PURPOSE: This study was aimed at investigating the role of betaadrenoceptor subtypes in mediating the relaxation and contraction of seminal vesicles in rabbits. MATERIALS AND METHODS: Relaxation or contractile responses of epithelium- removed muscle strips of a rabbit seminal vesicle, which were precontracted with 10-5M norepinephrine, to selective betasubtypes-adrenoceptor agonists were observed in an organ bath. The contractile responses induced by isoproterenol were also observed after application of selective antagonists. RESULTS: Isoproterenol showed a concentration-dependent contractile response, but the contractility was weaker than those with phenylephrine and norepinephrine. The betaselective-agonists(xamoterol for beta, clenbuterol for beta and BRL37344 for beta) alone evoked neither contraction nor relaxation. However, the beta- and beta-agonists inhibited the contraction of the precontracted strips with 10-5M norepinephrine, while the beta-agonist enhanced the contraction. The pretreatment with a beta-antagonist(ICI118551) reduced the tension of the strips developed by 10-4M isoproterenol, but the beta-(atenolol) and beta-(SR59230A) antagonists showed no changes in the response. CONCLUSIONS: beta- and beta-adrenoceptors seem to mediate the relaxation of the seminal vesicle, while the beta-adrenoceptor may have a supplementary role in contraction.
Baths ; Clenbuterol ; Isoproterenol ; Negotiating ; Norepinephrine ; Phenylephrine ; Rabbits ; Relaxation* ; Seminal Vesicles*

OBJECTIVETo explore the status of clenbuterol pollution in swine products in Beijing city in 2002.

METHODSEuropean Union method (EUR 15127-EN Cy2.3) was adopted to examine the samples. Samples were screened by enzyme-linked immunosorbent assay (ELISA) and confirmed by GC-MS. Detected limit of the method was 0.5 micro g/kg. Samples, including lung, liver, pork, kidney and urine of swine, were collected from slaughterhouses, refrigeratories and markets in 11 districts of Beijing.

RESULTSThe results indicated that 185 out of 1 379 samples were positive with an annual positive rate of 13.4%. The highest was 15.7% in lung of swine, followed by urine 15.2% and pork liver 14.0%.

CONCLUSIONRates of detection had decreased from 30.0% to 2.7% during 2002.

Animals ; China ; Clenbuterol ; analysis ; Drug Residues ; analysis ; Food Contamination ; analysis ; Humans ; Meat ; analysis ; Meat Products ; Swine

OBJECTIVETo establish a novel suspension microarray technology for the detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17-beta-estradiol (CAP, CL and E2).

METHODSThe three conjugates that veterinary drug coupled with bovine serum albumin (BSA) were synthesized and identified by ultraviolet (UV) spectrophotometry and mass spectrum. The veterinary drug conjugates were immobilized on the polystyrene fluorescent microspheres/beads. There were competitive reactions between the veterinary drugs in the aqueous phase and that on the beads for combination with their specific biotinylated monoclonal antibodies. The optimum amount of the veterinary drug conjugates and the antibodies were optimized and selected. The detective standard curves were plotted. The specificity and the unknown samples were also determined by grouping according to different concentrations of the interferes and the samples. Meantime, the different microstructures of the surfaces of the beads were also observed by scanning electron microscope.

RESULTSCouplings were completed between small molecular veterinary drugs and BSA. The amounts of the three conjugates and the antibodies were optimized. The detective standard curves of the suspension array and their corresponding coefficients of determination (R2) were good (R2 > 0.99). The detection ranges of the three veterinary drugs were (40.00 - 6.25) x 10(5) ng/L, (50.00-7.81) x 10(5) ng/L and 1.00 x 10(3) - 7.29 x 10(5) ng/L respectively. Simultaneously, the specific detection of the suspension microarray was excellent and did not indicate significant cross-reactions. Errors between the found and the real are in the range of 8.09% - 17.03%. It can be considered that the relative standard deviations were relatively small. Successful couplings were also directly confirmed by the observation for microstructures of the surfaces of the beads by scanning of electron microscope and laid good foundation for the following responses.

CONCLUSIONThe high-throughput suspension microarray should provide a novel method for multi-analysis of the veterinary drugs and have a wide applicative prospects with simple operation, sensitive, rapid and low cost.

Chloramphenicol ; analysis ; Clenbuterol ; analysis ; Drug Residues ; analysis ; Estradiol ; analysis ; Microarray Analysis ; methods ; Veterinary Drugs ; analysis

AIMTo examine the liver mechanism with which clenbuterol (CL) is explained how to affect growth metabolism.

METHODSThe technique of chronic poly catheter was used to study the effects of CL (0.8 mg/kg b w) on the hepatic flux of nitrogen, VFA and glucose in 4 sheep.

RESULTSThe urea-nitrogen flux in CL-treated period always was lower than that in control during 24 h. The average flux of urea-nitrogen in hepatic and portal vein were decreased by 16.86% (P < 0.01) and 15.51% (P < 0.05), respectively, compared with that of control. The peptide level in hepatic vein was decreased with the treatment of CL, average flux of peptide was decreased by 38.71% (P < 0.01). But the peptide level of portal vein in CL treatment period was similar to control. Moreover, VFA level in the portal vein was enhanced by CL, the average flux of acetate in portal vein was increased by 19.49% (P < 0.01). No difference of VFA level in hepatic vein was noted between CL-treated period and control. In addition, the glucose flux in hepatic vein was obviously increased with CL treatment, the average flux of glucose was increased by 25.96% (P < 0.01). And glucose flux in portal vein was also elevated during CL-treated period.

CONCLUSIONCL can affect growth metabolism of animal with increasing nitrogen deposition, improving absorption and utilization of VFA and enhancing glucose synthesis in sheep liver.

Animals ; Clenbuterol ; pharmacology ; Fatty Acids, Volatile ; metabolism ; Glucose ; metabolism ; Liver ; drug effects ; metabolism ; Sheep

OBJECTIVETo study clinical features, treatment and curative effects in children with acute clenbuterol poisoning, in order to provide a basis for early diagnosis and treatment.

METHODSClinical data of 28 hospitalized children with acute clenbuterol poisoning in April 2011 were retrospectively studied.

RESULTSOf the 28 patients, there were 15 males and 13 females, aged 1 to 13 years (mean age 6.5±4.8 years). Vomiting, palpitations and limb shaking were found as main clinical manifestations in the patients. Main changes of blood biochemical included hypokalemia, lactic acidosis, hyperglycemia, hypsocreatinkinase. Snus tachycardia and S-T segment depression were observed on ECG. Patients' symptoms were gradually alleviated after 12-78 hours by use of beta blockers, potassium supplement, protecting the heart and other symptomatic and supportive treatment. Blood biochemical indexes were improved after 48 hours of admission. All of the patients were cured after 5 days. The symptoms of the patients do not longer occur during a follow up of half a month.

CONCLUSIONSAcute clenbuterol poisoning is characterized by vomiting, palpitations, limb shaking, hypokalemia, lactic acidosis and tachycardia in children. An early effective treatment of this disease can improve prognosis in children.

Acute Disease ; Adolescent ; Adrenergic beta-Agonists ; poisoning ; Child ; Child, Preschool ; Clenbuterol ; poisoning ; Electrocardiography ; Female ; Humans ; Infant ; Male ; Retrospective Studies

OBJECTIVETo identify the self-preparation monoclonal antibody which target to clenbuterol, and set up the standard curve to clenbuterol (CL) detection.

METHODSThe affinity constants and activity of the monoclonal antibody which target to CL were determined by ELISA. ELISA was also used to confirm whether the monoclonal antibody had any across-reaction with BSA and CL analogues. The rat ascites which contains the monoclonal antibody target to CL was purified by (NH4)2SO4 salt-out method and further by affinity column. At last, the CL detection standard curve which based on indirect competition ELISA was established.

RESULTSThe ELISA experiment showed that the antibody titer was 10(6) and the monoclonal antibody affinity constants was 2.90 x 10(10) L/mol. The result of the indirect competition ELISA confirmed that the monoclonal antibody had no cross-reaction with BSA and a few kind of CL analogue. CL detection standard curve based on indirect competition ELISA was established, which R2 was 0.9812, and the lowest detectable limit was 1.0 ng/ml.

CONCLUSIONThe standard curve based on indirectly competitioning ELISA was established. The self-preparation monoclonal antibody which target to CL has high affinity and high specific to CL, which had established the foundation to the advanced development of the CL immune test paper and CL ELISA kit.

Animals ; Antibodies, Monoclonal ; chemistry ; isolation & purification ; Antibody Affinity ; Clenbuterol ; immunology ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay ; Limit of Detection ; Rats

AIMTo study the effects of beta 2-adrenergic receptor-selective agonist clenbuterol on nitrogen metabolism and glucose-6-phosphate dehydrogenase activity of rat hepatocyte and its pharmacological mechanism.

METHODSBiochemical methods were used to study the influence of clenbuterol on urea-nitrogen concentration of hepatocyte culture medium, 3H-leucine incorporation into hepatocyte, insulin-like growth factor I (IGF-I) production and glucose-6-phosphate dehydrogenase (G6PDH) activity of rat hepatocyte.

RESULTSThe results showed that urea-nitrogen production by cultured rat hepatocytes was markedly affected with clenbuterol treatment (1 x 10(-6) mol.L-1), urea-nitrogen concentration of culture medium was decreased by 25.51% (P < 0.05) compared with control. The inhibitory effect of hepatocyte urea-nitrogen production of clenbuterol was blocked by propranolol, a beta-adrenoreceptor antagonist (1 x 10(-6) mol.L-1), but hepatocyte urea-nitrogen level was not affected with propranolol treatment only (P > 0.05). The content of 3H-leucine incorporation in rat hepatocyte was significantly increased by 23.35% (P < 0.05) with clenbuterol-treatment (1 x 10(-6) mol.L-1), and the enhanced effect of 3H-leucine incorporation into hepatocyte was antagonized by propranolol (1 x 10(-6) mol.L-1. The level of 3H-leucine incorporation of rat hepatocyte was not influenced by propranolol alone. IGF-I production of rat hepatocyte might be affected by clenbuterol. IGF-I concentration of culture medium was increased by 39.46% with clenbuterol (1 x 10(-6) mol.L-1), but no significant difference was found compared with the control (P > 0.05). Moreover, G6PDH activity of rat hepatocyte was significantly decreased by 43.36% (P < 0.05) with clenbuterol treatment (1 x 10(-6) mol.L-1), and the declined effect of clenbuterol was antagonized by propranolol. G6PDH activity of rat hepatocyte was not affected on condition that propranolol was administered alone (P > 0.05).

CONCLUSIONIt is suggested that clenbuterol may regulate nitrogen and fat metabolism by means of increasing nitrogen retention and protein synthesis, and decreasing G6PDH activity of rat hepatocyte for pharmacological effects.

Adrenergic beta-2 Receptor Agonists ; Animals ; Cells, Cultured ; Clenbuterol ; pharmacology ; Glucosephosphate Dehydrogenase ; metabolism ; Hepatocytes ; drug effects ; enzymology ; metabolism ; Nitrogen ; metabolism ; Rats ; Rats, Sprague-Dawley

To construct the recombinant vector pBV220-scFv and express anti-clenbuterol (CBL) scFv antibody in Escherichia coli, we amplified the scFv gene using plasmid pCANTABSE-CBL as a template, recombined it with pPICZalphaA, then amplified the scFv-His-tag gene from plasmid pPICZalphaA-scFv and linked it with expression plasmid pBV220. We identified the recombinant plasmid by restrictive enzyme digestion, PCR amplification and sequence analysis. Finally, we transformed the recombinant vector into E. coli DH5alpha that was temperature-induced and expressed recombinant protein. We identified the recombinant protein by SDS-PAGE, Western blotting and indirect competitive ELISA. The results show that recombinant plasmid pBV220-scFv contained the inserted fragment with highest homology about 99.8%. The expression of scFv induced by temperature show 37 kD Mw and anti-His-tag mAb recognized-activity by SDS-PAGE and Western blotting respectively, and could competitively combine with CBL, the IC50 is 4.55 ng/mL. The recombinant plasmid pBV220-scFv is constructed and expresses the scFv gene of CBL in E. coli successfully. This study suggests the corresponding immunoassay methods could be set up by the recombinant scFv.
Antibodies ; immunology ; Clenbuterol ; immunology ; Cloning, Molecular ; Genetic Vectors ; genetics ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

AIMTo detect the residual clenbuterol in pork meat and liver using HPLC with Coulometric electrode array system.

METHODSHomogenized meat or liver sample was treated with 1 mol x L(-1) hydrochloric acid and centrifuged, the fat existing in meat or liver tissue was removed by diethyl ether. The pH of the remaining aqueous layer was adjusted to 10.8 +/- 0.2 or 11.6 +/- 0.2 for meat or liver and liquid-liquid extraction with diethyl ether was followed. The ether extract was evaporated to dryness, the residue was dissolved in the mobile phase. The mobile phase A consisted of 50 mmol x L(-1) phosphoric acid-30 mmol x L(-1) triethylamine and was adjusted to pH 4.0 with 2 mol x L(-1) sodium hydroxide solution. The mobile phase B consisted of methanol-acetonitrile (30:45). A mixture of mobile phase A and B (80:20) was used in the method. A four electrode array module was selected for quantitation, the electrode potentials were set at 450, 600, 650 and 680 mV respectively.

RESULTSThe two calibration curves for meat and liver showed good linearity between 1.88 - 60.16 ng x g(-1), the detection limit of clenbuterol was 1.2 ng x g(-1).

CONCLUSIONThis method using HPLC-electrochemical detection is reproducible, and the sensitivity is good enough for the determination of clenbuterol in meat and liver.

Animal Feed ; analysis ; Animals ; Chromatography, High Pressure Liquid ; methods ; Clenbuterol ; analysis ; Drug Residues ; analysis ; Electrochemistry ; methods ; Electrodes ; Liver ; chemistry ; Meat ; analysis ; Swine

AIMTo obtain Clenbuterol monoclonal antibodies.

METHODSClenbuterol complete antigen was prepared with diazotization method. BALB/c mice was immunized with subtractive immunization, Clenbuterol monoclonal antibody was prepared with rule hybridoma technique.

RESULTSThe mice obtained tolerance to BSA by subtractive immunization. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%, and the specific clenbuterol monoclonal antibody was obtained at last.

CONCLUSIONMonoclonal antibodies to micromolecule contaminant be prepared by subtractive immunization, could decrease the workload in the bolting of monoclonal antibodies, and increase the chance to obtain the antibody of expected.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Clenbuterol ; immunology ; Female ; Hybridomas ; metabolism ; Immunization ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Serum Albumin, Bovine ; immunology