Stereoselectivity of porcine beta-adrenergic receptors for ractopamine stereoisomers

Ractopamine HCl is a beta-adrenergic receptor ((betaAR) ligand approved for use in swine to enhance carcass leanness. Ractopamine is produced commercially as a mixture of four stereoisomers (RR, RS, SR, SS). In order to determine which stereoisomers are active in the pig and whether they exhibit betaAR subtype selectivity, receptor affinity and adenylyl cyclase activation were determined using cloned porcine beta1- and beta2AR expressed in Chinese hamster ovary (CHO) cells. Dissociation constants (Kd) were determined by competitive displacement of [125I]iodocyanopindolol binding by ractopamine stereoisomers. The RR isomer had the highest affinity for both beta1- and betaAR (Kd of 29 and 26 nM, respectively). Dissociation constants for the other stereoisomers were higher (RS = 463 and 78 nM, SR = 3,230 and 831 nM, SS = 16,600 and 3,530 nM for the beta1- and beta2AR, respectively) relative to the RR stereoisomer. Isoproterenol stimulated adenylyl cyclase activity 600% relative to basal rates in CHO cells, regardless of betaAR subtype. Ractopamine stereoisomers did not significantly (P > 0.05) stimulate adenylyl cyclase through the beta1AR at moderate (near Kd) or high (10(-4) M) concentrations. In contrast, the RR isomer increased adenylyl cyclase activity 200 to 300% relative to basal rates through the beta2AR at moderate and hiconcentrations; the SR stereoisomer increased adenylyl cyclase activity nearly 100%. Neither the RS nor SS stereoisomers were effective in activating adenylyl cyclase activity through the beta2AR. A pattern of stereoselective activation similar to that for adenylyl cyclase also was exhibited for lipolysis using porcine adipocytes. The RR stereoisomer was equal to isoproterenol in stimulating lipolysis, whereas the SR isomer was 50% as effective; the RS and SR stereoisomers did not stimulate lipolysis in porcine adipocytes. The porcine betaAR exhibited stereoselectivity toward ractopamine stereoisomers with the RR isomer exhibiting the highest affinity for the (beta1- and beta2AR. In contrast, ractopamine stereoisomers seemed to be more effective at eliciting adenosine cyclic 3',5'-phosphate responses from beta2AR than beta1AR. The RR isomer ilikely the functional stereoisomer of ractopamine, but its effectiveness may be compromised by the presence of competing isomers, in particular the RS stereoisomer.     

Manipulation of porcine carcass composition by ractopamine

The effect of dietary ractopamine and protein level on growth performance, individual muscle weight and carcass composition of finishing pigs were evaluated in two experiments. Twelve barrows and 12 gilts (Exp. 1) and 32 barrows (Exp. 2) with an average initial weight of 64 kg were penned individually and offered ractopamine at 0 or 20 ppm in diets containing 13 or 17% CP in 2 x 2 factorial experiments for 28 d. In both experiments, dietary ractopamine improved daily gain (P less than .1) and gain-to-feed ratio (P less than .05) at 17% dietary protein level but depressed these response criteria at 13% protein level. Leaf fat was reduced (P less than .05) and longissimus muscle depth was increased (P less than .1) by feeding ractopamine regardless of dietary CP concentration. Longissimus, psoas major, semitendinosus, biceps and quadriceps femoris (P less than .05) and tensor facia latae (P less than .1) muscles were 8 to 22% heavier with ractopamine feeding at 17% dietary CP level. Results from both trials suggest that ractopamine improves growth rate and carcass leanness at the higher dietary protein level but improves only carcass leanness at the lower protein level.     

Insulin binding to mouse adipocytes exposed to clenbuterol and ractopamine in vitro and in vivo

Insulin binding to mouse adipocytes was measured after in vitro (30 min) and in vivo (5 days) exposure to clenbuterol and ractopamine. At 10(-6) M, both agonists decreased insulin binding by 20-30% after a 30 min preincubation at each insulin concentration between 1 and 25 ng/ml. Binding was not decreased if propranolol was present. Scatchard plots suggested that decreased binding was due to a decrease in insulin receptor concentration. Insulin binding was decreased approximately 10% at agonist concentrations as low as 10(-13) M, but binding was not further decreased until concentrations exceeded 10(-9) M. Rate of gain was increased 2-fold by clenbuterol (10 mg/liter of drinking water) and 50% by 500 mg ractopamine/liter, but not by 50 mg ractopamine/liter. Clenbuterol and ractopamine (500 mg/liter) decreased fat pad weight but only clenbuterol increased hind limb muscle mass. Insulin binding following in vivo administration was not influenced by ractopamine at 50 mg/liter, but tended to be increased by clenbuterol and ractopamine at 500 mg/liter. The disparity in results between administering the beta-agonists in vitro or in vivo suggests that counter regulatory factors influenced insulin binding capacity in vivo. Results indicate that ractopamine and clenbuterol can decrease insulin binding to adipocytes but the relevance of this response to decreased fat accretion is not clear.     

盐酸克伦特罗的检测方法

本文综述了当前盐酸克伦特罗的检测技术——化学分析法、色谱技术、毛细管区带电泳、免疫分析技术和生物传感器技术及发展趋势。     

Determination of affinity of Pasteurella multocida isolates for porcine respiratory tract mucus, and partial characterization of the receptors

The ability of 25 Pasteurella multocida isolates to adhere in vitro to porcine respiratory tract mucus was examined. Microplate wells were coated with crude mucus preparation, then bacteria were added. After incubation, unbound bacteria were removed by washing, and the number of mucus-bound bacteria was estimated by quantitation of the adherent colony-forming units and by use of an ELISA. Pasteurella multocida had affinity to respiratory tract mucus, although significant differences were not observed in affinity of capsular type-A and type-D isolates. Preliminary characterization, using ultrafiltration, gel filtration chromatography, electrophoresis, and enzymatic treatments, indicated that the receptors may be a class of protein molecules of low molecular weight (less than 25,000). The origin of these receptors, however, is not known at this time.     

Regulation of porcine adipocyte metabolism by insulin and adenosine

The acute effects of insulin and adenosine on rates of lipolysis and lipogenesis in pig adipocytes were investigated to determine what limits the expression of the insulin response in vitro. Adenosine and insulin independently inhibited isoproterenol-stimulated lipolysis. Adenosine, acting through the pertussis toxin-sensitive G-protein Gi, was more effective than insulin and could completely inhibit lipolysis. Fatty acid synthesis from glucose was increased by both adenosine and insulin. Neutralization of endogenous adenosine with adenosine deaminase decreased basal rates of lipogenesis and increased the insulin response from 30 to 60% above basal. Neutralization of Gi with pertussis toxin further decreased the basal rate and increased the insulin response to 160% above basal. These data indicate that Gi, and the ligands that signal through Gi, stimulate glucose incorporation into fatty acids and can attenuate the insulin response. It seems likely that an exaggerated rate of glucose metabolism in the absence of insulin contributes to the inconsistent insulin responses exhibited in pig adipose tissue in vitro. These data also demonstrate that insulin and adenosine have major roles in regulating pig adipose tissue metabolism.     

饲料中莱克多巴胺的HPLC法测定

用碱性甲醇提取试样中的莱克多巴胺,经SLA固相萃取柱净化,浓缩后用2%乙酸溶液定容,过膜后用高效液相色谱-荧光检测法分离测定;色谱柱类型Waters Svmmetry C18柱,250mm×4.6mm(i.d.),粒径5μm;流动相为戊烷磺酸钠溶液:乙腈(体积比80:20);激发波长为226nm,发射波长为306nm;流速1ml/min;进样量50μl;饲料中莱克多巴胺的检测限为0.5mg/kg;莱克多巴胺的测定在0.02-0.50μg/ml范围内具有良好的线性关系,平均回收率在85.1%以上,RSD小于7.5%;方法简单,灵敏度高,可用于饲料中莱克多巴胺的含量测定。     

Tissue residues of ractopamine and urinary excretion of ractopamine and metabolites in animals treated for 7 days with dietary ractopamine

Ractopamine HCl is a beta-adrenergic leanness-enhancing agent recently approved for use in swine. Depletion of ractopamine in tissues, and elimination of ractopamine and its metabolites in urine, is of interest for the detection of off-label use. The objectives of this study were to measure the residues of ractopamine in livers and kidneys of cattle (n = 6), sheep (n = 6), and ducks (n = 9) after treatment with dietary ractopamine for seven (sheep, ducks) or eight (cattle) consecutive days and to measure the depletion of ractopamine from urine of cattle and sheep. Two cattle and sheep and three ducks were each slaughtered with withdrawal periods of 0, 3, and 7 d. Urine samples were collected daily from cattle and sheep. Tissue ractopamine concentrations were determined using the regulatory method (FDA approved) for ractopamine in swine tissues. Ractopamine residues in urine samples were measured before and after hydrolysis of conjugates. Analysis was performed with HPLC using fluorescence detection after liquid- (hydrolyzed samples) and(or) solid-phase extraction. No residues were detected in duck tissues. Liver residues in sheep averaged 24.0 and 2.6 ppb after 0- and 3-d withdrawal periods, respectively. Sheep liver residues after a 7-d withdrawal period were less than the limit of quantification (2.5 ppb). Sheep kidney residues were 65.1 and undetectable at 0- and at 3- and 7-d, withdrawal periods, respectively. Cattle liver residues were 9.3, 2.5, and undetectable after 0-, 3-, and 7-d withdrawal periods, respectively; kidney residues were 97.5, 3.4, and undetectable at the same respective withdrawal periods. Concentrations of parent ractopamine in sheep urine were 9.8+/-3.3 ppb on withdrawal d 0 and were below the LOQ (5 ppb) beyond the 2-d withdrawal period. After the hydrolysis of conjugates, ractopamine concentrations were 5,272+/-1,361 ppb on withdrawal d 0 and 178+/-78 ppb on withdrawal d 7. Ractopamine concentrations in cattle urine ranged from 164+/-61.7 ng/mL (withdrawal d 0) to below the LOQ (50 ppb) on withdrawal d 4. After the hydrolysis of conjugates in cattle urine, ractopamine concentrations were 4,129+/-2,351 ppb (withdrawal d 0) to below the LOQ (withdrawal d 6). These data indicate that after the hydrolysis of conjugates, ractopamine should be detectable in urine of sheep as long as 7 d after the last exposure to ractopamine and as long as 5 d after withdrawal in cattle.     

动物组织中莱克多巴胺残留的不确定度评定

为保证实验室测试结果的准确性和可靠性,依照国家计量技术规范JJF1059—199《测量不确定度评定与表示》,对气相色谱-质谱法测定动物组织中莱克多巴胺残留量进行了不确定度的评定。分析和量化了影响测定结果的各不确定度分量,得出了被测量的合成标准不确定度和扩展不确定度。当动物组织中莱克多巴胺残留量为17．72μg／kg时,其合成标准不确定度为0．47μg／kg,扩展不确定度为0．94μg／kg（k=2）。     

酶联免疫法测定饲料中盐酸克仑特罗含量

采用酶联免疫法测定饲料中盐酸克仑特罗的含量。方法的最低检测限为100μg/kg,线性范围0.1～2.7μg/L,r=0.9997。浓度为100、500、1000μg/kg的添加水平时,回收率分别为74％、81％和90％;板内变异系数<15.0％,板间变异系数<14.4％。此方法快速、灵敏、准确、经济,具有实际应用价值。     

高效液相色谱/质谱联用仪测定盐酸克仑特罗

利用高效液相色谱质谱检测器 ,建立了检测盐酸克仑特罗的方法。其中质谱 (SIR)检出限为 0 .0 1～ 5 μg/kg ,液相色谱中外检测器的检测限为 0 .3μg/kg ,最大吸收峰 2 96nm ,符合农业部规定动物性食品中的盐酸克仑特罗检测限 :1μg/kg。本方法可用于定量、定性检测     

ELISA法测定禽组织中克仑特罗残留量

用ELISA法对家禽血清、肌肉和肝脏等组织中克仑特罗的残留量进行了检测，肝脏和肌肉组织的回收率分别为82．4％和78．6％。GC-MS法证实，ELISA方法灵敏度高、快速有效，适用于禽类产品中克仑特罗残留量的筛选检测。     

Acute effects of beta-adrenergic agonists on porcine adipocyte metabolism in vitro

Backfat was obtained at slaughter from market weight hogs to study the acute effects of clenbuterol (CB), ractopamine (RAC) or epinephrine (EPI), in the presence and absence of theophylline (THEO) or adenosine deaminase (ADA), on rates of lipolysis and fatty acid synthesis in vitro. Only EPI increased lipolytic rate in the absence of THEO or ADA. In the presence of THEO or ADA, RAC and CB were lipolytic, although CB had a lower maximal response. With THEO present, RAC and EPI increased lipolysis with a similar potency and responsiveness. Lipolytic responses from all agonists were prevented by propranolol. Insulin stimulated glucose incorporation into fatty acids 50 to 100%; stimulated rates were not influenced by any agonist, either alone or in the presence of ADA. When THEO was present, EPI and RAC inhibited fatty acid synthesis approximately 50%. Clenbuterol was not inhibitory under any conditions. Results indicate that, under appropriate conditions, beta-adrenergic agents increase lipolysis and decrease lipogenesis in porcine adipocytes. Combined evidence suggests that lipolysis is more sensitive to beta-adrenergic stimulation than is insulin-stimulated lipogenesis. Finally, RAC and CB possess only partial agonist activity relative to EPI, CB being least active.     

盐酸克伦特罗快速检测卡的检测效果评价

主要考查盐酸克伦特罗快速检测卡的检测限、重现性、稳定性、检测范围等指标,同时考查假阳性与假阴性率,以及检测温度和光照对试纸条检测结果的影响。结果显示,该检测卡最低检出限为4ng/ml,pH值对检测结果影响不大,但不同的环境条件影响检测结果。     

饲料中盐酸克伦特罗的气相色谱-质谱法测定分析

探索了饲料中盐酸克伦特罗的气相色谱-质谱法测定方法。饲料试样中盐酸克伦特罗经0.01mol/l盐酸溶液提取,乙醚脱脂净化,乙酸乙酯提取后蒸发至干,用乙醇溶解后加样到氧化铝柱上,用0.01mol/l盐酸溶液洗脱,蒸发至干后,分别用三甲基氯硅烷(TMCS)和乙酸酐衍生,采用GC/MS/SIM方式进行测定,均达到了保留时间之差不大于2s(分别为0.002min,0.004min),匹配度大于800(分别为863,923)的定性要求,符合农业部行业标准NY/T468—2001。     

莱克多巴胺人工抗原的合成与鉴定

用戊二酸酐将盐酸莱克多巴胺活化成莱克多巴胺-戊二酸酐半醛,用混合酸酐法将莱克多巴胺-戊二酸酐半醛与KLH和BSA偶联,合成免疫原和包被抗原,经核磁共振法和紫外吸收法鉴定,偶联成功.用紫外吸收法测定免疫原和包被抗原的偶联比分别为24：1和2.5：1.用免疫原免疫新西兰白兔,采用间接ELISA法检测抗体效价,采用间接竞争ELISA法检测IC50值,获得了效价为1：6 000以上、IC50值为10 ng/mL的多克隆抗体,证明合成的人工免疫原具有免疫原性.