Animals by ELISA. To the Editor: Clenbuterol is a f3-adrenergic ... power of the analysis of hair content, ... the ELISA, according to the manufac- turer's directions.
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
