenine dinucleotide phosphate (NADP)-dependent reactions
plasma and organ concentrations of 1,4-BD were observed
compared to a single administration of 1,4-BD (Fung et al.
2008; Poldrugo et al. 1985; Poldrugo and Snead 1986). The
increase of 1,4-BD concentrations may be caused by a
competition of EtOH and 1,4-BD for degradation by ADH,
whereas EtOH has a somewhat higher affinity to rat liver
The other toxicological issue of 1,4-BD is caused by its
degradation product GHB and may induce serious effects as
well. Among them, coma and severe respiratory depression
are most prominent. Until now, the only remedy for GHB
intoxication is a supportive management of vital functions.
However, in case of 1,4-BD intoxications the inhibition of
ADH or ALDH is a potential target in order to prevent
further formation of the central depressing GHB. The
prevention of 1,4-BD toxicity by blocking the conversion
to GHB has been discussed before (Carai et al. 2002; Carai
et al. 2006; Dempsey et al. 2009; Megarbane et al. 2002).
Carai et al. showed that mice receiving a lethal amount of
1,4-BD were resuscitated by a timely application of
fomepizole (FOM) (Carai et al. 2006). This suggests the
benefit of the inhibition of 1,4-BD metabolism in case of a
1,4-BD intoxication. However, until now a risk–benefit
assessment concerning this issue has not been investigated
in humans, whereas the prolonged effect of 1,4-BD itself
must be taken into account especially.
In this study the effect of EtOH on 1,4-BD metabolism
by human liver enzymes was investigated in order to
evaluate the pharmacokinetic interaction. Besides EtOH,
the EtOH metabolite acetaldehyde (AL) was employed as
an inhibitor as well, in order to evaluate the interaction on
the second stage of this metabolic pathway. The second
purpose was to investigate the in vitro efficiency of
potential antidotes for a 1,4-BD intoxication which may
inhibit the GHB formation. Beside the potent ADH
inhibitors fomepizole and pyrazole (PYR) the ALDH
inhibitor disulfiram (DSF) and cimetidine (CIM), an H2-
antihistaminic with a putative ADH-inhibiting property,
were tested by determination of their half-maximal inhibitory
concentration (IC50). These inhibitors were chosen due to
ADH compared to 1,4-BD (Km 1,4-BD, 0.75 mM; Km EtOH
,
0.16–0.5 mM) (Fung et al. 2008; Lad and Leffert 1983;
Poldrugo and Snead 1986; Reynier 1969; Zorzano and
Herrera 1989). Seventy-five minute after administration of
both drugs the 1,4-BD levels in brain, liver, and kidney
increased significantly at least by factor 3.5 compared to
animals receiving 1,4-BD only (Poldrugo et al. 1985).
Furthermore, an increased mortality of rats was observed
after administration of both (EtOH+1,4-BD, 50% mortal-
ity) 1,4-BD (1 g/kg) and EtOH (3 g/kg) in comparison to a
single administration of each drug (EtOH, 0% mortality;
1,4-BD, 6% mortality). As cause for the high mortality both
raised 1,4-BD levels reaching the LC50 and the summation
of the effects of 1,4-BD and EtOH were discussed
(Poldrugo et al. 1985). Considering a supposable consump-
tion of 1,4-BD with EtOH as recreational drugs these
results are alarming (Snead and Gibson 2005). It was
observed that the blood EtOH concentration of rats was not
affected by administration of both drugs (Poldrugo and
Snead 1984; Poldrugo et al. 1985). In contrast to the
findings of Poldrugo and Snead a population pharmako-
kinetic model, which is based on in vivo experiments with
rats, showed mutual inhibition of metabolism between
1,4-BD and EtOH (Fung et al. 2008). Until now, the in
vitro metabolism of 1,4-BD as well as the inhibition of this
pathway by EtOH have never been investigated with
human enzymes or tissue extracts.

Naunyn-Schmied Arch Pharmacol (2011) 383:647–654
649
their comparatively low toxicity, well-known pharmacolog-
ical properties, and, except for CIM and PYR, their clinical
approval (Brent 2009; Carai et al. 2002; Dawidek-Pietryka et
al. 1998). CIM is approved for the inhibition of gastric acid
secretion.
differences of the activity of ADH or ALDH due to, e.g.,
polymorphisms must be taken into account as well.
Ethical concern
This study has been performed in accordance with the
declaration of Helsinki and has been approved by the ethics
committee of the University of Cologne. All relatives were
informed about the purpose of this study and gave written
consent.
Material and methods
Chemicals and reagents
1,4-BD (≥99%), 1,5-pentanediol (1,5-PD, ≥97%), GHB
sodium salt, FOM (99%), cimetidine, pyrazole (98%), and
disulfiram (≥99%, DSF) were purchased from Sigma Aldrich
(Steinheim, Germany). Nicotinamide adenine dinucleotide
(free acid, gradient grade I, 100%) was obtained from Roche
Diagnostics (Mannheim, Germany). The methanolic solution
of sixfold deuterated GHB sodium salt (GHBd6; 1 mg/ml) was
delivered by LGC Promochem (Wesel, Germany). Ethanol
(≥99%, EtOH), water (ROTISOLV, gradient grade), di-sodium
hydrogen phosphate anhydrous (≥99%, p.a.), and potassium di-
hydrogen phosphate (≥99%, p.a.) were acquired from Carl
Roth (Karlsruhe, Germany). Acetonitrile (gradient grade) and
methanol (LiChrosolv, gradient grade) were purchased from
Merck (Darmstadt, Germany). The silylation agent N-methyl-
N-trimethylsilyl-trifluoroacetamide (MSTFA) was obtained by
Macherey-Nagel (Düren, Germany). The Uptima BC assay
protein quantitation kit was from Interchim (Montluçon,
France).
Incubations
All incubations were performed in 1.5 ml Eppendorf cups.
For the enzymatic degradation of 1,4-BD to GHB the
incubation time and concentration of both cofactor (nicotin-
amide adenine dinucleotide) and protein were optimized.
The incubation experiments were performed with a ther-
moshaker (Eppendorf, Hamburg, Germany) at 37°C and
800 rpm. The final reaction mixtures contained 6.25 μl
cytosolic supernatant and enough buffer to fill the volume
to 250 μl. The final NAD concentration was 5 mM. The
reaction was started by addition of cytosolic supernatant.
Unless specified otherwise, stock solutions of cofactor,
substrate, and inhibitors were prepared with 100 mM
phosphate buffer (pH 7.4). The final protein concentration
and incubation time were 525 μg/ml and 20 min, respec-
tively, which were in the linear range of GHB formation for
each parameter.
The inhibition of 1,4-BD metabolism at substrate
concentrations of 3, 5, 10, 20, 40, and 80 mM by EtOH
was investigated without inhibitor and at three different
EtOH concentrations (0.5, 1, and 2 mM EtOH). Addition-
ally, acetaldehyde was employed as inhibitor (0.5, 1, and
2 mM AL). Here the following 1,4-BD concentrations were
used: 1, 2, 3, 5, 10, 20, 40, and 80 mM. All incubations
were performed fivefold. Calibrators were prepared without
cofactor but with cytosolic supernatant as matrix.
The IC50 of potential antidotes were determined at three
different substrate levels (0.5, 2, and 5 mM 1,4-BD). The
1,4-BD concentrations were chosen according to the
determined Michaelis–Menten constant (Km) and to pub-
lished 1,4-BD concentrations listed in case reports (Lora-
Tamayo et al. 2003; Megarbane et al. 2002; Duer et al.
2001; Zvosec et al. 2001). Apart from controls (without
inhibitor, 100% activity) incubations were performed with
individual ranges of inhibitor concentrations (FOM, 0.01–
10,000 μM; PYR, 0.1–100,000 μM; CIM, 25–10,000 μM;
DSF, 2.5–1,000 μM). DSF stock solutions were prepared in
acetonitrile, since this agent exhibits insufficient water
solubility. Here, the acetonitrile content in the assay mixture
was 2% [v/v]. In this case the control (without DSF)
contained 2% [v/v] acetonitrile as well. Here, all incuba-
Cytosolic supernatant of human livers
Samples of ten human livers (five female and five male
donors; age, 43–79 years) were collected during autopsy
within 72 h after death and were stored at −80°C until
preparation of the pooled supernatants. The records of each
case document that there was no obvious influence (e.g., by
medication or liver disease) on the relevant enzymatic
system. Immediately after defrosting, the liver samples
were homogenized in ice-cold 100 mM phosphate buffer
(pH 7.4) using a blender (ratio 1 g–3 ml). The homogenates
were centrifuged at 15,000×g for 30 min at 2°C. Afterwards
all homogenates were pooled. The obtained extract was
centrifuged at 120,000×g for 45 min at 2°C. The pooled
supernatant was aliquoted in 1.5 ml Eppendorf cups and
stored at −80°C until utilization. The photometric determi-
nation of the total protein concentration was performed
with a protein quantitation kit, which is based on a method
using bicinchoninic acid (Smith et al. 1985). The activity of
the relevant enzymes depends on both the postmortem interval
and storage conditions. Although the donor bodies did not
show any relevant decomposition, a partial inactivation of
enzymes cannot be excluded. Furthermore, interindividual

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tions were assayed threefold and the incubation time was
60 min. In additional experiments both the degradation of
5 mM 1,4-BD and the formation of GHB were investigated
simultaneously. Here somewhat different conditions were
chosen. Briefly, besides a higher protein concentration
(17.0 mg/ml) a prolonged incubation (0–240 min) was
applied. 1,5-PD was used as internal standard for determi-
nation of 1,4-BD.
The reaction was stopped by adding 1,000 μl of an
acetonitrile/methanol mixture (95:5 [v/v]) containing GHBd6
and 1,5-PD as internal standards to 200 μl of the reaction
mixture. Calibrators were stopped immediately after prepa-
ration. The mixture was centrifuged at 21,100×g for 5 min.
Afterwards 1,000 μl of the supernatants were evaporated to
dryness at 40°C under a nitrogen stream. The residue was
reconstituted in 100 μl MSTFA and derivatized at 60°C for
20 min. One microliter of each sample or calibrator was
injected into the gas chromatography–mass spectrometry
(GC-MS) instrument.
Fig. 3 Degradation of 1,4-BD (filled square) at an initial concentra-
tion of 5 mM and formation of GHB (filled circle) in cytosolic
supernatant of human liver over a period of 240 min. Drug
concentrations are given as mean standard deviation, (n=5). Error
bars are often smaller than the symbol
spectrometer was operated with electron impact ionization at
70 eV in segmented scan mode (mass ranges, m/z 114–120
and m/z 201–242). The electron multiplier was set to 1,550 V.
For the detection of GHB- and GHBd6-di-trimethylsilyl
derivatives, the following fragments were used: m/z 117,
204, and 233 for GHB (GHB-di-TMS); m/z 119, 206, and
239 for GHBd6 (GHBd6-di-TMS). Peak area ratios of m/z
233 and 239 were used for quantitation (Fig. 2). All steps
such as data acquisition, processing, and evaluation were
conducted with the instrument software Xcalibur 1.3. The
accuracy (bias) determined at GHB concentrations of 10, 100,
and 500 μM was 16.7%, −3.6%, and 2.6%. The interday
precision (relative standard deviation) was 9.9%, 3.1%, and
2.9%, respectively.
Gas chromatography–mass spectrometry
A Thermo Trace GC ultra gas chromatograph—coupled
with a Polaris Q ion trap mass spectrometer—was used for
analysis (Thermo Fisher Scientific, Dreieich, Germany).
The injection was performed splitless. The GC-MS system was
equipped with a J&W HP-1ms GC-capillary column (100%
polydimethylsiloxane; 15 m×0.25 mm×0.25 μm; Agilent
Technologies, Waldbronn, Germany). The temperatures of
the injector, transferline, and ion source were set to 280°C,
290°C, and 250°C respectively. Helium 4.6 was used as
carrier gas with a flow rate of 1.0 ml/min. The following
temperature program was applied: 1 min at 60°C, 10°C/min
to 130°C, 40°C/min to 290°C, held for 1 min. The mass
For detection of di-trimethylsilyl-derivatives of 1,4-BD
(1,4-BD-di-TMS; m/z 177 and 219) and 1,5-PD (1,5-PD-di-
TMS; m/z 177 and 233) besides GHB and GHBd6, the
Fig. 4 Lineweaver–Burk plot showing the effect of ethanol on 1,4-
BD metabolism by cytosolic supernatant of human liver in vitro: no
inhibitor (filled circle), 0.5 mM (filled square), 1 mM (filled inverted
triangle), and 2 mM ethanol (filled triangle). 1/Rate [(micromoles per
Fig. 2 Chromatogram of an incubation mixture showing the ion
traces of 1,4-BD (m/z 177 and 219), GHBd6 (m/z 119, 206, and 239),
GHB (m/z 117, 204, and 233), and 1,5-PD-di-TMS (m/z 177 and 233)
minute per milligram of protein)−1] is given as mean
standard
deviation, (n=5)

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651
EtOH inhibited the conversion of 1,4-BD to GHB at
different concentrations (3–80 mM). The enzyme kinetic
data were fitted to a competitive inhibition model by a
nonlinear regression analysis yielding a sufficient correla-
tion (R2=0.965; Fig. 4).
The apparent Km, Vmax, and Ki were 5.5 mM, 10.0 nmol/
min/mg, and 0.56 mM, respectively. Unlike EtOH its
metabolite AL did not inhibit 1,4-BD degradation in vitro.
Surprisingly, an increased formation of GHB was observed
here, which is visualized by the Michaelis–Menten plot
shown in Fig. 5.
At AL levels of 0.5, 1, and 2 mM Vmax was increased by
factor 2.8, 3.4, and 3.8, respectively. The Scatchard plot of
these data revealed a parabolic curve at a constant AL level
(2 mM) as shown in Fig. 6b (Hill coefficient, 1.4).
All tested antidotes inhibited the metabolism of 1,4-BD to
GHB in cytosolic supernatant of human liver in vitro (Fig. 7).
The IC50 of the tested drugs are summarized in Table 1.
In all used substrate concentrations, FOM was the most
potent inhibitor. This was significant compared to PYR and
DSF at all tested concentrations. PYR was less potent than
FOM, but showed an almost complete inhibition of GHB
formation from 1,4-BD at high concentrations like FOM.
Due to its limited water solubility, CIM was not used at
concentrations higher than 10 mM in the assay. Here a
saturation of its effects was not reached. Therefore, the
determination of its IC50 was performed approximatively.
CIM was the weakest inhibitor by far. DSF showed an
incomplete inhibition, with about 40% residual metabolic
activity after saturation of its effects. Acetonitrile, which was
used as solvent for DSF, caused a decrease of the enzymatic
activity of about 30% at a concentration of 2% [v/v].
Fig. 5 Michaelis–Menten plot of gamma-hydroxybutyric acid forma-
tion from 1,4-butanediol at different AL levels in vitro: 0 mM (filled
circle), 0.5 mM (filled square), 1 mM (filled inverted triangle), and
2 mM AL (filled triangle)
same temperature program was applied in full scan mode
(m/z 170–240).
Calculation of pharmacological parameters and data
analysis
The enzyme kinetic parameters Km, inhibition constant (Ki),
and maximum rate (Vmax) were calculated using a nonlinear
regression analysis program (SigmaPlot Enzyme Kinetics
Module 1.1, SigmaPlot software, Chicago, IL, USA). The
IC50 values were calculated according to a sigmoidal
logistic four-parametric regression model using the Origin
8.1 software (OriginLab Corporation, Northampton, MA,
USA). The statistical significance (p<0.05) of the differ-
ences was evaluated using a t test.
Results
Discussion
Preliminary results showed that GHB is stable in the incubation
mixture, which was assayed for a period of 120 min. Further
investigations showed that GHB is formed almost as fast as the
initial amount of the substrate is decreased in an in vitro assay
with a high protein content (Fig. 3).
The results show that the metabolism of 1,4-BD is inhibited
by EtOH in cytosolic supernatant of human liver in vitro,
which confirms the thesis of potential pharmacokinetic
interaction of both drugs in human. Dempsey et al.
Fig. 6 Scatchard plots of gamma-
hydroxybutyric acid formation
from 1,4-butanediol in vitro: a
without acetaldehyde, b parabolic
curve progression in the presence
of acetaldehyde (2 mM), mean
standard deviation (n=5)

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gamma-hydroxybutyraldehyde (Poldrugo and Snead
1986). Another explanation for the incomplete inhibition
by DSF might be a delayed onset of inhibition, since the
mechanism of DSF seems to be based on the formation of
an intramolecular disulfide bond. This was supported by
experiments with rat liver mitochondrial ALDH, whereas
the inhibition of this enzyme was time dependent with
42% of initial activity after 10 min of incubation with DSF
(Shen et al. 2000). Fifty minutes later about 16% of
ALDH activity were left.
The GHB formation from 1,4-BD was accelerated by AL
in rat liver extract (Poldrugo and Snead 1986). The authors
expected an inhibition of 1,4-BD metabolism due to a
competition of AL and gamma-hydroxybutyraldehyde for
degradation by ALDH. A further degradation of the formed
GHB in the assay was not ruled out by the authors.
Poldrugo et al. discussed an inhibition of GHB degradation
by AL, whereby the GHB concentrations could have been
increased compared to incubations without AL.
Fig. 7 Inhibition of GHB formation from 1,4-BD (0.5 mM) in
cytosolic supernatant of human liver in vitro, given as residual
enzymatic activity standard error of mean, (n=3): fomepizole (filled
circle), pyrazole (filled square), disulfiram (filled inverted triangle),
and cimetidine (filled triangle)
demonstrated involvement of ADH in human 1,4-BD
metabolism (Dempsey et al. 2009).
FOM is a strong and specific competitive inhibitor of
ADH (Theorell and Yonetani 1969). Due to the almost
complete inhibition of GHB formation caused by FOM, our
results suggest that ADH is the main enzyme for 1,4-BD
degradation in our experiments and most probably in
human. Our results showed that GHB is formed almost as
fast as 1,4-BD is degraded. This suggests that the first step
of 1,4-BD metabolism is rate limiting. This issue is known
for EtOH metabolism as well, on the condition that there is
no ALDH deficiency (Eriksson 1983; Tsukamoto et al.
1989). Therefore, and according to Poldrugo et al., it can be
concluded that the estimated enzyme kinetic parameters
represent the kinetic of the first step of 1,4-BD metabolism
catalyzed by ADH (Poldrugo and Snead 1986).
The 1,4-BD degradation to GHB in cytosolic rat liver
extract was decreased by DSF (Poldrugo and Snead
1986). An incomplete antagonism of the sedative effects
(sleep time, onset of sleep) mediated by 1,4-BD was
observed in mice pretreated with DSF (Carai et al. 2002).
In our study, DSF inhibited the GHB formation up to 60%.
Therefore our results confirm the involvement of another
enzyme, which is responsible for the degradation of
In the present study, AL caused an acceleration of 1,4-BD
metabolism to GHB in human liver as well. The explanation
suggested by Poldrugo et al. can be excluded here, because
there was no degradation of GHB in the incubation mixture
for at least 120 min (data not shown). Obviously, the enzymes
being responsible for GHB metabolism were not active here.
Our findings suggest that the increased formation of GHB
from 1,4-BD might be caused by a positive cooperativity at
the dimeric ADH where AL acts as modulator. This is
visualized by the Michaelis–Menten plot in Fig. 5. Interest-
ingly, the Scatchard plot at a constant AL level (2 mM)
reveals a parabolic curve progression shown in Fig. 6b,
which suggests the involvement of 1,4-BD in this interaction
as well. If AL is not present, the Scatchard plot suggests
absence of cooperativity or positive allosteric regulation, as
shown in Fig. 6a. Further investigations are necessary for
the elucidation of this phenomenon. However, this issue
seems to be irrelevant concerning the pharmacokinetic
interaction of 1,4-BD and EtOH, because an increase of
GHB formation was not observed if both drugs were
incubated. Furthermore, an in vivo accumulation of AL,
which could theoretically increase GHB formation, seems
unlikely due to the fast degradation of AL (Eriksson
1983; Tsukamoto et al. 1989). If an increase of total
reaction rate was observed, the slower of both reactions
must have been accelerated here. The reaction catalyzed
by ADH is obviously rate limiting. A cooperativity at
ALDH, which catalyzes the oxidation of gamma-
hydroxybutyraldehyde, appears to be unlikely since this
would most probably not cause an increase of the total
reaction rate.
Table 1 Comparison of IC50 of tested antidotes
1,4-BD
concentration,
[mM]
IC50 standard error of mean (n=3), [μM]
Fomepizole Pyrazole Disulfiram Cimetidine
0.5
2
2.0 0.2
3.6 0.1
28.0 0.8* 21.5 2.6* 4,643 650*
60.7 2.7* 22.8 1.2* 4,795 1,369
FOM was applied to six probands before administration of
a low dose of 1,4-BD (10 mg/kg) in order to prevent GHB
formation (Dempsey et al. 2009). Although FOM decreased
5
6.2 0.2 120.0 4.5* 25.7 2.7* 4,002 395*
*p<0.05, statistically significant difference compared to fomepizole

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Brent J (2009) Fomepizole for ethylene glycol and methanol
poisoning. N Engl J Med 360(21):2216–2223
GHB formation, no significant differences of the effects
(vital signs, oxygen saturation, and subjective effects) caused
by 1,4-BD (control group) or 1,4-BD preceded by FOM
administration were observed. However, the rescuing effect
of a timely FOM administration was investigated in mice,
which received a lethal dose of 1,4-BD before (Carai et al.
2006). Even 90 min after 1,4-BD administration all animals
were resuscitated by 30 mg FOM/kg. In another study, the
complete blocking of the sedative effects of 1,4-BD was
observed in mice pretreated with FOM (Carai et al. 2002). If
FOM was administered to mice after receiving 1,4-BD, a
shorter impairment of their performance was observed
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within 3 h, although it is unknown whether FOM influenced
the clinical course. Our results show that among the tested
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The inhibition of 1,4-BD metabolism appears reasonable
in order to block the formation of central depressing GHB
in case of an acute 1,4-BD intoxication. However, a
thorough risk–benefit assessment is necessary concerning
the supposable increase of 1,4-BD levels after inhibition of
ADH. Especially in case of a 1,4-BD and EtOH overdose
this approach might cause an unpredictable increase of both
1,4-BD and EtOH levels. This should be considered
thoroughly in each individual case. EtOH should not be used
as an ADH inhibitor regarding the potentially dangerous
interactions between EtOH and 1,4-BD (Poldrugo and Snead
1984; Poldrugo et al. 1985).
Acknowledgment The authors would like to thank the JÜRGEN
MANCHOT STIFTUNG for financial support of this study.
Conflict of interest The authors declare that they have no conflict of
interest.
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