Simultaneous Determination of Hexoestrol, Diethylstilbestrol, Estrone and 17-Beta-estradiol in Feed by Gas Chromatography-mass Spectrometry

A method was developed for the simultaneous determination of four kinds of estrogens （hexoestrol, diethylstilbestrol, estrone, and 17-beta-estradiol） in feed by gas chromatography-mass spectrometry （GC-MS）. After the sample was extracted by ethyl ether and cleaned-up on HLB phase extraction column, four kinds of estrogens were derived and quantified in gas chromatographymass spectrometry. The results showed that the linear detectable ranged from 2.5 ng· mL-1 to 250 ng· mL-1for hexoestrol and from 5 ng· mL-1 to 500 ng· mL-1 for three other estrogens with the correlation coefficients （R2） were no less than 0.990. The recoveries were in the range of 76.34%-96.33% and the relative standard deviation was no more than 22.7%. The limits of quantitation （LOQ） for all analytics were between 10 ug· kg^-1 and 20 ug· kg^-1. The method was accurate and sensitive and could meet the actual requirements for the analyses of feed samples.

Production and Characterization of Anti-estrone Monoclonal Antibody

Objective Determination of estrone (E1) levels has a significant meaning in evaluating physiological effect and diagnosing some diseases. In order to detect free E1 in biological fluids, a monoclonal antibody specific for E1 was prepared after the complete antigen of E1 was synthesized. The purified monoclonal antibody was fully characterized for later immunoassay. Methods 3-O-carboxymethyl ether derivative of E1 was synthesized and in turn coupled to bovine serum albumin (BSA) to form complete antigen E1-BSA. A monoclonal antibody (McAb) specific for E1 was produced both in vitro and in vivo by a hybridoma anti-E1. Anti-E1 was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse. The McAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western-blotting. The specificity of the immunoassay was investigated by determining the cross-reactions of E1 analogs when free E1 was detected by competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Results Analysis revealed that anti-E1 McAb (E1-McAb) was of the IgG1 type, the molecular weight of E1-McAb was 164 000 daltons. The affinity constant of E1-McAb with coated complete antigen was 8.2108L/mol. The linear range for free E1 determined by CI-ELISA was 10pg/mL^10ng/mL. The detection limit was 21.4 pg/mL (defined as twice the standard deviation of the blank). Conclusion The CI-ELISA developed with E1-McAb was both sensitive and specific. The prepared E1-McAb can be used in some immunoassays.

Oxidative Metabolism of Estrone Modified by Genistein and Bisphenol A in Rat Liver Microsomes

Genistein, the main isoflavone from soy, and bisphenol A （BPA）, a food contaminant, are considered ubiquitous xenoestrogens. Here we investigated the influence of genistein and BPA on estrone （El） metabolism in rat liver microsomes. Both substances inhibited the 2-hydroxylation and 16a-hydroxylation of E1, but in different degrees, thereby reducing the 2-OH-E1/16a-OH-E1 ratio,

Molecular Docking Studies of Estrone-Coumarin Derivatives as Aromatase and 17β-HSD1 Inhibitors Related to Hormone Receptor Positive (HR+) Breast Cancer

Hormone Receptor positive (HR+) breast cancer is the most common malignancy in women. New strategies in the treatments have targeted the estrogen biosynthesis pathways including the inhibition of the aromatase and 17β-HSD1 enzymes. The present work, describes the study of a new family of 9 hybrid compounds derived from estrone attached to a coumarin fragment, linked through different lengths of hydrocarbon chains. The activity of these compounds was evaluated by molecular docking with two relevant enzymes in breast cancer (HR+). It has been proposed nine compounds as 17β-HSD1 inhibitors and six as aromatase inhibitors. We found important interactions with key amino acids at the orthosteric site of each enzyme and their score values compared to the crystallographic ligand. The in silico analysis showed good score values in the proposed compounds, where the steroidal portion presented important interactions with Met374 and Tyr155 in aromatase and in 17β-HSD1 respectively. Highlighting Compounds 2, 5 and 8 with an aromatic ring at the C4 position of the coumarin moiety, which favored arene-H type interactions essential for protein-ligand recognition. In addition, the results related to the 17β-HSD1 enzyme demonstrated how the length of the linker influences the interaction;the best score was found for derivative 8 with a chain of 8 methylenes.

Antibody Production for a Rapid Fluorescence Polarization Immunoassay of Estrone

Estrone has been identified as a potential endocrine-disrupting chemical （EDC）[1]. Estrone is usually quantified by gas chromatography-mass spectrometry （GC-MS）, GC-MS/MS, high performance liquid chromatography （HPLC）, HPLC- MS, and HPLC-MS/MS, etc.[2-3]. Meanwhile, several immunoassays based on radioimmunoassay, enzyme linked immunosorbent assay （ELISA） or chemiluminescence immunoassay （CLIA） for determination of estrone in real samples have been developed[2＇4]. Although these methods are sensitive, they need multistage separation and are thus time-consuming and laborious. A very promising way for the simplification of immunoassays for routine applications is a shift from heterogeneous methods （with separation） to homogeneous assays （without separation）[5]. Fluorescence polarization immunoassay （FPIA） is one of the homogeneous techniques that meets the requirements of a simple, reliable, fast, and cost-effective analysis[6]. Therefore, the present study is focused on the development of FPIA in order to analyze estrone based on antibody production.

SYNTHESIS OF NEW ESTRONE DERIVATIVES OF AMINO ACIDS AND PEPTIDES

A series of estrone derivatives of amino acids and peptides haze been synthesized by different coupling reagents and the binding affinity of deblocked derivatives to the estrogen receptor of rats uteri have been measured by a comptetive radiometric bind- ing assay.

Oxidation of estrone by permanganate: Reaction kinetics and estrogenicity removal

Permanganate was used as an oxidant to control estrone in the present study. Kinetics was determined at pH 2.5-9.4 and temperature 15–40°C for the reaction of estrone with potassium permanganate. It was found that the reaction is second-order overall and first-order with respect to both estrone and permanganate. The second-order rate constant for the reaction at pH 5.8 and 25°C is 44.45 L mol-1 s-1. The reaction rate first decreased with the increase of pH in the range of 2.5-6.6 and then increased greatly with the increase of pH in the range of 6.6-9.4. In addition, the rate constant exponentially increased with the increase of reaction temperature. Removal of estrogenicity was also investigated during the degradation of estrone using yeast estrogen screen (YES). Results show that the estrogenicity increased in the initial 15 min of reaction and then decreased fast, with a removal rate of 73.8% within the 30 min of reaction. Results also demonstrate that the reaction rate between estrone and permanganate is faster in natural water background than in the ultra-pure water system. Permanganate oxidation is therefore a feasible option for removal of estrone in drinking water treatment processes. However, the contact time must be enough in order to remove estrone without causing the increase of estrogenicity.

A SIMPLE TWO-STEP SYNTHESIS OF Δ^(6(7))-ESTRONE

With the continuous developments in labeling techniques, assay methods of radioisotopes and their increasing applications in physiological and biochemical research, it has become both possible and necessary to supply highly active labeled estrogens. 6, 7-3H2-estrone and 6, 7-3H2-estradiol are the more widely used commercial products

雌酮硫酸钠的制备及其对小鼠心肌缺血再灌注损伤保护作用的研究

目的研究雌酮硫酸钠(Estrone sulfate sodium,ES)的制备方法及其对小鼠心肌缺血再灌注损伤的保护作用。方法(1)采用雌酮和氨基磺酸通过氨基磺酸化和离子置换法合成ES。采用核磁共振内标法计算ES的纯度,采用紫外光谱法、红外光谱法、高分辨质谱、核磁共振氢谱和核磁共振碳谱对ES进行结构分析。(2)建立雌鼠更年期模型,将小鼠随机分为溶媒组(蒸馏水0.094 mg/kg体重)、雌二醇组[(Estradiol,E2),给药剂量0.3 mg/kg体重]和ES组(给药剂量0.094 mg/kg体重)灌胃给药2周后建立小鼠心肌缺血/再灌注损伤模型,采用2,3,5-氯化三苯基四氮唑(TTC)染色法测量心梗面积,评估ES预处理对小鼠心肌损伤的影响。结果合成的ES纯度为66.6%,Tris含量30.8%,表征结果显示产物为雌酮硫酸钠。与溶媒组比较,E2降低小鼠心肌缺血/再灌注损伤面积,但是E2组差异不具有统计学差异,而ES组小鼠心肌缺血/再灌注损伤导致的心肌梗死面积减少,且差异具有统计学意义(P<0.05)。结论本合成方法能够得到纯度较高且稳定的ES,动物试验结果证明ES能够减少小鼠心肌缺血再灌注损伤后的心肌梗死面积。

珠江广州河段表层水中雌激素化合物的污染状况

分析了珠江广州河段枯水期表层水中雌激素化合物的污染状况.结果表明,在所有样品中均检测出了双酚A(BPA)和雌酮(E1),其浓度范围分别为97.8—540.6ng·l-1(中值为145.9ng·l-1)和2.5—8.2ng·l-1(中值为4.5ng·l-1);两种化合物的最高值都出现在前航道的东圃大桥,最低值则在黄埔水道的黄埔港,其空间分布主要与沿途生活污水和工业废水的排放有关.

高效液相色谱-串联质谱法测定海水中雌酮、雌二醇、雌三醇

建立了测定海水中雌酮、雌二醇和雌三醇的高效液相色谱-串联质谱的分析方法。样品的提取方法为固相萃取,流动相为乙腈和0.1%氨水,梯度洗脱,流速0.2 mL/min,运行时间10 min。质谱采用负离子扫描模式,定量的碎片离子分别是：雌酮：269.02/144.99;雌二醇：271.04/182.96;雌三醇：287.03/170.94。仪器检出限均为0.001 ng,方法检出限均为0.2 ng/L。回收率分别是78.0-110.0%,82.2-103.2%,76.4-95.1%。该方法适用于海水中雌激素类物质的检测。

血清性激素与乳腺密度和绝经后女性乳腺癌的相关性研究

目的：探讨绝经后女性血清雌激素和雄激素水平与乳腺密度和乳腺癌的相关性。方法选择2013年6月至2014年4月期间在哈尔滨医科大学附属第三医院接受乳腺肿瘤手术治疗的绝经后女性患者；应用ELISA试剂盒检测患者血清雌二醇、雌酮、睾酮和雄烯二酮水平；分析患者乳腺钼靶X线图像，以乳腺影像报告和数据系统BI-RADS标准对乳腺密度进行评估，按乳腺百分密度分成≤25％、26％～50％、51％～75％和＞75％共4个等级。结果符合筛选条件的绝经后女性患者包括90例乳腺癌和32例乳腺良性肿瘤患者，乳腺癌患者血清雌二醇水平高于乳腺良性肿瘤患者（ P＜0.05），两者血清雌酮、睾酮和雄烯二酮水平无明显差异（P＞0.05）；两者乳腺密度分布未显示出显著差异（P＞0.05）；相关性分析显示这些绝经后乳腺肿瘤患者血清睾酮与乳腺密度呈正相关（ P＜0.05）。结论绝经后女性血清雌二醇水平与乳腺癌发病相关，其代谢前体睾酮与乳腺密度呈正相关，提示绝经后女性性激素水平与乳腺密度和乳腺癌发病均相关。

枸杞多糖对自然衰老雌性大鼠卵巢保护作用机制的探讨

目的:研究枸杞多糖(Lyceum barbarum Polysaccharides,LBP)对自然衰老雌性大鼠雌二醇(E2)、孕酮(P)、IGF-Ⅰ、IGFR和IGFBP-1水平的影响,初步探讨枸杞多糖对自然衰老雌性大鼠作用的机制。方法:14月龄SD大鼠随机分为模型组、阳性对照组及枸杞多糖高、中、低剂量组,4月龄SD雌性大鼠为正常对照组。给药30 d后,取血及卵巢组织,放射免疫(RIA)法测定血清中雌二醇(E2)、孕酮(P)水平,酶免疫法测定血清中的IGF-Ⅰ与IGFR及卵巢组织中的IGFBP-1与IGFR水平。结果:卵巢功能衰退与激素(如雌激素、孕激素及IGF-Ⅰ)分泌能力呈正相关,而与IGFBP-1水平呈负相关。灌服不同剂量的LBP后能明显升高雌激素、孕激素及IGF-Ⅰ的水平,降低IGFBP-1水平,提示LBP能积极对抗卵巢功能的下降。结论:枸杞多糖对大鼠卵巢系统具有保护作用。

哌嗪雌酚酮对去卵巢大鼠骨代谢的作用

目的 研究哌嗪雌酚酮 (一种新合成雌激素哌嗪类衍生物 )对去卵巢大鼠骨代谢及子宫的作用。方法雌酚酮 (E) 0 75mg·kg- 1 ·d- 1 ,哌嗪雌酚酮 (P E) 1和 10mg·kg- 1 ·d- 1 ,ig共 90d ,骨形态计量学测量。结果 去卵巢大鼠子宫萎缩 ,血清胆固醇升高 ,骨量减少伴骨高转化的改变。E组与去卵巢组比 ,子宫重量增加 ,血清胆固醇降低 ,骨高转化降低。P E(1mg)组与去卵巢组比 ,子宫重量轻微增加 ,骨量增加 ,骨形成和骨吸收均维持在去卵巢组的高水平。P E(10mg)组比低剂量组增加更多的骨量 ,但其作用机理与雌酚酮相同 ,明显地降低骨转化。结论 哌嗪雌酚酮可预防去卵巢大鼠所致的骨丢失。低剂量 (1mg)

畜禽粪便改良土壤中E1和E2自然降解的影响因素

研究了E1(雌酮)和E2(17β-雌二醇)在牛粪、猪粪和鸡粪改良土壤自然降解过程中灭菌、光照和温度的影响以及泰乐菌素与吐温80的复合影响.对比灭菌和未灭菌环境中E1和E2的降解效果表明,灭菌能显著抑制E1的降解,而对E2的降解影响不显著;对比光照和黑暗环境中E1和E2的降解可知,光照对二者的影响不大;在前2种条件下,E1和E2的降解效果(以去除率计)不受粪便类型的影响.在0～35℃,雌激素E1和E2的去除率随温度的升高呈增大趋势,其中E1在牛粪改良土壤中的去除率由1.21%增至58.04%,E2的去除率由1.24%增至48.93%;环境中共存的吐温80能促进E1和E2的降解,当泰乐菌素与吐温80共存时,可有效促进E2的降解.

控温相变免疫分析测定雌酮

通过热引发聚合与氧化还原引发聚合 ,合成水凝胶 ,作为雌酮全抗原 (E1 BSA)载体 ;以异硫氰基荧光素标记雌酮的单克隆抗体 (McAb )作为示踪剂 ,建立了控温相变竞争免疫分析测定雌酮 (E1 )的方法并应用于分析血清样品。实验表明 :以热引发聚合合成的水凝胶为载体的荧光免疫分析方法具有更高的灵敏度 ,它的线性范围为 1 0～ 70 0 μg L ,检出限为 1 μg L(n=1 0 ,3倍空白 )。经水凝胶性质的比较 (包括LCST、非特异性吸附、抗原与抗体反应时间及固定化抗原活性 ) 。

高效液相色谱-串联质谱法测定青菜中雌酮、雌二醇和雌三醇残留

为了监测蔬菜被粪源类固醇激素污染状况,本试验建立同时测定青菜中雌三醇、17β-雌二醇、17α-雌二醇和雌酮残留量的高效液相色谱-串联质谱方法。青菜经乙腈超声提取,C18 固相萃取柱去除杂质,氮吹浓缩处理后,用梯度洗脱上机检测。结果显示：青菜中雌三醇、17β-雌二醇、17琢-雌二醇和雌酮得到很好的分离,标准曲线在10 ng/ ml至1 000 ng/ ml浓度范围内呈线性关系,相关系数逸0.999,各自的保留时间分别为2.6 min、8.2 min、8.9min 和9.9 min;当添加浓度为50 μg/ kg、100 μg/ kg和500 μg/ kg时,雌三醇、17β-雌二醇、17α-雌二醇和雌酮在青菜中的平均回收率分别为75. 15%-86.32%、73. 82%-83.21%、72. 01%-81.33% 和81. 08%-90.35%,相对标准偏差为1. 03% -6.19%,该方法17β-雌二醇和17α-雌二醇的定量限均为10 μg/ kg,雌三醇的定量限为5 μg/ kg,雌酮的定量限为1 μg/ kg。表明该方法适用于蔬菜中雌三醇、17β-雌二醇、17α-雌二醇和雌酮残留的同步快速检测。

液相色谱-串联质谱法检测动物肝肾组织中3种雌激素

建立了动物肝、肾组织中雌酮、17β-雌二醇、雌三醇药物残留检测的液相色谱-串联质谱(LC-MS/MS)分析方法。样品以叔丁基甲醚提取,于45℃下氮吹至干,用正己烷-二氯甲烷(6∶4,v/v)重新溶解,过硅胶固相萃取小柱净化,氮吹至干,用1 mL乙腈-水(7∶3,v/v)定容后测定,用Poroshell 120 EC-C18柱分离,以乙腈和水作为流动相进行梯度洗脱,电喷雾负离子模式电离,多反应监测模式检测,外标法定量。该方法对3种雌激素的线性范围均为1.0～20.0μg/kg,相关系数大于0.99,定量限为1.0μg/kg。在不同基质中,1.0、2.0、10.0μg/kg 3个添加水平的平均回收率范围为70.2%～114%,相对标准偏差范围为2.01%～14.5%。该方法具有快速简便、净化效果好、灵敏度高等特点,适用于动物肝、肾组织中雌激素的检测。

酶免疫法测定尿液生殖激素在正常和体外受精周期中的应用

目的 :建立一种用尿酶免疫方法监测尿生殖激素促卵泡素 (FSH β)、雌酮结合物 (E1C)、孕二醇 3 葡糖苷酸 (PdG)在正常月经周期和人工受精 (IVF)周期中的动态变化。方法 :对 11例正常生育年龄妇女用尿酶免疫法测定月经周期中尿FSH β、E1C、PdG水平 ,10例同时抽血用放射免疫法测定血FSH、雌二醇 (E2 )、孕激素 (P)水平。用尿酶免疫法测定 18例IVF周期治疗的患者尿FSH β、E1C、PdG水平的变化。结果 :10例正常生育年龄妇女 2 8天月经周期中血FSH、E2 、P与尿FSH β、E1C、PdG周期变化的一致性非常好 ,19个正常月经周期中尿生殖激素水平变化曲线也说明该点。 18例IVF周期由于持续大量促性腺激素释放激素对垂体的降调节作用 ,在排卵期无FSH和LH峰。结论 :酶免疫法测定尿生殖激素在IVF周期中。