基于肝微粒体和肝S9成分的食品接触材料中 五氯苯酚体外代谢分析

目的通过体外代谢方法,研究五氯苯酚(pentachlorophenol,PCP)的代谢情况。方法采用肝微粒体和肝S9成分2种体外代谢试剂模拟体内代谢模式,通过优化代谢反应条件,建立PCP、四氯苯醌(Cl4BQ)2种目标物的气相色谱-质谱联用(GC-MS)检测方法,对PCP进行体外代谢研究分析。结果体外代谢分析的最佳反应条件为代谢试剂浓度0.75 mg/m L、代谢反应时间4 h。结论在最优反应条件下分析比较2种试剂对PCP的代谢情况,验证了PCP的代谢产物为Cl4BQ,且在肝微粒体和肝S9成分中的代谢没有显著差异。     

食品包装材料中有害物质双酚A二缩水甘油醚 体外代谢研究

目的通过模拟体内代谢,对双酚A二缩水甘油醚(BADGE)的体外基本代谢情况进行研究。方法采用肝微粒体、肝S9 2种体外代谢试剂,通过模拟体内肝脏代谢,对BADGE的代谢行为及其代谢产物进行研究。通过代谢试剂浓度及代谢时间条件的优化,采用高效液相色谱-串联质谱(HPLC-MS/MS)作为检测手段对BADGE的体外代谢产物进行分析确证。结果体外代谢最佳孵化时间为60 min,最佳体外代谢试剂浓度为0.5mg/m L,在肝S9及肝微粒体2种体外代谢试剂的作用下,BADGE发生显著的代谢反应。结论本研究与传统的动物试验相比,节约了时间、精力,对食品包装材料的毒理学研究和安全性评价有重要的推动作用。     

食品包装材料中丙烯酰胺在大鼠肝微粒和S9中的体外代谢研究

目的研究食品包装材料中丙烯酰胺(acrylamide)的体外代谢情况,并确证其代谢产物。方法采用肝微粒体及肝S9体外温孵法,优化代谢条件,对丙烯酰胺进行了体外代谢研究,并用液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)检测并确证丙烯酰胺的代谢物。结果通过检测结果发现,丙烯酰胺分别在肝S9和肝微粒体作用下发生显著的代谢反应,其代谢产物之一为环氧丙酰胺。结论丙烯酰胺能通过代谢转化成毒性更强的环氧丙酰胺,因此,监控食品、饮水和周围环境中的丙烯酰胺的含量对于维护人们的健康具有重要的意义。     

绿原酸在肝微粒体和肠道菌群中的体外代谢研究

摘 要：目的 研究绿原酸在大鼠肝微粒体和肠道菌群中的代谢产物及代谢途径。方法 运用大鼠肝微粒体体外温孵法和大鼠离体粪便温孵法,采用超高效液相色谱串联四极杆飞行时间质谱(UPLC-Q-TOF/MS),色谱柱为Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 mm×100 mm),流动相为乙腈和0.1%甲酸水溶液梯度洗脱系统,流速为0.35 mL/min,离子源为电喷雾离子源(ESI),以负离子模式进行检测。采用Masslynx软件通过保留时间与一、二级质谱信息,进行成分分析。结果 大鼠肝微粒体温孵样品中有绿原酸原型成分和奎宁酸、咖啡酸2个代谢产物;粪便温孵样品中有绿原酸原型成分和奎宁酸、3-羟基苯丙酸、二氢咖啡酸、二氢阿魏酸、咖啡酸、O-甲基绿原酸6种代谢产物。结论 UPLC-Q-TOF/MS技术鉴定绿原酸及其代谢产物,操作便捷高效,结果准确全面,绿原酸在体外代谢的主要途径为氢化、甲基化、水解、去羟基化等。     

不同种属来源活化体系中CYP2A6酶活及其对烟碱代谢的动力学

为探讨10种不同种属来源活化体系中CYP2A6蛋白的表达水平与酶活性之间的关系,包括来自人体的细胞(BEAS-2B、HepG2和巨噬细胞)及肝微粒体(25供体混合性别人肝微粒体及男性人肝微粒体)、实验室常用不同动物肝微粒体或S9(雄性SD大鼠肝微粒体、雄性恒河猴肝微粒体、雄性比格犬肝微粒体及雄性大鼠诱导肝S9)和同源重组酶,通过免疫印迹法测定其CYP2A6蛋白表达水平,并以烟碱的代谢活化为基础,通过HPLC-MS/MS法测定其酶代谢动力学参数。结果表明:①10种活化体系中CYP2A6酶活具有明显差异,烟碱分别与25供体混合性别人肝微粒体、同源重组CYP2A6酶及雄性恒河猴肝微粒体共孵育时其浓度明显降低,另外7种活化体系中无酶活或活性较低;②CYP2A6蛋白表达水平与CYP2A6酶活具有一定的相关性。研究结果可为卷烟烟气体外毒理学评价中代谢活化体系的选择提供参考,也可为进一步了解CYP2A6基因多态性对烟碱代谢的影响提供体外实验数据。     

亚麻籽油中苯并(α)芘的脱除工艺

针对亚麻籽油中苯并（α）芘残留问题,采用了物理吸附法脱除亚麻籽油中的苯并（α）芘。以活性炭与活性白土为吸附剂,通过二者单独作用及其混合使用,比较三种吸附剂对苯并（α）芘的吸附速率,并采用超高效液相色谱-四级杆-飞行时间串联质谱法结合NIST标准图谱库对经脱除装置处理前后的亚麻籽油中不饱和脂肪酸成分含量进行了鉴定分析。结果表明活性白土的固定用量为4%时,苯并（α）芘脱除率将近达到40%;活性炭的用量为2%时,其脱除率达到83%;将二者串联使用时,脱除率可达到96%。另外苯并（α）芘浓度<15 μg/kg时使用活性白土吸附过滤;苯并（α）芘浓度为15~30 μg/kg时使用活性炭吸附过滤;苯并（α）芘浓度>30 μg/kg时使用二者串联吸附过滤,因此物理吸附法去除亚麻籽油中苯并（α）芘效果最好的是将活性白土与活性炭串联处理,其最佳条件为:活性粘土与活性炭串联比例为0.8%+4%,在此条件下去除率可达96%。同时利用吸附剂对亚麻籽油中不饱和脂肪酸并没有造成损失。综上,本文为亚麻籽油中苯并（α）芘的脱除提供了理论依据。     

植物油中苯并(α)芘的净化方法比较研究

通过试验,分析对比了GB 5009.27—2016中2种净化方法,发现采用分子印迹柱测定植物油中苯并(α)芘含量,比采用中性氧化铝柱,能大大缩短净化时间、节约试剂,重现性好,线性关系、精密度和准确度均能获得满意的结果,可为植物油中苯并(α)芘的检测提供参考。     

芘荧光探针应用于狭鳕鱼皮凝胶形成过程的可行性研究

在狭鳕鱼皮弹性水凝胶的动态形成过程中引入了芘荧光探针。结果表明,在6.67%的鱼皮明胶溶液中,芘单体的荧光强度和I1/I3值均随芘浓度的增大先升高后降低,当芘浓度为40μmol/L时,单体荧光强度达到最大值,而当芘浓度为20～60μmol/L时,I1/I3值均处于较高水平,数值相近;探针的IE/IM值随芘浓度的增大一直升高,当芘浓度为2～40μmol/L时,比值偏低。综合考虑上述三个因素,选用浓度为40μmol/L的芘作为荧光探针应用于狭鳕鱼皮水凝胶的动态形成过程。实验确定芘I1/I3值能够用来表征溶胶中分子间的交联情况,其凝胶时间点为7h;但是IE/IM值则无法表征分子间的交联。这为表征其它鱼皮凝胶的动态形成过程提供了重要基础。     

油橄榄叶羟基酪醇提取工艺优化及抗氧化研究

研究油橄榄叶中羟基酪醇(HT)的最佳提取工艺及其抗氧化活性。在单因素试验基础上,采用BoxBehnken中心组合设计原理,研究了提取温度、时间、盐酸浓度和液料比对羟基酪醇提取率的影响。比较了不同浓度羟基酪醇、维生素C、橄榄叶粗提物、白藜芦醇的还原能力、羟自由基清除能力以及DPPH·清除能力大小。以羟基酪醇提取率为响应值,利用Design Expert 7.0软件得到回归方程的预测模型并进行响应面分析;油橄榄叶羟基酪醇最佳提取条件为:液料比50.74︰1(m L/g),提取温度92.39℃,盐酸浓度0.14 mol/L,提取时间6.02 h。该条件下羟基酪醇的提取率达到0.58%。验证性试验表明,所得模拟方程能较好地预测试验的结果。体外抗氧化试验结果表明,HT具有较强的体外抗氧化活性,对DPPH·清除的能力超过了维生素C。     

改性Fenton试剂法氧化降解染色废水及其工艺优化

文章为了克服均相Fenton催化剂难以分离的缺点,采用羟基铁粉取代二价铁制成改性Fenton试剂,并使用改性Fenton试剂处理染色废水。探讨反应温度、反应时间、溶液pH值及羟基铁粉的用量等因素对处理效果的影响,并通过分析氧化降解前后染色废水溶液紫外-可见光谱曲线和COD值的变化情况来确定改性Fenton试剂处理染色废水的最佳工艺条件。根据实验结果得知最佳反应条件为:反应温度为室温,反应时间为30min~40min,溶液pH值为2~3,羟基铁粉的用量为1.0g。     

Studies on the mutagenicity of aniline in association with norharman in the Salmonella/mammalian microscome assay

The mutagenicity of aniline was investigated in association with norharman using Aroclor 1254–induced and uninduced liver S9 fractions. The non-mutagenicity of aniline in the Salmonella /mammalian microsome test was also re-examined under various experimental conditions. Aniline caused no increase in the number of revertants per plate in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 at concentrations of up to 5000 μg/plate. S9 preparations from rat liver, hamster liver, rat spleen, or hamster spleen were all ineffective in activating aniline to a mutagen in tests using various concentrations of S9 in S9 mix. In contrast to the results with aniline alone, mixtures of aniline and norharman were mutagenic in strain TA1538 in the presence of S9 fractions from liver of rats pretreated with Aroclor 1254. The observed mutagenicity decreased as a function of the concentration of S9 in S9 mix. In the presence of uninduced liver S9 preparations, the mutagenicity of the mixture of the two compounds was slight (three-fold).     

缺乏VB6对大鼠必需脂肪酸代谢的影响

以大鼠为实验动物,用高蛋白质饲料成功诱导缺乏VB6的生理状态,分析了缺乏VB6对必需脂肪酸代谢的影响。实验结果表明:缺乏维生素B6实验大鼠肝脏微粒体的△6去饱和酶活性大幅度降低、ω-6系和ω-3系必需脂肪酸代谢受到严重阻碍,肝脏和血浆脂质中出现代谢起始物的蓄积和代谢产物的显著减少,ω-3系还出现中间产物EPA的增加。     

Temporal effects of ruminal infusion of propionic acid on hepatic metabolism in cows in the postpartum period

A faster rate of infusion of propionic acid into the rumen of cows in the postpartum period increased meal size compared with a slower rate of infusion in a previous experiment. Because propionate is anaplerotic and stimulates oxidation of acetyl coenzyme A (CoA) in the liver, and hepatic oxidation has been linked to satiety, this result was opposite to our expected response. We then hypothesized that the faster rate of infusion might have saturated the pathway for propionate metabolism in hepatocytes resulting in lower first-pass extraction by the liver. Because we were measuring feeding behavior, we could not sample blood and liver tissue over time in that experiment. Therefore, to determine the temporal effects of propionic acid (PA) infusion on hepatic metabolism and plasma metabolites over the time course of a meal, we infused 1.25 mol of PA (2.5 L of 0.5M PA) over 5 min (FST) or 15 min (SLW) into the rumen. We evaluated response to PA infusions both before feeding, when ruminal PA production by rumen microbes is lower and hepatic acetyl CoA concentration is greater, and 4 h after feeding, when PA production is greater and hepatic acetyl CoA concentration is lower. Blood and liver samples were collected before, and after 5, 15, and 30 min of infusion. Contrary to our hypothesis, the rate of PA infusion into the rumen did not affect plasma propionate concentration, indicating the FST effects on feeding behavior were not because of a limitation on propionate uptake by the liver. However, FST increased plasma glucose and insulin concentrations faster than SLW, resulting in a reduction in plasma nonesterified fatty acid concentration during the time frame of meals. Decreased plasma nonesterified fatty acid concentration during infusion likely decreased the supply of acetyl CoA for oxidation in the liver. The FST treatment also increased fumarate concentration at 5 min after the initiation of infusion but did not affect oxaloacetate concentration compared with SLW, consistent with a limitation to propionate metabolism at that reaction. A metabolic bottleneck at the malate dehydrogenase reaction for FST compared with SLW would further contribute to a reduction in hepatic oxidation within the time frame of a meal, allowing greater meal size, consistent with the hepatic oxidation theory and our previous results.     

Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads

We present an improved cytotoxicity test for reactive metabolites, in which the S9 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of S9-self-generated toxicants, microsomal lipid peroxides. The S9-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the S9-encapsulated gel microbeads was about 20% slower than that of bare S9, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the S9-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity.     

Integrated Remodeling of Gut–Liver Metabolism Induced by Moderate Protein Restriction Contributes to Improvement of Insulin Sensitivity

1 Scope

Protein restriction (PR) is beneficial for relieving metabolic disorders and aging‐related diseases. However, extreme PR could result in malnutrition due to severe deficiency of essential amino acids. Therefore, the effect of moderate PR on insulin sensitivity is investigated.

2 Methods and results

The growing and adult pigs are subjected to moderate PR by 15–30%. Plasma insulin concentration and insulin resistance index HOMA‐IR are significantly decreased upon moderate PR. Furthermore, IRS1/PI3K/AKT pathway in the basal state is enhanced in both liver and skeletal muscle. The adapted metabolism in the liver upon moderate PR is in support of improving insulin sensitivity. The liver shares a coordinated metabolic adaption in terms of energy metabolism and amino acid metabolism with the small intestine. Particularly, alteration of the metabolic footprint appeared in the portal venous blood, representing metabolites to be absorbed into liver after intestinal metabolism, is also in favor of improvement of insulin sensitivity.

3 Conclusion

In summary, the study proves that moderate PR could improve insulin sensitivity from childhood to adulthood in a pig model, and sheds a new light on the role of integrated remodeling of gut and liver metabolism in the improved insulin sensitivity induced by moderate PR.     

Investigation of transformation products of selected veterinary drugs generated by electrochemistry,microsomal assays,hydrolysis & photolysis

The knowledge of transformation pathways and transformation products of veterinary drugs is important for health, food and environmental matters. Residues (original veterinary drug and transformation products) are found in food products of animal origin and also in the environment (e.g., soil or surface water). Several transformation processes can alter the original veterinary drug, ranging from biotransformation in living organism to environmental degradation processes like photolysis, hydrolysis, or microbial processes. In this thesis, four veterinary drugs were investigated, three ionophore antibiotics Monensin, Salinomycin and Lasalocid and the macrocyclic lactone Moxidectin. Ionophore antibiotics are mainly used to cure and prevent coccidiosis in poultry especially prophylactic in broiler farming. Moxidectin is an antiparasitic drug and is used for the treatment of internal and external parasites in food- producing and companion animals. The main objective of this work was the usage of different laboratory approaches to generate and identify transformation products. The identification was conducted using high-resolution mass spectrometry (HR-MS). A major focus was put on the application of electrochemistry for simulation of transformation processes. The electrochemical reactor, equipped with a three-electrode flow-through cell, enabled the oxidation or reduction by applying a potential. Derived transformation products were analyzed by online coupling of the electrochemical reactor and a HR-MS and offline by liquid chromatography (LC) combined with HR-MS. The main modification reaction of the identified transformation products was different for each investigated veterinary drug. Monensin was showing decarboxylation and demethylation as main modification reactions, for Salinomycin mostly decarbonylation was occurring and for Lasalocid methylation was prevalent. For Moxidectin I observed oxidation (hydroxylation) reaction and adduct formation with solvent. In general, for salinomycin and Lasalocid more transient transformation products (online measurement) than stable transformation products (offline measurements) were detected. In contrast, the number of transformation products using online and offline measurements were identical for monensin and moxidectin. As a complementary approach, metabolism tests with rat or human liver microsomes were made for the ionophore antibiotics. Monensin was investigated by using rat liver microsomes and identified transformation products were based on decarboxylation and demethylation. Salinomycin and Lasalocid were converted by human and rat liver microsomes. For both substances were more transformation products found by using human liver microsomes. The transformation products of the rat liver microsome conversion were redundant, the transformation products were also found at the human liver microsome assay. Oxidation (hydroxylation) was found to be the main modification reaction for both. In addition, a frequent ion-exchange between sodium and potassium was identified. The last two experiments were performed for one substance each, the hydrolysis of monensin and the photolysis of moxidectin was investigated. The transformation products of the pH- dependent hydrolysis were based on ring-opening and dehydration. Moxidectin formed several transformation products by irradiation with UV-C light and main modification reactions were isomeric changes, (de-)hydration and changes of the methoxime moiety. In summary, transformation products of the four investigated veterinary drugs were generated by the different laboratory approaches. Most of the identified transformation products were identified for the first time. The resulting findings provide an understanding for clarifying the transformation behavior.     

酶改性大豆分离蛋白的制备及产品功能性的研究

为了改善碱溶酸沉法大豆分离蛋白产品(简称SPI)的功能性,采用有限酶解的方法,从6种蛋白酶中选出中性蛋白酶(Neutrase)作为改性用酶.通过单因素实验,分析了底物浓度,E/S,反应时间对水解度和氮收率的影响,并通过正交实验确定制备酶改性大豆分离蛋白(简称ESPI)的最佳工艺参数为:底物浓度为3%,E/S为1 200U/g,时间为60 min.得到的ESPI的蛋白含量与SPI相近,但NSI由SPI的90%提高到97%.而且在功能性方面,ESPI也得到了很好的改善,尤其是乳化性和起泡性.     

AGE RELATED CHANGES IN POULTRY BREAST MEAT COLLAGEN PYRIDINOLINE AND TEXTURE

Pyridwotine (PYR), a mature collagen crosslink, ‐was measured in Ross hen breast meat, Pectoralis major, age ranging from 20 to 540 days and its concentration varied between 0.009 to 0.101 mol/mol collagen, respectively. PYR concentration was inversely related to collagen solubility in muscle homogenates (P>0.05) and total collagen content increased from 0.448 to 0.568% from 20 to 540 days, respectively. Shear values analyzed by texturometer showed a direct relationship between PYR amount with advancing poultry aging irrespective of collagen quantity (P > 0.05). Finally, there was also a direct relationship between the increase of PYR concentration and collagen solubility measured in Ringer solution.