Full text of DOI:10.1093/chromsci/40.9.495

Journal of Chromatographic Science, Vol. 40, October 2002
Determination of the Norepinephrine Level by
High-Performance Liquid Chromatography to Assess
the Protective Effect of MAO-B Inhibitors Against
DSP-4 Toxicity
"
Diana Haberle*, Kálmán Magyar, and Éva Szöko
Department of Pharmacodynamics, Semmelweis University, Nagyvárad tér 4, Budapest, H-1089 Hungary
raphy–electron capture detection (EC) (3,4), gas chromatog-
Abstract
raphy–mass spectrometry (5,6), high-performance liquid chro-
matography (LC)–ultraviolet detection (7), LC–fluorometric
detection (8), LC–electrochemical detection (9,10), and
radioenzymatic assay methods (11,12). The LC–EC method is
popular because of its simplicity. It has been suggested that the
selectivity and sensitivity of this method can be further
enhanced by the modification of the detector assembly, thus
LC–EC became the method of choice (10). EC is intrinsically
highly sensitive and selective compared with other analytical
techniques, and the analysis of biological samples containing
a considerable amount of various contaminants is usually pos-
sible with minimum sample pretreatment. In this study an
LC–EC method was used to determine the neurotoxic effect of
the selective noradrenergic toxin N-(2-chloroethyl)-N-ethyl-2-
bromobenzylamine (DSP-4), which causes severe and long-
lasting depletion of NE content in various parts of a rodent
brain (13,14). We also investigated the possibilities to eliminate
DSP-4 toxicity.
Liquid chromatography (LC) combined with electrochemical
detection (EC) is suitable for measuring oxidizable biogenic amine
levels in small samples of brain tissue. The norepinephrine (NE)
content in mouse hippocampus after treatment with various
monoamine oxidase-B enzyme (MAO-B) inhibitors ([–]-deprenyl,
[+]-rasagiline, and the noradrenergic neurotoxin
N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine [DSP-4]) is
determined using an LC–EC method. Treatment with a single
intraperitoneal dose of (–)-deprenyl (selegiline) before DSP-4
administration markedly reduces the NE depleting effect of the
toxin, and (+)-rasagiline does not significantly modify the NE level
decreased by the neurotoxin. The MAO-B inhibitory potency of
(–)-deprenyl and (+)-rasagiline is also evaluated. Significantly
reduced MAO-B enzyme activity in mouse brain and liver is
measured 6 h after treatment with their single dose. (+)-Rasagiline
is found to be a more potent MAO-B inhibitor than (–)-deprenyl.
Introduction
Although the presence of norepinephrine (NE) in brain
tissue had been demonstrated biochemically in the 1950s and
its transmitter role suspected, detailed analysis of its neuronal
distribution only became possible when the fluorescent tech-
nique (based on the formation of a fluorescent derivative of cat-
echolamines when tissue is exposed to formaldehyde) was
devised by Falck and Hillarp (which is based on the formation
of a fluorescent derivative of catecholamines when tissue is
exposed to formaldehyde). Control noradrenergic systems orig-
inate from the pontine tegmentum, the locus ceruleus, and
locus subceruleus (1,2). Numerous analytical methods have
been elaborated to measure the NE level in different parts of
the brain. The NE level can be measured by gas chromatog-
Experimental
Chemicals
(–)-Deprenyl (N-methyl-N-propinyl-[2-phenyl-1-methyl]-
ethylammonium chloride) was obtained from Chinoin Phar-
maceutical Works (Budapest, Hungary). (+)-Rasagiline
(N-propinyl-1-amino-indane) and DSP-4 were kindly donated
by TEVA (Debrecen, Hungary) and Svante B. Ross (Södertälje,
•
Sweden), respectively. β-Phenylethylamine HCl (β-PEA) and
the chemicals for the HPLC–EC assay (perchloric acid, sodium-
bisulfite, 3,4-dihydroxybenzylamine [DHBA], methanol, dis-
odium-phosphate, citric acid, ethylenediaminetetraacetate
[EDTA], and octane-sulfonic acid) were obtained from Sigma
14
14
•
* Author to whom correspondence should be addressed.
(Budapest, Hungary). 2-Phenyl-(1- C)-ethylamine HCl ( C-
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495

Journal of Chromatographic Science, Vol. 40, October 2002
PEA) (specific activity of 41.8 mCi/mmol) was purchased from
Du Pont de Nemours GmbH (NEN Life Science Products,
Boston, MA).
deprenyl and (+)-rasagiline. Before treatment, animals were
deprived of food for 16 h, but they had free access to water. The
animals were sacrificed by decapitation 6 h after drug admin-
istration. The liver and brain were dissected on an ice-chilled
aluminum surface. The nuclei-free homogenate of the organs
was used for the determination of enzyme activity.
Animals
Male NMRI mice (20–25 g) (TOXICOOP, Budapest, Hun-
gary) were housed five in a cage in the animal house under a
12:12 light–dark cycle at 22°C 1°C. Tap water and standard
mice pellets were available ad libitum. Experiments were
started after an adaptation period of at least 2 days.
Determination of monoamine oxidase-B enzyme activity
Monoamine oxidase–B enzyme (MAO-B) activity was deter-
mined radiometrically based on the method of Wurtman and
Axelrod (15) with slight modification (16). 14C-PEA was used as
a substrate. Protein concentration was measured according to
the method of Lowry et al. (19).
Treatment and tissue preparation for the measurement of
the hippocampal NE level
DSP-4 was applied as a single 50-mg/kg intraperitoneal (i.p.)
injection. (–)-Deprenyl and (+)-rasagiline (1 mg/kg, i.p.) were
administered 1 h before DSP-4. In all cases mice were decapi-
tated after 7 days from the treatment with the toxin. The sub-
stances were dissolved in distilled water immediately before
administration. Control animals received the same volume of
vehicle. After the mice were sacrificed, the brain was removed
and the hippocampal region was dissected on an ice-chilled alu-
minum surface and stored at –80°C. The tissue samples were
sonicated for 30 s in 100 µL of cold 0.2M perchloric acid con-
taining 0.1% Na2S2O3 and 3 ng/20 µL DHBA as the internal
standard. After centrifugation for 20,000 rpm at +4°C for 20
min, the supernatant (20 µL) was removed and the concen-
tration of NE in the supernatant was determined by the LC–EC
method.
Statistics
In order to keep the resulting experimental error at a con-
stant level, data were analyzed by one-way analysis of variance
(Statistica 5.0 for Windows, Stat Soft Inc., Tulsa, OK) followed
by post hoc analysis. Each treatment group was compared
with each other by the method of least significant difference,
and the conventional level of p < 0.05 was used as the criterion
of significance.
Results
The NE content in mouse hippocampus was determined by
an LC–EC method. The chromatogram of the NE standard is
shown in Figure 1. A single i.p. injection of DSP-4 (50 mg/kg)
induced an 80–90% reduction of hippocampal NE content in
mice 1 week after treatment. An i.p. administration of 1 mg/kg
(–)-deprenyl 1 h before DSP-4 injection considerably reduced
the NE-depleting effect of the toxin. Pretreatment with the
other MAO-B inhibitor (1 mg/kg [+]-rasagiline) did not affect
the DSP-4-induced NE depletion (representative chro-
matograms after various treatments are shown in Figure 2).
The mean NE contents measured in the hippocampus after var-
ious treatment schedules are summarized in Table I.
Apparatus
The LC apparatus consisted of a pump (PU-1580) (Able &
Jasco, Budapest, Hungary), a Rheodyne injector (Able & Jasco)
with a 20-µL injector sample loop, a C18-filled guard column
(Hypersil ODS 5, 4 × 4 mm) (Sigma-Aldrich Kft., Budapest,
Hungary), and a SupelcoSil reverse-phase column (LC-18-DB,
3 µm, 25 x 4.6 mm) (Supelco, Sigma-Aldrich Kft.). A Decade
amperometric detector (Able & Jasco) with a glassy carbon
electrode (VT-03 analytical cell) was used for EC detection.
The inhibition of MAO-B enzyme activity in the brain and
liver measured 6 h after the i.p. treatment of mice with either
1 mg/kg (–)-deprenyl or (+)-rasagiline is shown in Figure 3.
Both compounds induced more than 80% enzyme inhibition in
the brain. In the liver, the MAO-B inhibition produced by (+)-
rasagiline was significantly higher.
Chromatographic conditions
The mobile phase (isocratic elution) contained 5% methanol
(v/v) in a 0.05M phosphate–citrate buffer of pH 3.2, which con-
tained 0.025mM EDTA and 1mM octanesulfonic acid. The flow
rate of the mobile phase was 1.0 mL/min and the injection
volume was 20 µL. External standards were regularly injected
in order to identify and calibrate the peaks from the injected
samples. The standards were diluted from a stock solution (1
mg/mL) with 0.2M perchloric acid containing 0.1% Na2S2O3.
The quantitative determination of each standard showed linear
regression at the concentration range from 0.1 ng/20 µL to 10
ng/20 µL, and the equation of the calibration line was y =
–0.0215 + 4.2573x and the correlation coefficient (R) = 0.9999.
The limit of detection was 0.1 ng/20 µL.
Standards
Time (min)
Figure 1. Representative chromatogram of NE standard (1 ng/20 µL) and
the internal standard DHBA (1 ng/20 µL). For details see chromatographic
conditions.
Treatment schedule to measure monoamine
oxidase-B enzyme activity
Mice were treated i.p. with single doses of 1 mg/kg (–)-
496

Journal of Chromatographic Science, Vol. 40, October 2002
disease (24), can provide protection against selective dopamin-
ergic (MPTP, 6-hydroxydopamine), cholinergic (AF64A), and
noradrenergic (DSP-4) toxins as well. According to this study,
(–)-deprenyl in a single 1-mg/kg i.p. dose 1 h before DSP-4
injection almost completely prevented the NE-depleting effect
Discussion
The LC–EC method combination has become the method of
choice for the determination of tissue biogenic amines and
their metabolites. This technique has the advantages of sen-
sitivity, selectivity, and is more favorably
applicable for the determination of low
amounts of catecholamines in biological
fluids (17,18) and tissue homogenates
(19,20). LC–EC has several attractive fea-
tures when compared with both classical
fluorescence and radioenzymatic
methods. The technique is rapid and
selective. The sensitivity approaches that
of the previous methods for most types of
samples. LC–EC combines the selectivity
of LC with that of electrochemistry,
resulting in a highly specific and sensitive
assay. This opens the field for the analysis
of a wide variety of pharmaceuticals,
agricultural chemicals, and industrial
intermediates as well. Some of these com-
pounds have physiological or patholog-
ical importance, and LC–EC is a useful
tool in neurotoxicological and neurophar-
macological works.
Control
The neurotoxin DSP-4 causes severe
and long-lasting depletion of NE content
in various parts of a rodent brain (13,14).
This effect is most prominent in brain
areas that obtain the majority of their NE
terminals from the locus ceruleus (21).
One of the targets of DSP-4 action seems
to be the carrier system of NE transport
into the neurons (22). Previously pub-
lished data showed that DSP-4 (a tertiary
amine) can spontaneously generate a qua-
ternary ammonium derivative in aqueous
media, whose positively charged struc-
ture was suggested to be responsible for
the long-lasting impairment of the NE
uptake process, which in turn causes
depletion of NE (22). The reduced amount
of NE after DSP-4 treatment can be ana-
lyzed reproducibly and accurately by
LC–EC.
(+)-Rasagiline + DSP-4
(–)-Deprenyl + DSP- 4
Previous studies showed that the neu-
rotoxic effect of DSP-4 can be inhibited by
pretreatment with NE uptake blockers,
such as desipramine, cocaine, or zimeli-
dine (13). We intended to study whether
any other (not NE uptake blocker) sub-
stances are capable of diminishing the
effect of DSP-4. (–)-Deprenyl, which is a
selective, irreversible inhibitor of the
MAO-B (23) and is widely used alone or in
Time (min)
Figure 2. Representative chromatograms of NE extracted from mice hippocampus after various treatment
schedule: (A) NE level after treatment with saline; (B) NE level 7 days after DSP-4 (50 mg/kg i.p.) treat-
ment; (C) NE level 7 days after pretreatment with (+)-rasagiline (1 mg/kg i.p.) and treatment with DSP-4
(50 mg/kg i.p.); (D) NE level 7 days after pretreatment with (–)-deprenyl (1 mg/kg i.p.) and treatment with
DSP-4 (50 mg/kg i.p., time between pretreatment and treatment was one hour in each case).
combination with
L-3,4-dihydroxyphenyl-
alanine in the treatment of Parkinson’s
497

Journal of Chromatographic Science, Vol. 40, October 2002
of the toxin. The protective effect of (–)-deprenyl against the
dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine can be explained by the MAO-B inhibitory potency
of the substance (25). Supposing that the MAO-B inhibitory
action of the compound is related to its protective effect against
DSP-4, other enzyme inhibitors should also be protective. (+)-
Rasagiline is more potent to inhibit MAO-B enzyme, and its
effect on brain MAO-B activity lasts longer than that of (–)-
deprenyl. However, (+)-rasagiline pretreatment did not influ-
ence the effect of the toxin significantly. Based on these results,
the effect of (–)-deprenyl against DSP-4 is unrelated to MAO-
B enzyme inhibition. This finding is in agreement with that of
Finegan (26) when (–)-deprenyl pretreatment 24 h before DSP-
4 was not effective in preventing NE depletion, although MAO-
B inhibition was still complete when the toxin was injected.
Several hypotheses have been proposed regarding the neu-
roprotective mechanism of (–)-deprenyl. One of them has sug-
gested that (–)-deprenyl can inhibit the uptake of biogenic
amines (27,28) and such an effect may be relevant in its pro-
tection against DSP-4 (12). (–)-Deprenyl is a structural analog
of (–)-methylamphetamine ([–]-MA) and is converted to (–)-
MA, (–)-desmethyldeprenyl, and (–)-amphetamine ([–]-A) by
CYP-450 enzymes in the liver (29–31). The main metabolites of
(–)-deprenyl, (–)-MA, and (–)-A are more effective uptake
blockers than the parent compound (32), but they can also
enhance the release of NE and dopamine from the nerve end-
ings (33). The possible contribution of the metabolites to the
protective effect of (–)-deprenyl against DSP-4-induced NE
depletion has also been suggested (34). The mechanism of the
protective effect of (–)-deprenyl needs to be further clarified.
Acknowledgments
This study was supported by the National Fund for Scientific
Research (OTKA) Grant No. T 025213.
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