连苯三酚在山羊体内的毒物动力及其临床病理学研究
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摘要
为了研究连苯三酚在山羊体内的毒物动力学及其对机体的毒性作用,建立了气相色谱测定连苯三酚在山羊血清中浓度的方法,研究了其在体内的动力学过程以及对家兔的临床病理学特征。主要结果如下。
1.气相色谱测定血清中连苯三酚方法的建立通过对固定液、衍生化试剂、进样口温度、检测器温度、升温方法、气体流速的优化,确立了最佳气相色谱条件:固定液采用6%的丁二酸乙二醇聚酯;衍生化试剂为N,O-双(三甲基硅烷基)三氟乙酰胺;柱温170℃维持3 min,20℃/min升温至190℃,维持3 min后降温7 min;进样口温度280℃,检测器温度280℃;氮气流速40 mL/min;氢气流速40 mL/min;空气流速400 mL/min。采用气相色谱法测定连苯三酚,血清中内源性成分不干扰连苯三酚的分离测定,连苯三酚的保留时间为4.3 min左右,血药浓度在16μg/mL～200μg/mL内,与其峰面积有良好的线性关系(r=0.9967),最低检测浓度10μg/mL。回收率80%～98.71%,日内精密度小于3.31%,日间精密度小于5.97%,符合体内药物测定要求。
2.连苯三酚在山羊体内的动力学研究山羊口服100 mg/kg连苯三酚后不同时间采血,测定血清连苯三酚浓度。连苯三酚在山羊体内的动力学过程符合有吸收因素的一室开放模型。消除半衰期t1/2为(2.4646±0.6472) h,吸收半衰期t1/2ka为(0.6237±0.0516)h。曲线下面积AUC为(973.7847±82.41)μg·mL~(-1)·h。达峰时间(Tmax)为(1.7181±0.2379)h,达峰浓度Cmax为(173.5979±17.7271)μg·mL~(-1)。连苯三酚在山羊体内为快速消除。
3.连苯三酚对家兔的临床病理学研究给家兔每周以100 mg·kg~(-1)、50 mg·kg~(-1)、25 mg·kg~(-1)的剂量灌服连苯三酚,连续4周。在每次给受试物后第7天自家兔心脏采血,分离取血清作为测定样本,试验结束后对其剖检,取心、肝、脾、肺、肾等脏器制作石蜡切片,进行组织病理学检查。结果表明,与正常对照组的相比,试验组血清丙氨酸氨基转移酶、门冬氨酸氨基转移酶、血清尿素氮、血肌酐明显升高,差异极显著或显著(P<0.01或P<0.05)。肾小管上皮细胞变性,脱落。肾小管肿胀,胞浆混浊,管腔狭窄。肝脏中央静脉扩张,充血,肝细胞肿胀变性。脾窦有轻微的淤血现象。心肌纤维和心肌细胞发生颗粒变性。肺泡壁毛细血管扩张,充血,肺泡壁有巨噬细胞浸润。
The gas chromatography method which could determine the concentration of pyrogallol in Serum was established in order to study toxicokinetics of pyrogallol and toxicity to animal body.The toxicokinetics process in goats and clinic pathology in rabbits were studied.The main results were as follows.
1. The gas chromatography(GC) method establishment Through optimizing fixed liquid, derived reagent, the injection chamber temperature, the Flame Ionization Detector (FID) chamber temperature, the method of arising temperature and gas velocity of flow, the optimal conditions of gas chromatography were determined: the fixed liquid for 6% polydieth-ylene glycol succinate(DEGS), derived reagent for N,O-Bis-(trimethylsilyl) trifluoroacetamide (BSTFA), column initial temperature for 170℃after 3 min rising to 190℃at the rate 20℃/min, and holding for 3 minutes, then falling for 7 minutes, the injection chamber temperature for 280℃, the FID chamber temperature for 280℃, the flow velocity of nitrogen gas for 40 mL/min, the flow velocity of hydrogen gas for 40 mL/min, the flow velocity of air for 400 mL/min. The endogenesis components of serum did not disturb the measure of pyrogallol by gas chromatography. The reservered time was about 4.3 min. The calibration curve was linear in the concentration range from 16μg.mL ~(-1) to 200μg.mL ~(-1) ( r = 0.9967) . The detection limit was 10μg.mL ~(-1) and the method recoveries were from 80% to 98.71%. The RSD within a day was less than 3.31%and the RSD between days was less than 5.97%, therefore these indexes were accorded with the request for drug measure.
2. The study of toxicokinetics The blood was plucked at different time post administrating orally Pyrogallol to goats at the dose of 100 mg·kg~(-1). The concentration of pyrogallol in Serum was determined by GC. The result of toxicokinetics showed that the toxicokinetics process of pyrogallol in goats was accorded with one compartment open model with absorption factor. The elimination half time (t1/2) was (2.4646±0.6472) h, which indicated that pyrogallol was quickly eliminated in goats body. The half-time of absorption phase(t1/2ka) was (0.6237±0.0516) h. The area under curve(AUC) was (973.7847±82.41)μg·mL~(-1)·h. The maximun time was (1.7181±0.2379) h and the maximum concentration(Cmax) was (173.5979±17.7271)μg·mL~(-1).
3. The study of Clinic pathology to rabbits The rabbits were administrated orally Pyrogallol at the dose of 100 mg·kg~(-1)and 50 mg·kg~(-1)and 25 mg·kg~(-1) once a week for four times. Every time, after administrating orally pyrogallol to the rabbits, the blood was plucked from heart on the seventh day. Serum was separated as sample.Then, the rabbits were dissected. The heart, liver, spleen, lung and kidney were collected and made paraffin section for investigation of their pathologic histology. The results of clinic pathology showed that alanine aminotransferase(ALT), aspartate aminotrasferase (AST), blood urea nitrogen(BUN)and creatinine of test groups comparing with that of control group were significantly different or different (P<0.01 or P<0.05). The epithelium cell of renal tubular was degenerated and falling off. Renal tubular was tumid. The renal tubular epithelium cytoplasm was turbid, and renal tubular antrum was cabined. The center veins of liver were dilated and congestive. Liver cells were tumid and degenerated. Spleen sinus had slight gore. Heart muscular fibers and cells were degenerated. Alveolus wall capillary vessels were dilated and congestive. Alveolus wall was infiltrated by macrophage.
1.气相色谱测定血清中连苯三酚方法的建立通过对固定液、衍生化试剂、进样口温度、检测器温度、升温方法、气体流速的优化,确立了最佳气相色谱条件:固定液采用6%的丁二酸乙二醇聚酯;衍生化试剂为N,O-双(三甲基硅烷基)三氟乙酰胺;柱温170℃维持3 min,20℃/min升温至190℃,维持3 min后降温7 min;进样口温度280℃,检测器温度280℃;氮气流速40 mL/min;氢气流速40 mL/min;空气流速400 mL/min。采用气相色谱法测定连苯三酚,血清中内源性成分不干扰连苯三酚的分离测定,连苯三酚的保留时间为4.3 min左右,血药浓度在16μg/mL～200μg/mL内,与其峰面积有良好的线性关系(r=0.9967),最低检测浓度10μg/mL。回收率80%～98.71%,日内精密度小于3.31%,日间精密度小于5.97%,符合体内药物测定要求。
2.连苯三酚在山羊体内的动力学研究山羊口服100 mg/kg连苯三酚后不同时间采血,测定血清连苯三酚浓度。连苯三酚在山羊体内的动力学过程符合有吸收因素的一室开放模型。消除半衰期t1/2为(2.4646±0.6472) h,吸收半衰期t1/2ka为(0.6237±0.0516)h。曲线下面积AUC为(973.7847±82.41)μg·mL~(-1)·h。达峰时间(Tmax)为(1.7181±0.2379)h,达峰浓度Cmax为(173.5979±17.7271)μg·mL~(-1)。连苯三酚在山羊体内为快速消除。
3.连苯三酚对家兔的临床病理学研究给家兔每周以100 mg·kg~(-1)、50 mg·kg~(-1)、25 mg·kg~(-1)的剂量灌服连苯三酚,连续4周。在每次给受试物后第7天自家兔心脏采血,分离取血清作为测定样本,试验结束后对其剖检,取心、肝、脾、肺、肾等脏器制作石蜡切片,进行组织病理学检查。结果表明,与正常对照组的相比,试验组血清丙氨酸氨基转移酶、门冬氨酸氨基转移酶、血清尿素氮、血肌酐明显升高,差异极显著或显著(P<0.01或P<0.05)。肾小管上皮细胞变性,脱落。肾小管肿胀,胞浆混浊,管腔狭窄。肝脏中央静脉扩张,充血,肝细胞肿胀变性。脾窦有轻微的淤血现象。心肌纤维和心肌细胞发生颗粒变性。肺泡壁毛细血管扩张,充血,肺泡壁有巨噬细胞浸润。
The gas chromatography method which could determine the concentration of pyrogallol in Serum was established in order to study toxicokinetics of pyrogallol and toxicity to animal body.The toxicokinetics process in goats and clinic pathology in rabbits were studied.The main results were as follows.
1. The gas chromatography(GC) method establishment Through optimizing fixed liquid, derived reagent, the injection chamber temperature, the Flame Ionization Detector (FID) chamber temperature, the method of arising temperature and gas velocity of flow, the optimal conditions of gas chromatography were determined: the fixed liquid for 6% polydieth-ylene glycol succinate(DEGS), derived reagent for N,O-Bis-(trimethylsilyl) trifluoroacetamide (BSTFA), column initial temperature for 170℃after 3 min rising to 190℃at the rate 20℃/min, and holding for 3 minutes, then falling for 7 minutes, the injection chamber temperature for 280℃, the FID chamber temperature for 280℃, the flow velocity of nitrogen gas for 40 mL/min, the flow velocity of hydrogen gas for 40 mL/min, the flow velocity of air for 400 mL/min. The endogenesis components of serum did not disturb the measure of pyrogallol by gas chromatography. The reservered time was about 4.3 min. The calibration curve was linear in the concentration range from 16μg.mL ~(-1) to 200μg.mL ~(-1) ( r = 0.9967) . The detection limit was 10μg.mL ~(-1) and the method recoveries were from 80% to 98.71%. The RSD within a day was less than 3.31%and the RSD between days was less than 5.97%, therefore these indexes were accorded with the request for drug measure.
2. The study of toxicokinetics The blood was plucked at different time post administrating orally Pyrogallol to goats at the dose of 100 mg·kg~(-1). The concentration of pyrogallol in Serum was determined by GC. The result of toxicokinetics showed that the toxicokinetics process of pyrogallol in goats was accorded with one compartment open model with absorption factor. The elimination half time (t1/2) was (2.4646±0.6472) h, which indicated that pyrogallol was quickly eliminated in goats body. The half-time of absorption phase(t1/2ka) was (0.6237±0.0516) h. The area under curve(AUC) was (973.7847±82.41)μg·mL~(-1)·h. The maximun time was (1.7181±0.2379) h and the maximum concentration(Cmax) was (173.5979±17.7271)μg·mL~(-1).
3. The study of Clinic pathology to rabbits The rabbits were administrated orally Pyrogallol at the dose of 100 mg·kg~(-1)and 50 mg·kg~(-1)and 25 mg·kg~(-1) once a week for four times. Every time, after administrating orally pyrogallol to the rabbits, the blood was plucked from heart on the seventh day. Serum was separated as sample.Then, the rabbits were dissected. The heart, liver, spleen, lung and kidney were collected and made paraffin section for investigation of their pathologic histology. The results of clinic pathology showed that alanine aminotransferase(ALT), aspartate aminotrasferase (AST), blood urea nitrogen(BUN)and creatinine of test groups comparing with that of control group were significantly different or different (P<0.01 or P<0.05). The epithelium cell of renal tubular was degenerated and falling off. Renal tubular was tumid. The renal tubular epithelium cytoplasm was turbid, and renal tubular antrum was cabined. The center veins of liver were dilated and congestive. Liver cells were tumid and degenerated. Spleen sinus had slight gore. Heart muscular fibers and cells were degenerated. Alveolus wall capillary vessels were dilated and congestive. Alveolus wall was infiltrated by macrophage.
引文
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