Clenbuterol and (3-Adrenergic
Drugs Detected in Hair of Treated
Animals by ELISA
To the Editor:
is a f3-adrenergic
Clenbuterol
agent
used therapeutically
to treat asthma
and bronchitis. In addition to its sympathomimetic stimulant
side effect,
its strong “anabolic-like”
properties
have been substantiated,
and illegal
uses as cattle growth
promoter
and
for muscling-in
in sport have been
reported (1, 2). Because its effects appear after chronic administration,
the
possibility
of drug accumulation
in
body compartments
should be considered. Tissues rich in melanin actively
take up clenbuterol,
and detection of
the drug in hair has been reported.’
Given
the retrospective
power of the
analysis of hair content, the availability of simple, rapid,
and sensitive
techniques
hair
tecting
tions
of
highly
relevant
previous
chronic
the drug.
One of the problems
sis is the
terial,
analysis
for clenbuterol
appear
in
for de-
administra-
in hair analy-
availability
of real-life
mai.e., hair in which the drug has
by physiological
mechanisms-in
contrast to reference
calibrations
prepared
by soaking or
supplementation.
The use of dosed
experimental animals
appears to be
an acceptable source of drug-containing hair samples. Here we present the
applicability
of a rapid ELISA methodology [originally developed to test
horse urine for clenbuterol
and, generically,
(3-agonists
(ELISA Technologies, Lexington, KY)] to detect denbuterol
and other (3-agonist drugs
(i.e., salbutamoh)
in hair obtained
from animals given the drugs under
controlled conditions.
Guinea
pigs received two-dosages
(low and high) of the drugs
for 15
days:
clenbuterol
(low dose, 0.015
mg/kg intramuscularly
every 2 days;
high dose, 0.12 mg/kg intraperitoneally daily) and salbutamol
(low dose,
0.28 mg/kg
intramuscularly
every
two days; high dose, 1.9 mg/kg intraperitoneally
daily). The drugs were
been
incorporated
‘Adam
A, Ayotte
C, Gervais
N,
Panoyan A, Dehehaut P, Beliveau L, Ong
H. Hair as a target site for the detection of
chenbuterol as drug residue. Presented at
2nd mt. Symp. on Hormone and Veterinary Drug Residue Analysis, Bruges,
1994. Abstract Book, p. 51.
administered
to separate
groups of
two animals each, one brown-haired
and one black-haired
guinea
pig in
each group. The brown-haired
animal
in the clenbuterol
group had a large
white hair spot, which allowed us to
obtain additional data for clenbuterol
accumulation
on white hair. Special
care was taken in the animals’ housing to avoid external contamination
by their own urines.
At the end of each study period,
hairs were obtained by plucking from
the animals. Hair samples (100 mg)
were washed in a 1 g/L sodium dodecyl sulfate solution (once) and in distilled water (3 times) and were then
digested in 2 mL of 2 mol/L NaOH for
30 mm at 80#{176}C.
We then added to the
digest 2 mL of the buffer from the
ELISA kit and adjusted the pH to 7.
After centrifugation,
20-.tL aliquots
of the supernates
were analyzed with
the ELISA, according to the manufacturer’s directions. Calibration
curves
were prepared with hair of untreated
animals
to which we had added
known
amounts
of clenbuterol
and
salbutamol;
the curves were linear
(logit B/B0 vs log concentration)
over
the range 0.01-1 ng of clenbuterol per
milligram
of hair (r = 0.984; denbuterol-specific
assay)
and
over
0.0 1-5 ng/mg hair for salbutamol
(r =
0.996; (3-agonists generic assay).
Clenbuterol
was
detected
and
quantified
in all samples
obtained
from animals treated with the drug
(Fig. 1). Concentrations
close to or
exceeding
0.1 ng/mg were obtained
only after
the high-dose
regimen,
with the black hair containing
substantially
higher amounts
than the
brown or white hair. Despite
the
larger dose of salbutamol,
its accumulation in hair was lower, being undetectable in the low-dose regimen.
Because results obtained by ELISA
methodologies
should be considered
only semiquantitative,
we also analyzed the hair extracts by gas chromatography-mass
spectrometry
(CC!
MS), using
the methyl
boronate
derivatives
of clenbuterol
and salbutamol (3, 4). Linear regression
of the
clenbuterol
results by ELISA (x) and
those by CC/MS (y) gave the equation
y = 0.89x - 0.01 (r = 0.994, S
=
18.3). The only two samples witi detectable salbutamol
by ELISA (high
dose) were quantified as 0.034 ng/mg
(brown) and 0.047 ng/mg (black) by
GC/MS. All these results
indicate
that ELISA semiquantification
gave a
relatively good initial estimate of the
real content of the hair samples.
We conclude that ELISA methodology after alkaline
digestion
of hair
appears
to be a useful method for
rapid and low-cost detection of denbuterol and other $3-agonists to identify illegal application
of these compounds.
We appreciate the financial support of
the Human Capital and Mobility Program
of the European
Union (project CHRXCT93-0274) and the Spanish Fondo de
Investigaciones
Sanitarias
(project FIS 94/
1376). Technical assistance was provided
by C.J. Sanchez and T. Smeyers.
References
1. Martinez Navarro JF. Food poisoning
related to consumption of illicit (3-agonist
in liver [Letter]. Lancet 1991;336:1311.
2. Muscling in on chenbuterol [Editoriall.
Lancet 1992;340:403.
3. Pohettini
A, Groppi
A, Ricossa
MC,
Concentration(ng/mg)
.402
0.4
Haircolour
Dwhite
O
0.3
Clenbuterol
Brown
#{149}
Black
0.2-
Saibutamol
0.1
Fig. 1. ELISA quantification of
clenbuterol and salbutamol in hair
obtained from treated guinea
pigs.
Lowdose
High dose
Low dose
High dose
ND, not detected.
CLINICAL CHEMISTRY, Vol. 41, No. 6, 1995
945
M. Gas chromatographic/electron impact mass spectrometric selective
Montagna
analysis
of clenbuterol in human and bovine urine. Biol Mass Spectrom 1993;22:457-61.
4. Zamecnik J. Use of cyclic boronates for
CC/MS screening
and quantitation
of
$3-adrenergic
blockers
and some bronchodilators. J Anal Toxicol 1990;14:132-6.
confirmatory
Aldo
1lnst.
Polettini’
Jordi Segura2’3
Gerard Gonzalez2
Xavier de J.a Torre2
Maria Montagna’
of Legal Med. (Univ. of Pavia)
Pavia, Italy
2lnst. Municipal
d’Invest.
Med.
IMIM- (JAB
Barcelona,
Spain
Address for correspondence:
Farmacol.
i Toxicol.,
Institut
Dept. de
Municipal
d’Investigaci#{243}
M#{232}dica
IMIM, Av. Dr.
Aiguader 80, 08003 Barcelona, Spain.
Reduced
Muscle Cell Phosphate
(PJ Without Hypophosphatemia in
Mild Dietary P Deprivation
To the Editor:
The plasma concentration
of inorganic phosphate [P11 has an ill-understood yet apparently important
influ-
ence on cellular metabolism
(1, 2),
although it bears no simple relationship to intracellular
[P1] (3, 4). Hypophosphatemia may be associated
with clinical abnormalities
of skeletal
muscle
(2, 5) and with muscle bioenergetic dysfunction
in vivo (5, 6) and
in vitro (7). Muscle cell [P1] can be
altered by reducing dietary intake of
P.. Muscle cell [P1], measured
by
chemical
assay
in freeze-clamped
muscle samples (i.e., total cell [P1]),
showed a marked decrease (45%)
in
rats subjected
to severe dietary P1
deprivation
(4-12 weeks of phosphorus at 0.25 g/kg of the diet vs 3.5 g/kg
in controls), while plasma P. was reduced by 66%
(7). A similar degree
of P, depletion in the mouse (0.9 g/kg
in diet vs 3.5 g/kg in controls, which
decreased
plasma [P1] by 54%) was
associated with slow recovery of phosphocreatine
(PCr) after exercise, suggesting
a defect of mitochrondrial
function; this effect was also demonstrable in isolated
mitochondria
(6).
This influence of P1 depletion on muscle
bioenergetics could explain the clinical
symptoms in some patients
with hypophosphatemia. However, hypophosphatemia does not always cause large
948
CLINICAL
CHEMISTRY,
Vol. 41, No. 6,
change in [ATP], even in hypophosphatemia
(6, 7, 11). Such a decrease
in cell [P1] without
a decrease
in
plasma [P1] suggests an alteration
of
the properties
of P1 transport
across
the cell membrane,
the simplest possibility being a reduction in the Na,P1
cotransport
that
accumulates
P,
against its free-energy gradient (12).
The relatively
high affinity
of this
transporter
for
extracellular
P1
should protect cell [P1] against large
changes
in plasma [P1] (12, 13). The
properties of this transporter
are also
in cytosolic [P.]. Muscle cell
P/ATP, measured
by 31P magnetic resonance
spectroscopy
(MRS) in human
subjects with renal P1 wasting,
was
much
less markedly
reduced
despite substantial
hypophosphatemia
(3,5,8).
The method
of measuring
muscle cell [P,] may explain some of the
differences
between
these studies. Total muscle P1 comprises
P1 in the cytosol, nucleus,
mitochondrial
matrix,
and other metabolically inaccessible regions of the cell. 31P MRS measures the
free cytosolic [P.], i.e., P1 that is freely
available for phosphorylation
of ADP.
To investigate the interrelationship
of plasma [P1], dietary phosphate
inchanges
take,
intracellular
apparently
[P1], and cell bioen-
ergetics, we used 31P MRS to study
muscle in vivo in Wistar rats subjected tomild dietary [P1] depletion (6
weeks of 2.6 g/kg dietary phosphorus
vs 5.0 g/kg in controls).
We studied
calf muscle in a 7-T magnet, measuring cell pH and the ratios of P1 and
PCr to ATP at rest and during 10 mm
of sciatic nerve stimulation
at 2 Hz
and subsequent
recovery (9). Resting
spectra were collected under fully relaxed conditions (15-s interpulse
delay); exercise and recovery spectra
were collected with 2-s interpulse
delay and the data were corrected for
incomplete
saturation
(9).
In P-deprived
animals
(Table 1),
we found no significant
change in
plasma [P1] compared with controls,
as shown previously
(10). Resting
muscle showed a 36% decrease
in
P1/ATP
and an 11% reduction
altered
in rats
made
ure-
mic by experimental
nephrectomy;
in
these rats, muscle cell [P1] is reduced
by 27% despite mild (30%) hyperphosphatemia,
possibly attributable
to circulating inhibitors
of the Na,
K4ATPase
(9). Rats injected for 4 days
with parathyroid
hormone
(PTH)
(11), in which the 42% decrease in
muscle cell [P1] exceeds the 22% decrease in plasma [P1], have a decrease
in cell [P1], possibly because of the
direct PTH inhibition
of the Na,Picotransporter
in muscle, similar
to its
well-known
action in renal tubular
epithelium.
What are the consequences of this
decrease in cell [Pt]? The decrease in
PCr/ATP suggests a decrease also in
cell [PCr]. This is consistent with the
need to keep constant the cytosolic
phosphorylation
potential,
[ATP]/
([ADP][P1]),
which is a controlling influence on resting mitochondrial ATP
synthesis; the decrease in [PCr] shifts
the creatine kinase equilibrium so as
to increase [ADP], which compensates for the decrease in [P.] (14).
Conversely,
an increase in resting
[PCr] is seen when cell [F1] increases
in response
to the
hyperphosphatemia
of chronic renal failure
(15).
The result of these changes in cell
in PCr/
ATP.
In response
to stimulation,
there was no significant
difference
in
the response of pH and PCr during
exercise or the rate of poststimulation
PCr recovery in P-deficient animals.
In resting muscle, the decrease in
P1/ATP strongly suggests a decrease
in free cytosolic [P1]; there was no
Table 1. Effect of P1depletion on rat muscle in vivo.
Mean
Plasma [Pj, mmoVL
Resting muscle (8)
P/ATP
PCr/ATP
Cell pH
0.53
3.66
t1,2, mm
SEM
Confr
± 0.10 (br
2.16
±
0.04
0.37
±
±
0.09
0.01
3.26
7.03
± 008b
±
0.01
6.96
±
0.01
0.48
1.3
± 0.04
± 0.2
7.04 ±
Stimulation (5)
Final cell pH
Finalrelative[PCr]c
PCr recovery
2.16
*
7.01 ± 0.01
0.53 ± 0.02
1.0 ± 0.1
P,-depleted
± 0.06(6)
#{149}
Numbers in parentheses refer to number of subjects.
b Sign ificantly different from controls (P <0.05) by Student’s unpaired f-test.
Changes in pH and [PCrJ
during stimulation were not significantly different between groups by repeated-measures analysis of
variance.
#{176}
Relativeto resting value.
1995